Volume 59 · Supplement 1 · 2009
Volume 59 · Supplement 1 · 2009
The XXXVI International Congress of Volume 59 · Supplement 1 · 2009 · pp 1–XX · pp 1 · 2009 Volume 59 · Supplement Physiological Sciences (IUPS2009) International Scientific Program Committees (ISPC) ISPC Chair Yoshihisa Kurachi Vice Chair Ole Petersen ISPC from IUPS Council Akimichi Kaneko (IUPS President) Irene Schulz (IUPS Vice President) Pierre Magistretti (IUPS Vice President) Malcolm Gordon (IUPS Treasurer)
ISPC IUPS2009 Members and Associated Members Proceedings of the XXXVI International Congress of Physiological Sciences (IUPS2009) Commission I Locomotion Commission VII Comparative Physiology: Hans Hoppeler, Masato Konishi, Hiroshi Nose Evolution, Adaptation & Environment Function of Life: Elements and Integration Commission II Circulation/Respiration Malcolm Gordon, Ken-ichi Honma, July 27–August 1, 2009, Kyoto, Japan Yung Earm, Makoto Suematsu, Itsuo Kodama Kazuyuki Kanosue Commission III Endocrine, Reproduction & Commission VIII Genomics & Biodiversity Development David Cook, Hideyuki Okano, Gozoh Tsujimoto Caroline McMillen, Yasuo Sakuma, Toshihiko Yada Commission IX Others Commission IV Neurobiology Ann Sefton, Peter Hunter, Osamu Matsuo, Quentin Pittman, Harunori Ohmori, Fumihiko Kajiya, Tadashi Isa, Tadaharu Tsumoto, Megumu Yoshimura Jun Tanji Commission V Secretion & Absorption Local Executives Irene Schulz, Miyako Takaki, Yoshikatsu Kanai Yasuo Mori, Ryuji Inoue Commission VI Molecular & Cellular Biology Cecilia Hidalgo, Yoshihiro Kubo, Katsuhiko Mikoshiba, Masahiro Sokabe, Yukiko Gotoh P1AM-1-1 P1AM-1-2 FAMILIAL ISOLATED CARDIAC CONDUCTION DEFECT THE EFFECTS OF INTERCELLULAR ELECTRICAL ASSOCIATED WITH A MUTATION IN THE CONNEXIN40 UNCOUPLING ON THE DYNAMICS OF SPIRAL GENE GJA5 REENTRY IN VENTRICULAR MYOCARDIUM OF Naomasa Makita1, Naokata Sumitomo2, Akiko Seki3, Nobuhisa Hagiwara3, ISOLATED RABBIT HEARTS Jean-Jacques Schott4, Hiroyuki Tsutsui1 Yoshio Takemoto1, Tomoyuki Suzuki1, Sara Kato1, Hiroki Takanari1, 1Department of Cardiovascular Medicine, Hokkaido University Graduate Mikio Morishima1, Yusuke Okuno1, Masahide Harada1, Haruo Honjo1, 2 School of Medicine, Japan, Department of Pediatrics and Child Health, Nihon 2 1 1 3 Ichiro Sakuma , Kaichiro Kamiya , Itsuo Kodama University School of Medicine, Tokyo, Japan, Department of Cardiology, Tokyo 1 4 Department of Cardiovascular Research, Research Institute of Environmental Women’s Medical University, Tokyo, Japan, INSERM U915, l'institut du thorax, 2 Medichine, Nagoya University, Japan, Graduate School of Engineering, Nantes, France University of Tokyo, Japan Isolated cardiac conduction defect (ICCD) is a hereditary lethal arrhythmia, characterized Background: Gap junction (GJ) remodeling associated with heart diseases increases the risk by slow conduction in the specialized conduction system without underlying structural heart of life-threatening ventricular tachyarrhythmia (ventricular fibrillation /tachycardia: VF/VT), diseases. We have genetically screened gap junction (GJ) genes for 139 ICCD probands. In but underlying mechanisms remain unclear. one ICCD family with two sudden death victims, we found a missense mutation Q58L in We investigated the effects of intercellular uncoupling on the dynamics of spiral-wave connexin40 (Cx40), a GJ predominantly expressed in the atrium and His-Purkinje system. (SW) reentry, which is the principle mechanism of VF/VT. Methods: Optical action The mutation carriers showed identical ECG of idioventricular rhythm and complete left potential signals were recorded from ventricles of Langendorff-perfused rabbit hearts. bundle branch block. GFP-tagged Q58L Cx40 transfected in GJ-deficient N2A cells showed Results: Inhibition of GJ channels by carbenoxolone (CBX, 30 μM) significantly decreased predominant expression in the intracellular space and impaired GJ formation. Intracellularly conduction velocity (by 26.4±9.0 %, n=11) and the space constant (by 35.7±7.9 % n=7) applied dye Lucifer yellow was readily transferred to the neighboring cells in wild type (WT), without affecting action potential duration. SW reentry in controls showed meandering along but its transfer was delayed in the Q58L. Furthermore, junctional conductance measured a functional block line (FBL, 5.80±0.42 mm, n=6). After CBX infusion, a stationary rotor by the dual whole-cell patch clamp was severely impaired in Q58L cell pairs (12.9±5.8 nS circulated around shorter FBL (1.74±0.16 mm, n=6). The incidence of sustained reentry for WT (n=4) vs. 1.2±0.7 nS for Q58L (n=5); p<0.01), suggesting that the genetic defect of episodes (>30 s) significantly increased from 22/73 to 53/67 after CBX, and average VT GJA5 disrupts cell-to-cell communication in the atrium and His-Purkinje system, leading to cycle length significantly increased from 132±12 ms to 149±23 ms (n=25, p<0.0001). manifest ICCD. In conclusion, present study has provided the first demonstration that GJA5 Conclusions: Intercellular electrical uncoupling of ventricular myocardium by inhibition of is the responsible gene for familiar ICCD. GJ channels stabilizes SW reentry in favor of its persistence.
P1AM-1-3 P1AM-1-4 REGIONAL HETEROGENEITY OF CONNEXIN-43 GENERATION OF THE ELECTROCARDIOGRAPHIC COUPLING PROMOTES SUSCEPTIBILITY TO T WAVE AND BODY SURFACE POTENTIAL REENTRANT ARRHYTHMIAS IN RAT CULTURED DISTRIBUTION IN FROGS AND PIKES MYOCYTE MONOLAYERS Marina Vaykshnorayte, Jan Azarov Hideo Tanaka, Takuo Nakagami, Tetsuro Takamatsu Laboratory of Cardiac Physiology, Institute of PhysiologyKomi Science Department of Pathology and cell Regulation, Kyoto Prefectural University Center, Urals Branch Division, Russian Academy of Sciences, Russia of Medicine, Japan The amphibian and the fish heart presents a convenient model to study the T wave Although altered connexin43 (Cx43) expression is known to promote genesis genesis as it has the only ventricle and hence the contributions of apex-to-base of tachyarrhythmias, precise mechanism of Cx43 remodeling underlying and transmural repolarization gradients can be compared. Potential mapping was arrhythmogenesis is unknown. To address this we studied changes in impulse done on eight pikes and nine frogs using 32 body surface, 24 epicardial and 40-72 propagation of confluent monolayers of neonatal rat cultured myocytes under intramural unipolar leads. The distribution of repolarization durations in frog and inhibition of intercellular coupling by dominant-negative (DN) Cx43 via adenoviral pike myocardium was similar for that the ARIs were shorter at the apex than at vector-mediated gene transfer for DNCx43-fused red fluorescence protein (RFP). A the base and that the largest transmural ARI gradient was found in the base and high-resolution fluorescence microscopy was used to visualize both the fluo4- and decreased toward the apex. However, the directions of the transmural gradients RFP-fluorescence intensities for measurements of Ca2+-transient propagation and were opposite: in pikes, the repolarization sequence proceeded from endocardial DNCx43 distribution, respectively. DNCx43 inhibition of the monolayers produced slowing of Ca2+ transient conduction velocity and preferential emergence of spiral- -to-epicardial, in frogs - epicardial-to-endocardial. Cardiac electric field on the wave reentrant arrhythmias. The monolayers showed regional conduction slowing and body surface correlated with the epicardial sequences of repolarization as well as subsequent generation of wave break, resulting in reentrant arrhythmias. Higher RFP with the potential distributions on the ventricular epicardium. The body surface fluorescence intensity was detected at the wave-break point than at the surrounding potential distributions during the T wave in both animal species studied were myocardium, indicating a culprit role of DNCx43 inhibition in the genesis of reentry. quite close to each other in spite of opposite transmural repolarization gradients In conclusion, regional heterogeneity in gap-junctional communication produces a providing evidence in support of the major role of apex-to-base repolarization wave break of conduction, leading to reentrant arrhythmias. pattern in the generation of the electrocardiographic T wave in pikes and frogs.
P1AM-1-5 P1AM-1-6 CARDIAC TISSUE SLICES FROM GUINEA-PIG HEARTS DIACYLGLYCEROL KINASE ζ RESTORES Gαq- AS A SUITABLE MODEL FOR PHARMACOLOGICAL INDUCED ATRIAL STRUCTURAL REMODELING IN AND PHYSIOLOGICAL INVESTIGATIONS TRANSGENIC MICE Alexandra Bussek1, Erich Wettwer1, Horst Lohmann2, Patrizia Camelliti3, Masamichi Hirose1, Yasuchika Takeishi2, Hisashi Shimojo3, 1 Ursula Ravens Takeshi Niizeki4, Tsutomu Nakada1, Isao Kubota1, Mitsuhiko Yamada1 1Pharmacology und Toxicology, Dresden University of Technology, Germany, 1 2 3 Department of Molecular Pharmacology, Shinshu University, Japan, Lohmann Neuropharmacological Consulting, Germany, Department of 2First Department of Internal Medicine, Fukushima Medical School, Physiology, Anatomy and Genetics, University of Oxford, UK Japan, 3Department of Pathology, Shinshu University, Japan, 4Cardiology, We established a cardiac tissue slice model from adult guinea-pig hearts for Pulmonology, and Nephrology, Yamagata University, Japan electrophysiological and pharmacological investigations. Cell orientation in living Previous study showed that diacylglycerol kinase ζ (DGKζ), which degenerates slices was studied in relation to the direction of the primary cutting plane. diacylglycerol (DAG), inhibits ventricular structural remodeling and rescues activated G Vertical and horizontal transmural and tangential slices (0.45 mm thick) were prepared protein αq (Gαq)-induced heart failure. However, whether DGKζ inhibits atrial structural from left ventricles. For cell orientation two-photon fluorescence mycroscopy was remodeling is still unknown. This study aimed to elucidate effects of DGKζ on the atrial used with Di-4-ANEPPS and CellTracker green for staining. Action potentials remodeling. (APs) elicited by electrical stimulation (1Hz) were recorded with intracellular micro- METHODS A transgenic mouse (Gαq-TG) with cardiac expression of activated Gαq and electrodes at 37°C. a double transgenic mouse (Gαq/DGKζ-TG) with cardiac overexpression of DGKζ and activated Gαq were created. Vertical transmural and horizontal slices did not show a major direction of cell RESULTS During electrocardiogram recording for 10 min, atrial fibrillation was observed in orientation in contrast to tangential slices with mainly longitudinal orientation. APs 5 of 11 anesthetized Gαq-TG mice but not in any wild type (WT) and Gαq/DGKζ-TG mice parameters at control were: resting membrane potential 84.8 ± 0.8 mV; amplitude (p< 0.05). Dilatation of the left atrium with thrombus formation was observed in 9 Gαq- 120.7 ± 0.8 mV; APs duration at 90% of repolarisation 169.9 ± 4.0 ms (59/22; slices/ TG hearts but not in any WT and Gαq/DGKζ-TG hearts. Moreover, the degree of extensive hearts). These parameters remain constant for at least 90 min of perfusion. APs were interstitial fibrosis in the left atrium was significantly greater in Gαq-TG hearts than that in prolonged by selective IKr blocker E4031 from 168.7 ± 5.7 ms to 228.9 ± 9.9 ms with WT and Gαq/DGKζ-TG hearts (p<0.05). 1μM; IC50 of 31 nM. We conclude that cardiac tissue slices from adult hearts are a CONCLUSION These results demonstrate that DGKζ inhibits Gαq-induced atrial structural suitable model for electrophysiological and pharmacological investigations. remodeling. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 121 P1AM-1-7 P1AM-1-8 OPTICAL MAPPING STUDY OF THE EXPERIMENTAL INCREASED SUSCEPTIBILITY TO ATRIAL TACHYARRYTHMIA IN THE ISOLATED RAT ATRIUM TACHYARRHYTHMIA IN HEARTS FROM RATS WITH USING A VOLTAGE-SENSITIVE DYE ELEVATED AFTERLOAD DUE TO BANDING OF THE TETSURO SAKAI ASCENDING AORTA Andrew Frederick James1, Shang Jin Kim1, Stephanie CM Choisy1, Department of Physiology, University of the Ryukyus School of Medicine, 1 2 1 Japan Lesley A Arberry , Sandra A Jones , Julian C Hancox 1Department of Physiology & Pharmacology, University of Bristol, UK, 2School of We have studied the experimental tachyarrhythmia in an isolated rat atrial Biological Sciences, University of Hull, UK preparation for the optical mapping of excitation spread. The atrial preparation Atrial fibrillation (AF) is the most common arrhythmia and is associated with was dissected from the adult rat heart, and an artificial hole was made in significant morbidity and mortality. Hypertension and left ventricular hypertrophy in the center of the preparation. The preparation was then stained with a fast patients are considered good epidemiological indicators of the risk of AF. To address merocyanine-rhodanine voltage-sensitive dye (NK2761). Using a multi-element the hypothesis that hypertension increases the risk of atrial tachyarrhythmia (AT), (16 X 16) photodiode array, the spread of excitation was assessed optically by we have examined the susceptibility to AT of excised perfused hearts from rats with timing the initiation of the action potential-related extrinsic absorption changes. elevated afterload induced surgically by banding of the ascending aorta. In comparison The electrical stimulation applied by a bipolar electrode evoked the sustained with sham-operated controls, rats subject to aortic banding (AoB) showed significant excitation with a fast rhythm, which we termed “experimental tachyarrhythmia” left ventricular hypertrophy and left atrial enlargement at 8, 14 and 20 weeks following (ET). We optically mapped the spatiotemporal patterns of the spread of excitation surgery. Unipolar electrograms were recorded from the left atrial epicardial surface during the initiation and the maintenance phases of ET. In most cases, a rotation of perfused hearts using a 5 x 5 electrode array, allowing measurements of effective refractory period (ERP), of conduction velocity (CV) and of inhomogeneity in of the excitatory wave around the artificial hole, i.e., a circus movement of the conduction. At 14 and 20 weeks following surgery, hearts from AoB rats showed an excitatory wave, was observed. On the other hand, the appearance of an ectopic increased incidence and duration of burst pacing-induced AT associated with greater pacemaker with a fast rhythm was also observed. In some examples, two ectopic inhomogeneity in conduction, without changes in ERP or CV, compared with shams. pacemakers appeared simultaneously. We consider that the experiment using the Taken together, our results demonstrate an increased susceptibility to AT and atrial improved preparation is a superb in vitro model of atrial arrhythmia. remodelling due to elevated afterload.
P1AM-1-9 P1AM-1-10 ECG BODY SURFACE MAPPING CHANGES IN TYPE 1 REFRACTORINESS DURING HYPOTHERMIA IN DIABETIC PATIENTS HIBERNATING AND ACTIVE GROUND SQUIRREL Otomar Kittnar1, Sabina Palova2, Marcela Szabo2, Jiri Charvat2, ATRIUM AND VENTRICLE Jaroslav Slavicek1, Mikulas Mlcek1, Eva Medova1 Vladislav Stephanovich Kuzmin, Denis V Abramochkin, Juriy V Egorov 1 Laboratory of Electrophysiology, Institute of Experimental Cardiology of Institute of Physiology, 1st Faculty of Medicine, Charles University in Prague, 2 Russian Cardiological Center, Russia Czech Republic, Department of Medicine, 2nd Medical Faculty, Charles University in Prague, Czech Republic During winter slipping body temperature in hibernating animals decrease to about 0C but their heart remain excitable and no any arrhythmias observe. In nonhibernating ECG body surface mapping (BSM) parameters in the 1st type diabetic patients (DM1) mammals decreasing of the temperature to 27-17C leads to arrhythmias and loss of are significantly abnormal comparing to healthy nondiabetic subjects. Hypothesis that the excitability. Probably, one of the reason of hibernators tolerance to cold-induced these changes are more pronounced in DM1 patients with autonomic neuropathy (AN) arrhythmias is long refractoriness in heart. was tested. The parameters of BSM were registered by diagnostic system Cardiag We estimated refractoriness duration (RD) in hibernating (HGS), in active Siberian 112.2 in 54 DM1 patients including 25 with AN and 30 control subjects. AN was ground squirrel (AGS) and in rat papillary muscle (PM) and atrium in temperature diagnosed according to Ewing criteria when two or more Ewing tests were abnormal. diapason 37-17C. Action potentials (AP) were obtained with use of microelectrode The maximum in depolarization isopotential maps was more positive until the 30th technique, RD were estimated with using of S1S2 technique in different pacing ms and more negative in the 50th ms, minimum more negative until the 40th ms of the intervals (200-500 ms). QRS, the repolarization isopotential map maximum more positive in the 10th ms and In PM and atria of HGS and AGS refractoriness increasing significantly with lowering of temperature. For example, in PM RD increase from 80+/-7ms at 37C to 320+/-23ms minimum more negative since the 50th to 80th ms from the point J, the amplitude of at 17C in HGS and from 52+/-7 ms to 240+/-14ms in AGS at pacing interval 500 ms. the Q wave more negative, the activation time of depolarization faster and QT duration In atria RD increase from 56+/-6ms at 37C to 217+/-18ms at 17C in HGS and from decreased in DM1 patients comparing to controls. The statistically more significant 57+/-7 ms to 161+/-15ms in AGS at same pacing interval. changes in BSM parameters and QT duration were present in DM1 patients with than Increase of RD with lowering of the temperature in ground squirrel heart was more without AN. The more pronounced changes of BSM parameters were detected in than in rat and this peculiarity may contribute to insusceptibility of hibernators DM1 patients with than without AN comparing to healthy subjects. myocardium to arrhythmias.
P1AM-1-11 P1AM-1-12 EFFECT OF APNOE/REOXYGENATION ON SOME ECG INCIDENCE OF CARDIAC ARRHYTHMIA IN PASSIVE PARAMETERS. CHRONOPHYSIOLOGICAL STUDY OPIUM SMOKING RABBITS WITH AND WITHOUT Pavol Svorc Jr.1, Ivana Bacova1, Imola Bracokova1, CARDIAC ISCHEMIA AND HYPERCHOLESTEROLEMIA 1 1 2 Erika Svorcova2 Siyavash Joukar , Hamid Najafipour , Fatemeh Mirzaeipour , 2 1Department of Physiology, Safarik University, Slovakia, 2Department of Hamid Reza Nasri 1 Hematolgy and Oncohematology, Louis Pasteur Teaching Hospital with Physiology and Physiology Research Center, Kerman University of 2 Health Care, Slovakia Medical Sciences, Iran, Department of Cardiology and Physiology Research Center, Kerman University of Medical Sciences, Kerman, Iran The aim of our study was refered to the effect of apnoe/reoxygenation on the diagnosticly important time intervals of ECG in dependence on the light-dark (LD) There is a conception among some people that opium has prevention or cycle. Experiments were performed in the anaesthetized rats (ketamine/xylazine ameliorating effects on cardiovascular diseases. We investigated the severity and 100mg/15mg/kg, i.m.) after the adaptation to LD cycle (12:12h). Ventilatory incidence of lethal cardiac arrhythmia, ventricular tachycardia and ventricular manoeuvres were performed by respiratory pump. Parameters of normal ventilation/ fibrillation, in opium smoking ischemic norm and hypercholesterolemic rabbits. reoxygenation VT=1ml/100g, respiratory rate 40-50breaths/min. The apnoe was Animals divided into two norm and hypercholesterolemic main groups fed simulated by the switching off respirator for 2 minutes. The significant LD differences with a normal or high cholesterol diet (2 weeks) with short (3 days) or long- in PQ interval were found (p<0,001) in the control values and after 30. and 60sec. term (4 weeks) passive opium smoking. Results showed that in ischemic hearts, Poster Session of apnoe. Reoxygenation recovered LD differences. Significant LD differences hypercholesterolemia along with long term opium, significantly increased the in QT interval were not found in the control values but only after 90. and 120sec. incidence and severity of arrhythmia compared with the relative control group of apnoic episode (p<0,001). Significant LD differences were detected only at the (p=0.01 in both cases). this susceptibility was not mediated by changes in QT end of reoxygenation. It is concluded that the predisposition of the myocardium to interval. The results suggest long-term passive opium smoking along with the ventricular arrhythmias resulting from disorders of the production and impulse hypercholesterolemia can be a predisposing factor for lethal arrhythmia after conduction is significantly influenced by LD cycle during apnoe/reoxygenation. myocardial ischemia. Dispersion of the refractory periods, presented by duration of QT interval, is independent on LD cycle during ventilatory manoeuvres. Key words: cardiac arrhythmia, hypercholesterolemia, ischemia, opium smoking
122 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-1-13 P1AM-1-14 ISCHEMIA AND REPERFUSION INDUCED CARDIAC ELECTROPHYSIOLOGY AND ARRHYTHMIA ARRHYTHMIAS: ROLE OF HYPEROXIC IN THE MOUSE: EFFECTS OF TEMPERATURE AND PRECONDITIONING KNOCK OUT OF RGS2 1,2,3 1 1 1 Khalil Pourkhalili1, Sohrab Hajizadeh2, Zahra Akbari1, Douglas L Jones , Peter Chidiac , Stephen Sims , Jari M. Tuomi 1 3 Departments of Physiology & Pharmacology, University of Western Ontario, Ali Khoshbaten Canada 2Department of Medicine, University of Western Ontario, Canada 3The 1 Department of Physiology, Bushehr University of Medical Sciences, Iran, Lawson Health Research Institute, London, Canada 2Department of Physiology, School of Medical Sciences, Tarbiat Modares 3 Background: My laboratory investigates arrhythmia mechanisms. Uninterpretable University, Tehran, Iran, Chemical Injuries Research Center, Baqiyatallah electrophysiological (EP) data on the mouse has been published. Parasympathetic input is University of Medical Sciences, Tehran, Iran stated to be via cholinergic M2-type receptors. RGS2 regulates M3 signaling. RGS2's role in Hyperoxa is known to protect the heart against necrosis and contractile dysfunction, but protection modulating cardiac EP has not been determined. against arrhythmias has not been well characterized. We hypothesized that exposure to hyperoxia Methods: We determined EP parameters [(ERP, VERP, AVNERP, WCL &AHC) in -/- o (H) reduces ischemia and reperfusion induced arrhythmias in isolated rats heart. Following 60 and C57BL/6 wild type & RGS2 knockout mice at 37 & 34 C body temperatures. Atrial burst 180 min of hyperoxia , hearts were isolated immediately (H60 and H180) or 24 hours afterward pacing determined atrial fibrillation (AF) inducibility, & ventricular stimulation determined (H60/24 and H180/24), and subjected to 30 min of regional ischemia followed by 120 min of AV nodal re-entrant tachycardia (AVNRT). Real-time RT-PCR quantified M2 and M3 reperfusion. Severity of arrhythmias were analyzed during ischemia and reperfusion. 60 and 180 receptor mRNA. o min of breathing hyperoxic gas induced significant protection against ischemia and reperfusion Results: AERP, AH, WCL, and AHCs were lower at 37 than 34 C while AVNERP were o induced arrhythmias. Total number of PVBs were markedly attenuated by hyperoxia especially in prolonged. AF was was more inducible at 37 than 34 C. Also, AVNRT was inducible H60 and H180 groups. VT and VF duration was also affected by hyperoxia. Hyperoxia reduced the ventricular but not atrial pacing. Atrial M3 was higher than M2 receptor mRNA, but there -/- number of VT episodes in ischemia and reperfusion phase. Accordingly, severity of arrhythmias was no differences between RGS2 & wild-type mice. o and infarct size was lower in hyperoxia treated groups. The effects were more pronounced in early Conclusions: Lower temperatures prolong EP, thus standardized EP are needed at 37 C. phase of hyperoxia exposure. These results indicate that hyperoxic preconditioning attenuates Arrhythmia inducibility indicates that mice are suitable for determining genetic and therapy ventricular ischemia and reperfusion induced arrhythmias in isolated rat heart, decreases infarct size effects on EP & arrhythmia. M3 is the dominantly expressed muscarinic receptor in the and improves postischemic heart function. atrium & modulates AV nodal function through M3 rectifier potassium channel, K,M3I .
P1AM-1-15 P1AM-1-16 NORADRENALIN INDUCES AUTOMATIC ELECTRICAL ADAPTIVE ABILITIES OF ORGANISM:GENERAL ACTIVITY IN CARDIAC MUSCLE OF RAT PULMONARY THEORETIC AND APPLIED ASPECTS VEINS Nick Vasilievich Malikov, Nadezda Vasilievna Bogdanovskya Ian Findlay, Nicolas Doisne, Pierre Cosnay, Veronique Maupoil Zaporizhja National University, Ukraine CNRS FRE 3092, University Francois Rabelais Tours, France Now in biology the experimental research aimed at studying the peculiarities Ectopic activity in cardiac muscle within pulmonary veins (PV) is of the adaptatiogenesis. In the course of present research we carried out the associated with the onset and the maintenance of atrial fibrillation. Here we medical examination of 3570 children of school age and 1632 employees of use microelectrode recording to show that noradrenalin (NA) can induce industrial enterprises two regions (Ukraine and Western Siberia), which differ automatic electrical activity in rat PV but not in left atrium (LA). in geographic and climatic characteristics. The membrane potential was lower in PV (-72 mV) than in LA (-84 mV). The analysis of experimental data allows to establish the considerable In PV 10 μM NA induced a hyperpolarisation (-7 mV) followed by a slowly influence of climate and geography on the level of general adaptive developing depolarisation (+20 mV). In LA it had little effect (-2 and +1 abilities of an organism, the character of intersexual and interregional mV). The hyperpolarisation was blocked by 5 μM atenolol and replicated by 0.1 μM isoprenaline. The depolarisation was blocked by 1 μM prazosine correlations according to this parameter. This is provided by the fact that and replicated by 1 μM cirazoline. In PV, NA triggered bursts of automatic the representatives of more heterogeneous population and of the region activity at ~-50 mV (period 56 s, duration 25 s, n=16). Bursts consisted of which is more extreme as to climate and geography (Western Siberia) were slow action potentials (dV/dt-max 6-13 V/s) whose frequency increased characterized by higher adaptive abilities of organism. Obviously, because from ~2 to ~5 Hz and then declined again. The diastolic membrane potential of wider set of adaptive subprogrammes which were formed as a result of progressively increased from -54 mV to -61 mV during a burst, then high migrative mobility of Siberian population, natural extremity of Western further hyperpolarised to -64 mV before the slow and essentially linear Siberia and existence of hereditary adaptive abilities which are passed from depolarisation that would trigger the next burst. woman to woman in Western Siberia the female representatives of this region Both alpha- and beta-adrenergic stimulation are required to induce bursting left the boys and men from same region behind as to the level of adaptive automatic electrical activity in cardiac muscle in rat PV. abilities in almost all cases.
P1AM-1-17 P1AM-2-1 GENISTEIN INHIBITS SYMPATHEIC OUTFOLW ELECTROPHYSIOLOGICAL AND PHARMACOLOGICAL THROUGH NITRIC OXIDE AND PROTEIN TYROSINE PROPERTIES OF THE ISOLATED GUINEA-PIG KINASE IN ROSTRAL VENTROLATERAL MEDULLA IN PULMONARY VEIN MYOCARDIUM ANESTHETIZED RATS Iyuki Namekata1, Yayoi Tsuneoka1, Takahiko Sugimoto1, Yuming Wu1, Huijuan Ma1, Xin Wang1, Depei Li2, Ruirong He1 Kiyoshi Takeda1, Toru Kawanishi2, Ryu Nakamura3, 1Department of Physiology, Hebei Medical University, China, 2Department Akira Takahara1, Hikaru Tanaka1 of Critical Care, The University of Texas, M. D. Anderson Cancer Center, 1Department of Pharmacology, Toho University Faculty of Pharmaceutical USA Sciences, Japan, 2Division of Drugs, National Institute of Health Sciences, We investigated the effect of microinjection of plant-derived diphenolic compounds Japan, 3Advanced Imaging Microscopy Department, Product Management genistein into RVLM on sympathetic outflow in anesthetized rats. Microinjection of Division of Microscopy, Carl Zeiss, Japan 2μl genistein (1, 10, and 100 μM) into the RVLM dose-dependently decreased renal Pulmonary veins contain a myocardial layer, whose electrical activity is considered to sympathetic activity, blood pressure, and heart rate with the maximum inhibition be involved in the genesis and maintenance of atrial fibrillation. We recorded the action at 100 μM. Pre-microinjection of 2μl tamoxifen (100 μmol/L) (a selective estrogen potential from the myocardial layer of the isolated guinea-pig pulmonary vein with glass response modifiers) into the RVLM potentiated the inhibitory effects of genistein microelectrode techniques, and studied its electrophysiological properties. Spontaneous (10 μmol/L) on BP, HR, and RSNA. Pre-microinjection of sodium orthovanadate (1 electrical activity was observed in 16 out of 119 of pulmonary vein preparations, whereas mmol/L) into RVLM, attenuated the inhibitory effects of genistein (10 μmol/L) on no spontaneous activity was detected in the left atria. The incidence in the left superior BP, HR and RSNA. Inhibition of nitric oxide production with pre-microinjection of pulmonary vein was relatively higher than that in the other veins. Spontaneous activity was L-NAME (100 μmol/L) into RVLM, attenuated the inhibitory effects of GST 10μmol/ inhibited by 0.1 μM ryanodine, 10 μM cyclopiazonic acid, or Na+/Ca2+ exchanger inhibition. L on BP, HR and RSNA. In addition, microinjection of a combination of L-NAME In pulmonary vein preparations with no spontaneous activity, delayed afterdepolarizations (100 μmol/L) and sodium orthovanadate (1 mmol/L) into the RVLM abolished were induced after the termination of burst pacing. Also, spontaneous activity could be GST (10 μmol/L)-induced inhibition of the BP, HR, and RSNA. These data suggest induced by 10 μM noradrenaline or 1 μM ouabain. These phenomena were not observed that microinjection GST into RVLM inhibits sympathetic outflow. These effects in left atrium. In conclusion, the guinea-pig pulmonary vein myocardium have a tendency may involve the production of NO and activation of protein tyrosine kinase, but not to show spontaneous electrical activity which is mediated by Ca2+ released from the activation of estrogen receptors. sarcoplasmic reticulum and the resulting activation of the Na+/Ca2+ exchanger. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 123 P1AM-2-2 P1AM-2-3 ASYMMETRICAL DISTRIBUTION OF ION CHANNELS REGULATION OF ARRHYTHMOGENIC AUTOMATICITY IN THE LAYERS OF HUMAN AND CANINE LEFT IN VENTRICLE OF THE DILATED CARDIOMYOPATHY VENTRICULAR MYOCARDIUM MOUSE MODEL Balazs Horvath, Gergely Szabo, Gabor Harmati, Norbert Szentandrassy, Nagomi Kurebayashi1, Takeshi Suzuki1,2, Takashi Murayama1, Tamas Banyasz, Janos Magyar, Peter Pal Nanasi 1,2 1 2 3 Hiroto Nishizawa , Akihito Chugun , Yuji Nakazato , Sachio Morimoto Department of Physiology, University of Debrecen, Medical and Health Science 1 Centre, Hungary Department of Pharmacology, Juntendo University School of Medicine, Japan, 2Department of Cardiology, Juntendo University School of Medicine, Japan, The object of this study was to compare the distribution of ionic currents and ion 3Department of Clinical Pharmacology, Kyushu University Graduate School of channel proteins in human and canine subepicardial (EPI) and midmyocardial (MID) Medicine, Japan layers of the left ventricular myocardium. Action potentials and ionic currents were measured on enzymatically isolated canine Dilated cardiomyopathy (DCM) is often associated with sudden death by malignant left ventricular cardiomyocytes. Quantity of ion channel proteins in both human and arrhythmias. Recently a knock-in mouse model of DCM has been created by a mutation canine preparations were determined by Western blotting. in cardiac troponin T (δK210). Because the model mice closely mimic human DCM, we We obtained that on EPI cells the amplitude of action potentials were smaller, the early studied properties of those hearts to know the mechanism of arrhythmia in DCM. Ventricular repolarization was greater and the action potentials were shorter compared to MID muscles were excised from WT and δK210 homo mutant hearts and loaded with rhod-2 and/or di-4-ANEPPS. Ca2+ and membrane potential signals were monitored using a laser myocytes. Among the studied ionic currents the densities of Ito and IKs were larger on scanning confocal microscope. Gene expression levels of ion channels and related proteins EPI than on MID cells. We found no significant EPI-MID differences in Ca,LI , IK1 and IKr. were determined by realtime RT-PCR. Left ventricle (LV) showed remarkably increased In the EPI layer of canine heart the quantity of Kv4.3, Kv1.4, KChIP2 and KCNQ1 automaticity and prolonged action potential duration. Correspondingly, a significant + proteins were significantly higher, and that of Nav1.5 and MinK much lower than in decreases in some of K channels and an increase in Cav3.1 were detected in the mutant LV. the MID region. Comparing the human and canine preparations they showed high Using confocal imaging, we further investigated effects of various arrhythmogenic factors similarities in the EPI-MID distribution of ion channel proteins. and anti-arrhythmogenic agents on Ca2+ and membrane potential signals in mutant LV. Our results prove that from electrophysiological point of view canine heart is an Our results suggest that ion channel remodeling make the δK210 hearts highly susceptible adequate model of human myocardium, and also draw attention to the electrical to various types of arrhythmogenic factors and that some kinds of agents can effectively heterogenity of the myocardium. suppress the automaticity.
P1AM-2-4 P1AM-2-5 CARDIAC ELECTROPHYSIOLOGICAL PROFILE OF IONIC MECHANISMS UNDERLYING THE ARTICAINE DEVELOPMENT OF VENTRICULAR ARRHYTHMIAS IN Gabor Harmati1, Gergely Szabo1, Peter Birinyi1, DILATED CARDIOMYOPATHY Adrienn Szabo2, Norbert Szentandrassy1, Balazs Horvath1, Takao Shioya1, Sachio Morimoto2, Tsuguhisa Ehara1 Tamas Banyasz1, Ildiko Marton2, Janos Magyar1, Peter Pal 1Department of Physiology, Faculty of Medicine, Saga University, Japan, 2 Nanasi1 Department of Clinical Pharmacology, Kyushu University Graduate School 1Department of Physiology, University of Debrecen Medical and Health of Medicine, Japan Science Center, Hungary, 2Department of Dentistry, University of Debrecen In dilated cardiomyopathy (DCM), occurrence of severe ventricular arrhythmias is the Medical and Health Science Center, Hungary major cause of death. To study cellular mechanisms underlying the arrhythmogenesis Background: In spite of its widespread clinical application of articaine there is little in DCM, we examined electrophysiological properties of single ventricular myocytes information on the cellular cardiac effects of the drug. In the present study, effects of isolated from a knock-in (KI) mouse model of hereditary DCM. The KI mice had a articaine on action potential morphology and the underlying ion currents were studied in ΔK210 mutation in cardiac troponin T genes (Tnnt2), which causes a hereditary DCM isolated canine ventricular cardiomyocytes. in humans (CMD1D: OMIM 601494). Our results show: 1) Ventricular myocytes Methods: Action potentials were recorded from the enzymatically dispersed cells. 2+ Conventional patch clamp and action potential voltage clamp arrangements were used to of the KI mice produced a larger Ca transient than those of the wild-type (WT) study the effects of articaine on the ion currents. littermates. 2) The action potential of KI cells had a longer duration and exhibited a Results: Articaine induced concentration-dependent changes in action potential configuration more prominent plateau phase than in WT cells. 3) β-adrenergic stimulation induced including shortening of the action potentials, reduction of their amplitude and maximum early afterdepolarizations and extrasystoles in KI cells, but not in WT cells. 4) In KI
velocity of depolarization (Vmax), suppression of early repolarization and depression of cells, the major outward current components: Ito and IK,slow had significantly smaller plateau. The EC50 value obtained for the Vmax block was162+/-30 uM. Under voltage clamp amplitudes than in WT cells, while the inward INaCa activated by the intracellular conditions a variety of ion currents were blocked by articaine: ICa, Ito, IK1, IKr and IKs. Ca2+ transient had a significantly larger amplitude. We conclude that these changes Conclusions: Articaine can modify cardiac action potentials and ion currents at concentrations higher than the therapeutic range. Since its suppressive effects on the in the membrane current systems favor a longer action potential duration, and will membrain currents are relatively well balanced, the articaine-induced changes on action facilitate the occurrence of afterdepolarizations that leads to the development of lethal potential morphology can be moderate even in case of intoxication. ventricular arrhythmias in DCM.
P1AM-2-6 P1AM-2-7 HEART REMODELING DURING THE TIME-COURSE BENEFICIAL EFFECTS WITH BETA-ADRENERGIC RECEPTOR BLOCKERS ON ALTERED OF CHAGAS’ DISEASE: ROLE OF NO-PI3KINASE AND 2+ IFN-γ PATHWAYS INTRACELLULAR Ca IN DIABETIC RAT HEART Danilo Roman Campos1, Hugo Leonardo Duarte1, Policarpo Sales-Junior1, Erkan Tuncay, Aytac A Seymen, Belma Turan Antonio Jose Natali2, Catherine Ropert3, Ricardo Tostes Gazzinelli1, Jader Department of Biophysics, Ankara University, Faculty of Medicine, Turkey 1 Santos Cruz The defects identified in the mechanical activity of the hearts from diabetic 1Biochemistry and Immunology, Federal University of Minas Gerais, Brazil, 2+ 2 3 animals include alteration of Ca signalling via changes in critical processes Department of Physical Education, Vicosa University, Brazil, Laboratory of that regulate intracellular Ca2+ concentration. Since propranolol (PROP) and Immunopathology, Rene Rachou Research Center, Brazil timolol (TIM) are nonselective agents with different beta-potency, we wanted Chagas' disease is one of the most common determinants of congestive heart failure and to examine the long-term effects (3 months) of these blockers, either PROP sudden death in Latin America . In order to understand the molecular mechanisms involved (25 mg/kg/day) or TIM (5 mg/kg/day) administrations (intragastrically), on in determining this cardiomyopathy, we used a murine model to study heart remodeling Ca2+ related mechanisms in cardiomyocytes from streptozotocin-induced during the time-course of Chagas’ disease. Using cardiomyocytes we evaluated, in acute diabetic rats. TIM treatment of diabetic rats restored significantly the altered and chronic phases: cell shortening (CS), outward potassium current (IK), L-type calcium parameters of Ca2+ transients, depressed Ca2+ loading of sarcoplasmic current (ICa), action potentials (AP) and intracellular calcium dynamics (ICD). In acute reticulum, and depressed L-type Ca2+ - currents as well as increased basal phase we found a reduction in CS as well as a smaller IK and ICa , increased AP duration 2+ Poster Session and larger SR calcium release. In the chronic phase we observed similar results, however, Ca level in electrically stimulated cardiomyocytes. The beneficial effect CS was smaller then in acute phase, indicating progressive cardiomyocyte dysfunction. with PROP was in comparable level with respect to those of TIM. Although We found that NO-PI3Kinase is the main pathway involved in ICD dysfunction. Using prevention of diabetes-induced altered cardiac function by beta-blockers infected IFN-γ -/- we found that cardiomyocytes presented similar results to non-infected might present a useful pharmacological strategy for the treatment of diabetic IFN-γ -/-, concerning CS, electrophysiology and ICD. Taking together, our data demonstrate cardiomyopathy, it can be clearly suggested that there are differences in that during the time-course of Chagas’ disease occur a remodeling in cardiomyocyte, the effects of individual beta-adrenergic blocking agent when particular including changes in CS, electrophysiology and ICD. Evidence is presented supporting the clinical benefits are addressed (Supported by TUBITAK-SBAG-107S427 and involvement of NO-PI3Kinase and IFN-γ pathways. TUBITAK-COST-107S304).
124 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-2-8 P1AM-2-9 CARDIAC ELECTRICAL REMODELING IN BRADYKININ INTEGRATED EXPERIMENTAL AND COMPUTATIONAL TYPE 2 RECEPTOR KNOCK-OUT MICE RESEARCH TOOLS FOR THE STUDY OF ACUTE Jader Cruz1, Danilo Roman-Campos1, Hugo L Duarte1, Eneas R Gomes2, ISCHEMIC EFFECTS ON CARDIAC MECHANO- Carlos H Castro2, Antonio Natali3, Silvia Guatimosim2, Alvair P Almeida2, ELECTRICAL INTERACTIONS 2 4 Jorge L Pesquero , Michael Bader T Alexander Quinn1, Blanca Rodriguez2, Peter Kohl1 1 2 Biochemistry and Immunology, UFMG, Brazil, Department of Physiology 1 3 4 Department of Physiology, Anatomy and Genetics, University of Oxford, and Biophysics, UFMG, Brazil, Department of Physiology, UFV, Brazil, Max- UK, 2Computing Laboratory, University of Oxford, UK Delbruck-Center for Molecular Medicine, Berlin-Buch, Germany Arrhythmias in myocardial ischemia represent a major cause of sudden cardiac death. It has been reported that kallikrein-kinin system is important in controlling cardiac Mechanisms are complex and still unknown, but mechano-electrical interactions are homeostasis. These effects are modulated mainly via B2 receptor activation. likely a key player. Here an integrated experimental and computational approach Therefore, the aim of this work was to verify the influence of kinin B2 receptor is proposed to investigate ischemia-induced changes in mechano-electric feedback deletion on baseline electrophysiology. We generated knock-out (KO) mice that have and their role in cardiac arrhythmias. Experiments are conducted using the ‘Soft no expression of B2 receptors (B2RKO), and we recorded cardiac cell contractility, + 2+ 2+ Tissue Impact Characterisation Kit’, a novel device that allows precise control of action potentials, K and Ca ionic currents. In addition, Ca transients from isolated local mechanical stimulation to isolated rabbit hearts under conditions of acute low- myocytes were recorded by confocal microscopy. The mean cellular shortening was flow global ischemia and pharmacological modulation of stretch-activated channels. significantly decreased in B2RKO cardiomyocytes compared to wild type (WT) cells. Electrical and mechanical ventricular activity is characterized by microelectrodes, The ventricular action potentials were lengthier in B2RKO cells than WT cells. The voltage- and ion-sensitive optical mapping, electrocardiograms, tissue deformation + 2+ outward K current-density was smaller as also observed by L-type Ca current. The monitoring, and intraventricular pressure measurement. Results are combined with mean amplitude of calcium transient was significantly decreased in B2RKO myocytes, computational modeling using anatomically-based representations of the rabbit compared with WT. These results suggested that the fundamental properties might ventricle for hypothesis formation, data interpretation, and investigation of underlying have been affected by the deletion of endogenous B2 receptor leading to cardiac mechanisms. This integrative research provides a unique opportunity to investigate failure and electrical remodeling. We conclude that B2 receptor might be involved the contribution of mechano-electrical interactions to lethal arrhythmias in myocardial with the control of cardiac contractility and electrical activity. ischemia.
P1AM-2-10 P1AM-2-11 THE POSSIBLE ROLE OF SLOW SODIUM CURRENT IN REGULATION OF THE CARDIAC SODIUM CHANNEL GENERATION OF TRUE PACEMAKER ACTIVITY NaV1.5 BY THE MAGUK PROTEIN SAP97: EVIDENCE Vladimir Golovko FOR MULTIPLE NaV1.5 INTERACTING COMPLEXES 1 1 2 Institute of Physiology, Komi Science Centre, the Ural's Branch, The Severine Petitprez , Anne-flore Zmoos , Stephane Hatem , Russian Academy of Sceinses, Russia Alain Coulombe2, Hatem Abriel1 1Department of Pharmacology and Toxicology, University of Lausanne, There are some facts supporting the participation of slow I Na in upstroke generation. 2 1.The substitution of 50% Na+ in the solution caused a decrease of dV/dt max from Switzerland, INSERM U621, Pierre et Marie Curie-Paris6 University , France
3.2 to 1.4 V/s in pacemaker cells and diastolic depolarization rate two times compared Nav1.5 is the voltage-gated sodium channel that initiates the cardiac action potential. with control. Finally action potential frequency generation was slowed from 180 to The 3 last amino-acids of Nav1.5 (SIV) constitute a PDZ-domain binding motif 122 beat/min (n=19) . known to interact with the syntrophin-dystrophin complex and PDZ domains found 2. Note that overshoot and dV/dt max action potentials cells in solution containing in proteins of the MAGUK family. Among their multiple roles, MAGUK proteins can 70 mM Na+ decreased accordingly to the reduction of external Na+ concentration target and cluster ion channels at the plasma membrane. [Na+]o , although exposure TTX (20 mkM) didn’t cause such effect. 3. A rapid change Pull-down experiments revealed that the association between SAP97 and Nav1.5 of [Na+]o in solution by Li+ blocked the upstroke, although diastolic depolarization depends on the PDZ-domain binding motif of Nav1.5. Silencing of SAP97 cells reached the threshold level. reduced the whole-cell sodium current measured in HEK293 stably expressing Nav1.5 4.Sinus node dysfunction results from genetic mutations encoding the cardiac Na- channels without decreasing the total protein amount. In control or silenced HEK293 channel (SCN 5A). cells, sodium current produced by Nav1.5 SIV-truncated channels was reduced 5. It has been found the messenger RNAs coding channels Nav 1.1 and Nav 1.5 in compared to WT. Immunostainings demonstrated the colocalisation of Nav1.5 and rabbit’s SA node. dystrophin specifically at lateral membranes, but not at the intercalated discs. 6.The existence of Na+ on the outside of cells with K+ domination in cytosol was We postulate that the interaction between Nav1.5 and SAP97 may be implicated in the initial condition for electrogenesis formation on external membrane in terms of correct localisation, turn-over and regulation of Nav1.5 in cardiomyocytes. This would evolution. support the hypothesis of the presence of two pools of Nav1.5 channels: one targeted at 7. Special attention will be paid to the facts which contradict the hypothesis of slow lateral membranes by the syntrophin-dystrophin complex, and another one targeted at Na-current participation in generation action potential upstroke. intercalated discs by SAP97.
P1AM-2-12 P1AM-2-13 KINETICS OF SLOW VDI AND FAST RECOVERY OF MOLECULAR MECHANISMS FOR THE REGULATION 2+ CaV1.3 KEEP ITS HIGH CHANNEL AVAILABILITY OF Ca SIGNALOSOME VIA LIPID BINDING PROTEIN UNDER PACEMAKER ACTION POTENTIAL IN ATRIA IN ATRIA 1 1 1 Hiroko Izumi-Nakaseko1, Shingo Murakami2, Masanori Ito1, Satomi Adachi-Akahane , Hiroko Izumi-Nakaseko , Masanori Ito , 2 1 Yoshihisa Kurachi2, Hiromichi Tsuru1, Satomi Adachi-Akahane1 Isao Naguro , Hiromichi Tsuru 1 1Pharmacology, School of Medicine, Faculty of Medicine, Toho University, Dept. Pharmacology, Sch. Medicine, Fac. Medicine, Toho University, 2 Japan, 2Div. Mol. Cell. Pharmacol., Dept. Pharmacol., Sch. Med., Osaka Japan, Laboratory of Cell Signaling, Graduate School of Pharmaceutical Univ., Osaka, Japan Sciences, the University of Tokyo, Japan 2+ Atrial myocytes express two subtypes of pore-forming α subunit of L-type Ca2+ Recent studies have shown that the impairment of L-type Ca channel function 1 is associated with arrhythmia such as atrial fibrillation. In addition to modulation channel, CaV1.2 and Ca V1.3. We have confirmed, in heterologous expression system of ion channel activities, manipulation of lipid metabolism system has been with β1a and α2/δ subunits, that CaV1.3 activated and inactivated at voltages lower 2+ shown to be a novel strategy for the treatment of atrial fibrillation. We found that, than those of CaV1.2 by approximately -15mV. The Ca -dependent inactivation in atria, phosphatidylcholine transfer protein-like protein (STARD10) interacts (CDI) kinetics of CaV1.2 and CaV1.3 were similar. However, the voltage-dependent 2+ inactivation (VDI) kinetics of CaV1.3 was significantly slower than that of CaV1.2. with the C-terminal region of the L-type Ca channel CaV1.2 in a subtype- Fitting analysis of VDI showed that the fast-inactivating component of CaV1.3 was specific manner. Mammalian START family proteins (STARD1-15) appear to smaller and the non-inactivating component was larger as compared to those of function in lipid transfer between intracellular compartments, lipid metabolism,
CaV1.2. In addition, the recovery from VDI was faster in CaV1.3 than in CaV1.2. The and modulation of signaling events. STARD10 turned out to modulate the action potential (AP) clamp experiments with pacemaker AP waveform of sinoatrial availability of CaV1.2. The knockdown of STARD10 in atrial myocytes resulted node suggested that CaV1.3 current contributes to the diastolic depolarization at phase in the shortening of action potential duration and an increase in the frequency of IV and AP duration. In addition, CaV1.3 maintained higher availability than CaV1.2 spontaneous action potentials. These results indicate that the excitability of atrial during repetitive action potential. These results indicate that the unique voltage- myocytes is modified through signaling complex that involves the L-type Ca2+ dependence and VDI kinetics of CaV1.3 contribute to maintain the availability of the channel and STARD10. The physiological and pathophysiological roles of lipid Ca2+ channel and the stability of pacemaker AP. transporting proteins in the heart will be discussed. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 125 P1AM-2-14 P1AM-2-15 THE TIME COURSE OF CALCIUM CURRENT BETWEEN IKr AND IKs CHANNEL SUPPRESSION CAUSES EARLY AFTERDEPOLARIZATIONS BY PREVENTING EPI AND ENDO CARDIOMYOCYTES CONTRIBUTES 2+ TO THE TRANSMURAL HETEROGENEITY OF ACTION DEACTIVATION OF L-TYPE Ca CHANNELS IN POTENTIAL VENTRICULAR MYOCYTES NARI KIM1, MARK B CANNELL2, PETER J HUNTER1 Mitsuhiko Yamada, Keisuke Ohta, Atsunori Niwa, Natsuko Tsujino, 1Auckland Bioengineering Institute, New Zealand, 2Department of Tsutomu Nakada, Masamichi Hirose Physiology, The University of Auckland, Auckland, New Zealand Dept. Mol. Pharmacol., Shinshu Univ. Sch. Med., Japan To clarify the transmural heterogeneity of action potential
of ICa,L had two peaks in both cell types. The amplitude of peak ICa,L was significantly examined with the action potential-clamp method, ICa,L currents decayed mainly smaller in ENDO than in EPI cells, while the integrated current was significantly due to inactivation in phase 2 and deactivation in phase 3 repolarization. When
larger in ENDO than in EPI cells. The second peak of ICa,L occurred at more positive EADs were induced by complete block of IKr channels with partial block of IKs potentials in ENDO cells. During rectangular pulses, peak ICa,L was significantly channels, repolarization of the action potential prior to EAD take-off failed to 2+ greater in EPI than in ENDO cells. The inactivation of ICa,L by Ca dependent increase IK1 currents and thus failed to completely deactivate ICa,L channels which mechanisms
The window of ICa,L was smaller in EPI than ENDO cells. We conclude that there are IKs channels were completely blocked, ICa,L channels were not deactivated and distinct differences in the actual time course of ICa,L during AP clamp between EPI and mediated sustained inward currents until the end of EADs. Under this condition, ENDO cells arising from the transmural heterogeneity of the action potential. This the recovery and reactivation of ICa,L channels were absent before EADs. difference should translate into subtle differences in Ca2+ metabolism between EPI and Therefore, an essential mechanism underlying EADs caused by suppression of ENDO cardiomyocytes. the delayed rectifiers is the failure to completely deactivate Ca,LI channels.
P1AM-2-16 P1AM-2-17 INVESTIGATION OF THE CALCIUM-DEPENDENT ENDOTHELIN-1 POTENTIATES L-TYPE Ca CURRENT MODULATION OF L-TYPE CALCIUM CURRENT IN BY ACTIVATING CaMKII IN RAT VENTRICULAR TROUT VENTRICULAR MYOCYTES MYOCYTES Fabien Brette1, Caroline Cros1, Daniel E Warren1, Holly A Shiels1, Kimiaki Komukai1, Jin O-Uchi2, Satoshi Morimoto1, Makoto Kawai1, 2 Laurent Salle Kenichi Hongo1, Michihiro Yoshimura1, Satoshi Kurihara3 1Faculty of Life Sciences, University of Manchester, UK, 2Laboratoire de 1Division of Cardiology, The Jikei University School of Medicine, Japan, 2Aab Physiologie, Universite de Caen, France Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine & Dentistry, USA, 3Department of Cell Physiology, In cardiac myocytes, Ca has a dual role upon of L-type calcium current (ICa) by either The Jikei University School of Medicine, Japan inactivating it (calcium-dependent inactivation, CDI) or facilitating it (calcium- dependent facilitation, CDF). In fish cardiac myocytes, it is unclear whether both Ca [Introduction] Endothelin-1 (ET-1) has a positive inotropic effect but details of the modulations exist. This study addresses this point. Trout ventricular myocytes were mechanisms have not been clarified. L-type Ca current (ICa) is one of the major determinants enzymatically isolated. I was recorded using whole cell patch clamp with Na- and of cardiac muscle contraction, but the effect of ET-1 on ICa is not clear. The response of ICa Ca to ET-1 depends on patch clamp configuration. In conventional whole cell with Ca buffer K-free solutions to avoid contaminating currents. With a low concentration of a slow in pipette solution, Ca-dependent effect cannot be observed. Thus, we hypothesized that Ca buffer (EGTA 2mM) in the pipette solution, ICa inactivated slowly (compared to ET-1 increases I via activation of Ca/calmodulin-dependent protein kinase II (CaMKII). mammalian cardiac myocytes): the time to reach 37% of peak current (T37) was 26.2± Ca [Methods] ICa of rat ventricular myocytes was measured using perforated patch clamp 2.4 ms (mean±SEM, n=14). CDF was absent in all cells studied. When a fast Ca buffer technique. Protein expression of endothelin receptor subtypes was examined by Western (BAPTA 10 mM) was present in the pipette solution, ICa decay was similar to the decay immunoblott analysis. [Results] ET-1 (10 nM) increased ICa without changing the current- in the presence of EGTA (T37: 25.4±1.5 ms, NS, t-test, n=9) and CDF was absent (n=9). voltage relation. This increase in ICa by ET-1 was blocked by KN-93 (0.5 μM), a CaMKII We quantified the relative contribution of CDI and sarcoplasmic reticulum (SR) CDI inhibitor, but not by KN-92 (0.5 μM), the inactive analogue of KN-93. The increase was
according to our published method, and estimated that CDI represents ~39% of total blocked by BQ-123 (1 μM), a selective ETA antagonist, but not by BQ-788 (1 μM), a ICa inactivation, and that SR Ca release causes ~12% of CDI. We conclude that in fish selective ETB receptor antagonist. In Western immunoblott analysis, ETA receptor, but not myocytes CDI play a role in ICa modulation but CDF is absent. ETB receptor was found in membrane fraction of rat ventricular myocytes. [Conclusion] Supported by the Welcome Trust ET-1 increases ICa via ETA receptor stimulation and CaMKII activation.
P1AM-2-18 P1AM-2-19 PDE2 INVOLVES IN A NON-GENOMIC REGULATION OF A NONSELECTIVE NON-STEROIDAL ANTI- CARDIAC L-TYPE CALCIUM CURRENTS INFLAMMATORY DRUGS (NSAID), DICLOFENAC 2+ Emika Kurobane1, Junko Kurokawa2, Takeshi Suzuki1, INHIBITS L-TYPE Ca CHANNEL IN CARDIAC Tetsushi Furukawa2 MYOCYTES 1 2 Seong-Geun Hong1, Oleg V Yarishkin1, Eun-Mi Hwang1, Hee-Jung Shin2, Division of Basic Biological Science, Keio University, Japan, Department 2 2 1 1 of Bioinformational Pharmacology, Tokyo Medical and Dental University, Hye-Joo Chung , Ho-Sang Jeong , Dawon Kang , Jaehee Han , Japan Jae-Yong Park1 1 We have demonstrated that non-genomic regulation of cardiac ion channels is related Department of Physiology, of Health Sciences, and Medical Research Center for Neural Dysfunction, Gyeongsang National University School of Medicine, to gender-differences in cardiac repolarization. In our previous study, we found that 2 2+ Korea, Division of Molecular Pharmacology, National Institute of Toxicological progesterone (P ) rapidly suppresses cardiac L-type Ca currents (I ) under a cAMP- 4 Ca,L Research, Korea Food and Drug Administration, Seoul , Korea stimulated condition via a non-genomic pathway, which may be related to the gender- difference of QT intervals and risks of arrhythmia. Because the I suppression was As a NSAID, diclofenac has been widely used as anti-inflammatory drugs, but also has serious Ca,L cardiovascular (CV) side effects. This study examined the molecular mechanism of diclofenac cGMP-dependent, and was not observed without cAMP inside, we hypothesized that as a strong risk factor for serious CV problems in single myocytes isolated from one-day old rat the regulation is involved in cAMP hydrolysis by cGMP-stimulated phosphodiesterase hearts. In this study to explore the diclofenac effect on ion channels, diclofenac was found to inhibit (PDE2). We therefore investigated effects of PDE inhibitors on the ICa,L suppression inward currents in cardiac myocytes. Under the physiological condition, the rapid transient and the Poster Session by P4 in patch-clamped guinea pig ventricular myocytes. Without inhibitors, a 7-min sustained inward components were elicited by step depolarizations from -100 mV. The sustained 2+ 2+ application of P4 at 100 nM significantly suppressed Ca,LI to 58±4 % (n=13), while ICa,L inward component was carried by Ca through L-type Ca channels (LTCC) by results; permeable to Ca2+ or Ba2+, activated at voltage higher than -50 mV, and blocked by 1 micromol nifedipine, an ran down to 84±2 % without P4 (n=5, time control). In contrast, either a non-specific 2+ PDE inhibitor, IBMX (0.1 mM), or a specific PDE2 inhibitor, EHNA (30 μM), L-type Ca channel blocker. At higher than 3 μM, diclofenac irreversibly inhibited LTCC in a dose- dependent manner (IC50 = 39.76+/-11.98 micromol). Diclofenac also depressed Ca2+ transients in abolished the suppression of ICa,L by the same application of P4 (IBMX; 76±7 %, n=9, a similar fashion with nifedipine. These results suggest that diclofenac inhibits LTCC and thereby EHNA; 84±5 %, n=7). These results suggest that the P4-stimulated cGMP elevation depresses Ca2+ transients generated by Ca2+-induced Ca2+ release from SR, leading to negative stimulates cAMP hydrolysis by PDE2, resulting in the suppression of the cAMP- inotropy on muscle contraction. Our first finding may account for the molecular mechanism of stimulated ICa,L. diclofenac to cause the heart failure.
126 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-2-20 P1AM-2-21 2+ 2+ EFFECT OF T-TYPE Ca CHANNEL BLOCKER ON Ca ANTI-ARRHYTHMIC EFFECTS OF KV11.1 CHANNEL HANDLING OF THE JUNCTIONAL SARCOPLASMIC ACTIVATORS RETICULUM(JSR) IN CARDIAC MUSCLE OF Soren-Peter Olesen, Rie S Hansen, Thomas G Diness, HYPERTENSIVE RAT Morten Grunnet Midori Tanaka, Tatsuo Akema Biomedical Sciences, University of Copenhagen, Denmark Department of Physiology, St. Marianna Univ. Sch. Med., Japan Kv11.1 channels (=hERG channels) are important for terminating the cardiac We previously reported the acceleration of the JSR Ca2+ repletion and an action potential, and block of the channels is known to cause arrhythmia. alternation of the Ca2+ -induced release of Ca2+ from JSR in young adult We have developed a series of Kv11.1 channel openers and addressed their spontaneously hypertensive rat(SHR). Recently, overexpression of T-type effects in arrhythmia models. The organic compound NS1643 (10 uM) Ca2+ channels (Cav3.x) has been found in genetic cardiomyopathy and activates human Kv11.1 channels by doubling the conductance, slowing hypertrophied rat hearts. However, the significance of increased Cav3.x in the inactivation, and shifting the inactivation curve rightward by 9 mV. The diseased hearts is obscure. To clarify the JSR Ca2+ handling mechanism compound is devoid of effect on inactivation-deficient Kv11.1. channels. In in cardiac muscle of hypertensive rat, we examined the effect of R(-)- isolated guinea pig myocytes NS1643 inhibits triggered after-depolarizations enantiomer of efonidipine, a selective blocker of Cav3.x, on the time course and prolongs the post-repolarization refractory time. In isolated guinea pig of short-term mechanical restitution (STMR) after varying magnitude hearts NS1643 shortens the monophasic action potentials and shifts the of twitch contraction with the papillary muscle of adult (20 weeks-old) restitution curve downwards without changing its slope. 10 uM compound SHR[(R(-)SHR)]. The slope of the STMR in R(-)SHR was constant and attenuates all ectopic beats caused by bradypacing the isolated hearts in independent of the magnitude of the preceding twitch, further similar to those hypokalemia. In live rabbits bradypaced during 3 weeks NS1643 likewise of both SHR and their age matched control(WKY), except that the slope inhibited all ectopic beats. In a mathematical model of the human cardiac after rested-state contraction in SHR decreased than the rest. In addition, the electrical activity the anti-arrhythmic effects of the NS1643 are due to latencies in R(-)SHR shortened than in SHR. The results suggest that R(-)- both the increased conductance and the shift in inactivation. In conclusion, enantiomer of efonidipine improves the delay in the onset of the JSR Ca2+ Kv11.1 channel activators show anti-arrhythmic properties in several models repletion and rescues impaired JSR function in adult SHR. primarily by attenuating triggered activity.
P1AM-2-22 P1AM-2-23 AIP1 FROM ARTEMISIA IWAYOMOGI REDUCES THE TARGETED S-NITRYSILATION OF THE KCNQ1 ACTION POTENTIAL DURATION BY ACTIVATION OF IKr CHANNEL IN THE HEART IN VENTRICULAR MYOCYTES Junko Kurokawa, Ken Asada, Tetsushi Furukawa Seong Woo Choi, Won Sun Park, Eun A Ko, Nari Kim, Jin Han Department of Bio-informational Pharmacology, Tokyo Medical and Dental Department of Physiology, NRL for Mitochondrial Signaling, CMDC, FIRST University, Japan Mitochondrial Research Group, Inje University, Korea Nitric Oxide (NO) regulates numerous cellular signaling pathways through We investigated the effects of a hot-water extract of Artemisia iwayomogi cGMP activation or/and s-nitrosylation. Recent works implicate that , a plant belonging to family Compositae, on cardiac ventricular delayed s-nitrosylation at the side chain of cysteine residue is a key mechanism of posttranslational modification. We have previously reported that NO regulates rectifier K+ current (IK) using the patch clamp technique. Application of 50 currents through the cardiac slowly-activating delayed rectifier potassium microg/ml of the carbohydrate fraction AIP1 reduced the action potential channel (IKs) independently of soluble guanylate cyclase activation. Here we duration (APD) by activating IK without significantly altering the resting demonstrate using a biotin-switch assay that NO s-nitrosylates Cys445 in the membrane potential. Pre-treatment with the rapidly activating delayed carboxyl-terminus of the alpha-subunit of the IKs channel, KCNQ1. A redox rectifier K+ current (IKr) inhibitor, E-4031 prolonged APD. However, motif flanking Cys445, and the interaction of KCNQ1 with calmodulin are additional application of AIP1 did not reduce APD. The inhibition of slowly required for preferential s-nitrosylation of Cys445. Patch-clamp experiment activating delayed rectifier K+ current (IKs) by chromanol 293B did not shows that S-nitrosylation of Cys445 modulates the KCNQ1/KCNE1 channel change the effect of AIP1. AIP1 did not significantly affect coronary arterial function. Our data provide a molecular basis of NO-mediated regulation tone or ion channels, even at the highest concentration of AIP1. In summary, of the IKs channel. This novel regulatory mechanism of the IKs channel may AIP1 reduces APD by activating IKr but not IKs. These results suggest a play a role in previously demonstrated NO-mediated phenomenon in cardiac novel clinical effect of the natural product AIP1 in the treatment of long QT electrophysiology, including shortening in action potential duration in syndrome. response to intracellular Ca2+ or sex hormones.
P1AM-2-24 P1AM-2-25 THE ROLE OF KCNE3 IN THE REGULATION OF REGULATION OF VOLUME-REGULATED CARDIAC KCNQ1 POTASSIUM CHANNEL OUTWARDLY RECTIFYING ANION CHANNELS BY Futoshi Toyoda, Wei-Guang Ding, Dimitar P Zankov, PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE IN Hiroshi Matsuura MOUSE VENTRICULAR CELLS Department of Physiology, Shiga University of Medical Science, Japan Kunihiko Ichishima, Shintaro Yamamoto, Tsuguhisa Ehara KCNQ1 potassium channel exhibits diverse functional properties by Physiology, Saga University Faculty of Medicine, Japan coassembling with the KCNE family of regulatory β-subunits comprising at least We examined the effect of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and five known members (KCNE1-5). It is well established that in heart KCNQ1 phosphatidylinositol 4,5-bisphosphate (PIP2) on the hypotonicity-activated interacts with KCNE1 to generate the slow delayed rectifier IKs current important chloride current that flows through volume-regulated outwardly rectifying anion for cardiac repolarization. Nevertheless, the involvement of multiple KCNE channel (VRAC) in mouse ventricular cells. The VRAC current was suppressed subunits in regulation of cardiac KCNQ1 channel has been of interest, because by intracellular application of LY294002 (a PI3K inhibitor), anti-PIP3 antibody all KCNE members are reportedly expressed in human heart. The present (PIP3-Ab) or anti-PIP2 antibody (PIP2-Ab). The VRAC currents attenuated by study addressed the question whether KCNE3 is involved in the regulation of LY294002 and PIP2-Ab were restored by intracellular application of excess cardiac KCNQ1 channels. Heterologous assembly of KCNQ1 and KCNE3 PIP3. These results suggest that PI3K-mediated PIP3 production from PIP2 is yielded constitutively active non-IKs potassium current, which was observed required to activate VRAC current. We found that an α1-adrenergic receptor even in the presence of lager amount of KCNE1. In primary cultured guinea- (α1AR) agonist, phenylephrine (PE), inhibited VRAC current in PLC-dependent pig cardiomyocytes, silencing of KCNE3 by small interfering RNA reduced an and PKC-independent manner. This inhibition did not occur in the presence of instantaneous HMR1556-sensitive current without changing IKs amplitude, which a subtype-specific α1A-receptor antagonist. In addition, m-3M3FBS (a putative was accompanied with the prolongation of action potential duration. Using PLC activator) also inhibited VRAC current. PE and m-3M3FBS unaffected computer simulation, the contribution of various amplitude of the KCNQ1/ VRAC current in cells dialyzed with excess PIP2. These findings suggested that KCNE3 current to cardiac action potential was determined. Taken together the PE-induced inhibition of VRAC current is related to PIP2 depletion caused by our results provided presumptive evidences for the mechanistic link between α1A-aderenergic activation of PLC. We propose that PIP3 and its precursor, PIP2, KCNQ1 and KCNE3 in the heart. are essential modulators for activation of VRAC. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 127 P1AM-3-1 P1AM-3-2 THE EFFECT OF CAFFEINE ON CARDIOMYOCYTES EFFECTS OF BEAT-TO-BEAT MODULATION OF Ca2+ ISOLATED FROM DIFFERENT AGE GROUPS CURRENT ON THE GAIN OF Ca2+-INDUCED Ca2+- Sarah Elizabeth Martin, Nilima Shukla, Hua Lin, Jamie Jeremy, RELEASE IN CARDIAC MUSCLE Saadeh Suleiman Moutaz El-Kadri1, George Hart1, Munir Hussain2 Bristol Heart Institute, University of Bristol, UK 1Department of Cardiology, University of Liverpool, UK, 2Medical The aim of this study was to determine whether the effect of caffeine on Biosciences Research Group, University of Bradford, UK 2+ 2+ 2+ 2+ Ca transients in isolated perfused rat cardiomyocytes is age-related. L-type Ca current (ICa) is thought to be the main trigger for Ca -induced Ca - Cardiomyocytes were enzymatically isolated from 14, 21, 28 days old and release (CICR) from the sarcoplasmic reticulum (SR) in cardiac muscle. We adult rat heart. Freshly isolated cardiomyocytes were loaded with Fura-2, investigated whether changes in unitary Ca2+ current during enhanced Ca2+ ο then perfused in a chamber under an inverted microscope at 37 C and channel open probability (Po) modulates CICR. Myocytes were voltage clamped stimulated at 0.2Hz. Ca2+ transients were monitored using photometry. (perforated patch clamp) and stimulated with 5 conditioning pulses from -40 to Addition of 20mM caffeine for 1 min induced the characteristic marked 0 mV (0.5 Hz, 35°C) to standardize SR Ca2+ load before applying test pulses to increase in Ca2+ transients amplitude that was followed by complete abolition different potentials. Using a rapid solution changer after the last conditioning 2+ of the transients as the sarcoplasmic reticulum (SR) is emptied. The effect pulse, unitary current was modulated by changing external [Ca ]o (from 1.0 to 2+ was seen in cardiomyocytes from all age groups. Upon washing out the 0.2 or 5.0 mM) and Po was increased using 2.5 μM FPL64176 (Ca channel 2+ 2+ caffeine, the SR gradually refills its Ca stores and transients are restored. agonist). FPL64176 and low [Ca ]o significantly altered ICa amplitude at most The rate of SR refilling was significantly slower in 14 day cardiomyocytes potentials tested (e.g. 72±4 % at 0 mV, n=7) without causing a parallel change compared to those from adult. At 14 days the SR is not fully developed and in SR Ca2+ release (93±4 % at 0 mV, P>0.05). A modest decrease was observed may have reduced ability to accumulate Ca2+. Refilling of the SR with Ca2+ only at positive potentials. The combined application of FPL64176 and high 2+ 2+ in early postnatal development depends on extracellular Ca coming via the [Ca ]o increased ICa amplitude by 225±10% (at 0 mV, n=10) with only a modest Na/Ca exchanger rather than the Ca2+ channel. The exchanger is likely to be increase in SR Ca2+ release (112±2 %), thereby effectively decreasing CICR slower in transporting Ca2+ across the sarcolemma which would explain the gain (52±2 %). This suggests that modification of SR Ca2+ load or RyR receptor slower rate of SR refilling. activity may be critical determinants of the gain of CICR
P1AM-3-3 P1AM-3-4 EFFECTS OF RAPIDLY DECREASING L-TYPE A MODEL OF THE CARDIAC SARCOPLASMIC/ 2+ 2+ Ca CURRENT (ICa) ON Ca RELEASE FROM THE ENDOPLASMIC RETICULUM CALCIUM ATPASE SARCOPLASMIC RETICULUM (SR) (SERCA) THAT IS SENSITIVE TO CELLULAR Moutaz El-Kadri1, George Hart1, Munir Hussain2 ENERGETICS 1 2 Department of Cardiology, University of Liverpool, UK, Medical Kenneth Tran1, Nicolas P Smith2, Denis S Loiselle3, Edmund J Crampin1 Biosciences Research Group, University of Bradford, UK 1Auckland Bioengineering Institute, University of Auckland, New Zealand, SR calcium-induced calcium release (CICR) is normally triggered by and is 2Computing laboratory, University of Oxford, UK, 3Department of Physiology, proportional to ICa amplitude. We assessed the effects of rapidly decreasing ICa University of Auckland, New Zealand 2+ 2+ 2+ amplitude on SR Ca release independently of changes in SR Ca load. ICa The cardiac sarcoplasmic/endoplasmic reticulum Ca (SERCA) pump is responsible 2+ and [Ca ]i transients were recorded using pulses from -40 to 0 mV. A train of for bringing about relaxation in the heart by actively sequestering Ca into the SR 5 conditioning pulses preceded each test pulse. A rapid solution changer was lumen after the initial Ca release. The pump requires an adequate supply of chemical 2+ used to reduce ICa amplitude by using low [Ca ]o, 0.5 μM nifedipine, or both. energy in the form of ATP hydrolysis to transport Ca against a large concentration 2+ Reducing [Ca ]o from 1.0 to 0.2 mM decreased ICa amplitude by 34 ± 5%, gradient. During ischemia, the energetic state of the cell becomes compromised, 2+ (n=10, P<0.05), whereas the amplitude of the accompanying [Ca ]i transient causing an adverse shift in the fine balance between energy demand and supply. remained at 98 ± 2% of control. Nifedipine produced a similar effect; ICa We have developed a mathematical model of the SERCA pump that consolidates 2+ amplitude decreased by 38 ± 5% (P<0.05) but [Ca ]i transient remained at 96 a variety of experimental data and the fundamental principles of free energy 2+ ± 4% of control. However, the combined use of low [Ca ]o and nifedipine was transduction into a consistent biophysical framework. The model has a reversal 2+ synergistic; ICa decreased to 22 ± 4% (P<0.001) and [Ca ]i transient to 47 ± 6% point, which is determined by the energy needs of the pump and the available free 2+ of control (P<0.001). The inhibition of both ICa and [Ca ]i transient amplitudes energy. The parameters of the model are simultaneously fitted to a range of data sets was not parallel leading to a 108 ± 20% (P<0.05) increase in CICR gain. Our which characterise the pump’s dependence on Ca2+, H+ and the metabolites MgATP, data show that some L-type Ca2+ channels may conduct current but not be MgADP and Pi. The SERCA model is integrated into a whole cell model of cardiac needed to trigger SR Ca2+ release. Subsequent changes in SR Ca2+ release may be electrophysiology, Ca dynamics and mitochondrial energetics to investigate the role of mediated by changes in SR load or local Ca2+ concentration SERCA in heart failure and ischemia.
P1AM-3-5 P1AM-3-6 TIME COURSE OF CALCIUM SPIKE ACTIVATION BY REGULATION OF DIASTOLIC CALCIUM LEAK BY THE CALCIUM CURRENT RYANODINE RECEPTOR GATING Ivan Zahradnik1, Alexandra Zahradnikova jr.2, Eva Polakova3, 1 2 3 1 3 Alexandra Zahradnikova , Ivan Valent , Ivan Zahradnik Jana Pavelkova , Alexandra Zahradnikova 1Laboratory of Molecular Biophysics, UMFG SAV, Slovakia, 2Department 1Laboratory of Electrophysiology, UMFG SAV, Slovakia, 2Laboratory of Cell 3 of Physical and Theoretical Chemistry, Comenius University, Slovakia, Morphology, UMFG SAV, Slovakia, Laboratory of Molecular Biophysics, 3 Laboratory of Electrophysiology, UMFG SAV, Slovakia UMFG SAV, Slovakia Calcium released in cardiac myocytes is triggered by calcium influx through the Diastolic Ca spark frequency, a key regulator of sarcoplasmic reticulum calcium plasma membrane calcium channels, but the relationship between the kinetics of content, is increased in the failing heart. This increase might contribute to the decrease calcium current and that of local calcium release activation is not known. of cardiac contractility as well as to the increase in the propensity to arrhythmias. We Local calcium release events (Ca-spikes) were evoked by calcium currents in voltage- present analytical calculation of diastolic spark frequency based on ryanodine receptor clamped isolated left ventricular myocytes excited by step depolarizations from -50 (RyR) open probability. Because of scarcity of experimental data on Ca2+ dependence to 0 mV. Ca-spikes were measured using 1 mM Oregon Green 488 BAPTA-5N as of spark frequency, the whole parameter space of RyR models, in which RyR opening the calcium indicator and 4 mM EGTA to limit calcium diffusion. The latencies and was coupled either to calcium binding, or to interaction between RyR monomers, amplitudes of calcium spikes were analyzed using the maximum likelihood method. was explored. Changes in any of the parameters of RyR gating (cytosolic calcium Two populations of Ca-spikes (early and late), differing in their latency and amplitude, Poster Session sensitivity, coupling between Ca binding and channel opening, interaction between were found. Early spikes had markedly higher amplitude and slightly slower time monomers, tendency for spontaneous channel opening, changes in the refractory course than late spikes. Both populations were activated with kinetics proportional 2+ period) evoked increase in spark frequency as well as increase in the apparent Ca to the kinetics of calcium current activation. The time constants of Ca-spike latency distribution were about 4- and 20-times slower than that of calcium current activation sensitivity of spark generation in the diastolic calcium concentration range of 0.01 - 0.4 for early and late spikes, respectively. μM. Our results confirm the importance of altered RyR gating in abnormal diastolic Our data show that both early and late spikes are activated by calcium channels, albeit spark generation and reveal that different mechanisms of RyR dysfunction may lead to with different coupling fidelity. a similar increase in diastolic calcium release, thus converging to the same symptoms. Supported by the grants VEGA 02/0102/08 and APVV-0139-06. Supported by APVV-0139-06
128 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-3-7 P1AM-3-8 REACTIVE OXYGEN SPECIES PRODUCTION PLAYS Na+, Mg2+, AND ADENINE NUCLEOTIDES MODULATE AN INTEGRAL ROLE IN BETA-ADRENERGIC-INDUCED THE Ca2+-, CAFFEINE-, AND RAPID COOLING- CARDIAC INOTROPY IN WILD-TYPE BUT NOT IN OB/ INDUCED Ca2+ RELEASE IN SKINNED FERRET OB MICE CARDIAC MUSCLES Daniel Claes Andersson1, Jeremy Fauconnier2, Takashi Yamada1, Etsuko Tanaka1, Satoshi Kurihara2 Rolf Wibom3, Abram Katz1, Hakan Westerblad1 1Health Service Center, Yokohama National University, Japan, 2Department 1Department of Physiology and Pharmacology, Karolinska Institutet, Sweden, of Cellular Physiology, The Jikei University School of Medicine, Japan 2INSERM U637, Department of Cardiovascular Physiopathology, University of 2+ 3 It is well known that Ca release from sarcoplasmic reticulum (SR) in Montpellier, France, Department of Laboratory Medicine, Karolinska Institutet, 2+ Stockholm, Sweden cardiac muscle is enhanced by adenine nucleotides and inhibited by Mg . In the previous studies, we demonstrated that intracellular Na+ enhances We studied the role of cellular reactive oxygen species (ROS) in the inotropy mechanism of Ca2+ release from SR in aequorin-injected ferret ventricular muscles. In the beta-adrenergic stimulation of wild type (WT) and insulin resistant ob/ob mouse cardiac cells. 2+ + 2+ 2+ present study, we demonstrate the modulation of Ca release by Na , Mg , Freshly isolated cardiac cells were used to study cytoplasmic Ca transients, cell shortening and adenine nucleotides in skinned myocardium. We measured the relative and mitochondrial superoxide production confocal microscopy and fluorescent indicators. 2+ 2+ Malondialdehyde (MDA) modification of proteins was detected with western blotting and amount of Ca released by Ca (pCa6), caffeine, and rapid cooling (RC) (from 24°C to 3°C), using a fluorescent Ca2+ indicator, fluo-3 in saponin- used as a marker of oxidative stress. Beta-adrenergic stimulation by isoproterenol (ISO, 100 + + + nM) increased mitochondrial ROS production in WT cardiac cells. WT hearts that were treated ferret ventricular muscles. Na was replaced with K , Li , and NMDG+. Mg2+ inhibited the Ca2+-induced Ca2+ release, and either Na+ or perfused with ISO for 30 min showed a twofold increase in MDA protein adducts relative 2+ + 2+ 2+ AMP antagonized the inhibitory effect of Mg . Na and AMP enhanced to control. ISO caused an increase in L-type Ca current density, Ca transient amplitude 2+ and contractility in WT cardiac cells and these increases were diminished by application of the caffeine- and RC-induced Ca release in a concentration-dependent 2+ the antioxidant N-acetylcysteine (NAC). In contrast, ROS production was not increased by manner in the presence of 1.5 mM free-Mg . On the other hand, ATP did 2+ + ISO in ob/ob cardiac cells and the stimulating effect of ISO on Ca2+ transient amplitude was not enhance the Ca release in the absence of Na . We concluded that the 2+ 2+ 2+ not affected by NAC. In conclusion, increased ROS production is an integral element in the inhibitory effect of Mg on the Ca -, caffeine- and RC-induced Ca release beta-adrenergic stimulation of WT but not insulin resistant ob/ob hearts. is antagonized by either Na+ or AMP.
P1AM-3-9 P1AM-3-10 K201 INHIBITS CAMKINASE BUT DOES NOT β-ADRENOCEPTOR STIMULATION INCREASED Ca MEDIATE ITS ACTION ON THE SARCOPLASMIC LEAK FROM SARCOPLASMIC RETICULUM WITHOUT RETICULUM OF ISOLATED RABBIT VENTRICULAR DISSOCIATION OF FKBP12.6 UNDER PHYSIOLOGICAL CARDIOMYOCYTES CONDITION 1 2 2 Elspeth Barbara Ann Elliott , Hisashi Hasumi , Ryuko Matsuda , Satoshi Morimoto1, Jin O-Uchi2, Makoto Kawai1, Kimiaki Komukai1, 2 3 1 Noboru Kaneko , Susan Currie , Christopher M Loughrey , Hiroyuki Sasaki3, Michihiro Yoshimura1, Kenichi Hongo1, Satoshi Kurihara4 4 Godfrey L Smith 1Division of Cardiology, Department of Internal Medicine, The Jikei University 1Division of Cell Sciences, Faculty of Veterinary Medicine, University of School of Medicine, Japan, 2Aab Cardiovascular Research Institute, Department Glasgow, UK, 2Department of Cardiolgy and Pneumology, Dokkyo University of Medicine, University of Rochester School of Medicine & Dentistry, USA, 3 Scool of Medicine, Japan, Strathclyde Institute of Pharmacy & Biomedical 3Division of Molecular Cell Biology, The Jikei University School of Medicine, 4 Sciences, University of Strathclyde, UK, Faculty of Biomedical and Life Japan, 4Department of Cell Physiology, The Jikei University School of Medicine, Sciences, University of Glasgow, UK Japan 2+ Previous studies showed that K201 inhibits spontaneous diastolic Ca release events (DREs) in 2+ 2+ 2+ In heart failure, β-adrenoceptor stimulation (β-ARS) is considered to increase diastolic Ca leak isolated heart cells via inhibition of both the SR Ca release channel and the SR Ca pump. One from ryanodine receptor (RyR) of sarcoplasmic reticulum (SR), leading to contractile dysfunction potential indirect mechanism is via inhibition of CaM kinase (CaMKII). Biochemical assay of and arrhythmia. β-ARS would dissociate the FK506 binding protein (FKBP) 12.6 from RyR CaMKII activity in isolated rabbit homogenates revealed that K201 (1μM) reduced CaMKII activity which is phosphorylated through the activation of protein kinase A (PKA). In the present study, we by 47±6% (P<0.005). Isolated rabbit cardiomyocytes perfused with modified Krebs with 100μM 2+ 2+ intended to identify whether FKBP12.6 dissociates from RyR when Ca leak from SR is increased Ca and 200nM isoproterenol (ISO) were incubated with and without (control) autocamtide-2- by β-ARS. Thin trabeculae obtained from mouse heart were permeabilized with saponin in the related inhibitory peptide (AIP,10μM,60min). Cells were then Fura-4 AM loaded, field stimulated 2+ 2+ o presence of 15 nM calyculin A after treatment with 1 μM isoproterenol (Iso). To estimate Ca (1.5Hz) and perfused with 1.8mM Ca and 200nM ISO (37 C). This resulted in DREs between 2+ 2+ 2+ leak from SR, remaining Ca in SR was released by caffeine (50 mM) and the released Ca was Ca transients. Initial population studies revealed that AIP incubation reduced spontaneous DRE 2+ frequency by ~50%. In single cell studies, perfusion with 3-4 min K201 (1μM) significantly reduced measured with fluo-3. Ca leak from SR was significantly increased accompanying the increase in DRE amplitude in the control group by 66% (n=8,p<0.01). DREs were abolished in the AIP-treated PKA-dependent phosphorylation of RyR at Serine2808 after Iso treatment. However, the amount 2+ group (n=4,p<0.01). These data indicate that AIP does not prevent the action of K201 on diastolic of FKBP12.6 did not alter even after Iso treatment. β-ARS increases Ca leak from cardiac Ca2+ release. Thus despite inhibiting CaMKII, K201 appears to act through a separate pathway to SR through PKA-dependent phosphorylation of RyR at Serine2808 without the dissociation of inhibit spontaneous Ca2+ release. FKBP12.6 under physiological condition.
P1AM-3-11 P1AM-3-12 EXCITATION-CONTRACTION COUPLING IN THE EFFECT OF INORGANIC PHOSPHATE ON 2+ CARDIOMYOCYTES ISOLATERD FROM HEARTS WITH MITOCHONDRIAL Ca DYNAMICS IN SINGLE CORONARY DISEASE PERMEABILIZED VENTRICULAR MYOCYTES OF RAT M.-Saadeh Suleiman, Anabelle Chase, Jihad Hawi, Christopher L Jeong-Hoon Lee, Jeong-Mi Ha, Min-Jeoung Kim, Chae-Hun Leem Jackson, Gianni D Angelini Department of Physiology, University of Ulsan College of Medicine, Korea Department of Clinical Science South Bristol, University of Bristol, UK Mitochondia has Ca2+ regulation mechanisms such as Ca2+-uniporter and Na+- 2+ 2+ Coronary artery disease with resultant myocardial ischaemia is responsible for major Ca exchanger. Mitochondria can absorb huge amount of Ca because of inorganic phosphate (Pi). In this study, we would like to see the effect of Pi clinical complications which can lead to sudden death. We have recently demonstrated 2+ 2+ that our apolipoprotein E knockout mouse model of atherosclerosis and plaque rupture on mitochondrial Ca dynamics. We measured NADH, mitochondrial Ca with Fura-2-FF, and mitochondrial potential with TMRE simultaneously. is also a relevant model of coronary artery disease and sudden death. One possibility 2+ 2+ for sudden death is a triggered ventricular arrhythmia. Unlike other pathologies Cytosolic Ca increased Ca -uptake kinetics in a dose-dependent manner with Pi. The removal of Pi greatly affected Ca2+ concentration dependent (e.g. hypertrophy) nothing is known about the cellular basis for triggered ventricular 2+ arrhythmia in a chronically ischaemic heart. In this study we used enzymatically Ca -uptake kinetics and a dose-dependent curve was shifted to the left. The 2+ isolated ventricular myocytes from control and diseased hearts to compare contractility application of Pi hyperpolarized Δψm and the addition of Ca depolarized (using edge tracking device) and Ca2+cycling (using Fura-2). Δψm rapidly. The depolarization was not related to MPTP. Since the 2+ 2+ Hearts with coronary disease have low myocardial ATP and high lactate which could depolarization occurred after Ca influx, matrix Ca and phosphate caused change the ionic homeostasis including Ca2+handling. Our preliminary data show the depolarization through unknown mechanism. In the absence of Pi, Na+- 2+ that unlike control hearts, myocytes from diseased hearts are heterogenous and show dependent Ca efflux was not changed. However, in the presence of Pi and 2+ impaired contractility and many of them show abnormal Ca2+ transients. Additionally, ATP, as Ca loading time increased, the efflux time constant was increased. 2+ we found disruption to SR function in myocytes from diseased hearts as shown by This effect may link Ca -phosphate complex formation in the matrix. From the response to caffeine. All these observations clearly indicate impairment in E-C these results, we can conclude Pi is an important regulator of mitochondrial coupling associated with progression of coronary disease. Ca2+ dynamics. This kind of Pi effect must have important role in ischemia- reperfusion conditions. (supported by MHWF) Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 129 P1AM-3-13 P1AM-3-14 NOVEL NADH CORRECTION METHOD FOR Ca2+ SENSITIVITY OF THE MYOFILAMENTS IS NOT 2+ MITOCHONDRIAL Ca MEASUREMENT WITH FURA- ALTERED BY THE 1,4 BENZOTHIAZEPINE DERIVATIVE 2-FF IN SINGLE PERMEABILIZED VENTRICULAR K201 IN ISOLATED ADULT RAT VENTRICULAR MYOCYTES OF RAT CARDIOMYOCYTES Min-Jung Kim, Jeong-Mi Ha, Jeong-Hoon Lee, Chae-Hun Leem Hisashi Hasumi1, Elspeth Barbara Ann Elliott2, Toshiya Matsuda1, Department of Physiology, University of Ulsa College of Medicine, Korea Naoyuki Otani1, Ryuko Matsuda1, Noboru Kaneko1, 2+ Fura-2 is a ratiometric fluorescent dye to measure Ca quantitatively. However, Godfrey Leven Smith3, Christopher Michael Loughrey2 Fura-2 was not an appropriate dye for the measurement of mitochondrial 1 2+ Department of Cardiology & Pneumology, Dokkyo Medical University, Ca because of the interference of NADH in the mitochondria and because 2 3 2+ Japan, Faculty of Veterinary Medicine, University of Glasgow, UK, Faculty of Ca dependent change of NADH in the mitochondria. These interferences of Biomedical and Life Sciences, University of Glasgow, UK limit ratiometric quantitative measurement of mitochondrial Ca2+. We used K201 modulates various cardiac Ca2+ handling proteins. Our previous work demonstrates that the principle that the emission intensity ratio of two different excitation lights 2+ K201 (1μM) reduces the Ca transient amplitude by 17% via inhibition of RyR2 and SERCA but must be constant. With this principle, we could develop a novel online NADH 2+ 2+ not cell shortening. To investigate if K201 alters myofilament sensitivity to Ca cardiomyocytes correction of Fura-2. When we measure mitochondria Ca with Fura-2-FF and were isolated by enzymatic digestion, loaded with Fura-4AM, perfused with modified Krebs Rhod-2 simultaneously, we found Rhod-2 showed much slower kinetics than solution (37οC) and field stimulated (6Hz). Cell fluorescence (340/380nm) and cell shortening 2+ Fura-2-FF. We thought Rhod-2 is not an appropriate dye to measure Ca even were measured simultaneously. Perfusion with a range of [Ca2+] from 1.8 to 0.5mM reduced + 2+ though its preferred loading characteristic. When we measured Na -dependent Ca transient amplitude (0.21±0.03 to 0.05±0.01 F340/380;n=12) and cell shortening (5.0±0.62 to 2+ Ca2+-efflux, half activation concentration of Na+ (Kh) was about 0.13 mM which 0.47±0.2μm;n=12). A subset of cells were perfused with 1.8mM [Ca ] containing 0.3, 1.0 and 3.0μM K201. Ca2+ transient amplitude was reduced from 0.22±0.01 in 1.8mM [Ca2+] to 0.09±0.01 was far different from the previous results (3.6 mM Saotome, 2005 and 4.4 mM 2+ 2+ F340/380 in 3.0μM K201;n=25;) as was cell shortening (4.8±0.37 in 1.8mM [Ca ] to 1.19±0.22 in Sedova, 2000). The maximum reachable Ca concentration in the mitochondria 2+ 2+ 3.0μM K201). The relationship between Ca transient amplitude and relative cell shortening was in any cytosolic Ca concentration is about 15 to 20 μM, which is far below the unchanged. A further subset of cells was perfused with 1.1mM [Ca2+] followed by 1.8mM [Ca2+] equilibrium concentration of electrochemical gradient. This method can be very +1μM K201. Ca2+ transient amplitude and cell shortening changed in parellel. These data suggest useful when NADH interference is expected. (supported by MHWF) that K201 has no effect on myofilament Ca2+ sensitivity.
P1AM-3-15 P1AM-3-16 EXPRESSION OF THE CARDIAC MUSCLE ROLE OF NOVEL MAP KINASE TNNI3K IN CARDIAC REGULATORY MOLECULES, TROPONIN T, I AND C IN MYOGENESIS AND IN DEVELOPMENT OF THE SHEEP HEART ACROSS LATE GESTATION CONTRACTILE FORCE 1 2 1 Janna L Morrison1, Giuseppe S Posterino2, Stacey Dunn1, Zhong-Fang Lai , Yu-Zhen Chen , Ke Huang , Kimberley J Botting1, William Wang1, H Forbes1 Shokei Kim-Mitsuyama1 1 1Sansom Institute, University of South Australia, Australia, 2Department of Department of Pharmacology and Molecular Therapeutics, Kumamoto 2 Zoology, Latrobe University, Melbourne, Australia University Graduate School of Medical Sciences, Japan, Department of Immunogenesis, Graduate School of Medical Sciences, Kumamoto During development, the fetal heart undergoes a progressive increase in the ability to University, Japan produce force related to evolving function. The aim of this study was to determine the expression of each of the major troponin molecules, T, I and C during late gestation We investigated effects of TNNI3K, a novel MAP kinase, on cardiomyogenesis and contractile force in the P19CL6-derived cardiomyocytes. P19CL6 cells transfected fetal heart development. At a range of gestational ages (110-140d; term, 150d), with pcDNA6-TNNI3K were induced to beating cardiomyocytes by incubating cells ewes (n=23) were humanely killed and the fetal heart was dissected, frozen and with 1% dimethylsulfoxide. The morphology and beating frequency of beating masses cardiomyocytes dissociated. Western blots were performed on heart tissue for cardiac were investigated and spontaneous beating activities of single cells were investigated isoforms of Troponins C, T and I (Abcam Inc, Cambridge USA). Band densities were by the whole-cell patch clamp technique. The contraction force of beating masses was calculated using Quantity One software relative to internal controls.Despite a decline investigated using computer software to collect and analyze the frequency and amplitude in the percentage of mononucleated cardiomyocytes with increasing gestational of optical signals obtained. TNNI3K-overexpression promoted cardiomyogenesis by age, there was no change in the expression of either troponin T or I. Troponin C, increasing number of beating masses and α-actinin-positive cells. Contractile force and however, showed a 40% increase in protein expression (P<0.04) after 120d gestation. beating frequency of the cells also increased in the TNNI3K overexpressing cells. In single Furthermore, this increase in troponin C was positively correlated with both gestational cardiomyocytes, TNNI3K increased the velocity of diastolic depolarization, prolonged action potential duration(P<0.05) and enhanced epinephrine response of spontaneous action age and fetal weight (P<0.04). These data show that changes in troponin C occur potentials. TNNI3K also suppressed phosphorylation of cTnI, suppressed Bax, p38, JNK or in late gestation tracking the timing of cardiomyocyte maturation. This may be the ERK-mediated apoptosis, and decreased the number of annexin-V+ cells. Our study indicates mechanism that supports our previous data showing little change in cardiac muscle that TNNI3K plays an important role in cardiomyogenesis and in regulation of cardiac contractility from 125 to 140d gestation. contractile function.
P1AM-4-1 P1AM-4-2 ROLES OF HYPERPOLARIZATION-ACTIVATED MODELLING OF β1-ADRENERGIC MODULATION OF CURRENT Ih IN SINOATRIAL NODE PACEMAKING: CARDIAC EXCITATION IN RAT SINOATRIAL NODE INSIGHTS FROM BIFURCATION ANALYSIS OF CELL MATHEMATICAL MODELS Tao Tao1, David Paterson2, Nic Smith1 1 2 3 Yasutaka Kurata , Hiroyuki Matsuda , Ichiro Hisatome , 1The Computing Laboratory, The University of Oxford, UK, 2Department of Toshishige Shibamoto1 Physiology, Anatomy & Genetics, The University of Oxford, UK 1Department of Physiology, Kanazawa Medical University, Japan, 2Nano- The heart is an efficient and effective electro-mechanical pump for supplying Medicine Merger Education Unit, Kyoto University Graduate School of Medicine, the continuous flow of blood that is fundamental for life. The crucial function is Japan, 3Division of Regenerative Medicine and Therapeutics, Tottori University Graduate School of Medical Science, Japan highly dependant on the regulation of the cardiac excitability by modulation of the autonomic nervous system. In this study, we employed a consistent model of To elucidate the roles of hyperpolarization-activated current (Ih) in sinoatrial node (SAN) sympathetic regulation of pace making in rat heart to represent the biochemical pacemaking, we theoretically investigated 1) the effects of Ih on bifurcations during hyperpolarization, 2) combined effects of I and Na+ channel current (I ) on pacemaker networks and unravel the complex and multi-scale mechanisms underlying the h Na generation of spontaneous excitation and electrical conduction. The primary rat robustness against hyperpolarization, and 3) whether Ih-dependent pacemaking is possible. Bifurcation analyses were performed for mathematical models of rabbit SAN cells; sinoatrial node model was developed on the basis of published experimental data equilibrium points (EP), periodic orbits, and their stability were determined as functions of (Shinagawa et al; Satoh; Heaton et al) and was incorporated by the β-adrenergic
Poster Session parameters. Structural stability to hyperpolarization was also evaluated by applications of signalling regulation (Saucerman et al; Himeno et al). This model contains bias currents (Ibias), acetylcholine or electrotonic loads. Unstable potential region determined most of β-adrenergic sensitive membrane currents including If, Ist, ICaL, IKs, with Ibias applications did not shrink as Ih diminished. In the periphery, Ih increased the critical which are regulated by β-adrenergic agonists; the β-adrenergic modulation on acetylcholine concentration and gap conductance for pacemaker cessation. Effect of INa on the Phospholamban and the Phosphoralation of RyR are also considered in the pacemaker robustness was greater in the Ih-incorporated system. In hyperpolarized cells, blocking I abolished pacemaking via saddle-node and Hopf bifurcations. These results model; the simulated action potential are consistent with those from experimental h recordings, and responds properly to the β-adrenergic stimulation. As far as we suggest that 1) Ih does not destabilize an EP but helps INa destabilize the EP, 2) Ih improves the structural stability to hyperpolarization, and 3) Ih-dependent pacemaking is possible in know, it is the first cellular model for rat SA node with the feature of sympathetic hyperpolarized cells. modulation of pace making.
130 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-4-3 P1AM-4-4 ERRATIC BEATS OF SINOATRIAL NODE INDUCED BY FUNCTIONAL STABILITY OF ARTIFICIAL BIOLOGICAL SYMPATHETIC HYPER-STIMULATION PACEMAKERS :A SIMULATION STUDY 1 1 2 Guo-Qiang WU , Cheng LI , Lin-Lin SHEN Maria Takeuchi, Aoi Miyabe, Yasuhiro Naito, Masaru Tomita 1 Mechanics and Engineering Science, Fudan University, China, Institute for Advanced Bioscience, Keio University, Japan 2Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, China Recent advances have demonstrated several approaches to creating artificial biological pacemaker (BP) cells from quiescent ventricular cells via Heart rate series of the patients with congestive heart failure are fraught adenoviral gene transfer, as an alternative to electronic pacemaker that have with erratic beats. Detailed inspection of ECG revealed some of these erratic potential risk of pacemaker pocket infections. The present study evaluated a beats are composed of merely sinus rhythms. Such erraticism is probably stability of BP cells against various changes in external environment, such ascribes to sinoatrial node behavior. To illustrate possible mechanism, we as plasma electrolyte concentration. We simulated suppression of K 2.1 applied a sinoatrial cell model to simulate the modulation of autonomic ir neurotransmitters. Three signal pathways were constructed to depict the channel current (IK1) and over expression of the hyperpolarization activated action potential (AP) formation. 1)The neuromuscular junction pathway cyclic-nucleotide (HCN) gated channel current (If) in a comprehensive transduces the ACh signal directly to Na channel; 2)Through muscarinic electrophysiological cardiomyocyte model, known as the Kyoto model (Matsuoka et al., 2003), on the basis of the E-CELL Simulation Environment receptor M2, ACh also activates the GTPase activity of Gi proteins, whose (SE). Our simulation reproduced well the reported behavior of the induced βγ-subunits further bind to KACh channels; 3)L-type calcium-(ICa,L), BP activity. We then compared a stability of BP cells with that of sinoatrial potassium-(IK), sodium-potassium ATPase (INaK) and hyperpolarization- activated currents (If) are activated by second messenger cAMP, which is (SA) node pacemaker against possible fluctuation in plasma electrolyte the production of ADC-catalyzed ATP, and modulated by interaction of NE- concentration. We evaluated a stability of pacemaker cells in a parameter activated Gs-protein positively and ACh-activated Gi-protein negatively. The sensitivity analysis. Induced BP activity was successfully simulated with simulation showed that the sympathetic hyper-stimulation led to larger cAMP E-CELL SE. Evaluation of the stability of BP showed that BP cells are production, and consequently resulted in a short followed by prolonged AP generally less stable than SA node cells against possible fluctuation in plasma durations. These beats formed erraticism in heartbeat variability. electrolyte concentration.
P1AM-4-5 P1AM-4-6
IN SILICO STUDY ON EFFECTS OF IKur BLOCK SIMULATION OF DEVELOPMENTAL CHANGES IN KINETICS ON PROLONGATION OF HUMAN APD ACTION POTENTIALS WITH VENTRICULAR CELL AFTER ATRIAL FIBRILLATION-INDUCED ELECTRICAL- MODELS REMODELING Hiromi Kumamoto, Hitomi I Sano, Yasuhiro Naito, Masaru Tomita Kenji Tsujimae, Shingo Murakami, Yoshihisa Kurachi Institute for Advanced Biosciences, Keio University, Japan Department of Pharmacology, Graduate School of Medicine, Osaka During cardiomyocyte development, early embryonic ventricular cells show University, Japan spontaneous activity that disappears at a later stage. Dramatic changes in Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice. action potential are mediated by developmental changes in individual ionic Pharmacological treatment with various anti-arrhythmic agents for the termination currents. To simulate the action potential of the rodent ventricular cell at or prevention of AF is not yet satisfactory. This is in part because the drugs may not three representative developmental stages, quantitative changes in the ionic be enough selective for the atrium and they often cause ventricular arrhythmias. The 2+ + currents, pumps, exchangers, and sarcoplasmic reticulum (SR) Ca kinetics ultra-rapid delayed rectifying K current (IKur) is found in the atrium but not in the were represented as relative activities, which were multiplied by conductance ventricle, and it has been recognized as a potentially promising target for anti-AF or conversion factors for individual ionic systems. The simulated action drugs which would be without ventricular proarrhythmia. Several new agents that potential of the early embryonic ventricular cell model exhibited spontaneous specifically block I have been developed. They block I in a voltage- and time- Kur Kur activity, which ceased in the simulated action potential of the late embryonic dependent manner. Here we use mathematical models of normal and electrically- remodeled human atrial action potentials to examine the effects of the blockade and neonatal ventricular cell models. The simulations with our models were kinetics of I on atrial action potential duration (APD). It was found that after AF able to reproduce action potentials that were consistent with the reported Kur characteristics of the cells in vitro. The action potential of rodent ventricular remodeling an IKur-blocker with fast onset can effectively prolong APD at any stimulus frequency, while a blocker with slow onset prolongs APD in a frequency-dependent cells at different developmental stages can be reproduced with common sets manner only when the recovery is slow. The results suggest that the voltage- and of mathematical equations by multiplying conductance or conversion factors 2+ time-dependence of IKurblockade should be taken into account in the testing of anti-AF for ionic currents, pumps, exchangers, and SR Ca kinetics by relative drugs. activities.
P1AM-4-7 P1AM-4-8 THE ROLE OF THE PURKINJE SYSTEM IN DRUG- THE ROLE OF STRUCTURAL BIFURCATION OF INDUCED ARRHYTHMIAS Z-DISKS ON REGULATING SPONTANEOUS CALCIUM Alberto Corrias, Kevin Burrage, Blanca Rodriguez ACTIVITIES IN CARDIAC MYOCYTES: A THEORETICAL Computing Laboratory, Oxford University, UK INVESTIGATION 1 2 2 1 1 Drug-induced unwanted side effects can cause lethal cardiac arrhythmias, Pan Li , Christian Soeller , Mark Cannell , Wenjie Wei , Xing Cai , 3 which are often related to repolarization abnormalities such as early after Arun V Holden 1Center of Biomedical Computing, Simula Research Laboratory, Norway, depolarizations (EADs). Important insights of in vitro experimental studies 2 indicate that Purkinje cells are more prone to experience drug-induced Department of Physiology, School of Medical Sciences, University of Auckland, New Zealand, 3Institute of Membrane and Systems Biology, Faculty of Biological EADs than ventricular cardiomyocytes. However, the role of Purkinje EADs Sciences, University of Leeds, UK in arrhythmogenesis is still uncertain. In particular, both suppression and The spatial organization of Calcium Release Units (CRUs) is important in understanding facilitation of EADs at the Purkinje-ventricular interface were observed in intracellular calcium dynamics [1], such as initiation and propagation of spontaneous calcium different experimental setups using different animal species. Here we present waves. The structural bifurcation of Z-disks [2] suggested a more homogeneous distribution of a 3D computational model of the rabbit ventricles, which incorporates CRUs in the centre of the cell than considered previously [3]. Here, we incorporated a symmetric geometrical and electrophysiological description of the Purkinje system profile of calcium spark [4] into a three-dimensional (3D) model of ventricular myocyte [5], and based on experimental data. Importantly, a novel mathematical model of the investigated the role of branching Z-disks on regulating spontaneous calcium activities, where the spatial distribution of CRUs was obtained from high resolution Confocal Imaging. Our simulation rabbit Purkinje cell action potential (AP) is developed and incorporated in the results suggested that the structural bifurcation of Z-disks can suppress spontaneous calcium 3D model. Purkinje and ventricular cell AP models are used to investigate activities and lead to isotropic calcium wave propagation. The conduction velocity of intracellular cellular mechanisms of drug-induced EADs, and validation is performed by calcium wave was estimated about 90 μm/s, which is consistent with experimental findings. comparison with isolated cell experimental data. The 3D tissue model is then [1] Izu et al, Biophys J (2006) 91: 95-112 [2] Soeller et al, PNAS (2007) 104: 14958-14963 used to investigate how cell-to-cell coupling, in particular at the Purkinje- [3] Izu et al, Biophys J (2001) 80: 103-20 ventricular interface, affects generation and propagation of EADs at the [4] Banyasz et al, Biophys J (2007) 92: 4458-4465 ventricular level. [5] Li et al, LNCS (2007) 4466: 180-189 Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 131 P1AM-4-9 P1AM-4-10 NUMERICAL STUDY OF THE EFFECT OF COUNTER- THE CONTRIBUTION OF GUYTON'S LARGE PULSATION BY A VENTRICULAR ASSIST DEVICE ON CIRCULATORY MODEL TO OUR UNDERSTANDING OF THE CORONARY AND SYSTEMIC CIRCULATIONS LONG-TERM ARTERIAL PRESSURE CONTROL Ki Moo Lim1, Seong Wook Choi1, In Su Kim1, Hyung Min Jun1, Jean-Pierre Montani1, Thomas H. Adair2, Bruce N. Van Vliet3 Gi Seok Jung2, Eun Bo Shim1 1Medicine/Physiology, University of Fribourg, Switzerland, 2Physiology 1 & Biophysics, University of Mississippi Medical Center, Jackson, USA, Mechanical & Biomedical Engineering, Kangwon National University, 3 Korea, 2Korea Artificial Organ Center; §Department of Biomedical Memorial University of Newfoundland, St. John’s, Canada Engineering, College of Medicine, Korea University, Korea In a large computer model of circulatory control, Guyton et al. demonstrated the We devised a computational model to investigate the hemodynamic effects importance of salt and water balance in setting the long-term blood pressure (BP) of a pulsatile left ventricular assist device (LVAD) on cardiovascular system. level. The model incorporates the empirically demonstrated concepts of blood flow The model consisted of 16 compartments and described the cardiovascular autoregulation (BFAR) and pressure-natriuresis (PN). However, the inherent model system, including the coronary circulation and pulsatile LVAD, and included complexity has led to two major misconceptions by some scientists, i.e. (1) that changes in BP must be linked to changes in total blood volume (BV), and (2) that a baroreflex regulatory system. Congestive heart failure was modeled by whole body BFAR is the cause of volume-loading hypertension (HT). Simulations decreasing the end-systolic elastance of the ventricles in the model. We with Guyton's model show that BP is not a function of total BV per se, but of the compared the hemodynamic response of three different operating conditions 'volume in excess' in the vascular tree, explaining why the pathogenesis of HT in for LVAD treatment: co-pulsation, counter-pulsation, and continuous flow states of low BV induced by sympathetic hyperactivity or angiotensin infusion is to find pulsatile effects on the model. The simulation results indicated that still consistent with the PN concept. Furthermore, we analyzed the role of BFAR counter-pulsation treatment resulted in a 2% higher coronary perfusion, 16% in 3 classical models of volume-loading HT (reduced renal mass-high salt intake, lower left ventricular peak pressure. Co-pulsation treatment gave an even aldosterone infusion and Goldblatt HT). In all 3 HT models, the absence of BFAR poorer response than continuous LVAD. These results suggest that counter- greatly increased the rise in cardiac output and BV associated with HT, but did not pulsation treatment has advantages over continuous LVAD or co-pulsating alter the final level of BP achieved. Thus, computer simulations with Guyton’s model LVAD in terms of the hemodynamic response of heart-failure patients. offer a unique way to test the role of central concepts in BP control.
P1AM-4-11 P1AM-4-12 QT DYNAMICS: NORMATIVE DATA AND DIFFERENCE OF RR AND QT-INTERVALS VARIABILITY PHYSIOLOGICAL IN CONGENITAL HEART DISEASES Hirofumi Kusuki1, Kayo Horio2, Marina Kuriki2, Misa Hosoi3, Seiko Mano3, 1 1 2 3 3 3 3 4 Marina Kuriki , Kayo Horio , Hirofumi Kusuki , Misa Hosoi , Seiko Mano , Hideaki Matsuura , Hirohisa Takasuga , Masayuki Fujino , Masafumi 3 3 4 5 5 2 2 Hideaki Matsuura , Hirohisa Takasuga , Masayuki Fujino , Miyata , Toshio Yamazaki , Shunji Nagaoka , Tadayoshi Hata 5 5 1 1 1 2 Masafumi Miyata , Toshio Yamazaki , Syunji Nagaoka , Tadayoshi Hata Department of Clinical Laboratory, Mie University Hospital, Japan, Graduate 1 3 Graduate School of Health Sciences, Fujita Health University, Japan, School of Health Sciences, Fujita Health University, Japan, Department of 2 3 Clinical Laboratory, Fujita Health University Hospital, Japan, 4Department of Department of Clinical Labolatory, Mie University Hospital, Japan, Department 5 of Clinical Labolatory, Fujita Health University Hospital, Japan, 4Department Pediatric, Toyokawa City Hospital, Japan, Department of Pediatric, Fujita 5 Health University Hospital, Japan of Pediatric, Toyokawa City Hospital, Japan, Department of Pediatric, Fujita Background The objective of this study was to observe aging-related changes by Health University Hospital, Japan determining the relationship between RR and QT variability with QT variability Objectives Congenital heart diseases with increased pulmonary blood flow caused by shunt in index(QTVI) and variability ratio(VR). atrial septal defect(ASD) and ventricular septal defect(VSD), are known to be accompanied by a Subjects and Methods The subjects were 167 children aged 0 to 7 years without heart reduction in heart rate variability. The objective of this study was to investigate shunt rates(Qp/Qs) disease. The standard deviation(SDNN) of the RR interval, average and variance of the heart and fluctuations in RR and QT-intervals by using the variability ratio(VR). rate, and average, standard deviation(SDQT) and variance of the QT of 120 sequential heart Subjects and methods The subjects were 20 children with ASD, 20 children with VSD and 100 beats were calculated to obtain QTVI and VR. These parameters were investigated between healthy children as control. ECG was recorded during echocardiography, and the RR and QT- the following groups: healthy students(n=20), 0-to 6-month-old, 7-to 12-month-old, 1-year- intervals were measured. The VR was obtained by calculating the SD of the RR-intervals(SDNN) old, 2-to 3-year-old, 4-to 5-year-old and 6-to 7-year-old. and the QT-intervals(SDQT). Qp/Qs were obtained using Doppler ultrasonography. Correlations Results 1) QTVI and VR decreased in accordance with aging until 4-to 5-year-old. 2) were investigated between Qp/Qs and VR, SDNN and SDQT. The statistical tests and the values Although SDNN increased from the 0-to 6-month-old to the 6-to 7-year-old, SDQT did not were determined significant at p<0.05. show a significant change. Results 1. In ASD and VSD, the VR showed significant increases compared with the control. 2. Discussion It was shown that maturity of the autonomic nervous system due to human SDNN was presenting a greater decrease in the ASD than in the control and VSD. 3. SDQT was growth increased the SDNN but decreased the QTVI and VR. The relationship between age presenting a greater increase in the VSD than in the control and ASD. and RR and QT variability can be an index to evaluate the functional state of the autonomic Discussion The results of this study indicate that the VR in children with heart disease accompanied nerve system and a proarrhythmic substrate in a child heart. by increased pulmonary blood flow is greater compared with that of healthy children.
P1AM-4-13 P1AM-4-14 OPTICAL MAPPING GUIDED CONTROL OF ORIGIN OF CARDIAC ALTERNANS ELECTRICAL SHOCK TIMING AND ITS APPLICATION Makoto Kodama, Komei Tanaka, Takeshi Kashimura, Makoto TO INTERACTION ANALYSIS OF SPIRAL WAVE Hoyano, Wataru Mitsuma, Hiroaki Obata, Hitoshi Tachikawa, DYNAMICS Masahiro Ito, Satoru Hirono, Haruo Hanawa, Yoshifusa Aizawa Ichiro Sakuma1, Naoki Tomii1, Yuhei Takata1, Tatsuhiko Arafune2, Department of Internal Medicine 1, Division of Cardiology, Niigata Takahiro Yamaguchi3, Nitaro Shibata4, Haruo Honjo5, Kaichiro Kamiya5, University Graduate School of Medical and Dental Sciences, Japan 5 Itsuo Kodama Cardiac alternans is a possible linker between heart failure and ventricular 1Department of Precision Engineering, School of Engineering, The University 2 fibrillation. Interval-force relationship (IFR) is an intrinsic principle of the of Tokyo, Japan, National Institute Of Advanced Industrial Science And regulation of myocardial contractility. In this study, we analyzed IFR during Technology, Japan, 3Toyota Central R&D Labs., Japan, 4Shinjuku Mitsui Building Clinic, Japan, 5Research Institute of Environmental Medicine, Nagoya University, mechanical alternans. [Methods] We measured left ventricular dP/dt under Japan programmed stimulation which was constructed by 8 or 9 beats of basic cycle We developed an optical mapping system that measures action potentials and delivers an electrical length stimuli followed by extra-stimuli of decreasing coupling intervals. The stimulation near the core of a spiral reentry on a Langendorff perfused isolated rabbit heart. We dP/dt of the extra-beats were plotted and fitted to the mono-exponential equation set 16 measurement points on a circular line surrounding the stimulation electrode. Fluorescence (mechanical restitution). A time constant was calculated from the fitted equation signals were continuously monitored at these points with a high speed camera (Dalster, Dalsa) with curves for each patient. [Results] We were able to obtain adequate mechanical longpass filter (cut-off wavelength 600nm) with 490 fps. Changes in fluorescence intensity at each Poster Session restitution curves after both strong and weak beats from 7 patients. The restitution of monitoring point were converted into changes in phase. Discontinuity of phases at monitoring points shows existence of the spiral core in the circle. We could deliver 25 electrical shocks to curves after weak beats steeply rose compared with those after strong beats. The near the core of a spiral reentry among 65 trials. We could visualize the interaction of pre-existing time constant and the expected refractory time of the curves after weak beats wave and virtual electrode phenomen. In one case, two phase singularity (PS) originated from were shorter than those after strong beats. The expected maximum dP/dt did not two hyper polarized area. One of these two PS collided with the original wave. The remaining PS differ after strong and weak beats. [Conclusion] Mechanical restitution properties constitutes a new spiral wave reentry at different location. This observed phenomena are consistent with computer simulation results by Ahihara (Ashihara: J of Cardiovasc Electrophys ,2004, 15, were different after strong and weak beats during mechanical alternans. This 226-2339) It demonstrated that spiral wave can be shifted by applying weak electrical shocks near implies that mechanical alternans originates from alternating changes of the core. myocardial Ca2+ cycling property.
132 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-4-15 P1AM-4-16 ABNORMAL RESPONSE OF STROKE VOLUME AND ACUTE NORMOBARIC HYPOXIA ATTENUATED CARDIAC OUTPUT DURING EXERCISE IN CARDIAC STROKE VOLUME RESPONSE DURING EXERCISE PATIENTS Taira Fukuda1, Taketeru Maegawa2, Akihiro Matsumoto3, 1 2 1 Taira Fukuda , Akihiro Matsumoto , Miwa Kurano-Meguro , Yutaka Komatsu2, Toshiaki Nakajima1, Ryozo Nagai1, 1 1 3 3 Haruhito Takano , Haruko Iida , Taketeru Maegawa , Yutaka Komatsu , 2 3 1 1 1 Takashi Kawahara Takashi Kawahara , Yasunobu Hirata , Ryozo Nagai , Toshiaki Nakajima 1 1 Cardiovascular Department of Internal Medicine, University of Tokyo, Cardiovascular Department of Internal Medicine, University of Tokyo, Japan, 2 3 2 3 , Japan, Japan Women’s College of Physical Education, Japan, Japan Institute of Japan, Japan Institute of Sports Sciences Japan Women’s College Sports Sciences, Japan of Physical Education, Japan Backgrounds and Purpose Although response of stroke volume (SV) and cardiac output (CO) Backgrounds and Purpose Although acute hypoxia might induce abnormality in during exercise might be a good index of cardiac function, their response during exercise cardiac function, its effect on stroke volume (SV) and cardiac output (CO) response was still unclear because of the technical difficulty in their non-invasive measurement. The during exercise was unclear. The present study was conducted to reveal whether acute present study was conducted to reveal whether their response during exercise was abnormal normobaric hypoxia might alter SV and CO response during exercise. Methods and in cardiac patients. Methods and Results Nine cardiac patients and nine normals performed Results Nine healthy male subjects performed symptom-limited bicycle exercise symptom-limited bicycle exercise. A novel thoracic impedance method was used to measure testing under normobaric normoxic and normobaric hypoxic conditions (O2: 14.4%). A SV and CO during exercise. SV and CO were lower in cardiac patients than those in normals novel thoracic impedance method was used to measure SV and CO during exercise. In at submaximal and maximal exercise (SV at maximal exercise: 116±9 [SE] vs. 166±10 hypoxia, not only VO2 max but also CO at maximal exercise was significantly reduced ml, p<0.005). SV reached to its maximal levels during submaximal exercise in the most of (CO: 26.7±2.1 [SE] vs. 30.0±1.8 l/min, p<0.05). Acute hypoxia shifted downward the cardiac patients, whereas it progressively increased until maximal exercise in the most of curve of CO in relation to heart rate (HR), as well as SV in relation to HR. In hypoxia, normals. In cardiac patients, CO at maximal exercise was correlated with SV at maximal 44% subjects attained their highest SV at maximal exercise, although in normoxia, exercise (r = 0.93, p<0.001). Conclusions Cardiac patients had attenuated increment of 89% subjects did. Conclusions Acute normobaric hypoxia corresponding to an altitude SV and CO during exercise and SV reached to its plateau level earlier than did in normals. of 3,000 m attenuated the increment of SV and CO during exercise, and SV reached Furthermore, attenuated CO response was mainly due to the attenuated SV response during its plateau level earlier in hypoxia than that did in normoxia. Thus, acute normobaric exercise. hypoxia might induce cardiac dysfunction during exercise.
P1AM-4-17 P1AM-4-18 ISCHEMIC POSTCONDITIONING PROTECTS HEART INTERLEUKIN-2 PROTECTS CARDIAC FUNCTION FROM HYPOXIA-REOXYGENATION INJURY BY 2+ + AGAINST EARLY DIABETIC INJURY VIA ENHANCING OPENING MITOCHONDRIAL Ca -ACTIVATED K ANTI-OXIDATIVE ABILITY CHANNELS Ling-Bo Qian1, Hui-Ping Wang1, He Huang2, Qiang Xia1 Chunhong Jin, Jinrong Wu, Takao Okada 1Department of Physiology, Zhejiang University School of Medicine, Department of physiology, Juntendo University School of Medicine, Japan China, 2Department of Cardiology, Sir Run Run Shaw Hospital, Zhejiang 2+ + Opening of mitochondrial Ca activated K (mitoKCa) channels has been shown University School of Medicine, China to induce cardioprotection by preconditioning. However, it is unclear whether Cardiovascular complications are the most principal cause of death in diabetes. The protective effect of ischemic postconditioning (IPT) also via opening of mitoKCa aim of the present study is to explore the effect and mechanism of interleukin-2 (IL-2) channels. Male SD rat hearts were excised and perfused with Langendorrf on the cardiovascular functions in early diabetic rats. A diabetic Sprague-Dawley rat manner. For hypoxia-reoxygenation experiment, hearts were made hypoxic for 45 model was established by injection of a single dose of streptozotocin (STZ, 60 mg/kg, min and then reoxygenated for 30 min (control: CT). Ischemic postconditioning i.p.). SOD and GSH-Px activity in myocardium were measured spectrophotometrically. (IPT) was attained by 3 cycles of 10 sec reperfusion and 10 sec ischemia before Left ventricular hemodynamic parameters were recorded in vivo. Pathological changes reoxygenation in the absence and presence of paxilline (Pax; mitoKCa blocker, were assessed by HE and Masson’s trichrome staining. Cardiac Bcl-2 and TGF-β1 1μM) or 5-hydroxydeanoate (5-HD; mitoKATP blocker, 100 μM). Isolated rat mRNA expression was determined by RT-PCR method. IL-2 treatment (5000 or myocytes were loaded with tetramethylrhodamine methyl ester (TMRE, 2 μM). 50000 U/kg/d, s.c.) for 7 weeks had no effect on blood glucose level, but markedly Laser illumination of TMRE was performed to cause opening of mitochondria inhibited the increase of ventricular weight and the decrease of LVDP and ±dP/dtmax permeability transition pore (mPTP). MitoKCa opener NS1619 (NS, 30μM) was in diabetic rats. The myocardial fiber disorder, the perivascular fibrosis, the reduction administered immediately before the laser illumination. Intensity of TMRE of SOD and GSH-Px activities, and the down expression of Bcl-2 and up expression fluorescence and cell length were monitored. Pax or 5-HD abolished the IPT of TGF-β1 mRNA in diabetic myocardium were all significantly attenuated by high induced cardioprotection. NS1619 prevented the decline of TMRE fluorescence IL-2. The results indicate that IL-2 improves early diabetic cardiac function, which intensity and shortening of cell length caused by mPTP opening. We conclude may be related to increased activity of antioxidant enzymes, and regulation of Bcl-2 that IPT protects heart against reoxygenation via opening of mitoKCa channels. and TGF-β1 expressions.
P1AM-4-19 P1AM-4-20 TARGETING CALPAIN/CALPASTATIN TO PROTECT THE EFFECT OF As2 AND As4 ON THE HEART ACTION CARDIOMYOCYTES FROM HYPERGLYCEMIA- POTENTIAL DURATION INDUCED APOPTOSIS Mi Eun Kim, Won Sun Park, Nari Kim, Jin Han Tianqing Peng1, Ying Li1, Limei Shan1, Yanwen Li2, Huaqing Zhu1, 1 Department of Physiology, NRL for mitochondria signaling, CVDC, FIRST Malcolm Arnold Mitochondria Research Group, Inje University, Korea 1Critical Illness Research, Department of Medicine, Lawson Health Research Institute, University of Western Ontario, Canada, 2Department of As2 is well known for the anti-cancer chemical, however, its side effect for Microbiology, Imperial College London, London, UK tachyarrhythmia by inhibition of delayed rectifier K+ channels induces the This study was to investigate the role of calpain/calpastatin in cardiac apoptosis cardiac sudden death in some cases. Therefore, to overcome the side effect induced by hyperglycemia. In cultured adult rat cardiomyocytes, high glucose (33 of As2, we synthesized the As4 and tested the heart rate and action potential + + mM) increased calpain-1 activity, down-regulated Na /K ATPase activity and induced duration (APD). Short term extracellular application of As2 and As4 increased apoptosis. These effects of high glucose were abolished by various calpain inhibitors, the heart rate and reduced the APD. Intracellular application of As2 increased calpain-1 siRNA or calpastatin overexpression. The inhibitory effect of calpain inhibition on cardiomyocyte apoptosis was abrogated by ouabain, a selective inhibitor the APD in proportion of 20%, which could induce the long QT interval and of Na+/K+ ATPase. Furthermore, blocking gp91phox-NADPH oxidase prevented high tachyarrhythmia. However, application of As4 intracellulary increased the APD glucose-induced calpain activity and apoptosis. In a mouse model of streptozotocin- only in proportion of 5%. These results suggest that the side effect on increase induced diabetes, inhibition of calpain increased the Na+/K+ ATPase activity and of APD by As4 is smaller than that of As2. We could not address the detailed decreased apoptosis in the heart. To investigate the functional significance of calpain, mechanism for reduction ration of increase of APD by As4, It could be beneficial isolated hearts were subjected to globlal ischemia/reperfusion (I/R). I/R induced more to treat of cancer by reduction of risk. severe heart dysfunction in hyperglycemic than normoglycemic hearts, which was prevented by overexpression of calpastatin in transgenic mice. In summary, NADPH oxidase-mediated calpain-1 activation induces cardiac apoptosis in hyperglycemia via down-regulation of the Na+/K+ ATPase activity. Overexpression of calpastatin prevents I/R-induced heart dysfunction in hyperglycemia. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 133 P1AM-4-21 P1AM-5-1 MELATONIN REVERSES IMPAIRMENT OF ACTIVITY-SENSITIVE SYNAPTIC PALMITOYLATION CARDIOVASCULAR FUNCTIONS BY MERCURY OF PSD-95 IS INVOLVED IN AMPA RECEPTOR INTOXICATION HOMEOSTASIS Mitali Jindal Jun Noritake, Yuko Fukata, Naoto Matsuda, Tsuyoshi Iwanaga, Department of Physiology, Vallabhbhai Patel Chest Institute, University of Masaki Fukata Delhi, India Division of Membrane Physiology Department of Cell Physiology, National Mechanisms of impairment of neural control of arterial blood pressure Institute for Physiological Sciences, Japan after heavy metal intoxication are ambiguous. The aim of our study was to PSD-95, a postsynaptic scaffolding protein, determines the number of AMPA investigate the toxic effects of organic Hg after acute and chronic exposure on receptors at excitatory postsynapse. Postsynaptic targeting of PSD-95 is the cardiovascular system and to evaluate the efficacy of melatonin, a potent regulated by palmitoylation, which is a common posttranslational lipid antioxidant and free-radical scavenger against Hg intoxication. Wistar albino modification and regulates the membrane targeting of proteins. We recently rats were anesthetized and administered, a single dose of MeHg (2.5mg/kg, i.v.) for acute exposure. Dose (0.5mg/kg/day) for one month was used for chronic identified a subset of DHHC proteins (DHHC2, 3, 7, and 15) as PSD-95- exposure. Changes in systolic, diastolic, mean arterial pressure, heart rate, palmitoylating enzymes. Here, we report that blocking synaptic activity left ventricular pressure (LVP), LVPdp/dt, LVPdp/dtmax, cardiac output and rapidly augments PSD-95 palmitoylation and recruits synaptic AMPA baroreflex sensitivity were recorded, analyzed and compared with the sham- receptors. A dendritically localized palmitoyl-transferase DHHC2, but operated group. In acute Hg exposed group, melatonin (10mg/kg, i.v.) and in not the Golgi-resident DHHC3, mediates this activity-sensitive PSD-95 chronic group, (4.0ug/ml) for one month reversed the baroreflex impairment palmitoylation. Upon activity blockade, DHHC2 translocates to the caused by Hg. Baroreflex sensitivity (BRS) improved significantly (p< 0.05) after postsynaptic density to mediate this effect. Furthermore the knockdown of antioxidant treatment. The impaired haemodynamic parameters and baroreflex DHHC2 or PSD-95 completely abolished the increase of mEPSC amplitude sensitivity may be repaired by melatonin supplementation suggesting that upon activity blockade. These results suggest that dynamic recruitment of cardiovascular functional changes by mercury exposure are mediated through protein palmitoylation machinery plays a central role in regulating synaptic generation of reactive oxygen species. homeostasis.
P1AM-5-2 P1AM-5-3 THE MODIFICATION OF CEREBELLAR AMPA RESCUE OF ABNORMAL PHENOTYPES IN δ2 RECEPTOR EXPRESSION BY MOTOR EXERCISE GLUTAMATE-RECEPTOR-NULL MICE BY THE Hiroshi Maejima, Syuhei Sakano, Takuya Otani, Tomoyuki Kurose, EXTRACELLULAR N-TERMINAL DOMAIN Masataka Deie Takashi Torashima1, Akira Iizuka1, Hajime Horiuchi1, Kazuhiro Mitsumura1, Graduate School of Health Sciences, Hiroshima University, Japan Miwako Yamazaki2, Chiho Koyama1, Kiyohiko Takayama1, Masae Iino1, 2 1 It has been reported long term depression (LTD) at Purkinje cell synapses Masahiko Watanabe , Hirokazu Hirai underlie various forms of motor learning in the cerebellum. Cerebellar Purkinje 1Department of Neurophysiology, Gunma University, Japan, 2Department of neurons contain AMPA receptors with a high proportion of GluR2 but few Anatomy and Embryology, Hokkaido University Graduate School of Medicine, GluR1 subunits. Physical exercises facilitate motor skills based on motor Japan learning. In this study, we assessed the effects of two types of motor exercises on The δ2 glutamate receptor (GluRδ2) is expressed predominantly in Purkinje cells. the expression of AMPA receptors. Knock-out of the GluRδ2 gene results in impaired synaptogenesis and failure of long- 7 weeks old rats were assigned to control group, treadmill running group, and term depression (LTD) at parallel fiber/Purkinje cell synapses, resulting in severe balance exercise group. In treadmill running, rats ran 50 minutes every day at 20 ataxia. However, whether GluRδ2 works as a ligand-gated ion channel and how it is m/min. In balance exercise, rats were imposed 10 times of rotor rod walking at activated are unknown. To clarify those issues, we produced GluRδ2 constructs that 20 rpm. After the interventions, cerebellar cortex was deprived for quantitative lacked conventional S1S2 ligand-binding domains and the ion channel pore. These PCR method to measure expressions of mRNA of GluR1, GluR2, PSD-95, and GluRδ2 constructs contained only the extracellular N-terminal domain (NTD) and/or BDNF. the intracellular C-terminal domain (CTD) linked with a transmembrane domain. Our After 4 weeks intervention, GluR2 mRNA expressions in both exercise groups results indicate that lentivector-mediated expression of the GluRδ2 deletion constructs were significantly lower than control group. in Purkinje cells of immature GluRδ2-null mice reliably rescued ataxia, the expression Our data showed exercises modified the expression of AMPA receptor (GluR2) of LTD, and the developmental removal of CF synapses on Purkinje cell dendrites. depending on intervention term. It was suggested that such modification is related These results suggest that GluRδ2 does not function as either a glutamate-binding to long adaptation and stabilization of motor learning by exercises. protein or an ion channel, and that the NTD together with the CTD regulates the function of GluRδ2.
P1AM-5-4 P1AM-5-5 DEVELOPMENTAL CHANGES OF PRESYNAPTIC MATURATION OF AMPA RECEPTORS ACTIVITY GLYCINE RECEPTOR EXPRESSION ON HIPPOCAMPAL IS REGULATED BY EXPRESSION OF DREBRIN, MOSSY FIBER BOUTONS AN ACTIN-BINDING PROTEIN, IN CULTURED Hisahiko Kubota1,2, Henrik Alle1, Heinrich Betz2, Jorg Geiger1 HIPPOCAMPAL NEURONS 1Indepedent Hertie Research Group, Max-Planck Institute for Brain Research, Kenichi Kato1, Hiroyuki Yamazaki2, Tomoaki Shirao2, Yuko Sekino3 2 Germany, Department of Neurochemistry, Max-Planck Insitute for Brain 1CREST, JST and Department of Neurobiology and Behavior, Gunma Research, Germany University Graduate School of Medicine, Japan, 2Department of Using direct recordings from mossy fiber boutons (MFBs) in rat hippocampal slices Neurobiology and Behavior, Gunma University Graduate School of O 3 (34 C, VH=-80mV), we demonstrate strychnine sensitive glycine-receptor-mediated Medicine, Japan, Division of Neuronal Network, Institute of Medical presynaptic currents. In symmetrical chloride conditions, puff-application of glycine Science, University of Tokyo, Tokyo, Japan, and CREST, JST, Japan to MFBs evoked inward currents which decreased significantly in amplitude during Regulation of AMPA receptors (AMPARs) activity is one crucial mechanism for synaptic postnatal development. Single channel recordings revealed a channel conductance of plasticity. Since actin-dependent alteration in spine shapes is correlated with changes in the presynaptic glycine receptor of about 53 pS (P=12). Using a K-gluconate-based the strength of synaptic signals, actin reorganization during synaptic plasticity could be internal solution with a low chloride concentration, eEPSC amplitudes recorded in involved in regulation of AMPARs activity. In this study, we examined the role of drebrin, a CA3 pyramidal cells (0.1 Hz stimulation at DG or DG hilus) show a slight increase spine resident actin-binding protein, in AMPARs recruitment during synaptic development in the presence of glycine, and exhibit a prolonged and significant enhancement after in rat hippocampal cultured neurons. We transfected drebrin-targeted shRNA and control Poster Session glycine washout. 1μM DCG-IV (mGluR-II agonist) reduced eEPSC amplitudes to control (a scrambled sequence shRNA) vectors at 8 DIV using calcium phosphate methods 71 % indicating that these eEPSCs are largely generated by mossy fiber boutons. In and analyzed at 16-17 DIV by perforated whole-cell patch-clamp configuration. AMPAR- the additional presence of 3μM strychnine, eEPSC amplitudes were not changed by mediated miniature EPSCs (mEPSCs) amplitudes recorded from drebrin knockdown (KD) glycine and wash of glycine. neurons (13.8 ± 1.0 pA, n = 9) were significantly larger than that from control neurons (9.4 ± This marked development decline of presynaptic glycine receptor expression and 1.1 pA, n = 6) (P = 0.01). In addition, mEPSCs decay time constant of drebrin KD neurons glycine receptor-mediated modulation of glutamatergic neurotransmission suggests (5.9 ± 0.4 ms, n = 9) was shorter than that of control neurons (8.7 ± 0.9 ms, n = 6) (P = 0.02). a specific role of presynaptic glycine receptors in the maturation of the hippocampal Our data indicate that drebrin expression is involved in regulation of AMPAR activity, such mossy fiber system. as recruitment and modulation during synaptic development.
134 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-5-6 P1AM-5-7 NEURONAL PHENOTYPE SPECIFIC MEMBRANE DEVELOPMENT AND FUNCTION OF GLUTAMATE RESPONSE VIA POST-SYNAPTIC METABOTROPIC RECEPTOR CLUSTERING IN THE CALYX OF HELD GLUTAMATE RECEPTORS ON 2nd-ORDER SYNAPSES BARORECEPTOR NEURONS OF RATS Naomi Kamasawa, Ko Matsui, Timotheus Budisantoso, Shin-ichi Sekizawa, Andrea G Bechtold, Rick Tham, Ryuichi Shigemoto Ann C Bonham Division of Cerebral Structure, National Institute for Physiological Sciences, Department of Pharmacology, University of California Davis, USA Japan The calyx of Held synapse in the auditory pathway is finely tuned for rapid signal We have shown that postsynaptic group I metabotropic glutamate receptors (mGluRs) transmission. Dynamic changes occur in pre and postsynaptic factors during the depolarize the membrane potential on nucleus tractus solitarius (NTS) neurons + 2+ first three postnatal weeks in rodents to accomplish this feature. However, whether through Na -Ca exchanger mechanism while group II mGluRs hyperpolarize it. arrangement of postsynaptic ionotropic glutamate receptors is also developmentally Since NTS neurons are classified into two phenotypes based on firing behavior from regulated has not been well addressed. As calyx terminal directly encases the hyperpolarized potential, rapid or delayed onset spiking (RS or DS, respectively), postsynaptic cell body, large continuous postsynaptic membrane can be obtained we tested the hypothesis that postsynaptic mGluR response could be depending in freeze-fractured replicas, and SDS-digested immunogold labeling revealed on phenotypes. The data were obtained from anatomically identified 2nd-order 2D-distribution of glutamate receptors. At P7, labeling for AMPA receptors baroreceptor neurons in a brainstem slice with blockade of ionotropic GluRs and (AMPARs) was abundant on loosely packed intramembrane particle (IMP) clusters GABAA-Rs. Higher concentration of a potent and highly selective group II mGluR but also was sparsely located on the surrounding membrane, whereas NMDAR was agonist, L-CCG-1 (100μM), hyperpolarized membrane potential in RS phenotype (-3.7 localized on ~70% of the IMP clusters. At the time of the onset of hearing (P24), ±1.8mV) but depolarized in DS type (+5.3±1.0mV), The depolarization was blocked dense AMPAR but not NMDAR labeling was observed in tightly packed IMP clusters by group I mGluR antagonist, LY 367385 (100μM). Group II mGluR induced- with few extrasynaptic labeling. Using electrophysiological mEPSC recordings from + + hyperpolarization was diminished by Na /K -ATPase inhibitor, ouabain (100μM). postsynaptic MNTB neurons, corresponding changes in AMPAR and NMDAR The data suggest that postsynaptic group I mGluRs may specifically locate on DS activation were observed. Whether the dense packing of receptor molecules contributes phenotype of NTS neurons while group II mGluRs could hyperpolarize the cell to the ultrafast signal transmission required for the precise sound localization is being through a mechanism involving Na+/K+ ATPase regardless of phneotypes. assessed.
P1AM-5-8 P1AM-5-9 SEX DIFFERENCE OF THE EFFECT OF NEONATAL SEROTONIN FACILITATES AMPA RECEPTOR SOCIAL ISOLATION ON EXPERIENCE DRIVEN TRAFFICKING IN THE DEVELOPING BARREL CORTEX SYNAPTIC DELIVERY OF AMPA RECEPTORS IN RAT OF RATS WITH VISUAL DEPRIVATION BARREL CORTEX Susumu Jitsuki, Takuya Takahashi Department of Physiology, Yokohama City University Graduate School of Hirobumi Tada, Tomoyuki Miyazaki, Kasane Komiya, Medicine, Japan Takuya Takahashi Loss of one modality leads to plastic changes of other remaining modalities. Department of Physiology, Yokohama City University, Japan Synaptic delivery of GluR1-containing AMPA receptors into synapses is crucial Although sex differences of the response to stress have been well known, for neural plasticity. Here we show that visual deprivation drives GluR1 into molecular mechanisms are still poorly understood. Here we show that neonatal synapses formed from layer 4 to 2/3 in the rat barrel cortex at adolescent age when normal whisker stimulation no longer deliver GluR1 into these synapses. social isolation disrupts experience-driven delivery of GluR1 containing AMPA Extracellular serotonin is increased in the barrel cortex by the loss of visual receptors to synapse in barrel cortex of males but not females. Furthermore, we function. Antagonists of serotonin receptors prevent visual deprivation- driven found that injection of glucocorticoid disrupts experience dependent synaptic synaptic GluR1 trafficking in the barrel cortex. Direct application of serotonin GluR1 trafficking in the barrel cortex of male but not female rats. Moreover, drives GluR1 into synapses in the barrel cortex of rats with intact vision. we showed that disruption of GluR1 trafficking with social isolation was not Furthermore, visual deprivation sharpens functional whisker-barrel map. These prevented in masuculinized female rats. Our findings suggested that female rats results suggest that loss of visual function leads to the improvement of whisker- could have molecular machinery which protects social isolation stress induced barrel function by serotonin mediated facilitation of experience-driven synaptic disruption of synaptic AMPA receptors delivery. incorporation of GluR1.
P1AM-5-10 P1AM-5-11 ANALYSIS OF SYNAPTIC TRANSMISSION MEDIATED DIFFERENTIAL REGULATION OF SYNAPSE BY AMPAR CHANNEL WITH TARP(γ7) FORMATION AND PLASTICITY BY GluRδ2 IN THE Akiko Miyamoto1, Yoko Nakazato1, Akihito Kawaguchi1, CEREBELLUM 1 2 1 1 2 1 Wataru Kakegawa , Taisuke Miyazaki , Kazuhisa Kohda , Chika Toyoshima , Tetsuhiko Sasaki , Hiroshi Kojima 1 1 1 1 Keiko Matsuda , Kyoichi Emi , Junko Motohashi , Department of Intelligent Information Systems, Tamagawa University, 2 1 Japan, 2Honeybee Science Research Center, Tamagawa University, Japan Masahiko Watanabe , Michisuke Yuzaki 1Department of Physiology, Keio University, Japan, 2Department of Changes in synaptic efficacy are thought to be substrates of learning and memory. Anatomy, Hokkaido University, Japan We focused on Transmembrane AMPA Regulatory Proteins (TARPs) which The δ2 glutamate receptor (GluRδ2), which is predominantly expressed at cerebellar regulate AMPA receptor channels in the postsynaptic membrane and would parallel fiber (PF)-Purkinje cell synapses, plays two crucial roles in the cerebellum: cause synaptic plasticity. Especially, the functional role of γ7 ( one of the TARPs) formation of PF synapses and long-term depression (LTD). Although LTD absolutely is known to be abundant in cerebellar cortex. However, its functional role has requires the signaling via the C-terminus of GluRδ2, the mechanisms by which GluRδ2 not been understood completely. In order to elucidate the role of γ7, firstly we regulates the PF synaptogenesis have remained unclear. Here, we show that the extracellular N-terminal domain (NTD) of GluRδ2 is required for PF synaptogenesis introduced this protein together with GFP in to hippocampal cells in culture by by injecting the Sindbis virus carrying wild-type (GluRδ2wt) and mutant GluRδ2 constructing the plasmid. Then, we examined its function and localization at into the subarachnoidal space on GluRδ2-null cerebella. Remarkably, expression of the synaptic regions of the cells by electrophysiological recording and uncaging GluRδ2wt, but not a mutant GluRδ2 lacking the NTD (GluRδ2dNTD), rapidly induced stimulation. Moreover, we tested whether this protein would have an important PF synapse formation and rescued gross motor discoordination in adult GluRδ2-null role in synaptic plasticity or not. mice just 1 d after injection. In addition, although the kainate receptor GluR6 did not induce PF synaptogenesis, a chimeric GluR6 that contained the NTD of GluRδ2 did. In contrast, LTD was restored in GluRδ2-null Purkinje cells expressing GluRδ2dNTD. Taken together, GluRδ2 differently regulates PF synaptogenesis and cerebellar LTD through the NTD and the C-terminal end, respectively. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 135 P1AM-5-12 P1AM-5-13 MONOCARBOXYLATE TRANSPORTER-2 (MCT2) AGONIST BINDING DOMAINS AT THE REGULATES SUBCELLULAR LOCALIZATION AND EXTRACELLULAR LOOP OF P2X3 RECEPTOR SURFACE EXPRESSION OF GLUTAMATE RECEPTOR-2 SUBUNITS (GluR2) Peter Paul Illes, Patrizia Rubini, Mandy Bodnar, Erzsebet Kato, Fumihiko Maekawa1, Takashi Tsuboi2, Toshihiko Yada1, Helke Groeger-Arndt Luc Pellerin3 Department of Pharmacology, University of Leipzig, Germany 1Department of Physiology, Division of Integrated Physiology, Jichi Medical It has been suggested that the pain-relevant P2X3 receptor subunit exhibits at University, Japan, 2Department of Life Sciences, Graduate School of 3 its extracellular loop four nucleotide binding domains (NBD1-4). In the present Arts and Sciences, University of Tokyo, Japan, Institute of Physiology, experiments, we consecutively replaced all conserved amino acids of the University of Lausanne, Switzerland NBDs by alanin and expressed the wild-type and mutant receptors in HEK293 It has been reported that neuronal monocarboxylate transporter MCT2 not only works cells. The selective agonist alpha,beta-methylene ATP produced in these cells as a lactate transporter, but also plays a role as a binding partner of AMPA receptor membrane currents and increases of intracellular free calcium. It has previously subunit GluR2. Thus, we aimed to elucidate how MCT2 regulates subcellular localization been suggested that several positively charged amino acids in the extracellular and surface expression of GluR2 by visualizing fluorescence-tagged proteins in vitro. loop of P2X receptors are essential for the binding of the negatively charged Neuroblastoma Neuro2A cells and mouse primary cortical neurons were transfected with phosphate groups of ATP or its analogues. This suggestion could be confirmed in plasmids expressing Venus-fused GluR2 and mStrawberry fused with or without MCT2 the present experiments by using the K63A, K176A, R281A, R295A and K299A (mStb-MCT2 and mStb, respectively) and were observed under confocal microscope. Venus- GluR2 was widely distributed within cytoplasm when co-transfected with control mStb. mutants. Then, the question arose whether the neighbouring conserved amino In contrast, Venus-GluR2 and mStb-MCT2 formed clusters in the perinuclear organella in acids of each nucleotide binding domain also contribute to agonist binding. In which mStb-MCT2 was intrinsically localized. In addition, both total and plasma membrane fact, substitution of G66 but not of G302 markedly depressed the sensitivity surface levels of Venus-GluR2 were significantly reduced by expression of mStb-MCT2, of the P2X3 receptor to alpha,beta-meATP. In addition, the substitution of indicating the possibility that MCT2 plays an inhibitory role in plasma membrane expression the conserved amino acids K65, T172, N279, F280 and of the non-conserved of GluR2. Taken these results together into consideration, it is suggested that MCT2 regulates K284 all induced an clear-cut decrease of receptor-sensitivity, as indicated by a intracellular distribution of GluR2 and thereby possibly alters glutamatergic transmission. corresponding increase of EC50 values.
P1AM-5-14 P1AM-5-16 ANALGESIC ω-CONOTOXINS CVIE AND CVIF TRANSIENT RECEPTOR POTENTIAL (TRP) A1 SELECTIVELY BLOCK N-TYPE CALCIUM CHANNELS CHANNEL PRESENTS IN NUCLEUS TRACTUS IN A REVERSIBLE AND VOLTAGE-DEPENDENT SOLITARIES AND FACILITATES GLUTAMATE RELEASE MANNER Boram Sun1, Sung-Il Bang2, Young-Ho Jin2, Byung-Il Min3 1 1 1 1 David J. Adams , Geza Berecki , Leonid Motin , Alison Haythornthwaite , 1 2 2 3 Department of East-west medicine, KyungHee University, Korea, Richard J. Lewis , Paul F. Alewood , Macdonald J. Christie 2Department of Physiology, School of medicine, KyungHee University, 1Queensland Brain Institute, The University of Queensland, Australia, 3 2 3 Seoul, Korea, Department of Physiology, School of medicine, Department Institute for Molecular Bioscience, University of Queensland, Australia, Pain of East-West medicine, Graduate school, KyungHee University, Seoul, Management Research Institute, University of Sydney, Australia Korea Two new ω-conotoxins, CVIE and CVIF, were discovered following a PCR screen of a Conus catus cDNA library. Both peptides potently displaced 125I-GVIA binding to rat Transient receptor potential (TRP) A1 channel responds to low temperature, noxious brain membrane. In Xenopus oocytes, CVIE and CVIF inhibited Ba2+ currents through chemical and pungent ingredients of various spices. In response to such physical and chemical stimulations, TRPA1 channel facilitates Ca2+ influx in the sensory recombinant N-type (α1B, β3, and α2δ1) calcium channels with IC50 values of 2.6 and 19.9 nM, respectively. Recoveries from block were voltage dependent, indicating a afferent nerve and potentiates signal transmission between sensory and CNS neurons. higher affinity of ω-conotoxins to channels in the inactivated state. Neither CVIE nor Many spices, which containing TRPA1 agonists, used for improving taste daily CVIF had any effect on P/Q-, R-, or L-type calcium channels. In rat DRG neurons, and discovered some of them have beneficiary effect for health by lowering blood CVIE and CVIF potently and selectively inhibited N- but not T-type Ca2+ channels and pressure and body weight. TRPA1 agonist mediated positive effects are not related to the reversibility of block was voltage dependent increasing with hyperpolarization. In well known TRPA1 channel’s nociceptive function in sensory afferent nerve. TRPA1 rat spinal cord slices, CVIE and CVIF reversibly inhibited excitatory monosynaptic channels in the visceral afferent nerve may have closely related with autonomic transmission between primary afferents and dorsal horn superficial lamina neurons. regulation and energy metabolism. However TRPA1 channel effect on the visceral In the rat chronic constriction injury model of neuropathic pain, both peptides (3 nerve activity and synaptic transmission in NTS has been not well studied yet. Here, nM) significantly reduced allodynic behaviour. These selective N-type Ca2+ channel we tested TRPA1 channel expression on the segregated group of NTS neuron using the ω-conotoxins are potentially useful neurophysiological tools and inhibitors of patch clamp method for the understanding functional contribution of TRPA1 channel nociceptive signaling with therapeutic implications. on autonomic regulation and energy homeostasis
P1AM-6-1 P1AM-6-2 β1- AND β2-ADRENERGIC ACTIVATIONS ENHANCE BI-DIRECTIONAL REGULATION OF CEREBELLAR EXCITATORY SYNAPTIC TRANSMISSION OF THE SYNAPTIC PLASTICITY THROUGH INTER-GPCR MEDIAL PREFRONTAL CORTEX OF RATS INTERPLAY 1 2 2 2 Ze-Jun Feng, Chun-Lei Zhang, Feng Yi, Xue-Han Zhang, Yuji Kamikubo , Takeshi Shimomura , Yosuke Fujita , Toshihide Tabata , Takashi Sakurai1, Kenkichi Fukurotani2, Masanobu Kano3 Bao-Ming Li 1Department of Cellular and Molecular Pharmacology, Graduate School of Insititute of Neurobiology, Fudan University, China Medicine, Juntendo University, Japan, 2Laboratory for Neural Information Technology, Graduate School of Science and Engineering, University of β-Adrenoceptors (β-ARs) are widely distributed throughout the central nervous 3 Toyama, Japan, Department of Neurophysiology, Graduate School of Medicine, system. Our previous work showed that β-AR activation facilitates excitatory University of Tokyo, Japan synaptic transmission in pyramidal cells of the medial prefrontal cortex On the surface of many central neurons, various G protein-coupled receptors (GPCRs) (mPFC). However, little is known about the β1- and β2-AR regulations of display overlapping distributions and/or form heteromeric complexes. Type-1 metabotropic synaptic transmission in the mPFC. The present study investigated β1- and β2- glutamate receptor (mGluR1) is a Gq/11 protein-coupled receptor which triggers induction AR modulation of glutamate synaptic transmission in layer V/VI pyramidal of long-term depression (LTD) of cerebellar parallel fiber-Purkinje cell synapses. Using cells of the rat mPFC. Our results show that, 1) stimulation of β1-ARs does not cultured mouse Purkinje cells, we found that co-localization of mGluR1 with Gi/o protein- affect the frequency nor the amplitude of mEPSCs, and stimulation of β2-ARs coupled gamma-amino butyric acid and adenosine A1 receptors (GABABR and A1R, respectively) and functional interaction between these GPCRs.
Poster Session increases the frequency but not amplitude of mEPSCs; 2) stimulation of β2-ARs When activated with submicromolar doses of agonists, GABABR increased mGluR1's suppresses the paired-pulse ratio of eEPSC, and stimulation of β1-ARs does not; glutamate-responsiveness while A1R decreased. Both of these effects were independent and 3) stimulations of β1- and β2-ARs both enhance the amplitude of eEPSC of Gi/o protein. When activated with higher doses of the agonists, GABABR augmented and non-MDA-R current and NMDA-induced currents. Taken together, these mGluR1's glutamate-responsiveness continuously while A1R augmented transiently. Both results suggest that β1- and β2-AR activations both facilitate excitatory synaptic of these effects required Gi/o protein. GABABR activation leads to facilitation of LTD of glutamate-responsiveness of Purkinje cells in cultures as well as in cerebellar slices. By transmission in mPFC pyramidal cells. contrast, A1R activation impeded induction of LTD. These findings suggest that GABABR and A1R together may serve as a bi-directional regulator of cerebellar synaptic plasticity.
136 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-6-3 P1AM-6-4 IS MEMORY TRANSFER REPRODUCIBLE IN VITRO? COMMON SIGNALING MECHANISMS FOR LONG- A LONG-LASTING SYNAPTIC ENHANCEMENT AFTER TERM POTENTIATION AND DOPAMINE-INDUCED REPEATED LTP INDUCTION IN CULTURED BRAIN SYNAPTIC ENHANCEMENT AT THE HIPPOCAMPAL SLICE MOSSY FIBER SYNAPSES Kazuyuki Iijima, Yuki Oe, Keiko Tominaga-Yoshino, Akihiko Ogura Nobuyuki Obara1, Satoshi Fukuda2, Haruyuki Kamiya1 Graduate school of Frontier Biosciences, Osaka University, Japan 1Department of Neurobiology, Hokkaido University Graduate School of 2 Memory consolidation is assumed to be established by structural changes of Medicine, Sapporo, Japan, Department of Otolaryngology-Head and Neck synapses. We previously reported that the repeated induction of LTP with proper Surgery, Hokkaido University Graduate School of Medicine, Sapporo, intervals led to a slowly-developing long-lasting enhancement in synaptic Japan strength coupled with an increase in synapse density in CA3-CA1 pathway in It was recently reported that dopamine selectively enhanced the synaptic transmission cultured rat hippocampal slices. We named this phenomenon RISE (Repeated- at the hippocampal mossy fiber synapses by activation of presynaptic 1D -like receptors LTP-Induced Synaptic Enhancement) to discriminate it from the conventional and subsequent increase in cAMP levels within the nerve terminals. Since increase in single LTP and proposed as an in vitro model system for the analysis of the presynaptic cAMP levels has been also suggested to mediate the induction of long- structural plasticity. It is also believed that the information tentatively stored term potentiation (LTP) at this synapse, we wondered whether the dopamine effects share some common signaling pathways with tetanus-induced LTP. In this study, we in the hippocampus is transferred to the cerebral cortex where final storage is examined the effects of prior application of dopamine on LTP induction at the mossy established. To confirm this information transfer is reproducible in vitro, we fiber synapses in hippocampal slices made from young mice (from two to three weeks prepared here a stable culture of the hippocampus with maintained synaptic old). As reported before, dopamine (100 μM) elicited robust enhancement of field connection to the entorhinal cortex. The repeated induction of LTP by chemical EPSPs evoked by mossy fiber stimulation. Tetanic stimulation (100 Hz for 1 sec) means using forskolin in this culture produced a synaptic enhancement in the in the presence of NMDA receptor blocker D-AP5 (25 μM) also caused LTP, but cortical part instead of the hippocampal part, as revealed by a 2-dimensional the magnitudes of potentiation were significantly smaller than those without prior membrane potential assay using a voltage-sensitive dye. Since the synaptic application of dopamine. These results suggested that dopamine-induced synaptic enhancement occurred in the hippocampus when it was cultured alone, the above enhancement is induced, at least in part, by the common signaling mechanisms with result suggests that the transfer was reproduced in vitro. teatanus-inducd presynaptic LTP at this synapse.
P1AM-6-5 P1AM-6-6 DYNAMIC ROLES OF PROTEINS IN SYNAPTIC KCC2 A SYNCHRONIZING FACTOR IN SYNAPTIC ADHERENS JUNCTION MATURATION 1 2 1 Hidekazu Tanaka, Kazuaki Takafuji, Shushi Nagamori, Claudio Rivera , Hans Gerd Nothwang , Olaya Llano , 1 Yoshikatsu Kanai Anastasia Ludwig 1Institute of Biotechnology, University of Helsinki, Finland, 2Institut fur Department of Pharmacology, Osaka University School of Medicine, Japan Biologie und Umweltwissenschaften, Carl von Ossietzky Universitat, Synapse is an adhesive junction specialized for chemical communication Oldenburg, Germany with neurotransmitters and their receptors. Synaptic cell adhesion molecules A common assumption is that the maturation of both inhibitory and excitatory not only hold pre- and post-synaptic membranes in close apposition, but neurotransmission is tightly synchronized during development. However, also play highly dynamic roles during synaptic transmission and plasticity. very little is known about the molecular mechanism involved. Our recent For example, N-cadherin undergoes conformational change upon synaptic data identified the neuron specific K-Cl cotransporter KCC2 as a potential stimulation. At the same time, the steady-state endocytosis of N-cadherin synchronizing factor. This transporter plays a pivotal role in the maturation slows down, resulting in the stabilization of N-cadherin on cell surface. of inhibitory synapses. Its developmental activation sets the gradual shift in These modifications of synaptic adhesive machinery are probably responsible GABA/glycine mediated responses from depolarizing to hyperpolarizing. Our for the activity-induced remodeling of synaptic architecture, as well as the recent results show that in addition KCC2 plays a crucial role in the formation synaptic plasticity. To elucidate the mechanism how this synaptic adhesive of dendritic spines as well as functional glutamatergic synapses. This role is independent of its chloride extrusion activity. machinery respond to synaptic stimulation, and how it modulates the Here, we will present data on the interaction of KCC2 with the dendritic spine efficiency of synaptic transmission, we tried to identify a set of proteins cytoskeleton as well as with important intra-spine proteins with established involved in the synaptic adhesive machinery. We set out to identify proteins functions in spine morphology and stability of glutamatergic synapses. These co-immunoprecipitated with N-cadherin from cultured rat hippocampal results further suggest that KCC2 plays a key role in the developmental neurons. synchronization of inhibitory and excitatory neurotransmission.
P1AM-6-7 P1AM-6-8 THE FLUCTUATION OF THE VESICULAR RELEASE ANALYSIS OF CLIMBING FIBER INPUTS TO PURKINJE PROBABILITY IN DEVELOPING HIPPOCAMPAL CELLS IN THE DEVELOPING RAT CEREBELLUM IN AUTAPTIC SYNAPSES VIVO Shutaro Katsurabayashi, Sachiko Nakano, Kotaro Takasaki, Yoshinobu Kawamura, Hisako Nakayama, Kazuo Kitamura, Kenichi Mishima, Katsunori Iwasaki, Michihiro Fujiwara Masanobu Kano Department of Neuropharmacology, Fukuoka University, Japan Department of Neurophysiology, The University of Tokyo, Japan Coordinated synaptic transmission correlates with functional maturation In the developing cerebellum, transition from multiple to single climbing fiber (CF) including formation of newly active synapses. This aspect is accompanied innervation of Purkinje cell (PC) occurs during the first three postnatal weeks. To with changes in the quantal size (q), the number of release sites (N) and the examine what pattern of activity of PC in vivo is related to the maturation of CF, probability of release (P). However, little is known whether vesicular release we made whole-cell recordings from PCs in anesthetized rats at P4-P17. We found probability (Pvr) fluctuates with age. Here we compared synaptic parameters that a spontaneous burst spiking (BS), known to be a typical immature CF response, using autaptic single neural cultures on "microdot" of astrocyte among 7-9 was induced by a burst of EPSPs at P4-P7. Complex spikes (CSs), which represent day in vitro (DIV), 13-15 DIV and 21-27 DIV. During the age of culture mature CF inputs, appeared at P8. CSs occurred with preceding multiple EPSPs at excitatory postsynaptic currents (EPSCs) increased in response to increasing P8-P10, and the number of such EPSPs decreased with age. CSs occurred without the size of the readily releasable pool of vesicles (RRP). Interestingly, the preceding EPSPs after P11. We assessed developmental changes in the number of CFs Pvr calculated by dividing the EPSC charge by the response of hypertonic innervating each PC in slice preparation, which corresponded well with the decrease sucrose (500 mM) showed a higher value at 7-9 DIV, and was lowered after in the number of EPSPs preceding BS/CS. This result suggests that the disappearance 13 DIV. As the fraction of action potential-evoked phasic and asynchronous of EPSPs preceding BS/CS may reflect elimination of surplus CFs. Importantly, the release during high frequency stimulation (20 Hz) is constant with the age amplitude of EPSP was inversely correlated with the time between the EPSP peak and of culture, the lowering Pvr is unlikely due to change in the heterogeneous the BS/CS onset, and the EPSP just preceding BS/CS selectively became larger with populations of vesicles. We are currently exploring precise mechanisms for age. These results suggest that developmental changes in the strength of CF synapses Poster Session lowering the Pvr with functional synaptic maturation. are dependent on the timing of CF input relative to BS/CS
IUPS 2009 July 27 - August 1, 2009 in Kyoto 137 P1AM-6-9 P1AM-6-10 TRANSLOCATION OF THE STRONGEST CLIMBING A PKCγ MUTATIONS IN SPINOCEREBELLAR TAXIA FIBER TO PURKINJE CELL DENDRITES AFTER TYPE 14 IMPAIRS PKC FUNCTIONS IN CEREBELLAR SYNAPTIC COMPETITION ON THE SOMA IN PURKINJE CELLS IN VIVO DEVELOPING CREBELLUM Anton Nikolaevich Shuvaev1, Hirokazu Hirai1, Norio Sakai2, Kouichi Hashimoto, Masanobu Kano Takahiro Seki2, Masae Iino1, Hajime Horiuchi1 Department of Neurophysiology, University of Tokyo, Japan 1Department of Neurophysiology, Gunma University, Japan, 2Department In the newborn mouse cerebellum, multiple climbing fibers (CFs) innervate the of Molecular and Pharmacological Neuroscience, Graduate School of soma of each Purkinje cell (PC). Then, CFs extend their synapses to PC dendrites Biomedical Sciences, Hiroshima University, Japan (CF translocation). However, its mechanisms have not been well understood. The spinocerebellar ataxia type 14 (SCA14) that is characterized by a slowly We estimated the extent of CF translocation by analyzing the 10-90% rise times progressive cerebellar ataxia is caused by a mutation of PKCγ . A major cell type of quantal EPSCs (qEPSCs) originating from single CFs. Under whole-cell affected by SCA14 is Purkinje cell, significantly linked to high levels of expression recording from PC somata in cerebellar slices, single CFs were stimulated in the of PKCγ. Knock-out studies showed that PKCγ was critical for developmental 2+ presence of Sr and qEPSCs due to asynchronous glutamate release from the elimination of climbing fiber (CF) synapses from Purkinje cells. However, it is still stimulated CF terminals were recorded. After P7, each PC is known to be either unclear how mutations of PKCγ in SCA14 affect Purkinje cells in vivo. Here, using mono-innervated by a strong CF (CF-mono) or multiply innervated by a strong lentiviral vectors we expressed a mutant (S119P) in Purkinje cells of postnatal day (P) CF (CF-multi-S) plus a few weaker CFs (CF-multi-W). We found that the qEPSC 6 mice and examined the influence on Purkinje cell by electrophysiology. Lentivector- rise times for CF-multi-S and CF-multi-W were identical at P7-P8. Then, the injected mice were sacrificed at P25 or later. All Purkinje cells that expressed mutant fractions of qEPSC with slow rise time for CF-mono and CF-multi-S increased PKCγ formed diffuse aggregates in soma and dendritic processes, while only ~5% progressively until P14, suggesting that the number of qEPSCs originating from of Purkinje cells that expressed wild-type PKCγ by lentivectors formed aggregates. synaptic terminals on dendrites increased with age for the strongest CFs. In Moreover, significantly higher ratio of Purkinje cells expressing mutant PKCγ , but not contrast, the qEPSC rise times for CF-multi-W were unchanged. These results wild type PKCγ , showed persistent innervation by multiple CFs. These results suggest suggest that competition among multiple CFs occurs on the PC soma until P8, that the mutant PKCγ disrupts functions of PKC in a dominant-negative fashion, and then the “winner” CFs selectively translocate to PC dendrites. presumably by forming heteromeric aggregates with wild-type PKC.
P1AM-6-11 P1AM-6-12 EFFECT OF PRENATAL TREATMENT OF JIN GUEI REVERSAL OF HIPPOCAMPAL NEURONAL DANG GUEI SAO ON CELL PROLIFERATION IN MATURATION BY CHRONIC INHIBITION OF THE HIPPOCAMPUS AND SPARTIAL MEMORY OF SEROTONIN REUPTAKE OFFSPRING Katsunori Kobayashi, Yumiko Ikeda, Atsushi Sakai, Byung Soo Ahn1, Byung- Il Min1, Chang- Ju Kim2 Hidenori Suzuki 1East-west medicine, Kyung-hee University, Korea, 2Department of Physiology, Pharmacology, Nippon Medical School, Japan Kyung hee University, Korea Serotonin is known to regulate the generation of new neurons in the dentate Background : JGDS is regarded as a medicinal herb during the period of maternity in east gyrus of the adult hippocampus. Chronic treatments with selective serotonin north Asia. Steady attention has focused on the biological functions and health benefits of reuptake inhibitors (SSRIs) can facilitate this adult neurogenesis, which has been JGDS as a gynecologic major medicine. suggested to be a cellular basis underlying behavioral effects of these drugs. OBJECTIVE : Our purpose was to determine whether prenatal exposure to the JGDS affects However, it is not clear how additional new neurons that constitute only a small neurogenesis in the hippocampus and spatial, learning memory of mouse offspring. proportion of the entire dentate neuronal population can significantly affect brain STUDY DESIGN : Female rats mate with male rats.Female rats were randomly divided into functions. In the present study, we found that chronic treatments of adult mice four groups: the control group, the 10 mg/kg JGDS group, the 50 mg/kg JGDS group, and the 100 mg/kg JGDS group. with the SSRI fluoxetine cause a dramatic decrease in the expression of markers From the 15th day of pregnancy, rats of all groups were subcutaneously injected with 100 for the mature granule cells. In the fluoxetine-treated mice, remarkable synaptic mg/kg BrdU, once a day 30 min before injection of with JGDS until delivery.After birth, facilitation that characterizes the mature dentate-to-CA3 signal transmission the offspring in each group was left undisturbed together with the respective mother. Spatial is strongly reduced, and, conversely, active somatic membrane properties learning test was performed on the 42 days after birth. NADPH-d and BrdU & NeuN was resembling those of the immature granule cells are induced. These changes done to estimate neurogenesis. cannot be explained by an increase in newly generated immature neurons, Results : JGDS during pregnancy significantly enhances the spatial memory of offspring and but represent phenotypic rejuvenation or dematuration of the mature neurons. increases hippocampal neurogenesis of offspring and increases NOS expression of offspring. Our results suggest that the SSRI treatment reverses terminal differentiation/ Conclusions : These results shows that JGDS during the gestational period improves the maturation of the dentate granule cells, thereby substantially altering the function memory capability of offspring by increasing neurogenesis. of the dentate-to-CA3 neuronal system.
P1AM-7-1 P1AM-7-2
Kv3 POTASSIUM CHANNELS ARE RESPONSIBLE ACTIVATION OF THE SIGMA RECEPTOR 1 FOR FAST FIRING PATTERN OF THE RAT RETINAL MODULATES NMDA RECEPTORS IN RAT RETINAL GANGLION CELLS GANGLION CELLS Svetlana Fedulova, K Kuznetsov, V Maslov, N Veselovsky Xin-Jun Zhang, Lei-Lei Liu, Yong-Mei Zhong, Xiong-Li Yang Bogomoletz Institute of Physiology, Ukraine Institute of Neurobiology, Institutes of Brain Science, Fudan University, China Retinal ganglion cells (RGCs) are the ultimate output neurones transmitting afferent information from the retina to the CNS. Intrinsic firing properties Sigma receptors (σRs) represent unique nonopiate, nonphencyclidine binding of mature (1 month old) rat RGCs were studied in retinal flat-mounted sites in mammalian nervous systems. There is evidence that ligands of σRs preparations. Changes of firing frequency and parameters of individual have robust neuroprotective effects. In the present work, we investigated how σR1 is expressed in the rat retina and how activation of σR1 modulates NMDA APs caused by potassium channels blockers 4-aminopyridine (4-AP) (0.2 receptors in retinal ganglion cells (GCs). By immunocytochemistry, we show that mM) and tetraethylammonium (TEA) (0.5 mM) were analysed. 4-AP and σR1 is strongly expressed in amacrine cells and GCs. Labeling for σR1 is also TEA reduced maximal steady firing frequency from 43.2 ± 6.0 to 18.4 ± observed in both outer and inner plexiform layers. Using whole-cell recordings 3.0 Hz (n=3) and from 31.9 ± 3.7 to 18.7 ± 0.9 (n=3) respectively, whereas in retinal slice preparations, we first characterized NMDA receptor-mediated AP half-time increased from 1.2 ± 0.2 to 3.0 ± 0.5 ms (4-AP, n=4) and from currents of GCs. A robust inward current is induced from GCs with focal puff of
Poster Session 1.4 ± 0.1 to 2 ± 0.1 ms (TEA, n=6). Potassium channel antagonists reduced 1 mM NMDA to the dendrites or with puff of 120 mM KCl onto the somata of maximal membrane repolarization rate from 97.0 ± 23.9 to 31.8 ± 8.6 mV/ms associated bipolar cells in the absence of Mg2+. The current could be completely (4-AP, n=4) and from 75.2 ± 5.6 to 44.0 ± 2.6 mV/ms (TEA, n=6). Single blocked by D-AP5, a specific NMDA receptor antagonist. We further show that cell RT_PCR demonstrated high level of expression of Kv3.1 and/or Kv3.2 the σR1 agonist PRE-084 suppresses NMDA-induced currents from OFF type subunits. Simultaneous expression of both Kv3.1 and Kv3.2 subunits occurred GCs and the effect could be reversed by the σR1 antagonist haloperidol. These in 3 out of 12 cells, whereas 9 out of 12 cells expressed only one of these results suggest that the σR1-mediated neuroprotective effects on GCs may be, at subunits. We concluded that Kv3 potassium channels are expressed in the least in part, due to the suppression of NMDA receptors caused by activation of tonic rat RGCs and are essential for their fast firing pattern generation. σR1.
138 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-7-3 P1AM-7-4 PROTON PUMP INHIBITOR SUPPRESSES NEGATIVE PRESYNAPTIC MODULATION BY SOMATOSTATIN IN FEEDBACK SIGNAL FROM HORIZONTAL CELLS TO CULTURED RAT RETINAL GABAERGIC AMACRINE CONES IN THE CARP RETINA CELLS Masahiro Yamada1, Kazunori Yamamoto1, Nilton Liuji Kamiji1, Jiang-Bin Ke, Yong-Mei Zhong, Xiong-Li Yang, Zhongfeng Wang Riho Hatano2, Hiroshi Jouhou3, Shiro Usui1, Akimichi Kaneko4 Institute of Neurobiology, Institutes of Brain Science&State Key Laboratory of Medical Neurobiology, Fudan University, China 1Neuroinformatics Lab., Riken Brain Scinece Institute, Japan, 2Department of Biological Sciences, Tokyo Metropolitan University, Japan, 3Astellas Somatostatin (SRIF) is widely distributed in the CNS and exerts its actions Pharma. Inc., Japan, 4School of Health Science, Kio University, Japan via specific receptors (ssts). In a previous work we showed the co-localization of SRIF with sst5 in both dopaminergic and cholinergic amacrine cells (ACs) Centre-surround receptive field formation and opponent colour signalling was believed to in rats. In the present work, we investigate how SRIF modulates presynaptic be mediated by GABAergic feedback from horizontal cells (HCs) to cones. This GABA activity of cultured retinal GABAergic ACs. SRIF reduced the frequency of hypothesis was doubted by the fact that picrotoxin did not suppress the feedback signal. + GABA-mediated miniature inhibitory postsynaptic currents (mIPSCs), which Hirasawa & Kaneko (2003) proposed a H -mediated negative feedback hypothesis. Jouhou et al. (2007) detected depolarization-dependent H+ release from HCs, which was blocked by could be reversed by co-application of the sst5 antagonist BIM 23056 and bafilomycin A1, a specific inhibitor of V-ATPase +(H pump), and showed its localization at the sst2 antagonist CYN-154806, but did not affect the kinetics of mIPSCs. HC plasma membrane surface. By intracellular recordings of light-adapted retinae, effects Elevation of [Ca2+]o increased the frequency of mIPSCs, and all mIPSCs of 400 nM bafilomycin or pH buffers (20 mM HEPES or 15 mM Tris) were compared with disappeared after elimination of extracellular Ca2+. In addition, CdCl2 and those of control bicarbonate Ringer of the same pH. The drug-added Ringers depolarized the nimodipine suppressed mIPSCs. All these results reveal the extracellular H1 HC dark membrane potential, increased the light response and suppressed the response Ca2+ dependence of mIPSCs. Furthermore, forskolin enhanced the frequency sag, a slow depolarizing response component assumed to be generated by the feedback of mIPSCs, while the PKA inhibitor Rp-cAMP reduced it. In the presence signal. The test Ringers suppressed also depolarizing red response of H2 HCs evoked by of Rp-cAMP, SRIF had no effect on mIPSCs. It is therefore concluded that the negative feedback from H1 HCs to cone photoreceptor. These results suggest that H+ SRIF may inhibit the Ca2+ influx through transmembrane Ca2+ channels released from HCs works as the feedback mediator from HCs to cones, supporting the H+ by activating sst5 and/or sst2 receptors, which results in a reduction of the feedback hypothesis. The feedback signal was quantified by modelling. presynaptic release of GABA from retinal ACs via the cAMP-PKA pathway.
P1AM-7-5 P1AM-7-6 IMMUNOHISTOLOGICAL LOCALIZATION OF PROTON THE POSTNATAL EXPRESSION OF HISTAMINE PUMP (V-ATPase) IN THE OUTER RETINA OF RECEPTORS IN THE GERBIL RETINA GOLDFISH AND MONKEY Hideki Imada1, Masahiro Kokubo2, Mahito Ohkuma1, Hiroshi Jouhou1, Kenji Nakano2, Shuuji Ishikawa2, Kouichi Sano2, Toshi-aki Kato3, Ei-ichi Miyachi1 2 3 3 Takao Shishido , Kazunori Yamamoto , Masahiro Yamada , 1Department of Physiology, School of Medicine, Fujita Health University, 4 Japan, 2Joint Research Laboratory, Fujita Health University, Japan, Akimichi Kaneko 3 1Drug development toxicology, Astellas Pharma Inc., Japan, 2Astellas Department of Clinical Laboratory Science, Fujita Health University Research Technologies Co.,Ltd., Japan, 3Neuroinformatics, Riken Brain College, Japan Science Institute, Japan, 4School of Health Science, Kio University, Japan In the central nervous system, histamine is known to act on three major types It has been hypothesized that proton mediates the negative feedback from horizontal cells of G-protein-coupled receptors; histamine H1 receptor (H1R), histamine H2 (HCs) to cone photoreceptors (PRs) forming the antagonistic center-surround receptive receptor (H2R) and histamine H3 receptor (H3R). The localization of the histamine field in the outer retina. We studied the localization of V-ATPase by immunohistochemical receptors in the gerbil retinae in age from 1 to 350 days were examined using analyses of the fish and monkey retina by using antibodies against V-ATPase under the immunocytochemical method. The animals were perfused intra-cardially with a fluorescence and the immuno-electron microscopy. Immunoreactivity (IR) against V-ATPase mixture of 4% paraformaldehyde in 0.1M sodium phosphate buffer. After the removed was localized in PR synaptic terminals and HCs. Glutamic acid decarboxylase-IR was co- eyeballs were frozen with the liquid nitrogen, they were cut with 16 μm section using a localized with the V-ATPase IR in the plasma membrane of fish H1 type HCs. Electron cryostat. The sections were examined by ABC immunocytochemical staining method. microscopic observation of the invaginating synapse of the goldfish and monkey retina The fura-2 based calcium imaging was also performed at the adult gerbil retina. The revealed strong V-ATPase-IR in the cytoplasm of cone terminals and weak IR in the lateral preparations were superfused at 1 ml/min with Control, 100 μM histamine- and 100 2+ elements of the triad (representing HC dendrites). Differential expression of B1 and B2 μM glutamate-containing solution. Increases of intracellular Ca concentration were isoforms of V-ATPase B subunit was observed in monkey cone pedicles, B1 in the HC observed in the ganglion cells. H1R was labeled in the ganglion cells from P (postnatal membrane whereas B2 in the membrane of synaptic vesicles of PRs. These results provide day) 2 to P350. H2R was labeled in the ganglion cells from P8 to P26. H3R was further support to the notion that V-ATPase of HC plasma membrane is the molecular labeled in the ganglion cells from P6 to P70. At adult, H1R was seen in the ganglion machinery of proton efflux leading to HC-cone negative feedback in the goldfish and cells, while H2R or H3R was not seen. These results suggest that H1R is one of the monkey retina. functional receptors in the adult gerbil retina.
P1AM-7-7 P1AM-7-8 OPTIMIZATION OF THE SYNAPTIC TRANSMISSION BY FRACTION OF OPEN CHANNELS IN ELECTRICAL BODY TEMPERATURE IN THE MOUSE RETINA SYNAPSES BETWEEN HOMOLOGOUS RETINAL NEURONS Fuminobu Tamalu, Shu-Ichi Watanabe Soh Hidaka Department of Physiology, Saitama Medical University, Japan Department of Physiology, Fujita University of Health Sciences, Japan Synaptic transmission from rods to rod bipolar cells is mediated by a Homologous gap junctions of retinal horizontal, amacrine and ganglion cells metabotropic glutamate receptor 6 (mGluR6) in the mammalian retina. In (Hidaka et al., J. Neurosci., 2004) were addressed to establish the relationship the dark, rods release glutamate by which rod bipolar cells hyperpolarize; between connexin channel distribution and electrical coupling measured conversely, in response to light, reduced release of glutamate from rods by simultaneous recordings. Channel opening in electrical synapses was opens a non-selective cation channel and depolarizes the rod bipolar cells, investigated with protocols using multiplex arrays of methodologies: dual from which glutamate is released to post-synaptic AII amacrine cells. We patch clamp, intracellular labeling, connexin immunocytochemistry, freeze fracturing and high-voltage electron microscopy (08HVEM-06, NIPS, found that in response to the temperature higher than 25 degree C in the Okazaki) of the cells. Junctional conductance was measured in cell pairs. light condition, the rod bipolar cells were depolarized and an amplitude and Number of gap junctions between labeled cells and the size of gap junction frequency of EPSCs in AII amacrine cells dramatically increased. However, plaques were examined by electron microscopy. Measuring the packing L-AP4, a selective agonist of the mGluR6, completely inhibited the EPSCs density of connexons in replicated tissue of the intramembrane distribution, in AII amacrine cells regardless of the temperature. These data raise the a total number of connexons in a pair was estimated. When a single channel possibility that body temperature may optimize the dynamic range of the conductance of the specific connexin is present, a total of gap junction conductance in a pair was evaluated. The measured conductance could allow membrane potential in rod bipolar cells by increasing the open probability of us to identify the fraction of active channels as 0.3% for horizontal cells, the non-selective cation channels. 0.1% for amacrine cells, and 0.6% for ganglion cells, respectively. The small fraction suggests that possible positive neuromodulation could give rise to a greater proportion of active channels. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 139 P1AM-7-9 P1AM-7-10 MATHEMATICAL PREDICTION METHODS OF SUPPRESSION OF RECIPROCAL SYNAPTIC DISORDER DYNAMICS IN CHILDREN AFFECTED BY CURRENTS BY GROUP II METABOTROPIC CORTICAL VISUAL IMPAIRMENTS USING VISUAL GLUTAMATE RECEPTORS IN THE MOUSE EVOKED POTENTIALS ACCESSORY OLFACTORY BULB Stelian V. Sarlea1, Adela E. Joanta2, Roxana Cziker2, Andreea Seceleanu2, Mutsuo Taniguchi, Hideto Kaba Remus Moldovan2 Department of Phsiology, Kochi Medical School, Japan 1Department of Physiology, Iuliu Hatieganu University of Medicine and 2 By measuring the reciprocal synaptic currents from mitral cells in the accessory Pharmacy, Cluj-Napoca, Romania, Department of Physiology, "Iuliu Hatieganu" olfactory bulb (AOB), we have demonstrated that an agonist for group II metabotropic University of Medicine and Pharmacy, Cluj-Napoca, Romania glutamate receptors (mGluR2/mGluR3), DCG-IV, suppressed dendrodendritic It is a great challenge to identify early manifestations of visual impairment. Visual Evoked inhibition (DDI) whereas the mGluR2/mGluR3 antagonist LY341495 enhanced it. Potential (VEP) is an electrophysiological technique which can be extracted from the The effects of these drugs were markedly impaired by genetic ablation of mGluR2, recorded electroencephalogram. It is a method which uses light stimuls (uniform flash) and indicating that DCG-IV-mediated suppression of DDI is mediated by mGluR2. pattern stimuls (repeting or onset/offset). The retina transforms the information from light In the present study, in order to conduct further investigation on the role to electricity and transmits the information to the brain, where the informations are analyzed of mGluR2 in the synaptic transmission, AOB slices were prepared from and integrated. By the acquiring VEP we propose to quantify the wave variation obtained 23- to 36-day-old Balb/c mice. Miniature EPSCs (mEPSCs) were recorded following the computer analyzes, in both physiological and pathological situations. For this, from granule cells in slice preparations with the patch-clamp technique in we used Biopac MP100/MP150 software, which uses Fast Fourier series analysis. Those are whole-cell configuration (holding potential, -70 mV). DCG-IV reduced the linear operators which have the possibility of iteration from -∞ to +∞, by multiple function decomposition into continuous equational spectrums. They are very useful operators for frequency of mEPSCs with slight reduction of the amplitudes. Together with analyzing all species of waves, quantifying each of their components and the acquired the results reported by another group that DCG-IV decreases the synaptic experience will be used for preventing the unfortunate evolution of analyzed children. We transmission from granule cells to mitral cells in the AOB, the present results also aim to develop a database from acquired waves using VEP, for identifying the border suggest that mGluR2/mGluR3 can modulate the synaptic transmission not between physiology and pathology at children suffering of visual problems and to prevent only from granule to mitral cells but also from mitral to granule cells in the further aggravation of the disease. AOB.
P1AM-7-11 P1AM-7-12 ACTIVITY-DEPENDENT SYNAPSE FORMATION OF MODULATION OF OSCILLATORY NEURONAL MITRAL/TUFTED CELLS IN THE MOUSE OLFACTORY SYNCHRONIZATION BY NEUROTRANSMITTER AND BULB NEUROPEPTIDE IN SLUG OLFACTORY CENTER Nobuko Inoue, Hitoshi Sakano Suguru Kobayashi, Mariko Hattori, Ryota Matsuo, Etsuro Ito Department of Biochemistry, Tokyo University, Japan Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Japan In the mouse olfactory system, each olfactory sensory neuron (OSN) expresses one functional odorant receptor (OR) gene. Furthermore, OSNs expressing Oscillatory neuronal activity is common in the olfactory system of both the same OR species converge their axons to a specific pair of glomeruli in the vertebrates and invertebrates. In terrestrial molluscs, spontaneous oscillations are observed in the procerebral (PC) lobe of the cerebral ganglia, where olfactory bulb (OB). In the OB, OSNs form synapses with Mitral/Tufted cells the olfactory information is processed. Recently, we showed that gamma- (M/T cells) within glomeruli. amino butyric acid (GABA) and Phe-Met-Arg-Phe-NH (FMRFamide) In order to study the effects of neuronal input from OSNs on M/T cell 2 immunopositive neurons exist in the PC, and these neurons may be involved development, we focused on Semaphorin-7a (Sema-7a), which is known to play in the oscillatory neural network of the PC. In the present study, we examined important roles both in the immune and central nervous system. Here we report the effects of GABA and FMRFamide on the periodic oscillation of local that Narris-occlusion treatment down-regulated the expression level of Sema- field potential (LFP) in the PC and discussed the roles of these neurons in 7a in OSNs and delayed the synapse formation of OSNs with M/T cells. We the oscillatory neural network. 1. GABA increased the frequency of the also found that the Sema-7a receptor PlexinC1, but not Integrinβ1, is strongly LFP oscillation, but its prolonged application decreased the frequency. 2. expressed in M/T cells’ dendrites in the neonatal period. In the Sema-7a knock FMRFamide strongly decreased the frequency of the LFP oscillation via out mice, synapse formation of M/T cell dendrites with OSN axons appear to be the metabotropic receptors. 3. GABA-induced intracellular Ca2+ increase delayed. These results suggest that Sema-7a is involved in the synapse formation was observed in the PC neurons. These results suggest that GABAergic and of M/T cells with OSN axons in an activity-dependent manner. FMRFamidergic synaptic transmission is involved in the oscillatory neural network of the PC, and it may change the odor-evoked LFP frequency.
P1AM-7-13 P1AM-7-14 DENDRITIC ARBORIZATION OF THE 8TH CRANIAL AXONAL MECHANISMS UNDERLYING PRECISE NERVE OF TELEOST SACCULAR SENSORY TEMPORAL CODING IN AVIAN COCHLEAR NUCLEUS EPITHELIA: A COMPARATIVE STUDY Hiroshi Kuba1, Harunori Ohmori2 1Career-Path Promotion Unit for Young Life Scientists, Kyoto University Gordon Michael Selckmann, John U Ramcharitar 2 Biology, St. Mary's College of Maryland, USA Graduate School of Medicine, Japan, Department of Physiology, Kyoto University Graduate School of Medicine, Japan This study investigated patterns of dendritic arborization of the eighth How the axonal distribution of Na+ channels affects the precision of spike cranial nerve on saccular sensory epithelia of several teleosts including two timing is not well understood. We addressed this question in auditory relay hearing generalists, spot (Leiostomus xanthurus) and white perch (Morone neurons of avian nucleus magnocellularis that precisely encode timing americana), as well as one species with hearing specializations (Atlantic information at each characteristic frequency (CF) by processing CF-specific croaker, Micropogonias undulatus). Dendritic arbors were visualized using patterns of synaptic inputs. We found that neurons varied axonal Na+- osmium tetroxide and significant interspecific differences in arborization channel density and distribution according to the distribution of synaptic patterns were observed. The simplest pattern was seen in white perch which inputs, thereby ensuring precise spike timing across CF. Low-CF neurons exhibited the lowest total numbers of arbors. On the other hand, spot, a had a higher density of Na+ channels within a longer axonal stretch than sciaenid species with more expansion of the rostral end of the saccular high/middle-CF neurons, and showed a larger spike amplitude and whole- + Poster Session epithelium than white perch, had more primary branches of the eighth nerve. cell Na current. Computer simulation revealed that for low CF neurons, The most complex arrangement of dendritic arbors was observed in croaker. a high density of Na+ channels was crucial for preserving spike timing In this sciaenid which has the largest degree of rostral expansion among the because it overcame Na+ current inactivation and K+ current activation during fishes investigated, more primary and secondary branches of the eighth nerve compound EPSPs evoked by converging small inputs. In contrast, fewer were observed. Also, croaker exhibited overlap of arbor terminals, a feature channels were sufficient to generate a spike with high precision in response absent in both white perch and spot. The prediction of increased dendritic to an EPSP induced by a single massive input in the high/middle CF region. arbor complexity as a teleost’s auditory system gets more sophisticated, is Thus, the axonal Na+-channel distribution is adjusted to optimize neuronal supported by these data. responses at a precise auditory relay nucleus.
140 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-8-1 P1AM-8-2 ACTION POTENTIAL CODE MODULATES SYNAPTIC COCAINE AND ACTION POTENTIAL CODE TRANSMISSION IN A MOUSE BRAIN SLICE MODULATES DOPAMINE RELEASE IN MICE STRIATUM Zhuan Zhou1, Bo Zhang1, A Yang2, XY Liu1, CX Zhang1, LY Wang2 IN VIVO 1 1 1 1 1 1 1Institute of Molecular Medicine, Peking University, China, 2Department of Zhuan Zhou , PL Zuo , XJ Kang , XY Zhang , MY Song , LL Liu , Physiology, University of Toronto, Toronto, Canada. Q Lei1, JL Gu1, SR Wang1, W Yao1, CX Zhang1, H Gu2 1 2 The calyx of held, a large glutamatergic synapse, can be recorded by dual Institute of Molecular Medicine, Peking University, China, Ohio State patch-clamping recordings on presynaptic membrane capacitance (Cm) University, Columbus, USA and postsynaptic EPSCs in mouse brainstem slice. In a previous study, Dopamine (DA) is a crucial neurotransmitter crucial for fundamental brain function we showed that action potential (AP) code modulates secretion in isolated and diseases. Micro electrochemical carbon fiber microelectrode (CFE) can record adrenal chromaffin cells (Duan et al, JNS, 2003). The stimulus code was DA release from brain. Action potential (AP) induced secretion of DA in mouse brain defined by a AP “code” F(N,m,f,d) with “N” (number of total APs), “m” striatum in vivo. The Stimulus pattern is defined as AP code [N, m, f, d] (N = total (number of bursts), “f” (frequency), “d” (interval between two bursts). Here stimulating number, m = burst-number, f = frequency, d = inter-burst interval) (Duan we show that AP codes strongly modulate both presynaptic secretion signals et al, JNS, 2003). In wide type mice (WT), with fixed N, evoked DA release was (Cm) and postsynaptic EPSCs. F(100,m,100Hz, 1s)-induced signal is 59% strongly modulated by m, f and d. In contrast to N and f, which regulate DA release by [Ca2+]i accumulation, m and/or d may modulate secretion by recycling vesicle pool. (presynaptic Cm) and 39% (EPSC) larger at m = 4 vs. 1. Statistically, the To test this hypothesis, we used a knock-in mice (KI) with the DA transporter (DAT) EPSC “burst effect” is insensitive to AMPA-R desensitization, but sensitive 2+ insensitive to cocaine. In KI vs. WT mice, both amplitude and kinetics of DA release to low [Ca ]o, [EGTA]i, temperature, and age. According to our simulation was drastically changed following given AP code. The effect of AP burst number ([144, data of presynaptic Cm, the “burst effect” is produced jointly by depletion 2+ 2+ m, 80Hz, 0.5s], m = 1 vs. 16 ), or “m-effect”, on DA release is increased in KI vs. WT and recycling of readily releasable vesicle pool (RRP), Ca entry, Ca mice. Cocaine increased AP-induced DA release for blocking DAT in WT but not KI buffer, and endocytosis. This “burst effect” in the synaptic transmission is mice. In addition, we examined effects of cocaine on “m-effect” in KI and WT mice. less insensitive to coding parameters “N”, “f” and “d” than that in chromaffin We propose that cocaine affects not only DAT, but also presynaptic DA vesicle pool in cells. mice striatum.
P1AM-8-3 P1AM-8-4 THE C-TERMINAL DOMAIN OF BRUCHPILOT V-SNARES REGULATE THE STRENGTH OF QUANTAL ACCELERATES VESICLE SUPPLY AT ACTIVE ZONES SYNAPTIC TRANSMISSION Robert Johannes Kittel1,3,4, Stefan Hallermann1,3, Carolin Wichmann2,4, Dieter Bruns, Raul Guzman, Ivonne Schwarz Wernher Fouquet2, Sara Mertel2, David Owald2, Marcus Dyba5, Department of Physiology, University of Saarland, Germany Stefan Eimer4, Stephan J. Sigrist2,3, Manfred Heckmann1,3 Neurotransmitter release relies on rapid exocytosis of small synaptic vesicles 1Carl-Ludwig-Institute of Physiology, Medical Faculty, University of Leipzig, but the presynaptic mechanisms that ensure fast transmitter discharge are Germany 2Rudolf Virchow Center, DFG Research Center for Experimental unknown. Membrane-bridging interactions of SNARE proteins play an 3 Biomedicine, University of Wuerzburg, Germany Institute for Clinical essential role in exocytosis. Here we show that a short intramolecular distance 4 Neurobiology, Medical Faculty, University of Wuerzburg, Germany European between the complex-forming SNARE domain and the transmembrane Neuroscience Institute-Goettingen, University of Goettingen, Germany domain of Synaptobrevin II is required for rapid neurotransmitter release. 5Research & Development, Leica Microsystems CMS GmbH, Germany For this, we expressed Synaptobrevin II mutant proteins carrying an Fast vesicle replenishment during sustained neuronal activity has been linked to large extended juxtamembrane region in hippocampal neurons that are genetically electron-dense structures at active zones (AZs) of tonic synapses but has also been reported deficient for Syb II. We find that tight coupling between the complex- for phasic synapses with less prominent AZ-structures. Here, we studied both the structure forming SNARE motif and the TMD of Syb II governs amplitude and timing and the function of the Drosophila AZ-protein Bruchpilot (BRP), which displays homologies of the action potential evoked response. Furthermore, lengthening of sybII’s to mammalian CAST. We demonstrate that loss of BRP impairs vesicle supply at the AZ without affecting the size of the readily releasable vesicle pool. juxtamembrane region slows the time course of quantal glutamatergic EPSCs In mutants lacking only the last 16 c-terminal amino acids of the 1740 amino acid BRP providing evidence that SNAREs exert force on membranes to mediate protein, the number of vesicles surrounding the AZ was reduced and synaptic depression submillisecond release of neurotransmitter from small synaptic vesicles. was increased. Consistent with findings in synapsin mutants and in animals with reduced numbers of AZ calcium channels, these results demonstrate that the c-terminal domain of BRP accelerates vesicle supply at the AZ of a phasic synapse.
P1AM-8-5 P1AM-8-6 Ca2+-INDEPENDENT SNARE BINDING OF THE MECHANISM OF RELIABLE SYNAPTIC SYNAPTOTAGMIN I THROUGH INTERACTION TRANSMISSION IN LAYER 4 OF THE VISUAL CORTEX BETWEEN ITS C2B DOMAIN AND SYNTAXIN 1 Chao-Hua Huang, Takeshi Sakaba 1 1 1 Toshio Masumoto , Tei-ichi Nishiki , Iori Omori , Department of Membrane Biophysics, Max-Planck Institute for Biophysical Kazuhito Tomizawa2, Hideki Matsui1 Chemistry, Germany 1Department of Physiology, Okayama University, Japan, 2Department of Neurons in layer 4 of the primary visual cortex (V1) play an important role Molecular Physiology, Kumamoto University, Japan in transferring signals from thalamus to other layers of V1. For sensory As SNARE-mediated membrane fusion is constitutively active, SNARE complex signals, the short-term plasticity of cortical synapses determines the temporal formation is thought to be clamped to prevent synaptic vesicle exocytosis until Ca2+ resolution and how reliable the signal can be transferred within a neuronal influx into presynaptic nerve terminals. One of the candidates for such a clamp circuit. Here, we have determined three parameters which can be strong is synaptotagmin I. To investigate Ca2+-independent binding of synaptotagmin to indicators of short-term plasticity: the quantal size (q), the size of readily SNARE complexes, native proteins were immunoprecipitated from rat brain extract releasable vesicle pool (N) and the release probability (p) in excitatory and analyzed by immunoblotting. Three SNARE proteins, syntaxin 1, SNAP-25 connections within layer 4 of V1 in mice. Compared to the excitatory and synaptobrevin II were coprecipitated with synaptotagmin in the absence of synapses in the somatosensory cortex, those in V1 show relatively low Ca2+. Recombinant synaptotagmin and syntaxin expressed in HEK293 cells directly 2+ release probability and have a larger pool size. This suggests that excitatory bound each other in a Ca -independent manner and the binding was abolished connections within layer 4 can transmit high frequency signals more reliably in the presence of 1 M NaCl. Synaptotagmin has positively-charged polylysine regions in its C domains. Replacing of these lysine residues in the C B domain with than those in the somatosensory cortex. It also implies that in different brain 2 2 regions, synaptic transmission is tuned in a different manner to perform glutamine inhibited syntaxin binding by ~90%, but not in the C2A. Synaptotagmin binding of syntaxin was reduced by mutating of aspartate and glutamate residues in specific tasks. an amino-terminal half of the SNARE motif of syntaxin. These results indicate that 2+ synaptotagmin I binds syntaxin 1 through its C2B domain in the absence of Ca . Thus, before Ca2+ influx, synaptotagmin I could act on SNARE complexes through syntaxin 1 binding. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 141 P1AM-9-1 P1AM-9-2 ROLE OF NICOTINIC ACETYLCHOLINE RECEPTORS PREFERENTIAL EXPRESSION OF M1 mAChR IN ON SYNAPTIC TRANSMISSION IN THE VENTROBASAL DENDRITES AND SPINES OF CORTICAL PRINCIPAL THALAMIC COMPLEX CELLS AND ITS ANATOMICAL EVIDENCE FOR Yasuyuki Nagumo1, Yuichi Takeuchi2, Yoriko Kawakami1, VOLUME TRANSMISSION 2 1 Keiji Imoto , Mariko Miyata Miwako Yamasaki, Masahiko Watanabe 1Department of Physiology, Tokyo Women's Medical University, Japan, 2 Department of Anatomy, Hokkaido University Graduate School of Department of Information Physiology, National Institute for Physiological Medicine, Japan Sciences, Japan The M1 subtype of muscarinic acetylcholine receptor (M1) is a Gq/11-protein The rodent ventrobasal thalamic complex (VB) receives somatosensory information coupled receptor, and its activation exerts various effects on neural functions and through the medial lemniscus synapses and from the neocortex layer VI via activities. In the present study, we examined cellular and subcellular distribution of corticothalamic (CT) synapses. The VB also receives cholinergic systems from the M1 in the cerebral cortex and hippocampal CA1 region by immunofluorescence and brainstem. However, little is known about the role of nicotinic acetylcholine receptors preembedding immunoelectron microscopy. We found that M1 was preferentially (nAChRs) in VB synapses. Here, we investigate the modulatory mechanism of expressed in somatodendritic compartments of pyramidal cells, but not GAD67/65- nAChRs on synaptic transmissions in the mouse VB relay cells. Bath-application of ACh elicits the direct postsynaptic inward current and reduces amplitudes of EPSC in positive interneurons. Immunoparticles for M1 were heavily deposited on the CT synapses. These depressions accompanied with an increase of paired-plus ratio and plasma membrane of spines and thin dendrites of pyramidal cells, whereas those on coefficient variation; these are presynaptic parameters. However, ACh has no effect on somata and nerve terminals were almost at the background level. Analysis by double EPSC in medial lemniscus synapses. DMPP, a nAChR agonist, but not a muscarinic labeling for choline transporter (CHT) and M1 revealed that M1 was distributed AChR agonist, mimics the effect of ACh. Pretreatment of a α4β2 nAChR antagonist, without any special association with CHT-positive cholinergic fibers. Moreover, but not a α7 nAChR antagonist, suppresses the effect of ACh. Furthermore, the CT cholinergic varicosities rarely made “classical” synapses with M1-positive spines or antidromic population spike in somatosensory cortex layer VI has no effect by ACh. dendritic shafts, being consistent with the concept of “volume transmission”. These Thus, these results suggest that the α4β2 nAChRs contribute to reduce the transmitter results suggest that M1 mAChR is strategically allocated to distal somatodendritic release selectively at CT synapses. α4β2 nAChRs also are main nAChRs to directly compartments of cortical principal neurons, and thereby modulated by ambient ACh in depolarize in VB relay cells. the cerebral cortex and hippocampal CA1.
P1AM-9-3 P1AM-9-4 GATING ENERGY CHANGES IN THE ACETYLCHOLINE CEVIMELINE ENHANCES THE EXCITABILITY IN THE RECEPTOR-CHANNEL PORE RAT SUPERIOR SALIVATORY NUCLEUS NEURONS Archana Jha, Prasad Purohit, Snehal Jadey, Shaweta Gupta, Hirotaka Ueda1, Yoshihiro Mitoh1, Hiroyuki Ichikawa2, Anthony Auerbach Motoi Kobashi1, Takashi Yamashiro3, Ryuji Matsuo1 Department Physiology and Biophysics, SUNY, Buffalo, USA 1Department of Oral Physiology, 2Department of Oral Function and Agonist binding to cys-loop receptors leads to ion flux through the channel. Anatomy, 3Department of Orthodontics, Okayama University, Japan To explore the link between binding and gating, we measured the properties [Introduction]Cevimeline is an agonist of muscarinic receptors, and used of acetylcholine receptors with mutations in the pore-lining, M2 helix. For as therapeutic drug for the xerostomia. As for the mechanism of salivation each mutant we quantified the diliganded gating equilibrium constant (E ; 2 induced by Cevimeline, it is considered that Cevimeline directly stimulates the change in the energy difference between Closed and Open states), the the peripheral muscarinic receptors on the salivary glands. However, it relative effect on O vs. C (phi; the character of the perturbed residue at the transition state) and the single-channel current amplitude (i ). Positions 9’, is unknown that the effect of Cevimeline for superior salivatory (SS) o neurons which control the submandibular salivation. In the present study, 12’, 13’and 16’ showed the largest changes in E2. In the α and ε subunits, most M2 residues had an intermediate phi (0.63 and 0.54), and in all subunits we examined the effects of Cevimeline on SS neurons using the whole-cell the pore-facing residues 9’ and 12’ had a low phi(0.32). In ε,R substitutions patch-clamp technique in brain slices. [Methods]In neonatal Wistar rats (6-10 days), the SS neurons were retrogradely labelled by fluorescent dye applied progressively reduced io: 2’>5’>7’>10’>12’>16’. These results complement the structures of ELIC and GLIC and are consistent with the idea that in into the chorda-lingual nurve. Whole-cell recordings were obtained from closed AChRs, extra- and intracellular water is separated by a hydrophobic labelled cells, and miniture excitatory postsynaptic currents (mEPSCs) were region (M2, 5’-16’). We hypothesize that midway through the channel- examined. [Results and Discussion]Cevimeline induced the inward currents opening process the M2 helices tilt inwardly, to disrupt this barrier and allow dose-dependently and increased the frequency of mEPSCs. Therefore, it is the water compartments to fuse. Later, the pore-facing M2 residues may suggested that Cevimeline enhances the excitability via postsynaptic and experience a second energy change, from hydration. presynaptic muscarinic receptors in the rat SS neurons.
P1AM-9-5 P1AM-9-6 MONOAMINERGIC CROSS-TALK AND MODULATORY ENHANCEMENT OF GABAERGIC INTERNEURON ACTION OF SYNAPTIC TRANSMISSION IN THE DEEP EXCITABILITY BY DOPAMINE IN NEONATAL MOUSE CEREBELLAR NUCLEI HIPPOCAMPUS Fumihito Saitow, Hidenori Suzuki Keiju Nakagawa1, Yoichi Ogawa2, Hiroki Yoshino1, Department of Pharmacology, Nippon Medical School, Japan Yoshinobu Noriyama1, Masayuki Yamashita2, Toshifumi Kishimoto1 Modulation of the synaptic transmission at the deep cerebellar nuclei 1Department of Psychiatry, Nara Medical University, Japan, 2Department of (DCN), a main output of the cerebellum, plays crucial roles in regulating Physiology I, Nara Medical University, Japan the information flow processed from the cerebellar cortex. In this study, we examined a role of both noradrenaline (NA) and dopamine (DA) on the We have reported that dopamine profoundly suppresses GABAergic transmission in synaptic transmission using rat cerebellar slices. We obtained stimulation- the neonatal rat hippocampus by a presynaptic manner via phosphatidylinositol (PI)- evoked postsynaptic current (PSC) from DCN neurons by whole-cell patch- linked D1-like receptor (Neuroscience 2006). In this study, we studied the effect of clamp recording. Bath application of NA or DA decreased the amplitude dopamine on GABAergic interneurons in the neonatal mouse hippocampus (postnatal of glutamatergic-PSC but not GABAergic-PSC. Pharmacological study day 0-14) by whole-cell patch clamp recordings from CA1 pyramidal cells. The amplitude of evoked GABA -PSCs was decreased by dopamine. In contrast to the revealed that presynaptic α2-adrenergic (α2AR) and D2-like receptors (D2R) A were responsible for the NA- and DA-induced modulations, respectively. effect, the frequency of spontaneous GABAA-PSCs was significantly increased by
Poster Session DA decreased the amplitude of glutamatergic-PSC in all tested-synapses, dopamine. SKF83959, a selective agonist for PI-linked D1-like receptor, mimicked the whereas ~40% of synapses did not show the effects by the application of a action of dopamine. The enhancement effect on spontaneous GABAA-PSC frequency selective D2R agonist quinpirole. Regarding the extent of inhibitory effect, was abolished by pre-application of a D1-like receptor antagonist SKF83566, not there was a strong correlation between NA- and DA-induced inhibitory by a D2-like receptor antagonist sulpiride. Furthermore, the effect was attenuated by actions. Moreover, an α2AR antagonist RS79948 partly inhibited the DA- pre-application of a phospholipase C (PLC) inhibitor U73122, not by a proteinkinase induced inhibitory action. Taken together, we suggested that 1) the mossy A (PKA) inhibitor H89. TTX-resistant miniature GABAA-PSCs were not affected fibers innervating the DCN had heterogeneity in the expression of D2R; 2) by dopamine. These results suggest that dopamine may increase the excitability of DA affected not only D2R but also α2AR at the mossy fiber terminals. GABAergic interneurons by activating PI-linked D1-like receptors via PLC pathway.
142 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-9-7 P1AM-10-1 MECHANISMS OF SEROTONERGIC SENSITIZATION NEURONAL SYNCHRONIZATION AND NETWORK INDUCED BY SUSTAINED MDMA EXPOSURE IN RAT TOPOLOGY IN HIPPOCAMPAL CA3 MICROCIRCUITS ORGANOTYPIC MESENCEPHALIC SLICE CULTURES Naoya Takahashi, Takuya Sasaki, Norio Matsuki, Yuji Ikegaya Takayuki Nakagawa, Megumi Higuchi, Kazuki Nagayasu, Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Yuichi Suzuki, Yumi Yatani, Hisashi Shirakawa, Shuji Kaneko Sciences, University of Tokyo, Japan Department of Molecular Pharmacology, Graduate School of Spontaneous spike synchronization plays a crucial role for the information Pharmaceutical Sciences, Kyoto University, Japan processing in brain. In a microcircuit level, however, it is unknown how A body of evidence suggests the involvement of the raphe serotonergic neurons neuronal synchrony is shaped and acts functionally in the surrounding in the psychological actions of MDMA. In this study, we examined the effects of circuit. To address the mechanisms of synchronous firing among neurons, we sustained exposure of rat organotypic mesencephalic slice culture including the performed whole-cell patch-clamp recordings simultaneously from multiple raphe nuclei to MDMA. Immunostaining study revealed TPH-positive serotonergic neurons were abundant in the slice cultures. Acute treatment with MDMA for 30 neurons that showed spontaneous activity in hippocampal CA3 networks ex min increased extracellular 5-HT release. Sustained MDMA exposure for 4 days vivo. Both excitatory and inhibitory synaptic inputs were strongly correlated followed by challenge after a 1-day withdrawal period augmented the MDMA- between neuron pairs that frequently emitted synchronized spikes, while induced 5-HT release in a concentration-dependent manner, while it had no effect only inhibitory inputs were related between poorly synchronized pairs. To on 5-HT tissue content and TPH immunoreactivity. The augmented 5-HT release elucidate the network topology responsible for the coherent spontaneous 2+ was partially attenuated by citalopram, and was Ca -dependent and tetrodotoxin- activity, a reverse optical probing technique was used to manifest the wiring sensitive. Sustained MDMA exposure augmented redistribution of VMAT-2 from 3 patterns of synaptic connectivity among neurons within a CA3 microcircuit. cytoplasmic to membrane-associated vesicles. However, [ H]5-HT efflux was not Synaptically coupled neuron pairs tended to receive the converging facilitated in synaptosome prepared from sustained MDMA-exposed slice cultures. These data suggest that sustained MDMA exposure induces serotonergic sensitization projections from common presynaptic CA3 neurons and tended to terminate without serotonergic neurotoxicity, which is due to augmentation of secretory vesicle- preferentially on common CA1 postsynaptic neurons. These data suggest dependent 5-HT release by increased serotonergic activity, rather than reverse that the network is effectively woven to produce synchronous activity and transport-mediated 5-HT efflux. transfer the information to the next layer.
P1AM-10-2 P1AM-10-3 LARGE-SCALE MAPPING OF SYNAPSTIC COOPERATION OF NMDA AND METABOTROPIC CONNECTIVITY IN HIPPOCAMPAL SLICE CULTURES GLUTAMATE RECEPTORS Takuya Sasaki, Genki Minamisawa, Naoya Takahashi, Sylvain RAMA, Alexei Semyanov Norio Matsuki, Yuji Ikegaya Unit of Extrasynaptic Transmission, RIKEN BSI, Japan Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical N-methyl-D-aspartate receptors (NMDARs) are implicated in many Sciences, The University of Tokyo, Japan physiological processes such as synaptic and dendritic plasticity or Neural circuits are composed of a myriad number of synapses, yet it is cytotoxicity. They are present in both synaptic and extrasynaptic mostly unknown about their integrated wiring structures and the fundamental compartments. Their activation requires presence of agonist (glutamate) properties of individual synaptic transmission. The main reason is attributed and relief of voltage dependent Mg2+ block. AMPA receptor dependent to the lack of appropriate techniques to search the synaptic connections. depolarization is sufficient to relief Mg2+ block of synaptic NMDARs. We introduce a new method to detect the presence of synaptic connections in However, processes involved in relief of Mg2+ block of extrasynaptic hundreds of neuron pairs in a hippocampal slice culture. The spatiotemporal NMDARs are unclear. Using two-photon Ca2+ imaging combined with whole firing patterns of CA3 neurons in response to local application of glutamate cell recordings we investigated Ca2+ transients in aspiny parts of dendrites were monitored with functional multineuron calcium imaging (fMCI), and voltages in soma of CA1 pyramidal neurons. We found that EPSPs an optical technique that monitors neuronal population activity with induced by electrical stimulation of Schaffer collaterals do not produce single cell resolution, and excitatory postsynaptic currents (EPSCs) were Ca2+ transients in dendrites of these neurons suggesting no recruitment of simultaneously whole-cell recorded from given CA1 pyramidal neurons. By extrasynaptic NMDARs during synaptic events. Then we tested if activation sorting the neurons that exhibited calcium events immediately before or at of high affinity metabotropic glutamate receptors (mGluRs) can provide the same time of EPSCs, we identified the putative presynaptic cells in CA3 necessary depolarization. Surprisingly, activation of mGluRs reduced Ca2+ region. This probing method also allows us to achieve simultaneous double transients mediated by synaptic NMDARs. Now we are testing if activation or triple whole-cell recordings from neurons with multiple monosynaptic of mGluRs affects extrasynaptic NMDARs. The coupling of mGluRs and connections. Our technique is expected to be a powerful and versatile tool for NMDARs could be the mechanism by which changes in ambient glutamate our understanding of the neural circuits at the single synapse level. modulate neuronal activity.
P1AM-10-4 P1AM-10-5 TWO MODES OF Ca2+ DYNAMICS IN CNS NEURONS SIMULTANEOUS IMAGING OF THE MOTILITY OF N Charles Harata, Yasuhiro Kakazu, Kirsty M Goodman, PRESYNAPTIC VARICOSITIES AND POSTSYNAPTIC Jin-Young Koh SCAFFOLDING PROTEINS AT INHIBITORY SYNAPSES 1 1 2 Department of Molecular Physiology & Biophysics, University of Iowa, Toshihiko Kuriu , Shiro Konishi , Yuchio Yanagawa Carver College of Medicine, USA 1Department of Neurophysiology, Tokushima Bunri University, Kagawa 2 Intracellular Ca2+ serves as a key second messenger in many cell types. School of Pharmaceutical Sciences, Japan, Department of Genetic and A temporary rise in intracellular Ca2+ concentration (Ca2+ transient) in Behavioral Neuroscience, Gunma University Graduate School of Medicine, neurons regulates events such as ion channel opening, protein kinase Japan activity and synaptic plasticity, to name a few. Recent findings indicate that This study aimed at visualizing the morphology and dynamics of inhibitory synapses. ion channels are distributed heterogeneously within different subcellular For this purpose, we used two distinct presynaptic and postsynaptic probes: one is regions of a single neuron. It is thus critical to understand the spatial and genetically Venus-labeled inhibitory neurons as a presynaptic marker and the other temporal properties of Ca2+ transients. In the present study, we characterized mCherry-tagged gephyrin, a postsynaptic scaffolding protein, as a postsynaptic Ca2+ transients in dendrites and somata of neurons of the rat brain. We marker. Using primary culture of mouse hippocampal neurons and dual wavelength found that caffeine (10 mM) stimulated Ca2+-induced Ca2+ release (CICR). fluorescence microscopy, we found close contacts of Venus-positive varicosities with 2+ mCherry-labeled gephyrin clusters in the dendritic shafts of dissociated pyramidal When caffeine was applied over all surfaces of a neuron, Ca transients neurons. To study the dynamics of inhibitory synaptic contacts, we exploited were first observed in the dendrite and then in the soma. Ca2+ transients 2+ + time-lapse imaging which revealed that: (1) inhibitory varicosities and gephyrin led to membrane hyperpolarization by way of Ca -activated K currents. clusters underwent profound changes in the shape and movements, (2) the extents When neurons were instead depolarized, by applying local, extracellular 2+ of morphological changes and movements depended on the culture days, and (3) electrical stimulation to the plasma membrane of soma, Ca transients were the motility indexes of the varicosity and its counterpart gephyrin cluster were well likewise observed in both the dendrite and soma. However, the onsets were correlated. These results indicate that the presynaptic varicosity undergoes a marked indistinguishable in the two regions at a 1- to 2-ms scale by rapid imaging. morphological change, and the postsynaptic scaffolding protein gephyrin actively 2+ The diverse patterns of Ca transients will have different functional impacts moves at the inhibitory synapse without losing the contact with the presynaptic on a neuron. varicosity. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 143 P1AM-10-6 P1AM-10-7 FUNCTIONAL RELEASE COMPETENT UNITS AT TRACKING OF FLUORESCENTLY LABELED BDNF A CENTRAL SYNAPSE REVEALED BY OPTICAL ON CEREBELLAR BERGMANN GLIA USING SINGLE GLUTAMATE IMAGING MOLECULE LIVE IMAGING TECHNIQUE Hirokazu Sakamoto, Shigeyuki Namiki, Sho Iinuma, Kenzo Hirose Maki Koike-Tani, Tomomi Tani Department of Cell Physiology, Nagoya University, Grad. Sch. of Medicine, Research Institute for Electronic Science/ Nanosystems Physiology, Japan Hokkaido University, Japan The number of release units (N) at a central synapse is a key determinant Brain Derived Neurotrophic Factor (BDNF) is a member of neurotrophin for synaptic efficacy, yet functional entities of release units have not family found in central nervous system. In cerebellum, BDNF works as a been revealed. In this study, we measured glutamate released from single retrograde modulator of synaptic transmission between parallel fiber and hippocampal synapses by optical glutamate imaging. We show that the Purkinje cell. To visualize the role of BDNF in cerebellum, a fluorescently labeled BDNF (Cy3-BDNF) was synthesized and was applied to the amount of glutamate released had a large trial to trial variability at individual cerebellar slice preparation from 15 day chick embryo. Cy3-BDNF (5 nM) synapses, although hippocampal synapses typically contain only one active was applied to the brain slice by bath application, and observed by wide field, zone. By using variance-mean (V-M) analysis based on a simple binomial critically illuminated epi-fluorescent microscope. Fluorescent was observed model, we determined the N-value at individual synapses. Combining high- not to Purkinje cells, but specifically to Bergmann glial cells. The fluorescent frequency stimulus trains and deconvolution analysis, we estimated the size BDNF clusters showed a bidirectional translocation along the axis of glial of readily releasable vesicles pool (RRP). Interestingly, although the N-value projection. It is reported that BDNF induce increase of intracellular Ca2+ and the size of RRP strongly correlated, the size of RRP is usually larger than concentration by releasing calcium from intracellular stores; truncated TrkB the N-value. These results indicate that limited vesicles of RRP were released receptors are predominately expressed on astrocytes, however, its function is by a single presynaptic action potential. Therefore, the release probability unclear. Our live image observation of BDNF behavior on slice preparation of individual vesicles is heterogeneous within an active zone; a subset of in combination with calcium imaging or patch clamp technique suggests that releasable vesicles lined up in active zone functions as apparent release Bergmann glia express high affinity TrkB receptors on its projection and competent units. interact with surrounding neurons via calcium signaling.
P1AM-10-8 P1AM-10-9 A RECOMBINANT ANTIBODY TO TRACK MORPHOLOGICAL CHARACTERIZATION OF LEFT ENDOGENOUS PALMITOYLATED PSD-95 AND RIGHT HIPPOCAMPAL CA1 TO CA1 PYRAMIDAL Masaki Fukata1, Ariane Dimitrov2, Tsuyoshi Iwanaga1, CELL SYNAPSES Franck Perez2, Yuko Fukata1 Ryouske Kawakami, Ryuichi Shigemoto 1Department of Cell Physiology, National Institute for Physiological Division of Cerebral Structure, National Institute for Physiological Science, Sciences, Japan, 2Centre National de la Recherche Scientifique, Unite Japan Mixte de Recherche 144 and Institut Curie Section Recherche, France Previously we found a left-right asymmetry in mouse hippocampal ipsilateral Protein palmitoylation, a reversible lipid modification, regulates the dynamic CA3-CA1 synapses. Synaptic NMDA receptor NR2B and AMPA receptor GluR1 trafficking and function of proteins. The postsynaptic density-protein-95 (PSD-95) subunits were denser in the left and right CA1 stratum radiatum, respectively. represents a major palmitoylated protein in brain. This lipid modification is critical Also, the GluR1-dense synapses in the right were 1.5 times larger than NR2B- for PSD-95 clustering of AMPA-type glutamate receptor at excitatory synapses. We dense synapses in the left. These two types of synapses were distributed have recently found that activity-sensitive PSD-95 palmitoylation mediates synaptic depending on the side of input origin since we found the opposite asymmetry in scaling in hippocampal neurons. Here, we attempted to develop a biosensor to the contralateral CA3-CA1 synapses. For building up such asymmetrical circuits, specifically monitor the palmitoylation state of endogenous PSD-95 in living neurons. CA1 pyramidal cells need to recognize some side-specific cues expressed By antibody phage display method using stoichiometrically palmitoylated PSD-95 in the CA3 projection. In the present study, to examine if such cues are also as an antigen, we selected a recombinant antibody, clone PF11. PF11 specifically expressed in the CA1 projection fibers, we focused on the ipsilateral synaptic bound to PSD-95 palmitoylated by DHHC2 palmitoylating enzyme, but hardly bound connections between CA1 pyramidal cells. To identify CA1-CA1 synapses, we to non-palmitoylated PSD-95 in HEK293 cells. After GFP-tagging and intracellular injected GFP-expressing lentivirus into the unilateral CA1 area in the left and expression in hippocampal neurons, GFP-PF11 specifically tracked synaptic clusters right hippocampus. In the spines making synapses with GFP-labeled ipsilateral of endogenous PSD-95. Treatment of neurons with 2- bromopalmitate, an inhibitor CA1 axon collateral terminals, PSD areas and spine volumes were measured by of protein palmitoylation, relocalized PF11-GFP from postsynaptic membrane into electron microscopic reconstruction analysis. The NR2B component of CA1- dendrites. These results strongly suggest that PF11 can visualize palmitoylation CA1 synaptic response was also examined by electrophysiological analysis using dynamics of endogenous PSD-95 in living neurons. lentivirus-mediated expression of channelrhodopsin in CA1 pyramidal cells.
P1AM-10-10 P1AM-10-11 CYTOARCHITECTONIC REGULATES MOLECULAR LEFT-RIGHT ASYMMETY OF THE HIPPOCAMPAL SIGNALING IN SPINES OF CEREBELLAR PURKINJE SYNAPSES WITH DIFFERENTIAL SUBUNIT NEURONS ALLOCATION OF GLUTAMATE RECEPTORS Thomas Launey1, Tsutomu Hashikawa2, Yoshiko Takagishi3, Masao Ito4 1 1 2 1 Yoshiaki Shinohara , Hajime Hirase , Ryuichi Shigemoto Launey Research Unit for Molecular Neurocybernetics, RIKEN Brain 1 2 Science Institute, Japan, 2Research Resources Center Support Unit for Hirase Research Unit, RIKEN Brain Science Institute, Japan, National Neuromorphological Analysis, RIKEN Brain Science Institute, Japan, 3Research Institute for Physiological Sciences, Japan 4 Institute for Environmental Medicine, Nagoya University, Japan, Lab. Memory Left-right asymmetry of the brain has been studied mostly through and Learning, RIKEN Brain Science Institute, Japan psychological examination and functional imaging in primates, leaving The synaptic spine represents a highly adapted structure for cellular signaling. Its its molecular and synaptic aspects largely unaddressed. Here, we show constricted neck acts as a barrier to diffusion of cytoplasmic proteins and second that hippocampal CA1 pyramidal cell synapses differ in size, shape, and messengers while its convoluted organelles create micro-domains with high density of reacting molecules, influencing the speed of intermolecular reactions. glutamate receptor expression depending on the laterality of presynaptic We used 3D reconstruction of electron micrographs from serially sectioned Purkinje origin. CA1 synapses receiving neuronal input from the right CA3 pyramidal cell (PC) dendrites to analyze the subcellular morphological features of the spine, cells are larger, have more perforated PSD and a GluR1 expression level at synapse between PC and parallel fibers (PF). We measured the shape and relative twice higher than those receiving input from the left CA3. The synaptic
Poster Session position in the spine of the post-synaptic density, the spine apparatus (SA) and density of GluR1 increases as the size of a synapse increases, whereas that organelles that may impede molecular diffusion in the spine and shaft. of NR2B decreases due to the relatively constant NR2B expression in CA1 We show that the PSD is systematically located on the side of the spine head rather regardless of synapse size. Densities of other major glutamate receptor than at the apex, irrespective of the spine shape and incidence angle of the PFs. The subunits show no correlation with synapse size, thus resulting in higher morphology of the SA appeared to be optimized to maximize its surface facing the PSD and facilitate signal transduction. Similar evaluation in rat mutant devoid of spine net expression in synapses having right input. Our study demonstrates apparatus demonstrated that this structure plays a central role as organizer of spine universal left-right asymmetry of hippocampal synapses with a fundamental morphology. In-silico simulation of the IP3 cascade in spine confirmed the key role of relationship between synaptic area and the expression of glutamate receptor morphology in regulating molecular signal transduction. subunits.
144 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-11-1 P1AM-11-2 SYNTABULIN, A LINKER OF ANTEROGRADE ELECTROPHYSIOLOGICAL AND BEHAVIORAL CARGO, IS REQUIRED FOR MAINTAINING SYNAPTIC EFFECTS OF SUBSTANCE P IN GLOBUS PALLIDUS TRANSMISSION Lei Chen1, Qiao-Ling Cui1, Wing-Ho Yung2 Huan Ma1, Sumiko Mochida2 1Department of Physiology, Qingdao University, China, 2Department of 1Department of Neurobiology, School of Medicine, Shanghai Jiaotong Physiology, The Chinese University of Hong Kong, Hong Kong, China University, China, 2Department of Physiology, Tokyo Medical University, The globus pallidus plays a significant role in movement regulation. Japan Substance P has been demonstrated to modulate neuronal activity in a Syntabulin, identified in immature neurons as a linker molecule that attaches cargo number of brain regions via neurokinin-1 receptors. To determine the effects to a motor protein KIF5B enabling axonal transport of mitochondria, syntaxin, and of substance P in globus pallidus, whole-cell patch-clamp recordings, in vivo bassoon, should play important roles in mature neurons. To explore its functional extracellular recordings and behavioral test were performed in the present significance in synaptic transmission, we examined synaptic transmission in cultured study. Neurokinin-1 receptor agonist, [Sar9, Met(O2)11] substance P (SM- rat superior cervical ganglion neurons in which syntabulin was knocked down with its SP), depolarized globus pallidus neurons and increased their firing rates. siRNA or syntabulin binding to KIF5B was perturbed by overexpression of its binding Furthermore, SM-SP significantly increased the frequency of spontaneous domain. Syntabulin dysfunction slowed down maturation of synaptic coupling, and inhibitory postsynaptic currents, but only induced a transient increase in reduced basal and highly active synaptic transmission in mature synapses. Reduction the frequency of miniature inhibitory postsynaptic currents. Consistent of basal synaptic transmission is due to decrease in the readily releasable pool (RRP) with in vitro results, our in vivo extracellular recordings revealed that size, and is partially rescued by syntaxin overexpression. Reduction of highly active micropressure ejection of SM-SP increased the spontaneous firing rate synaptic transmission, such as paired-pulse facilitation, augmentation and post-tetanic of pallidal neurons in a concentration-dependent manner. In line with potentiation, is due to a decrease in replenishment of the RRP with synaptic vesicles, electrophysiological recordings, in behaving rats, unilateral microinjection of and partially rescued by ATP supplement. Thus, syntabulin supports basal synaptic SM-SP led to contralateral deflection in the presence of systemic haloperidol transmission and presynaptic short-term plasticity by mediating axonal transports of administration. Based on the direct excitatory effects of SM-SP on pallidal syntaxin and mitochondria that maintain the readily releasable synaptic vesicles in the neurons, we hypothesize that substance P in globus pallidus may be involved presynaptic terminal. in its treatment of Parkinson’s disease.
P1AM-11-3 P1AM-11-4 CAFFEINE-INDUCED MODULATION OF ACTION ROLE OF HEPARANASE IN SYNAPTIC PLASTICITY AT POTENTIAL WAVEFORM AT THE HIPPOCAMPAL THE HIPPOCAMPUS AND VESTIBULAR NUCLEUS MOSSY FIBER AXONS Chun-Wai Ma1, Yi Zhang2, Wai-Chun Cham2, Ying-Shing Chan1, 2 Masataka Yaginuma, Haruyuki Kamiya Daisy Kwok-Yan Shum 1Department of Physiology, The University of Hong Kong, Hong Kong, Department of Neurobiology, Hokkaido University School of Medicine, 2 Japan Department of Biochemistry, The University of Hong Kong, Hong Kong Recently we reported that the ryanodine-sensitive Ca2+ stores exist in Long-term potentiation (LTP) in the hippocampus was shown to be perturbed by the hippocampal mossy fiber axons, and caffeine, a potent drug inducing bath treatment of brain slices with bacterial heparitinase. We hypothesized that Ca2+ release from ryanodine-sensitive stores, causes robust enhancement heparanase is the mammalian counterpart that serves a role in synaptic plasticity by cleaving peri-synaptic heparan sulfates. We then looked for cells that express of transmitter release from the mossy fiber terminals. Large increase in 2+ heparanase in the hippocampus and the vestibular nucleus, tissues where LTP presynaptic Ca levels by caffeine may facilitate transmitter release by direct was demonstrated. In both cases, heparanase-immunoreactivity was localized to actions on the release machinery and by additional effects on the waveform of perinuclear organelles of neurons. Next, we prepared recombinant proheparanase presynaptic action potentials. In this study, we addressed whether application from medium conditioned by a heparanase-overexpressing 3T3 H1641 line. of caffeine affects the waveform of fiber volleys, compound action potentials Proheparanase is the precursor of the enzymatically active, mature heparanase. of axons recorded extracellularly, in mouse hippocampal slice preparations. Treatment of hippocampal brain slices with the pro-form resulted in decrease Application of 10 mM caffeine caused reversible broadening of fiber volley of the basal excitatory postsynaptic field potential. Work is ongoing to decipher waveforms. Small increase in the latency or the amplitude of fiber volleys whether the pro-form and the mature, active enzyme play differential roles in 2+ 2+ was induced by caffeine, in either Ca -containing or Ca -free perfusing the regulation of synaptic efficacy. To this end, attempts are made to recover the 2+ solution. These results suggest that increase in Ca levels by intracellular active, mature heparanase from lysates of the overexpressing cell line. Similar release from ryanodine receptors modulates the excitability of mossy fiber strategies are applied to brain slice preparations of the medial vestibular nucleus axons, possibly by affecting voltage-dependent conductance involving in to find if synaptic responseness is also perturbed by pro- and mature forms of shaping of the action potential waveform at the mossy fiber axons. heparanase. [Supported by HKRGC]
P1AM-11-5 P1AM-11-6 SYNAPTIC METAPLASTICITY IN THE HYPOTHALAMUS Cbln1 REGULATES INHIBITORY SYNAPTIC FOLLOWING IN VIVO CHALLENGE TRANSMISSION ON CEREBELLAR PURKINJE CELLS Aya Ito-Ishida1, Eriko Miura2, Taisuke Miyazaki2, Tetsushi Sadakata3, Brent J Kuzmiski, Quentin J Pittman, Jaideep S Bains 3 1 3 Ritsuko Katoh-Semba , Keiko Matsuda , Teiichi Furuichi , Masahiko Department of Physiology & Biophysics, University of Calgary, Canada Watanabe2, Michisuke Yuzaki1 1 2 Presynaptic metabotropic glutamate receptors (mGluRs) provide Department of Physiology, Keio University, Japan, Department of Anatomy and Embryology, Graduate School of Medicine, Hokakido University, Japan, negative feedback at excitatory synapses. At glutamatergic synapses onto 3Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, Japan hypothalamic magnocellular neurosecretory cells (MNCs), group III mGluRs Cbln1, a glycoprotein that belongs to C1q family proteins, is secreted from cerebellar are functionally downregulated by noradrenaline (NA). This loss of function granule cells. We showed previously that Cbln1 strongly induces formation of parallel may favor the induction of activity-dependent long-term potentiation of fiber-Purkinje cell synapses in the adult brain. Although its importance on excitatory glutamate synapses. Here we show that the functional state of presynaptic synapses was established, the effect of Cbln1 on inhibitory synapses remained unclear. group III mGluRs is impacted by physiological manipulations in vivo and Here, we analyzed anatomical and physiological properties of inhibitory synapses this determines whether these synapses exhibit metaplasticity. Similar to the on Purkinje cells in cbln1-/- mice. Immunohistochemistry of the brain showed increased density of vesicular γ-aminobutyric acid transporter positive puncta in the effects of NA on synapses in slices prepared from naive animals, glutamate molecular layer of the cerebellum. Whole-cell patch-clamp recordings from Purkinje transmission onto MNCs is depressed and group III mGluR function is cells revealed increase in both frequency and amplitude of miniature inhibitory post- compromised following in vivo volume depletion. In both conditions, loss synaptic currents (mIPSCs). When the acute slices from cbln1-/- mice were treated of mGluR function is permissive for the induction of activity-dependent with recombinant Cbln1 in aCSF for 3 hrs, mIPSC amplitude was significantly synaptic potentiation. These findings provide the first clear demonstration decreased, while an inactive form of Cbln1 lacked these effects. Increase in the amplitude of mIPSCs was also reversed by inhibition of BDNF with K252a and Fc- that inactivation of presynaptic mGluRs unmasks a metaplastic state that TrkB, while these inhibitors lacked effects on wild-type cells. These results indicate may facilitate the re-setting of homeostatic networks following a successful that Cbln1 also regulates inhibitory synaptic transmission through modulation BDNF response to an acute physiological challenge. signaling pathways in cerebellar Purkinje cells. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 145 P1AM-11-7 P1AM-11-8 MULTISENSORY INTERACTIONS IN SINGLE STATE-DEPENDENT MODULATION OF FEEDBACK CEREBELLAR GRANULE CELLS IN VIVO INHIBITION IN CORTICAL PYRAMIDAL NEURON Misa Shimuta, Taro Ishikawa, Michael Hausser Jie Zhu, Yousheng Shu Wolfson Institute for Biomedical Research, University College London, UK Department of neuroscience, State Key Laboratory of Neuroscience, Institute of Neuroscience, Chinese Academy of Sciences, China Each cerebellar granule cell receives multiple (~4) mossy fiber inputs. It is not known if mossy fibers terminating on a single granule cell can carry It has been shown that membrane potential changes in the presynaptic soma sensory signals of different modalities and how these signals are integrated could modulate intracortical synaptic transmission triggered by action potentials. in granule cells. We recorded excitatory postsynaptic currents (EPSCs) However, little is known about the effect of such modulation on the operation of local circuitry. Here we demonstrate that membrane potential changes in a single evoked by somatosensory (air puff to the face), auditory (white noise) and layer V pyramidal neuron control the amount of feedback inhibition received visual (flashing light) stimulation from granule cells in the Crus I and II and by its neighboring neurons. Modest depolarization increases the amplitude and the paraflocculus of rats (P19-24) under ketamine-xylazine anaesthesia. A integral, shortens the latency and reduces the jitter of the disynaptic inhibitory subpopulation of granule cells (16%) exhibited responses to stimulation of potential evoked by a burst of action potentials (15-20 APs, 100-200 Hz). The two different sensory modalities. Activation of different sensory modalities low-threshold spiking (LTS) interneuron, which has been shown to mediate the triggered EPSCs with significantly different mean amplitudes in some disynaptic inhibition, may contribute to the facilitation of this recurrent inhibition. of these neurons (5/12). The number of EPSCs evoked by simultaneous Presynaptic depolarization increases the summated excitatory postsynaptic bimodal stimulation was a sublinear summation of those evoked by two potentials (induced by a train of action potentials), thus increases the number unimodal stmuli. These results indicate that a single granule cell can receive of action potentials generated in LTS interneuron, and consequently enhances separate mossy fibers conveying sensory signals from different modalities. inhibitory postsynaptic responses in nearby pyramidal neurons. These results Furthermore, cross-modal suppression can be observed, indicating that indicate that cortical feedback inhibition can be modulated in a state-dependent interaction between sensory modalities in the cerebellar cortex can take place manner, and may contribute to the balance of excitation and inhibition within a at, or before the granule cell relay. cortical network.
P1AM-11-9 P1AM-11-10 DEEP CORTICAL INTERNEURONS CONTROL RECYCLING OF D1 RECEPTOR BY G-PROTEIN RHO DENDRITIC ENCODING OF SENSORY STIMULI AND PHOSPHOLIPASE D THROUGH VESICULAR Masanori Murayama, Enrique Perez-Garci, Thomas Nevian, TRAFFICKING TO THE PLASMA MEMBRANE IN Tobias Bock, Walter Senn, Matthew Evan Larkum APLYSIA NEURONS 1 1 1 2 Department of Physiology, University of Bern, Switzerland Satoshi Kawasaki , Shingo Kimura , Jin Ochiai , Reiko Fujita , 1 Layer 5 (L5) neocortical pyramidal neurons have active dendrites which Kazuhiko Sasaki 1 2 increase the effectiveness of distal input in a highly non-linear fashion. Department of Physiology, Iwate Medical University, Japan, Department of Here we show, however, that the population response of L5 apical dendrites Chemistry, Iwate Medical University, Japan We previously reported that monomeric G protein Rho and subsequent phospholipase is nearly linear with respect to somatosensory stimulus intensity. We + recorded dendritic calcium responses to sensory input in the upper layers D (PLD) regulate the D1-receptor induced Na current response in the ganglion cells of the hindlimb area of the primary somatosensory cortex using a fiberoptic of Aplysia. Present study examined possible role of these molecules in regulation of the D1 receptor recycling. Application of methyl-β-cyclodextrin and Filipin, inhibitors for system in anesthetized and awake rats. The liner slope of the stimulus- caveolae-mediated endocytosis, markedly augmented the response, but dancylcadaverine, response function was under the control of a particular subset of dendritic inhibitor for clathrin-dependent endocytosis, had insignificant effect. The DA-induced targeting inhibitory neurons activated by synaptic inputs predominantly response was significantly suppressed after the application of monensin, an inhibitor of in L5. Recordings from single apical tuft dendrites in vitro showed that recycling. Application of dynamin inhibitory peptide or Dynasore, inhibitors for dynamin, activity in disynaptically coupled L5 pyramidal neurons directly blocks the markedly suppressed the response to DA. Intracellular application of N-ethylmaleimide or initiation of dendritic calcium spikes in neighboring pyramidal neurons. The C-terminal peptide of SNAP-25 (SNAP-25ct), blockers for SNARE-dependent membrane results constitute a functional description of a cortical microcircuit in awake fusion, markedly depressed the response. Furthermore, augmenting effect of RhoGEF on the response disappeared when examined after the monensin or SNAP-25ct. These results animals that relies on the active properties of L5 pyramidal dendrites and suggest that D1 receptor may be recycled through caveolae-mediated endocytosis, dynamin- their exquisite sensitivity to inhibition. We conclude that these interneurons dependent vesicular budding from recycling endosome, and SNARE-dependent vesicular dynamically modulate the slope and threshold of the dendritic response fusion to the plasma membrane, the fusion of which is mediated by Rho and subsequent function to match the physiologically relevant input range. PLD.
P1AM-11-11 P1AM-11-12 MODULATION OF HIPPOCAMPAL INHIBITORY IMMUNOHISTOCHEMICAL LOCALIZATION OF SYNAPTIC TRANSMISSION THROUGH PROTEASE- MOLECULES FOR 2-ARACHIDONOYLGLYCEROL- ACTIVATED RECEPTOR 1 MEDIATED RETROGRADE SIGNALING IN THE MOUSE Yuki Hashimotodani1, Takako Ohno-Shosaku2, Masanobu Kano3 DENTATE GYRUS 1Department of pharmacology, University of Tokyo, Japan, 2Department Motokazu Uchigashima, Masahiko Watanabe of Impairment Study, Kanazawa University, Japan, 3Department of Department of Anatomy, Hokkaido University School of Medicine, Japan Neurophysiology, University of Tokyo, Japan 2-Arachidonoylglycerol (2-AG) retrogradely suppresses neurotransmission Protease-activated receptors (PARs) are G protein-coupled receptors that are activated through the activation of presynaptic cannabinoid receptor CB1. In by proteolytic cleavage of a specific site within their extracellular N terminus. PAR1, the dentate gyrus, 2-AG-mediated retrograde suppression is thought to one of four subtypes of PARs (PAR1-PAR4), is widely expressed in the brain, and has contribute to prevention of epilepsy. In the present study, we examined been suggested to play a variety of roles in pathological and physiological conditions. immunohistochemical distribution of 2-AG synthetic enzyme diacylglycerol However, the roles of PAR1 in modulation of synaptic transmission have not been lipase-α (DAGLα), CB1, and 2-AG-degrading enzyme monoacylglycerol well investigated. In this study, we examined effects of PAR1 activation on inhibitory lipase (MGL). Immunoreactivity for DAGLα was abundantly distributed in postsynaptic currents (IPSCs) in cultured hippocampal neurons. Application of the somatodendritic elements of granule cells and accumulated in their spines. PAR1 agonist TFLLR induced a transient suppression of IPSCs, which was blocked by At granule cell synapses, CB1 was expressed highly in parvalbumin-negative Poster Session the PAR1 antagonist SCH79797. The second application of TFLLR failed to suppress inhibitory terminals and also moderately in mossy cell excitatory terminals. IPSCs, indicating severe desensitization. Thrombin, an endogenous PAR1 activator, MGL was high in some inhibitory terminals, including CB1-positive ones, caused a similar suppression of IPSCs. When TFLLR and thrombin were sequentially and also in astrocytic processes surrounding granule cell synapses. From applied, the effect of the secondly applied drug was diminished irrespective of the this molecular arrangement, granule cell's excitability can be modulated order, confirming the involvement of the same receptor. The PAR1-mediated IPSC bidirectionally via 2-AG-mediated suppressions of excitatory recurrent suppression was accompanied by an increase in the paired-pulse ratio. From these inputs from mossy cells and of inhibitory inputs from local interneurons. results, we conclude that activation of PAR1 presynaptically suppresses the inhibitory Furthermore, MGL in terminals and astrocytes may contribute to restrict transmission in hippocampal neurons. spatial and temporal extents of 2-AG-mediated signaling.
146 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-11-13 P1AM-11-14 MODULATION OF EVOKED SYNCHRONOUS AND OSCILLATION OF THE END PLATE POTENTIAL ASYNCHRONOUS RELEASE OF MEDIATOR AT Toshifumi Kumai MAMMALIAN'S SYNAPSE UNDER CHANGES OF Oral Medicine, Matsumoto Dental University, Japan CALCIUM METABOLISM Monopolar surface electromyograms (EMGs) were recorded in response Alexandr Lvovich Vasin, Dmitry V. Samigullin, Ellya A. Bukharaeva to their quick contractions from eight sites of each of the masseter, Laboratory of Biophysics of Synaptic Processes, Kazan Institute of temporal, brachioradialis, and palmaris longus muscles of human subjects Biochemistry and Biophysics, Russia using traditional disc electrodes lined along the muscles. (The reference The correlation between changes of quantal content and time course of various electrode was putted on a place suitable for the recordings for each muscle.) components of secretion was studied under different concentrations of calcium, Component of the slow wave was extracted from the raw recordings for calcium buffers, strontium and specific calcium channel blockers. Quanta were each muscle using digital low-path filter (0-30Hz). The extracted signals classified as synchronous (within3 ms) and asynchronous (from 8 to 50 ms) release deflected either negatively or positively depending on the recording sites. after presynaptic action potential. After increase of stimulation frequency from 0.5 The differences in the polarity and magnitude with the recording sites seem to 15 Hz the rise of quantal content and frequency of asynchronous release was to reflect the sink-source relation of the end plate current, where the site accompanied by changes in time course of secretion. Exogenous calcium chelators showing most negative deflection would correspond to the neuromuscular have decreased the facilitation of asynchronous release but not synchronous junction. Further the negative slow potential for each muscle exhibited small component. The reduced extracellular Ca2+ has decreased the facilitation of both oscillation. The oscillation emerged more clearly in the strong contractions. synchronous and asynchronous release whereas blockade of P/Q calcium channels has changed the facilitation of synchronous release only. Blockade of potassium The oscillation of the end plate potential must be the result of the inhibitory channels has leaded to increase in facillitation of synchronous release, but addition of effect of the muscle spindle system on the motor neuron activity to control L-type calcium channel blocker eliminated this effect. The replacement of calcium the strength of muscle contraction. by strontium has increased facilitation of both release components. The data support a hypothesis about different mechanisms governing time course of synchronous and asynchronous components of release and their various roles in synaptic plasticity.
P1AM-11-15 P1AM-12-1 NITRIC OXIDE INHIBITS NICOTINE-INDUCED PLACE PLC/PKC PATHWAY IS INVOLVED IN MELATONIN- PREFERENCE IN RATS: THE ROLE OF BASOLATERAL MEDIATED POTENTIATION OF GLYCINE CURRENTS NUCLEUS OF AMYGDALA AND CA1 AREA OF IN RAT RETINAL GANGLION CELLS HIPPOCAMPUS Wen-Jie Zhao, Min Zhang, Shi-Xiang Jiang, Xiong-Li Yang, Cyrus Jalili1, Yousof Sadeghi2, Hedayat Sahraei3 Zhongfeng Wang 1Department of Anatomy, Faculty of Medicine, Kermanshah University of Institute of Neurobiology, Institutes of Brain Science&State Key Laboratory Medical Sciences, Kermanshah, Iran, 2Department of Anatomy, Faculty of of Medical Neurobiology, Fudan University, China Medicine, Shaheed Beheshti University of Medical Sciences, Tehran, Iran, Melatonin (MEL) is involved in the regulation of various physiological processes. 3Applied Neuroscience Research Center, Baqiyatallah (a.s.) University of Recently, it has been shown that MEL exerts neuromodulatory roles. Our previous Medical Sciences, Tehran, Iran work demonstrated that MEL potentiates glycine-induced currents in rat retinal ganglion cells (RGCs) via activating the MT2 receptor. Here we further investigated It has been shown that nitric oxide may be involved in nicotine dependence and/or reward. the possible intracellular signaling pathways underlying the MEL effect by using In the present study, effects of intra-basolateral nucleus of amygdala and hippocampal CA1 patch-clamp techniques in acutely dissociated RGCs. The results showed that the area injections of L-arginine and L-NAME on the acquisition and expression of nicotine- potentiation of glycine currents by MEL was blocked by D609, a phospholipase induced place preference in male Wistar rats (W: 220-250 g) were examined. Results showed C (PLC) inhibitor. The PKC activator phorbol 12-myristate 13-acetate (PMA), that intra-basolateral amygdala injactions of L-arginine (1, 5 and 10 mg/rat) and L-NAME on the other hand, mimicked the potentiation of glycine currents by MEL and (1, 5 and 10 mg/rat) did not change both acquisition and expression of nicotine place MEL didn’t further boost the enhanced currents. Furthermore, the PKC inhibitor preference. On the other hand, injection of L-arginine (1, 5 and 10 mg/kg) in the CA1 area of bisindolylmaleimide IV (Bis IV) eliminated the MEL-induced potentiation of glycine hippocampus increases where as L-NAME administration reduces the acquisition of nicotine currents. In contrast, application of 8-Br-cAMP or 8-Br-cGMP failed to mimic the place preference. However, the drugs could not change the expression of nicotine place MEL effect. Similarly, both H-89, a PKA inhibitor, and KT5823, a PKG inhibitor conditioning.It could be concluded that nitric oxide can interfere with reward properties of didn’t alter the MEL-induced potentiation. All these results suggest that the activation nicotine in the CA1 area of hippocampus but it seem that it fails to induce any effects in the of the PLC/PKC pathway, coupled to the MT2 receptor, could mediate the MEL effect basolateral amygdala as investigated by place preference paradigm. on the glycine currents of rat RGCs. (WJZ and MZ contributed equally to this work).
P1AM-12-2 P1AM-12-3 PROPERTIES OF NECK PROPRIOCEPTIVE CONTROLLING THE DOMINANT TIME CONSTANT RESPONSES OF PURSUIT NEURONS IN THE CAUDAL IN CONE PHOTORECEPTORS: THE ROLE OF Ca2+ PART OF THE FRONTAL EYE FIELDS (FEF) OF ALERT CONCENTRATION MONKEYS Jingjing Zang, Hugh R Matthews Hiroshi Saito, Teppei Akao, Sergei Kurkin, Kikuro Fukushima Physiological Laboratory,Department of Physiology, Development and Department of Physiology, Hokkaido University School of Medicine, Japan Neuroscience, University of Cambridge, UK The smooth-pursuit system moves the eyes accurately to track slowly moving In rods previous studies have indicated that the time constant that dominates 2+ objects. Normally, the head moves on the stationary trunk. Neck proprioceptive response recovery is governed by transducin shutoff and insensitive to Ca i. inputs provide information about head movement relative to the trunk. We In contrast, our recent results indicate that in salamander red-sensitive cones 2+ examined properties of 102 FEF horizontal pursuit neurons that responded to the shutoff of the phototransduction cascade is sensitive to Ca i and instead passive horizontal trunk rotation (±10°) on the stationary head when monkeys dominated by photopigment quenching. We used suction pipette recording and fixated a stationary spot in space. During sinusoidal trunk rotation at different rapid superfusion with 0Ca2+/0Na+ solution to progressively delay the light- 2+ frequencies (0.3-1.0Hz,±10°), the amplitude of discharge modulation increased induced fall of Ca i in salamander UV and blue-sensitive cones, and found 2+ as the peak trunk velocity increased. Velocity sensitivity calculated by linear that cascade quenching remained sensitive to Ca i throughout the response. regression ranged 0.29-0.57sp/s/°/s. During velocity step trunk rotation at 20 Furthermore, the dominant time constant for response termination was prolonged 2+ °/s, about half of FEF pursuit neurons tested (48%=25/52) exhibited only when Ca i was not allowed to fall. These results are similar to those obtained velocity-related discharge modulation, 12%(=6/52) exhibited only position- previously from red cones and indicate that dominance of response recovery related discharge, and 40%(=21/52) exhibited both. The shortest latency of neck by photopigment quenching is a general feature of cone phototransduction. responses to the onset of trunk rotation was 21 ms and the modal value was 75 Additionally, when the pH of the external solution was altered without allowing 2+ ms. Discharge modulation during smooth-pursuit and neck velocity responses Ca i to vary, the dominant time constant for red cone response recovery changed added linearly. Our results suggest that neck proprioceptive signals carry head in a graded manner, shortening with reduction and lengthening with increase in velocity relative to the trunk and contribute to modulation of FEF pursuit neurons pH. This observation suggests that red cone photopigment quenching may be during smooth-pursuit when the head moves on the trunk. speeded by the occupancy of a proton binding site. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 147 P1AM-13-1 P1AM-13-2 TASTE RESPONSIVENESS OF TYPE II AND III TASTE SODIUM CHLORIDE-INDUCED TASTE RESPONSES IN BUD CELLS IN MOUSE FUNGIFORM PAPILLAE MORPHOLOGICALLY IDENTIFIED FROG TASTE CELLS 1 1 1 1 Ryusuke Yoshida , Toshiaki Yasuo , Yoshihiro Murata , Masashi Jyotaki , Hideyuki Fukami, Kazuhisa Okuda-Akabane, Yasuyuki Kitada Yuchio Yanagawa2, Kunihiko Obata3, Hiroshi Ueno4, Sami Damak5, 6 1 Department of Oral Physiology, School of Dentistry, Iwate Medical Robert F. Margolskee , Yuzo Ninomiya University, Japan 1 Sect. of Oral Neurosci., Grad. Sch. of Dental Sci., Kyushu University, Japan, + 2 3 4 Depolarization by Na influx through cation channels leading to voltage- Grad Sch. of Med., Gumma Univ., Japan, RIKEN, Japan, Nara Women’s 2+ Univ., Japan, 5Nestle Research Center, Switzerland, 6Mount Sinai Sch. of Med, gated Ca currents has been thought to be involved in salt taste reception. USA However, the salt taste transduction has not fully been understood. We used 2+ Four types of cells, Type I ~III and basal cells, exist in a taste bud. Among them, Type patch pipettes filled with Ca -sensitive dye for calcium imaging and cell II cells express sweet, bitter and umami taste receptors and transduction components, type identification in slice preparations of bullfrog (Rana catesbeiana) taste suggesting that Type II cells may be taste receptor cells responsible for these taste discs. An isotonic solution of 110 mM NaCl + 2 mM NiCl2 (Na-Ni) was qualities. Type III cells possess putative sour taste receptors, suggesting that these cells used as a NaCl stimulating, because addition of NiCl2 to NaCl solutions has may be sour taste receptor cells. However, physiological responses of Type II and III been reported to enhance the neural responses to NaCl in the frog. The Na- cells to taste stimuli are not clear. In this study, we investigated response properties of Ni solution applied to the apical portion of taste cells elicited both inward the specified taste cells by using gustducin-GFP, TRPM5-GFP (Type II) and GAD67- 2+ currents and [Ca ]i increase in type III cells but not to other types of cells at GFP (Type III) mice. Both types of cells generated action potentials in response to a holding potential of -80 mV and in the Ca2+ free bath solution, suggesting taste stimuli, suggesting that these cells may transmit taste information to gustatory 2+ that the increase of [Ca ]i is due to release from intracellular stores. Only nerve fibers. Type II cells responded to sweet, bitter and umami stimuli whereas 2+ Type III cells responded to sour stimuli and electrolytes. Single cell RT-PCR analysis type III cells have been reported to have voltage-gated Ca channels and demonstrated that sweet sensitive cells expressed sweet taste receptor (T1r2/T1r3) make synaptic-like contacts with afferent axons. We speculate that a salty transduction uses a receptor-related second messenger pathway: release and sour sensitive cells expressed sour taste receptor (PKD2L1). These results suggest 2+ that both type II and Type III cells may be taste responsive cells that are devoted to of Ca from intracellular stores, cation channels activated by intracellular different taste qualities. Supported by KAKENHI 18077004, 18109013 (YN) and calcium (depolarization) and transmitter release via conventional synaptic 19791367 (RY). mechanism.
P1AM-13-3 P1AM-13-4 TASTE TRANSMISSION PATHWAY FOR BITTER TASTE ANION MODULATION OF SALT RESPONSES IN THE IN THE FROG TASTE DISCS: ANALYSIS OF QUININE- FROG GUSTATORY NERVE SENSITIVE FIBERS IN THE GLOSSOPHARYNGEAL Kazuhisa Okuda-Akabane, Hideyuki Fukami, Yasuyuki Kitada NERVE Department of Oral Physiology, School of Dentistry, Iwate Medical Kinya Narita, Yasuyuki Kitada University, Japan Department of Oral Physiology, Iwate Medical University, Japan Ca2+-sensitive fibers (Ca-fiber) of the frog glossopharyngeal nerve respond Vertebrate taste buds contain diverse types of cells. It has been shown that to Na-salts. Competitive inhibition of responses to Na-salts by Ca2+ has been some taste cells communicate with taste cells within taste buds via gap reported previously. This implies that there are specific receptors for Na+ and junctions or paracrine secretions. The cell-cell communications in taste buds that anions are not directly stimulatory. In this study, we examined anion would reflect neural responses. There are quinine-sensitive fibers (Q-fibers) modulation of Na+-induced responses in Ca-fibers in the bullfrog. Addition and calcium-sensitive fibers (Ca2+-fibers) in the frog glossopharyngeal 2+ of 1 mM NiCl2 to Na-salt solutions has been reported to enhance responses to nerve. Q-fibers show phasic responses to quinine-HCl and Ca -fibers Na-salts in Ca-fibers. Thus, we used stimulating solutions containing 1 mM show sustained responses to CaCl2. The different characteristics of the two 2+ NiCl throughout the experiment. The magnitude of responses to 100 mM responses suggest that Q-fibers and Ca -fibers innervate the different types 2 2+ NaCl (Stand-R) decreased with increasing concentrations of several Na-salts of taste cells. If quinine receptor cells communicate with Ca receptor cells, neural responses to quinine would be modulated by Ca2+ taste stimulation. In added to 100 mM NaCl. The order of effectiveness of anions in decreasing Stand-R was 5’-nucleotides >> glutamate > gluconate > sulfate. The results this study, we examined whether quinine responses of single Q-fibers in the + frog are altered by Ca2+ stimulation. Antidromic unitary impulses of Q-fibers suggest that these anions inhibited Na -induced responses. We found that Ca- 2+ fibers rarely responded to tetraethylammonium Cl (TEA-Cl). Thus, effect of and Ca -fibers of bullfrogs (Rana catesbeiana) were recorded from a single - fungiform papilla drawn into a suction electrode. Quinine responses (latency Cl was examined by addtion of TEA-Cl to 100 mM NaCl. The magnitude of responses and frequency of impulses) of single Q-fibers were not altered of Stand-R increased with increasing concentrations of TEA-Cl, suggesting by Ca2+ stimulation. The results suggest that there are no communications that Cl- has an enhancing effect on Na+-induced responses. It appears that between quinine receptor cells and Ca2+ receptor cells in taste organs. enhancement by Cl- is responsible for NaCl being the most potent stimulus.
P1AM-14-1 P1AM-14-2 IMPLICATION OF MEMBRANE RAFT IN RAT ACETYLCHOLINE AFFECTS THE SIGNAL ENCODING OLFACTORY SIGNAL TRANSDUCTION IN NEWT OLFACTORY RECEPTOR CELLS Atsushi Nakamura, Hiroyuki Takeda, Tadashi Nakamura Mahito Ohkuma, Fusao Kawai, Ei-ichi Miyachi Department of Applied Physics and Chemistry, The University of Electro- Department of Physiology, School of Medicine, Fujita Health University, Communications, Japan Japan Lipid rafts are detergent-resistant membrane microdomains enriched in cholesterol and sphingolipids. A variety of signaling components are The olfactory epithelium is innervated by efferent neurites and the olfactory localized to rafts, which are thought to function as coordinated signaling receptor cells express muscarinic receptors. These observations raise the platforms in cells. To investigate a role of the lipid rafts in olfactory signal possibility that acetylcholine could affect odor responses of the olfactory transduction, we examined localization of olfactory-specific G protein, receptor cells. Here we investigated the effect of acetylcholine on newt Golf and adenylate cyclase type III (ACIII) to lipid raft. Olfactory sensory olfactory receptor cells, using the whole-cell version of the patch-clamp cilia were prepared from rat olfactory epithelium by the calcium shock technique. Under current clamp condition, bath-applied 100 μM carbachol, method. After incubation of cilia suspensions with or without odorants, an agonist of acetylcholine receptor, lowered spike threshold from 5.3 ± 0.6 detergent-resistant membrane fractions (raft) were obtained by density to 3.8 ± 0.5 pA. Furthermore, the maximum spike frequency was increased gradient centrifugation. The localization of Golf and ACIII in the fractions from 9.1 ± 1.4 to 11.0 ± 1.3 spikes/s by carbachol. Under voltage clamp Poster Session was analyzed by immunoblot. Both of these molecules were found in the condition, carbachol increased the peak amplitudes of a voltage-gated T-type raft fraction prepared without stimulation with odorants. On the other hand, calcium current by 39% and that of a voltage-gated sodium current by 32%. stimulation with odorants caused the translocation of a significant portion of These results indicate that carbachol directly modulates spike generation in Golf to the non-raft fraction in spite of no noticeable change of distribution of ACIII. Taken together, we speculate that rafts provide platforms for the olfactory receptor cells. Because T-type calcium current is known to lower assembly of signaling complexes in olfactory transduction, and that the the threshold in olfactory receptor cells, we suggest that acetylcholine, which translocation of Golf from the raft induced by stimulation contributes to shut- is released from the efferent fibers, may enhance odorant sensitivity by down the olfactory response. lowering the threshold of spike generation in olfactory receptor cells.
148 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-14-3 P1AM-14-4 INSECT OLFACTORY RECEPTOR COMPLEX ARE Ca2+-ATPase AS A REGULATOR OF Ca2+ HOMEOSTASIS ODOR-GATED ION CHANNELS REGULATING IN VERTEBRATE OLFACTORY SENSORY NEURONS- RECEPTOR POTENTIALS OF OLFACTORY RECEPTOR TWO EXTRUSION MECHANISMS INSTEAD OF ONE? NEURONS 1 2 1 1 2 1 Salome Antolin , Joahnnes Reisert , Hugh R Matthews Koji Sato , Maurizio Pellegrino , Takao Nakagawa , 1 1 2 1 Department of Physiology, Development and Neuroscience, University of Tatsuro Nakagawa , Vosshall B. Leslie , Kazushige Touhara Cambridge, UK, 2Monell Chemical Senses Center, Philadelphia, USA 1Department of Integrated Biosciences, The University of Tokyo, Japan, 2 In olfactory sensory neurons (OSNs), the odour response culminates in the opening of Laboratory of Neurogenetics and Behavior, The Rockefeller University, 2+ 2+ USA cyclic nucleotide-gated channels, increasing ciliary [Ca ]i and opening excitatory Ca - activated Cl- channels which greatly augment the depolarising receptor current. When In vertebrates, binding of odorants to olfactory receptors (ORs) results in activation of [cAMP] is elevated by exposure of the cilia to the phosphodiesterase inhibitor IBMX, the G-protein/adenylyl cyclase signaling cascade. This in turn leads to the activation 2+ of cyclic-nucleotide gated channels and subsequently generates a depolarizing the decay of current upon its removal is governed by the extrusion of Ca and the 2+ - 2+ receptor potential. In insects, ORs consist of a heteromeric complex of unknown consequent closure of Ca -activated Cl channels. Hitherto, Ca extrusion has been stoichiometry but comprise at least one variable odorant-binding subunit and one thought to depend principally on Na+-Ca2+ exchange. By simultaneously recording 2+ insect ubiquitous Or83b family subunit. We demonstrated that insect ORs are ligand- the electrical response with a suction pipette and [Ca ]i with fluo-4, we show that in activated nonselective cation channels. Heterologous cells expressing silkmoth, fruitfly 2+ OSNs from the fire salamander the recovery of the response is only modestly affected or mosquito heteromeric OR complexes showed extracellular Ca influx and cation- by the removal of external Na+ but instead is considerably prolonged by vanadate and non-selective ion conductance on stimulation with an odorant. Odor-evoked OR carboxyeosin, blockers of Ca2+-ATPase. These results provide functional evidence currents are independent of known G-protein-coupled second messenger pathways. + 2+ Direct evidence for odorant-gated channels was obtained by outside-out patch-clamp for the existence of an additional Na -independent Ca removal mechanism, which recording. Stimulation with particular odorants resulted in either increase or decrease instead plays the dominant role in response termination in this species. This is in in the spontaneous channel open probability. The various open probabilities of odor- stark contrast to the situation in frog and mouse, in which the contribution of such + + 2+ gated ion channels appear to account for the bipolar properties of insect olfactory a Na -independent mechanism to response recovery when Na -Ca exchange is receptor neurons. incapacitated is very limited.
P1AM-14-5 P1AM-14-6 SINGLE NEURON RESPONSES TO PERIPHERAL REGULATION OF OLFACTORY SYSTEMS BY X11 PRESENTATION OF ODORANTS IN MAIN OLFACTORY PROTEIN BULB AND ANTERIOR PIRIFORM CORTEX OF Makoto Kashiwayanagi1, Takeshi Osako2, Takashi Narukawa2, 2 1 1 ANESTHETIZED RATS Yuhki Saito , Naoya Kamiyama , Tomohiro Noguchi , 2 Hyun Joo Lee1, Hyung Geol Ham1, Xue Mei Jin1, Yiran Lang1, Toshihara Suzuki 1 Julia Komissarova1, Jaewon Yu2, Seop Hyeong Park2, Hyung-Cheul Shin1 Department of Sensory Physiology, Asahikawa Medical College, Japan, 2Graduate School of Pharmaceutical Sciences, Hokkaido University, Japan 1Department of Physiology, College of Medicine. Hallym University, Korea, 2College of Information & Electronic Engineering, Hallym University, Korea X11 is a neuronal adaptor protein suppressing the generation of amyloid-protein. Multi-channel single unit recordings were done in the main olfactory bulb (MOB) and the A decrease in the ability of olfactory function is one of typical pothogeneis of anterior piriform cortex (aPC) of SD rats to determine neural responses to various odorants. Alzheimer disease. X11 protein has been shown to be expressed in the olfactory Methyl methacrylate monomer (MMA) and rat's urine were used as odorants and they were bulb and accessory olfactory bulb. Pheromones contained in urine induce presented in front of rat's nose for varying durations with a retractable cotton probe. Averaged excitation of vomeronasal sensory neurons and neurons in the accessory olfactory activity of the MOB units (n=263) was 12.9+0.5Hz. Spontaneous responses were facilitated bulb (Inamura et al., 1999a, 1999b). In the present study, we explored Fos- by 7.8±2.7% (n=94, above ±25%: n=7, below -25:n=5) with rat's urine and they were immunoreactive (Fos-ir) structures in the AOB of X-11 deficient mice after the activated by 25.8±5.20% (n=116, above +25%:n=52, below -25%:n=16) with MMA. Cotton vomeronasal organ was exposed to conspecific urine to explore the role of X11 probe itself did not change the MOB activity (1.4±1.6%,n=56). Averaged activity of the aPC in the olfactory system. Exposure of the vomeronasal organ of female mice to units (n=207) was 16.4±0.8Hz. Spontaneous response of aPC was altered by 3.1±1.1% (n=98, male urine led to the appearance of many Fos-ir cells in the AOB. It is expected above +25%:n=4) with rat's urine and they were activated by 4.8±1.6% (n=70, above +25%: that responses of X11-deficient mice to urinary pheromone is weaker than those n=7) with MMA. Cotton probe itself did not change the aPC activity (0.11±0.9%,n=98). The of wild mice because amyloid proteins accumulate in the brain. However, the results suggest that strategy of encoding odorants in the MOB might be different from that density of Fos-ir cells in the AOB after exposure of X11-deficient female to male in the aPC of SD rats. This study was supported by grants to HCSHIN(Hallym Univ.-2008, urine was higher than that after exposure of wild females. These results suggest KRF-8R07-0201-064-S000100, MEST-M103KV010023-08K2201-02310, KOSEF-R11-20 that X11 protein regulate olfactory function not only via amyloid-protein but also 00-075-01003-02008). via unknown pathways.
P1AM-15-1 P1AM-15-2 CONTRIBUTION OF Ca2+ PERMEABLE AMPA VOLTAGE FLUCTUATIONS OF CALCIUM STORE IN 2+ RETINAL NEUROEPITHELIUM RECEPTORS TO DENDRITIC Ca SIGNALS IN Masayuki Yamashita MIDBRAIN DOPAMINE NEURONS Department of Physiology 1, Nara Medical University, Japan Jin Young Jang, Myoung Kyu Park Department of Physiology, Sungkyunkwan University, Korea Synchronous Ca oscillation occurs in neuroepithelium. However, the Glutamate is a major excitatory neurotransmitter in the midbrain dopamine mechanism for synchronization of Ca increases between cells remains neurons which activates AMPA and NMDA receptors. Dendrites of midbrain unclear. Recently, synchronous oscillatory changes in the membrane dopamine neurons have rare spines and thus do not possess a clear morphological potential of intracellular Ca sores were recorded using an organelle-specific basis for synapse-specific compartmentalization. Therefore, in aspiny dendrites voltage-sensitive dye, and a capacitative electrical coupling model has of the dopamine neurons, Ca2+-permeable AMPA receptors (CP-AMPAR) might been proposed (Yamashita, 2006). Electrophysiological and Ca-sensitive play a major role in input-specific synaptic plasticity by confining calcium fluorescence measurements in the neuroepithelium of embryonic chick retina events within a microdomain. However, little is known about Ca2+ signals and showed that synchronous Ca oscillation was caused by releases of Ca from roles of CP-AMPA receptors in dendrites of the midbrain dopamine neurons. 2+ Ca stores without any evidence of action potentials in neuroepithelial cells or Therefore, we investigated expression of CP-AMPAR and Ca signals in the newborn neurons. High-speed fluorescence measurement of store membrane acutely dissociated dopamine neurons from the rat midbrain. Single cell RT- potential surprisingly revealed that the synchronous oscillatory changes in PCR experiments showed that all mRNAs for GluR1, 2, 3 and 4 subtypes were expressed in the dopamine neurons. Application of glutamate and AMPA the store potential were periodic repeats of a burst of high-frequency voltage evoked inward currents at -60 mV membrane potential together with increases fluctuations. The burst coincided with a Ca increase. The present study in cytosolic Ca2+ levels at dendrites, which were significantly inhibited by a suggests that synchronization of Ca release is mediated by the high-frequency specific CP-AMPAR blocker, Philanthotoxin. From these results, we conclude fluctuation in the store potential. Close apposition of the store membrane that, contribution of CP-AMPA receptors to the glutamate-induced currents and and plasma membrane in an epithelial structure would allow capacitative Ca2+ influx along a dendrite increases with distance from the soma in the SNc coupling across the cells. dopamine neurons. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 149 P1AM-15-3 P1AM-15-4 REGULATION OF SPONTANEOUS FIRING ACTIVITIES EFFECTS OF INTRACELLULAR Ca2+ LEVEL ON BY DENDRITIC LOCAL Ca2+ EVENTS IN THE CHANNEL MODULATION INDUCED BY ACTIVATION MIDBRAIN DOPAMINE NEURONS OF MUSCARINIC RECEPTORS IN RAT CULTURED Shin Hye Kim, Ki Bum Um, Myoung Kyu Park HIPPOCAMPAL NEURONS Department of Physiology, Sungkyunkwan University, Korea Takako Ohno-Shosaku, Ryousuke Echigo, Kohsuke Kashima, Midbrain dopamine neurons have multiple dendrites in which local and Hanae Minami, Miho Muranishi, Yuiko Nishikawa, Hiro-omi global Ca2+ events occur. Although it was known that dendritic Ca2+ signals Taguchi, Megumi Watanabe play critical roles in generating excitability in the midbrain dopamine Department of Impairment Study, Graduate School of Medical Science, neurons, their regulatory mechanisms of spontaneous firing are not clear Kanazawa University, Japan 2+ yet. Therefore, we investigated how Ca changes in dendrites regulate Recent studies revealed that the activation of PLCβ driven by Gq-coupled receptors is spontaneous firing and determine firing patterns in the midbrain dopamine dependent on intracellular Ca2+ concentration in a physiological range. It is then expected neurons. that postsynaptic responses evoked by the receptor-PLCβ signaling should be dependent 2+ on the postsynaptic Ca2+ level. To test this possibility, we examined effects of changing In spontaneously firing dopamine neurons, the basal [Ca ]c level was correlated with 2+ the firing, indicating that spontaneous firing is an important source for continuous the Ca concentration of pipette solutions on channel modulation induced by activation of 2+ 2+ 2+ muscarinic receptors in rat cultured hippocampal neurons, using the patch-clamp whole-cell Ca influx. Gradual or stepwise increases in [Ca ]c in soma by uncaging caged Ca compound NP-EGTA proportionally decreased the spontaneous firing rate, indicating method. Application of the muscarinic agonist oxotremorine-M induced depolarization in 2+ most of the neurons. We found that this depolarization was caused by suppression of certain that Ca levels are inversely correlated with firing rates and patterns. Local uncaging + types of K channels and activation of non-selective cation channels. These modulations of of NP-EGTA in the limited areas of the proximal or distal dendrites also increased 2+ ion channels were suppressed by the treatment with the PLC inhibitor U73122. When the Ca in the uncaged area, inhibited spontaneous firings, and hyperpolarized membrane Ca2+ concentration of the pipette solution was decreased from pCa7 to pCa9, the both types potential. The inhibition was not weaker when uncaging area were moved along the of modulations were attenuated, at least when the neurons were exposed to nominally Ca2+- 2+ direction of the distal dendrite. From these data, we conclude that distal dendritic Ca free external solutions. From these results, we concluded that the PLC-dependent channel
events are enough signals to regulate spontaneous firings in the midbrain dopamine modulations driven by muscarinic receptors, probably M1 and M3 receptors, are actually neurons. Ca2+-dependent in hippocampal neurons.
P1AM-15-5 P1AM-15-6 PROPERTIES OF THE NEURAL AND GLIAL COINCIDENCE DETECTION OF PRE- AND SPONTANEOUS CALCIUM TRANSIENTS IN STRIATUM POSTSYNAPTIC ACTIVITY BY Ca2+ SENSOR PROTEIN Makoto Osanai, Yuichi Yaguchi, Naohiro Yamada, Tetsuya Yagi HIPPOCALCIN Division of Electrical, Electronic and Information Engineering, Graduate Pavel Belan1, Volodymir Cherkas1, Alexandr Dovgan1, School of Engineering, Osaka University, Japan Daniel Fitzgerald2, Alexie Tepikin2, Robert D Burgoyne2 The striatum plays an important role in linking cortical activity to basal 1 2+ Department of General Physiology of Nervous System, Bogomoletz ganglia outputs. We conducted the Ca imaging study on the striatal slice of Institute of Physiology, Ukraine, 2Physiol., Univ. of Liverpool, Liverpool, UK GFAP-GFP mice, which expressed GFP in astrocytes. 2+ Hippocalcin (HPCA) is a calcium sensor protein that is involved in many signal Long lasting spontaneous [Ca ] transients, which lasted up to about 300 i transduction pathways in hippocampal neurons. At the same time biophysical s, were observed in both GFP positive cells (astrocytes) and GFP negative mechanisms of HPCA signaling underlying these pathways have not been profoundly cells (putative neurons). Some properties of the individual spontaneous 2+ studied. In this work we have examined if synaptic glutamate receptor activation can [Ca ]i transients in putative neurons were different from those of astrocytes. result in HPCA signaling in a neuronal dendritic tree. Thapsigargin, an intracellular Ca2+ store depletor, greatly reduced the 2+ 2+ Trains of presynaptic stimuli induced excitatory postsynaptic potentials (EPSPs) in [Ca ]i transients in both neurons and astrocytes. Therefore, these [Ca ]i 2+ 2+ postsynaptic neurons leading to fast (1-4 s) HPCA-YFP translocation to many sites in transients were mainly due to Ca release from an intracellular Ca store. a neuronal dendritic tree. The translocation took place in all-or-none fashion depending 2+ 2+ Administration of tetrodotoxin (TTX) blocked little the [Ca ]i transients on the number of stimuli in a train. We have shown that Ca influx via synaptic 2+ 2+ in the both types of cells, but the some parameters of the individual [Ca ]i NMDARs in which Mg block is relieved by membrane depolarization is a main transients in neurons (not in astrocytes), were altered by TTX administration. reason for HPCA-YFP translocation. Subthreshold presynaptic stimulation and series 2+ Thus, action potential related process might be concern with the [Ca ]i of postsynaptically induced action potentials applied separately did not result in any transients in neurons but not in astrocytes. Dopamine or dopamine receptor HPCA-YFP translocation whereas their simultaneous application evoked the robust 2+ agonists blocked the spontaneous [Ca ]i transients in the both types of cells. HPCA-YFP translocation. Thus, we have shown that hippocalcin may serve as a Therefore, the dopamine signal might contribute to the expression of the coincidence detector in dendrites of hippocampal neurons decoding simultaneous pre- 2+ [Ca ]i transients. and postsynaptic activity, into specific translocation to its plasma membrane targets.
P1AM-15-7 P1AM-15-8 CALCIUM- AND HIPPOCALCIN-DEPENDENT Arf1 AND SUBSEQUENT PLD REGULATES + MEMBRANE TRANSFER OF CREATINE KINASE B RECYCLING OF D2 RECEPTOR COUPLED WITH K - Masaaki Kobayashi, Ken Takamatsu CURRENT RESPONSE IN THE GANGLION CELLS OF Department of Physiology, Toho University School of Medicine, Japan APLYSIA Jin Ochiai1, Satoshi Kawasaki1, Shingo Kimura1, Reiko Fujita2, Hippocalcin (Hpca) is a member of the neuronal calcium sensor family 1 predominantly expressed in the hippocampal neurons and acts as a Kazuhiko Sasaki 1Department of Physiology, School of Medicine, Iwate Medical University, multifunctional modulator in the calcium-signaling underlying memory 2 Japan, Department of Chemistry, Center for Liberal Arts & Science, Iwate formation. We have previously identified creatine kinase (CK)-B subunit as Medical University, Japan a Hpca-associated protein in the rat brain. Here we characterized binding Recently, small G-protein ARf family and their effector phospholipase D (PLD) are profile between Hpca and CK-B. Immunoprecipitation study revealed that shown to regulate rcycling and endocytosis of the receptors in addition to regulation of recombinant Hpca associated with CK-B in the presence of calcium. Filter vesicular trafficking between the intracellular organelles. Using isolated Aplysia neurons, overlay method using biotinylated CK-B demonstrated that CK-B bound with we examined possible involvement of recycling of D2 dopamine (DA) receptor to the Hpca responding to increase in free calcium concentration in range of 0.5-30 plasma membrane after endocytosis in regulation of the DA-induced K+ current response. μM in vitro. Myristoylation of Hpca was not required for its association with Intracellular application of N-terminal peptides of the Arf1 but not Arf6 significantly suppressed the K+ current response to DA. Either intracellular application of α -synuclein Poster Session CK-B, but required for calcium-dependent membrane transfer of CK-B. + Calcium sensitivity of the membrane transfer of CK-B was examined in the or extracellular application of 1-butanol, inhibitors of PLD, markedly depressed the K current response to DA. Intracellular application of dynamin inhibitory peptide gradually presence of Hpca (10 μM) and half-maximal translocation was observed at + about 5 M free calcium in vitro. The calcium-dependent membrane transfer augmented the DA-induced K current response. On the other hand, extracellular application of monensin, an inhibitor of vesicular recycling, gradually suppressed the DA-induced of CK-B was diminished in the hippocampus of Hpca-deficient mice. These K+ current response. Furthermore, effect of monensin occluded the effect of α-synuclein results indicate that Hpca associates with CK-B and transfers it to the plasma when examined after the application of monensin. These results suggest that Arf1 and PLD membrane in a calcium-dependent manner, suggesting that Hpca plays an regulate the DA-induced K+ current response through recycling of the DA receptor to the important role in local energy shuttling system. plasma membrane.
150 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-15-9 P1AM-15-10 CONTRIBUTION OF CALCIUM-INDUCED CALCIUM ACTIVATION OF THE Na+/H+ EXCHANGER-1 BY RELEASE FOR PROPAGATION OF γPKC EXTRACELLULAR SIGNAL-RELATED KINASE DURING TRANSLOCATION ALONG THE DENDRITES OF GLUTAMATE EXCITOTOXICITY CEREBELLAR PURKINJE CELL Bo Kyung Lee1, Sunkyung Lee2, Kyu Yang Yi2, Sung Eun Yoo2, Norihiro Katayama1, Norio Sakai2, Naoaki Saito3, Kyung Hee Lee3, You-Sun Kim3, Soo Hwan Lee1, Eun Joo Baik1, 1 4 Mitsuyuki Nakao1, Hiroshi Tsubokawa4 Chang-Hyun Moon , Yi-Sook Jung 1 1Department of Physiology and Brain Korea 21 for Medical Science, Ajou Graduate School of Information Sciences, Tohoku University, Japan, 2 2 3 University School of Medicine, Korea, Medical Science Division, Korea Grad Schl Biomed Sci, Hiroshima Univ, Japan, Biosignal Res Ctr, Kobe 3 4 Research Institute of Chemical Technology, Daejon, Korea, Research Center, Univ, Japan, Fucul Health Sci, Tohoku Fukushi Univ, Japan 4 Yuyu Inc. GyeongGi Bio-Center, Suwon, Korea, Department of Physiology and Protein kinase C (PKC) plays important role in controlling neuronal Brain Korea 21 for Molecular Science and Technology Ajou University, Korea excitability. However, little is known about spatiotemporal profiles of PKC Na+/H+ exchanger-1 (NHE-1) activity plays an important role in neuronal damage in both in vitro activation in CNS neurons. To address this issue, we have generated brain- and in vivo ischemic models. Ischemic neuronal death caused by excitotoxic levels of glutamate region specific and inducible γPKC-GFP transgenic mice using tetracyclin- is dependent on mitochondrial Ca2+ influx via glutamate receptor and NHE-1 activation, however regulated system, and found that in cerebellar Purkinje cells electrical the regulation of NHE-1 following glutamate excitotoxicity are not well understood. In the present study, we demonstrated that extracellular signal-related kinases (ERK) play a role in stimulation stimulation of parallel fibers evoked not only local translocation but also of NHE-1 induced by glutamate. The expression of p-ERK was significantly increased during slow propagation of the translocation along dendritic arbor. Calcium glutamate exposure and it was blocked by MEK inhibitor U0126. U0126 abolished phosphorylation imaging studies have shown that the parallel fiber stimulation also induces and activation of NHE-1 induced by glutamate in cultured neurons. Moreover, neuroprotection slowly propagating calcium wave, which could lead propagation of PKC was observed with U0126 or KR-33028, a potent NHE-1 inhibitor following glutamate translocation. To understand mechanism underlying the PKC wave, we excitotoxicity. Our results suggest that ERK is intimately involved in regulation of the NHE-1 induced by glutamate, possibly through serine phosphorylation of the carboxyl-terminal cytosolic constructed a compartmental model of Purkinje cell with dendrites. Computer domain, which subsequently contributes to neuronal damage. This work was supported by a grant simulations suggest the important contribution of calcium-induced calcium (CBM31-B3003-01-02-00) from the Center for Biological Modulators of the 21C Frontier R&D release system for the propagation of calcium wave along dendrites. Program of the Ministry of Science and Technology, and a grant Yuyu Inc.
P1AM-15-11 P1AM-15-12 LONG-TERM HYPERPORALIZATION INDUCES RATE AND TEMPORAL NEURAL CODES IN DEEP ABRUPT INCREASE IN CURRENT INFLUX AND CEREBELLAR NEURONS TRANSLATE PURKINJE FLUCTUATION IN HIPPOCAMPAL CA1 PYRAMIDAL CELL INHIBITORY INPUT NEURONS REZA TADAYON NEJAD, W. Hamish Mehaffey, Hiroyuki Ohta, Yoshiaki Sato, Kazuo Kato, Takehito Kemuriyama, Kusala M. Jayasuriya, Ray W. Turner Megumi Tandai-Hiruma, Satoshi Maruyama, Yasuhiro Nishida Department of Neuroscience, University of Calgary, Canada Department of Physiology, National Defense Medical College, Japan The DCN is a critical motor center responsible for translating Purkinje cell Previous studies indicate that hyperpolarization causes smaller fluctuation of inhibitory input into final spike patterns appropriate for control of movement. membrane current. We, however, observed that a prolonged hyperpolarization (step Large diameter DCN cells exhibit either a Weak Burst (WB) or Transient Burst or triangle, longer than 100sec) induced abrupt increases of inward current (more than (TB) phenotype. Here we identify how DCN cells use rebound burst properties 100 pA) and time-varying current fluctuation (more than 5 times, peek-to-peek). To to encode Purkinje cell inhibitory input in the in vitro rat DCN slice preparation investigate the mechanisms for the long-term hyperpolarization-induced current influx in response to 100 Hz trains of 5-30 stimuli. Rebound discharge was quantified in and fluctuation (LHIF), we measured the current of rat hippocampal CA1 pyramidal terms of frequency, duration, number of spikes, and first spike latency. Identical neurons in normal artificial cerebrospinal fluid (aCSF) using the whole-cell patch- trains of Purkinje cell input evoked markedly different responses in TB and WB clamp method in voltage-clamp mode. Frequencies of emergence of LHIF increased as the hyperporalization became deeper or longer. A short term Fourier transformation cells. Stimulus trains evoked a rebound burst in TB cells only after a minimal analysis showed the remarkable spatio-temporal properties of the fluctuations in the number or intensity of IPSP while WB cells responded with a graded increase low frequency domain. The application of 100 nM TTX and calcium-free aCSF did and lower frequency of rebound. Both cells displayed an equivalent and graded not show any significant effects for the emergence of LHIF. On the other hand, 1 mM shift in first spike latency in relation to stimulus number or intensity. cesium and 0.1 mM carbachol enhanced or depressed LHIF. These results indicate that These results suggest that TB cells can exhibit feature detection to a specific long-term hyperpolarization probably modulate some voltage-dependent ion channels threshold of inhibition while WB cell rebound discharge indicates a role in rate to induce LHIF. We consider that these mechanisms jack up a membrane voltage of an coding. Both cells exhibit temporal coding through first spike latency shifts, excessively hyperpolarized neuron. potentially contributing to the known timing function of cerebellar processing.
P1AM-15-13 P1AM-15-14 EFFECTS OF TRPV1 AND TRPV4 ON LPS-INDUCED JNK PHOSPHORYLATION OF Hes1 STABILIZES Hes1 2+ FEVER AND THE CONTENT OF cAMP AND [Ca ]i IN AND POTENTIATES ITS REPRESSION ACTIVITY ON RAT HYPOTHALAMUS GluR1 TRANSCRIPTION IN CULTURED CORTICAL Yu Cao, Lan Lan Wang, Le Wang, Xin Qin NEURONS 1 2 Department of Physiology, China Medical University, China Cheng-Hsiung Lin , Eminy Lee 1Graduate Institute of Life Sciences, National Defense Medical Center, Aim: To explore the effects of TRPV1 and TRPV4 on the febrile response in 2 ras, and their possible central mechanisms in thermoregulation. Methods: We Taiwan, Division of Neuroscience, Institute of Biomedical Sciences, made the fever model with lipopolysaccharide in rats. Capsazepine(CPZ), the Academia Sinica, Taiwan antagonist of TRPV1 and ruthenium red (RR), the antagonist of TRPV4 were The glutamate receptor subunit GluR1 plays an important role in excitatory synaptic used respectively. Then changes in body temperature(∆T)were continually transmission in neurons. In analyzing the 2kb GluR1 promoter, we found seven N 2+ boxes and four E boxes in rat GluR1 promoter. Hairy and Enhancer of Split1 (Hes1) observed, and the expression of TRPV4, content of cAMP and [Ca ]i in hypothalamus were measured by molecular biology techniques. Result: is a mammalian transcription repressor that regulates neuronal differentiation by Compared with LPS group, the amplitude of ∆T decreased in CPZ+LPS binding to N box or preventing other transcription factors from binding to E box. To 2+ examine whether Hes1 regulates GluR1 promoter activity through N box and E box, group. The content of cAMP was the higest and [Ca ]i lowest in CPZ+LPS group compared with other three groups(LPS, CPZ and control group) at Hes1 plasmid and GluR1 promoter construct were co-transfected to cultured cortical neurons. Results showed that Hes1 could bind these N boxes and repress GluR1 the peak of fever. While in RR+LPS group, the amplitude of ∆T and content 2+ promoter activity by about 25-fold. Interestingly, the regulation of Hes1 expression in of cAMP and [Ca ]i all decreased. And the expression of TRPV4 in RR cortical neurons is Notch-independent but c-Jun N-terminal kinase (JNK)-dependent. and RR+LPS group were lower than that of control group. Given RR solely Activation of JNK increased Hes1 protein level and decreased GluR1 mRNA and decreased the temperature. Conclusion: TRPV1 may be involved in LPS- 2+ protein level. However, the JNK inhibitor, sp600125, decreased Hes1 protein level induced fever by changing [Ca ]ii in hypothalamus to limit the increasing but enhanced GluR1 protein level. Further, JNK could interact with Hes1 and amplitude of ∆T; and TRPV4 may participate in the maintenance of phosphorylate Hes1 directly. JNK Phosphorylation of Hes1 increased the stability normothermia. of Hes1 and potentiated its repression ability. Our results indicate that JNK regulates 2+ Poster Session Key Words:fever; TRPV1; TRPV4; LPS; cAMP; [Ca ]i GluR1 promoter activity through phosphorylation and stabilization of Hes1.
IUPS 2009 July 27 - August 1, 2009 in Kyoto 151 P1AM-15-15 P1AM-15-16 DIFFERENTIAL RECOVERY OF BACKGROUND CHANGES IN MOVEMENT RELATED CORTICAL AND EVOKED AFFERENT ACTIVITY IN THE FROG NEUROMAGNETIC FIELDS EVOKED BY FINGER SEMICIRCULAR CANAL FOLLOWING OTOTOXIC VOLUNTARY MOVEMENT Toshio Soma1, Hideaki Onishi1, Mineo Oyama2, Makoto Oishi3, INSULT 4 Ivo Prigioni, Donatella Contini, Giancarlo Russo Kameyana Shigeki 1Department of Physical Therapy, Niigata University of Health and Welfare, Department of Physiology, University of Pavia, Italy Japan, 2Department of Occupational Therapy, Niigata University of Health The present study was aimed to define the damage and recovery of pre- and and Welfare, Japan, 3Niigata University Medical and Dental Hospital, postsynaptic activity in the frog crista ampullaris after ototoxic insult with gentamicin Japan, 4National Nishi-Niigata Central Hospital, Japan (GM). We found that the endoampullar potential (receptor potential) recorded from PURPOSE: The purpose of this study is to define the positional relations of somatotopic the isolated canal was abolished 6 days after GM administration and that it recovered organizations between the movement related cortical fields (MRCFs) during voluntary close to the control 20 days after treatment. Consistently with the presynaptic damage, movements and the somatosensory evoked fields (SEFs) of the dominant nerves that control the afferent activity from the ampullar nerve was abolished 6 days after GM treatment, the muscle movements. METHODS: After obtained informed consent, ten healthy volunteers participated in this while the recovery of background and evoked afferent activity showed a different present study. MRCFs and SEFs data were recorded using a 306-channel whole head MEG sequence. Background activity reappeared 7-8 days after GM treatment and in this system (Neuromag, Elekta) in a magnetically shielded room. MRCFs were measured during period it was not driven by canal mechanical stimulation. In addition, background three types of voluntary movement of the right fingers: index finger flexion (Index-flex), activity reached control values around 15 days after the ototoxic insult, while the index finger extension (Index-ext) and little finger abduction (Little-ab). The median, radial evoked activity was detected 9-10 days after GM treatment and appeared normal close and ulna nerves were electrically stimulated to measure the SEFs, and the positional relations to 20 days. Intracellular recordings from single canal afferents confirmed the above between MRCFs and SEFs were investigated. RESULTS: In all subject, the position of equivalent current dipole showed the reference mentioned results. The present study show that the frog semicircular canal rapidly and which Index-flex and median nerve, Index-ext and radial nerve, Little-ab and ulna nerve completely recovers its pre -and postsynaptic function following severe ototoxic insult coped with each. and that during the recovery afferent transmission at rest becomes functional before CONCLUSION: It was found that the somatotopic organizations of MRCFs and SEFs have the mechano-transduction function. approximate positional relations within each subject.
P1AM-15-17 P1AM-16-1 IMAGING STEREOSPECIFIC UPTAKE OF GLUCOSE BDNF IS A NOVEL ADIPOKINE THAT SUPPRESSES USING FLUORESCENT D- AND L-GLUCOSE PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) MRNA ANALOGUES EXPRESSION IN ADIPOCYTES THROUGH TRKB-T1 Katsuya Yamada1, Toshihiro Yamamoto2, RECEPTOR Seiji Watanabe3, Yuji Nishiuchi2, Tadashi Teshima2, Hideaki Matsuoka4, Suni Inoue-Lee, Atsushi Suzuki, Chitoku Toda, Maya Esaki, Yusuke Fujino, Sechiko Suga5 Kumiko Saito, Tetsuya Shiuchi, Shiki Okamoto, Yasuhiko Minokoshi 1Department of Physiolgy, Hirosaki Universtiy Graduate School of Medicine, Dept. Developmental Physiology, National Institute for Physiological Sciences, Japan, 2Saito Research Center, Peptide Institute, Inc. Japan, 3Department Japan of Anatomical Science, Hirosaki University Graduate School of Medicine, Brain-derived neurotrophic factor (BDNF) plays pivotal roles in brain development and Japan, 4Department of Biotechnology and Life Science, Faculty of Technology, 5 neuronal plasticity. Recent studies further revealed that BDNF regulates whole body Tokyo University of Agriculture and Technology, Japan, Center of Research & Education for Lifelong Learning, Hirosaki University, Japan energy metabolism through the hypothalamus-sympathetic nervous axis. However, the regulatory role of BDNF in peripheral tissues remains unclear. Here we report that An essential sugar, D-glucose is one of the most important energy sources for the survival BDNF and its receptor TrkB-T1 express in matured adipocytes of mice and suppress of various organisms, from E. coli to mammals. For many years, glucose transport activity 14 mRNA expression of PAI-1, an adipocytokine which exacerbates atheroscrelogenesis. has been monitored by radiolabeled tracers such as [ C] 2-deoxy-D-glucose. However, they BDNF also suppressed PAI-1 mRNA expression in 3T3-L1 adipocytes. We identified cannot be used for time-lapse monitoring of glucose uptake at the single-cell level due to their poor spatial and temporal resolution. To overcome the difficulty, a fluorescent D-glucose that BDNF-induced suppression of PAI-1 mRNA was mediated by both PI3-K/Akt/ derivative 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose [2-NBDG] FoxO1 and MEK/Erk/S6K/FoxO1 pathways via TrkB-T1, by using specific inhibitors has been widely used, but no strong control substance for evaluating 2-NBDG uptake or siRNA for the signaling molecules. ChIP analysis also revealed that Foxo1-binding has been available. Recently, we synthesized a new fluorescent derivative of 2-deoxy-L- to the promoter region of PAI-1 gene decreased in response to BDNF. In obese mice glucose, 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-L-glucose [2-NBDLG]. (ob/ob and diet-induced obese mice), BDNF expression increased in WAT, but PAI-1 Some other derivatives have been developed as well. We show here effectiveness of using mRNA expression was enormously high. BDNF-induced phosphorylation of Akt, Erk such transporter-recognizable (D-isomer) and unrecognizable (L-isomer) analogues in and FoxO1 significantly reduced in WAT of obese mice. These results indicate BDNF combination for evaluating stereospecific transport of glucose in live cells. These methods is a novel adipokine that regulates PAI-1 mRNA expression in adipocytes in normal should also provide valuable information on the ligand-transporter interactions. mice, whereas the response weakens and BDNF resistance occurs in obese mice.
P1AM-16-2 P1AM-16-3 cAMP IS IMPORTANT TO NERVE GROWTH FACTOR- CALCITONIN GENE-RELATED PEPTIDE- AND INDUCED DIFFERENTIATION OF PC-12 CELLS ADRENOMEDULLIN-INDUCED FACILITATION OF Mei-Chih Chen1, Guan-Shun Lee2, Paulus S. Wang1, Ho Lin2 CALCIUM CURRENT IN SUBMANDIBULAR GANGLION 1Physiology, National Yang-Ming University, Taiwan, 2Department of Life Takayuki Endoh, Yoshiyuki Shibukawa, Tetsu Yamamoto, Sciences, National Chung Hsing University, Taichung, Taiwan Masakazu Tazaki It had been reported that Cdk5 and p35 are important factors in the Department of Physiology, Tokyo Dental College, Japan development of central nervous system and in neurodegeneration. In the field The control of saliva secretion is mainly under parasympathetic control. of neurobiology, PC-12 cell line is a popular tool to investigate neuronal The submandibular ganglion (SMG) is a parasympathetic ganglion which differentiation. The induction of p35 expression and subsequent Cdk5 receives inputs from preganglionic cholinergic neurons, and innervates the activation triggered by nerve-growth factor (NGF) have been found as a submandibular salivary gland. The calcitonin gene-related peptide (CGRP) major cause of PC-12 differentiation. Our findings indicated that cAMP is a 37-amino-acid peptide that was initially described to be generated was also able to induce p35 protein expression in PC-12 cells. Interestingly, by alternative splicing of calcitonin gene. Adrenomedullin (ADM) is a 52-amino-acid peptide originally isolated from a human pheochromocytoma. the intracellular accumulation of cAMP was found after NGF treatment. It is structurally and functionally related to the CGRP, amylin peptide family. The NGF-dependent increase of p35 protein expression was enhanced by The purpose of this study was to investigate the effects of CGRP and ADM
Poster Session cAMP and blocked by cAMP antagonist, and both the NGF-induced p35- on voltage-dependent calcium current (I ) in the SMG. Application of CGRP upregulation and PC-12 differentiation were prevented by incubation with Ba caused facilitation of IBa in a concentration-dependent manner. Convergence adenylyl cyclase inhibitor. In addition, the involvement of cAMP in the NGF- or nonadditivity between CGRP and ADM pathways was investigated by induced decrease in proliferation and increase in G1 phase accumulation the co-application of agonists and by comparing the facilitation of IBa with imply that NGF triggers differentiation through cAMP production. Taken that produced by each of these agonists alone. The mean facilitation of IBa together, our data provide evidence indicating that cAMP is involved in NGF- induced by CGRP was unaltered by its coapplication with ADM, suggesting dependent pathway and possibly plays a vital role in neuronal differentiation. that the actions of CGRP and ADM are not additive, and further suggesting convergence in these facilitation pathways.
152 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-16-4 P1AM-16-5 ELECTROPHYSIOLOGICAL EFFECTS OF GHRELIN MODULATION OF HIPOCAMPAL SYNAPTIC AND SOMATOSTATIN ON HYPOTHALAMIC ARCUATE CURRENTS AND INTRACELLULAR Ca2+ LEVELS BY NUCLEUS NEURONS IN INFANTILE RATS: AN IN SOMATOSTATIN ANALOGUES VITRO STUDY Karoly Orban-Kis1, Karoly Antal2, Agnes Simon2, Gyorgy Keri3, Kyohei Mori, Juhyon Kim, Kazuki Nakajima, Kazuo Sasaki Tibor Szilagyi1, Julianna Kardos2, Zsuzsa Emri2 Division of Bio-Information Engineering, University of Toyama, Japan 1Department of Physiology, University of Medicine and Pharmacy Targu Mures, Roumania, 2Department of Neurochemistry, Institute of Growth hormone (GH) secretion from the pituitary is partly and indirectly 3 regulated by GH-releasing hormone (GHRH)-containing neurons in the Biomolecular Chemistry, Budapest, Hungary, Peptide Biochemistry hypothalamic arcuate nucleus (ARC), and these neurons express GH Research Group, Semmelweis University, Budapest, Hungary secretagogue (GHS) and somatostatin (SRIF) receptors. Hypothalamic GHS Somatostatin (SST) mediates its diverse physiological effects in the hippocampus through a family of four G-protein-coupled receptors (sstr1-sstr4) showing distinct subcellular and SRIF receptor expression is seen in infantile rats. Thus, using brain 125 slices of infantile rats we recorded extracellular electrical activity from the localization. TT-232 was shown to displace [ I]SST specifically bound to hippocampal ventromedial part of the ARC, and electrophysiological effects of ghrelin, an synaptic membranes with an IC50 value of 0.1nM. To identify those sstr subtypes that are endogenous ligand for GHS receptor, and SRIF were examined. Of 21 ARC activated by TT-232, pharmacologically isolated excitatory (EPSC) and inhibitory (IPSC) postsynaptic currents have been recorded from CA3 hippocampal cells. SST decreased the neurons, 20 were excited by ghrelin and inhibited by SRIF in normal artificial frequency of mEPSCs (32.9±15.9%) without affecting their amplitude and did not affect cerebrospinal fluid (ACSF), and the remaining 1 was inhibited by SRIF mIPSCs, but decreased both the frequency (75.8±1.0%) and amplitude (28.61±16.7%) of alone. Similar percentages of responses to ghrelin and/or SRIF were also sEPSCs and also decreased the frequency (52.65±3.0%) and the amplitude (39.87±10.4%) obtained in ARC neurosecretary neurons that were identified by antidromic of sIPSCs. Effects of TT-232 on EPSCs were similar to the effect of SST: TT-232 decreased activation to median eminence (ME) stimulation. The excitation and only mEPSC frequency (37.9±10.1%) and decreased sEPSC frequency and amplitude. inhibition induced by ghrelin and SRIF, respectively, were dose-dependent, In contrast to SST, TT-232 did not affect mIPSCs and decreased only sIPSCs frequency 2+ 2+ and persisted in low-Ca /high-Mg ACSF. These results suggest that even (39.71%). In slices loaded with the cell-permeable form of a Ca2+ ion-sensitive dye Fluo- in infantile rats ghrelin excites and SRIF inhibits ARC neurons, possibly 4AM only SST and sandostatin (but not TT-232) induced intracellular Ca2+ ion transients in GHRH-containing neurons that project to the ME, to regulate GH secretion. hilar or CA3 cells.
P1AM-16-6 P1AM-17-1 OREXIN(ORX)-INDUCED INTRACELLULAR Ca2+ CATIONIC GRADIENT REVERSAL AND ELEVATION IN ANTERODORSAL PART NEURONS OF CYTOSKELETAL INDEPENDENT VOLUME THE RAT MEDIAL AMYGDALA NUCLEUS (MeAD) REGULATORY PATHWAYS DEFINE AN EARLY STAGE Shinya Miura, Kazuki Nakajima, Kazuo Sasaki OF APOPTOSIS IN LYMPHOID CELLS Division of Bio-Information Engineering, University of Toyama, Japan Carl David Bortner, Maria I. Sifre, John A. Cidlowski ORX plays crucial roles in the regulation of sleep-wakefulness, feeding and Laboratory of Signal Transduction, National Institute of Environmental reproductive behavior. ORX neurons project to the MeAD that expresses Health Sciences, NIH, USA ORX receptors. Recent studies indicate that the MeAD can serve as a relevant Apoptotic volume decrease (AVD) is a ubiquitous feature of programmed cell death. part for the general arousal or nonspecific activation of social behaviors. We studied the role of the cytoskeleton during AVD using Jurkat cells treated with Therefore, we examined effects of ORX-A and ORX-B on intracellualar Ca2+ 2+ 2+ either Fas ligand or UV. Two discrete stages of AVD were observed upon disruption ([Ca ]i) changes in MeAD neurons using Ca imaging technique with Fura- 2+ of the actin cytoskeleton using cytochalasin B, with each stage accompanied by 2AM since changes in [Ca ]i regulate gene expression, neurotransmitter specific apoptotic characteristics. Sorting of apoptotic cells combined with ion release, plasticity, neuronal excitability and enzyme activity. ORX-A 2+ concentration measurements showed the primary stage of AVD is characterized by and ORX-B increased [Ca ]i dose-dependently with a similar potency in an early reversal of the intracellular Na+ and K+ ions. During the primary stage of 2+ normal artificial cerebrospinal fluid (ACSF). Elevation of [Ca ]i by ORX-B AVD, the normal intracellular Na+ concentration increases from 12 mM to 113.6 persisted in the presence of tetrodotoxin, showing a postsynaptic nature of mM, whereas the K+ concentration decreases from 139.5 mM to 30 mM. This early 2+ ORX-B effect. ORX-B-induced [Ca ]i elevation was significantly reduced ionic reversal is associated with a 20 to 40% decrease in cell volume, externalization 2+ in nifedipine-containing ACSF, suggesting that extracellular Ca pass into of phosphatidylserine, loss of the mitochondrial membrane potential, PARP cleavage, 2+ 2+ the cytoplasm through L-type Ca channel. In Ca -free ACSF, ORX-B and nuclear condensation. In contrast, disruption of the actin cytoskeleton prevents 2+ 2+ increased [Ca ]i in some extent, but the increase was blocked in Ca -free/ a secondary stage of AVD in apoptotic cells, characterized by a loss of both K+ and dantrolene-containing ACSF, indicating that ORX-B induce a release of Ca2+ Na+ that resulted in an 80 to 85% loss in cell volume, DNA degradation, and apoptotic from endoplasmic reticulum. These results suggest an involvement of ORX body formation. Thus AVD occurs in two distinct stages with the earliest stage 2+ in the regulation of [Ca ]i of MeAD neurons. reflecting a unique reversal of the intracellular cationic gradient.
P1AM-17-2 P1AM-17-3 ACTIVATION OF VOLUME-SENSITIVE CHLORIDE EXPRESSION OF TRPM7 CHANNELS SWITCHES CHANNELS AND CISPLATIN-INDUCED APOPTOSIS IN ACIDOSIS-INDUCED CELL DEATH FROM APOPTOSIS NASOPHARYNGEAL CARCINOMA CELLS TO NECROSIS IN HUMAN EPITHELIAL CELLS Liwei Wang1, Linyan Zhu2, Zhiquang Bai1, Linjie Yang3, Xiaoya Yang4, Tomohiro Numata1, Yasunobu Okada2 1 5 2 Haifeng Zhang , Tim JC Jacob , Lixin Chen 1Department of Synthetic Chemistry and Biological Chemistry, Kyoto 1Department of Physiology, Medical College, Jinan University, Guangzhou, University, Japan, 2National Institute for Physiological Sciences, Japan China, 2Department of Pharmacology, Medical College, Jinan University, Guangzhou, China, 3Department of Pharmacology, Medical College, Jinan In human epithelial HeLa cells, we recently showed molecular and functional University, Guangzhou; Guangxi Medical University, China, 4School of Nursing, expression of TRPM7. The inward current of TRPM7 was dramatically enhanced Guangzhou Medical College, China, 5Cardiff School of Biosciences, Cardiff by a decrease in extracellular pH due to reduced block by extracellular divalent University, UK cations. Upon exposure to acidic solution (pH 6 or 4), HeLa cells exhibited Cell shrinkage is the early hallmark of apoptosis induced by various agents in many cell persistent swelling. After 1-h incubation in acidic solution, HeLa cells largely types. Here we investigated the activation mechanisms of Cl- currents induced by the suffered from necrotic cell injury characterized by increased propidium iodide chemotherapy drug cisplatin (cDDP) in nasopharngeal carcinoma CNE-2Z cells. Exposition (PI) stainability as well as lack of both nuclear condensation (NC) and caspase-3 to cDDP activated a Cl- current, shrank cells in a few minutes and induced apoptosis. Cl- activation. Treatment with siRNA for TRPM7 suppressed acidification-induced channel blockers, NPPB and tamoxifen inhibited the current, prevented cell shrinkage and inward currents. After 1-h exposure to acidic solution, HeLa cells transfected - - - protected cells from apoptosis. The current, with permeability sequence of I > Br > Cl > with TRPM7 siRNA showed cell shrinkage, whereas mock-transfected HeLa gluconate, was outward-rectified, dependent on intracellular ATP and sensitive to NPPB, cells showed cell swelling. On the basis of observations of PI stainability, NC and tamoxifen and hypertonic cell shrinkage. Suramin, a wide-spectrum purinergic antagonist, caspase-3 activation, acidosis-induced cell death in siRNA- and mock-transfected inhibited the current and prevented current activation. The current was also blocked by reactive blue 2, a selective P2Y receptor antagonist. Incubation with ATPase suppressed the HeLa cells was judged to be due to apoptosis and necrosis, respectively. Also, current. Depletion of free cytoplasmic Ca2+ delayed the activation and increased the time acidosis-induced necrosis was observed in TRPM7-transfected, but not mock- to reach the peak. The data suggest that the volume-sensitive Cl- channel activation and the transfected, HEK293T cells. Therefore, it is concluded that the TRPM7 channel associated cell shrinkage are key processes of cDDP-induced apoptosis. Purinergic receptor plays a key role in switching acidosis-induced cell injury from apoptotic to pathways and Ca2+ signalling are involved in activation of the channels. necrotic cell death in human epithelial cells. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 153 P1AM-17-4 P1AM-17-5 AQUAPORIN 5 MEDIATED WATER PERMEABILITY THE APOPTOTIC VOLUME DECREASE IS AN AND ITS ROLE IN APOPTOTIC VOLUME DECREASE IN UPSTREAM EVENT OF p38 ACTIVATION DURING EAT CELLS STAUROSPORINE-INDUCED APOPTOSIS IN HELA Carl Frederik Hansen, Else K Hoffmann, Kristian A Poulsen CELLS 1 1 2 Department of Biology, Section of Cell- and Developmental Biology, Yuichi Hasegawa , Takahiro Shimizu , Nobuyuki Takahashi , University of Copenhagen, Denmark Yasunobu Okada1 Loss of cell volume during apoptosis, termed apoptotic volume decrease 1Department of Cell Physiology, National Institute for Physiological 2 (AVD), is a highly conserved morphological feature of programmed cell Sciences, Japan, Division of Food Science and Biotechnology, Graduate death that is due to an early loss of intracellular ions and water. While the School of Agriculture, Kyoto Univ., Kyoto, Japan mechanism of ionic redistribution in AVD has been widely studied, little is Cell shrinkage, called apoptotic volume decrease (AVD), is a pivotal event of known about the role of the water permeability. apoptosis. AVD is known to be induced by activation of the volume-sensitive To study aquaporin (AQP)-mediated water permeability and its role in outwardly rectifying Cl- channel (VSOR). On the other hand, activation of AVD we developed a cell culture model based on EATCs. EATCs are the MAP kinase cascade also known to play a critical role in apoptosis. In the chosen as they express only mRNA transcripts for AQP5, AQP3 and AQP9, present study, we investigated the relationship between AVD and MAP kinase the expression of AQP5 being by far the most dominant (elevated about cascade activation during the process of staurosporine-induced apoptosis in 60-fold compared to the others) and are easy to maintain and manipulate in HeLa cells. VSOR blockers inhibited staurosporine-induced activation of the lab. Subsequently, stable EATCs with AQP5 micro RNAi knockdown caspase-3/7 and cell death. VSOR blockers also inhibited phosphorylation (miR-AQP5) and AQP5 over-expression (AQP5ex) has been generated. of p38 and JNK. Moreover, neither application of a p38 inhibitor SB203580 Respectively, these cell lines has either very low or high expression of AQP5, nor siRNA-induced knock-down of ASK1 expression was found to inhibit have altered growth rates and diverge distinctly morphologically particularly the AVD event. Next, it was found that sustained osmotic cell shrinkage per with regard to cell volume. We are currently evaluating changes in water se triggered p38 phosphorylation, caspase activation and cell death. Thus, permeability in miR-AQP5 and AQP5ex EATCs following osmotic challenge it is concluded that AVD precedes MAP kinase activation in HeLa cells and investigating how AQP5 is involved in drug-induced AVD. undergoing staurosporine-induced apoptosis.
P1AM-17-6 P1AM-17-7 P53 ONE OF THE MAJOR PLAYERS FROM OSMOTIC IκB KINASE β /NF-κB PATHWAY PROTECTS HEARTS STRESS TO APOPTOSIS FROM HEMODYNAMIC STRESS MEDIATED THROUGH Maria Stine Enghoff1, Kenneth Skou2, Else Kay Hoffmann1 REGULATING MANGANESE SUPEROXIDE DISMUTASE 1Biological institute, University of Copenhagen, Denmark, 2Institute of EXPRESSION Cancer Biology, Denmark Shungo Hikoso1, Osamu Yamaguchi1, Toshihiro Takeda1, Shigemiki 1 2 1 3 1 Apoptotic Volume decrease is a hallmark in apoptosis and apoptosis can be Omiya , Keiichiro Suzuki , Kazuhiko Nishida , Michael Karin , Kinya Otsu 1 induced by hypertonic cell shrinkage via the following signalling events: cell Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Japan, 2Department of Biochemistry, Hyogo College of Medicine, shrinkage activates Rac, which activates p38, followed by phosphorylation 3 Japan, Department of Pharmacology and Pathology, University of California, at S15 of p53 and finally by Caspase 3 activation(1). However, p53 but USA not p38, follows a biphasic activation pattern with increasing osmolarity Objectives: Cardiomyocyte death plays an important role in the pathogenesis of heart indicating additional control mechanisms for p53 activation than p38. We failure. The NF-κB signaling pathway has been known to regulate cell death. In this study, thus investigated whether DNA damage and ribosomal stress are also factors we aimed to clarify the in vivo role of NF-κB pathway in the pathogenesis of heart failure involved in the p53 activation. ATM (ataxia-telangiectasia mutated) known using cardiac-specific IKKβ-deficient mice (CKO). Methods and Results:We subjected to be activated by DNA damage and to phosphorylate p53 at serine 15 was CKO and control littermates (CTRL) to pressure overload by means of transverse aortic activated during hypertonic stress and inhibition of ATM with Wortmanin constriction (TAC). One week after TAC, CKO showed cardiac dilation, dysfunction abolished p53 phosphorylation. ATM and p53 followed a similar biphasic and lung congestion, characteristics of heart failure. The number of apoptotic cells had activation pattern with increasing osmolarity suggesting a role for ATM. significantly increased in CKO hearts after TAC. The expression levels of manganse To investigate the possible role of ribosomal stress in p53 activation low superoxide dismutase (MnSOD) mRNA and protein in CKO after TAC were significantly attenuated than those in CTRL. Moreover, the levels of oxidative stress and JNK activation concentrations of Actinomycin D was implied, which increased the level of in CKO after TAC had increased compared with those in CTRL. Isoproterenol-induced cell p53 but not its Ser 15 phosphorylation. We now study whether ribosomal death of isolated adult CKO cardiomyocytes was significantly prevented with the treatment stress and hypertonic stress are additive or not. Using Cycloheximide we of a MnSOD mimetic or JNK inhibitor. Conclusion:IKKβ/NF-κB signaling pathway in have shown that the breakdown of p53 is biphasic at increasing osmolarities. cardiomyocytes plays a protective role mediated through the attenuation of oxidative stress 1. Friis MB et al. 2005. J. Physiol. 567.427-443. and JNK activation in response to pressure overload.
P1AM-17-8 P1AM-17-9 BIOLOGICAL SIGNIFICANCE OF SGK1 DEPENDENT Vibrio parahaemolyticus SERINE PROTEASE INDUCES TRANSPORT REGULATION EPITHELIAL CELLS DAMAGE VIA PROTEASE- Florian Lang1, Teresa F. Ackermann1, Daniela S. Kempe1, ACTIVATED RECEPTOR-2 Krishna M. Boini1, Volker Vallon2 Shwu-Fen Pan 1Department of Physiology, Eberhard-Karls-University of Tuebingen, Department of Biotechnology, Ming Chuan University, Taiwan 2 Germany, Department of Medicine & Pharmacology, UCSD & VASDHCS, Recently, it has been shown that a serine protease derived from Vibrio San Diego, USA parahaemolyticus is toxic for mice and causes bleeding of many organs. In The serum and glucocorticoid inducible-kinase-1 (SGK1) participates in a wide this study, crude extracts of wild type and serine protease gene mutant strain variety of functions including stimulation of ion channels (e.g. ENaC, SCN5A, were prepared and incubated with epithelial cells for an appropriate time for TRPV5, ROMK, KCNE1/KCNQ1, KCNQ4, Kv1.3, Kv1.5, Kv4.3, CFTR, ClC2, testing the cytopathic effects of serine protease. The results of MTS assay ClCKb, GluR6), carriers (e.g. NHE3, NKCC2, NCC, GLUT1, SGLT1, NaPiIIb, + + and cellular morphology shown that a considerable level of toxicity toward NaDC1, SMIT, ASCT2. SN1, EAAT1-5, CreaT), and the Na /K -ATPase. SGK1 the cells was observed after the treatment with crude extracts of wild type expression is stimulated by ischemia, cell shrinkage and several hormones including strain. Cytotoxicity was significantly reduced when crude extracts prepared gluco- and mineralocorticoids. SGK1 is activated by insulin, growth factors and from serine protease gene mutant strain. However, No significant effect oxidative stress. SGK participates in the regulation of transport, hormone release, was observed with the release of lactate dehydrogenase (LDH). Meanwhile, neuroexcitability, inflammation, cell proliferation and apoptosis. SGK1 contributes to Poster Session regulation of gastric acid secretion, intestinal NHE and nutrient transport, renal Na+ the number of TUNEL-positive cells was markedly increased when cells retention and K+ elimination, salt appetite, insulin-dependent salt sensitivity of blood incubated with extracts of wild type strain in comparison with extracts of pressure, salt sensitivity of peripheral glucose uptake, cardiac action potential as well mutant strain. Meanwhile, extracts of wild type strain-induced cell apoptosis as fibrosis during mineralocorticoid and salt excess. A common (3-5% prevalence was inhibited by pertussis toxin (a PAR2 inhibitor) and neutralizing antibody in Caucasians, 10% in Africans) SGK1 gene variant is associated with obesity, to PAR2. These results reveal that V. parahaemolyticus -derived protease hypertension and development of diabetes. SGK1 may further participate in tumor activates epithelial cells apoptosis through PAR2, suggesting an important growth, neurodegeneration, allergy, viral infection, peptic ulcer and the sequelae of role for PAR2 in the modulation of GI inflammation associated with V. ischemia. parahaemolyticus.
154 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-17-10 P1AM-17-11 MEMBRANE GLYCOPROTEINS DURING CELL CELLULAR AGGREGATION FACILITATES MATRIX- DEATH: HOW AND WHY THEY APPEAR ON THE CELL DETACHMENT INDUCED APOPTOSIS IN MDCK CELLS SURFACE? Yumiko Emoto Rostyslav Bilyy, Andrew Tomyn, Rostyslav Stoika Department of Biology, Kyushu University, Japan Department of Regulation of Cell Proliferation and Apoptosis, Institute of Normal epithelial cells require contact with extracellular matrix to survive. Cell Biology, NAS of Ukraine, Ukraine When these cells are detached from the matrix, they induce apoptosis, Recently, we (Bilyy et al, Cytometry, 2003; Bilyy, Stoika, Autoimmunity 2007) which is termed anoikis. Anoikis plays important roles in the physiological and others (Heyder et al, Cytometry 2003) have found that programmed cell induction of apoptosis during development and maintenance of tissue death (apoptosis) is accompanied by an increase in levels of plasma membrane homeostasis in the organism. In several cell lines, cellular aggregations in mannose- and galactose containing glycoproteins (GP), and simultaneous suspension culture have been shown to suppress anoikis. Here, we describe decrease in levels of sialil-containing GPs. Here we addressed a question on the the effects of cellular aggregation on anoikis in Madin-Darby canine kidney mechanisms of such GP redistribution, we considered few hypothesis, namely: (MDCK) cells. Suspension cultures of MDCK cells grown under conditions a) de novo GP synthesis - for this aim inhibitors of early stages in GP synthesis and processing, inhibitor of GP traffic between Golgi compartments as well as known to induce extensive cellular aggregation were less able to re-attach inhibitor of transcription and translation were used; b) modification of preexisting to culture dishes, exhibited higher caspase-8 activity, and contained more plasma membrane glycoproteins. Obtained results suggest that the redistribution sub-G1 cells than suspension cultures with less cellular aggregation. When of plasma membrane GPs observed at apoptosis is caused by modification of suspension cultures of MDCK cells were separated into aggregated cells and preexisting GPs on cell surface, and not by de novo GP synthesis. Activation of single cells, the aggregated cells had low caspase-8 activity regardless of membrane-associated neuraminidase activity was proved to be a primary cause of suspension conditions, whereas single cells had higher caspase-8 activity that plasma membrane glycoprotein redistribution during apoptosis, specific enzymes increased with increasing degree of aggregation. These results suggest that involved in glycoprotein redistribution were also determined. Involvement of cell-cell interactions facilitate anoikis in cellular aggregates of suspended these GP in the cell-to-cell interaction during physiological clearance of apoptotic MDCK cells. cell by macrophages was demonstrated.
P1AM-17-12 P1AM-17-13 COMPARATIVE EVALUATION OF THE ANTIPROLIFERATIVE EFFECTS OF BUFALIN ON EFFECTS OF ALPHA-TOCOPHERYL IN USE ANDROGEN-INDEPENDENT HUMAN PROSTATE OF ANTIPROLIFERATIVE AGENTS ON ECV304 CANCER IN VITRO AND IN VIVO ENDOTHELIAL CELLS Ching-Han Yu1, Yen-Jui Chang2, Ru-Lian Hsu3, Paulus S. Wang4 1 2 1 Serap Erdem Kuruca , Gul Ozcan Arican , Sabriye Karadenizli , 1Department of Physiology, National Yang-Ming University, Taiwan, 2Department 2 1 2 of Physiology, National Yang-Ming University, Taiwan,; Department of Ugur Serbest , Beyza Muzaffer Cetin , Kadriye Akgun Dar 3 1 2 Ophthalmology, Taipei City Hospital, Taiwan, Department of Physiology, Department of Physiology, Istanbul University, Turkey, Department of 4 National Yang-Ming University, Taiwan, Department of Physiology, National Biology, Istanbul University, Turkey Yang-Ming University, Taiwan,; Department of Medical Research and Education, This study was carries out to evaluate the effects of alpha-tocopheryl combine Taipei City Hospital, Taiwan antiproliferative agents frequently used in glaucoma surgery, differently Bufalin is one of the components in Chinese medicine chansu, and used as anticancer agent investigating side effects on endothelial cells. Effects antiproliferative agents on leukemia cells. In the present study, the proapoptotic and antigrowth effects of bufalin on have been investigated to treat alpha-tocopheryl single applying 5 minutes (short the androgen-independent DU145 and PC3 human prostate cancer cell lines and xenografted term) and along 10 days (long term) repeated with interval two days on human mouse model were investigated. Bufalin showed the antigrowth effects on both cell lines ECV304 endothelial cells. Paclitaxel, mitomycin-C, 5-fluorouracil are used as after 24-hr treatment detected by MTT assay. At the concentrations of 10-5 M, bufalin antiproliferative agents. Apoptotic index, mitotic index, Bcl-2/Bax expression increased the annexin-V staining to approximately 80% and 82% in DU145 and PC3 cells, and histologic examination are determined in all groups. Photomicrographs respectively. DU145- and PC3-xenografted mice were used to examine the antitumor effects of different groups after each treatment revealed characteristic morphologic of bufalin in vivo. After injection of bufalin, the tumor growth was significantly suppressed in both animal models. At the end of the in vivo study, bufalin injection resulted in 79% changes when compared to controls. Although increase toxic effects of inhibition on tumor volume, and 72% suppression on tumor weight in DU145-xenografted paclitaxel and mitomycin-C with combination alpha-tocopheryl the highest mice. Similar results were found in the PC3-xenografted mice. In tumor tissue, the protein cytotoxicity was seen in paclitaxel group. It was’nt found additive increase expressions of the proliferation cell nuclear antigen (PCNA) and the endothelial cell marker in 5-fluorouracil+alpha-tocopheryl group compared to controls. As a results, (CD31) were also decreased by bufalin treatment in both animal models. These data treatment alone or combine 5-fluorouracil and alpha-tocopheryl long and short suggested that bufalin showed antiproliferative and antitumor growth effects in androgen- term are safety than paclitaxel and mitomycin-C in glaucoma surgery. independent prostate cancer in vitro and in vivo.
P1AM-17-14 P1AM-17-15 INCREASED APOPTOSIS AND DECREASED eNOS- NAOFEN, A NOVEL WD-REPEAT CONTAINING VASCULAR INJURY IN EC-SOD DEFICIENT MICE IS PROTEIN, MEDIATES THE SPONTANEOUS AND PREVENTED BY HO-1: ROLE OF ADIPONECTIN TUMOR NECROSIS FACTOR-ALPHA-INDUCED Tomoko Kawakami, Kazuyoshi Inoue, Dong Hyun Kim, APOPTOSIS IN HEK293 CELLS Ming Li, Luca Vanella, Nader G. Abraham Guo-Gang Feng, Lei Huang, Jun-Hua Fan, Fumio Kondo, Department of Pharmacology, New York Medical College, USA Naohisa Ishikawa Department of Pharmacology, Aichi Medical University School of Medicine, EC-SOD is important in cytoprotection by decreasing superoxide levels. Thus, EC- Japan SOD deficiency may also affect heme oxygenase (HO) levels and anti-apoptotic signaling. We examined EC-SOD deletion on HO-1/HO-2 protein, HO activity, Naofen has recently been cloned from rat’s brain/spinal cord cDNA library using an eNOS and serum adiponectin levels in STZ-treated mice. EC-SOD deletion decreased anti-shigatoxin (STX)-2 antibody. Reportedly, STX-2 may induce apoptosis in many types of cells. In the present study, we evaluated roles of naofen in the spontaneous the basal levels of eNOS, BCL-XL and pAKT proteins although no change in Akt and tumor necrosis factor (TNF)-α-induced apoptosis. Results: 1. Transient over- protein expression was observed. EC-SOD deletion increased apoptotic signaling expression of naofen in HEK293 cells induced the caspase-8, -9 and -3 activities. 2. kinase-1 (ASK-1)(p<0.01) and HO-1 protein but decreased HO activity. In STZ- Stable naofen over-expression (FLAG-NF-S) HEK293 cells elicited rather higher treated EC-SOD (-/-) mice there was marked renal impairment reflected by an increase caspase-3 activation, and spontaneous apoptosis when examined using a flow in acute tubular damage and microvascular damage. HO activity was decreased cytometry. 3. Treatments with TNF-α enhanced the expression of naofen mRNA with a concomitant increase in superoxide anion. Upregulation of HO-1 by CoPP in a dose-dependent manner. In addition, the FLAG-NF-S cells showed extremely administration in EC-SOD (-/-) mice after development of diabetes produced an higher susceptibility to TNF-α-induced caspase-3 activation and apoptosis. 4. The increase in adiponectin (p<0.05) and prevented the increase in tubulointerstitial and knock-down of naofen expression in the FLAG-NF-S cells with specific siRNA, microvascular damage. Therefore, EC-SOD deficiency exacerbates STZ-induced which effectively inhibited the naofen mRNA expression, significantly reduced both vascular damage and morphological injury, HO-1 upregulation prevents morphological caspase-3 activities and apoptosis caused by TNF-α. We conclude that naofen is an damage. The basal level of EC-SOD participate in maintaining vascular integrity and important cytoplasmic protein, which may promote the spontaneous apoptosis and in cellular defense mechanisms against oxidative stress by regulating eNOS, pAKT, play an important regulatory role in mediating the TNF-α-stimulated apoptosis via ASK-1 and adiponectin. activating the caspase-3. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 155 P1AM-17-16 P1AM-17-17 GENES AND GENE NETWORKS RESPONSIVE TO GENE NETWORKS INVOLVED IN THE APOPTOSIS LOW-INTENSITY PULSED ULTRASOUND IN HUMAN INDUCED BY HEAT STRESS IN HUMAN LYMPHOMA LYMPHOMA U937 CELLS U937 CELLS Yoshiaki Tabuchi1, Yukihiro Furusawa2, Ichiro Takasaki1, Yukihiro Furusawa1,2, Yoshiaki Tabuchi3, Ichiro Takasaki3, 2 4 Shigehito Wada2, Qing-Li Zhao2, Takashi Kondo2 Shigehito Wada , Kenzo Ohtsuka 1 2 1Life Science Research Center, University of Toyama, Japan, 2Graduate Department of Radiology, Toyama University, Japan, Grad. Sch. Med 3 School of Medicine and Pharmaceutical Sciences, University of Toyama, & Pharm. Sci. (Med), Univ. Toyama, Toyama, Japan, Life Sci. Res. Ctr., 4 Japan Univ. Toyama, Toyama, Japan, Dep. Envir. Biol., Chubu Univ., Aichi, Ultrasound (US) has been widely used for diagnosis and therapy in many medical Japan fields. It has been indicated that fairly intense US induces cell killing, cell lysis, loss Heat stress (HS) induces complex cellular responses and changes in signal transduction. of viability, and loss of clonogenicity. Moreover, accumulating evidence indicates Hyperthermia induced by HS has been used for many years to treat a variety of malignant that intense US as well as low-intensity pulsed US (LIPUS) induce apoptosis in tumors. Here, to clarify the detailed mechanisms by which HS induces apoptosis in human human leukemia cell lines. Here, to clarify the detailed molecular mechanism leukemia U937 cells, we performed global-scale time course microarray analysis. We also underlying cellular responses to LIPUS, gene expression patterns and gene networks explored the functional relationships among the candidate genes using Ingenuity Pathway in human lymphoma U937 cells were examined using global-scale microarrays and Analysis tools. U937 cells were treated with HS at 42C for 90 min or 44C for 15 min, and computational gene expression analysis tools. Six hours after LIPUS treatment (0.3 W/ then cultured at 37C. Six hours after the HS, apoptosis (approximately 20%) was observed, cm2 for 1 min), apoptosis (approximately 15%) with minimal cell lysis was observed. and an elevation of the protein expression of heat shock proteins including Hsp27, Hsp40 LIPUS down-regulated 193 genes and up-regulated 201 genes by >1.5-fold. For down- and Hsp70 was detected following the activation of heat shock factor-1. A large number of regulated genes, a significant genetic network was associated with cellular growth genes that were differentially expressed were identified in cells with HS using GeneChip and proliferation, gene expression, or cellular development. For up-regulated genes, a system. In addition, computational gene expression analysis demonstrated that significant significant genetic network was associated with cellular movement, cell morphology, and temperature-specific gene networks were associated with cellular function and and cell death. The present results indicate that LIPUS affects the expression of many maintenance, cellular growth and proliferation, or apoptosis. These findings will provide a genes and will provide novel insight into the biomolecular mechanisms of LIPU in basis for understanding the detailed molecular mechanisms of apoptosis induced by HS in therapeutic application for cancer therapy. cells.
P1AM-17-18 P1AM-17-19 IN VIVO AND IN VITRO EVIDEENCES FOR INCREASES REGULATION OF THE RENOPROTECTIVE ACTIONS IN THE EXPRESSION OF NAOFEN IN THE KINDEY OF OF AATF BY AKT1 DIABETIC RATS Qing Guo, Jun Xie, Chelsea Baker Lei Huang1, Guo-Gang Feng1, Jun-Hua Fan2, Jun An3, Yuko Sato4, Department of Physiology, The University of Oklahoma Health Sciences Shuji Kurokawa4, Tooru Komatsu4, Fumio Kondo1, Naohisa Ishikawa1 Center, USA 1Department of Pharmacology, Aichi Medical University School of Medicine, AATF (apoptosis antagonizing transcription factor) was initially identified 2 Japan, Department of Gastroenterology, Nagoya University Graduate School as an interaction partner of DAP-like kinase, a member of the DAP (Death of Medicine, Japan, 3Division of Gastroenterological Surgery, Department of 4 Associated Protein) kinase family of pro-apoptotic kinases. We found that Surgery, Aichi Medical University School of Medicine, Japan, Department of Anesthesiology, Aichi Medical University School of Medicine, Japan levels of AATF expression in renal tubular epithelial cells were significantly altered in an in vitro model of renal injury induced by ischemia/reperfusion Recently, we have reported the mediating roles of naofen, a novel WD-repeat protein, (I/R). In renal tubule epithelial cells, RNAi-mediated silencing of AATF over-expression in the spontaneous apoptosis as well as in the tumor necrosis factor-α- exacerbated, while overexpression of AATF ameliorated mitochondrial stimulated apoptosis. The present study was undertaken to evaluate the roles of naofen especially in the in-vivo studies utilizing streptozotocin-induced diabetic rats and also dysfunction, oxidative damage, and apoptotic death induced by I/R. These in the in-vitro studies for evaluating the effects of high glucose on normal rat kidney results identify AATF as a novel cytoprotective factor in renal tubular epithelial (NRK52E) cells. Contemporary increases in naofen and transforming growth cells. Recently, we performed in vitro phosphorylation assays, and found that AATF is a phosphorylation substrate of Akt1/PKBα, a serine/threonine factor (TGF)-β1 mRNA expressions were obtained in the kidney of diabetic rats and also in the NRK52E cells treated with high glucose. Since naofen over-expression had protein kinase that plays important roles in promoting cell survival and elicited the activation of caspases, the elevation of caspase-3 activities was observed inhibiting apoptosis. Furthermore, we found that phosphorylation of AATF in the diabetic kidney, and also in the high glucose treated NRK52E cells. Treatment by Akt1 significantly ameliorated apoptotic cell death in renal tubule
with TGF-β1 in NRK52E cells enhanced naofen mRNA expressions from the 48 to 72 cells. These results suggest that AATF is a novel phosphorylation target of h incubation after a temporal decrease in 24 h. These results indicate that naofen is an Akt1, and that phosphorylation of AATF by Akt1 may represent a critical important cytosolic protein which promotes the spontaneous apoptosis in the kidney of mechanism of signal transduction mediated by Akt1 in regulating renal cell diabetic rats through hyperglycemia. survival.
P1AM-17-20 P1AM-17-21 RENOPROTECTIVE ACTIONS OF AATF: MODULATION EFFECTS OF VITAMINS E AND D3 ON THE CELL OF BCL-2 MEDIATED TRANSCRIPTION VIA DEATH AND REMYELINATION OF RAT HIPPOCAMPUS INTERACTION WITH PAR-4 FOLLOWING LOCAL INJECTION OF ETHIDIUM Jun Xie, Qing Guo BROMIDE Department of Physiology, The University of Oklahoma Health Sciences Mahdi Goudarzvand, Mohammad Javan, Javad Mirnajafi-Zadeh, Center, USA Taki Tiraihi AATF (apoptosis antagonizing transcription factor) is a leucine zipper protein Madical Physiology, Tarbiat Modares University, Iran which was identified as an interaction partner of DAP-like kinase, a member Vitamins E and D3 are recommended for patients suffering from neurodegenerative of the DAP (Death Associated Protein)-kinase family of pro-apoptotic serine/ disease, while the exact mechanism of their action is not well understood. Here, we threonine kinases. Par-4 (prostate apoptosis response-4) is a leucine zipper have tried to study the effect of vitamins E and D3 on cell death and remyelination, protein that regulates apoptosis. We reported Par-4 promotes the death of following ethidium bromide (EB) induced insult in rat hippocampous. EB was directly renal tubular epithelial cells following ischemia/reperfusion induced injury injected into the rat’s dentate gyrus. Animals received 100mg/kg vitamin E (for 2,7 (I/R). AATF binds with Par-4 via the leucine zipper domain, leading to a or 28 days), 5μg/kg vitamin D3 (for 2,7 or 28 days) or their combination for 7 days. blockade of the pro-apoptotic signaling mediated by Par-4. Par-4-dependent The expression of Caspase-3 and myelin basic protein (MBP) as the indices of cell cell-death seems to be mediated by decreased bcl-2 transcription. We found death and myelination were measured using western blot. While the administration of Poster Session that overexpression of Par-4 effectively reduced the adaptive increase in EB alone increased the expression of caspase-3, vitamins E, D3 and their combined Bcl-2 levels in the early stages of I/R injury in renal tubule cells. In pull administration, decreased the caspased-3 expression than control group.The expression down assays using specific sequence on the bcl-2 P1 promoter as bait, of MBP was reduced on day 2 post-lesion but reversed on days 7 and 28. Vitamins E we found a significantly increased Par-4 binding to the bcl-2 promoter in and D3 reversed the expression of MBP in EB treated animals on days 2 and 7; and proximal tubular cells following I/R. Binding of Par-4 to the bcl-2 promoter on day 28 increased its expression to higher levels, compares with control. Combined was significantly reduced in cells transfected with AATF. These results administration of vitamins E and D3 for 7 days increased MBP expression.These suggest that AATF confers renoprotective actions by regulating Par-4 results indicated that vitamins E and D3 could decrease apoptosis in the demyelinating mediated nuclear transcription of bcl-2. condition.
156 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-17-22 P1AM-17-23 EFFECT OF LIPOPOLYSACCHARIDE ON THE LIFE- THE INHIBITORY EFFECT AND INDUCTION OF SPAN OF MOUSE PRIMARY-CULTURED MICROGLIA APOPTOSIS OF CAFFEICACID GE ON THE GROWTH Yoko S Kaneko1, Akira Nakashima1, Keiji Mori1, OF H22 IN MICE Toshiharu Nagatsu2, Ikuko Nagatsu3, Akira Ota1 Yueyan Huang1, Chun Xiao2 1 2 1Department of Physiology, 2Department of Pharmacology, 3Department of Medical School, Jiaxing College, China, Traditional Chinese Medical Anatomy, Fujita Health University, Japan College of Jiangxi, China Microglia play many important roles in the physiology and pathophysiology of AIM To evaluate the antitumor effect of Caffeic-acid Ge on tumor bearing the central nervous system. Although activated microglia have been implicated in mice, investigate its possible mechanisms. Methods The tumor inhibitory effect the recognition and phagocytic removal of degenerating neurons, such activation of caffeicacid Ge on H22 tumor bearing mice was studied. Morphological should be tightly regulated, since prolonged activation could damage normal transformation induced from Caffeic-acid Ge was examined by electronic scan neurons. We report that mouse primary-cultured microglia, which are destined microscope and MG-P staining. Apoptosis-related protein level of Bax and to die in a few days under ordinary culture conditions, are able to live for more Bcl-2 was determined by immunohistochemistry. The MTT assay was applied to than 1 month when kept activated by lipopolysaccharide (LPS). Primary-cultured study the antitumor activities of Caffeic-acid Ge in H22 cell line. Results A dose microglia treated with sub-lethal doses of LPS showed a ramified shape with dependent inhibitory effect in the caffeic acid-germanium treated groups was enlarged somas and thickened cell bodies. LPS treatment of cells stimulated the found to be 37.4%, 40.4%, 57.0% (ranging from low dose to high dose treatment, transcription of the antiapoptotic factor Bcl-xL at 8 h and enhanced its protein respectively) (P<0.01). Characteristic morphological features of apoptosis were level at 1 day. Although the treatment also increased the mRNA expression indicated from MG-P staining histopathology and electronic scan microscope in level of proapoptotic factor BAX, the level of BAX protein was not affected caffeic-acid Ge treated H22 cells. Results from immunohistochemistry indicated at all. Furthermore, microtubule-associated light chain 3, a marker protein of a corresponding down-regulation of bcl-2 and up-regulation of bax in Caffeic- autophagy, decreased at 3 h after the exposure to LPS. These data indicate acid Ge treated H22 tumor tissue . The MTT results suggested that Cafffeic-acid that the optimal dose of LPS can suppress the induction of both apoptosis and Ge at concentrations 1 ug/ml, 10 ug/ml, and 100 ug/ml could inhibit H22 cell autophagy in primary-cultured microglia, allowing them to stay alive for more viability by 18.81%, 26.87%, 37.01% respectively. Conclusion Caffeic-acid Ge than 1 month. exhibited antiproliferative activity in vitro and in vitro.
P1AM-17-24 P1AM-17-25 THE SYNERGISTIC EFFECTS OF PACLITAXEL CREATION OF MUTANT LYMPHOTOXIN-ALPHAS WITH AND RETINOIC ACID INDUCED APOPTOSIS OF ENHANCED BIOACTIVITIES BY PHAGE DISPLAY DU145 CELLS BY STIMULATION CDK5 AND P53 TECHNIQUE EXPRESSTION Yasuo Yoshioka1, Hikaru Watanabe2, Yasuo Tsutsumi3, Wei Huan Huang1, Hung-Shou Kuo1, Chi-Kuan Chen2, Hua-Keng Yohei Mukai2, Naoki Okada2, Shinsaku Nakagawa2 Ke2, Ho Lin1 1The Center for Advanced Medical Engineering and Informatics, Osaka 2 1Department of Life Sciences, National Chung Hsing University, Taiwan, University, Japan, Department of Biotechnology and Therapeutics, 2National Taichung First Senior High School, Taichung, Taiwan Graduate School of Pharmaceutical Sciences, Osaka University, Japan, 3Department of Toxicology, Graduate School of Pharmaceutical Sciences, Cyclin-dependent kinase 5 (Cdk5) and its activator, p35, have been reported in Osaka University, Japan neuronal survival and apoptosis. Our previous results indicate that Cdk5 could Lymphotoxin-alpha (LT) has been considered as a promising new drug for cancer and be overactivated by digoxin treatment and led to apoptosis of prostate cancer immunotherapy because of a strong specific cytotoxicity to tumor cells and immune cells. Paclitaxel, extracted from the tree bark of“Taxus brevifolia”is a microtubule activating action. Serious drawbacks of LT as a clinical antitumor agent are the low stability stabilizing agent and was found to extend half-life of p35 in Alzheimer’s research. and efficacy in vivo. Here, to overcome these problems, we attempted to create mutant LTs On the other hand, retinoic acid (RA) is one of potent form of vitamin A derivatives. with lowered isoelectric point and enhanced bioactivities compared to wild type LT (wtLT). Our previous results also showed that RA induces apoptosis of HeLa cells through To create mutant LTs, a phage library displaying mutant LTs with randomized sequences in Cdk5 overactivation. Here, we try to investigate the effects of combination treatment place of the all lysine residues was prepared. The library was subjected to several rounds of of paclitaxel and RA in prostate cancer cell line (DU145). The results indicated that panning and we succeeded in obtaining bioactive lysine-deficient mutant LTs, Mut1, Mut2 24-h combination treatment could significantly decrease cell growth comparing to and Mut3. By in vitro cytotoxic assay using HEp-2 cells, bioactivities of mutant LTs were individual treatment. In addition, combination treatment increased the accumulation found to be 30-, 7- and 6-fold higher than that of wtLT, respectively. The mutant LTs showed in sub G1 phase. Interestingly, we found combination treatment could significantly higher bioactivity against HT29.14S and MCF-7 cells as well. In addition, we showed that up-regulate protein expressions of Cdk5, p35, and p53. Taken together, our results mutant LTs induced the strong and rapid activation of caspase compared to wtLT. These indicated that Cdk5/p35 possibly play important roles in the synergistic effect of results indicated that mutant LTs would be promising anticancer agents. Now, we are paclitaxel and RA on DU145 apoptosis. examining the mechanism of strong bioactivity of mutant LTs.
P1AM-17-26 P1AM-17-27 THE PHOTODYNAMIC ACTIVITY OF EVALUATION OF ss-DNA CNT EFFECTS ON HELA, 5,10,15,20-TETRAKIS-(METHOXYPHENYL)- HEPG2 AND PANC-1 CELL LINES IN VITRO PORPHYRINS AND THEIR ZINC COMPOUND IN RATS Teodora Mocan1, Lucian C Mocan2, Dana M Todea-Iancu1, BEARING WALKER CARCINOSARCOMA Brindusa Diaconu3, Stefania Simon4, Dan Lupu4, Adriana Gabriela Filip1, Simona V Clichici1, Doina Daicoviciu1, 5 4 2 2 1 3 4 Alexandru S Biris , Alexandru R Biris , Cornel Iancu Rodica M Ion , Adriana V Muresan , Corina Tatomir , Liliana Rogojan , 1 1 1 Physiology, University of Medicine and Pharmacy "Iuliu Hatieganu" Remus Moldovan , Diana Olteanu 2 1 Cluj-Napoca, Roumania, Emergency Hospital “O.Fodor” Cluj-Napoca, Department of Physiology, University of Medicine and Pharmacy Iuliu 3 2 Romania., Emergency Hospital “O.Fodor” Cluj-Napoca, Romania, Hatieganu Cluj Napoca, Roumania, National R&D Institute of Chemistry and 4 3 National Institute of Molecular and Isotopic Technologies, Cluj-Napoca, Petrochemistry - ICECHIM, Bucuresti, Romania, Oncologic Institute Prof. Dr. 5 I. Chiricuta Cluj Napoca, Romania, 4Department of Morphopatology, District Romania, Nanotechnology Center, University of Arkansas, Little Rock, Hospital Cluj Napoca, Romania USA Photodynamic therapy (PDT) mediated oxidative stress causes direct tumor cell damage as well Introduction: The use of single wall carbon nanotubes (SWCNTs) as bioactive as microvascular injury. In addition, tumor eradication also arises from an acute inflammatory molecules is still in early research stages but their unique physical and chemical response featured by an increased level of cytokines in the tumor. To improve this treatment new properties hold great hopes for chemotherapeutic delivery, cancer targeting and photosensitizers are being synthesized and tested. Our study evaluates the effects of PDT with 5,10,15,20-tetrakis- (methoxyphenyl)-porphyrins (TMPP) and their zinc compounds (ZnTMPP) imaging. Material and Methods: In regards with the above aspects, the death rates of on the tumoral levels of ROS, IL4 and TNFα and on the activity of caspase-3 and MMP in Wistar malign cells HeLa, HepG2 , PANC-1 upon incubation in medium with single strand rats bearing carcinosarcoma 256. Rats were randomly divided into five groups: group 1 - no DNA functionalized carbon nanotubes (CNT-DNA aqueous solution) at different treatment, group 2 - only irradiated, group 3 - 5-aminolevulinic acid (5-ALA) 250mg/kg b.w; concentrations (1mg/L, 5 mg/L, 10mg/L, 20mg/L) were analyzed. group 4 - TMPP 10mg/kg b.w, group 5 - ZnTMPP 10mg/kg b. w. The tumors were irradiated for 15 Results: We report low rates of necroses among the three cell lines with no statistical minutes with LASER light (100J/cm2, 10kHz, 685nm) 24h after drug administration. Our results differences between different concentrations, nor the cell type. (p>0,005) show an important increase in the levels of ROS (malondialdehyde: ZnTMPP: 1,09±0,30 vs. 0,34 ± 0,03 nmoles/mg protein, p<0,01), MMP2 was activated and TNFα was released. The activity of Conclusions: Using methods of cellular biology, we demonstrate here that CNT-DNA caspase-3 wasn’t influenced. We may conclude that the effects of PDT with TMPP and ZnTMPP are harmless to different cell lines. Our findings could lead to a new generation of are mediated by cellular membrane damage and the activation of the innate immune system. active biomolecules with effective response against cancer. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 157 P1AM-17-28 P1AM-17-29 ARSENIC KILLS PROSTATE CANCER CELLS CDK5 IS INVOLVED IN RETINOIC ACID AFFECTED THROUGH CDK5 OVERACTIVATION CELL FATE OF DU145 CELLS AND HELA CELLS Ho Lin1, Mei-Chih Chen2 Hung-Shou Kuo, Shuen-Chi You, Chien-Te Ku, Ho Lin 1Department of Life Sciences, National Chung Hsing University, Taiwan, Department of Life Sciences, National Chung Hsing University, Taiwan 2 Department of Physiology, National Yang Ming University, Taipei, Taiwan Cdk5 is a small serine/threonine protein kinase which belongs to Cdk family. Arsenic had been believed to involve the carcinogenesis of prostate cancer Unlike other Cdk members, Cdk5 is known to be irrelevant in cell cycle so and hormone disruption might be one of the causes. Our previous results far. Cdk5 kinase activity is regulated by binding with its activator, p35. Our indicate that drug treatment (digoxin) could lead to apoptosis of prostate previous results indicate that Cdk5 and p35 are involved in drugs-induced cancer cells through Cdk5 overactivation, in which the regular activator apoptosis of prostate cancer cells. Retinoic acid (RA) is one of vitamin for Cdk5, p35, was cleaved into more potent one, p25. Here, our latest A-related compounds. Because of its potency on biological functions, it has data showed that arsenic also caused p35 cleavage and subsequently Cdk5 been widely studied on its novel actions including the ability to inhibit cancer overactivation in prostate cancer cells. Similarly, this arsenic-induced cell growth and to induce apoptosis. Here, we report that RA treatment stimulatory effect of Cdk5 resulted in apoptosis of prostate cancer cells decreased the growth of human prostate cancer cell line-DU145 and cervical accompanying with the degradation of androgen receptor in LNCaP cells, cancer cell line-HeLa, and Cdk5 activity contributed to these inhibitory which suggests that androgen stimulation for prostate cancer survival could effects. We identified that RA-induced growth inhibition was correlated to be eliminated after arsenic treatment. In addition, STAT3 and Akt proteins, G1 phase arrest in DU145 cells and RA-induced apoptosis of HeLa cells was which are general survival signals, were also degraded after treatment of observed by detecting cell cycle distribution of sub G1 phase. In addition, our arsenic. In conclusion, although long-term exposure of arsenic possibly results also indicated that RA-induced HeLa apoptosis was Cdk5 activity- causes prostate cancer, our present results provide evidence indicating that in dependent by annexin V staining . In conclusion, we provide the evidence vitro and short-term treatments of arsenic could lead to apoptosis in prostate implying that Cdk5 and p35 might play important roles in RA-induced cancer cell lines. This finding might be interesting to carcinogenesis in DU145 growth inhibition and HeLa apoptosis and the detail mechanisms still microenvironment of the prostate gland. need further investigation.
P1AM-18-1 P1AM-18-2 CARDIOMYOCYTE DEATH AFTER SIMULATED CARDIOMYOCYTE INJURY AND DEATH AFTER ISCHEMIA/REPERFUSION IS DEPENDENT ON pHo, SIMULATED ISCHEMIA/REPERFUSION: ROLES OF YET INSENSITIVE TO INHIBITION OF THE Na+/H+ THE Ca2+-INDEPENDENT PHOSPHOLIPASE A2, PLA2- EXCHANGER, NHE1 VI Ann-Dorit Andersen, Kristian A Poulsen, Henrik Klingberg, Ann-Dorit Andersen, Kristian A Poulsen, Henrik Salling, Ian H Lambert, Stine F Pedersen Ian H Lambert, Stine F Pedersen Biology, University of Copenhagen, Denmark Biology, University of Copenhagen, Denmark 2+ In vivo, cardiac ischemia is associated with marked reduction of intra- (pHi) and The Ca -independent phospholipase A2 VI (PLA2-VI) is pH sensitive and has been extracellular pH (pHo), as a consequence of inadequate blood flow and increased assigned both protective and detrimental roles in cardiac ischemia/reperfusion. We glycolytic metabolism. Here, we investigated the effect of pHo on injury and death of previously showed that extracellular pH (pHo) during simulated ischemia (SI/R) affects HL-1 mouse cardiomyocytes. Cells were exposed to 5 h simulated ischemia (SI) at the mode of cell death after subsequent reperfusion (SI/R). Here, we address the role
pHo 6 and 7.4, alone or followed by 4 or 8 h reperfusion at pHo 7.4 (SI/R). Cytochrome of PLA2-VI in injury and death in HL-1 mouse cardiomyocytes after 5 h SI at pHo 6.0 c release and caspase-3 activation were attenuated after acidic compared to neutral SI, and 7.4 followed by 4 or 8 h reperfusion (SI/R), and ask whether pHo regulates PLA2- while necrotic death was similar in the two conditions. Both neutral and acidic SI were VI in SI/R. HL-1 cardiomyocytes expressed several splice variants of PLA2-VIA associated with reversible loss of F-actin and cortactin integrity. Inhibition by EIPA and -VIB. The PLA2-VIA mRNA level was reduced during SI and upregulated after of the Na+/H+ exchanger NHE1 which has been implicated in cell injury and death acidic yet not after neutral SI/R. Partial perinuclear translocation of PLA2-VIA was in SI/R slightly reduced stress fibre content yet had no significant effect on necrosis seen after neutral yet not after acidic SI/R. As previously shown, SI/R induced necrotic
after SI at pHo 6 or 7.4. NHE1 mRNA and protein levels were reduced and subcellular death, cytoskeletal disruption, and pHo-dependent caspase-3 activation, mitochondrial localization of NHE1 disrupted in SI. The NHE1 mRNA level was upregulated fragmentation, and cytochrome c release. Inhibition of PLA2-VI activity by bromoenol after acidic SI, yet not neutral SI. In conclusion, the mode of cell death and NHE1 lactone (BEL, 10 μM) disrupted F-actin, cortactin, and mitochondrial integrity. BEL
expression were dependent on pHo during SI, yet NHE1 activity did not contribute treatment elicited necrosis in normoxia and after acidic, yet not after neutral SI. In to SI/R induced necrosis. Necrosis-independent roles of NHE1 in control of death/ conclusion, PLA2-VI expression, localization, and role in cell death in SI/R are
survival balance in SI/R are under investigation. sensitive to pHo during SI.
P1AM-18-3 P1AM-18-4 ROLE OF ADENOSINE RECEPTOR 3 IN ALTERATIONS OF CARDIAC CONNEXIN 43 UNDER CARDIOMYOCYTE APOPTOSIS AND HYPOXIC STRESS: ROLES OF ACTIVATOR OF G EXTRACELLULAR MATRIX ACCUMULATION PROTEIN SIGNALING 8 AND G-βγ Guangyi Bai, Eun Jung Lee, Lak Shin Jeong, Hunjoo Ha Jiao Qibin, Motohiko Sato, Hiroko Suzuki, Reiko Kurotani, College of Pharmacy and Division of Life & Pharmaceutical Sciences, Yoshihiro Ishikawa Ewha Womans University, Seoul, Korea Cardiovascular Research Institute, Yokohama City University School of Medicine, Japan Adenosine, released rapidly during cardiac ischemia, is a potent cardioprotective mediator. Adenosine receptors are G protein-coupled receptors and subdivided into 4 Connexin 43 (CX43) is a less selective channel of cardiomyocytes exchanges small molecules. Recently, we identified CX43 was associated with Activator of G-protein types: A , A , A , and A . The specific aim of our present study was to evaluate the 1 2A 2B 3 signaling 8 (AGS8) involved in hypoxia-induced apoptosis of neonatal cardiomyocytes effect of newly synthesized selective A3 adenosine receptor (A3AR) agonists (LJ1867 (NCM). Here, we characterized a novel regulation of CX43 by AGS8 and its partner and LJ1869), 4`-thionucleoside analogues, on H9c2 cardiomyocyte apoptosis and Gβγ under hypoxic stress. Phosphorylations of CX43 (Ser262, Tyr265, Ser368) were extracellular matrix (ECM) accumulation. The effect was compared with 2-chloro- stimulated by expressed AGS8-Gβγ in cells, however, attenuated by kinase inhibitor,
N6-cyclopentyladenosine (CCPA), a well known cardioprotective A1AR agonist. H2O2 staurosporine. Phosphorylated CX43 (P-CX43) was internalized following exposure increased annexin V staining in a dose dependent manner. Angiotensin II and TGF-β1 of NCM to repetitive hypoxia (R-HP) (30min 1% O2 in 30min interval, 3times). Poster Session significantly increased fibronectin (ED-A) and collagen I protein expression. LJ1868 This event was blocked by treatment of staurosporine, Gβγ inhibitor or AGS8siRNA, and LJ1869 inhibited H O -induced annexin V staining in a dose dependent manner. suggesting involvement of AGS8-Gβγ mediated phosphorylation in internalization 2 2 of CX43. Requirement of proteasome inhibitor to visualize internalized P-CX43 Both LJ1868 and LJ1869 at 1 μM inhibited H O - induced apoptosis and angiotensin 2 2 suggested that phosphorylation led to internalization and degradation of CX43 under II- and TGF-β1-induced fibronectin (ED-A) and collagen I upregulation as much as hypoxic stress. Permeability of CX43 determined by dye transfer (Lucifer Yellow) 1 μM CCPA. RT-PCR revealed A3AR mRNA expression in H9c2 cells. These results into NCM was clearly decreased following R-HP, however, AGSsiRNA blocked it. demonstrate that A3AR plays a protective role in apoptosis and ECM accumulation in These data indicated unrecognized regulation of CX43 by AGS8-Gβγ. Such regulatory
cardiomyocytes and suggest A3AR agonists as a new cardioprotective agent. mechanism may influence survival of cardiomyocytes under hypoxic stress.
158 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-18-5 P1AM-18-6 REQUIREMENT OF RECEPTOR-INDEPENDENT EFFECT OF HYPOXIA AND GLUCOSE DEPRIVATION G-PROTEIN ACTIVATOR, ACTIVATOR OF G PROTEIN ON AMPK ACTIVITY IN HUMAN LUNG EPITHELIAL SIGNALING 8 FOR HYPOXIA-INDUCED APOPTOSIS OF CELL LINES CARDIOMYOCYTES Chong Da Tan, Hiten K Patel, Deborah L Baines Motohiko Sato1, Takashi Honda1, Qibin Jiao1, Reiko Kurotani1, Eiji Toyota2, Division of Basic Medical Sciences, St. George's University of London, UK Stephen M Lanier3, Yoshihiro Ishikawa1 1 We previously showed that pharmacological activation of adenosine Cardiovascular Research Institute, Yokohama City University School of monophosphate activated protein kinase (AMPK) inhibited Na+ transport in Medicine, Japan, 2Cardiology, Kyokuto Hospital, Japan, 3Pharmacology, MUSC, Charleston, USA H441 human airway epithelial cells. Exposure to physiological changes such as hypoxia (3% O2, 5% CO2) for 1 hour or glucose deprivation (< 0.4 mM) Activator of G-protein signaling 8 (AGS8) is a novel receptor-independent G-protein for 2 hours increased phospho/total acetyl CoA carboxylase (ACC) (a target activator identified from a cDNA library of rat hearts subjected to repetitive transient ischemia. AGS8 was up-regulated in the ischemic myocardium and regulated Gβγ signaling for AMPK mediated phosphorylation) as analysed by western blotting from in cell. Induction of AGS8 may suggest involvement of AGS8 in cellular events determining 0.06 ± 0.04 to 1.02 ± 0.11 and 0.93 ± 0.3 to 3.5 ± 0.8 densitometry units, P < survival or death of cells in the face of ischemia. To address this issue, we examined 0.05, n = 4 and n = 3, respectively in H441 cells. In addition, phospho/total influence of AGS8 on survival of cultured neonatal cardiomyocyte (NCM) following AMPK was correspondingly increased by both treatments. STO-609 (53 exposure of cells to hypoxia (6 or 24 h, 1%O2) or hypoxia(6h)/reoxygenation(18h). μM) an inhibitor of CaMKK (an upstream kinase of AMPK) inhibited basal Interestingly, AGS8siRNA completely blocked apoptosis induced by both of hypoxic phospho/total AMPK from 1.7 ± 0.5 to 0.5 ± 0.05 densitometry units, P = protocols, suggesting a requirement of AGS8 for hypoxia mediated death of NCM. AGS8 0.01, n = 4 but had no effect on basal phospho/total ACC. In the presence of was enriched in the cell-cell interfaces and assembled the complex with channel protein STO-609, an apparent rise in phopho/total AMPK and ACC remained after connexin 43 (CX43). AGS8 stimulated phosphorylation of CX43 in a Gβγ dependent manner and influenced the level of CX43. AGS8 also influenced intracellular distribution hypoxia and glucose deprivation but the effect of did not reach significance of CX43 under repetitive hypoxia. These data indicated requirement of AGS8 for apoptosis n = 3. These data indicate that glucose deprivation and hypoxia result in of NCM and regulation of CX43 under hypoxic stress. AGS8-mediated regulation of CX43 activation of AMPK in H441 cells and that CaMKK may play a role in may reorganize cells to be sensitive for hypoxic stress and play a critical role in the fate of phosphorylation of AMPK in these cells. NCM. Supported by the BBSRC and St George’s University of London.
P1AM-18-7 P1AM-18-8 OXYSIN, 2-OXOGLUTARATE AND IRON DEPENDENT ESSENTIAL ROLE OF P38 MAPK IN CASPASE- DIOXYGENASE FAMILY, FUNCTION IN HYPOXIC CELL INDEPENDENT, PHOSPHOLIPASE A2-DEPENDENT DEATH PATHWAY CELL DEATH UNDER HYPOXIA/LOW GLUCOSE Ken Saito, Masayuki Matsushita CONDITIONS 1 2 1 1 Brain Stroke Group, Mitsubishi Kagaku Institute of Life Sciences, Japan Mamoru Aoto , Koei Shinzawa , Yoji Suzuki , Nobutaka Ohkubo , Noriaki Mitsuda1, Yoshihide Tsujimoto2 It has been known that HIF-1alpha plays a central role as the regulator 1Department of Physiology, Graduate School of Medicine, Ehime of hypoxic response, which was modulated by the PHDs and FIH of 2 University, Japan, Laboratory of Molecular Genetics, Department of hydroxylases family. These hydroxylases belong to the 2-oxoglutarate Medical Genetics, Osaka University Medical School, Japan and iron dependent dioxygenases and are the key molecules for cellular adaptation in the change of oxygen concentration. In order to the discovery The mechanisms of cell death induced by hypoxia or ischemia are not yet fully understood. We have previously demonstrated that cell death induced of the genes for hypoxic cell survival, we performed a cell based screening by hypoxia/low glucose occurs independently of caspases, is associated with using the siRNA libraries of the 2-oxoglutarate and iron dependent nuclear shrinkage, and is mediated by phospholipase A2 (PLA2). dioxygenases. In 24 target genes of this family, the cell silencing Oxysin gene Here, we show that p38 MAPK is activated under hypoxia/low glucose showed the resistance for OGD cell death. Furthermore, we have used cDNA conditions. A selective inhibitor of p38 MAPK or reduction of p38α protein microarray analysis of the Oxysin knock out cells to search for changes in prevents hypoxia/low glucose-induced cell death. The p38 MAPK inhibitor gene expression. We revealed that p11 mRNA level dramatically decreased in abolishes PLA2 activation by hypoxia/low glucose, indicating that p38 Oxysin knock out cells. In hypoxia, Oxysin wild type cells increased the p11 MAPK acts upstream of PLA2. The antioxidant N-acetyl-cysteine inhibits mRNA level and overexpression of p11 induced cell death. Taken together, activation of p38 MAPK and cell death induced by hypoxia/low glucose, it is suggested that Oxysin is related to the hypoxic cell death through the indicating that reactive oxygen species (ROS) are responsible for p38 MAPK regulation of p11 expression. activation. These results demonstrate that the ROS -> p38 MAPK -> PLA2 signaling axis has a crucial role in caspase-independent cell death induced by hypoxia/low glucose stress.
P1AM-18-9 P1AM-18-10 EFFECTS OF QUERCETIN ON APOPTOSIS AND iNOS HYPOXIA PROMOTES EXERCISE-MEDIATED GENE EXPRESSION ACCOMPLISHED WITH REAL ANTITUMOR CYTOTOXICITY OF NATURAL KILLER TIME PCR ON RAT KIDNEY CELL 1 2 3 4 Aysegul Kucuk , M.Kenan Kinaci , Tulay Koken , Murat Tosun , Jong-Shyan Wang 5 6 Deniz Uren , Nilufer Erkasap Graduate Institute of Rehabilitation Science and Center for Healthy Aging 1 2 Department of Physiology, Dumlupinar University, Turkey, Department Research, Chang Gung University, Taiwan of Physiology, Eskisehir Osmangazi University, Turkey, 3Department of Biochemistry, Afyon Kocatepe University, Turkey, 4Department of Histology, This study was to investigate how hypoxic exercise affects natural killer cell (NK) Afyon Kocatepe University,Turkey, 5ATQ Biotechnology,Turkey, 6Department of subset mobilization and anti-nasopharyngeal carcinoma (NPC) cytotoxicity of NK. Physiology, Eskisehir Osmangazi University,Turkey Fifteen sedentary males randomly engaged in two normoxic exercise [i.e., strenuous The aim of this study is to evaluate the effects of quercetin on apoptosis and iNOS gene exercise (SE) and moderate exercise (ME)] and two hypoxic exercise (i.e., ME while expression on kidney in I/R induced rats. exposed to 12%O2 and 15%O2), as well as, exposed to two hypoxic conditions (i.e., 42 Sprague-Dawley rats were divided in 3 groups. Control group, I/R group(2 hours resting while exposed to 12%O2 and 15%O2). Results showed that normoxic SE, ischemia-6 hours reperfusion) and Quercetin+I/R group (50 mg/kg quercetin, i.p, 1 hour but not ME, elicited a large mobilization of senescent/activated NKs into the blood, before the ischemia). MDA and GSH levels were analyzed by biochemical methods. MDA levels were whereas NK count and perforin/granzyme B/interferon-gamma levels, NK-NPC significantly decreased in quercetin group compaired to the I/R group, however GSH binding and NK-induced CD95 expression and PS exposure on NPC were enhanced levels were increased with quercetin treatment according to the I/R group. Histological in response to the SE. Exposure to 15%O2 or 12%O2 did not induce a redistribution evaluation of hematoxylene eosine stained tissue sections in the I/R group showed edema of blood NK subset populations, and the two hypoxic exposures also unaltered in vascularisations and local areas of inflammatory cell infiltration. The number of apoptotic terms of NK-induced NPC apoptotic responses. However, both 15%O2 and 12%O2 cells in the Quercetin+I/R group was determined to be less than the apoptotic cells in the hypoxic ME increased NK cytotoxic protein levels, NK-NPC binding and anti-NPC I/R group. iNOS gene expression analysis by RT- PCR showed that the expression of iNOS cytotoxicity of NKs, whereas only 12%O hypoxic ME simultaneously enhanced gene (target gene) was increased in the I/R group, but when the concentration was calculated 2 relative to the GAPDH (reference gene), the differences were not statistically significant. senescent/activated NKs mobilization into the bloodstream. We conclude that severe Quercetin, decreased apoptotic cells according to I/R group, so these data revealed that hypoxia simultaneously increases NK subset mobilization and anti-NPC cytotoxicity quercetin has protective effects on renal tissue I/R injury. of NK during exercise. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 159 P1AM-18-11 P1AM-18-12 POSSIBLE PROTECTIVE EFFECT OF CELL SWELLING- PHOSPHORYLATION OF p53 AT Ser15 AND Ser20 INDUCED PEPTIDE SECRETION AGAINST ISCHEMIA INDUCED BY HYPOXIA REGULATES THE GADD45β REPERFUSION INJURY GENE PROMOTER IN CARDIOMYOCYTES 1 1 2 2 Vladimir Strbak , Zuzana Bacova , Jana Galcikova , Jana Matejikova , 1 1 1 1 2 1 1 3 Mi-Young Kim , Eun Ji Seo , Dong Ha Lee , Young-Ae Kim , Eun Joo Kim , Martina Orecna , Roman Hafko , Tana Ravingerova 3 3 3 1 1 Hye Soo Kim , Hea-Young Cho , Eun Yong Chung , Chang-Hyun Moon , Dept Neurohumoral Regulations, Institute of Experimental Endocrinology, 1 1 1 2 Soo Hwan Lee , Eun Joo Baik , Yi-Sook Jung Slovak Academy of Sciences, Slovakia, Institute of Cardiovascular Research, 1 2 3 Department of Physiology, School of Medicine, Ajou University, Korea, Korea Slovak Academy of Sciences, Bratislava, Slovakia, Institute of Cardiovascular 3 Research Slovak Academy of Sciences, Bratislava, Slovakia Institute of Toxicology, KRICT, Yusong, Taejon, Korea, Korea Food and Drug Swelling-induced peptide secretion represents cellular reaction when material stored Administration, Tongilro, Eunpyeong-gu, Seoul, Korea in secretory vesicles is expulsed from various types of cells. Dynamics of secretion is In this study, we performed DNA microarray to identify patterns of expression changes indistinguishable from that induced by specific secretagogue. It possesses, however, in genes, especially focusing on apoptotic genes, in both in vivo rat heart and in vitro specific features indicating unique signaling pathway: it bypasses Ca2+ involving cardiomyocyte models of transient ischemia/hypoxia, and we found that three apoptotic steps and conventional intracellular signal mediators and is resistant to physiological genes, ATF3, GADD45β and DDIT4, were up-regulated in both in vivo and in vitro models. inhibitors. Pathophysiological implications: shift to anaerobic glycolysis and The results from the transfection of H9c2 cells with Gadd45β siRNA and GADD45β plasmid production of metabolites in ischemia increase intracellular osmolarity producing suggested the potential role of GADD45β as an important mediator of myocardial ischemic cell swelling. Products released after swelling could participate in the development injury. To evaluate whether Gadd45β is a transcriptional target of p53, we examined effects of ischemia injury; the others could be mediators of local or remote preconditioning of p53 on Gadd45β-Luc, reporter of Gadd45β. Gadd45β-Luc expression in H9c2 cells was when factors released at the place of ischemia have protective effect. Perfusion of enhanced by overexpression of p53 in a dose-dependent manner, but not by transfection isolated rat heart with hyperosmolar high glucose medium followed by washout with mutant p53, p53(S15A) or p53(S20A). Furthermore, the mRNA and protein levels with isosmolar medium (relative hyposmotic stress) prior to ischemia substantially of Gadd45β were increased by overexpression of p53 in a dose-dependent manner. In decreased postischemic contractile dysfunction and size of myocardial infarction. conclusion, the present study reveals that the transcriptional factor p53 transactivates Protective effect of factors released during washout period might participate in the downstream effector gene, GADD45β. GADD45β protein products may mediate apoptotic mechanism of preconditioning. Supported by projects: VEGA 2/6158/26, APVV cell death. This work was supported by Brain Korea 21 for Molecular Science and 0235-06, 2/0173/08, APVV VVCE-0064-07 CENDO and CEKVY. Technology, Ajou University and grant from the Korea Food and Drug Administration.
P1AM-18-13 P1AM-18-14 HYDROGEN-RICH SALINE PROTECTS AGAINST HYDROGEN-RICH WATER THERAPY ON NEONATAL INTESTINAL ISCHEMIA/REPERFUSION INJURY IN HYPOXIA-ISCHEMIA RAT: SHORT AND LONG-TERM RATS EFFECTS Xuejun Sun1, Yuan Hongbin2, Mao Yanfei3, Sun Xuejun1 Xuejun Sun, Sun Xuejun, Cai Jian Mei, Kang Zhimin 1Diving Medicine, Second Military Medical University, China, 2Department Diving Medicine, Second Military Medical University, China of Anaesthesiology, Changzheng Hospital, Second Military Medical Cerebral hypoxia/ischemia (HI) represents a major cause of brain damage in University, Shanghai, China, 3Department of SICU, Xinhua Hospital, the term newborn. This study aimed to examine the short and long-term effect Shanghai Jiaotong University School of Medicine, Shanghai, China of hydrogen-rich normal saline water therapy in an established neonatal HI rat pup model. Seven-day-old rat pups were subjected to left common carotid Hydrogen gas was reported to reduce reactive oxygen species and alleviate ο cerebral, myocardial and hepatic ischemia/reperfusion (I/R) injuries. We studied artery ligation and then 90 min hypoxia (8% oxygen at 37 C). H2 saturated the effect of hydrogen-rich saline, which was easier for clinical application, on water was administered by peritoneal injection (5 ml/kg) immediately and again 8 h after HI insult. 24 h after hydrogen-rich water treament, the intestinal I/R injury. In male Sprague Dawley rats, intestinal injury was the pups were decapitated and brain injury was assessed by2,3,5- induced by clamping the superior mesenteric artery for 45 minutes, followed by triphenyltetrazoliumchloride (TTC), Nissl, and TUNEL staining, caspase-3 120 min reperfusion. Hydrogen-rich saline or vehicle physiological saline (5 activity, content of MDA as well as Iba-1 immunochemistry in the brain. ml/kg) was administered via intravenous infusion 10 minutes before reperfusion, 5 w after therapy, the long-term effects were assessed by the function test, respectively. The intestine damage was detected microscopically and was Spontaneous activity test, and morris water maze test. The results indicated assessed by Chiu score system after I/R injury. In addition, serum DAO activity, that proper amount of hydrogen-rich water therapy reduced the infarct ratio tissue MDA and MPO activity, serum TNF-α, IL-1β and IL-6 levels were all and increased the number of survival cells via suppressing the caspase increased significantly by I/R injury. Hydrogen-rich saline reduced these tissue activity, inhibiting oxidation and inflammation. This strategy also improved injury markers and relieved morphological intestinal injury. In conclusion, the long-term function of brain which proved that peritoneal injection of Hydrogen-rich saline protected the small intestine against I/R injury possibly by hydrogen-rich water was a good practice in the clinical treatment of HI or reduction of inflammation and oxidative stress. other ischemia-related diseases.
P1AM-19-1 P1AM-19-2 EFFECTS OF S-ADENOSYLHOMOCYSTEINE AND BEHAVIOR OF INTRACELLULAR VESICLE AND HOMOCYSTEINE ON DNA DAMAGE AND CELL CYTOSKELETON AFTER AMPHOTERICIN B VIABILITY IN MURINE HEPATIC AND MICROGLIA CELL TREATMENT LINES Masao Miyake, Akihiro Hazama 1 2 2 2 Tsai-Hsiu Yang , Wen-Yueh Ho , Kuen-Lin Leu , Chia-Chyuan Liu Department of Cellular and Integrative Physiology, Fukushima Medical 1Department of Health and Nutrition, Chia-Nan University of Pharmacy and University School of Medicine, Japan 2 Science, Taiwan, Department of Cosmetic Science, Chia-Nan University The sodium ionophore, amphotericin B (amB) causes HeLa cells to of Pharmacy and Science, Taiwan appear necrosis with morphological change after two-hour incubation. The limited research has been performed regarding the s-adenosylhomocysteine We visualized this cell death with propidium iodide (PI), and showed (SAH)-evoked cell damage in hepatic and neuronal cells. In this study we assessed that the ion replacement of extracelluar fluid and ion channel blockers effects of SAH or homocysteine (Hcy) in hepatic and neuronal cells on cell viability could suppress necrosis. However, PI can detect cells which has cracked and DNA damage, and attempted to find the underlying influences. Cell viability cell membrane only, there was less information about what was going and DNA damage were evaluated in BNL CL.2 cell line and BV-2 cell line with on during the morphological change after amB treatment. In this study, SAH or Hcy treatment for 48 hours. Effects of SAH or Hcy on lipid peroxidation we visualized intracellular vesicles with acridine orange, lysotracker and and DNA methylation were also measured in both cell lines. SAH or Hcy dose lysosensor to measure vesicular pH level. Incubation with amB neutralized dependently inhibited cell viability and enhanced DNA damage in both types of cells. Poster Session lysosomal pH, same as that with Bafilomycin A1. At the same time, vesicles Furthermore, SAH treatment markedly increased intracellular SAH levels and DNA hypomethylation, the effect of SAH was much stronger than that of Hcy. The findings moved vigorously after 20 minutes incubation with amB. And this vesicle fluidization occurred in dose-dependent manner with amB. When Cl- was were also obtained that Hcy significantly induced lipid peroxidation. Results show - that SAH might cause cellular DNA damage in hepatic and microglia cells by DNA replaced to Gluconate in extracellular fluid, vesicle neutralization and hypomethylation, further resulting in irreversible DNA damage and decrement of fluidization were inhibited effectively, as a result, necrotic cell death was cell viability. Additionally, higher Hcy could induce cellular DNA damage through suppressed. Accordingly, we report behavior of cytoskeleton and membrane increased lipid peroxidation and DNA hypomethylation. We suggest that SAH is more trafficking with fluorescent probe here, because their failure might contribute informative to cell damage than that of Hcy in hepatic and microglia cells. to realize amB-induced necrosis.
160 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-20-1 P1AM-20-2 SEASONAL VARIATIONS IN HORMONAL ACTIONS TOXICOLOGICAL AND PHYSIOLOGICAL EFFECTS ON GLUCOSE METABOLISM IN COMMON AFRICAN OF SOME PESTICIDES ON LARVAL STAGES OF SILK TOAD-BUFO REGULARIS WORM AKIN ABDUL-RAZAQ ALADA, Hoda Metwaly Nasr GRACE OLUFUNMILAYO ISEHUNWA Department of Pest control and environmental protection, Faculty of PHYSIOLOGY, UNIVERSITY OF IBADAN, Nigeria Agriculture, Alexandria University, Damanhour branch, Egypt The effects of adrenaline, cortisol, and glucagon on the blood glucose, liver Toxicity of selected pesticides was tested against third instars larval stages and muscle glycogen during the rainy and dry seasons were studied on fasted of Bombyx mori, series of concentration were prepared according to the adult toads. The toads were administered adrenaline, cortisol, glucagon, or recommended and half recommended doses of pesticides. Mortality Percent amphibian saline respectively. Blood glucose, liver and muscle glycogen was calculated after 24 and 48 hrs of treatment. The fresh mulberry leaves were determined using glucose oxidase and anthrone reagent methods were treated with the tested pesticides. The third larval instars were fed on the respectively. Data were analyzed for statistical significance using Student treated leaves one time during its life duration. Results showed that AChE and t test. The basal blood glucose 61.8±1.4 mg per dl, during the rainy season phenoloxidase activities were inhibited in vivo and inhibition was calculated for was significantly higher than 51.6±0.8 mg per dl in the dry season. Basal the survived larval. Percentage of pupation and cocoons shells were determined. liver glycogen 325.3±27.3 mg per 100 g, and muscle glycogen, 165.5±13.7 Results indicated that diazinon was the most effective one on the percent of mg per 100 g, during rainy season were significantly less than the basal liver mortality after 24 and 48 hrs from treatment followed by chloropirifos and glycogen 757.5±89.2 mg per 100 g, and muscle glycogen 265.3±36.5 mg kingbo (diazinon was 97% , 99% ,chloropirifos was 91% , 86% while kingbo per 100 g in the dry season. Adrenaline, cortisol and glucagon produced was 59% , 63% ). Chloropirifos was the most effective one inhibited AChE greater hyperglycemia during rainy season than the dry season. The liver followed by Diazinon and kingbo. Inhibition percentage was 92%, 89% and and muscle glycogen contents of 250.5± 13.20 to 376.10± 15.46 in response 61%, respectively. On the other hand, kingbo caused a higher percentage of to the hormones during the rainy season were lower than 567.12± 23.12 to phenoloxidase inhibition followed by chloropirifos and diazinon (85%, 68% and 840. 6± 17.3 in dry season. The study showed that seasonal conditions affect 65%, respectively). In conclusion, most parameters were in agree with mode of hormonal actions on carbohydrate metabolism in toads. action of tested pesticides and were discussed through the results.
P1AM-20-3 P1AM-20-4 EFFECT OF INTERMEDIATE-FREQUENCY MAGNETIC THE OXIDATIVE STRESS STATUS OF NINE DIFFERENT FIELD ON SEED GROWTH RATE BIRD SPECIES Isao Kayano, Seiichi Mochizuki Mirella Mariia Kanerva, Miia Koivula, Tapio Eeva, Mikko Nikinmaa Department of Medical Engineering, Kawasaki University of Medical Department of Biology, University of Turku, Finland Welfare, Japan Environmentally derived stress can affect the health, survival and In our preliminary studies, it was suggested that exposure to an intermediate- reproduction of individual organisms and populations. Environmental frequency magnetic field accelerate the growth of seeds. However, no oxidative stress, i.e. generation of reactive oxygen species to such an extent detailed studies have been performed on effects of magnetic-flux density as that the antioxidant defence systems of an organism is disturbed, can be well as exposure time on seed-growth promotion. Here, we thus aimed at caused by e.g. contamination by many metal ions or enhanced toxicant investigating effects of these factors on the growth of seeds. White daikon metabolism utilizing the Ah-receptor pathway. Some natural compounds radish sprouts seeds were used for hydroponic cultivation under exposure using this pathway may also generate free radicals and cause oxidative stress, to the 25 kHz magnetic field (10 or 20 μT), generated by a Helmholtz coil. Three groups were studied: 1) ‘pre-sprout exposure’ group was exposed which can thus be generated by changes in food web and or food utilized. to the magnetic field for 48 hours from the beginning, 2) ‘post-sprout Both are affected by, e.g., climate change. exposure’ group started to be exposed after 48 hours, and 3) ‘control’ group Information about species differences of antioxidant defence systems which was without any exposure (n=58 in each group). The average growth rates may result from different foods utilized are completely lacking. Because between 48 and 78 hours were calculated and compared among these groups. of this, we studied the oxidative stress status of nine different passerine The growth rates in the ‘pre-sprout exposure’ group were significantly higher bird species with different feeding and migration habits. The following (1.43±0.58 mm/h, mean±SD; p<0.001) than other two groups for 10 μT (0.89 parameters were measured: the ratio of reduced and oxidized glutathione, ±0.44 mm/h) and 20 μT (0.87±0.45 mm/h). Therefore, it is suggested that glutathione metabolism related enzymes, catalase, superoxide dismutase and the exposure to intermediate-frequency magnetic fields before sprout have EROD activities. Different species groups, e.g., granivores and insectivores retentive accelerating effects on the seed growth rates even after cessation of as well as migratory birds and non-migratory birds, were compared to get an exposure. indication of how these life history traits affect the redox systems of birds.
P1AM-20-5 P1AM-20-6 NF-KB MEDIATES THE DIVERSITY OF CELLULAR FLUORIDE INTOXICATION INDUCED OXIDATIVE IGF-I/IGFBP-1 EXPRESSION BY HYPOXIA IN TIBETAN DAMAGE AND BIOCHEMICAL PERTURBATIONS IN PLATEAU MAMMALS AND LABORATORY MICE RATS: ROLE OF GLUTATHIONE AND CURCUMIN Chen Xue-Qun, Chen Xue-Qun, Wang Shi-Jun, Liu Yu, Fatma Mohamady Eldemerdash, Mokhtar Ibrahim Yousef Mao Jin-Yan, Du Ji-Zeng Department of Environmental Studies, Institute of Graduate Studies and Research, Alexandria University, Egypt Physiology, Zhejiang University, China Ochotona curzoniae(O.c), Microtus oeconomus(M.o) and Myospalax baileyi(M. Oxidative damage is involved in fluoride-related toxicity. The present study b) are native mammals at Tibetan plateau in China. We address NF-kB involvement examined protective effects of glutathione and curcumin against sodium fluoride in hypoxia-induced diversity of IGF-I/IGFBP-1 expression in the mammals. (NaF) toxicity in male rats. Seven rats per group (and a control group) were IGF-I/IGFBP-1 of brain and liver was tested 6h after CoCl2 or mixed gas hypoxia. exposed to 300 mg glutathione/kg BW, 15 mg curcumin/kg BW; 10 mg NaF/kg PDTC(NF-κB inhibitor) was pre-injected before hypoxia. Results were: Liver IGF-I BW; glutathione plus NaF (300 mg/kg BW+ 10 mg/kg BW) and curcumin plus in mice, M. o, and M. b was increased dramatically, but not in O.c; IGFBP-1 in mice, NaF (15 mg/kg BW+10 mg/kg BW), respectively. Rats were orally administered O.c, and M.b markedly enhanced, not in M.o; A pre-injection with PDTC before their respective doses daily for one month. Results showed that NaF increased hypoxia reversed hypoxia-enhanced liver IGF-I in mice, M.o, and M.b; A pre-injection TBARS in plasma and organs. While, the activities of GST, SOD and the content of PDTC also reversed the hypoxia-enhanced IGFBP-1 both in O.c and M.b, partly in of sulfhydryl groups were decreased in plasma and organs. The activities of mice; hypoxia only increased IGF-ImRNA in brain of O.c but not in mice and M.o; phosphatases, aminotransferases, lactate dehydrogenase were decreased in mixed gas hypoxia did not change IGF-I levels of brain of all mammals and CoCl2- liver and testes due to NaF administration, whereas increased in plasma. Also, hypoxia only reduced mice brain IGF-I; Both types of hypoxia stimulated hepatic acetylcholinesterase was decreased. NaF caused an increase in urea and bilirubin NF-kB nucleus translocation of mice and M.b, and NF-kB was higher in M.b than in plasma. On the other hand, NaF decreased plasma creatine kinase, total protein in mice. The data suggest that the diversity (a “multi-model”) of IGF-I/IGFBP-1 and albumin. Glutathione and curcumin alone or in combination with NaF expression in response to hypoxia and NF-κB involves in transcriptional regulation. reduced the levels of TBARS and increased antioxidant enzymes activities. It This work was supported by the NSFC (30770807,30870300) and Program “973” (No. can be concluded that glutathione and curcumin have antioxidant and beneficial 2006CB504100). influences in protecting against the harmful effects of NaF. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 161 P1AM-20-7 P1AM-20-8 HYPOXIC RESEARCH AND MOUNTAIN MEDICINE IN AMELIORATION OF ACUTE TUBULAR NECROSIS IN CHINA ISCHEMIC ACUTE RENAL FAILURE WAS IMPAIRED Ming Fan IN MICE LUCKING HYPOXIA INDUCIBLE FACTOR-1α Department of Brain Protection and Neural Plasticity, Institute of Basic GENE Medical Sciences, China Shuhei Tomita, Kunihisa Yamaguchi, Keisuke Ishizawa, Hypoxic Research and Mountain Medicine are specially interested in Yuko Imamura, Yayoi Fukuhara, Koichiro Tsuchiya, China. Studies are taken out on various levels. At the level of Colonia, epidemiologic surveys on different reaction to hypoxia of plateaus resident Toshiaki Tamaki and people from plains showed that the former group has better adaptability Department of Pharmacology, University of Tokushima, Japan concerning acute hypoxia, but overcompensation-induced chronic mountain Renal responses in the hypoxic microenvironment under progression of ischemic renal sickness (CMS) existed. International Society of High Altitude Medicine injury are still not well understood, although the characteristics of the cellular response agreed with our diagnostic criteria as QINGHAI score. Measures promoting in kidney likely influence the resulting pathologic alterations. Hypoxia inducible acclimation in person involved in the Tibet railroad construction have also factor-1α (HIF-1α) is a transcription factor that regulates gene expression in response been carried out. We got zero mountain sick mortality in 5 years. At the to hypoxia to maintain physiological oxygen homeostasis. To investigate how HIF- individual level, studies on neuro-endocrine mechanisms related to hypoxic 1α contributes to the progression of ischemic renal injury, we examined the kidneys acclimation and adaptation demonstrating the critical role of CRF in this induced by renal ischemia-reperfusion injury (IRI) model in heterozygous mice process. Studies concerning hypothermic and hypoxic combined actions lacking Hif-1α gene. The mutant mice showed a prolonged elevation of BUN levels showed that acclimation to hypoxia exacerbates hypothermic injury. At the up to 72hr after initiation of the reperfusion, compared to the control mice. Consistent cellular level, studies focused on energy-consuming ion channels, energy- with this observation, morphological analysis showed that tubular injury score 72hr producing mitochondria and signal pathways. At the molecular level, we after IRI was also elevated in the mutant mice. The number of proliferating cells focused on oxygen-carrier proteins and molecular markers involved in evaluated by the expression of Ki-67 was decreased in the mutant kidney, compared hypoxic acclimation. Future interests may involve the the cognition-related to that in the controls. These results suggest that HIF-1α has protective role against research, de-acclimatization mechanisms and intervention. ischemia of the kidney and may contribute to a recovery from acute tubular necrosis.
P1AM-20-9 P1AM-20-10 EFFECTS OF REPEATED LIGHT ACCELERATION EXISTENCE OF THE NEURONS RESPONDING TO EXPOSURES ON BRAIN TISSUE-OXYGEN BAROMETRIC PRESSURE DECREASE IN THE CONCENTRATION IN RATS VESTIBULAR NUCLEI OF RATS Satoshi Maruyama1, Syuhei Tokuyama2, Tomoko Manabe3, Tomohumi 1 1 2 4 1 1 1 Megumi Funakubo , Jun Sato , Kazue Mizumura Takahata , Kazuo Kato , Yoshiaki Sato , Takehito Kemuriyama , 1 1 1 1 Futuristic Environmental Simulation Center, Res. Inst. Environ. Med., Megumi Tandai-Hiruma , Hiroyuki Ota , Yasuhiro Nishida Nagoya Univ., Japan, 2Div. Stress Recognition and Response, Res. Inst. 1 2 Department of Physiology, National Defense Medical College, Japan, Medical Environ. Med., Nagoya Univ., Japan department, Oita University, Japan, 3Nursing school, National Defense Medical College, Japan, 4Medical squadron, Central AC & W Air Base Group, ASDF, Complaints of chronic pain patients often increase when the low-pressure system Japan approaches. We have demonstrated that artificially lowering the barometric pressure Whether or not light acceleration exposure could increase the +Gz tolerance was (LP) within the range of weather change intensifies pain-related behaviors in investigated. Male Sprague-Dawly rats were randomly assigned the +Gz-exposure (Exp) neuropathic rats. Furthermore, chemical lesion of inner ear organs inhibited this group and control (Ctrl) group. All rats were anesthetized with pentobarbital sodium for aggravating effect, suggesting that the inner ear is involved in detecting LP. In the the conditionings, which were the exposures to +Gz stress (+1.5Gz, 5 min, three times per present study, we extracellularly recorded neural activities in the vestibular nuclei day, 5 days over 9 days) by a centrifuge for the Exp-group, or the same procedures without with a glass microelectrode and examined the effect of LP (40 hPa/ 8 min) in normal +Gz exposure for the Ctrl-group. Two days after the all conditionings, the effects on arterial anesthetized rats. A set of stimuli (electrical stimulation of the vestibular nerve, rotation pressure at the level of the brainstem (APLB), central venous pressure (CVP), heart rate of the body or caloric test) was also applied to characterize neurons. Location of the (HR), and the brain-tissue PO2 levels in the cortex and the hippocampus were estimated. neurons was histologically confirmed after the experiment. Seven out of 20 recorded No significant difference was observed in these values between both groups before the vestibular neurons increased their discharge frequency upon LP. There were three conditionings. After the conditionings, APLB and cortical PO during +3Gz stresses in Exp 2 response patterns: 1) discharge rate increased along LP and vice versa, 2) discharge rats were significantly higher than those in Ctrl rats. Hippocampal PO2 during +3Gz stresses in Exp rats was, however, lower than that in Ctrl rats, even though no significant different increased along LP but remained increased upon normalization of the pressure, 3) was found in CVP or HR between the two groups. These results suggest that repeated fluctuating discharge frequency was stabilized during LP. Our present results indicate acceleration exposures could increase +Gz tolerance although the effect of the conditionings that the vestibular neurons have an important role in detecting barometric pressure on the brain may be dependent on the brain areas. change
P1AM-20-11 P1AM-20-12 INCREASED DERMAL OXYGEN PRESSURE INVESTIGATION OF GENOTOXICITY OF ULTRAVIOLET ATTENUATES ULTRAVIOLET B-INDUCED DERMAL LASER IRRADIATION WRINKLE FORMATION Osvaldas Ruksenas1, Vaidotas Morkunas2, Mikas Vengris3, 1 2 1 1 Shigeo Kawada , Naokata Ishii , Masaru Ohtani , Chiho Fukusaki Romas Danielius3, Egle Danieliene4 1Department of Human and Engineered Environmental Studies, The 1 2 Department of Biochemistry-Biophysics, Vilnius University, Lithuania, University of Tokyo, Japan, Department of Life Sciences, The University 2Department of Botany-Genetics, Vilnius University, Lithuania, 3Department of Tokyo, Japan of Quantum Electronics, Vilnius University, Lithuania, 4Private Practice, Dermal aging includes physiological aging and photoaging. Of these, the latter is Lithuania considered to primarily contribute to dermal aging characterized by wrinkle formation. Ultraviolet (UV)-mediated DNA damage in various tissues has been This study reports that increased dermal oxygen pressure attenuates ultraviolet B (UVB)-induced wrinkle formation. Twenty-four male albino hairless HOS:HR-1 well documented. However, research on the damaging effect of UV-laser mice (8 weeks old) were divided into three groups: control, exposure to UVB (UVB), irradiation on the DNA of live organisms is scarce, even though this is of and exposure to UVB followed by hyperoxia (UVB+HO). The UVB and UVB+HO interest because UV-lasers increasingly are used in cosmetics and surgical groups were exposed to UVB irradiation 3 times/week for 5 weeks, and after each treatment. In this study we investigated the effect of new UV femtosecond irradiation, only the UVB+HO group was exposed to 90% O2 for 90 min. The crystalline laser irradiation on the DNA of murine bone marrow cells using control group was exposed to neither UVB nor hyperoxia. The UVB and UVB+HO
Poster Session Comet assay. Results show strong genotoxical impact of laser irradiation. groups developed prominent dermal wrinkles at the end of the experimental period. Even exposure to the smallest investigated intensity irradiation (0.105 J/cm2) In addition, the dermal wrinkle severity and dermal vessel density were higher in caused extremely significant (P < 0.0001) increase in DNA strand breaks of the UVB group compared with the control and UVB+HO groups. The vascular endothelial growth factor (VEGF) protein concentrations in the UVB and UVB+HO bone marrow cells: mean ± SEM tail moment scores (10 gels per treatment, groups showed significant increases compared with the control group. Moreover, the with 100 irradiated cells scored per gel) was 23.85 ± 3.64 compared with 2.38 UVB group tended to have higher VEGF concentrations compared with the UVB+HO ± 0.51 for controls. Investigation of effects of different intensity irradiation group. Therefore, these results suggested that increased dermal oxygen pressure showed dosage-effect relationship. It was distorted by highest intensity attenuates the dermal photoaging. irradiation (4.2 J/cm2) that caused mass degradation of DNA.
162 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-20-13 P1AM-20-14 EFFECTS OF ZINC-DEFICIENCY ON THE EFFECTS OF DIHYDROCAPSAICIN AND CAPSAICIN DISTRIBUTION OF RAT WHITE BLOOD CELLS ON THE DISTRIBUTION OF WHITE BLOOD CELLS IN Yu Kawashima1, Yui Someya1, Jun Tanihata1, Shogo Sato1, RATS 1 1 1 1 Fuuun Kawano1, Ken Shirato1, Mariko Sugiyama1, Sachiko Shunta Akimoto , Jun Tanihata , Fuuun Kawano , Shogo Sato , 1 1 1 2 Nomura2, Kaoru Tachiyashiki3, Kazuhiko Imaizumi1 Yumi Takei , Ken Shirato , Yui Someya , Sachiko Nomura , 3 1 1Faculty of Human Sciences, Waseda University, Japan, 2Graduate School Kaoru Tachiyashiki , Kazuhiko Imaizumi 3 of Medicine, Osaka University, Japan Department of Living and Health 1Faculty of Human Sciences, Waseda University, Japan, 2Graduate School Sciences, Joetsu University of Education, Japan of Medicine, Osaka University, Japan, 3Department of Natural and Health Zinc is known to play an important role for immune-functions. However, the effects Sciences, Joetsu University of Education, Japan of Zn-deficiency (ZnD) on immune response system are still unknown. Therefore, The acute effects of dihydrocapsaicin (DHC) and capsaicin (CAP) on the number of the effects of ZnD on the number of total white blood cells (WBCs), neutrophil, white blood cells (WBCs), neutrophil, eosinophil, basophil, monocyte, lymphocytes (T eosinophil, basophil, monocyte, T lymphocyte, B lymphocyte and NK cell were lymphocyte, B lymphocyte and NK cell), and serum corticosterone levels were studied in studied in rats. The weaned male SD rats were divided into zinc deficient diet (ZDD: rats. Male SD rats were divided into DHC (3.0 mg/kg BW), CAP (3.0 mg/kg BW) and the 0.7mg zinc/kg diet) group and the control diet (CON: 34.8mg zinc/kg diet) group, control (CON) groups. The number of total WBCs was 1.30-1.42 times higher in DHC and were pair-fed for 4 weeks. The number of lymphocyte subsets, visceral organ group than in CON group at 6-12 hours after the administration.. The number of neutrophil weights, serum zinc and corticosterone were also determined. ZnD increased duration- was 1.41-1.62 times higher in DHC group than in CON group at 6-12 hours. The number dependently the number of total white blood cells, granulocytes (neutrophil, eosinophil of lymphocytes was 0.61-0.70 times lower in DHC and CAP groups than in CON group at and basophil) and monocyte in 2-4 weeks without changing the number of T 3 hour. The number of T lymphocyte and B lymphocyte was 0.54-0.74 times lower in DHC lymphocyte, B lymphocyte and NK cell. The relative weights of thymus and adrenals group than in CON group. However, CAP did not change the number of T lymphocyte and were 0.63 times lower and 1.60 times higher in ZDD group than in CON group, B lymphocyte. There were no significant changes of the number of NK cells among three respectively. Serum corticosterone level was 1.46 times higher in ZDD group than in groups. CAP and DHC did not change the number of monocyte, eosinophil and basophil. In CON group. These results suggest that ZnD induces stress responses and the responses conclusion, capsaicinoids decreases the number of acquired immunity cells, and increases the may be in part participated in increased actions of the number of granulocytes and number of total WBCs and neutrophil without changing the number of monocyte, eosinophil monocyte during ZnD, and induces thymus atrophy and adrenal hypertrophy. and basophil. The magnitidue of these effects are relatively higher in DHC than in CAP.
P1AM-20-15 P1AM-20-16 SUBACUTE EFFECTS OF CAPSAICINOIDS ON THE ACUTE EFFECTS OF ALLYL ISOTHIOCYANATE ON NUMBER OF WHITE BLOOD CELLS AND STRESS PLASMA FFA AND GLYCEROL CONCENTRATIONS ORGAN MASS IN RATS AND THE NUMBER OF WHITE BLOOD CELLS IN RATS 1 1 1 1 Shoko Aritoshi , Jun Tanihata , Mari Kumazawa , Shogo Sato , Yuko Sakakibara1, Jun Tanihata1, Shogo Sato1, Fuuun Kawano1, 1 1 1 1 Fuuun Kawano , Yumi Takei , Takamasa Ban , Haruka Yamada , 1 1 2 1 2 1 Takamasa Ban , Yumi Takei , Kaoru Tachiyashiki ,Kazuhiko Imaizumi Kaoru Tachiyashiki , Kazuhiko Imaizumi 1Faculty of Human Sciences, Waseda University, Japan, 2Department of Living 1 2 Faculty of Human Sciences, Waseda University, Japan, Department of and Health Sciences, Joetsu University of Education, Japan Living and Health Sciences, Joetsu University of Education, Japan Wasabi is one of the popular spices grown in Japan and used as seasoning for dishes such as Capsaicinoids such as capsaicin (CAP) and dihydrocapsaicin (DHC) are responsible for sushi, sashimi and soba. The recent study showed that allyl isothiocyanate (AITC), a main up to about 90% of the total pungency of pepper fruits. Although these capsaicinoids pungent component of wasabi, increased adrenaline secretion via activation of the sensory affect immune-response system through various factors such as adrenal hormones and nerves and adrenal sympathetic nerves. However, little information is available on AITC- neuropeptides, capsaicinoids-induced change of the number and distribution of white blood induced lipolytic actions and immune response effects. In the present study, therefore, the cells (WBCs) is still unknown. Therefore, effects of CAP and DHC (3mg/kg BW/day for 10 acute effects of administration of AITC (20mg/kg BW, s.c.) on plasma free fatty acid (FFA) days, s.c.) on the number of WBCs were studied. Male adult SD rats were divided into CAP, and glycerol concentrations, and the number of white blood cells (WBCs) were studied. DHC and control (CON) groups. CAP increased the number of neutrophil and eosinophil to Male SD rats were divided into AITC and control (CON) groups. AITC increased plasma 2.14 and 2.90 times, and decreased the number of total WBCs, lymphocytes and monocyte to 0.77, 0.70 and 0.50 times, respectively, as compared with CON group. Similar tendencies FFA and glycerol concentrations to 1.24-1.27 and 1.32 times, respectively, as compared were observed in the case of DHC. No effects of CAP and DHC on the number of basophil with CON group. AITC decreased the number of total WBCs, eosinophil and lymphocytes were observed. Furthermore, CAP and DHC decreased thymus weight and increased adrenal to 0.70-0.79, 0.39-0.42 and 0.69-0.82 times, respectively, and increased the number of weight, suggesting that capsaicinoids induced thymic atrophy and adrenal hypertrophy. basophil to 1.85 times, as compared with CON group. No effects of AITC on the number These responses may be associated with the redistribution of WBCs. In conclusion, the of neutrophil and monocyte were observed. In conclusion, AITC induced lipolytic actions, capsaicinoids decreased the number of total WBCs, lymphocytes and monocyte and decreased the number of WBCs, eosinophil and lymphocytes, and increased the number of increased the number of neutrophil and eosinophil without changing the number of basophil. basophil without changing the number of neutrophil and monocyte.
P1AM-20-17 P1AM-20-18 PSYCHOEMOTIONAL PROBLEMS ON THE TO INVESTIGATE SOME AFFECTIVE FACTORS OF THE WORKPLACE: PREVALENCE AND ASSOCIATION OCCUPATIONAL FATIGUE OF THE ADMINISTRATIVE WITH CARDIOVASCULAR RISK FACTORS PERSONNEL Anna Kontsevaya, Kalinina Anna Lili Zhang1, Kejian Liu2, Fuqiang Liu2, Lingling Zeng1, Qi Ren1, Department of prevention in primary care, State research centre for Zhengyue Li1 preventive medicine, Russia 1Clinical Medicine, Tongji Medical College of Huazhong University of Objective: to study the prevalence of main psychoemotional disturbances in the Science and Technology, China, 2Institute of Occupational Medicine, Tongji working collective of research institute and its association with main cardiovascular Medical College, Huazhong University of Science and Technology, China risk factors. Methods: Survey with computer-based system of questionnaires (HADS questionnaire, We aimed to investigate some affective factors of the occupational fatigue Reeder inventory) was performed. Cardiovascular risk level was estimated by of the administrative personnel. Life Quality Comprehensive Scale of standard criteria (SCORE). The study was conducted in working collective of research Occupational Multitude was adopted to carry out questionnaires among 133 workers (n=467). cases of the administrative personnel who were working in the hospital, the Results. The prevalence of high stress by Reeder inventory was 7,8% in men and university and government organization in Wuhan City. The results showed 11,4% in women. The prevalence of high stress by self-estimate was 43,8% in men that an obvious fatigue score difference was found between men and women. and 52,2% in women. Anxiety was revealed in 36,3% men and in 38,9% women. The mean score of women (4.80±0.74, n=44) was higher than that of men Depression prevalence was 5,8% in men and 6,3% in women. Anxiety was associated (2.63±0.34, n=89). The difference had statistical significance (ANOVA, with increased body mass, abdominal obesity, low physical activity and hypertension, P<0.05). We concluded that the occupational fatigue degree might be related and as a result with high cardiovascular risk. Conclusion. The main psychoemotional disturbance in the working collective of to the gender of the administrative personnel. Women have stronger feelings research workers is anxiety. Workers do not feel the difference with stress and of fatigue or higher sensitivity than men do. But the difference has no consider all their psychoemotional disturbances as high stress. Anxiety is associated statistical significance among ages and educational levels of administrative with increased cardiovascular risk factors and increased risk. Workplace preventive personnel. It is a very important task to improve the work environment and interventions are necessary for decreasing anxiety and cardiovascular risk. reduce overstress for professional women. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 163 P1AM-20-19 P1AM-21-1 ACTIVATION OF TRANSIENT RECEPTOR POTENTIAL INVOLVEMENT OF TRPM8 IN THERMOREGULATION ANKYRIN 1 (TRPA1) BY HYDROGEN PEROXIDE INDUCED BY SKIN TEMPERATURE 1 1 1 Hiroshi Hosokawa1, Yosuke Sawada1, Shingo Maegawa1, Hiroshi Hosokawa , Koji Tajino , Shingo Maegawa , 2 1 Kiyoshi Matsumura2, Shigeo Kobayashi1 Kiyoshi Matsumura , Shigeo Kobayashi 1 1 Graduate school of Informatics, Department of Intelligence Science and Graduate school of Informatics, Department of Intelligence Science and 2 Technology, Kyoto University, Japan, 2Faculty of Information Science and Technology, Kyoto University, Japan, Faculty of Information Science and Technology, Osaka Institute of Technology, Japan Technology, Osaka Institute of Technology, Japan In mammals, a decrease of skin temperature triggers behavioural and autonomic heat- Hydrogen peroxide (H O ) contained in industrial products is also generated 2 2 gain response. The decrease of skin temperature is detected on coetaneous sensory within the cells. H2O2 causes pain, but it has not been elucidated how H2O2 nerve fiber. However, a molecule, which expresses in sensory nerve and detects activates sensory neurons in the pain pathway. Here we show that Transient the decrease of skin temperature, is still unknown. The cooling-activated cation Receptor Potential Ankyrin-1 (TRPA1), expressed by sensory neurons in the channel, Transient Receptor Potential Melastatin-8 (TRPM8), is a one-candidate 2+ pain pathway, is a receptor for H2O2. H2O2 activated TRPA1 to induce Ca molecule for skin temperature detection. Therefore, we examine the involvement of
influx and elicit non-selective cation currents. These effects of H2O2 were TRPM8 in thermoregulatory response in this study. When L-menthol, an agonist of mimicked by both reactive oxygen species and reactive nitrogen species. TRPM8, applied to skin, mice showed increase of core temperature but not TRPM8- deficient mice. L-menthol application induced a rise in oxygen consumption, Cysteine reducing agents suppressed H2O2-induced TRPA1 activation 2+ shivering-like muscle activity, tail skin vasoconstriction, and heat-seeking behavior. whereas cysteine oxidizing agents activated TRPA1. H O caused Ca influx 2 2 In a cold environment, the core temperature of TRPM8-deficent mice is decreased in a subset of dorsal root ganglia (DRG) neurons which also responded but not wildtype. TRPM8-deficent mice did not show heat-generating response of to a chemically TRPA1 ligand. TRPA1 deficient mice did not show pain brown adipocyte, tail skin vasoconstriction, and heat seeking behaviour in a cold behaviour, which caused by cutaneous injection of H2O2. These results environment. These results indicate that TRPM8 is involved in thermoregulation
indicated that TRPA1 might be involved in sensation of pain caused by H2O2. induced by a decrease of skin temperature.
P1AM-21-2 P1AM-21-3 DOUBLE TRACING OF PROSTAGLANDIN EP3 FEVER ASSOCIATED WITH INTRACEREBRAL RECEPTOR-EXPRESSING PREOPTIC NEURONS HEMORRHAGE; INVESTIGATION OF ITS MOLECULAR INNERVATING DORSOMEDIAL HYPOTHALAMUS AND MECHANISM IN A RAT MODEL ROSTRAL RAPHE PALLIDUS Aiko Hori1, Tomoko Yamamoto1, Kiyoshi Matsumura2, Yoshiko Nakamura, Kazuhiro Nakamura, Shaun F Morrison Hiroshi Hosokawa1, Shigeo Kobayashi1 Oregon National Primate Research Center, Oregon Health & Science 1Graduate School of Informatics, Kyoto University, Japan, 2Faculty of University, USA Information Science and Technology, Osaka Institute of Technology, Japan
Fever is mediated by a prostaglandin E2 action on its EP3 receptors (EP3R) located Fever is common in patients with intracerebral hemorrhage (ICH), although its
on preoptic area (POA) neurons. Previous findings suggest that the febrile signals molecular mechanism is unknown. We examined if PGE2 is involved in fever are transmitted from EP3R-expressing POA neurons via a direct projection to the associated with ICH as in the case of fever in infectious/inflammatory diseases. First, dorsomedial hypothalamus (DMH) to activate thermogenesis in brown adipose tissue we established a rat model of ICH-induced hyperthermia, in which collagenase was (BAT) and via another direct projection to the rostral raphe pallidus (rRPa) to reduce injected into one side of the preoptic area. In this model, abdominal temperature
heat dissipation through skin vasoconstriction. To examine how EP3R-expressing (Tab) started to rise at around 3 h after injection of collagenase and the hyperthermia POA neurons control the two effectors, we performed a double-tracing experiment, in persisted over 24 h. Hemorrhage was evident when the brains were examined at 4
which two types of cholera toxin b-subunit (CTb), a retrograde tracer, with different h and 28 h after collagenase injection. At these time points, PGE2 contents in both fluorophores were injected into the DMH and the rRPa of rats and the resulting cerebrospinal fluid and brain tissue were higher in collagenase-injected rats than retrogradely-labeled, EP3R-immunopositive neurons in the POA were identified with those in saline-injected ones. Intraperitoneal injection of diclofenac, a non-specific confocal microscopy. Many EP3R-positive neurons were labeled with CTb from cyclooxygenase (COX) inhibitor, suppressed collagenase-induced hyperthermia on
either the DMH or the rRPa, however, very few EP3R-positive neurons were labeled both the first and second days. NS398, a COX-2 specific inhibitor, suppressed bothab T with both CTbs. The paucity of the EP3R-expressing neurons innervating both the and PGE2 increase when administered on the second day. However, NS398 exerted DMH and rRPa suggests that febrile signals are sent independently from the POA to less suppressive effects when administered on the first day. These results indicate
these caudal brain regions and is consistent with different populations of POA neurons that PGE2 is involved in ICH-induced fever, and PGE2 is synthesized by the action of controlling BAT thermogenesis and skin vasoconstriction during fever. COX-1 on the first day and COX-2 on the second day.
P1AM-21-4 P1AM-21-5 MECHANISM OF ENHANCED LIPOPOLYSACCHARIDE SELECTIVE BRAIN COOLING DURING FEVER IN (LPS)-INDUCED FEVER BY EXPOSURE TO STRESSFUL SHEEP AMBIENT TEMPERATURES IN RATS Michael J McKinley, Michael L Mathai, Frank Weissenborn Tadashi Uno, Junko Saito, Masako Togawa Howard Florey Institute, University of Melbourne, Australia Department of Biometeorology, Yamanashi Institute of Environmental Sciences, Japan Selective brain cooling (SBC) occurs in many species of mammal so that the temperature of the brain is less than the core body temperature. However, Our previous studies revealed that intraperitoneal LPS-induced fevers after exposure to the alternatively changing and constant cold stressful ambient the physiological conditions during which SBC is engaged in animals is temperature for 2days were significantly enhanced over the control group still unresolved. It has been proposed that the major function of SBC is exposed to constant 25 degrees in rats. Plasma levels of TNFα 3 h after LPS to reduce body fluid loss by panting or sweating. We have investigated in in animals exposed to stressful ambient temperatures, tended to be higher than sheep (n = 4) whether SBC occurs during fever caused by systemic injection those exposed to constant 25 degrees temperature. Furthermore, The LPS- of lipolysaccharide (LPS; 25ug/kg). We measured simultaneously the stimulated peritoneal macrophages produced significantly higher amounts of temperatures of carotid blood (Tc) and within the brain (Tb). Prior to injection TNFα in animals exposed to alternatively changing ambient temperatures than in of LPS, Tb was 0.2 to 0.4 degree C higher than Tc (39.1 to 39.5 degrees C). those exposed to a constant ambient temperature of 25 degrees. We considered Tc and Tb began to increase within 45 min, with Tb remaining greater than Poster Session that intrinsic endotoxin from enterobacterum leaks into blood circulation by Tc during the initial phase of the fever. However, by 3h after LPS injection, stressful ambient temperature, and increases cytokine release from macrophages Tc continued to increase (the second phase of fever) to approximately 41.6 resulting in enhanced fever. The aim of the present study was to ravel underlying mechanisms of the enhanced LPS-induced fever in animals exposed to stressful degrees C, while Tb began to fall. Tb fell below Tc by up to 1.4 degrees ambient temperatures. For this purpose, we examined that whether enhancement C during the subsequent 1.5 to 4 h. Thus SBC was observed in all 4 sheep of TNFα production from peritoneal macrophages after stressful ambient during the peak of the second phase of fever and the defervescence. Panting temperature exposure was disappeared by oral pre administration of polymyxin B, occurred during defervescence. These data show that SBC is engaged during neutralized endotoxin activity. fever and defervescence but it does not prevent panting-induced fluid loss.
164 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-21-6 P1AM-21-7 PROTECTIVE EFFECTS OF LATERAL PARABRACHIAL NEURONS MEDIATE THE GERANYLGERANYLACETONE ON DIFFERENT HEAT THERMOSENSORY AFFERENT SIGNALING REQUIRED EXPOSED MODEL IN RATS FOR HEAT DEFENSE Yong-Qi Zhao1, You-E Yan1, Ming-Fen Wei2, Shu-Hong Liu1, Kazuhiro Nakamura, Shaun F Morrison You-Fei Guan2, Ming Fan1 Oregon National Primate Research Center, Oregon Health & Science 1 University, USA Department of Brain Protection and Plasticity, Beijing Institute of Basic Medical Sciences, China, 2ψDepartment of Physiology and To drive the rapid thermoregulatory responses necessary to maintain body Pathophysiology, Health Science Center of Peking University, China temperature during changes in ambient temperature, thermal information from This work was performed to evaluate the protective effects of geranylgeranylacetone the skin ascends to the preoptic area (POA), which then sends command signals (GGA) on unremitting heat exposure and heatstroke in rats. After core temperature (Tco) to peripheral effectors. We have recently shown that neurons in the external was measured, rats were exposed to heat environment (41.0~41.5˚C) to their death or for lateral part of the lateral parabrachial nucleus (LPBel) mediate cool signaling exactly 35 min to induce heatstroke. In the GGA group, rats were orally given GGA (100 from the skin to the POA. Here, we show that a population of neurons in the mg/ kg, daily and consecutively for 3 days) before the start of heat exposure. After 35-min dorsal part of the LPB (LPBd) is activated by skin warming and projects directly heat exposure, the animals were allowed to recover at room temperature (24˚C). Tco and the to the POA. Mimicking skin warming, activation of rat LPBd neurons inhibited survival time in heat environment or in room temperature were observed. Results showed the spontaneous activity of sympathetic cutaneous vasoconstrictor (CVC) that in heat exposure group, Tco rose quickly after rats were put into the heat chamber, and component of the sural nerve innervating the hind paw and evoked an increase in it took about 24 min to reach 42.1˚C and the survival time was 46 min. In GGA group, the tail skin temperature―responses consistent with increased body heat dissipation. time to reach 42.1˚C for Tco was slightly prolonged to 26 min while GGA pretreatment has Inactivation of LPBd neurons or blockade of glutamate receptors in the LPBd no effect on the basal temperature, the time from Tco reaching 42.1˚C to animal death and eliminated the skin warming-induced inhibition of the spontaneous sural CVC the survival time in heat environment. Compared to heat exposure group, the survival time activity. These results indicate that different populations of neurons in the LPB in room temperature in GGA group was prolonged from 20 to 195 min (P<0.01). It indicated mediate the afferent pathways for cool and warm signals from the skin to the that GGA has not protective effects on unremitting heat exposed rats but prolonged the POA and that LPBd neurons transmit the skin warming signals to the POA that survival time in room temperature after rat heatstroke. are required to defend body temperature in hot environments.
P1AM-21-8 P1AM-21-9 COLD CAPILLARIES - ANGIOGENESIS IN ECTO- AND ACITVATION OF AMYGDALA α-2 ADRENERGIC ENDOTHERMS SUBJECTED TO LOW TEMPERATURES RECEPTORS INHIBITS SYMPATHETIC CUTANEOUS Stuart Egginton VASOCONSTRICTOR ALERTING RESPONSE (SCVARS) Department of Physiology, University of Birmingham, UK IN RATS Cold exposure of either extremities or body core will increase fluid viscosity, Keerthi Kulasekara, Rodrigo C Menezes, placing extra strain on the cardiovascular system. However, there will be Youichirou YoYo Ootsuka, William W Blessing reciprocal changes in oxygen solubility and metabolic rate. Angiogenesis Department of Human Physiology, Flinders University, Australia in skeletal and cardiac muscle would therefore appear to be paradoxical. Sudden salient events selectively activate sympathetic neuronal outflow to In mammals, the response to a simulated onset of winter is to conserve thermoregulatory cutaneous vascular beds, reduce blood flow. We call the effects the microcirculation and maintain a constant capillary to fibre ratio (C:F), SCVARs [1]. Our previous work indicates that the amygdala coordinates SCVARs [2], implying either an unaltered vascular bed or angiogenesis matched by muscle and that SCVARs are inhibited by antipsychotic drugs with affinity for monoamine hyperplasia, while chronic acclimation to low environmental temperature receptors, including α-2 adrenergic receptors [1]. In the present study we investigated induces a variable degree of muscle atrophy, which in turn increases capillary whether clonidine, an α-2adrenergic receptor agonist, inhibits SCVARs, and whether density (CD). In fishes, cold-induced angiogenesis results in an increase the amygdala is a site where clonidine acts. Male Sprague-Dawley rats (n=13) were in C:F, but also a cold-induced fibre hypertrophy that is accompanied by a chronically implanted with tail artery Doppler flow probes and bilateral guide cannulae powerful angiogenic response such that CD is much less sensitive to changes directed towards the central amygdala. After a week clonidine was injected i.p. (10, 50, 100μg/kg), or into the amygdala (5, 10, 20 nmol/200 nl). Then 20-30 min later rats in fibre size. Angiogenesis may be initiated by changes in the physical were subjected to five standard alerting stimuli [1]. SCVARs were dose dependently environment due to endothelial cell mechanotransduction. It is likely that in inhibited by either i.p. clonidine (F(1,17)=63.6, P<0.0001) or intra-amygdala mammals the metabolic consequences of cold exposure increases the luminal (F(1,17)=52.8, P<0.0001) clonidine. Effects of clonidine were substantially reduced by shear stress, while in fishes the stimulus for angiogenesis is abluminal stretch pretreatment with idazoxan, an α-2 receptor antagonist. Thus α-2 adrenergic receptors following an increase in fibre size. The consequences for tissue oxygenation in the amygdala contribute to SCVARs. [1] Psychopharmacol 2005, 181, 518-28. [2] are explored. AmJPhysiol 1997, 272, 208-16.
P1AM-21-10 P1AM-21-11 β-3 ADRENERGIC RECEPTORS ANTAGONIST THE CONTRIBUTION OF BETA-3 ADRENERGIC SR59230A INHIBITS ULTRADIAN RHYTHMICITY IN RECEPTOR IN BROWN ADIPOSE TISSUE DURING BROWN ADIPOSE TISSUE (BAT) THERMOGENESIS IN HIBERNATION CONSCIOUS RAT Naoya Kitao, Masaaki Hashimoto Youichirou YoYo Ootsuka, Rodrigo C Menezes, Department of Physiology, Asahikawa Medical College, Japan William W Blessing Brown adipose tissue (BAT), which was originally referred to as "the Department of Human Physiology, Flinders University, Australia hibernation gland", is an adaptive thermogenic organ. BAT thermogenesis BAT thermogenesis contributes to increases in body and brain temperature occurring is activated mainly via β3 adrenergic receptor but continuous adrenergic in an ultradian 90 min rhythm during the 12 hour waking period in rats (Society for stimulation (e.g. cold acclimation) causes the functional desensitization. Neurosci. 2008, Abstract, 83.2.). BAT thermogenesis is under sympathetic neural control, with noradrenaline acting on β-3 adrenergic receptors. We have assessed the In hibernation, which is usually induced by the prolonged stimulation of effect of SR59230A, a β-3 adrenergic receptor antagonist on BAT, brain and body cold and short photoperiod, BAT is believed to be a predominant heater temperature. Under isoflurane anesthesia Sprague-Dawley Rats were instrumented during arousal, but the BAT mechanism working under exteme low body with thermistors for continuous measurement of BAT, brain and body temperatures. temperature is not well known. In this study, we investigate the role of the β3 Wires from probes were connected to a headpiece fixed to the skull. At least one week adrenergic receptor in BAT during arousal from hibernation in vitro using a o later, conscious unrestrained rats were placed in a quiet constant temperature (24 C) selective antagonist SR59230A. Experiments were performed in hibernating environment with 12 hour light/dark cycle. Temperature signals were obtained from and non-hibernating hamsters. Noradrenaline induced BAT thremogenesis the headpiece via flexible cable and swivel device. Administration of SR59230A (5 was partially inhibited by i.v. administration of this antagonist. BAT was mg/kg i.p.) but not vehicle, eliminated the next expected ultradian rhythmic increase, and decreased basal BAT temperature by 0.6±0.2oC (P<0.001,n=7), with similar dissected, cut in blocks and measured noradrenaline induced oxygen effects on brain and body temperature. These results provide further evidence that consumption with or without adrenergic antagonists. The possibility how sudden increases in heat production by BAT make a major contribution to 90 min much β3 adrenergic receptor is contributed to hibernation will be discussed ultradian rhythms in brain and body temperature. based on the data acquired. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 165 P1AM-21-12 P1AM-21-13 METABOLIC MODULATORS SHOW ONLY MODEST SPINAL SEROTONERGIC NEUROTRANSMISSION EFFECTS ON THE DEFENCE OF CORE TEMPERATURE CONTRIBUTES TO EMG ACTIVATION DURING SKIN DURING ACUTE COOLING COOLING-EVOKED SHIVERING David Hauton, Andrew M Coney Shaun F. Morrison, Kazuhiro Nakamura Department of Physiology, School of Clinical and Experimental Medicine, Oregon National Primate Research Center, Oregon Health & Science University of Birmingham, UK University, USA Defence of core temperature presents considerable energy demands on Shivering thermogenesis contributes to the maintenance of body core temperature the cold-exposed rodent yet the contribution of non-esterified fatty acid in a cold environment and can be initiated by a fall in skin temperature. We (NEFA) to thermogenesis is unknown. Wistar rats were fed fenofibrate investigated the potential role of spinal serotonergic neurotransmission in the (0.1% w/w in diet) or cold acclimated to 4C over 4weeks. Under chloralose- skin-cooling-evoked increases in nuchal EMG in isoflurane-anesthetized rats. eurathane anaesthesia fatty acid metabolism (in vivo) was estimated by The cold-evoked EMG activation was reversed by inhibition of neuronal activity simultaneous infusion of two NEFA tracers; metabolisable 3H-palmitic in the rostral raphe pallidus (rRPa), a site of serotonergic neurons projecting to acid and non-metabolisable 14C-bromopalmitic acid. Fenofibrate did not the spinal cord. Disinhibition of rRPa neurons with bicuculline injection elicited effect on the rate of cooling, dichloroacetate (DCA) infusion increased the a prolonged increase in EMG. The amplitude of cold-evoked increases in EMG rate of cooling by ~33% (P<0.05). Cold acclimation decreased the rate of was reduced by >60% following the 5-HT2A antagonist, ketanserin (2 mg/kg, iv). cooling ~60% (P<0.001). Fenofibrate decreased plasma palmitate clearance The 5-HT2A agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI, 100 μg/kg, iv), by ~33% (P<0.05), DCA decreased palmitate clearance by 80% (P<0.01). elicited a prolonged increase in EMG in intact rats and in rats with cervical spinal Cold acclimation increased palmitate clearance 3-fold (P<0.001). Fatty acid cord transections that eliminated the EMG response to bicuculline injection into β-oxidation rates mirrored palmitate clearance. Palmitate uptake by brown rRPa. These data are consistent with a significant role for the excitation of rRPa adipose tissue (BAT) was 6-fold higher in cold-acclimated rats (P<0.001) neurons in shivering and for the resulting activation of spinal 5-HT2A receptors however 90% (P<0.01) was utilised for the defence of core temperature. This in the cooling-evoked increase in EMG that produces shivering thermogenesis. highlights the tissue-specific importance of NEFA for thermogenesis and the Supported by USPHS NIH grant NS40987 and by the Japan Society for the defence of core temperature. Promotion of Science.
P1AM-21-14 P1AM-21-15 ADMINISTRATION OF 17-β ESTRADIOL IN THE MEDIAL BODY TEMPERATURE AND ACTIVITY CORRELATES PREOPTIC AREA SUPPRESSES HEAT DISSIPATION OF THE MENSTRUAL CYCLE IN BABOONS, PAPIO DURING COLD EXPOSURE IN OVARIECTOMIZED HAMADRYAS RATS Trevor Tapiwa Nyakudya1, Andrea Fuller1, Leith CR Meyer1, 2 1 Yuki Uchida1, Madoka Tohi1, Ken Tokizawa1, Kei Nagashima2 Shane K Maloney , Duncan Mitchell 1Laboratory of Integrative Physiology, Faculty of Human Sciences, Waseda 1School of Physiology, Brain Function Research Group, University of the University, Japan, 2Laboratory of Integrative Physiology, Faculty of Human Witwatersrand, South Africa, 2Physiology, University of Western Australia, Sciences, Waseda University, Consolidated Research Institute for Advanced Australia Science and Medical Care, Waseda University, Japan Accurate determination of the phases of the menstrual cycle in primates is important BACKGROUND It has been reported that estrogen is associated with menopausal for understanding their thermoregulatory function and reproductive success. We disorders in thermoregulation such as hot flush and cold constitution, although investigated the relationship between body temperature, activity, faecal and urinary the mechanism remains unclear yet. We reported that ovariectomized (OVX) rats hormones, and external reproductive changes over the menstrual cycle in baboons. could not maintain body core temperature (Tcore) during cold exposure, but systemic Miniature thermometric data loggers were surgically implanted intra-abdominally administration of 17-β estradiol (E2) restored the response. Administration of E2 in the and activity loggers subcutaneously on the abdomen in five adult female baboons. medial preoptic area (MPO) also prevented a reduction of Tcore in the cold, but did not Faecal and urinary samples were collected daily for hormone assays. The length of affect UCP1 mRNA level in the interscapular brown adipose tissues, suggesting less the menstrual cycle, determined from daily observations of menstrual bleeding and effect on heat production response. AIM We tested the hypothesis that, in OVX rats, anogenital swelling, was 37.6±4.5 days (mean±SD). Baboons exhibited a regular the local E2 administration in the MPO minimizes heat loss in the cold. METHODS 24-h rhythm of body temperature and activity. Mean daily body temperature during ο o An OVX rat was placed at 10 or 25 C for 2 h, 48 h after the administration of E2 or the luteal phase (37.7±0.1 C) was significantly higher than during the ovulatory o o cholesterol through a thin pipe placed in the MPO. Tcore and tail temperature (Ttail) were phase (37.5±0.1 C), but not different to the follicular phase (37.4±0.4 C). Activity continuously measured by telemetry and thermography. RESULTS Both the groups followed a similar pattern, with mean 24-h activity almost twice as high in the luteal ο showed a decrease in Ttail at 10 C: greater in the E2 group than in the cholesterol compared to the ovulatory phase. We have characterised correlates of the menstrual group. CONCLUTION E2 administration in the MPO suppressed heat loss from the cycle in baboons and shown, for the first time in any species, a rhythm of both body tail. temperature and activity over the menstrual cycle.
P1AM-21-16 P1AM-21-17 THE EFFECT OF SEASON AND PASSIVE HEAT EFFECTS OF AGING ON SEVERAL HORMONES IN STRESS ON THERMOREGULATORY RESPONSE IN ASSOCIATION WITH TEMPERATURE AND METABOLIC YOUNG HEALTHY VOLUNTEERS REGULATION IN DIFFERENT SEASONS Dominika Kanikowska, Maki Sato, Yoko Inukai, Yuuki Shimizu, Maki Sato, Dominika Kanikowska, Yoko Inukai, Yuuki Shimizu, Naoki Nishimura, Satoshi Iwase, Junichi Sugenoya Naoki Nishimura, Satoshi Iwase, Junichi Sugenoya Department of Physiology, Aichi Medical University, School of Medicine, Department of Physiology, Aichi Medical University, Japan Japan With change in the season, the patterns of body temperature regulation as We investigated the seasonal changes on hormonal and autonomic nervous systems. well as hormonal regulation might be modified. The purpose of this study 8 volunteers were subjected for the experiment at four times of the year - around the is to examine the seasonal changes in various hormones in association with o vernal and autumnal equinoxes and the summer and winter solstices at latitude 35 temperature and metabolic regulation (ADH, TSH, fT3, fT4 and leptin) in N. Seasonal changes in the thermoregulatory responses were assessed by measuring young (21±1 yrs; mean±SD) and elder subjects (69±5 yrs; mean±SD). At core and skin temperatures, sweat rate and blood flow during immersion of the leg in each season, blood was taken in a sitting position at rest after the subject o hot water (mild heat at 42oC) for 30 min. The following parameters were analyzed: stayed for 30 min or more in the climatic chamber set at 26 C and 50%RH, antidiuretic hormone (ADH), angiotensin II, aldosterone, plasma renin activity and the blood osmolality and hormone concentrations were measured. The o (PRA). Tympanic temperature during water immersion showed significant differences average of atmospheric temperature in experimental days were 15-20 C in o o o Poster Session between seasons. Sweat rate and the skin blood flow were significantly higher in spring, 25-30 C in summer, 15-23 C in autumn and 5-10 C in winter. We summer compared with the other seasons. The concentration of ADH and aldosterone found that: 1) ADH concentration was significantly higher in elder than in was significantly higher in summer compared to the other seasons. Concentrations younger in winter; 2) osmolality in elder subject was significantly higher of angiotensin II and PRA did not show seasonal variations. The results of this study in winter than in summer; 3) concentration of leptin in elder subjects was indicate that concentrations of ADH and aldosterone as well the sweat rate increased significantly higher in winter than in other seasons; 4) concentration of TSH, in summer compared with the other seasons, suggesting that the change in those fT3 and fT4 in elder subjects was not significantly different with seasons. It parameters are attributable to heat acclimatization induced by natural hot climate was suggested that hormonal regulation was reduced in elder subjects in during the summer. winter.
166 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1AM-21-18 P1AM-21-19 HEAT ACCLIMATIZATION IN HOT SUMMER FOR TEN AUGMENTATION OF HUMAN SWEAT GLAND WEEKS SUPPRESS THE SENSITIVITY OF SWEATING SENSITIVITY TO ACETYLCHOLINE APPLIED Jeong Beom Lee1, Young Ki Min1, Young Oh Shin2, Hun Mo Yang1 IONTOPHORETICALLY 1Physiology, College of Medicine, Soonchunhyang University, Korea, Jeong Beom Lee, Young Oh Shin, Jeong Beom Lee, 2Department of Physiology, College of Medicine, Soonchunhyang Young Ki Min, Hun Mo Yang University, Korea Physiology, College of Medicine, Soonchunhyang University, Korea To determine the peripheral mechanisms involved in thermal sweating during the hot The aim of this study was to determine the peripheral sudomotor adaptive summers in July before acclimatization and after acclimatization in September, we responses attributed to indirectly activated (AXR) and directly activated evaluated the sweating response to acetylcholine (ACh), a primary neurotransmitter (DIR) in endurance-trained n=16 subjects (ETS) and control n=20 subjects involved in peripheral sudomotor sensitivity, in healthy subjects (n=10). The (CS). The Quantitative sudomotor axon reflex test (QSART) was performed quantitative sudomotor axon reflex test (QSART), at 2 mA for 5 min with 10% ACh, to determine AXR mediated by nicotinic receptor activation (axon reflex- was applied to determine the directly activated (DIR) and axon reflex-mediated mediated sweating) and DIR mediated by muscarinic receptor activation (AXR) sweating responses during ACh iontophoresis. The AXR sweat onset-time by during ACh iontophoresis, in which 10% ACh was applied at 2 mA for 5 the axon reflex was 1.50±0.32 min and 1.84±0.46 min before acclimatization in July min. The sweat onset time of the AXR1 mediated by nicotinic receptor and after acclimatization in September, respectively (P<0.01). The sweat volume of activity was shorter in the ETS than the CS (P<0.001). The volume of AXR1, the AXR(1) by the axon reflex was 1.45±0.53 mg/cm2 and 0.98±0.24 mg/cm2 before AXR2 and DIR was higher in the ETS than the CS (P<0.001). Furthermore, acclimatization in July and after acclimatization in September, respectively (P<0.001). the sweat gland density and single gland output were higher in the ETS The sweat volume of the DIR was 5.88±1.33 mg/cm2 and 4.98±0.94 mg/cm2 before than the CS. These findings suggest that the ETS had augmented peripheral and after acclimatization in July and September, respectively (P<0.01). These results sudomotor sensitivity to ACh, and higher thermal sweating (AXR1, AXR2 indicate that exposure to hot summer weather and acclimatization resulted in a lower and DIR) resulting from a combination of higher single sweat gland output sweat output due to a combination of lower sweat volume (AXR and DIR) and a and shorter time of onset of AXR1 from the higher number of activated sweat delayed action of the onset time of the AXR. conditions. glands than the CS.
P1AM-21-20 P1AM-21-21 THERMAL AND NON-THERMAL SWEATING: WHAT IMPROVEMENT OF HEMODYNAMICS BY A HEAT-AND YOU SEE IS NOT NECESSARILY WHAT YOU GET STEAM-GENERATING SHEET APPLICATION TO THE Christiano Antonio Machado-Moreira1, Robert J Barry2, NECK AND ITS INFLUENCE ON AUTONOMIC NERVE Maarten J Vosselman1, Rafael M Ruest1, Nigel AS Taylor1 ACTIVITY 1 2 School of Health Sciences, University of Wollongong, Australia, School of Yoshinao Nagashima1, Michihito Igaki1, Atsushi Suzuki1, Psychology, University of Wollongong, Australia 1 1 1 Yukihiro Yada , Shuuichi Tsuchiya , Toshiyuki Suzuki , Thermal and psychological sweating were simultaneously measured (dorsal and 2 Sachiko Oh-ishi volar fingers) using ventilated capsules and skin conductance methods in 15 1 2 subjects during two experiments (26oC). Trial 1 (N=7) involved passive heating Tokyo Research Laboratories, Kao Corporation, Japan, Kitasato (perfusion suit, feet immersion), whilst in trial 2 (N=8), sweating was elicited University, Japan using painful stimulation. Thermal sweating was evident only from the dorsal Effects of a heat-and steam-generating sheet (HSG sheet) applied to the neck on systemic surface (both methods), although it was detected 4.4 min earlier from changes in hemodynamics and autonomic nerve activity were investigated. A HSG sheet, was applied skin conductance (P<0.05). That is, while primary sweat secretion was evident, to the neck of healthy adults performing VDT work, and the skin temperature of the neck, sweat did not immediately reach the skin surface. In experiment 2, pain-induced scapular region, palm, and dorsum of the foot, skin blood flow measured by the laser Doppler sweating was evident from both surfaces, with each technique. However, peak method, total hemoglobin measured by near infrared spectrometry, analysis of ECG R-R skin conductance occurred at least 20 s prior to peak sweat rate (P<0.05). interval variation, and baroreceptor reflex employing the sequence method were investigated. Despite these differences, data from both techniques were significantly correlated HSG sheet application prevented a reduction of the skin temperature and blood flow in the during each experiment (P<0.05). The skin conductance technique has a much palm and dorsum of the foot, and markedly elevated the neck and scapular skin temperature greater temporal sensitivity at low secretion rates, and ductal reabsorption may and total hemoglobin level. On analysis of the ECG R-R interval, LF/HF tended to decrease, prevent the sweat capsule method from detecting such secretion. Thus, a failure suggesting that sympathetic tone decreased. The baroreceptor reflex was significantly to observe sweating from sweat capsules may not mean an absent sudomotor promoted, suggesting that parasympathetic tone increased. These findings suggested that the response, but a failure to detect secretion even though sympathetic drive has application of a HSG sheet to the neck improved the peripheral hemodynamics and inhibited initiated primary sweat production. sympathetic nerve activity, leading to the dominance of parasympathetic nerve activity.
P1AM-21-22 P1PM-1-1 EFFECTS OF ABDOMINAL WARMING USING A HEAT- BAROREFLEX FUNCTION APPEARS TO INITIATE AND STEAM- GENERATING SHEET ON MENSTRUAL COMPENSATORY MECHANISM FOR CARDIAC PAINS AUTONOMIC MODULATION IN PATIENTS WITH Takayoshi Hosono1, Yukari Takashima1, Yu Taguchi1, Haruka ANGINA PECTORIS 1 1 1 2 Akeno , Akiko Miyano , Yuki Morita , Ichiro Sakamoto , Kishore K Deepak1, Rita Khadka2, Rajiv Narang3, Ashok K Jaryal1, 2 2 2 Kyoko Tagami , Yuki Hidaka , Atsushi Suzuki Chetan D Patel4, Ravindra M Pandey5 1 Department of Biomedical Engineering, Osaka Electro-Communication 1Department of Physiology, All India Institute of Medical Sciences (AIIMS), 2 University, Japan, Kao Corporation, Japan India, 2Department of Physiology, B. P. Koirala Institute of Health Sciences, 3 The effects of a heat- and steam-generating (HSG) sheet with an area of 156 cm2 on Nepal, Department of Cardiology, All India Institute of Medical Sciences, India, 4Department of Nuclear Medicine, All India Institute of Medical Sciences, India, the relief of dysmenorrhea were investigated. The HSG sheet generates a heat around 5 40 °C for 5 to 8 hrs. Phase 1. Twenty-six women (mean: 21.1 yrs) were included in Department of Biostatistics, All India Institute of Medical Sciences, India phase 1. Each subjectively scored her own menstrual pains on a scale from 0 to 3 just Changes in arterial blood pressure (BP) are known to modulate cardiac autonomic tone through before, 1 hr after, and 5 - 8 hrs after the abdominal application of the HSG sheet on the baroreflex functions. The role of baroreflex in modulation of cardiac autonomic tone in angina 2nd day of menstruation. Fifty-four percent and all of the subjects reported pain relief patients remains unknown. In this study we assessed short-term heart rate variability (HRV), blood after 1 hr and 5 - 8 hrs the HSG sheet application, respectively. Phase 2. Eight women pressure variability (BPV), and spontaneous BRS in 33 consecutive male patients, age 54.91± (mean: 22 yrs) were included in phase 2. We applied the HSG sheet to the abdomen 7.43yrs with perfusion defects, assessed with Thallium-201 SPECT and 30 healthy controls, age on the 2nd day of menstruation for 5-8 hrs, and measured the systolic (Vmax) and 48±6.77yrs. Spearman's rank correlation was used to find the association between HRV and BPV. The controls showed correlation between BPV and HRV (time domain rMSSD r=0.541, p=0.002), diastolic (Vmin) velocities of blood flow in uterine arteries using Doppler flowmetry which was impaired in patients (r=0.099, p=0.584). In controls the sympathetic (LF) and vagal (HF) by ultrasonography just before and after HSG sheet application. We evaluated the powers for only SBP variability showed association with LF(r=0.447, p=0.009) and HF(r=0.486, resistance index (RI), (Vmax-Vmin)/Vmax, known to be well-correlated with vascular p=0.004) powers of HRV, whereas in patients, the LF and HF power for SBP, DBP and MBP all resistance. The mean RI decreased In 5 of the subjects whose menstrual pains were showed association with LF and HF of HRV. In conclusion the beat-to-beat HRV-BPV association relieved by the HSG sheet application. These results imply that HSG sheet application was impaired in angina patients with myocardial hypo-perfusion, however association between may relieve menstrual pains in a large proportion of women by decreasing uterine oscillations of HRV and SBP, DBP or MBP were present. Thus it appears that baroreflex mediates vascular resistance. compensatory cardiac autonomic modulation in angina patients. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 167 P1PM-1-2 P1PM-1-3 ANGIOTENSION-(1-7) AND ANGIOTENSIN II IN ROS IN ROSTRAL VENTROLATERAL MEDULLA ROSTRAL VENTROLATERAL MEDULLA MODULATE MEDIATE THE EFFECTS OF ANGIOTENSIN II IN CARDIAC SYMPATHETIC AFFERENT REFLEX AND PARAVENTRICULAR NUCLEUS ON CARDIAC SYMPATHETIC ACTIVITY SYMPATHETIC AFFERENT REFLEX Guo-Qing Zhu, Zhen Shi, Li-Min Zhou, Yao Xu, Ying Han, Guo-Qing Zhu1, Ming-Kui Zhong2, Juan Gao1, Shu-Juan Zhang1, 1 1 Feng Zhang, Jing Li, Xing-Ya Gao Ning Yuan , Zhi-Dan Fan 1 2 Department of Physiology, Nanjing Medical University, China Department of Physiology, Nanjing Medical University, China, Department The present study was to determine whether angiotensin-(1-7) and angiotensin II in of Physiology, Nanjing Medical University, ;Department of Physiology, rostral ventrolateral medulla (RVLM) modulate cardiac sympathetic afferent reflex Anhui Medical University, China (CSAR) and sympathetic activity in anesthetized SD rats. Following sinoaortic The present study was to investigate whether reactive oxygen species (ROS) in rostral denervation and vagotomy, the CSAR was evaluated with the renal sympathetic ventrolateral medulla (RVLM) mediate the effects of microinjection of angiotensin II (Ang nerve activity (RSNA) response to ventricular epicardial application of capsaicin. II) into the paraventricular nucleus (PVN) in anesthetized SD rats. Following sinoaortic Microinjection of angiotension-(1-7) or angiotensin II into RVLM enhanced the denervation and vagotomy, cardiac sympathetic afferent reflex (CSAR) was evaluated with CSAR and increased the RSNA and mean arterial pressure (MAP). The effects of the renal sympathetic nerve activity (RSNA) response to ventricular epicardial application angiotension-(1-7) were abolished by pretreatment with Mas receptor antagonist of capsaicin. Bilateral PVN microinjection of Ang II enhanced the CSAR and RSNA and A-779 but not by AT1 receptor antagonist losartan, while the effects of angiotensin increased arterial pressure, which were significantly attenuated by the pretreatment with II were abolished by losartan but not by A-779. Microinjection of A-779 into the bilateral RVLM microinjection of tempol (a superoxide dismutase mimic) or polyethylene RVLM attenuated the CSAR and decreased the RSNA and MAP, but losartan had no glycol-superoxide dismutase (PEG-SOD, an analogue of endogenous superoxide dismutase). significant effect on the CSAR, RSNA and MAP. These results suggest that CSAR- Moreover, the effects of Ang II in the PVN were also attenuated by the pretreatment with enhancing and sympatho-excitatory effects of angiotension-(1-7) and angiotensin RVLM microinjection of NAD(P)H oxidase inhibitors apocynin or phenylarsine oxide (PAO). II in the RVLM is mediated by Mas receptors and AT1 receptors, respectively. The The findings suggest that the ROS in the RVLM mediate the effects of Ang II in the PVN Mas receptors in the RVLM involve in tonic control of the CSAR, RSNA and MAP. on the CSAR, RSNA and arterial pressure. The NAD(P)H oxidase-derived ROS in RVLM The study was supported by Chinese National Natural Science Fund (30670768 & plays an important role in modulating the CSAR. The study was supported by Chinese 30870908). National Natural Science Funds (30670768 & 30870908).
P1PM-1-4 P1PM-1-5 ON MECHANISMS OF GENUINE (IDIOPATHIC) HIGH-CALCIUM DIET REDUCES BLOOD PRESSURE HYPERTENSION AND ARTERIAL MOTOR AND BLOOD VOLUME VIA IMPROVED DIURETIC OSCILLATIONS RESPONSE IN ORAL CONTRACEPTIVE-TREATED Michael Ch. Michailov1, Eva Neu1, Ursula Welscher1, Gerald Werner2, FEMALE RATS Janka Foltinova3, Tatjana Senn4, Walter Seidenbusch4 Rhoda Robinson, Lawrence A Olatunji, Ayodele O Soladoye 1Pharmaco-Physiology, Inst. Umweltmedizin c/o ICSD e.V. & Univ. Erl.- Department of Physiology, University of Ilorin, Nigeria Nuernberg, Germany, 2Fac. Med., Univ. Goettingen, Germany, 3Inst. Embryology, Cardiovascular complications are the main clinical challenges among users Univ. Bratislava, Slovakia, 4Inst. Exp. Physik, Univ. Innsbruck, Austria of oral contraceptive (OC) formulations. Interventions that enhance diuretic INTRODUCTION & METHOD: Pathogenesis of hypertension is not clarified (Strahlenther. response have been shown to reduce volume-dependent hypertension and the 151:549, 1976). RESULTS (recent/earlier): Augmented excitability of blood pressure (BP) associated risk factors. The aim of the present study was to investigate the regulatory system: 1. CNS (formatio reticularis/hypothalamus: transformation of depressor- influence of increasing dietary calcium from 0.9 to 3.0% on the development dR of ACH & central electr vagal stim (55Hz/2ms/5s/5V) into biphasic dR/pR pressor of OC-induced hypertension and associated changes in female Sprague- reaction by nicotine/MEG 30/300 mg/kg (n=150). 2. Sympathetic preganglionic neurons Dawley rats treated with a combination of OC steroids (1 μg ethinyl estradiol (cholinergic) in normal/spinal (decapitated) rats dR/pR; pR-potentiation of non- (AHR602, and 10 μg norgestrel; orally.) daily for 10 weeks. Results showed that OC MCN-A343) & Nicotine-like (N, DMPP) ganglion-stimulating agents, vasopressin by administration led to significant increases in blood pressure, blood volume MEG. 3. Vascular effector: appearance of periodic contractions (phasic 0.5-2 & tonic and cardiac weight. Conversely, OC caused significant reductions in body 0.1-0.2/min), induced by vasoactive agents (PGs/5-HT/angiotensinII; human renal/uterine weight, urinary excretion of water, plasma levels of calcium, 17β-oestradiol arteries, rat aorta). CONCLUSION: Similar to drugs exo-/endogenic substances probably and progesterone. Increased dietary calcium attenuated the OC-induced could sensitize BP regulatory central, spinal, peripheral structures (1-3) causing idiopathic elevated blood pressure and the associated changes in blood volume, cardiac hypertension (independent of renal), leading to angio-cardial/cerebral spasms (infarct & weight, plasma calcium and urinary excretion of water. The results indicate apoplexy) via biphasic dR/pR. Dedicated to birthdays & moral support Hon ICSD-members, that increased calcium intake abrogated the development of hypertension and * Nobel L. Profs Sir J Black*/GB, Dausset*, Lehn*/France, Karle*/USA, R Levy-M.*/Italy, associated increased blood volume and cardiac weight during OC use via Tonegawa*/Japan, Haeder, Michel*, M.-Mohnssen, Neher*, Stuehmer, Weiss, Welss/FRG, improved diuretic response.
P1PM-1-6 P1PM-1-7 ORAL CONTRACEPTIVE-SENSITIVE HYPERTENSION CIRCADIAN VARIATIONS IN ARTERIAL PRESSURE, RESULTING FROM IMPAIRED NITRIC OXIDE HEART RATE AND LOCOMOTOR ACTIVITY IN SYNTHESIS IS ASSOCIATED WITH ENHANCED WATER CONGENIC RATS RETENTION IN RATS Hiroshi Kawamura1, Hiromi Mitsubayashi1, Noboru Saito2, Isaiah Oluwatobi Owolabi, Lawrence A Olatunji, Yoshinori Ishiyama3, Toshihito Kadota3, Tohru Nabika4 1Department of Medicine, Nippon Dental University School of Life Dentistry, Ayodele O Soladoye 2 3 Japan, Miyazaki Medical Center, Miyazaki, Japan, Fuji Biomedix, Department of Physiology, University of Ilorin, Nigeria Kobuchizawa, Yamanashi, Japan, 4Department of Functional Pathology, Oral contraceptive (OC) is associated with hypertension in humans and in rats, Shimane University Faculty of Medicine, Izumo, Japan although mechanisms responsible are still unclear. This study sought to investigate Objective: The congenic rats (SHRSPwch1.0) were derived from stroke-prone SHR/ the effects of OC administration on hypertension resulting from nitric oxide (NO) Izumo (SHRSP/Izm) and Wistar-Kyoto rat/Izumo (WKY/Izm). We studied the circadian synthesis inhibition in female Sprague-Dawley rats. Rats were given ethinyl estradiol variations of systolic arterial pressure (SAP), heart rate (HR) and locomotor activity (ACT) and norgestrel and were treated with NO synthase inhibitor, NG-nitro-L-arginine in SHRSPwch1.0. We also studied the effect of central selective noradrenergic neurotoxin, methyl ester (L-NAME) or drinking water alone in the drinking water for 6 weeks. DSP-4 (N-2-chloroethyl-N-ethyl-2-bromobenzylamine HCl). OC treatment alone led to a significant increase in blood pressure and positive water Methods: We used ten male mature SHRSPwch1.0 and six age-sex matched SHRSP/Izm balance. L-NAME treatment alone resulted in a significant increased blood pressure for the control. SAP, HR and ACT were monitored using radio-telemetry, and circadian Poster Session without significant positive water balance. Concomitant OC and L-NAME treatment variations in SAP, HR and ACT were analyzed using the maximum entropy method. Results: As expected, SAP in SHRSPwch1.0 was lower than in SHRSP/Izm (194±9 vs. 229 produced significant increases in blood pressure and water balance. These magnitudes ±15mmHg, P<0.001). HR in SHRSPwch1.0 was slower than in SHRSP/Izm (310±9 vs. 381 of increases were significantly greater than those observed in rats treated with OC ±45 beats/min, P<0.004). The circadian variations of SAP, HR and ACT in SHRSPwch1.0 or L-NAME alone. OC did not affect NO biosynthesis with or without concurrent were clearly evident, compared to SHRSP/Izm. 24-hour periodicities were dominant in SAP, L-NAME treatment. OC and/or L-NAME treatment did not significantly affect heart HR and ACT of SHRSPwch1.0, compared to those in SHRSP/Izm. DSP-4 did not affect the rate, cardiac weight, plasma sodium, glomerular filtration rate and urinary sodium 24-hour periodicities of SAP, HR and ACT in SHRSPwch1.0. output. These data demonstrate that OC administration aggravated hypertension during Conclusions: The circadian variations of SAP, HR and ACT appear better maintained in NO synthesis inhibition, via enhanced water retention. SHRSPwch1.0, compared to SHRSP/Izm.
168 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-1-8 P1PM-1-9 CARDIOVASCULAR AND HAEMATOLOGIC CHANGES CARDIOVASCULAR AUTONOMIC MODULATION FOLLOWING CHRONIC EXPOSURE TO PETROLEUM INCREASES WITH SEVERITY OF MYOCARDIAL PRODUCT ISCHEMIA Chikodi Nnanyelu Anigbogu, Oladimeji S Joseph Rita Khadka1, Ashok K Jaryal2, Chetan D Patel3, Rajiv Narang4, Department of Physiology, University of Lagos, Nigeria Kishore K Deepak2 1 Cardiovascular and haematological changes following chronic exposure to Department of Physiology, B. P. Koirala Institute of Health Sciences (BPKIHS), Nepal, 2Department of Physiology, All India Institute of Medical Sciences, India, petrol were investigated in Sprague-Dawley rats. Rats were given 2.5ml/kg 3 Department of Nuclear Medicine, All India Institute of Medical Sciences, India, of petrol and saline orally for 2 weeks while the controls received normal 4Department of Cardiology, All India Institute of Medical Sciences, India saline only. The animals were then anaesthetized and cannulated for blood The cardiac autonomic tone reduces with severity of coronary obstructions. Moderate to pressure measurement and evaluation of haematological parameters. Results severe coronary obstructions lead to myocardial ischemia (MI) also fixed perfusion defects show that petrol caused a fall in systolic, diastolic and mean arterial pressures (FPD) (scarred myocardium). We studied whether cardiovascular autonomic tone alters with (97.71±9.23, 60.14±6.45, 72.6±7.28mmHg respectively) and a decrease severity of MI or FPD. The short-term heart rate variability (HRV) was assessed for cardiac in heart rate (p<0.05). Pulse pressure was not significantly higher in petrol autonomic tone and blood pressure variability for vascular autonomic tone in 33 consecutive treated group. Baroreflex sensitivity tested by carotid occlusion was lower in male patients, age 54.9±7.4 with perfusion defects as assessed with Thallium-201 myocardial the petroleum exposed rats (p<0.05). Haemoglobin concentration increased perfusion SPECT. The summed difference scores (SDS) were calculated for MI and summed in the petroleum treated rats (14.7±0.58g/100ml) compared to the control rest scores (SRS) for FPD. Spearman’s rank correlation was used to find association of rats (11.9±0.58g/100ml) (p<0.05), though the packed cell volume (PCV) cardiovascular autonomic tone with perfusion defects. The HRV was inversely related with SRS (LF power r= -0.524, p=0.002) and positively with SDS. The SBP variability was was not significantly higher (39.2±2.13% compared with 38.5±1.9%). Mean inversely related with SRS (SDNN r= -0.383, p=0.003) and positively with SDS (Total corpuscular haemoglobin concentration (MCHC) appeared elevated but this power r=0.563, p=0.026). In conclusion the cardiovascular autonomic tone decreases was not statistically significant (38.02±2.10 and 31.61±2.67: p>0.05). These with severity of fixed perfusion defects, in contrast increases with severity of myocardial results show that petroleum fraction ingestion altered cardiovascular function ischemia. Thus it suggests that myocardial ischemia and scarred myocardium both have and increased haemoglobin concentration. opposite effect on the cardiovascular autonomic modulation.
P1PM-1-10 P1PM-1-11 REDUCED HEART RATE COMPLEXITY IN PATIENTS LINEAR AND NONLINEAR BAROREFLEX ANALYSIS IN WITH TYPE 1 DIABETES MELLITUS YOUNG PATIENTS WITH TYPE 1 DIABETES MELLITUS Michal Javorka1, Andrea Calkovska1, Zuzana Turianikova1, Michal Javorka1, Zuzana Lazarova1, Ingrid Tonhajzerova1, Ingrid Tonhajzerova1, Jana Javorkova2, Kamil Javorka1, Zuzana Turianikova1, Natasa Honzikova2, Bohumil Fiser2, 1 1 3 Mathias Baumert3 Andrea Calkovska , Kamil Javorka , Mathias Baumert 1 1Department of Physiology, Jessenius Faculty of Medicine, Comenius Department of Physiology, Jessenius Faculty of Medicine, Comenius University, 2 Slovakia, 2Department of Physiology, Faculty of Medicine, Masaryk University, University, Slovakia, Clinic of Children and Adolescents, Martin Teaching 3 Hospital, Martin, Slovakia, 3Centre for Biomedical Engineering, The Brno, Czech Republic, School of Electrical & Electronic Engineering, The University of Adelaide, Australia University of Adelaide, Adelaide, Australia We hypothesized that nonlinear methods for the quantification of synchronization between blood The aim of this study was to test whether new heart rate variability (HRV) complexity pressure (BP) and heart rate (HR) are more sensitive to baroreflex impairment in patients with measures provide diagnostic information regarding early subclinical autonomic dysfunction subclinical autonomic dysfunction in Type 1 diabetes mellitus (DM) than standard baroreflex in diabetes mellitus (DM). sensitivity (BRS) indices. HRV in Type 1 DM patients (n = 17, 10f, 7m) aged 12.9-31.5 years was compared to a The aim of study was to apply novel synchronization indices to HR and BP data and to compare control group of 17 healthy matched probands. The length of R-R intervals was measured their performance to BRS methods. over 1 hr. In addition to linear measures, we assessed HRV complexity measures, including We recorded beat-to-beat HR and systolic BP in 14 patients with DM and 14 matched healthy multiscale entropy (MSE), compression entropy and various symbolic dynamic measures controls. The analysis included BRS methods (sequence, cross-correlation and cross-spectral (Shannon and Renyi entropies, normalized complexity index (NCI), pattern classification). methods) and nonlinear synchronization approaches (information domain synchronization index, HRV magnitude was significantly reduced in patients with DM. Several HRV complexity cross-multiscale entropy, information-based synchronization index). parameters (MSE at scales 2-4, Renyi entropy, NCI) were also significantly reduced in We found well-preserved baroreflex sensitivity in young patients with DM accompanied by an diabetics. MSE indices and compression entropy did not correlate with linear measures. increased time delay within the baroreflex loop and a decrease in the synchronization between BP The magnitude and complexity of HRV are reduced in young patients with DM, indicating and HR fluctuations. vagal dysfunction. The quantification of HRV complexity in combination with its magnitude Non-linear baroreflex assessment might provide additional information on the baroreflex may provide an improved diagnostic tool for cardiovascular autonomic neuropathy in DM. impairment while BRS indices are less sensitive to autonomic dysregulation in DM. This study was supported by grants VEGA no. 1/0064/08, MVTS Austr/SR/UK/08 and CR/ This study was supported by grants VEGA 1/0064/08, MVTS Austr/SR/UK/08 and CR/SR/UK/08 SR/UK/08 and by the Australian Research Council (# DP0663345). and by the Australian Research Council (# DP0663345).
P1PM-1-12 P1PM-1-13 EFFECTS OF OBESITY ON HEART RATE VARIABILITY MECHANISMS INVOLVED IN ARTERIAL BLOOD IN ADULTS PRESSURE REDUCTION INDUCED BY SWIM TRAINING Wilaiwan Khrisanapant1, Watchara Boonsawat2, IN MALE TWO-KIDNEY ONE-CLIP RENOVASCULAR Jaruwan Plaengdee1, Orapin Pasurivong1, Uraiwan Zaeoue1 HYPERTENSIVE RATS 1Department of Physiology, Khon Kaen University, Thailand, 2Department Alisa Suvannapura, Sivaporn Muaddech of Medicine, Faculty of Medicine, Khon Kaen University, Thailand Department of Physiology, Faculty of Science, Prince of Songkla University, Hat-Yai, Songkhla, Thailand Cardiac autonomic activity (CAA) in obesity remains controversial. The Swim training was recently shown to reduce mean arterial pressure (MAP) in two-kidney present study aimed to examine CAA using time- and frequency- domain one-clip renovascular hypertensive rats (2K1C). The exact mechanisms involved in MAP heart rate variability (HRV) analysis in 7 normotensive obese (OB) (22.5±2.9 reduction were not clear. This study aimed to investigate the mechanisms involved in swim- y) and 7 healthy lean (21.1±3.0 y) males. None of the subjects had evidence training induced MAP reduction in male 2K1C rats. of cardiorespiratory disease. Supine EKG was recorded continuously for Methods Male Wistar rats (180-200 g) were divided into two sham-operated groups 5 min after 10 min of rest in the same position. The spectral power was (sedentary group or SO and swim-training group or SOX), and two 2K1C groups (sedentary quantified in total power (TP), very low-frequency (VLF) power, low- group or RVH and swim-training group or RVHX). 2K1C renovascular hypertension was frequency (LF) power, high-frequency (HF) power and LF/HF ratio. LF, HF induced by placing a silver clip (0.2 mm diameter) on the left renal artery. Swim training and LF/HF represent primarily sympathetic activity with a minor influence was performed twice daily (45 min/session, 5 days/week for 6 weeks). from parasympathetic activity, parasympathetic activity and sympathovagal Results Swim training significantly reduced MAP in RVHX compared with RVH. Captopril produced less reduction in MAP in RVHX compared with RVH. L-NAME induced less balance, respectively. The OB had significantly higher systolic and diastolic MAP increase in both RVH and RVHX compared with SOX. RVHX had significantly higher blood pressures (BP). Overall HRV values were not significantly lower in vasodilatory responses to acetylcholine than those of RVH, but no differences in responses to OB. The significantly lower HF normalized units and higher LF/HF ratio but sodium nitroprussdide were found. not the time-domain parameter of parasympathetic activity were observed Conclusion These results suggest that swim training reduced MAP in male 2K1C partly by in OB. We found that obesity unfavorably affects cardiac autonomic activity attenuation of the activity of the renin-angiotensin system, and by enhancing endothelium- and BP by reducing parasympathetic activation. dependent vasodilation. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 169 P1PM-1-14 P1PM-2-1 EFFECTS OF CHRONIC INTERMITTENT HYPOBARIC INCREASED ABERRANT SPLICING OF Cav1.2 HYPOXIA ON RENAL SYMPATHETIC NERVE ACTIVITY CHANNELS IN NEONATAL RAT VENTRICLES IN ANESTHETIC RATS Ping Liao, Tuck Wah Soong Yi Zhang1, Fang Cui1, Zhao-Nian Zhou2 Physiology, Yong Loo Lin School of Medicine, National University of 1Department of Physiology, Hebei Medical University, China, 2Shanghai Singapore, Singapore 2+ 2+ Institutes for Biological Sciences, Chinese Academy of Sciences, China The Ca influx through L-type Ca channels Cav1.2 is pivotal for the The purpose of present study is to investigate the effects of CIHH on renal sympathetic excitation-contraction coupling in the mature mammalian myocardium via a process known as Ca2+ induced Ca2+ release (CICR). However, the efferent nerve activity and the underlying mechanism in anesthetic rats. 2+ Male Sprague-Dawley rats were used. The CIHH rats were exposed to simulated high- contraction of fetal or newborn hearts depends mainly on Ca influx through L-type Ca channels. CICR is absent in fetal or newborn hearts. Numerous altitude hypoxia in a hypobaric chamber mimicking 5000 m altitude (O2: 11.1%) 6 h 2+ per day for 14 d, 28 d and 42 d, respectively. Before and during acute hypoxia, blood studies addressed the changes of Ca currents during ontogeny, but the pressure (BP), heart rate (HR) and renal sympathetic efferent nerve activity (RSNA) structural changes of Cav1.2 channels are missing. We investigate whether in anesthetic rats were recorded simultaneously. The activities of baroreflex and the alternative splicing of Cav1.2 channels is altered during development. chemoreflex were measured. One day old neonatal ventricles and adult ventricles from Wistar rats were Results showed: 1. There was no difference in HR and BP between CIHH and CON studied. We found aberrant splicing of Cav1.2 channels significantly higher in animals under basic condition. 2. During acute hypoxia, decreasing of BP was slighted, neonatal ventricles. Mutually exclusive exons 21 and 22 were both deleted or but increasing of HR and RSNA was enhanced in CIHH rats. 3 Removing the buffer included in neonatal ventricles at higher rate than adult ventricles. Exon 33 nerves augmented the hypoxic decreasing of BP, attenuated the hypoxic increasing of with a new acceptor to generate a 104 bp longer version was found high in HR and inhibited RSNA. 4 The activities of baroreflex and chemoreflex in CIHH rats neonatal hearts. This novel exon results in frame shift and a stop codon was were increased markedly. found within exon 33. Electrophysiology showed that these aberrant channels Conclusion: CIHH diminishes acute hypoxic decreasing of BP, augments acute could not conduct calcium ions. However, coexpression with functional hypoxic increasing of HR and RSNA, which related with the peripheral baroreflex and channels will yield a dominant negative effect to down regulate current chemoreflex. density.
P1PM-2-2 P1PM-2-3 EFFECT OF LEVOSIMENDAN ON ISOMETRIC TWITCH ANTI-CHOLESTEROL DRUG STATINS AFFECT AND SLOW FORCE RESPONSE IN RAT PAPILLARY STRETCH-INDUCED CALCIUM INFLUX IN THE MUSCLE CARDIOMYOCYTE CELL LINE H9c2 Oleg Lookin, Yuri Protsenko Ken Takahashi, Marei Omori, Masahiro Sokabe Institute of immunology and physiology, Russia Department of Physiology, Nagoya University Graduate School of Medicine, Japan We tested hypothesis that additional Ca2+ dissociating from TnC might contribute to slow force response (SFR) in cardiac muscle. For that we used levosimendan (LVS) as Statins are commonly used for treatment of hypercholesterolaemia in a a calcium sensitizer. number of patients. They are also known to have antiarrhythmic effects, Rat right ventricular papillary muscles (n=7, Krebs solution, 250C, pacing interval but the mechanisms of their action are not completely understood. As the
3 s) were subjected to sudden stretch from 85% to 95% of LMAX, with simultaneous incidence of atrial fibrillation correlates with atrial stretch, we hypothesized measurement of force and calcium transients during 15 minutes in control solution and that statins affect the mechanosensitivity of the cardiac myocyte. To test this at 0.1, 1 and 10 μM LVS. hypothesis, we measured the stretch-induced calcium response of myocytes. At any studied concentration LVS decreased twitch force (at ~7%, ~10% and ~35% The rat cardiac myoblast H9c2 cells were grown in elastic silicone chambers. relatively control under 0.1, 1 and 10 μM LVS, respectively) on any stage of SFR, but A transient 15%-stretch was applied to the cells and their calcium response more pronouncedly at prestretch length and in lesser degree immediately after stretch. was observed using the calcium indicator, Fura2. 10 min application of 100 It was found that LVS affects SFR with high variability. 2 of 7 preparations showed μM-simvastatin, 100 μM-lovastatin and 100 μM-cerivastatin significantly dose-dependent increase of SFR after LVS application. In the rest of the measurements inhibited stretch-induced calcium increase by 74.3%, 39.8% and 36.4%, LVS did not modify SFR. Additionally, LVS in any concentration did not change peak respectively. Reduced stretch response by cerivastatin was completely calcium at any stage of SFR, but tended to slow down calcium relaxation at level recovered by washing out the drug. After washing out simvastatin and higher than ~50% of peak calcium and to accelerate it at low level. lovastatin, we observed partial recovery from the diminished calcium As the result, due to changing of affinity of Ca-ions to TnC and thus the number response by stretching. These results suggest that statins diminish the of force-generating cross bridges, LVS modifies SFR to sudden stretch in rat mechanosensitivity of the heart in the short-term, presumably by directly myocardium. affecting the stretch-sensing mechanism in the cell.
P1PM-2-4 P1PM-2-5 MEF2 DIRECTLY REGULATES THE TRANSCRIPTION EFFECTS OF THE DOXAPRAM HCL ON THE CARDIAC OF HCN4, CARDIAC PACEMAKER CHANNEL CONDUCTION SYSTEM Makoto Takano1, Kuniko Shimazaki1, Shin-ichi Muramatsu2, Hiroko Nomura1, Tadayoshi Hata2, Hirofumi Kusuki3, Tetsumi Irie4, Koichiro Kuwahara3 Mitsuru Irikura4, Yoshie Kochiyama4, Akiko Tanaka4, Shunji Nagaoka5 1 1Department of Physiology, Jichi Medical University, Japan, 2Division Department of Pharmacology, School of Health Sciences, Fujita Health University, Japan, 2Department of Clinical Pathophysiology, School of Health of Neurology, Department of Medicine, Jichi Medical University, Japan, 3 3 Sciences, Fujita Health University, Japan, Department of Clinical Laboratory, Department of Medicine and Clinical Sciences, Graduate School of 4 Mie University, Japan, Department of Clinical Chemistry and Informatics, Medicine, Kyoto University, Japan Graduate School of Medical and Pharmaceutical Sciences, Kumamoto Hcn4 gene is known to encode the hyperpolarization activated, cyclic nucleotide University, Japan, 5Department of Physiology, School of Health Sciences, Fujita sensitive channel. HCN4 is also known as a molecular maker of cardiac Health University, Japan sino-atrial node. To investigate its transcriptional mechanism, we screened Background: In a series of experiments, we have shown that the doxapram HCl inhibited evolutionally conserved non-coding sequences which are often involved in the cardiac conduction system under urethane anesthesia in the adult wister rats. The the regulation of gene expression. The VISTA Enhancer Browser identified purpose of the present study is to evaluate the effect of doxapram HCl on the cardiac conduction system under sevoflurane anesthesia which has been clinically used. Materials 16 regions within Hcn4 locus, and one of such region was found to possess and Methods: ECG and respiratory movements were recorded by Biopac MP-35 system. prominent enhancer activity (> 30-fold) on Hcn4 promoter. Subsequent The doxapram HCl was repeatedly injected into the right femoral vein with an appropriate
Poster Session mutation analysis revealed that its enhancer function was dependent on AP1- interval in each animal. The effect of the drug was evaluated by measuring RR, PQ and QT and MEF2-binding sequences. EMSA and ChIP experiments confirmed that intervals of ECG. The concentration of the drug was measured with the high performance AP1 and MEF2 proteins bound this site. Overexpression of dominant negative liquid chromatography. Results: 1) RR interval was dose dependently prolonged with MEF2 mutant inhibited the enhancer activity, and decreased mRNA of Hcn4 in administration of the doxapram HCl. 2) PQ interval showed no change in any dose of the doxapram HCl. 3) QTc value was significantly prolonged by administration of the drug (3.0 the myocytes isolated from the inflow tract of fetal rat heart. Furthermore, the mg/kg and 10 mg/kg) as compared with the control. 4) The concentration of doxapram amplitude of hyperpolarization activated current was significantly decreased by HCl in the blood was dose-dependently increased. Conclusion: We should carefully pay an the transfection of dominant negative MEF2. Our results suggest that the novel attention to QT prolongation when we use the doxapram HCl for an accelerant of respiration enhancer play a critical in the transcription of Hcn4 in the heart. under the sevoflurane anesthesia.
170 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-2-6 P1PM-2-7 EFFECTS OF SIMULTANEOUS SITE-TIMING IMPROVEMENT OF MULTI-CHANNEL BIOTELEMETRY OPTIMIZATION WITH BIVENTRICULAR PACING SYSTEM FOR MONITORING THE NEURAL SIGNALS DURING ACUTE VENTRICULAR FAILURE ARE Naoki Nishiura, Ishio Ninomiya PATHOLOGY DEPENDENT Cardiac Physiology, Nat. Cardio-Vascular CTR. RES. INS., Japan 1 2 3 T Alexander Quinn , Santos E Cabreriza , Marc E Richmond , We had been developed the biotelemetry system for recording the neural 4 5 2 Alan D Weinberg , Jeffrey W Holmes , Henry M Spotnitz signals. In this time, multichannel telemeter, which enabled to record 1Department of Physiology, Anatomy and Genetics, University of Oxford, UK, 2 3 simultaneously 4 channels of neural signals, was developed. We improved Department of Surgery, Columbia University, USA, Department of Pediatrics, the modulation and demodulation circuits to transmit high sub-carrier Columbia University, USA, 4Department of Biostatistics, Columbia University, USA, 5Departments of Biomedical Engineering and Medicine, University of frequency modulation pulse train about 60kHz. This telemetry system has Virginia, USA employed FM-PWM (frequency modulation- pulse width modulation) Hypothesis: Effects of simultaneous left ventricular pacing site (LVPS)-interventricular pacing method, and applied multi-sampling rate for multiplex the input signals delay (VVD) optimization with biventricular pacing (BiVP) differ in acute LV and right ventricular depend on the individually frequency band. At the neural amplifiers, three (RV) failure. Methods: In open-chest pigs, BiVP was performed with right atrial and RV leads and OP400 were used for the head-amplifier and one OPA4348 was used for a LV pacing array. Acute LV volume overload (LVVO) was induced by 30% regurgitant flow via an the output stage. OPA4277 was used for the DC amplifier. Other biological aortic-LV conduit (n=6) and RV pressure overload (RVPO) by doubling peak RV systolic pressure signals were amplified by the OP400 and OPA4348. To reduce the size with a pulmonary snare (n=6). 54 LVPS-VVD combinations were tested. Cardiac output (CO) was and weight, small packed IC was employed. Last year, we reported the evaluated by aortic flow probe, interventricular synchrony by RV-LV pressure diagram area (APP), and ventricular function by dP/dtmax. Effects were assessed by 3-factor ANOVA. Results: In LVVO, digital signal processing system (modulation and demodulation circuits). optimization improved all functional indices (CO: +8±2%; APP: -0.45±0.03; LV dP/dtmax: +9±3%; Almost all circuits were not change except sub-carrier oscillation frequency. RV dP/dtmax: +12±3%), with a different optimum LVPS-VVD combination for each index. Function Discrete elements, small 60KHz crystal and CMOS inverter constructed this was also improved in RVPO, however changes (CO: +5±2%; APP: +0.32±0.03; LV dP/dtmax: no oscillation. On the receiver, two TA7302 were used for the IF amplifier and effect; RV dP/dtmax: +20±5%) and optimum settings differed from those in LVVO (CO: p<0.001; APP: p<0.001; LV dP/dtmax: p=0.035; RV dP/dtmax: p<0.001). Conclusion: Effects of LVPS-VVD TA7031P for the discriminator. As the result, this system could transmit the optimization during acute ventricular failure are pathology dependent. data and demodulated the input signals.
P1PM-2-8 P1PM-2-9 A REGIONAL GRADIENT OF INTRINSIC RHYTHMICITY CHEWING REDUCES STRESS INDUCED ARRHYTHMIA DEPICTED IN EMBRYONIC CULTURED MULTIPLE- So Koizumi1, Yumie Ono2, Kenichi Sasaguri1, Sadao Sato1, HEARTS Minoru Onozuka2 1 2 3 4 Tetsuro Sakai , Akihiko Hirota , Toshihiko Yada , Hitoshi Komuro , 1 5 Dept of Craniofacial Growth & Development Dentistry, Kanagawa Dental Kohtaro Kamino College, Japan, 2Dept of Physiology and Neuroscience, Kanagawa Dental 1 Department of Physiology, University of the Ryukyus School of Medicine, College, Yokosuka, Japan Japan, 2Department of Physiology, Shimane University, School of Medicine, Japan, 3Division of Integrative Physiology, Department of Physiology, Jichi To address the involvement of chewing in response of autonomic nervous system Medical School, Japan, 4Department of Neurosciences/NC30, Lerner Research (ANS) to stress, we measured electrocardiogram (ECG) changes in male SD Institute, The Cleveland Clinic Foundation, USA, 5Department of Physiology, rats, which underwent immobilization stress for 30 min. Animals were divided Tokyo Medical and Dental University School of Medicine, Japan into two groups: (stressed group; ST), and to chew a wooden stick during the We have used optical methods to examine the spatial gradient of intrinsic ryhthmicity in immobilization (stressed and chewing group; SC). We recorded ECG through the early stage multiple-heart chick embryos which were induced experimentally in whole the whole experiment using radiotelemetry. The SC showed significantly higher embryo culture. The embryos were cut microsurgically through the tissue of the anterior heart rate (HR) during stress exposure (567 ±8 bpm, n=4) than in the ST (528±6 intestinal portal at the 5- to early 7-somite developmental stage. Spontaneous electrical activity in 4 to 6 segmented hearts, during the 7- to 10-somite stages of development, bpm, n=4, p<0.05). However, recovery of the increased HR to the pre-stress level were monitored simultaneously by means of multiple-site optical recordings of membrane was shorter in SC (74±23 min and over 4 hr in SC and ST, respectively). When potential activity. Each segment of the heart exhibited its own inherent rhythmicity. In the occurrence of arrhythmia was manually determined and quantified as number quadruple-hearts, the order of the rhythmicity was often [left-caudal segment] > [right- of events. Arrhythmias (mostly ventricular premature beats, VPBs) appeared caudal segment] > [left-cephalic segment] > [right-cephalic segment]; the heart rate in the more frequently in ST (7±2 times) than SC (4±1 times) during post-stress period. left-caudal segment was often faster than that in the other segments. An atypical pattern of Furthermore, a significant positive correlation was seen between recovery time “bursting” rhythm was observed in the cephalic segments, suggesting that, in these segments, the development of the rhythmicity is relatively poor. These findings strongly emphasize of HR and occurrence of arrhythmia (r = .878, P < .004) .The results suggest the concept that, in the early phases of cardiogenesis, the formation of a regional gradient of that chewing under stress condition might relieve stress-induced arrhythmia by pacemaker activity results in the functional self-organization of the pacemaking area. reducing post-stress activation of the ANS.
P1PM-2-10 P1PM-2-11 THE EFFECTS OF ADRENALECTOMY AND NERVE CONDUCTION VARIABLES AND HEART AUTONOMIC BLOCKADES ON THE EXERCISE- RATE VARIABILITY: A POSSIBLE PHYSIOLOGICAL INDUCED TACHYCARDIA IN CONSCIOUS RATS CORRELATION IN AEROBIC PLAYERS Rie Wakasugi, Tomoko Nakamoto, Kanji Matsukawa Pradeep Bhandari, Bishnu Hari Paudel, Paras Nath Singh, Department of Physiology, Hiroshima University, Japan Asim Das Heart rate (HR) during exercise is controlled by cardiac sympathetic efferent Department of Physiology, BP Koirala Institute of Health Sciences, Nepal nerve activity (CSNA), cardiac vagal efferent nerve activity (CVNA) and This study correlated standard sensory & mixed nerve conduction variables plasma catecholamines. To examine their contribution to the exercise- (NCV) & short-term heart rate variability (HRV) of consenting professional induced tachycardia by adrenalectomy (ADX) and autonomic blockades, aerobic male players (21.8±2.5 years, n=31) compared to matched healthy non- we measured HR during treadmill exercise (20 m/min for 30 min) in 13 players (22.9±2.6 years, n=30). Players showed negative correlation between left conscious rats. Baseline HR did not differ between intact and ADX rats (367 tibial F-wave latency & HRV high frequency peak (HFP, r=-.36, p<.05), & sural vs. 350 beats/min, respectively), suggesting no significant role of adrenal sensory nerve action potential (SSNAP) duration & low frequency (LF) power catecholamines on the baseline HR. Since the baseline HR was increased to (r=-.38, p<.05). Right common peroneal (CP) compound muscle action potential 428 beats/min by atropine (1.5 mg/kg iv) and decreased to 341 beats/min by (CMAP) conduction velocity & SSNAP latency were positively correlated with atenolol (3 mg/kg iv), CVNA more determined the baseline HR than CSNA HFP (r=.43, p<.05). SSNAP amplitude was positively correlated with LF peak and adrenal catecholamines. In contrast, the exercise-induced tachycardia, (r=.36, p<.05) & its conduction velocity with LF power (r=.4, p<.05). In non- which was 102 beats/min in the intact condition, was blunted by atenolol (30 players, left tibial CMAP duration was positively correlated with LF peak (r=0.5, beats/min) or atropine (25 beats/min) and slightly decreased by ADX (10 p<.01) & negatively with LF peak (r=-.38, p<.05). Left CP CMAP latency was beats/min). The HR increase at the onset of exercise was the same between positively correlated with NN50 (r=.37, p<.05), pNN50 (r=.38, p<.05) & LF intact and ADX conditions, but was more blunted by atenolol than atropine. power (r=.38, p<.05); & its F-wave latency with RMSSD (r=.37, p<.05), NN50 In conclusion, the exercise-induced tachycardia is initially produced by a (r=.4, p<.05), & pNN50 (r=.38, p<.05). Physical training decreases correlation prompt increase in CSNA and then controlled by a decrease in CVNA and an between somatic motor function & HRV but increases between somatic sensory increase in adrenal catecholamines as well. function & HRV. Physical training changes the NCV quicker than HRV. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 171 P1PM-2-12 P1PM-2-13 HEART RATE VARIABILITY DURING FOLLICULAR AND A NEWLY PROPOSED METHOD, A DELAYED LUTEAL PHASES OF MENSTRUAL CYCLE LORENZ MAP, DETECTS THE CHAOTIC NATURE OF Dmitry Alekseevich Dimitriev, Elena Vladimirovna Saperova HEARTBEATS Anatomy, Physiology and Hygiene Department, Chuvash State Motohisa Osaka Pedagogical University named after I. I. Yakovlev, Russia Department of Basic Science, Nippon Veterinary and Life Science The purpose of the present study was to evaluate the influence of the normal University, Japan menstrual cycle on heart rate (HR) and heart rate variability (HRV). 14 The dynamics of heart rate control had been under debate whether the healthy, young woman were studied during the follicular phase and luteal dynamics is chaos or not. We propose a new method, a delayed Lorenz map, phase over three month. HRV was recorded for 5 minutes while subjects were in order to detect determinism and instability geometrically. The Lorenz map
resting in a supine position. We found pronounced changes in HR during the zn+1 = f(zn), which projects a local maximum zn to the next local maximum menstrual cycle with a minimum in the follicular phase and maximum in the zn+1 of the time series, is useful for such a thin attractor as Lorenz attractor, luteal phase (Mann-Whitney test (U)=1008.00; p<0.0001). HF component of but is not applicable for such a fat attractor as the circulation regulatory
HRV, which reflects only parasympathetic activity, decreased more during the dynamics. It is shown that a T-delayed Lorenz map zn+T = fT(zn), which luteal phase than follicular phase (U=1557.00; p=0.0016). The LF/HF ratio, projects a local maximum zn to the T-delayed local maximum zn+T, is more reflecting the balance of autonomic nerve activities, in the luteal phase was useful. It is found that the appearance of a parabolic image in a T-delayed significantly higher than that in the follicular phase (U=1818.50; p=0.04). Lorenz map is a sign of chaos. The parabolic images appear by applying a Futhermore, SDNN was significantly higher in the follicular phase compared T-delayed Lorenz map to the time series of heartbeats recorded for over 100 to luteal phase (U=1808.50; p=0.04). This findings indicate that sympathetic minutes in normal conscious unrestrained rats (7 each). nervous activity in the luteal phase is significantly greater than in the follicular phase whereas parasympathetic nervous activity is predominant in the follicular phase. Some endogenous female sex hormones may be responsible for these changes in the cardiac autonomic innervation.
P1PM-2-14 P1PM-3-1 AUTONOMIC AND CARDIOVASCULAR RESPONSES ENDOGENOUS ACTIVATION OF 5-HT1A RECEPTORS TO STIMULATION OF MIDBRAIN DOPAMINERGIC SUPPRESSES MUSCLE MECHANOSENSITIVE REFLEX NEURONS IN ANESTHETIZED RATS IN THE CONSCIOUS CONDITION Tomoko Nakamoto1, Kanji Matsukawa1, Rie Wakasugi1, Nan Liang1, Kanji Matsukawa, Manami Shimizu, Akito Kadowaki, Britt L Wilson2, Jouji Horiuchi3 Tomoko Nakamoto, Nan Liang, Rie Wakasugi 1Department of Physiology, Hiroshima University, Japan, 2Department of Department of Physiology, Hiroshima University, Japan Pharmacology, Physiology and Neuroscience, University of South Caroline, 3 We have shown that the cardiovascular response to passive stretch of skeletal School of Medicine, USA, Department of Physiology, University of Sydney, muscle is suppressed in the conscious state. To test a hypothesis if the brain Australia serotonergic system causes the suppression of muscle mechanoreflex, we Heart rate (HR) has a positive linear relationship with arterial blood pressure (AP) during examined the effects of antagonists and agonists of 5-HT1A. and 5-HT2A. receptors 24-hour period in humans, suggesting that the major changes in HR are not controlled by on the responses in heart rate (HR) and arterial pressure (AP) during passive arterial baroreflex but by central command coupled with voluntary activities in daily life. stretch of the hindlimb using conscious cats. We confirmed that the hindlimb Since the positive AP-HR relationship was lost or weakened in both idiopathetic Parkinson’ passive stretch did not alter HR and AP. IV injection of an antagonist of 5-HT1A. s disease patients and vascular Parkinson’s patients, we hypothesized that the midbrain receptor (NAN-190) augmented the reflex cardiovascular response during stretch, dopaminergic system plays a role in producing central command signals for autonomic control of the cardiovascular system. To examine this hypothesis, we investigated whereas an agonist of 5-HT1A. receptor and an antagonist or agonist of 5-HT2A. the autonomic and cardiovascular responses to electrical and chemical stimulation of receptor did not modulate them. After administering pentobarbital (25mg/kg iv), dopaminergic neurons in the midbrain, in particular the ventral tegmental area (VTA) in the identical hindlimb stretch could induce a greater cardiovascular response. anesthetized rats. Microinjection of bicuculline methochloride (2mM, 60-180 nl) increased Injection of an agonist of 5-HT1A. receptor (buspirone) blunted the reflex HR and AP. Electrical stimulation of VTA for 30 s (100 μA, 50 Hz, 1ms in duration) caused cardiovascular response during stretch and a subsequent injection of NAN-190 a large increase in limb blood flow, suggesting vasodilatation of skeletal muscle blood reversed the blunted effect. Neither antagonist nor agonist of 5-HT2A. receptors vessels. The present findings are in favor of our hypothesis that the midbrain dopaminergic modulated the cardiovascular response to passive stretch in the anesthetized state. neurons are closely by linked with generating central command responsible for the We conclude that endogenous activation of 5-HT1A. receptors suppresses muscle cardiovascular adjustment during exercise. mechanoreflex in the conscious state.
P1PM-3-2 P1PM-3-3 EFFECTS OF THE FIRST TISSUE-SPECIFIC HEART RATE VARIABILITY AND BETA-2 ADRENERGIC ACETYLCHOLINESTERASE INHIBITOR C-547 IN THE RECEPTOR HAPLOTYPE IN HUMANS RAT ATRIUM John H Eisenach1, Tasha L Pike1, Emma C Hart1, 1 2 2 Denis Valerievich Abramochkin , Konstantin A Petrov , Leniz F Nurullin , 1 2 1 3 2 4 John E Schmidt , Stephen T Turner , Michael J Joyner Lilia O Yagodina , Eugen E Nikolsky , Leonid V Rosenshtraukh 1 1 Department of Anesthesiology, Mayo Clinic College of Medicine, USA, Department of Human and Animal Physiology, Moscow State University, 2 Russia, 2Kazan Institute of Biochemistry and Biophysics, Russian Academy of Department of Medicine, Mayo Clinic College of Medicine, USA Sciences, Kazan, Russia, 3Institute of Organic and Physical Chemistry, Russian 4 Polymorphic variation in the beta-2 adrenergic receptor gene (ADRB2) influences Academy of Sciences, Kazan, Russia, Institute of Experimental Cardiology, cardiovascular traits. In 149 healthy males from Kyoto, Japan, heart rate variability Moscow, Russia (HRV) indices were influenced by Arg16/Gly and Gln27/Glu polymorphisms; the The novel acetylcholinesterase (AChE) inhibitor C-547 is one of the alkylammonium allele frequency of Glu27 was low and there were no Glu27 homozygotes. In contrast, derivatives of 6-methyluracil. We have studied the effects of C-547 on action potential (AP) and sinus rhythm (SR) in the rat isolated right atrium and compared this effects with those the allele frequency of Glu27 is much higher in the U.S. From an ongoing physiologic of organophosphorous AChE inhibitor armin and carbamate inhibitor neostigmine. Both phenotyping protocol, we evaluated HRV in healthy men and women, age 18-40, from armin (10-7, 10-6, 10-5M) and neostigmine (10-7, 10-6, 5X10-6M) produced a decrease in AP Rochester, MN who were grouped by homozygous haplotype: Arg16+Gln27 (n=32), duration and slowing of SR. These effects were abolished by atropine, and are attributable Gly16+Gln27 (n=6), and Gly16+Glu27 (n = 33). Following instrumentation with -9 -7
Poster Session to the accumulation of acetylcholine (ACh) in myocardium. However, C-547 (10 -10 M) ECG, brachial arterial line, and pneumobelt, subjects underwent quiet supine rest for had no such effects. We have tested several hypothesis concerning such low sensitivity -8 -6 analysis of RR intervals from 5-10 min measuring windows by spectral analysis using of myocardium to C-547. C-547 (10 M) decreased effects of exogenous ACh (10 M) fast Fourier transform. LF and HF were analyzed by geometric mean, natural log more than twofold. This suggests that C-547 can block muscarinic receptors, partially or (ln), and normalized units (nu). There was evidence to suggest an effect of haplotype completely compensating for its anticholinesterase action. We have measured the inhibition on the LF mean, LFln, HF mean, and HFln, with the highest values occurring in the constant (kI) of C-547 and neostigmine in extracts of heart and sceletal muscle. KI of C-547 on cardiac AChE is 10000-fold higher than on extensor digitorum longus muscle AChE, Gly16+Gln27 group (PANOVA < 0.05) but these effects were not apparent for LFnu, while there is no such difference for neostigmine. Thus, C-547 is a tissue-specific inhibitor of HFnu, or LF/HF ratio. We conclude HRV may be related to ADRB2 haplotype in skeletal muscle AChE that has little effects on heart AChE. some populations, and more studies are needed to replicate these findings.
172 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-3-4 P1PM-3-5 IL-6 MICROINJECTED INTO THE NUCLEUS TRACTUS COMPLEX CARDIOVASCULAR ACTIONS OF SOLITARII ATTENUATES BARORECEPTOR REFLEX α-ADRENERGIC RECPTORS EXPRESSED IN THE FUNCTION IN RATS NUCLEUS TRACTUS SOLITARII OF RATS Miwa Takagishi1, Hidefumi Waki2, Mohammad ER Bhuiyan2, Mohammad ER Bhuiyan, Hidefumi Waki, Miwa Takagishi, Sabine S Gouraud2, He Cui2, Akira Kohsaka2, Toshiya Yamazaki2, Sabine S Gouraud, He Cui, Toshiya Yamazaki, Akira Kohsaka, Julian FR Paton3, Masanobu Maeda2 Masanobu Maeda 1 Department of Physiology,Wakayama Medical University ; Department of Department of Physiology, Wakayama Medical University, Japan Therapeutic Health Promotion, Kansai University of Health Sciences, Japan, 2Department of Physiology, Wakayama Medical University, Japan, 3Department Although both α-1 and α-2 adrenergic receptors are known to be expressed in the of Physiology & Pharmacology, University of Bristol, UK nucleus tractus solitarii (NTS), the functional significance of these receptors is still not fully established. In this study, we microinjected α-1 and α-2 adrenergic receptor Recent studies have demonstrated that an abnormal gene expression profile of interleukin-6 agonists into the NTS of urethane-anesthetized Wister rats to study the cardiovascular (IL-6) in the nucleus tractus solitarii (NTS), a pivotal region for regulating arterial pressure, effects in response to their activation. When the α-1 adrenergic receptor agonist may be related to the development of neurogenic hypertension. However, the functional phenylephrine was microinjected into the area where barosensitive neurons are role of IL-6 in the NTS remains unknown. In the present study, we have tested whether IL-6 affects cardiovascular control at the level of the NTS. IL-6 (1 fmol, 10 fmol, 100 fmol ) was dominantly located (baro-NTS), mean arterial pressure (MAP) and heart rate (HR) microinjected into the NTS of Wistar rats (250-350g) under urethane-anesthesia. Although were elevated. When tested in the area where chemosensitive neurons are dominantly the baseline levels of arterial pressure and heart rate did not change by IL-6 injections, located (chemo-NTS), however, MAP and HR were decreased. In contrast, the cardiac baroreflex in response to increased arterial pressure was dose-dependently microinjection of the α-2 adrenergic receptor agonist clonidine into either the baro- or attenuated. In addition IL-6 (100 fmol) microinjections also attenuated L-glutamate induced chemo-NTS decreased MAP and HR. Moreover, we immunohistochemically found bradycardia at the level of the NTS. Immunohistochemical detection of IL-6 demonstrated that cardiovascular responses induced by α-1 adrenergic receptors may be mediated that IL-6 staining was dominantly observed in neurons within the NTS. These findings by neurons while those induced by α-2 adrenergic receptors may be mediated by suggest that IL-6 within the NTS may play an important role for regulating cardiovascular astrocytes in the NTS. These results suggest that both types of α-adrenergic receptor in control via modulation of input signals from baroreceptor afferents. Whether the abnormal the NTS may be involved in regulating cardiovascular homeostasis via modulation of gene expression of IL-6 in the NTS is pro-hypertensive remains to be resolved. input signals from baroreceptor and chemoreceptor afferents.
P1PM-3-6 P1PM-3-7 STUDY ON HEART RATE VARIABILITY IN STUDY ON AUTONOMIC FUNCTION STATUS AND ADOLESCENT MALE ATHLETES THEIR RELATION WITH SERUM ESTROGEN AND Noorzahan Begum1, Masud Alom2, Sultana Ferdousi1, PROGESTERONE LEVELS IN POSTMENOPAUSAL Shelina Begum1, Taskina Ali1 WOMEN 1 2 1 1 1Physiology, Bangabandhu Sheikh Mujib Medical University, Dhaka, Sultana Ferdousi , Latifa dil Afrin , Noorzahan Begum , Shelina Begum , 1 Bangladesh, 2Sylhet, Bangladesh Taskina Ali 1Department of Physiology, Bangabandhu Sheikh Mujib Medical University, ABSTRACT 2 Dhaka, Bangladesh, Moulana Bhasani Medical College, Uttara, Dhaka, Background: Cardiac autonomic activities deteriorate with age, obesity, Bangladesh sedentary life style . Objective: To observe the heart rate variability in male adolescent athletes to find out the influence of regular physical exercise on ABSTRACT Autonomic nerve functions were studied in30 apparently healthy postmenopausal women cardiac autonomic activities. Method: This cross sectional study was carried out on aged 45-60 years and 30 premenopausal women with age 20-30 years. Five cardiovascular 62 adolescent male athletes aged 12-18 years, in the Department of Physiology reflex tests including heart rate response to valsalva maneuver, heart rate response to deep in 2008. For comparison 30 age, sex ,BMI matched apparently healthy sedentary breathing, heart rate response to standing and sustained handgrip test and BP response to subjects were also studied. HRV parameters were assessed by Polygraph . Data standing were performed and serum estrogen and progesterone levels were measured. were analyzed by Idependent-Samples t-test and Pearson’s correlation coefficient Correlations of autonomic function parameters with hormone levels and duration of tests. Results: Mean resting heart rate, the LFnu and the LF/HF ratio were menopause were observed. For statistical analysis, ANOVA and Multiple regression analysis significantly lower in the athletes. Mean R-R interval, SDNN, total power, HFnu were used. Results: Mean resting SBP and DBP were significantly higher in postmenopausal and VLF,LF,HF were significantly higher in athletes . The variance, LF and HF women. Significantly decreased value of HR response to deep breathing and HR response to standing were observed in postmenopausal women. Again, significant positive correlations in athletes and LF/HF ratio in nonathletes showed significant positive correlation of parasympathetic function parameters and non significant negative correlations of and LF/HF ratio in athletes and HF power in nonathletes significant negative sympathetic function parameters with estrogen were observed. Postmenopausal women had correlation with age and BMI.. Conclusion:Regular physical exercise increases varying degrees of autonomic neuropathy (66.66%), and had short duration of menopause. cardiac parasympathetic predominance with shifting of sympatho-vagal balance Conclusion: altered autonomic function along with autonomic neuropathy may occur in towards parasympathetic. postmenopausal women depending on duration of menopause
P1PM-3-8 P1PM-3-9 THE AUTONOMIC NERVOUS RESPONSES DURING ACTIVATION OF CARDIAC SYMPATHETIC THE VOLUNTARY ABDOMINAL BREATHING IN THE OUTFLOW, BUT NOT VAGAL WITHDRAWAL, DURING ELDERLY SPONTANEOUS MOTOR ACTIVITY IN DECEREBRATE Michiko Tanaka1, Mou Nagasaka1, Tomoko Yano1, CATS Toshio Kobayashi2, Yoshikazu Sakakibara3 Akito Kadowaki, Kanji Matsukawa, Tomoko Nakamoto, Nan Liang, 1Department of Nursing, Miyazaki Prefectural Nursing University, Japan, Rie Wakasugi, Manami Shimizu 2Hiroshima University, Japan, 3Kanazawa Institute of Technology, Japan Department of Physiology, Graduate Shcool of Health Sciences, Hiroshima The purpose of this study is to elucidate the effects of voluntary abdominal University, Japan breathing using the diaphragm on the autonomic nervous and cardiovascular It has been thought that cardiac acceleration the start of exercise is evoked by responses in the elderly. The subjects carried out the spontaneous or withdrawal of cardiac vagal nerve activity (CVNA), but not by activation of abdominal breathing at 6 breaths/min for 15 min after 5min spontaneous cardiac sympathetic nerve activity (CSNA). To clarify whether this assumption is breathing. Cardiovascular responses during the abdominal breathing were true, we directly measured CVNA and CSNA during spontaneous motor activity similar to those responses during the spontaneous breathing. Heart rate, in 4 decerebrate cats immobilized with neuromuscular blockade. The activity was blood pressure and pressure rate product (SBP×HR) during the abdominal confirmed by measuring motor discharges of the tibial nerve tied peripherally. As soon breathing were gradually decreased, but not significantly. Using the Lorenz as spontaneous motor activity occurred, CSNA increased by 96 ± 13% at 1 s from the onset of the activity. In contrast, CVNA gradually increased by 71 ± 12%. Heart plot methods of the heart rate variability, we analyzed the autonomic rate and mean arterial pressure increased by 6 ± 1 beats/min and 22 ± 2 mmHg. On nervous responses during the abdominal breathing. The abdominal breathing the other hand, during passive stretch of the hindlimb, CSNA gradually increased by increased log(L×T), which indicated the activities in parasympathetic nervous 72 ± 28% at 22 s from the onset of stretch, while CVNA gradually decreased by 46 response compared to that of resting state before the abdominal breathing, ± 15%. Not the increase in CSNA but the decrease in CVNA was sustained until the but not significantly. Urine noradrenaline and adrenaline concentrations after end of stretch. In conclusion, augmentation of cardiac sympathetic outflow by central the spontaneous and the abdominal breathing were lower than before the command, but not vagal withdrawal, contributes to the cardiovascular adaptation experiment. It is suggested that the voluntary abdominal breathing using the during spontaneous motor activity, whereas a muscle mechanoreflex causes an diaphragm might induce beneficial in management of the stress to the elderly. increase in CSNA and a decrease in CVNA. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 173 P1PM-3-10 P1PM-4-1 DEVELOPMENT OF BASAL/INTRINSIC HEART RATE VANILLOID RECEPTOR 1 MEDIATES THE REFLEX AND AUTONOMIC NERVOUS SYSTEM IN MICE CARDIORESPIRATORY ALTERATIONS EVOKED BY EVALUATED BY USING A PIEZOELECTRIC (PZT) INTRA-ARTERIAL INJECTION OF MESOBUTHUS SENSOR TAMULUS VENOM Shinichi Sato, Kyoichi Ono Sanjeev Kumar Singh, Shripad B. Deshpande Department of Physiology, Akita University, Japan Department of Physiology, Institute of Medical Sciences, Banaras Hindu Piezoelectric (PZT) sensor enabled a noninvasive measurement of basal/intrinsic University, India heart rate (HR) of freely-moving newborn and adult mice without inducing Present study was conducted to examine the role of long lasting stimulation of unpreferable HR response due to experimental condition (Sato, 2008). Basal HR perivascular nociceptors and the afferent pathways involved in mediating the at immediately after birth (320 b/m) increased in an exponential-like curve to cardiorespiratory reflexes evoked by intra-arterial (i.a.) injection of Indian red scorpion ~700 b/m during the first two postnatal weeks, whereas intrinsic HR measured (Buthus tamulus; BT) venom. The reflexes were elicited by injecting the venom in under autonomic blockade (~300 b/m) remained unchanged during P0-P5, the distal end of femoral artery in urethane anesthetized rats. The effect of venom on then increased rapidly to ~500 b/m (= adult intrinsic HR) during P5-P9. This blood pressure (recorded from the proximal end of the same artery), heart rate, and suggests that postnatal increase in HR is caused by both adrenergic stimulation respiration rate was recorded for 60 min. After the i.a. injection of venom, there was and intrinsic pacemaker activity of the heart. Furthermore, we found that immediate hyperventilatory (~2 s), intermedaite hypertensive (~40 s) and delayed newborn mice show freezing behavior in response to attaching ECG electrodes bradycardiac responses (~5 min). The maximal responses were seen at 1 mg/kg of with concomitant decreasing-HR response that varied depending on postnatal venom. The venom-induced responses were markedly attenuated after pretreatment day during the first two postnatal weeks. In addition, mice exhibited a vagally- with the capsazepine (vanilloid receptor 1 antagonist). However, the ipsilateral mediated bradycardia during sleep, which progressively increased its amplitude neurotomy or lignocaine pretreatment partially attenuated the blood pressure and with age from P5. These parasympathetic control of HR appeared and matured respiratory responses but not the heart rate. Hence, a separate pathway is proposed for during P4-P9 coincident with the period of the steep elevation of intrinsic HR. the regulation of heart rate changes. The data provide evidences for the involvement The present study has revealed several features in development of HR and of vanilloid receptor 1 and the afferents running mainly through the ipsilateral somatic autonomic nervous system of mice from newborn to adult. nerve in mediating the vasosensory responses.
P1PM-4-2 P1PM-4-3 DEVELOPMENT AND PREVENTION OF LEFT MULTI-FUNCTIONAL AND HIGH-THROUGHPUT VENTRICULAR DIASTOLIC FAILURE IN OLD PHYSIOLOGICAL PROFILING OF ENGINEERED MYOCARDIAL INFARCTED RAT HEART TISSUES FOR DRUG DEVELOPMENT Naoyuki Otani, Setsu Nishino, Naohisa Nasuno, Yu Nishi, Vy Lam, Nazanin Tabesh, Anesa Badic, Matthew Staniszewski, Kazuhiko Oda, Shigeru Toyoda, Shichiro Abe, Isao Taguchi, Tetsuro Wakatsuki Matsuda Ryuko, Noboru Kaneko Biotechnology and Bioengineering Center, Medical College of Wisconsin, Department of Cardiology and Pneumology, Dokkyo Medical University, USA Japan Mitochondrial metabolic activities support physiological functions of tissues and Diastolic failure was induced with norepinephrine in rats with old myocardial organs. Therefore, drugs and toxic compounds that alter mitochondrial metabolism infarction to investigate the pattern of development of old myocardial infarct and can also alter tissue and organ physiology, e.g. contractility, and thus human improvement of this condition by K201, a 1,4-benzothiazepine derivative that has physiology. We developed a high-throughput physiological profiling system that a stabilizing effect on the ryanodine receptor. Ten to 13 weeks after ligation of the measures contractility, mitochondrial activity, and viability of engineered tissues (ETs) coronary artery, norepinephrine (20 μg/kg/min) was administered for 30 minutes. that mimic connective and myocardial tissues in vitro. Contractility of miniaturized K201 (0.1 mg/kg/min) was co-administered for 10 minutes during administration ETs, in 96-well format and treated with a library of compounds, is measured using a of norepinephrine in some rats. HR, LVSP, LVDP, LVEDP, +dP/dt and -dP/dt were robotic system: PalpatorTM. Concurrently, a plate reader measures the mitochondrial measured in the K201 group (n=5), non-K201 group (n=5), and a sham-infarct group membrane potential (MMP) with the fluorescent dye TMRE and cellular viability with (n=3). Positive correlations of infarction size with LVDP and LVEDP were observed MTT assay. The MMP uncoupler dinitrophenol (DNP, 450 μM) reduced contractility after administration of norepinephrine, and K201 significantly decreased HR, LVSP, (69%) and MMP (35%) of cardiac ETs. DNP also dose-dependently reduced
LVDP and LVEDP and also improved the percentage diastolic capacity compared contractility and MMP of connective ETs with EC50 of 55 and 109 μM respectively. with the non-K201 group. These results indicate that K201 improves norepinephrine- DNP did not affect viability by 24 hr. The toxic pesticide rotenone, however, reduced
induced diastolic failure in the infarcted heart and that the functional mechanism contractility, MMP, and viability with EC50 of 0.6, 2.4, and 0.4 μM respectively. 2+ may involve reduction of intracellular Ca loading due to α1 receptor blocking and Further validation of ET-based high-throughput profiling system and its applications in ryanodine receptor stabilization. drug discovery research will be discussed.
P1PM-4-4 P1PM-4-5 BIOLOGICAL MEANING OF ALKALINE PHOSPHATASE GENE EXPRESSION PROFILES OF MAJOR AS AN INDEX FOR ARTERIOLAR CAPILLARY CYTOKINES AND CHEMOKINES IN THE NUCLEUS Akira Taka1, Tomiyasu Koyama2 TRACTUS SOLITARII OF THE SPONTANEOUSLY 1 2 HYPERTENSIVE RAT Sapporo Aoba Academy of Holistic Medicine, Japan, Hokkaido University, 1 1 1 Japan, Hidefumi Waki , Sabine S Gouraud , Akira Kohsaka , Julian FR Paton2, Masanobu Maeda1 Alkaline phosphatase (ALP) is expressed on the endothelial cells of 1 Department of Physiology, Wakayama Medical University, Japan, the arteriolar sides of coronary capillaries as shown by an infusion of 2Department of Physiology & Pharmacology, Bristol University, UK microspheres into the coronary artery in rats. A single injection of vasopressin Since the nucleus tractus solitarii (NTS) is a pivotal region for regulating the set- and/or a transient coronary occlusion followed by reperfusion caused point of arterial pressure, we proposed a role for it in the development of neurogenic significant increases in capillary portions expressing ALP. These results hypertension. Recent studies have suggested that pro-inflammatory molecules are suggest a meaningful role of ALP on coronary microvessels through an highly expressed in the NTS of an animal model of human essential hypertension - unknown pathway. However, there is a difficulty in understanding that ALP the spontaneously hypertensive rat (SHR), compared to normotensive rats (WKY). activity can be observed only in the alkaline medium. Recently Chesler et al. Based on this evidence, we hypothesized that inflammatory mediators such as cytokines are up-regulated in the NTS of SHR. In the present study, we have screened reported a pH rise in the interstitial space of electrically stimulated rat brains.
Poster Session for abnormally expressed inflammatory mediators in the NTS of SHR using the RT2 Assuming such a pH rise in the cardiac tissues a following interpretation Profiler PCR arrays, which were designed to specifically target major cytokines/ of the biological role of endothelial ALP seems possible. Adenosine related chemokines. In addition, for further confirmation of abnormal inflammatory condition substances are squeezed out from cardiac tissues by the cardiac contraction within the NTS of SHR, we identified gene expression levels of an inflammatory and decomposed to vasodilatory adenosine by ALP in localised regions near marker, gp39 precursor. The specific PCR array revealed that gene expression of IL-6, IFNa1, OX40L, growth differentiation factor 9, CCL5, CCL9 and CCR1 were found endothelial cells and cause the vasodilation in the posterior blood stream. to be differentially expressed in the NTS of SHR compared to the WKY. Moreover, gp39 gene was found to be 3-fold up-regulated in the NTS of SHR. These data further confirmed that the NTS of the SHR exhibits a specific inflammatory state.
174 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-4-6 P1PM-4-7 INCREASED GENE EXPRESSION OF ANTI-APOPTOTIC AEROBIC CAPACITY AND CENTRAL OBESITY ARE FACTORS IN THE NUCLEUS TRACTUS SOLITARII OF INDEPENDENT CARDIOVASCULAR RISK FACTORS IN SPONTANEOUSLY HYPERTENSIVE RAT DYSLIPIDEMIA Sabine Gouraud1, Hidefumi Waki1, Akira Kohsaka1, Jatuporn Wichitsranoi1, Naruemon Leelayuwat2, Nongnuch Settasatian3 2 1 1Department of Biomedical Sciences, Khon Kaen University, Thailand, Julian F. R. Paton , Masanobu Maeda 2 1 Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Department of Physiology, Wakayama Medical University, Japan, 3 2 Kaen, Thailand, Faculty of Associated Medical Sciences, Khon Kaen University, Department of Physiology & Pharmacology, University of Bristol, UK Khon Kaen, Thailand Since the nucleus tractus solitarii (NTS) is a pivotal region for regulating the set- To investigate relationships between aerobic capacity, serum lipid profiles and central obesity point of arterial pressure, we proposed a role for it in the development of neurogenic in dyslipidemia hypertension. Recent studies have suggested that the NTS of the spontaneously One-hundred and twenty-four dyslipidemia aged 47.9±0.7 years and 128 healthy sedentary hypertensive rat (SHR) exhibits a specific inflammatory state (Waki et al, subjects aged 44.7±0.7 years in urban, Thailand were recruited. Blood sample was obtained Hypertension, 2007; Waki et al, Auton Neurosci, 2008). As a consequence of the after a 12-hour fast and analyzed for lipid profiles. Anthropometric and body composition specific condition, we hypothesized that gene expression levels of apoptosis factors parameters were also investigated. Each subject underwent exercise test for determination of are altered in the NTS of SHR, compared to normotensive Wistar-Kyoto rat. In this aerobic capacity. study we have performed the RT2 Profiler PCR arrays targeting apoptosis related Body mass index, waist circumference, and waist to hip circumference ratio were factors to test this hypothesis. We have identified that the gene expression of the death significantly higher in dyslipidemia than those in healthy sedentary subjects. Aerobic capacity was significantly lower in dyslipidemia (21.5±0.7ml/kg/min, p<0.01) than that in receptor Fas (CD95/Apo-1) and the cysteine protease caspase 12 (Casp12) were the healthy subjects (24.4±0.7ml/kg/min). Based on Pearson's correlation analysis, aerobic down-regulated while the neuronal apoptosis inhibitory protein (NAIP) was found capacity was inversely related to serum low density lipoprotein cholesterol concentration up-regulated in the NTS of SHR. These results suggest that as a consequence of the (r=-0.16, p<0.05) and positively related to serum high density lipoprotein cholesterol specific inflammatory state, gene expression of anti-apoptotic factors may be increased concentration in dyslpidemia (r=0.36, p<0.01). However, aerobic capacity was not related to to promote cell survival in the NTS of SHR. Whether the abnormal gene expression waist circumference. contribute to the hypertensive state via alteration of neuronal circuitry regulating This study demonstrates that aerobic capacity and central obesity are independent cardiovascular autonomic activity needs to be elucidated. cardiovascular risk factors in dyslipidemia.
P1PM-4-8 P1PM-4-9 METABOLIC AND CARDIOVASCULAR RESPONSES TO CHANGES IN HEART RATE VARIABILITY INDICES ARM AND LEG EXERCISE IN ELITE KAYAKERS TO LABORATORY STRESSOR AFTER BREATHING Takashi Migita1, Mauro Marzorati2, Simone Porcelli2, TRAINING IN CHRONIC PAIN PATIENTS 1 2 1 Michele Belletti2, Paolo Cerretelli2, Claudio Marconi2 John E Schmidt , Heather M. Tonyan , William M. Hooten , 3 2 1Institute of Health and Sports Science, Kurume University, Japan, 2IBFM- Kevin I. Reid , Michael M. Joyner Physiology, Italian National Research Council, Italy 1Department of Psychiatry and Psychology, Mayo Clinic, USA, 2Department of Anesthesiology, Mayo Clinic, USA, 3Department of Dentistry, Mayo We compared the metabolic (V’O2; mechanical power, w’; blood lactate concentration, [La]) and cardiovascular (heart rate, HR; stroke volume, SV) Clinic, USA responses to arm cranking (AC) and leg pedaling (LP) in 6 top level kayakers The main objectives of this study were to determine if training and practice in a brief during incremental work (IW) up to exhaustion and in the transition from rest to focused breathing technique caused improvements in heart rate variability indices 3 submaximal constant-load (40, 60, and 80% of AC and LP w’peak) exercises after a standard laboratory stressor (cold pressor). The participants for this study were (CLE). At IW exhaustion, w’peak during AC and LP was 160 and 280 watt, chronic pain patients diagnosed with fibromyalgia (n=20) or masticatory myofascial pain (n=10). Study participants completed an initial laboratory assessment including respectively, whereas V’O2, [La], HR, and SV were almost alike (3.5 l min-1, 6-7 mM, 180 b min-1, and 160 ml, respectively). During CLE no significant a diaphragmatic breathing training session. Participants were instructed to practice differences were found between AC and LP: a) steady-state V’O and HR the technique for three ten-minute sessions daily, and returned to the lab for a second 2 assessment after two-weeks. Post-stressor HRV indices before and after training increased with intensity up to 80% of w’peak, whereas SV appeared to level off were compared using ANOVA. Significant differences were found on SDNN, total at 60%; b) mean response time and the time constant of phase II of the kinetics HRV power, LF/HF ratio, CCV-LF, and CCV-HF (all p’s<.05). Baroreflex power was of V’O2 readjustment at the onset of AC and LP CLE showed the tendency to marginally significant at p<.06. Study results suggest that practice of diaphragmatic increase similarly, at least up to 60% of w’peak. By contrast, gross efficiency breathing caused significant changes in physiological response to a standard laboratory during CLE increased with exercise intensity being significantly lower in AC stressor in chronic pain patients. In particular, changes in HRV indices may represent a than LP (0.13 and 0.19, respectively). In conclusion, during AC kayakers have significant improvement in post-training self-regulatory strength suggesting enhanced metabolic and cardiovascular responses to maximal and submaximal exercise inhibitory ability of sympathetic tone and improved physiological reactivity in daily similar to LP. AC was less efficient, whence the lower w’peak value attained. functioning.
P1PM-4-10 P1PM-4-11 COMPLETING THE ANALYSIS THROUGH RELATIONSHIP BETWEEN CORONARY ARTERY PHYSIOLOGICAL ASSESSMENT OF CARDIAC STENOSIS AND A WAVEFORM OF GRAFT FLOW FUNCTION Atsutoshi Hatada1, Yosihta Okamura1, Kentarou Honda1, Anil Kottam, Blair Poetschke Akira Kohsaka2, Hidefumi Waki2, Masanobu Maeda2 Department of Engineering, Scisense Inc., Canada 1Department of Thoracic and Cardiovascular Surgery, Wakayama Medical 2 It is increasingly evident that molecular research must be viewed in the University, Japan, Department of Physiology, Wakayama Medical context of Systems Biology. All levels of research, from genetic to whole University, Japan organ, must now be considered as a system. Object: We investigated the relationship of targeted, between stenosed rate of coronary artey and a fast Fourier transformation (FFT) analysis of the graft At the whole organ level, the major challenge in cardiac research is to successfully waveform. quantify cardiac function in rodent models due to the small size of the hearts and the Methods: Using 7 swine to undergo coronary artery bypass grafting (CABG) (left rapid heart rates (up to 700 beats per minute). Approaches such as ultrasonic crystals, mammary artery to left coronary artery branch (LAD)), we prepared the models MRI and Echo have been used to measure instantaneous LV volume with varying of coronary artery stenosis to ligate LAD. Graft flow tracing was obtained degrees of success. These technologies all have severe limitations, particularly during intraoperatively to analyze the waveform using FFT under stable hemodynamics. dynamic maneuvers, such as transient occlusion of the inferior vena cava or aorta, that We defined high frequency area of FFT analysis as Ha and low frequency are as are required to generate load independent indices of contractility. Now a new modality exists to assess function by using pressure-volume loop analysis Hb . Ha was calculated to add from the power of 6th harmonics to the power generated from a new generation of conductance catheter technology. Admittance of 10th harmonics. In the same way, Hb was the sum from the power of 1st Derived Volume technology provides repeatable data, lower standard deviations and harmonics to the power of 5th harmonics. avoids error prone techniques such as saline bolus injections. Results: Significant difference of Hb/Ha was observed between the group of When combined into a single micro-probe this new type of pressure-conductance 0% stenosed rate, the one of 50%, the one of 75% and the one of 100% (p<0.05). catheter adds the final dimension to molecular research through accurate assessment of Conclusion: Our findings indicated that there are a relationship between the graft ESPVR, PRSW and many other important variables. waveform and the stenosed rate of coronary artery. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 175 P1PM-5-1 P1PM-5-2 A COMPARISON OF STRATEGY SELECTION IN BEHAVIORAL AND HISTOCHEMICAL CONSEQUENCES SPATIAL MEMORY TASKS AFTER LESIONS OF OF SELECTIVE IMMUNOLESIONS IN DISCRETE DISCRETE REGIONS OF THE BASAL FOREBRAIN REGIONS OF THE BASAL FOREBRAIN CHOLINERGIC CHOLINERGIC SYSTEM SYSTEM Maia Burjanadze, Manana Dashniani, George Maglakelidze, George Maglakelidze, Liana Bakradze, Nato Kotaria Gela Beselia Behavior and Cognitive Function, Beritashvili Inst. of Physiology, Georgia Department of Behavior and Cognitive Functions, I.Beritashvili Institute of Adult male outbred rats were subjected to bilateral lesions of the cholinergic Physiology, Georgia neurons in the nucleus basalis magnocellularis (NBM) and medial septal area In this experiment the ability of medial septal (MS) and Nucleus basalis (MS) by injection of 192-IgG-saporin. Control rats received an equivalent magnocellularis (NBM) ibotenic acid lesioned and sham-operated rats to learn amount of mouse saporin. All rats were tested for strutegy (place, cue) selection the location of a visible, as well as submerged platform in a water maze has been in a water-maze task. Examination of the AChE stained sections showed that investigated. Sham-operated rats acquired both the visible and hidden platform after injections of 192 IgG saporin into the MS, animals exhibited significantly versions of the task, but when required to choose between the spatial location they less AChE staining in hipocampus as compared to sections obtained from sham- had learned and the visible platform in a new location majority of them swam first to operated animals (T= 4.08, p< 0,01). After injections of 192 IgG saporin into the the old spatial location. An overview of the data from both competition trials for each NBM there was weak damage to the hippocampus (15, 25%). The behavioral group show that the sham-operated rats in 21 trials out of 24 competition test trials results showed that sham-operated rats acquired both the visible and hidden used place strategy, while MS-lesioned ones used this strategy in 14 trials and NBM- lesioned rats used this strategy in 6 trials only. Decreased place-bias in NBM and MS- platform versions of the task, but when required to choose between the spatial lesioned rats compared to the sham-operated rats was significant (P<0.05). Sham- location they had learned and the visible platform in a new location majority of operated rats identified as place responders had significantly more accurate searches them swam first to the old spatial location. Decreased place-bias in NBM and during hidden platform training, providing additional evidence of their effective use of MS immunolesioned rats compared to the sham-operated rats was not significant a place learning strategy than MS and NBM-lesioned rats. These findings suggest that (P>0.05). The behavioral effects of the cholinergic lesions do not support the in MS and NBM-lesioned rats behavior was not affected by spatial information and idea of a substantial implication of the basal forebrain cholinergic system in the responding to local reinforced cues was enhanced. memory processes assessed in this study.
P1PM-5-3 P1PM-5-4 EFFECTS OF MEDIAL SEPTAL LESIONS ON LEARNING REQUIRES SYNAPTIC DELIVERY OF AMPA ACGUISITION OF A PLACE AND CUE WATER MAZE RECEPTORS AT SCHAFFER COLLATERAL SYNAPSES TASK IN THE DORSAL HIPPOCAMPUS 1 2 2 Temur Naneishvili , Maia Burjanadze , Gela Beselia , Dai Mitsushima, Kouji Ishihara, Yoshinori Kamiya, 2 3 Nino Chkhikvishvili , Mzia Zhvania Takuya Takahashi 1 Precise and Natural Science faculty, Akaki Tsereteli St. Univers, Georgia, Physiology, Yokohama City University School of Medicine, Japan 2Behavior and Cognitive Function, Beritashvili Inst. of Physiol., Tbilisi, Georgia, 3Cell Morphology and Biochemistry, Beritashvili Inst. of Physiol., By combining Herpes virus-mediated in vivo gene delivery with in vitro patch-clamp Tbilisi, Georgia recordings, we found that hippocampal-dependent inhibitory avoidance learning drives recombinant GluR1, an AMPA receptor subunit, into synapses formed between In this experiment the ability of medial septal electrolytic, selective ACh lesioned CA3 and CA1 pyramidal neurons. In contrast, bilateral expression in the dorsal (by immunotoxin 192 IgG-saporin) and sham-operated rats, to learn the location of a hippocampus with GluR1 cytoplasmic tail, a construct that inhibits synaptic delivery visible, as well as submerged platform in a water maze was investigated. A total of 36 of endogenous AMPA receptors, successfully impaired learning without changing the male outbred albino rats were used in the study.Sham-operated rats acquired both the visible and hidden platform versions of the task, but when required to choose between latency to enter a novel dark box or the amount of 24-hour spontaneous locomotor the spatial location they had learned and the visible platform in a new location, activity. Similarly, the expression of mutated form of membrane proxymal region, majority of them swam first to the old spatial location. The medial septal electrolytic a much smaller fragment of GluR1 cytoplasmic tail, impaired learning. Further, lesioned rats acquired the visible platform version of the water maze task but failed acetylcholine release in the dorsal hippocampus increases during learning, and to learn the platform location in space. When the visible platform was moved to a scopolamine pretreatment blocked the learning-induced synaptic delivery of GluR1. new location they often swam directly to it. The medial septal selective ACh lesioned Consistently, scopolamine pretreatment blocked the increase in AMPA/NMDA ratio rats, as well as sham-operated, acquired the platform location in space. These findings and impaired learning. These results suggest that hippocampal-dependent learning suggest that the septo-hippocampal system is essential for accurate spatial learning, but requires synaptic delivery of AMPA receptors at Schaffer collateral synapses in the raised the unexpected possibility that hippocampal ACh is not essential for all types of dorsal hippocampus, and endogenous acetylcholine release during learning mediates hippocampal-dependent memory. the synaptic delivery in behaving rats.
P1PM-5-5 P1PM-5-6 MODULATION OF CONTRALATERAL AND DIFFERENTIAL TIME COURSES OF SENSORY IPSILATERAL MOTOR CORTEX BY PROLONGED AND MOTOR RELEARNING AFTER A UNILATERAL UNILATERAL MUSCLE ACTIVITY WITH AND WITHOUT SENSORIMOTOR CORTEX LESION IN RATS SENSORY INPUT Hidekazu Kaneko1, Hiroshi Tamura2, Takahiro Kawashima3, 1 2 1 Monica Ilieva Christova , Dietmaer Rafolt , Eugen Gallasch Shinya S Suzuki1 1 2 Department of Physiology, Medical University Graz, Austria, Center of 1Institute for Human Science and Biomedical Engineering, National Institute Byomedical Engineering and Physics, Medical University of Vienna, Austria of Advanced Industrial Science and Technology (AIST), Japan, 2Laboratory Interactions between the sensory and motor system open a perspective to induce for Cognitive Neuroscience, Graduate School of Frontier Biosciences, Osaka neuroplastical changes using different afferent stimulation parameters. University, Japan, 3Department of Electrical and Electronic Engineering, To investigate such effects on contralateral (cM1) and ipsilateral (iM1) motor cortex Toyohashi University of Technology, Japan two protocols were tested: isotonic muscle activity alone (A) and in presence of Rehabilitation of sensorimotor functions after brain lesions involves a process similar to superimposed vibration (A+V). learning. We analyzed the effects of a unilateral lesion of the rat sensorimotor cortex on Vibration (0.5 mm, 60 Hz) was applied to the right index finger while subjects the reversal learning of a choice reaction time task. Rats were trained to respond quickly performed isotonic abduction for 10min. Single and paired-pulse transcranial to a tactile stimulus delivered to its left or right forepaw by moving the stimulated forepaw magnetic stimulation was applied to left and right M1 in separate sessions, motor (compatible) or the unstimulated forepaw (incompatible). By examining performance on evoked potentials (MEP) were recorded from right and left first dorsal interosseus m., compatible and incompatible trials, we were able to differentiate the effects of the lesion on Poster Session respectively. MEP excitability changes were assessed pre, during and post A and A+V. sensory processing and motor execution. Those rats that had learned the both compatible Effect of A+V: significant increase in MEP recruitment and reduced intracortical and incompatible tasks were lesioned in the forepaw area of the left or right sensorimotor inhibition lasting up to 30 min post A+V in cM1 and unchanged excitability in iM1; cortex. Following the recovery of rats' task performance, the compatibility condition was nonsignificant MEP facilitation in iM1 during A+V. Effect of A: nonsignificant MEP reversed. After the reversal, relearning was slower for the contralesional forepaw than for the facilitation in cM1 and iM1 post A; significantly increased excitability in iM1 during A. ipsilesional one. This relearning progressed more slowly when the contralesional forepaw Unilateral hand muscle activation with and without sensory input modulate both was used for motor execution than when it was used for sensory detection. These results M1 simultaneously in different manner mostly via transcallosal interactions and suggest that motor relearning was preceded by sensory relearning after a sensorimotor cortex GABAergic transmissions. lesion. This possibility may have important implications for clinical neurorehabilitation.
176 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-5-7 P1PM-5-8 CONDITIONED FLAVOR PREFERENCE LEARNING DIFFERENTIAL EFFECTS OF NMDA RECEPTOR INDUCED BY INTRAGASTRIC INFUSION OF BLOCKADE IN THE PREFRONTAL CORTEX AND GLUTAMATE IN RATS HIPPOCAMPUS ON SPATIAL WORKING MEMORY IN Akira Uematsu, Tomokazu Tsurugizawa, Hisayuki Uneyama, RATS 1 2 1 Kunio Torii Kazuko Hayashi , Toru Yoshihara , Yukio Ichitani 1 Institute of Life Sciences, Ajinomoto Co., Inc., Japan Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan, 2Advanced Science Research Center, Kanazawa Postingestive consequences as well as oronasal cues are the important factors on University, Japan the preference for foods and fluids. This study demonstrates that the postingestive To investigate effects of N-methyl-D-aspartate (NMDA) receptor blockade in the effect of glutamate by using conditioned flavor preference paradigm. Adult male medial prefrontal cortex (mPFC) and the dorsal hippocampus (dHPC) on spatial Sprague-Dawley rats with chronic intragastric (IG) cannula were given daily 30 working memory in rats, we used two versions of 8-arm radial maze task. In the min one-bottle training to drink a flavored solution (CS+) paired with IG infusion standard task, rats were trained to obtain food reward without reentering the arms of nutrient solution and another flavored solution (CS-) paired with IG water that had been previously visited within a trial. After the animals acquired the task, infusion on alternate 8 days. Nutrient solution was given 60mM monosodium they received bilateral microinjections of NMDA receptor antagonist DL-2-amino- L-glutamate, NaCl, or glucose. Before and after conditioning, rats received 30 5-phosphonopentanoic acid (AP5) into either the mPFC or dHPC. NMDA receptor min two-bottle choice tests for CS+ and CS- solution. Before conditioning, all blockade in the dHPC caused severe deficits in performance, while that in the mPFC groups had no higher preference for the CS+ solution. During conditioning, the did not. In the delay-interposed task, one trial was divided into two phases by a 2-h intake of CS+ at the last half of the session was significantly higher compared to delay: up to the fourth correct choice on the standard task (the first phase) and the rest of the trial (the second phase). Well-trained rats were injected with AP5 into the mPFC the first half of the session in MSG group, but not in NaCl and glucose groups. or dHPC 15 min prior to the first phase, immediately after the first phase, or 15 min After conditioned, MSG group showed significantly higher intake and preference prior to the second phase. These three conditions were applied to assess the function for the CS+ solution (p<0.05), while the NaCl and glucose group did not show of NMDA receptors in encoding, retention and retrieval processes, respectively. The any significant intake and preference for the CS+ solution. These results indicate results suggest that activation of NMDA receptors in each structure is required in that glutamate without its metabolized calorie has a positive post-ingestive effect. different processes of spatial working memory.
P1PM-5-9 P1PM-5-10 OREXIN IMPAIRS SPATIAL LEARNING AND MEMORY COMPARISON OF EFFECTS ON HIPPOCAMPAL ADULT Yutaka Oomura1, Shuji Aou2, Koji Fukunaga3, Kazuo Sasaki4 NEUROGENESIS BY TOOL-USE LEARNING AND 1Department of Integrative Physiology, Faculty of Medicine, Kyushu SPATIAL LEARNING IN RODENT DEGU (OCTODON University, Japan, 2Kitakyushu Institute of technology, Japan, 3Department DEGU) 1 2 1 3 of Pharmacology, Faculty of Pharmacy, Tohoku University, Sendai, Japan, Noriko Kumazawa , Hiroshi Hama , Eriko Kariya , Atsushi Miyawaki , 1 4Life-information, Toyama University, Japan Atsushi Iriki 1Lab. for Symbolic Cognitive Development, BSI, RIKEN, Japan, 2Lab. for Cell Glucose-ensitive neurons in the lateral hypothalamic area produce orexin-A 3 Function Dynamics, BSI, RIKEN, Japan, Lab. for Cell Function Dynamics, BSI, ( orx )and send their axons to the hippocampus, which predominantly RIKEN, Japan expresses orx receptor 1 showing a higher sensitivity to orx. We studied Tool-use is defined as the manipulation of inanimate object to change the spatial position or the effect of orx on the rat hippocampal spatial learning and memory form a separate object and considered as an origin of human intelligence. performance. The results of the Morris water maze test show that 1.0 and The rodent degus have clever hands, diurnal behaviors and unique social vocal 10 nmol orx, when administered icv, retarded spatial learning and memory communications. We focused on such a unique rodent degu and demonstrated for the first preformance. A probe test examined after training of water maze task also time that degu can be trained to use tool, T-shaped rake. We trained degus to use a rake-like tool with their forelimbs to retrieve otherwise out-of-reach rewards. In the final level, the showed an impairment. The in vitro hippocampal slice experiments showed food was displaced horizontally from the tip so that the degu has to move the tool laterally that 1.0 and 10 nmol orx applied to the perfusate produced a dose dependent before pulling in. All of animals successfully advanced to the final level after an average of suppression of LTP, but not LTD. The paired pulse facilitation experimes 39.7 days of training (success rate: >75%, for 3 sessions). indicated that the effects of orx were post-synaptic and not due to presynaptic Hippocampal dentate gyrus is known to generate new neurons throughout adulthood. Such transmitter release . These results indicate that orx impairs spatial leraning adult neurogenesis has been known to be enhanced during hippocampus-dependent learning and memory processes in rodents. We found markedly increased number of newborn neurons and memory performance and these impairments can be attributed to a in the dentate gyrus of tool use-trained, compared to sham-trained, degus. Furthermore, we suppression of LTP in the Schaffer colateral-CA1 hippocampal synapses. compared efffects on adult neurogenesis by tool-use learning and spatial learning, 8-arm radial maze, to examine whether such phenomena are specific for tool-use learning.
P1PM-5-11 P1PM-5-12 INFLUENCE OF RAT PREFRONTAL SPONTANEOUS THE EFFECTS OF ORBITOFRONTAL CORTEX LOCAL FIELD POTENTIAL POWERS BEFORE CUE LESIONS ON ANTICIPATORY CONTRAST OF PRESENTATION ON REACTION TIME IN OPERANT CONSUMING SUCROSE SOLUTIONS DISCRIMINATION TASK Wea Lun Lin, Ruey-Ming Liao Yoshinori Izaki, Sei-etsu Fujiwara, Tatsuo Akema Department of Psychology, National Chengchi University, Taiwan Department of Physiology, St. Marianna University School of Medicine, As presented by two sweet solutions in a sequential order on each day, the Japan rat learns to suppress its intake of the first solution as if the second solution Our previous study has shown that spontaneous local field potential (LFP) is relatively preferred. This behavioral response is termed anticipatory activity in a specific frequency band (including the gamma band) in the rat contrast effect (ACE). Although ACE is presumed to be established via medial prefrontal cortex (PFC) influences the responses evoked by hippocampal several behavioral processes involved, very little is known about the electrical stimulation under urethane-anesthesia. Based on these findings, we underlying neural mechanism. The present study, thus, investigated the role hypothesized that spontaneous PFC LFP activity could also influence behavioral of orbitofrontal cortex (OFC) in the acquisition of ACE. Rats were assigned responses to stimuli such as cue signals for reward. To investigate this possibility, the present experiment examined correlations between reaction times in lever- to receive bilateral infusions with ibotanate or vehicle into OFC before press responses to reward cue stimuli in a discrimination task and spontaneous being exposed to ACE procedures. In each day of which, a 3-min access to PFC LFP activities recorded immediately before the cue presentation. We 4% sucrose solution, a 30-sec interval, and a 3-min access to either the 4% demonstrated that significant correlations between the reaction time and the PFC sucrose solution or a more preferred 32% sucrose solution were sequentially LFP powers of a specific frequency band of the higher gamma band immediately presented. The results showed that the rats with OFC lesion significantly before the cue presentation. These results indicate that the gamma band activities failed to acquire ACE in comparing to the sham lesion controls completed in the PFC LFPs influence lever-press responses to discrimination cues. acquiring ACE in 4 days. These data suggest that the OFC is involved to the Spontaneous LFP activities in specific frequency bands and in specific brain acquisition of ACE on comparing the rewards of different values leading to region may have a role in information processing in behavior related to learning withhold certain responses and to anticipate a more valuable reward to come and memory. later on. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 177 P1PM-5-13 P1PM-5-14 LOSS OF MOTIVATION INDUCED BY IN VIVO SOCIAL LEARNING FOR AVOIDING BEHAVIOR IN DELIVERY OF SPECIFIC ANTIBODY TO GluR1 RATS C-TERMINUS INTO HIPPOCAMPAL NEURONS VIA Akira Masuda, Shuji Aou HVJ-E VECTOR Department of Brain Science and Engineering, Kyushu Institute of 1 2 3 1 Technology, Japan Makoto Saji , Yumi Okumura , Haruna Tomizawa , Masanori Ogata , Kazuko Noda1, Nobuyuki Suzuki1, Hisanao Akita1 Social learning assists adaptation to environment by transmitting information 1Department of Physiology, School of Allied Health Sciences, Kitasato University, between animals. According to the previous studies, rats transmit information Japan, 2Division of Brain Science, Graduate School of Medical Science, Kitasato about avoidance to other conspecifics. However, it is not known how the University, Japan, 3Division of Brain Science, Graduate School of Medical avoiding behavior is modified by environmental change (i.e. safe situation to Science, Kitasato- University, Japan dangerous situation, and vice versa). Moreover, the dependency of learning C-terminus of AMPA receptor GluR1 is considered to play a role in plasticity (LTP/LTD) experience is not studied systematically (interaction between learned or by regulating synaptic incorporation of AMPA receptor or its internalization. However, unlearned subjects and their learned or unlearned partners). Here, we little is known on the function of GluR1 C-terminus in vivo study. To clarify the functional investigated how the social learning of the avoiding behavior was influenced role of GluR1 C-terminus in memory formation, we performed in vivo delivery of specific by situation changes by focusing on the avoidance-learning experience of the antibody against GluR1 C-terminus into hippocampal CA1 neurons via HVJ-E vector in rat subjects. We found that avoiding behavior of experienced rats was inhibited brain. During 5-6 days after the antibody-delivery, water maze test and open field test are by inexperienced partners under the safe situation, and facilitated by conducted in antibody-treated (AN), vehicle (vacant vector)-treated (VE) and non-treated experienced partners under the dangerous situation. The avoiding behavior of (NT) rats. In water maze, AN rats displayed reduced searching behavior resulting in no inexperienced rats, however, was not affected by any social circumstances. accomplishment of spatial learning and marked delay in accomplishment of skill learning These results suggest that social learning can be updated in the changing for staying long on the platform, while VE rats displayed defective spatial memory. In open environments, and the previous experience is the crucial factor for social field, AN rats showed normal adaptive behaviors as well as VE and NT rats. We will discuss learning of avoiding behavior in rats. The brain areas that involve in the that the abnormal behaviors seen in AN rats due to loss of motivation may be interpreted by social learning of avoidance behavior will be discussed with the results of disruption of interaction between GluR1 phosphrylation sites and protein phosphatases. lesion study.
P1PM-5-15 P1PM-5-16 HEMEROCALLIS (AKINOWASUREGUSA) IMPROVES MORINDA CITRIFOLIA SUPPRESSES REDUCTION IN MEMORY IN MICE LEARNING AND MEMORY INDUCED BY RESTRAINT Kayoko Uezu1, Susan A Farr2, Eiko Uezu3, Atsuko Sano4, William A Banks2, STRESS IN MICE James F Flood2, Jhon E Morley5 1 1 2 1 1 2 Junko Muto , So Hong Lee , Toshio Mikami , Makoto Ohno Department of Nutritional Health, Kwassui Women's College, Japan, Geriatric 1Graduate School of Health and Sport Science, Nippon Sport Science Research Education and Clinical Center (GRECC), VA Medical Center, St. 2 Louis, MO, USA, 3Faculty of Education, University of the Ryukyus, Japan, University, Japan, Department of Health and Sports Science, Nippon 4Department of Integrative Physiology, The University of Tokushima, Japan, Medical School, Japan 5 Department of Internal Medicine, St. Louis University School of Medicine, St. The purpose of this study was to investigate the preventive effects of Morinda citrifolia Louis, MO, USA (Noni) fruit, which has antioxidative activities, on stress-induced impairment of The flowers of Hemerocallis fulva L.var sempervirens M.Hotta (Akinowasuregusa cognitive function. Male ICR mice were divided into 4 groups: Control (C), Restraint ), a Hemerocallis genus of the lily family, have been utilized as food in Okinawa, (R), Restraint+Noni (RN), and Restraint+vitamin E (RE). R, RN and RV mice were Japan. The leaves and roots are also valued as medical herb. Some old books mention subjected to 8h of restraint stress 6 days weekly for 7 weeks. During this period, antipyretic actions, diuretic actions, and alleviation of insomnia as the major effects RN and RV mice were given feed containing either Noni or vitamin E, respectively. of Hemerocallis. Previously we reported that slow wave sleep and paradoxical sleep At Week 4, a water maze test (MWT) and open field test (OFT) were performed to of the mice fed Hemerocallis increased (Effects of Hemerocallis on sleep in mice, measure cognitive function and physical activity. At Week 7, brains were isolated for Uezu E, Psychiatry Clin Neurosci, 1998). In this study, we examined the effects of immunohistochemical analysis using CD31 antibody to estimate the area covered flowers of Hemerocallis on memory in mice. The water extracts from Hemerocallis was given intracerebro-ventricular of CD-1 young male mice immediately after T- by blood vessels in the dentate gyrus (DG) of the hippocampus. Time to reach the maze training. Memory retention was tested one week later. The retention test scores platform in the WMT was shorter in RN mice than in R mice. Rearing frequency in in the Hemerocallis groups were significantly lower than in the control group. We also OFT and blood vessel area were significantly lower in R and RV mice than in C mice; analyzed the concentration of carotenoid and GABA in the flowers. The high levels of however, no such differences were found between C and RN mice, suggesting that cryptoxanthin and GABA were detected. These results suggest that Hemerocallis may administration of Noni suppresses stress-induced impairment of cognitive function and improve memory in mice, and its effects may be caused by carotenoid or GABA in activity; and that this preventive effect may be related to suppression of stress-induced the flowers. decrease in blood vessel area in the DG.
P1PM-5-17 P1PM-5-18 HIGHLY EXPRESSED-HIPPOCAMPAL SPAR ROLES OF NITRIC OXIDE IN SHORT-TERM MEMORY ENHANCES SPATIAL LEARNING AND MEMORY IN FORMATION IN EARTHWORMS NORMAL AND HYPOXIA-EXPOSED NEONATAL MICE Takehiko Mochida1, Kosuke Watanabe1, Mamiko Suzuki1, 1 1 1 1 Du Ji-Zeng , Lu Xin-Jiang , Chen Xue-Qun , Weng Jian , Toshinobu Shimoi1, Hiroto Ogawa2, Koji Hotta1, Kotaro Oka1 1 2 Zhang Heng-Yi , Pak Daniel T 1Center for Biosciences and Informatics, School of Fundamental Science 1Department of Physiology, Zhejiang University, China, 2Department of and Technology, Keio University, Japan, 2Department of Biological Pharmacology, Georgetown University School of Medicine, USA Sciences, Faculty of Science, Hokkaido University, Japan SPAR (a spine-associated RapGAP) forms a complex with PSD-95 and Eathworms, Eisenia fetida, don’t respond to weak vibration (conditioned stimulus, NMDA receptors (NMDARs) and morphologically regulates dendritic spines CS), but do to strong light (unconditioned stimulus, US). After paired application of that may contribute to memory storage. This paper reports that neonatal CS and US, only weak vibration induces contraction behavior (classical conditioning). exposure to mild intermittent hypoxia (IH) for 4 weeks markedly enhances We also succeeded to induce short-term memory (STM) or long-term memory (LTM) spatial learning in mice and is associated with SPAR, a regulator of dendritic selectively by changing the intertrial interval (ITI) of the pairings of stimulus. Nitric spine morphology in hippocampal neurons. Both SPAR expression and long- oxide (NO) has been known to a neuromodulator by learning and memory in the term potentiation (LTP) were significantly increased in the hippocampus nervous system of a wide variety of animals. We demonstrated that NO is produced
Poster Session during IH in postnatal mice compared with normoxic groups. SPAR antisense by serotonin stimulus in ventral nerve cord (VNC). In this study, we investigate the infusion into the hippocampal formation disrupted SPAR expression, leading roles of NO in short-term memory formation. Several chemicals for changing NO to impairment of spatial learning and reduction of hippocampal LTP in IH- concentration are injected into abdominal cavity of the earthworm before conditioning treated and normoxic mice. This study provides the first evidence that SPAR and STM formation was evaluated. We found NO production is requisite for STM is functionally required for synaptic plasticity and IH-induced enhancement formation. Deficient NO production by L-NAME and Carboxy-PTIO inhibite of spatial learning and memory in developing mice.This work is supported by STM formation. On the other hand, surplus NO production by SNAP and serotonin the grant from NSFC (No. 30393130; 30770807; 30870300) and the National accelerate STM formation. And STM formation by serotonin is mediated by NO. Basic Research Program “973” (No. 2006CB504100). These results indicate that STM formation is thought mediated NO-cGMP pathway.
178 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-5-19 P1PM-5-20 EFFECTS OF SOCIAL ISOLATION ON NERVE ENHANCEMENT OF C. elegans CHEMOTAXIS TO EXCITABILITY AND AGGRESSION IN DROSOPHILA: SODIUM ACETATE AFTER PREEXPOSURE TO THE IMPLICATIONS ON REDOX STATE FROM HK AND SAME CHEMICAL IS MODULATED BY DOPAMINE GSTS1 MUTANTS Tetsuya Matsuura, Takayuki Oda, Genta Hayashi, Atsushi Ueda, Chun-Fang Wu Daisuke Sugisaki, Mitsuyuki Ichinose Department of Biology, University of Iowa, USA Department of Welfare Engineering, Iwate University, Japan Our previous study showed that adult female flies show higher frequency In this study, we revealed an enhancement of the chemotactic response of wild- of aggressive behaviors when reared in isolation. In this study, we found type nematodes Caenorhabditis elegans (N2) to water-soluble sodium acetate markedly enhanced larval motoneuron excitability when larvae were reared (Na-Ace) after preexposure to Na-Ace. The chemotaxis index of nonexposed in isolation. Upon repetitive stimulation, group-reared WT larvae displayed a control nematodes in 1.0 M Na-Ace was approximately 0.7, whereas that of gradual increase in synaptic transmission, whereas isolation-reared WT larvae nematodes preexposed to 1.0 M Na-Ace for 90 min as a training precedure was approximately 0.9 (p<0.05). To clarity the mechanism, several mutants were showed an explosive increase in the amount of transmitter release coupled used. bas-1 mutants having a defect of serotonin secretion showed almost the with supernumerary firing of motor axons. In contrast, mutations of two same results as N2. However, cat-1 and cat-2 mutants, which were defective in genes, Hyperkinetic (Hk) and glutathione S-transferase-S1 (gsts1) altered the the secretions of both serotonin / dopamine and only dopamine, respectively, did response to social isolation. Hk and gsts1 mutants displayed increased adult not show an increase in chemotactic response to Na-Ace by the preexposure to female aggression and larval neuromuscular hyperexcitability even when Na-Ace. cat-1 and cat-2 mutants preexposed to Na-Ace with 40 mM dopamine reared in group. Significantly, these mutant phenotypes were not enhanced during the preexposure period showed an increase in chemotactic response to further by isolation rearing. Mutations of these two genes are implicated in Na-Ace. When cat-1 and bas-1 mutants were preexposed to Na-Ace with various reactive oxygen species (ROS) metabolism and have been shown to alter serotonin concentrations during the preexposure period, there was no significant neuronal ROS levels at neuromuscular junctions. Our data suggest potential difference in the response to Na-Ace between the control and preexposed involvement of ROS regulation in the cellular processes underlying neuronal mutants. These results suggest that the enhancement of chemotactic response to hyperexcitability and aggressive behaviors induced by social isolation. Na-Ace after preexposure to Na-Ace is modulated by the presence of dopamine.
P1PM-5-21 P1PM-5-22 EFFECT OF INTRAHIPPOCAMPAL INJECTION OF REGULARITIES OF THE SPATIAL MEMORY ALUMINUM ON ACTIVE AVOIDANCE LEARNING IN DEVELOPMENT IN PRESCHOOL CHILDREN ADULT MALE RATS Manana Dashniani1, Nino Chckikvishvili1, Rodam Aragveli1, Alireza Sarkaki1, Saleh Zahedi Asl2, Raheleh Assaei3 Ada Noselidze1, Temur Naneishvili2 1 Physiology, Physiology Research Center, Ahwaz Jondishapour University 1Behavior and cognitive function, I. Beritashvili Institute of Physiology, of Medical Sciences, Iran, 2Institute of Endocrinology and Metabolism, 2 3 Georgia, Precise and Natural science faculty, Akaki Tsereteli St. Univers., Shaheed Beheshti University of Medical Sciences, Tehran, Iran, Department Kutaisi, Georgia of Physiology, Medicine Faculty, Lorestan University of Medical Sciences, Khoramabad, Lorestan, Iran In order to assess development of the egocentric system of the spatial short- Aim of this work was to study the effect of intrahippocampal injection of different doses of term memory in the children (n=66) of different ages (24-60 months) the AlCl3 in adult male rats on active avoidance learning. 35 adult male Wistar rats (250-300g) Inverted Delayed Reaction test has been used. Altering conditions of testing were used into five groups: 1) Control, 2) Test-I received daily 1υl AlCl3 1%, pH =7.2, the factors were determined, which allowed modeling different loads onto the 3); test-II received daily 1υl AlCl3 0.5%, pH =3.4, 4); sham-I received daily 1υl aCSF, pH mechanisms of dead reckoning in the egocentric system of the spatial short- =7.2, 5); sham-II received daily 1υl aCSF, pH =3.4. All rats in test and sham groups treated term memory. It was found that in the children aged 24-36 months regularities 10 minutes before training. Animals were anaesthetized with ketamine HCl/xylazine and of performance of the Inverted Delayed Reaction test significantly differ in underwent a stereotaxic surgery for implant of two stainless steel guide cannula into the conditions of different loads onto the mechanisms of dead reckoning; the hippocampus bilaterally. Every day 10 minutes after above treatments all rats were used to children aged 36-60 months do not show sensitivity to different loads. In the assess the spatial learning performing using Y-maze. Criterion correct response (CCR) was children aged 42±4 months functional elimination of any of the sensory system 90% in last session of training. (visual, kinesthetic, vestibular) during rotation significantly deteriorated results of There were no significant differences between training sessions to receiving CCR in control, sham-I and sham-II groups. Cognition in animals received AlCl3 1%, pH =7.2 was impaired the Inverted Delayed Reaction test performance, while in the children aged 60± significantly with compare to other groups (*P<0.0001). 4 months number of correct responses decreased if two or three sensory systems Our results show that intrahippocampal injection of AlCl3 1%, causes active avoidance were eliminated simultaneously. The data obtained permit to conclude that the learning impairment significantly. formation of the dead reckoning mechanisms starts in an age of 24 months and in Keywords: Al3, Hippocampus, Active avoidance learning, Y-maze, Rat the period of 24-60 months its further ramification does occur.
P1PM-5-23 P1PM-5-24 REDUCED MOTOR CORTEX PLASTICITY FOLLOWING EXAMINATION OF MULTIPLE UNIT ACTIVITY-BASED INHIBITORY RTMS IN OLDER ADULTS CROSS-CORRELOGRAM AS A POSSIBLE MEASURE Gabrielle Todd1, Thomas E Kimber2, Michael C Ridding1, OF RAT HIPPOCAMPUS-PREFRONTAL FUNCTIONAL John G Semmler1 CONNECTIVITY 1School of Molecular and Biomedical Science, The University of Adelaide, Sei-etsu Fujiwara, Tatsuo Akema, Yoshinori Izaki 2 Australia, Neurology Unit, Royal Adelaide Hospital, Australia Department of Physiology, St. Marianna University, School of Medicine, Ageing is accompanied by diminished practice-dependent plasticity. We Japan investigated the effect of age on another plasticity inducing paradigm, repetitive Recently, we reported that multiple unit activities (MUAs) in the hippocampus transcranial magnetic stimulation (rTMS). Healthy young (n=15; 25±4 yrs) (Hip) and prefrontal cortex (PFC) could show significant cross-correlograms and old (n=15; 67±5 yrs) adults participated in 2 experiments. Motor evoked (CCs), suggesting that the MUA-based CCs can be used as a measure of functional potentials (MEPs) were measured in the first dorsal interosseus (FDI) during connectivity between these brain regions. To investigate this possibility, we analyzed a set of 15 single stimuli. Subjects then received either real or sham inhibitory MUA-based CCs and then examined the relationship between features of the CCs rTMS over the FDI motor area. Subthreshold trains of stimuli were applied at and characteristics in evoked responses of PFC by Hip stimulation using urethane- a frequency of 6 Hz for 5 s and repeated every 30 s for 10 mins. MEPs were anesthetized rats. Analysis of MUA-based CCs revealed two types of la patterns; again measured at 0, 15, and 30 mins post-rTMS. In young adults, there was a from Hip to PFC (type Hip-PFC) and from PFC to Hip (type PFC-Hip). In type Hip- significant main effect of experiment (P=0.044) and time (P<0.001) on MEP PFC, the evoked responses showed negative components with latencies suggesting area. MEP area was approximately 20% smaller with real rTMS than with sham monosynaptic projection from Hip to PFC, but negative components with longer rTMS (P<0.044) and MEP area was suppressed by 19-28% for 30 mins after latencies in type PFC-Hip. Furthermore, in type Hip-PFC, the peak heights of CCs rTMS (P<0.009), suggesting reduced motor cortical excitability. In contrast, showed significantly correlations with burst counts of MUAs and inverse correlations no significant main effect of experiment or time on MEP area was observed in with degrees of evoked responses. These results indicated that MUA-based CCs at old adults. These results suggest that advancing age is associated with reduced least in type Hip-PFC may relate to information processing using the Hip-PFC direct efficacy of rTMS. This work has important implications for the potential pathway, supporting the idea that the MUA-based CC could be used as a measurement therapeutic use of rTMS in stroke and neurological disease. of functional connectivity. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 179 P1PM-6-1 P1PM-6-2 OXYTOCIN-INDUCED SYNAPTIC PLASTICITY IN HDAC INHIBITION FACILITATES AVERSIVE THE OLFACTORY BULB IS REQUIRED FOR THE OLFACTORY LEARNING IN YOUNG RATS INDUCTION OF MATERNAL BEHAVIOUR Fumino Okutani, Hideto Kaba Hideto Kaba, Guang-Zhe Huang, Jing-Ji Zhang, Guo-Zhong Yu, Department of Physiology, Kochi Medical School, Japan Fumino Okutani Epigenetic mechanisms have an important role in synaptic plasticity and Department of Physiology, Kochi Medical School, Japan memory formation. Chromatin structure is remodeled by modification of In virgin rats, pup odours are aversive and maternal behaviour is not elicited histone acetylation during the early stages of long-term memory formation. until this aversion is overcome. We have shown that oxytocin (OT) released Preweanling young rats prior to eye opening depend on somatosensory at parturition modulates olfactory inputs at the level of the olfactory bulb (OB) and olfactory function for survival, as they can learn their dam’s odor and thereby facilitates maternal behaviour. By using primary OB cultures and approach her. In order to establish olfactory learning, the pairing of and whole-cell patch-clamp recording techniques, we have demonstrated odor and somatosensory stimulation is crucial. We have previously shown that OT facilitates spontaneous EPSCs in granule cells by both pre- and that synaptic plasticity in the OB underlies aversive olfactory learning. postsynaptic mechanisms. In this study we show that intrabulbar infusions of Behavioral pharmacology shows that long-term, but not short-term, olfactory the GABAA receptor antagonist bicuculline, which has been shown to induce synaptic plasticity, induce the rapid onset of maternal behaviour in oestrogen- memory required activation of CREB. Western blot analyses reveal that primed virgin female rats. This raises the possibility that OT augmentation expression of P-MAPK/ERK was increased for 1 hour after odor-shock of the glutamatergic transmission may induce synaptic plasticity that may training, followed by increase of P-CREB lasting for 6 hours. Therefore we be important for the induction of maternal behaviour. We have further tested examined if intrabulbar infusion of a histone deacetylase (HDAC) inhibitor this possibility in OB slices. Pairing of lateral olfactory tract stimulation and has a facilitatory effect on aversive olfactory learning in young rats. Animals depolarization of the granule cell is subthreshold for the induction of long- infused with an HDAC inhibitor during or 2 hours after odor-only training term potentiation (LTP), but when OT is added, robust LTP is induced. Taken show aversion at the odor preference test on the next day, but infusion 8 together, these results suggest that OT-induced LTP at the mitral-to-granule hours after training has no effect. These results show that HDAC inhibition cell synapse is required for the induction of maternal behaviour. facilitates synaptic plasticity in the OB.
P1PM-6-3 P1PM-6-4 MUSCARINIC RECEPTOR ACTIVATION POTENTIATES DYNAMIC SNIFFING PATTERNS DURING OLFACTORY TRANSMISSION FROM GRANULE CELLS TO MITRAL DECISION MAKING BY FREELY BEHAVING MICE CELLS IN THE MOUSE ACCESSORY OLFACTORY IMPLANTED WITH A NOVEL TELEMETRY DEVICE BULB Koichi Matsumura, Alan Gelperin Yoshito Takahashi, Hideto Kaba Monell Chemical Senses Center, USA Department of physiology, Kochi University, Japan Sniffing is the first step in gathering information about the olfactory world. The accessory olfactory bulb (AOB) receives extensive centrifugal projections The mode of sniffing impacts the processing of information in the olfactory that act to modulate the flow of pheromonal information. Dendrodendritic system as well as in decision making via olfaction. So far, sniffing patterns synapses between mitral and granule cells are likely targets for this modulation. have been recorded from anesthetized animals or awake animals with a wired Modulation of dendrodendritic synapses by noradrenergic inputs plays a key sensor and cannula implanted into the nasal cavity. However, anesthesia role in the formation of pheromonal memory in mice. The AOB also receives and the restraints of a tether limit the kinds of experiments that can be done. cholinergic inputs from the basal forebrain. However, little is known about We record sniffing signals by using tiny pressure sensors implanted into the the actions of acetylcholine at the cellular levels in the AOB. Therefore, we investigated the action of the cholinergic agonist carbachol on transmission from chest cavity and connected to wireless telemetry devices in the abdominal granule to mitral cells with the use of whole-cell patch-clamp recordings in slice cavity, which allow us to use freely behaving animals for measurements preparations. Application of carbachol (100 microM) produced an increase in of sniffing dynamics during odor sampling and odor decision making. We report a variety of sniffing patterns of mice during odor decision making, the frequency of GABAA receptor-mediated spontaneous currents (so called mIPSCs) recorded from mitral cells. This effect was significantly reduced by the and the implications of the sniffing patterns for the processing of sensory muscarinic receptor type-1 (M1) antagonist pirenzepine (10 microM), but not by input in the olfactory bulb. Our results suggest that information processing the M3 antagonist 4-DAMP (300 nM) or the M2 and M4 antagonist himbacine in the olfactory system is highly dynamic during behavior and is variable (10microM). Carbachol challenge did not affect the responses of mitral cells depending on task demands. When combined with single unit recording from to puff application of GABA. These results demonstrate that M1 receptors olfactory bulb or olfactory cortex, the wireless sniffing monitor will augment activation increase spontaneous release of GABA from granule cells. tests of the dynamic properties of olfactory information processing.
P1PM-6-5 P1PM-6-6 THE STIMULATION OF MU-OPIOID RECEPTORS IN THE ROLES OF THE PREFRONTAL CORTEX IN RETRIEVAL VENTRAL PALLIDUM PRODUCES A PALATABILITY AND EXTINCTION OF TASTE AVERSION LEARNING IN SHIFT OF A CONDITIONED AVERSIVE TASTE IN RATS MICE Tadashi Inui1, Takashi Yamamoto2, Tsuyoshi Shimura1 Yasunobu Yasoshima, Tsuyoshi Shimura 1Division of Behavioral Physiology, Department of Behavioral Sciences, Department of Behavioral Physiology, Graduate School of Human Graduate School of Human Sciences, Osaka University, Japan, 2Faculty of Sciences, Osaka University, Japan Health Science, Kio University, Japan When ingestion of a novel taste stimulus (conditioned stimulus, CS) is followed To clarify the role of the opioidergic system in the ventral pallidum (VP) in the by visceral malaise (unconditioned stimulus), animals avoid ingesting the palatability shift of a taste stimulus from ingestive to aversive in conditioned taste CS thereafter. This learned avoidance to the CS is called as conditioned taste aversion (CTA), we examined the effects of the microinjections of mu-opioid aversion (CTA). Repeated exposures of the CS alone without US (extinction receptors agonist D-Ala2-N-Me-Phe4-Glycol5-enkephalin (DAMGO) into the VP procedure) facilitate extinction of CTA. Although the prefrontal cortex is on the palatability of the CS after the development of CTA using a taste reactivity suggested to be activated during CTA extinction, its role remains fully unsolved. test (TR test) and a single-bottle intake test. On the conditioning day, rats received Here, we examined the roles of the medial prefrontal cortex (mPFC) in the a pairing of 5 mM saccharin solution (CS) with 0.15 M lithium chloride. Two days memory retrieval and extinction of CTA in C57BL/6 mice. First, when the mPFC after the conditioning, DAMGO (10 or 100 g) or vehicle was bilaterally injected into was destroyed using an excitotoxin, extinction of CTA in one-bottle voluntary Poster Session the VP just before the presentation of the CS. We counted the ingestive (e.g. tongue drinking test was significantly impaired, resulting in long-term maintenance of protrusion) and aversive responses (e.g. chin rubbing) in the TR test and measured the CTA. Second, the same lesions reduced the latency to reject the CS in the taste consumption of the CS in the single-bottle test. In the TR test, the injections of 10 ng reactivity test. Third, microinfusions of a GABAA receptor agonist muscimol DAMGO significantly decreased the aversive responses. In the single-bottle test, the into the mPFC prior to the same test did also decrease the latency to reject the DAMGO-injected group showed significantly higher intake of the CS than the vehicle- CS. These results suggest that the mPFC attenuates learned aversive behavior injected group. These results suggest that the administration of DAMGO into the VP to the CS in each memory retrieval, indicating that the possible attenuating reduced the aversion to the CS. The opioidergic receptors in the VP may be involved effect on learned aversion elicited by the mPFC may cumulatively play a role in in the palatability of an aversive CS in CTA. facilitation of extinction of CTA memory.
180 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-6-7 P1PM-6-8 HIPPOCAMPAL NEURONAL RESPONSES TO INFLUENCE OF MUSCARINIC ACETYLCHOLINE GUSTATORY STIMULI IN DIFFERENT PLACES RECEPTORS ON SUPPRESSION MECHANISM OF Takashi Kondoh1, Anh S Ho2, Etsuro Hori2, Phuong HT Nguyen2, CONDITIONED TASTE AVERSION BY GRAVITY Susumu Urakawa2, Kunio Torii1, Taketoshi Ono2, Hisao Nishijo2 DISTURBANCE 1Institute of Life Sciences, Ajinomoto Co., Inc., Japan, 2System Emotional Yumi Baba1, Harumi Tsuchida2, Yumi Fujiwara2, Minoru Matsui3, Sci., Grad. Sch. Med. Pharm. Sci., Univ. Toyama, Japan Hiroko Fujiwara2, Ryohei Satoh4, Takenori Miyamoto1 Neuroanatomical studies have suggested that hippocampal formation (HF) 1Division of Material and Biology Science, Japan Women's University, Japan, 2Laboratory of Behavior Neuroscience, Faculty of Science, Japan Women’s receives information from all sensory modalities including taste. However, 3 4 there is no electrophysiological evidence to support it. We recorded CA1 University, Japan, Faculty of Pharmacy, Chiba Institute Science, Japan, Department of Physiology, School of Medicine Kitasato University, Japan HF neurons from freely behaving rats during performance of a visually- guided licking task in 2 different places (white or black triangular chambers). The conditioned taste aversion (CTA) paradigm was employed in order to examine the effect of modification of gravitational force on acquisition and retention of emotional learning and When a cue lamp on, the rats were required to press a bar to trigger a tube memory. The gravity disturbance was applied to male mice between 0.1% sodium saccharin to protrude into the chambers for 3 s. During this period, the rats could lick as a conditioned stimulus (CS) and an i.p. injection of 0.15 M LiCl as an unconditioned water or one of 6 sapid solutions [NaCl (salty), sucrose (sweet), citric acid stimulus (US). Under the gravity disturbance, both acquisition and retention were suppressed. (sour), quinine HCl (bitter), monosodium L-glutamate (MSG, umami), and We performed the same experiment with mice lacking for muscarinic acetylcholine receptor a mixture of MSG with disodium 5’-inosinate (IMP) (MSG+IMP, umami)]. (mAChR) M1 and/or M3 subtypes (M1KO, M3KO, M1/M3KO). When M1KO and M3KO Of a total 285 pyramidal and theta neurons recorded, 137 neurons responded mice were used, the CTA was suppressed both acquisition and retention, whereas M1/M3KO during licking, and responses of 62 of these cells were greater to sapid mice acquired the CTA memory. When a nonspecific blocker of mAChR, scopolamine solutions than to water (taste neurons). Multivariate analyses of the taste was injected to the anterior paraventricular nucleus of thalamus, the CTA remains to be suppressed. However, the CTA acquisition was not suppressed by scopolamine injection neurons suggested that taste quality in the HF might be encoded based on to dorsal 3rd ventricle. These results suggest that low gravitational force prevents the hedonic value. Furthermore, the activity of most taste neurons was chamber- CTA memory formation through suppression of association process between CS and US. specific. These results implicate the HF in guiding appetitive behaviors such Furthermore, both M1 and M3 receptors in the paraventricular nucleus of thalamus play an as conditioned place preference. important role on the detection mechanism of the gravitational disturbance.
P1PM-6-9 P1PM-6-10 A TELENCEPHALIC NEURAL CIRCUIT CRITICAL FOR EXPERIENCE-DRIVEN RETRACTION OF VISUAL IMPRINTING IN CHICKS GENICULOCORTICAL AXONS FOLLOWING CORTICAL Tomoharu Nakamori1, Tomoharu Nakamori1, Katsushige Sato2, INACTIVATION BY PREVENTING SYNAPTIC Kohichi Tanaka1, Hiroko Ohki-Hamazaki1 TRANSMISSION 1Department of Molecular neuroscience, Tokyo Medical and Dental Kana Watanabe, Yu Morishima, Masahito Toigawa, Yoshio Hata 2 University, Japan, Physiology, Tokyo Medical and Dental University, Japan Division of Integrative Bioscience, Tottori University Graduate School of During a few days after hatching, chicks recognize and memorize a moving Medical Sciences, Japan object which they see frequently. They distinguish the object from others and In the visual system, the geniculocortical axons serving an open eye significantly follow it. In the visual imprinting, two telencephalic regions, the visual Wulst retract when cortical neurons are pharmacologically inhibited by a GABA receptor (VW) and the intermediate medial mesopallium (IMM) are known to have agonist, muscimol, suggesting that presynaptic activity can lead to the retraction of critical roles. the axons in the absence of postsynaptic activity. However, local synaptic interaction We postulated that the visual information should be transmitted from the VW may underlie the retraction of active axons in the inhibited cortex because muscimol to the IMM, and performed anatomical and physiological studies to elucidate application leaves transmitter release intact. Therefore, we determined whether the connection between these two regions. We found the VW-IMM neural experience-driven axon retraction could occur in the visual cortex inactivated by circuit was activated by the visual imprinting. Moreover, the behavioral blocking synaptic inputs. We inactivated the primary visual cortex of kittens by performance level of the imprinting positively correlated with the activity suppressing synaptic transmission with cortical injections of botulinum neurotoxin of this VW-IMM circuit. In the imprinted chicks, this activation could be type E and analyzed the axon morphology in the kittens with normal vision and those detected at 7 days after hatching, even after the closure of the critical period deprived of vision binocularly. We found that the axons in the animals with normal of the visual imprinting. The NMDA receptors containing NR2B subunit vision showed a significant retraction in the inactivated cortex, whereas the axons in were critical for this neuronal activation and the establishment of imprinting. the binocularly deprived animals were preserved. These results suggest that the axon From these results, we concluded that the visual imprinting requires the retraction in the inactivated cortex can proceed in the absence of synaptic transmission. activation of the VW-IMM circuit and this circuit may also contribute to the Therefore, presynaptic mechanisms play an important role in the experience-driven maintenance of the imprinting memory. refinement of geniculocortical axons.
P1PM-6-11 P1PM-6-12 MOLECULAR MECHANISMS CONTROLLING THE EXPERIENCE-DEPENDENT ORIENTATION PLASTICITY POLARITY OF LONG-TERM MODIFICATION AT IN THE MOUSE VISUAL CORTEX INHIBITORY SYNAPSES OF VISUAL CORTICAL Takamasa Yoshida, Toshiki Tani, Shigeru Tanaka PYRAMIDAL NEURONS Laboratory for Visual Neurocomputing, RIKEN Brain Science Institute, Tahamina Begum, Faruque Reza, Yumiko Yushimura, Japan Shoko Horibe, Yukio Komatsu To date, ocular dominance plasticity in the mouse visual cortex has been Department of Neuroscience, RIEM, Nagoya University, Japan deeply investigated. In contrast, plasticity of orientation selectivity remains High-frequency stimulation (HFS) of presynaptic fibers can induce long-term to be clarified. Previous studies using juvenile cats and rats reared under potentiation (LTP) or depression (LTD) at inhibitory synapses of layer 2/3 pyramidal single-orientation exposure revealed the over-representation of the exposed neurons in developing mouse visual cortex. In this study, we investigated the molecular orientation in the visual cortex (Tanaka et al., Neuroimage, 2006; Ohashi et mechanism controlling the polarity of these modifications. Inhibitory postsynaptic al., Neurosci.Res, 2007). To examine whether orientation selectivity in the currents were recorded using the whole-cell patch-clamp recording method under mouse visual cortex is also changeable depending on visual experience, we pharmacological blockade of non-NMDA and NMDA receptors. HFS was applied reared juvenile mice under single-orientation exposure by cylindrical-lens- during the temporal washout of the antagonists. LTD of inhibition occurred when HFS fitted goggles, which transmit only vertical orientation. Goggle rearing was was applied in the current clamp mode at the resting membrane potential, whereas started at P21-23 for four mice. Immediately after 1-week goggle rearing, we LTP occurred when HFS was applied in the voltage-clamp mode at -70 mV. LTP was performed optical imaging of intrinsic signals in the visual cortex under the blocked or converted into LTD when inhibitors for PLC (U-73122) or IP3 receptors (2APB) were loaded into postsynaptic cells. LTD was blocked or converted into monocular stimulation of drifting gratings in 6 orientations. Nineteen normal LTP when the NMDA receptor channel blocker MK801 or the calmodulin (CAM) mice (P20-33) were also recorded as a control group. In the goggle-reared inhibitor calmidazolium was loaded into postsynaptic cells. These results suggest that mice, the distribution of preferred orientations showed a prominent peak at the activation level of postsynaptic IP3 receptors regulates the polarity of modification. vertical orientation, whereas in the normal mice, preferred orientations were The Ca-CAM complex formed by NMDA receptor-mediated Ca2+ entry may inhibit biased toward horizontal orientation. The present study demonstrated that LTP induction, probably via the inhibition of IP3 receptors. orientation selectivity is modifiable by postnatal visual experience in mice. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 181 P1PM-6-13 P1PM-6-14 EXPOSURE TO ORTHOGONAL ORIENTATIONS IN PLASTIC CHANGES IN THE ANTERIOR AUDITORY THE TWO EYES REORGANIZES ORIENTATION MAPS FIIELD INDUCED BY DISCRIMINATION LEARNING OF ROBUSTLY IN KITTEN VISUAL CORTEX SYNTHETIC VOWELS IN RATS Toshiki Tani, Shigeru Tanaka Masaharu Kudoh Laboratory for Visual Neurocomputing, RIKEN Brain Science Institute, Department of Physiology, School of Medicine, Teikyo University, Japan Japan We have reported that discrimination learning of synthetic vowels with We have previously reported that the marked over-representation of an exposed multiple formants was impaired by bilateral partial lesions of the rostral orientation was induced by 1- or 2-week orientation-restricted visual experience or dorsal auditory cortex (AC) in rats, while lesions of the primary AC with head-mounted goggles concordant in the two eyes during development. had no clear effects. In the present study, plastic changes of AC responses However, the over-representation of the exposed orientation somewhat to synthetic vowels by discrimination leaning were investigated using decreased after prolonged goggle rearing. In the present study, to examine the flavoprotein autofluorescence imaging. Water-deprived rats were trained effect of exposure to orthogonal orientations in the two eyes on the alteration of to discriminate between two synthetic vowels. Licking a spout during orientation maps, we performed optical imaging of intrinsic signals in the visual presentation of one (S+) was rewarded with water while the other (S-) cortex of cats reared with discordant goggles. In the cats exposed to vertical and was not. In naive rats, pure tones evoked clear flavoprotein fluorescence horizontal orientations for the respective eyes, cortical domains occupied by the responses in the primary AC. However, synthetic formants or vowels evoked representations of the exposed orientations closely agree with ocular dominance prominent responses in the anterior auditory field (AAF), which corresponds patches. Intriguingly, the over-representation of the exposed orientation for each to the site of rostral AC lesions. In rats that had achieved the discrimination, eye tended to be preserved robustly after prolonged goggle rearing. Compared the synthetic vowels used as S+ elicited stronger fluorescence responses than with the reduction of over-representation in prolonged rearing with concordant those used as S- in the AAF. These findings indicate that the AAF plays a goggles, it is suggested that in cats reared with the discordant goggles, the pivotal role in discrimination learning of vowels in rats. segregation of exposed orientations into ocular dominance patches diminishes interocular suppressive interaction, resulting in the preservation of the marked over-representation.
P1PM-7-1 P1PM-7-2 A CRITICAL GENOMIC ELEMENT FOR SYNAPTIC INVOLVEMENT OF RYANODINE RECEPTORS IN LTP ACTIVITY-DEPENDENT EXPRESSION OF Arc/Arg3.1 OF C-FIBBER-EVOKED FIELD POTENTIAL IN THE Hiroyuki Okuno, Takashi Kawashima, Mio Nonaka, Nan Kyo, SPINAL CORD IN RATS Haruhiko Bito Ning Lu, Pei-Fen Wang, Yu-Qiu Zhang, Zhi-Qi Zhao Department of Neurochemistry, University of Tokyo Graduate School of Neurobiology Institute, Fudan University, China Medicine, Japan Lines of studies have shown that spinal LTP is associated with a central sensitivity The activity-regulated neuronal gene Arc/Arg3.1 is widely used as one of the of nociceptive input. Our previous work has demonstrated that NO and cGMP were most reliable molecular markers for intense synaptic activity in vivo. However, involved in spinal LTP. Here we investigated the possible involvement of ryanodine the cis-acting elements responsible for such stringent activity-dependence have receptors (RyRs), one of downstream targets of NO, in spinal LTP and the expression not been firmly identified. In this study, we combined luciferase reporter assays of RyRs in the spinal cord and dorsal root ganglion (DRG) neurons. in cultured cortical neurons and comparative genome mapping to identify the critical synaptic activity-responsive elements (SARE) of the Arc/Arg3.1 gene. We At high doses, ryanodine (as an antagonist of RyRs) strikingly blocked the induction found a major SARE as a unique 100 bp element located at >5-kb upstream of of LTP of C-fiber-evoked field potentials in the spinal dorsal horn in vivo, but did the Arc/Arg3.1 transcription initiation site. This single element, when positioned not affect the maintenance of LTP. Either BayK 8644 (an agonist of L-type calcium immediately upstream of a minimal promoter, was necessary and sufficient channel) or cyclic ADP-ribose (an endogenous agonist of ryanodine receptor) to replicate crucial properties of endogenous Arc/Arg3.1's transcriptional completely reversed ryanodine-induced inhibition on spinal LTP. RyR subtypes RyR1 regulation. We further identified the major determinants of SARE as a unique and RyR3 were located predominantly in spinal laminae IIinner or I and co-expressed cluster of neuronal activity-dependent cis-regulatory elements consisting of respectively with isolectin B4 (IB4) or calcitonin gene-related peptide (CGRP), closely localized binding sites for CREB, MEF2, and SRF. Consistently, a specific markers for primary nociceptive neurons, in both spinal cord and DRG SARE reporter could readily trace and mark an ensemble of cells that have neurons. However RyRs did not colocalized with neuronal (NeuN), dendritic (MAP2) experienced intense activity in the recent past in vivo. These results indicate that and glial (GFAP and OX42) markers in the spinal dorsal horn. SARE mediates a predominant component of the synapse-to-nucleus signaling in These results suggest that RyRs in the primary afferent terminals are involved in the synaptically activated neurons. induction of spinal LTP.
P1PM-7-3 P1PM-7-4 COCAINE REGULATES ERM PROTEINS AND RhoA THE ROLE OF KINASE ACTIVITY OF Ca2+/CALMODULIN- SIGNALING IN THE NUCLEUS ACCUMBENS DEPENDENT PROTEIN KINASE II IN HIPPOCAMPAL Wha Young Kim1, So Ra Shin1, Seungwoo Kim1, Songhee Jeon2, SYNAPTIC PLASTICITY AND LEARNING 1 Yoko Yamagata1, Shizuka Kobayashi2, Tatsuya Umeda3, Akihiro Inoue3, Hiroyuki Jeong-Hoon Kim 4 5 5 6 1 Sakagami , Masahiro Fukaya , Masahiko Watanabe , Nobuhiko Hatanaka , Department of Physiology, Yonsei University College of Medicine, Korea, 7 8 9 1 2 Masako Totsuka , Takeshi Yagi , Kunihiko Oabata , Keiji Imoto , Yuchio Medical Science Research Center, Dongguk University Research Institute 10 2 11 Yanagawa , Toshiya Manabe , Shigeo Okabe of Biotechnology, Korea 1Department of Information Physiology, National Institute for Physiological Sciences, Japan, The nucleus accumbens (NAcc) is an important neuronal substrate mediating 2Division of Neuronal Network, Institute of Medical Science, University of Tokyo, Japan, 3Department of Cell Biology, Tokyo Medical and Dental University, Japan, 4Department of the effects of drugs of abuse. Cocaine induces structural plasticity in this site 5 Anatomy, Kitasato University School of Medicine, Japan, Department of Anatomy, Hokkaido possibly via regulation of the actin cytoskeleton. The ezrin-radixin-moesin (ERM) University School of Medicine, Japan, 6Division of System Neurophysiology, National proteins have been implicated in cell-shape determination by crosslinking actin Institute for Physiological Sciences, Japan, 7CREST, Japan Science and Technology Agency, 8 9 to plasma membranes. RhoA, a small GTPase protein, is the key regulator of Japan, Graduate School of Frontier Biosciences, Osaka University, Japan, Laboratory of Neurochemistry, National Institute for Physiological Sciences, Japan, 10Department of Genetic actin cytoskeletal dynamics and it also regulates the activity of ERM proteins. and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Japan, Here we show first time that the phosphorylation levels of ERM proteins are 11Department of Cellular Neurobiology, Graduate School of Medicine, University of Tokyo, dose- and time-dependently decreased in the NAcc by a single injection of Japan 2+ Poster Session cocaine (15 or 30 mg/kg, i.p.). Further, we show that the amount of active RhoA Ca /calmodulin-dependent protein kinase II (CaMKII) is one of the most abundant protein kinases in the central nervous system and plays important roles in neuronal functions, especially in hippocampal synaptic is also significantly reduced in this site by cocaine. In keeping with this, bilateral plasticity. Since CaMKII has many protein properties, such as enzymatic activity, autophosphorylation at microinjections of the Rho kinase inhibitors, Y27632 (1.0 or 10.0 μg/0.5 μl/side) different sites, calmodulin-binding capacity, multimer-formation to make up a holoenzyme and interaction or RKI II (0.5 or 2.0 μg/0.5 μl/side), in the NAcc also caused a decrease of with other proteins, it is essential to analyze each functional role in order to advance our understanding of its physiological significance in vivo. We focused on the roles of enzymatic activity of CaMKIIα (the phosphorylated ERM protein levels in this site. Together, these results suggest major forebrain isoform), engineered the kinase-dead CaMKIIα (K42R) knock-in mouse, and analyzed that cocaine reduces phosphorylated ERM levels in the NAcc by making down- hippocampal synaptic plasticity and behavioral learning. In this meeting, we will present basic characteristics of the K42R mouse, cell biological studies using hippocampal neuronal culture, electrophysiological regulation of RhoA-Rho kinase signaling, which may importantly contribute to experiments using acute hippocampal slices and behavioral experiments for hippocampus-dependent synaptic morphological changes in the NAcc leading to drug addiction. learning, and discuss the physiological importance of kinase activity of CaMKIIα in vivo.
182 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-7-5 P1PM-7-6 CONSTITUTIVE ACTIVE CaMKKα TRANSGENIC SPECIFIC ROLE OF NEURONAL ISOFORM OF MOUSE SHOWS DEFICITS OF LONG-TERM DREBRIN IN HIPPOCAMPAL SYNAPTIC PLASTICITY HIPPOCAMPUS-DEPENDENT MEMORY AND FEAR MEMORY Taku Kaitsuka1, Sheng T Li2, Keizo Takao3, Tsuyoshi Miyakawa3, Masayuki Nobuhiko Kojima, Hiroki Yasuda, Kenji Hanamura, Hiroyuki Matsushita1 Yamazaki, Tomoaki Shirao 1 Research Group of Brain Stroke, Mitsubishi Kagaku Institute of Life Sciences, Department of Neurobiology and Behavior, Gunma University, Japan Japan, 2Institute of Neuroscience, Shanghai Jiaotong University, China, 3Genetic Engineering and Functional Genomics Unit, Horizontal Medical Research Drebrin is a major F-actin-binding protein, and has two isoforms E and A. Organization, Graduate School of Medicine, Kyoto University, Japan Drebrin A is neuron-specific and localized in dendritic spines, while drebirn Ca2+/calmodulin (CaM) kinase cascade is activated by voltage-dependent Ca2+cannel. E is expressed in a variety of cells. During neuronal development the isoform CaMKK, most upstream kinase of CaM kinase cascade, phosphorylates and conversion of drebrin E to A occurs concomitantly with synapse formation. In activates both CaMKI and CaMKIV, resulted in activation of CREB-dependent vitro study suggests that drebrin A is critical for development and plasticity of gene transcription. By using transgenic techniques, we created mutant mice in which dendritic spines. To examine this idea we generated drebrin A-specific KO mice constitutively active form of CaMKKα is expressed specifically in forebrain. In these by gene targeting of the exon utilized only in drebrin A. In adult KO mouse brain mice, although performance was normal in anxiety-like behavior and social interaction, E isoform was expressed instead of A and immunohistochemical distribution specific impairments were shown in long-term hippocampus-dependent memory, of drebrin was not changed. The density and length of dendritic spines of including spatial learning and contextual fear conditioning. At hippocampal shaffer the cortical pyramidal neurons were comparable to that in wild-type mice, collateral-CA1 synapses, input-output curve was weak and LTP was significantly suggesting that spine formation in KO mice can be substituted by drebrin E. KO diminished in mutant mice. In cultured neurons of these mice, phosphorylation of mice showed no apparent abnormalities in general behavior, but impairment of CaMKI was significantly increased in basal states, but activity range of CaMKI hippocampus-dependent contextual fear memory. We then analyzed hippocampal phosphorylation by BDNF and KCl stimulation was significantly diminished in LTP and found age-dependent impairment. The LTP amplitude significantly mutant mice. Our results define a critical role for CaMKKα in synaptic plasticity and decreased in KO mice older than 28 weeks old, whereas it was not altered in hippocampus-dependent memory and suggested that a novel mechanistic distinction mice younger than 9 weeks. Thus, our data suggest specific role of drebrin A in between short-term and long-term memory consolidation. synaptic plasticity underlying hippocampus-dependent fear memory.
P1PM-7-7 P1PM-7-8 REMODELING OF POSTSYNAPTIC GLUTAMATE MOLECULAR EVIDENCE FOR SYNAPTIC TAGGING RECEPTOR ORGANIZATION IN AWAKE RATS HYPOTHESIS REVEALED BY FREEZE-FRACTURE REPLICA Daisuke Okada, Kaoru Inokuchi, Fumiko Ozawa LABELING Mitsubishi Kagaku Institute of Life Sciences and JST, CREST, Japan Yugo Fukazawa1, Makoto Itakura2, Masami Takahashi2, Yoshito Saitoh3, 3 4 5 The late-phase synaptic plasticity is assumed to be the cellular basis of Kaoru Inokuchi , Elek Molnar , Ryuichi Shigemoto 1 long-term memory and depends on the synthesis of new repertoires of Dev. Cereberal Structure, National Institute for Physiological Sciences, SOKENDAI, Japan, 2Department of Biochemistry, Kitasato University, Japan, proteins. The late-phase plasticity is input-specific, implying spatially 3MITILS, Japan, 4Anatomy, MRC Centre for Synaptic Plasticity, Univ. Bristol, restricted functioning of the somatically-synthesized proteins in the synapses UK, 5Dev. Cereberal Structure, National Institute for Physiological Sciences, expressing early-phase plasticity; however, the mechanism underlying the SOKENDAI, SORST, Japan input-specificity of late-phase plasticity is not known. The synaptic tagging A modification of synaptic efficacy is thought be relevant to learning and memory. The hypothesis predicts that early-phase plasticity marks the synapse with a glutamatergic synaptic transmission is a major excitatory input in the central nervous system and hypothetical synaptic tag and that soma-derived new proteins spread cell- is mainly mediated by ionotropic glutamate receptors. Long-term potentiation of synaptic efficacy (LTP) in the hippocampus has been reported to be caused largely by increasing number of AMPA- widely before functioning exclusively in the tagged synapses. It is necessary type ionotropic glutamate receptors (AMPAR) in the postsynaptic plasma membrane specialization. to specify the tagging activity for the molecular identification of the synaptic Therefore, in order to understand cellular mechanisms underlying learning and memory, it is tag. Here we found that soma-derived Vesl-1S (Homer-1a) protein, which is important to know precise localization of AMPARs and their dynamics before and after the LTP a late-phase plasticity-related synaptic protein, prevailed in every dendrite, induction. We, here, employed the unilateral dentate gyrus (DG) -LTP in the hippocampus of freely moving and did not enter spines unless the N-methyl-D-aspartate receptor was rats, and distribution of AMPARs in single synapses was analyzed by SDS-digested freeze-fracture activated. The spine entry activity was was input-specific, persistent, protein- replica labeling, which quantitatively visualizes 2-dimensional distribution of the membrane synthesis-independent and interacted with soma-to-dendrite transport. These proteins at ~ 20 nm resolution. Our results indicated that induction of LTP increases and decreases results support the concept of synaptic tagging and suggest that the activity- AMPAR density in synaptic and extrasynaptic membranes, respectively. Similar changes were observed in rats exploring in a new environment for 45 min. These results suggest robust dependent regulation of spine entry functions as a synaptic tag. remodeling of AMPAR organization in awake animals.
P1PM-7-9 P1PM-7-10 AN INTEGRATIVE KINETIC MODEL OF AMPA- DENDRITIC SPINE CALCIUM SIGNALS ASSOCIATED RECEPTORS CONSTITUTIVE TRAFFICKING AND LTD/ WITH SPIKE-TIMING DEPENDENT SYNAPTIC LTP IN CEREBELLAR PURKINJE CELL PLASTICITY IN MEDIUM SPINY NEURONS IN THE Kazuhiko Yamaguchi MOUSE NEOSTRIATUM Laboratory for Memory and Learning, Brain Science Institute, RIKEN, Tomomi Shindou, Mayumi Ochi-Shindou, Jeffery R Wickens Japan Neurobiology Unit, Okinawa Institute of Science and Technology, Japan Synaptic plasticity underlying motor learning is mediated by an activity- The striatum is a central component of the basal ganglia and a major site dependent regulation of AMPA-type glutamate receptors (AMPARs) trafficking of convergence of glutamatergic afferents from the cerebral cortex and at parallel fiber (PF)-Purkinje cell (PC) synapse in the mammalian cerebellum. thalamus. These glutamatergic afferents terminate on spiny projection However, whether basal synaptic expression of AMPARs was regulated by neurons which are the principal output neurons of the striatum. Recent similar trafficking was elusive, and kinetic model which could integratively studies suggest that the temporal association of cortical and striatal activity explain constitutive and activity-dependent AMPARs’ trafficking was not can influence both the magnitude and direction of change in corticostriatal constructed. Based on electrophysiological data obtained from rat cerebellar synaptic efficacy. Many pieces of evidence suggest that the dynamics of spine slice, I develop an integrative kinetic model, which is composed of three pools of Ca2+ concentration play an important role in such spike-timing dependent AMPARs, namely, “internal mobile pool (IMP)”, “synaptic mobile pool (SMP)” plasticity (STDP). In this study we measured the effects of STDP induction and “synaptic stable pool (SSP)”. Transitions between these pools are described protocols on dendritic spine Ca2+ signals in mouse brain slices using patch by 4 rate constants. Constitutive exocytosis of AMPARs is fitted by a model clamp recordings combined with two-photon microscopy. Pre-post protocols with synaptic slots for AMPARs. LTP is demonstrated to share the same synaptic (three action potentials 10 ms after EPSP) resulted in greater Ca2+ transients slots. While, conjunction of PF-stimulation and somatic depolarization elicits in dendritic spines than those induced by post-pre protocols or postsynaptic LTD from TeTx-resistant, stable AMPARs pool, indicating that enhancement of action potentials alone. In our hands pre-post protocols caused long-term destabilizing rate constant is involved in LTD-induction. However, concomitant depression and post-pre protocols produced no change in EPSPs. Our results increase in endocytotic or decrease in exocytotic rate constant is predicted from suggest that increases in dendritic spine Ca2+ concentration are associated the kinetic model of AMPARs’ trafficking at PF-PC synapse. with STDP in the spiny projection neurons of the striatum. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 183 P1PM-7-11 P1PM-8-1 TRPC-MEDIATED CALCIUM ENTRY AS A CALCIUM LONG-TERM SYNAPTIC PLASTICITY INDUCED IN THE SOURCE OF Ca2+-ASSISTED ENDOCANNABINOID PRIMATE HIPPOCAMPUS RELEASE IN CEREBELLAR PARALLEL FIBER- Ryoi Tamura1, Satoshi Eifuku1, Michiya Sugimori1, Teruko Uwano1, PURKINJE CELL SYNAPSE Taketoshi Ono2 Won Seok Chang, Ji Young Kim, Hong Goo Chae, Jun Kim, 1Integrative Neuroscience, University of Toyama, Japan, 2University of Sang Jeong Kim Toyama, Japan Department of Physiology, Seoul National University, Korea Long-term potentiation (LTP) and long-term depression (LTD), 2 forms of Endogenous cannabinoids are considered to function as diffusible and short-lived long-term changes in synaptic weights, have been studied extensively in the modulators that transmit signals retrogradely from postsynaptic to presynaptic rodent hippocampus. However, there has been no information about LTP or neurons. To investigate this effect of endocannabinoid on the parallel fiber- LTD induced in the primate hippocampus in vivo. In the present study we Purkinje cell excitatory synapse, we made whole-cell patch clamp recordings implanted stimulation and recording electrodes in the perforant pathway and from cerebellar Purkinje cells of rat. We confirmed that brief depolarization and dentate gyrus, and gave high-frequency stimulation (HFS) or low-frequency tetanic stimulation of parallel fiber transiently suppressed both AMPA current stimulation (LFS) to test whether LTP or LTD could be induced in the primate and mGluR-induced slow currents in voltage clamp mode and also confirmed hippocampus. As results, some HFS conditions induced LTP, while all the LHS similar phenomenon in current clamp mode. Adding cannabinoid1 receptor conditions tested in this study failed to induce LTD. The induction of LTP was blocker AM251, and TRPC channel blocker SKF96365 and ruthenium red blocked by systemic administration of ketamine. Furthermore, we compared the blocked the tetanus-induced suppression of excitatory postsynaptic current and induction and maintenance of LTP between primates and rodents. Although LTP SKF96365 inhibited tetanus-induced postsynaptic calcium transient. SKF96365 was induced by HFS to a similar degree across the species, it was much more neither blocked depolarization-induced suppression of excitatory postsynaptic stably maintained in monkeys compared with that induced in rodents. These data current and postsynaptic calcium transient, nor cannabinoid agonist-mediated indicate that 1) LTP can be a ubiquitous model for long-term changes in synaptic suppression of excitatory postsynaptic current, however. Our data suggest that weights, and 2) the difference of LTP maintenance between the species is activation of TRPC channel may be involved in the endocannabinoid-dependent involved in the specific difference of hippocampal-dependent duration necessary depression of parallel fiber-Purkinje cell synapse. for memory consolidation.
P1PM-8-2 P1PM-8-3 EFFECT OF PRENATAL TREATMENT OF REHMANNIAE EFFECT OF PRENATAL TREATMENT OF CNIDIUM RADIX PREPARAT ON SPATIAL MEMORY ABILITY OFFICINALE MAKINO ON CELL PROLIFERATION AND NEUROGENESIS IN THE HIPPOCAMPUS OF IN THE HIPPOCAMPUS AND SPATIAL, LEARNING OFFSPRING MEMORY OF OFFSPRING Bong-Soo Lim1, Byung Soo Ahn1, Dae-Il Lee1, Seong-Kyu Kim1, Ji-Young Seong Kyou Kim1, Min Byung-il1, Bae Hyun-soo2, Ahn Byung-soo1, Lee Kim1, Hyunsu Bae2, Byung-Il Min1 Dae-il1, LIm Bong-soo1 1Department of East-West Medicine, Graduate School, Kyung Hee University, 1Department of Neruogenesis, Graduate school of Kyung-hee University, Korea, Korea, 2Department of Physiology, College of Oriental Medicine, Kyung Hee 2Department of Physiology, College of Oriental Medicine, Kyung Hee University, University, Korea Korea Brain development occurs crucially in fetal, neonatal and early infant period. Rehmanniae Radix OBJECTIVE : Our purpose was to determine whether prenatal exposure to the Cnidium officinale Preparat(RR) has the function of supplementing essential energy, dominating growth, development MAKINO(COM) affects cell proliferation in the hippocampus and spatial, learning memory of and reproduction. mouse offspring. Extract powder of RR(100, 200 and 400mg/kg) was administered orally to mother rats on the STUDY DESIGN : Dem mice were randomly assigned to receive daily doses of 50, 100, 200 15-17th days after mating for 3days. While, water was supplied to control group(CON). mg/kg per day COM (n = 6), while normal control animals (n = 2), stressed control animals (n = BrdU was injected to mother rats for same period(i.p.). 2) received an equal amount of normal saline daily for 3 days on the 15-17th days after mating. To estimate spatial memory ability, radial-arm maze test was performed twice to the 6week-old On the other hand, normal saline was supplied to stressed and non stressed control group. For offspring rats. The offspring rats were sacrificed and BrdU and NeuN immunohistochemistry was stress treatment, pregnant rats were restrained in small cylindrical cages made of transparent done to estimate neurogenesis. plastic during 180 min, and BrdU was injected to mother rats for same period (50mg/kg, i.p.). To In the 1st trial of arm maze test, the latency of RR200 was lower than that of CON and RR100. In estimate spatial, learning memory ability, radial-arm maze task and passive avoidance test were the 2nd trial, the latency of RR400 and RR200 was lower than that of CON. The reduction of the performed to the 6week-old, 78 offspring rats. The offspring rats were sacrificed and BrdU & NeuN latency of RR400 was higher than that of CON. immunohistochemistry was done to estimate neurogenesis. BrdU+ and NeuN+ cells in dentate gyrus of hippocampus of RR400 and RR200 were more than RESULTS : those of CON. 1) COM treatment with high dose during pregnancy enhances the spatial and learning memory of In conclusion, maternal treatment of RR during pregnancy improves spatial memory ability and offspring. neurogenesis of hippocampal DG of the offspring. It suggests that prenatal treatment of RR may 2) COM treatment with high does during pregnancy significantly increases hippocampal improve fetal brain development. neurogenesis of offspring.
P1PM-8-4 P1PM-8-5 INDUCTION- AND TRAINING-PROTOCOL DEPENDENT LATE PHASE OF LTP INDUCED BY CO- INVOLVEMENT OF NR2B-NMDARS IN SYNAPTIC APPLICATION OF NMDA AND THE ANTAGONIST POTENTIATION AND MEMORY IN HIPPOCAMPAL CA1 OF NR2B-CONTAINING NMDA RECEPTORS IN RAT REGION OF RATS HIPPOCAMPUS Xue-Han Zhang1, Long-Jun Wu2, Ming Ren2, Feng Yi1, Min Zhuo2, Bao- Nobuyuki Suzuki1, Arata Oh-Nishi1, Shozo Satoh1, Masanori Ogata2, Ming Li1 Makoto Saji1 1Institute of Neurobiology, Fudan University, China, 2Department of Physiology, 1Division of Brain Sciences, Kitasato University Graduate School of Medical Faculty of Medicine, University of Toronto, 1 King's College Circle, Canada Science, Japan, 2Department of Physiology, School of Allied Health Sciences, Long-term potentiation (LTP) in the hippocampal CA1 region requires the activation of Kitasato University, Japan N-methyl- D-aspartate receptors (NMDARs). Studies using genetic and pharmacological Activation of NMDA glutamate receptors (NMDARs) is required for long-term potentiation approaches have reported inconsistent results of the requirement of NR2B-containing (LTP) of excitatory synaptic transmission at hippocampal CA1 synapses, the proposed NMDARs in LTP in the CA1 region. Pharmacological studies showed that NR2B-containing cellular mechanisms of learning and memory. We demonstrate that a bath co-application NMDARs are not required for LTP, while genetic studies reported that over-expression of of a low concentration of NMDA, an agonist of NMDARs, and the selective antagonist NR2B-NMDARs enhances LTP and hippocampus-dependent memory. Here, we provide of NR2B-containing NMDARs, Ro25-6981, to hippocampal slices from young adult rats evidence showing that the functional role of NR2B-NMDARs in hippocampal LTP and produced a slowly developing LTP persisting at least for 6 hr following a transient depression of synaptic transmission in CA1 synapses. The LTP was likely to occur at postsynaptic
Poster Session behavioral memory depends on LTP-inducing and behavior training protocols. (1) Inhibition of NR2B-NMDARs with the NR2B selective antagonist ifenprodil or site and was initiated by activation of NMDARs, and its development required cAMP- Ro25-6981 suppressed LTP induced by spike-timing protocol, with no impact on LTP dependent protein kinase (PKA) activation and protein synthesis. This chemically induced induced by pairing protocol. LTP and the tetanus-induced late phase of LTP (L-LTP) were mutually occluding, suggesting (2) Pre-training intra-CA1 infusion of ifenprodil or Ro25-6981 impaired the formation of a common expression mechanism. Thus, we have demonstrated that a brief bath co- contextual fear memory induced by five CS-US pairings, with no effect on the memory application of NMDA with Ro25-6981 to a slice offers an alternative to electrical stimulation induced by one CS-US pairing. as a stimulation method to induce L-LTP. This chemically induced LTP may be useful for (3) Intra-CA1 infusion of ifenprodil or Ro25-6981impaired the spatial working memory in assessing the biochemical correlates and the molecular aspects of the expression mechanism delayed matching-to-place watermaze task, but not in T-maze delayed alternation task. for L-LTP that has been proven to correlate to hippocampal long-term memory.
184 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-8-6 P1PM-8-7 PROLONGED LOW-FREQUENCY STIMULATION TETRAETHYLAMMONIUM-INDUCED LONG-TERM INDUCES MITOCHONDRIA-DEPENDENT SHORT- PLASTICITY AT HIPPOCAMPAL MOSSY FIBER-CA3 TERM POTENTIATION AT HIPPOCAMPAL CA3-CA1 SYNAPSES SYNAPSES Etsuko Suzuki1, Takashi Okada2 1 2 Takayuki Morimoto1, Ayako M Watabe2, Toshiya Manabe2 Department of Psychology, Senshu University, Japan, Department of 1Division of Neuronal Network, Department of Basic Medical Sciences, Institute Psychology, Sophia University, Japan of Medical Sciences, University of Tokyo, Japan, 2Division of Neuronal Network, Tetraethylammonium (TEA), a blocker of K+ channels, induces long-term Department of Basic Medical Sciences, Institute of Medical Science, University potentiation (LTP). This form of LTP in hippocampal CA1 and dentate gyrus of Tokyo; CREST, JST, Japan is reportedly independent of NMDA receptor activation, but requires voltage- Synaptic plasticity is considered to be involved in the formation of certain types of dependent Ca2+ channel (VDCC) activation. In this study, the effect of TEA memory. While molecular mechanisms underlying long-term plasticity are intensively application and its characteristics in mossy fiber (MF) -CA3 synapses were studied, those of short-term plasticity are still a topic of debate. Here, we examined the examined. Hippocampal slices were obtained from three-week-old male Wistar posttetanic potentiation (PTP) induced by a tetanus (100Hz for 1s), responses to low- rats and extracellular field excitatory postsynaptic potentials (fEPSPs) were frequency stimulation (LFS: 5Hz, 3min) and the paired-pulse ratio (PPR) to elucidate the recorded from MF-CA3 synapses. The application of TEA (25 mM) for 12 min molecular mechanism of presynaptic short-term plasticity at the CA3-CA1 synapse in induced LTP irrespective of the presence of NMDA receptor antagonist, D-AP5 mouse hippocampal slices. We found that, during the LFS, fiber volleys and excitatory (50 μM). LTP induction by TEA was inhibited in the presence of T-type VDCC postsynaptic potentials changed dynamically: although the fiber volley was reduced during the LFS, LFS induced short-term potentiation (LFS-STP), which surmounted the fiber blocker, mibefradil (1 μM) or penfluridol (0.5 μM). A decreased paired-pulse volley depression. Surprisingly, the time course of the PPR after the LFS was comparable to ratio was observed after LTP, suggesting an increase in neurotransmitter release. that after the tetanus, implying some common mechanism mediating PTP and LFS-STP. In LTP was not induced by TEA in the presence of the GABAA receptor blocker fact, both forms of plasticity were suppressed by tetraphenylphosphonium, which inhibits picrotoxin (100 μM). Since this facilitative effect of GABA was reversed in + + - mitochondrial Ca2+ efflux, suggesting that mitochondrial regulation of presynaptic calcium is the presence of the Na -K -Cl co-transporter blocker bumetanide (1 μM), the - involved in some forms of short-term plasticity. These results suggest that mitochondria are a GABAergic facilitative effect may be due to the relatively higher intracellular Cl new player in dynamic presynaptic regulation of synaptic efficacy. concentration at MF terminals.
P1PM-9-1 P1PM-9-2 PRENATAL EXPOSURE TO DIESEL EXHAUST INTRACELLULAR MECHANISMS UNDERLYING THE PARTICLES PRODUCE LEARNING DEFICITS INHIBITORY EFFECT Of α-2A-ADRENOCEPTOR ASSOCIATED WITH IMPAIRMENT OF NMDA STIMULATION ON EXCITATORY SYNAPTIC RECEPTOR TRANSMISSION IN mPFC Satoshi Yokota1, Akira Sato1, Keisuke Mizuo2, Kayo Akiyama3, 3 1 Feng Yi, Xuehan Zhang, Baoming Li Den’etsu Sutoo , Ken Takeda 1 Institute of Neurobiology, Fudan University, China Department of Hygiene chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Japan, 2Research Center for Health Science Stimulation of α-2A-adrenoceptors (ARs) in the prefrontal cortex (PFC) of Nanoparticles, Tokyo University of Science, Japan, 3Institute of Medical produces a beneficial effect on cognitive functions such as working memory. Science, University of Tsukuba, Japan α-2-adrenergic agonist guanfacine has been used experimentally and Diesel exhaust particles (DEP) are a major component of airborne particulate matter. It has clinically for treatment of psychiatric disorders such as attention-deficit/ previously been reported that prenatal exposure to diesel exhaust (DE) has a negative impact hyperactivity disorder (ADHD). A previous study in our laboratory showed on several physiological systems related to respiratory and reproductive functions. Recently, that stimulation of α-2A-adrenoceptors suppresses excitatory synaptic we have further shown that prenatal exposure to DE has deleterious pathological effects on the central nervous system, including the hippocampus and cortex. However, little is known transmission in the medial prefrontal cortex (mPFC) of rat. However, the about the effects of prenatal exposure to DEP to the hippocampal functions. In the present underlying mechanism is still unclear. In the present study, we recorded study, effects of prenatal exposure to DEP on spatial learning and memory were investigated evoked excitatory postsynaptic current (eEPSC) in the mPFC of rats, using the Morris water maze test. In addition, expression of NMDA receptor (NMDAR) using whole-cell patch-clamp recording technique. Our results show that subunit NR2A and NR2B, which have a critical role for the spatial learning and memory, stimulation of α-2A-AR inhibits excitatory synaptic transmission in the were measured using immunohistochemistry. Prenatal exposure to DEP induced a longer mPFC through cAMP-PKA-PP1-AMPA-R and cAMP-PKA-PP1-CAMKII- escape latency during trial sessions. Furthermore, immunohistochemical analysis revealed that prenatal exposure to DEP significantly decreased NR2A expression in the hippocampus. AMPA-R signaling pathways. These results suggest that fetal exposure to DEP might exacerbate spatial learning and memory function via reduction of NMDAR activity.
P1PM-9-3 P1PM-9-4 NEUROPROTECTION OF [GLY14]-HUMANIN ON EFFECTS OF AMYLOID β-PEPTIDE ON nAChRs- AMYLOID β PROTEIN-INDUCED IMPAIRMENT OF MEDIATED CURRENTS IN ACUTELY ISOLATED RAT LONG-TERM POTENTIATION AND NMDA RECEPTOR HIPPOCAMPAL CA1 NEURONS MEDIATED CURRENTS Meina Wu, Jinshun Qi Fen Guo, Na Mei Wu, Fang Jun Zhang, Yi Xin Li, Shun Jin Qi Department of Neurobiology, Shanxi Medical University, China Department of Neurobiology, Shanxi Medical University, China Amyloid β protein (Aβ) is responsible for the deficit of learning and memory The novel neuroprotection of Humanin (HN), especially its derivative HNG, in AD and has high affinity with α7 and α4β2 subtypes of nAChRs. The against Alzheimer's disease-related insults such as amyloid β (Aβ) protein present study recorded the nAChRs-mediated whole cell membrane currents has been reported. However, it is still short of electrophysiological evidence and investigated the effect of Aβ fragments on nAChRs in acutely isolated rat for the protection of HN on synaptic plasticity. The mechanisms underlying hippocampal neurons. Our results showed that: 1) application of different Aβ the neuroprotection of HN remain unknown. The present study examined fragments alone did not directly induce any membrane current; 2) Nicotine the effects of HNG on Aβ-induced suppression of long-term potentiation effectively evoked inward currents in a dose-dependent manner; 3) Choline (LTP) in vivo and its possible mechanism. The effects of Aβ and HNG on the whole cell currents mediated by NMDAR, involved in the induction and Epibatidine all evoked whole cell currents with different desensitization of LTP, were also explored in cultured neurons. We found that: Aβ25-35 characteristics; 4) pretreatment with Aβ25-35 or Aβ31-35 reversibly significantly inhibited hippocampal LTP and NMDAR currents; HNG suppressed the Nicotine-induced current in a concentration-dependent pretreatment effectively prevented the Aβ-induced suppression of LTP and manner; 5) the whole cell currents evoked by Choline and Epibatidine were the impairment of NMDAR currents in a dose-dependent manner; Genistein, also suppressed by pretreatment with Aβ31-35. These results indicate that: 1) a tyrosine kinase inhibitor, nearly completely abolished the protective action both α7 and α4β2 nAChRs exist on the membrane of hippocampal neurons; of HNG on LTP and NMDAR currents. Therefore, we conclude that HNG 2) nAChRs including α7 and α4β2 subtypes may be the biological targets of could protect against Aβ-induced LTP deficit and reverse the impairment of Aβ molecules in AD process; 3) sequence 31-35 in Aβ molecule might be a NMDAR current induced by Aβ, and the tyrosine kinase pathway might be shorter active center of full length of Aβ responsible for the suppression of involved in the neuroprotective actions of HNG. nicotinic currents. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 185 P1PM-9-5 P1PM-9-6 α2* NICOTINIC ACETHYLCHOLINE RECEPTORS AND POSSIBLE INVOLVEMENT OF proBDNF-P75NTR LTP INDUCTION IN THE HIPPOCAMPAL CA1 REGION SIGNALS IN LTD-REPETITION OPERATED SYNAPTIC Sakura Nakauchi1, Jorge Riera1, Ryuta Kawashima1, Katumi SUPPRESSION, A FORM OF LONG-LASTING Sumikawa2 SYNAPTIC PLASTICITY 1 2 1 1IDAC, Tohoku University, Japan, 2Department of Neurobiology and Yoshihiro Egashira , Masami Kojima , Keiko Tominaga-Yoshino , Akihiko 1 Behavior, University of California, Irvine, USA Ogura 1 2 GABAergic interneurons have a central role in the control of synaptic plasticity Graduate School of Frontier Biosciences, Osaka University, Japan, Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and hippocampus-dependent learning. Many of these interneurons express and Technology, Japan different subtypes of nAChRs. In addition, nicotine promotes the induction of Synapse formation and elimination in the adult brain are assumed to be the cellular bases LTP at the Schaffer collateral (SC) pathway. However, the identities and locations for memory consolidation. However, the mechanisms underlying such structural plasticity of these subtypes responsible for this effect as well as the underlying mechanisms are not fully understood. We previously reported that the repeated induction (>3 times) largely remain to be determined. The α7 nAChR is the most abundant subtype of mGluR-dependent LTD led to the long-lasting reduction in synaptic strength as well 2+ in the hippocampus and is highly Ca -permeable; thus, has been proposed to as synapse density in CA3-CA1 pathway in cultured hippocampal slices. We call this participate in synaptic plasticity. In this study, we found that nicotine-dependent phenomenon LOSS (LTD-repetition-Operated Synaptic Suppression) to distinguish it facilitation of LTP induction in mice was prevented in the presence of DHβE, but from LTD itself. We found that LOSS depended on new protein synthesis in response to not MLA. Moreover, nicotine facilitated the induction of LTP in α7 knockout (KO) LTD-inducing stimuli, having a limited time window for the synthesis. These findings mice, whereas such facilitation was absent in α2 KO mice. The α2* nAChR, endorse LOSS as a good in vitro model system for the analysis of structural plasticity (as well as RISE, repetitive-LTP-induced synaptic enhancement, an apparently mirror-image the most sparsely expressed nAChR subtype in the brain, shows a distinct phenomenon of LOSS). Since proBDNF-p75NTR (low-affinity neurotrophin receptor) localization in a subset of GABAergic interneurons in the stratum oriens/alveus. signaling is suggested to be involved in apoptosis and synaptic plasticity including LTD, Thus, our results suggest that α2* nAChRs can serve as a molecular switch for we hypothesized its involvement in LOSS. We performed quantitative analyses on the gating the circuit activity via sustained excitation of distinct interneurons during expression levels of mature BDNF, proBDNF, TrkB and p75NTR with special respect to the nicotine exposure, thereby facilitating LTP induction at the SC pathway. times of mGluR activation. The results were largely consistent to the hypothesis.
P1PM-9-7 P1PM-9-8 TWO NOVEL SEROTONIN-MODULATING PROTEINS LIGHT DEPRIVATION IMPROVES MELATONIN INVOLVED IN MEMORY FORMATION IN OPPOSITE RELATED SUPPRESSION OF HIPPOCAMPAL WAY PLASTICITY Arif Aliovsad Mekhtiev Sayyed Alireza Talaei Zavareh, Mahmoud Salami Department of Ecological Physiology & Toxicology, Institute of Physiology, Department of Physiology, Kashan University of Medical Sciences, Iran Azerbaijan In early postnatal life sensory inputs influence development and function of The novel serotonin-modulating SMP-69 protein (Mr 69 kDa and pI 6,0) the brain.long-term potentiation (LTP), is affected by sensory deprivation was purified from the rat brains. Its inactivation by the antibodies leads in neocortex. This study is devoted to assess if dark rearing and melatonin to a significant impairment of memory consolidation in the conditioned influence LTP in the hippocampus. experiments were carried out on 2 groups passive avoidance model in rats, if they had been injected 24 h prior to the of 45 days old male Wistar rats kept in standard light/dark condition (Light learning session. Administration of the anti-SMP-69 antibodies brought to reared-LR) or in complete darkness (Dark reared- DR) since birth to testing. significant increase in the total RNA and protein synthesis in the rat brain cortex on 37% and 30%, correspondently. Electrophoresis fractionation of Each group, in turn, was divided to two, vehicle- and melatonin-treated, the water-soluble proteins of the rat brain cortex revealed approximately two- groups. Stimulating the Schaffer collaterals of CA3 area of hippocampus times upregulation of the 28th protein's fraction content under the influence extracellular postsynaptic potentials (EPSPs) were recorded in the CA1 of anti-SMP-69 antibodies. This protein fraction was purified from the area. then, the hippocampus was perfused by either saline or 2 micrograms rat brains and appeared to consist of two subunits of Mr 60 and 126 kDa. melatonin and EPSPs were recorded and LTP Induced. The light deprivation Intracerebral administrations of the purified protein into the rats 24 h prior to resulted in a significant augmentation in the amplitude of baseline responses. learning sessions in the passive avoidance model leads to memory processes Also, melatonin-induced increase in amplitude of the baseline recordings in impairment ("anticonsolidation protein"). On the basis of these data the either LR or DRs. Tetanic stimulation elicited LTP of EPSPs in both groups, existence of the "switch off" molecular mechanism in which SMP-69 protein robustly in the former. melatonin inhibited the production of LTP in the two induces memory formation, while anticonsolidation protein, in opposite, groups especially in the LR animals. concluded that higher level of neuronal blocks memory consolidationfor memory consolidation, is proposed. activity in the DR rats give rise to a lower level of LTP.
P1PM-9-9 P1PM-9-10 MELATONIN ATTENUATES LONG-TERM DOPAMINE D1 AGONIST FACILITATES INDUCTION POTENTIATION VIA INHIBITION OF NITRIC OXIDE OF LONG TERM DEPRESSION BY CALLOSAL LOW- SIGNALING PATHWAY IN RAT HIPPOCAMPUS FREQUENCY STIMULATION, IN RAT ANTERIOR Yoshiyuki Takahashi1, Takashi Okada2 CINGULATE CORTEX 1Graduate School of Human Sciences, Sophia University, Japan, Sokichi Sakuragi 2Department of Psychology, Faculty of Human Sciences, Sophia University, Department of School Nursing and Health Education, Aichi University of Japan Education, Japan Hippocampal long-term potentiation (LTP) is considered one of the candidate To test whether dopamine have some effects on synaptic plasticity in emotion physiological bases of memory, and some recent studies have tried to identify processing areas, I examined callosally evoked field potentials in coronal slices of rat the endocrine substances involved in the regulation of LTP. It is thought that a anterior cingulate cortex, when low-frequency stimulation (LFS) was applied to corpus pineal hormone, melatonin, may regulate LTP with circadian rhythm, because the callosum as a conditioning stimulus. Neither dopamine nor D2 agonist had significant secretion pattern of melatonin shows a circadian rhythm, and because melatonin effect on the synaptic plasticity, while D1 agonist, SKF-38393, facilitated long term receptors reportedly exist in the hippocampus. In this study, we examined the effects depression (LTD) induction. This facilitating effect of SKF on the LTD induction was of melatonin on LTP and the affected signaling cascade at the CA1 region in rat completely blocked by D1 antagonist, SCH-23390. The LFS induced LTD was not hippocampal slices electrophysiologically. Melatonin (50 μM) attenuated the degree blocked by application of metabotropic glutamatergic receptor antagonist, MCPG, Poster Session of LTP irrespective of the time that hippocampal slices were prepared. Then we or voltage gated calcium channel blocker, nifedipine, but blocked by application of investigated the effects of melatonin on the nitric oxide (NO) signaling pathway, NMDA receptor antagonist, APV. The facilitating effect of SKF on LTD induction which is important for LTP induction. Although melatonin (100 nM) attenuated LTP was not mimicked by forskolin, but partially mimicked by phorbol 12,13-didecanoate, and application of NO synthase (NOS) inhibitor (L-NAME, 100 μM) for blockade of which is known to activate intracellular PKC pathway. These results suggest that LTD NO signaling pathway also attenuated LTP, the attenuating effects of melatonin and in ACC would be induced via NMDAR, and facilitated by D1 agonist at least partially L-NAME were not additive. NO donor, DEA/NO (3 μM), rescued the attenuating via PKC related intracellular pathway. This type of facilitation of LTD induction might effects of melatonin and L-NAME on LTP. These findings suggest that melatonin have some contribution to the pathogenesis of schizophrenia or major depression, in attenuates hippocampal LTP through inhibition of the NO signaling pathway. which frontal lobe hypofunction was indicated.
186 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-9-11 P1PM-9-12 THE INFLUENCE OF MODULATING THE BACK- DOPAMINE MODULATES SPIKE-TIMING DEPENDENT PROPAGATING ACTION-POTENTIAL ON STDP IN SYNAPTIC PLASTICITY IN D1 RECEPTOR-POSITIVE HIPPOCAMPAL CA1 AREA SPINY NEURONS IN THE NEOSTRIATUM OF ADULT Masashi Kondo1, Yasuhiro Hukushima2, Minoru Tsukada2, Takeshi MICE Aihara1 Mayumi Shindou, Tomomi Shindou, Jeffery R Wickens 1Faculty of Engineering, Tamagawa University, Japan, 2Brain Science Neurobiology Research Unit, Okinawa Institute of Science Technology, institute, Tamagawa University, Tokyo, Japan Japan It is found that the profiles of spike-timing dependent synaptic plasticity Recent evidence suggests that spike-timing dependent plasticity (STDP) (STDP) were classified into two types, depending upon layer specific location exists in the corticostriatal pathway. In addition, dopamine (DA) has been along the dendrite in the CA1 area of a hippocampal slice. However, the suggested to play a crucial role in corticostriatal synaptic plasticity. Here we studied STDP of corticostriatal synapses in brain slices of adult mice interaction between information processing on different dendritic locations (2-5 month-old). Whole cell recordings were obtained from spiny projection is not clear. In this study, to investigate how the temporal information of neurons and synaptic responses were measured in response to stimulation of inputs to the proximal dendrite have an influence on information processing, the subcortical white matter. We found that long-term depression (LTD) was STDP, at the distal dendrite, stimulation was applied at the proximal dendrite induced by three action potentials occurring 10 msec after the EPSP (pre-post during STDP protocol at the distal dendrite. As the results, the information protocols), but post-pre protocols caused little change. We also examined processing at the distal dendrite was influenced depending on the relative effects of DA on STDP. The LTD evoked by pre-post protocols was reversed timing of BPAP and the input to the proximal dendrite. It is considered that by a D1 receptor agonist but not a D2 agonist. Modulation by the D1 agonist there is a causality delivered by BPAP as a carrier of temporal information only occurred if it was applied during the induction protocol. Furthermore, on a dendrite. This may be due to an effect on the coding process at the distal this LTD was observed in the D1-expressing spiny neurons but not the dendrite and could support the basis for a novel learning rule in the brain. D2-expressing spiny neurons. We suggest that the timing of DA receptor activation is important for modulation of corticostriatal STDP, especially in the D1-expressing spiny neurons.
P1PM-9-13 P1PM-9-14 THE NEURON SPECIFIC NF1 EXON 9a IS ESSENTIAL ROLE OF ENDOGENOUS CANNABINOID BREAKDOWN FOR THETA-FREQUENCY LTP AND LEARNING INHIBITOR ON HIPPOCAMPAL LTP IN RAT 1 2 Mohammad reza Hojjati , Geeske M. Van Woerden , Alcino J. Alireza Komaki1, Siamak Shahidi1, Fatemeh Kazemi1, Abdolrahman 3 2 Silva , Ype Elgersma Sarihi1, S. Mansour Malakouti1, Parisa Hasanein2, Reza Lashgari3, Abbas 3 1Department of Physiology, Shahrekord University of Medical Sciences, Haghparast 2 Iran, Department of Neuroscience, Erasmus University Medical 1Department of Physiology, Hamedan University of Medical Sciences, Iran, Center, The Netherlands, 3Department of Neurobiology, Psychiatry and 2Department of Biology, School of Basic Sceinces, Bu-ali Sina University, Psychology, University of Carolina, USA Hamadan, Iran, 3Neuroscience Research Center, Shaheed Beheshti University Neurofibromatosis type 1 (NF1) is one of the most common inherited single- of Medical Sciences, Tehran, Iran gene disorders affecting cognitive function. The NF1 gene encodes a large There is currently debate over the interaction between L-type voltage dependent Ca2+ amino acid protein called neurofibromin, which functions as a Ras GTP- channels and endocannabinoid system on the synaptic plasticity. In this study we examined ase activating protein (GAP). Although NF1 is expressed in all cells of the the effects of acute administration of URB597, as endocannabinoid breakdown inhibitor, brain, expression of NF1 containing the small alternatively spliced exon 9a following chronic administration of Verapamil, as Ca2+ channels blocker, on the evoked is restricted to neurons, and this isoform is particularly high expressed in potentials and LTP induction in the dentate gyrus of hippocampus. the forebrain. This exon is not part of the GAP domain and its function is Rats (Wistar) were treated i.p. with doses 10, 25, 50 mg/kg Verapamil for 13 days (once daily). Then following surgery, recording and stimulating electrodes were positioned in the unknown. To study the role of the NF1-Ex9a isoform in cognitive function, granular cells of dentate gyrus and perforant pathway respectively. After ensuring a steady we created a mouse lacking the NF1-Ex9a isoform. Here we show that mice state baseline response, single i.p injection of saline or URB597 (0.1, 0.3 mg/kg) was done. lacking NF1 exon 9a demonstrate spatial learning deficits in the Morris water The results show that there is difference between slopes of EPSP and population spikes in maze task and impaired LTP induced at theta frequency. Our finding suggest the Verapamil and URB597 groups after HFS. Decrease in LTP induction during chronic that exon 9a is essential for the function of NF1 in spatial learning and administration of Verapamil + acute URB597, is greater than other groups. hippocampal plasticity, and that the role of NF1 in neuronal function is not In conclusion, these results indicate that acute administration of endocannabinoid breakdown restricted to its function as a GAP protein. inhibitor, URB597, disrupt LTP induction in the dentate gyrus of hippocampus in rat. Also, there is an interaction between Ca2+ channels blocker and endocannabinoid system.
P1PM-9-15 P1PM-9-16 INVESTIGATION OF MODULATORY EFFECTS ON LTP LONG-TERM IN VITRO MORPHINE DISAPPEARS USING MULTIELECTRODE ARRAY (MEA) SYSTEM ENHANCEMENT OF PAIRED PULSE FACILLITATION Ji-Ho PARK1, Jung-A Shin1, Se-Ra Yang1, Min-Sook Son1, Gun- (PPF) IN WITHDRAWN SLICES Hoon Choi1, Do-Hyoung Kim2 Narges Hosseinmardi1, Yaghoub Fathollahi1, Naser Naghdi2, 1 1Department of Medical Science, Kyung Hee University, Korea, Mohammad Javan 2 Department of Biomedical Engineering, Hanyang University, Korea 1Department of Physiology, Tarbiat Modares University, Iran, 2Department In this study, a reliable and acceptable electrophysiological system was of Physiology, Pasteur Institute, Iran established for screening LTP modulatory potential materials in rat Addictive drugs are thought to alter normal brain function and cause the hippocampal slice using MEA systems. An organotypic slice culture was also remodeling of synaptic functions in areas important to memory and reward. adapted to meet better accuracy rather than acute slice. As the results, there Given the known effects of opiate on hippocampal function, we investigated were three different modulatory effects on LTP. Firstly, additional treatment the potential effect of chronic morphine treatment on baseline synaptic of Cassia tora extract increased fEPSP amplitude compare to the baseline of response and paired pulse index (PPI) at CA1 synapses of rat hippocampal tetanic bust induced LTP. It may relate to an improvement of memory as other slices. There was no significant difference in the baseline synaptic response researchers reported. Secondly, a slow declining post tetanic and inhibitory- of slices taken from control or dependent rats. Also morphine and naloxone like potential was appeared with treatment of ESP-102 (a novel extracts). had no effect on baseline responses. The mean PPI was <1 for the 10 and This data was similar to NMDA receptor antagonists treatment. Finally, 20 ms inter pulse intervals (IPIs) and >1 for 80 and 200 ms IPIs in both there was fast post-tetanic and inhibitory potential with a compound (a novel groups. A significant increase in PPI was observed in the dependent slices at extracts) treatment which may be related with elevation of the concentration. 80 ms IPI. This enhancement was disappeared in the present of morphine. Furthermore, it was investigated that NGF-induced the spatiotemporal LTP- In vitro morphine application caused an increase of PPI in 20 ms IPI in Like neuronal activity changes. Taken together, this study tried to establish dependent slices. These results suggest that chronic morphine treatment an accurate monitoring system for screening modulatory compounds on LTP alters mechanisms involved in PPF and paired pulse depression (PPD) and using a spatiotemporal analytic system. We hope it can help to investigate this alteration maybe a resetting in CA1 network due to chronic morphine more profound spatiotemporal changes on the synaptic plasiticity. treatment. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 187 P1PM-10-1 P1PM-10-2 BRAIN REGIONS RESPONSIBLE FOR THE CROSS- GENETIC TRACING AND CHARACTERIZATION OF MODAL REORGANIZATION OF CORTICAL CIRCUIT THE NEURONS IN THE NUCLEUS OF THE SOLITARY Taisuke Kawasaki, Hiroki Matsuki, Susumu Jitsuki, Takuya TRACT, RECEIVING SPECIFIC TASTE INPUT Takahashi Makoto Sugita1, Yoshiki Shiba2 Department of physiology, Yokohama City University Graduate School of 1PRESTO, JST, and Department of Oral Physiology, Hiroshima University, medicine, Japan Japan, 2Department of Oral Physiology, Hiroshima University, Japan Although sensory deprivation in one modality can lead to the improvement We recently applied a genetic approach to delineate the neuronal circuitries of remaining modalities, neuronal mechanisms underlying cross-modal of bitter and sweet/umami taste by selectively expressing the transsynaptic regulation of primary sensory cortex are poorly understood. Recently, tracer, tWGA-DsRed, in mT2R5- and mT1R3-expressing taste receptor cells, we found that AMPA receptors were delivered into layer4-2/3 synapses respectively. Locations of the neurons labeled by tWGA-DsRed originating in the barrel cortex of adolescent rats (P21-P23) with visual deprivation. from the specific taste receptor cells revealed segregation of bitter- and Furthermore, this is mediated by the increased secretion of serotonin in sweet-inputs in the gustatory area in the brain. Spatial distribution of the the barrel field (Jitsuki et al, preliminary data). However, it remains to be cell somata of tWGA-DsRed-labeled neurons suggested that the gustatory elucidated which brain regions control cross-modal regulation of barrel neurons dispersed in the solitary tract nuclei may be organized with sweet/ cortex specific serotonin secretion in animals with visual deprivation. Here umami inputs rostral and with bitter inputs caudal. In the present study, the we report brain areas responsible for the cross modal regulation of cortical neuronal types and the microstructure of tWGA-DsRed-labeled neurons in circuit. We monitored the expression of c-Fos in the brain of rats with visual the solitary tract nuclei were analyzed by immunohistochemistry and patch- deprivation. clamp recording, and by loading the fluorescent dye, lucifer yellow, in the labeled neurons. Large subsets of the neurons in the solitary tract nuclei, labeled by tWGA-DsRed originating from mT2R5-expressing taste receptor cells, receive glutamatergic synaptic transmission via the non-NMDA glutamate receptor, and are tyrosine hydroxylase-immunoreactive neurons.
P1PM-10-3 P1PM-10-4 THE WAY V1 FEEDBACK INFLUENCES AXON PROJECTION MAPS OF FOX-ODOR- SPATIOTEMPORAL RESPONSES IN LGN. A TMS RESPONSIVE MITRAL/TUFTED CELLS IN THE MOUSE STUDY OLFACTORY CORTEX Kei Igarashi1, Yukie Yamaguchi1, Myungho An1, Nao Ieki1, Shin Javier Cudeiro, Nelson Espinosa, Jorge Marino, Carmen de 2 3 4 4 Labra, Casto Rivadulla Nagayama , Nian Liu , Ko Kobayakawa , Reiko Kobayakawa , Yoshihiro Yoshihara5, Manabu Tanifuji5, Hitoshi Sakano4, Wei R Chen2, Kensaku Medicine-NEUROcom, University of A Coruna, Spain 1 Mori We have shown that sustained responses to flashed spots on X and Y lateral geniculate 1Department of Physiology, Graduate School of Medicine, University of Tokyo, nucleus (LGN) cells were reduced after application of Transcranial Magnetic Japan, 2Dept Neurobio&Anatomy, U Texas Med Sch, USA, 3Dept Neurobio, Yale Stimulation (TMS) at 1Hz on primary visual cortex (V1). Transient responses were Univ Med Sch, USA 4Dept Biochem&Biophys, Grad Sch Sci, Univ Tokyo, Japan, largely unaffected. Here we show, using a different experimental approach, that TMS 5RIKEN BSI, Japan at 1Hz induces subtle effects on transient responses depending on cell type. The glomerular sheet of the olfactory bulb (OB) forms odorant receptor maps. We have Experiments were performed in anesthetized and paralyzed adult cats.Visual previously shown that glomeruli in the dorsal (D) zone of the OB are required for innate stimulation (flashed bright and dark bars) was used to build the spatiotemporal aversive response to predator odors. Here we report the axonal projection pattern to the receptive fields of LGN neurons extracellularly recorded, both under control conditions olfactory cortex (OC) of individual mitral/tufted (M/T) cells associated with D-zone and during the simultaneous application of TMS on V1. glomeruli and responding to TMT (a compent of fox’s predator odor). TMT-responsive M/T X cells (n = 18) did show an increase in the latency to maximal response (mean = 1 cells were juxtacellularly labeled and then labeled axons were histochemically visualized and msec, p < 0.05) and a decrease of firing rate (mean = 10 %, p < 0.005). Analysis of 3D-reconstructed. The results showed that individual M/T cells extended axon collaterals to the receptive field shapes suggests that these changes were due to an increase in the multiple areas of the OC, including the anterior olfactory nucleus (AON), piriform cortex (PC), olfactory tubercle, tenia tecta, amygdaloid cortex, and lateral entorhinal cortex. Axon rate between peripheral and central amplitudes. Y cells (n = 25) showed no changes collaterals formed several high-density clusters at the stereotypical positions in the AON and regarding the spatiotemporal structure. Subsequent intervalogram analysis confirmed anterior PC, while they were distributed relatively widely in the posterior PC. These results the results. provide initial steps toward understanding how the fox-odor TMT signals are sent to specific These results indicate the existence of different cortical effects on X and Y cells regions of the OC to induce innate aversive behavioral responses. Distinct distribution transient response. patterns of axons for different cell types and for cells located in different domains in D-zone Supported by MEC and Conselleria de Educacion (Galicia) will be discussed.
P1PM-10-5 P1PM-10-6 PROCESSING OF FOOD ODOR INFORMATION IN THE ROLES OF PHASE-LOCKED INHIBITION FOR RAT OLFACTORY CORTEX PROCESSING INTERAURAL TIME DIFFERENCE CUE Ikue Yoshida, Kensaku Mori IN THE NUCLEUS LAMINARIS OF BIRDS Department of Physiology, the University of Tokyo, Japan Rei Yamada, Hiroko Okuda, Eri Nishino, Hiroshi Kuba, Takahiro Ishii, Harunori Ohmori Exploration, detection and evaluation of foods are behaviors that all necessary for appeasing animal’s appetite, and these behaviors are controlled Department of Physiology, Faculty of Medicine, Kyoto University, Japan by olfactory cues. However, it is not known about how olfactory signals Interaural time difference (ITD) is a cue for sound source localization and trigger or modulate these eating related behaviors. We previously reported first processed in nucleus laminaris (NL) in birds. NL neurons act as a that neurons in the dorsal part of the anterior piriform cortex (APC) of rats coincidence detector of bilateral phase-locked excitatory inputs and encode were tuned to specific odorant category profiles of fruits or vegetables. ITD. Relatively large temporal jitter is observed in the firing of low frequency Although the result suggests that APC neurons integrate signals from distinct auditory nerves, and the jitter should broaden the ITD tuning. In mammals that use low frequency sound for ITD coding, a phase-locked inhibition is odorant categories to detect food odors, it is not well understood how food proposed to increase the sharpness of ITD tuning. In contrast in birds, NL odor-responsive APC neurons integrate signals from multiple component receives sound level dependent inhibition from superior olivary nucleus odorants. Using single-unit recording methods, we here examined the odorant (SON) but this inhibition is poorly phase-locked. A presence of GABAergic
Poster Session selectivity of rat APC neurons that responded to fruits odor. Each fruits odor interneurons around NL is reported, but their roles are not known. In this responsive neurons showed excitatory responses to its component odorants. study, we found polysynaptic IPSC that followed EPSC with 1~2 ms latency 71% of 21 watermelon responsive neurons responded to multiple component in the low characteristic frequency (CF) NL neurons. IPSC was elicited even odorants. In addition to fruits odor responsive neurons, we examined odorant when SON was dissected out. This suggests that the low CF NL neurons also selectivity of rat food pellet responsive neurons. Some of these neurons receive phase-locked inhibition from some interneurons located near NL. responded to multiple components. Present result suggests that integration of Consistently, GABA positive neurons were found concentrated in the low CF diverse component odorants in the APC is a common way of processing food NL region and, their projection to NL was confirmed by photo-activation of odors. caged glutamate. We will examine the roles of phase-locked inhibition in NL.
188 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-10-7 P1PM-10-8 GAMMA BAND RESPONSES TO AM SOUNDS AT THE DIFFERENTIAL EFFECTS OF THE ACTIVITY OF AI TO AUDITORY CORTEX OF CONSCIOUS RATS THE BELT FIELDS IN THE GUINEA PIG AUDITORY Wan-Chun Liao, Paul W Poon CORTEX OBSERVED BY OPTICAL IMAGING Department of Physiology, National Cheng Kung University, Taiwan Ryota Numata, Shunji Sugimoto, Junsei Horikawa Evoked gamma band activity (viz., 30-200 Hz) is generally considered to Department of Knowledge-Based Information Engineering, Toyohashi reflect local fast synaptic and spike potentials related to cortical processing University of Technology, Japan of stimuli. Sound-evoked gamma activity is known to vary depending on Functional connections between the dorsal (AId) and middle (AIm) parts stimulus parameters, brain structures and animal species. It remains poorly of AI and the belt fields of the guinea pig auditory cortex were investigated understood whether the pattern of gamma response could be related to the using optical imaging and local cortical inhibition by muscimol (GABAA time-varying features of sounds, which are important building blocks of agonist) and activation by bicuculline (GABAA antagonist). The auditory speech signals. Here we studied gamma responses at the cerebral cortex cortex of a guinea pig under anesthesia was exposed and stained with a of conscious rats to sound stimuli with different time-varying or more voltage-sensitive dye. The flouresonent signals were recorded using a 100× specifically amplitude modulated (AM) feature. Chronic epi-dural electrodes 100 CMOS camera. Pure tones (2-16 kHz, 200-ms long, 5-ms cosine ramps) were first implanted surgically at the auditory fields on the rat cortex. After were used for stimuli. After inhibition of AId by muscimol, the activities of recovery from surgery, sound-evoked electro-corticograms to AM stimuli the dorsocaudal field (DC), the dorsocaudal belt (DCB) and the posterior were recorded. We presented at random intervals AM bursts amplitude belt (P) were decreased, and they were increased after activation of AId by modulated systematically from 20 to 200 Hz. Gamma responses were bicuculline. The activities of DCB and P were increased after inhibition of computed in the form of event-related-spectral-perturbation and displayed AIm and were decreased after activation of AIm. These results suggest that using EEGLAB and MATLAB programs. Results showed an apparent AId activity has excitatory effects to DC, DCB and P and AIm activity has demodulation of the AM time-profiles in the frequency range from 30 to160 inhibitory effects to DCB and P. The activities of AIm and AIv remained after Hz, which may or may not correlate with phase-locked evoked responses in inhibition of AId. The activities of AId and AIv remained after inhibition the averaged time waveform. of AIm. These results suggest that AI has the three regions receiving independent inputs from the thalamus.
P1PM-10-9 P1PM-10-10 EFFERENT CONNECTIONS OF AN AUDITORY AREA TEMPORAL INTERACTIONS BETWEEN AUDITORY IN THE INSULAR CORTEX: ANATOMICAL NODES AND SOMATOSENSORY INPUTS IN AUDITORY FOR AUDITORY, SOMATOSENSORY AND VISCERAL THALAMIC NUCLEI OF ANESTHETIZED AND AWAKE PROCESSING RATS Akihisa Kimura, Hiroki Imbe, Tomohiro Donishi Tomohiro Donishi, Akihisa Kimura, Hiroki Imbe Department of Physiology, Wakayama Medical University, Japan Department of Physiology, Wakayama Medical University, Japan We electrophysiologically delineated an auditory area in the caudal part of We determined how somatosensory inputs affect auditory thalamic cell activity the granular insular cortex of the rat and determined its efferent connections in anesthetized and awake rats. Under equithesin anesthesia, electrical stimuli or based on axonal labeling with biocytin. The auditory area projected to mechanical pinches on the hind paw increased and/or decreased unit discharges the rostral agranular insular cortex (r-AI), which plays a pivotal role in elicited by noise bursts in the medial geniculate body (MG), most notably those nociceptive processing. It also projected to the caudal end of the agranular recorded in the ventral division (MGV) of the MG. The effects were likely related insular cortex and sent feedback projections to cortical layer I of the primary with the time intervals between preceding electrical stimuli and noise bursts. In and secondary somatosensory areas. Corticofugal projections terminated most cases decrease was observed at short intervals (<20 ms), suggesting that in the medial division of the medial geniculate body (MG), somatosensory electrical stimuli suppressed acoustic activation in the ascending pathways to and visceral thalamic nuclei, and the rostral end of the auditory sector in the the MG. Increase took place at relatively long time intervals. In freely behaving thalamic reticular nucleus that overlaps with the somatosensory and visceral rats simply exposed to environmental noise, electrical foot shocks attenuated sectors. The ventral part of the caudate putamen and the external cortex of c-Fos expression in the MGV. Attenuation was also observed in the MGV and the inferior colliculus were also the main targets. The projection to the r-AI the medial division (MGM) of the MG when foot shocks were given before (the lateral part of the prefrontal cortex) is thought to constitute the main pure tone stimuli. Interestingly, the overall suppressive effect of foot shocks was stream of cortical auditory processing that originates from ventral auditory negated in the MGV when pure tone stimuli were given before foot shocks like area, the secondary auditory area receiving non-lemniscal thalamic inputs in the temporal sequence used for fear conditioning. These results suggest that from the dorsal division of the MG. The auditory area is considered pivotal the MGV, the primary auditory channel of the thalamus, could be also involved for cortical and thalamic integration of auditory, somatic and visceral inputs. in cross-modal sensory processing.
P1PM-10-11 P1PM-10-12 FUNCTIONAL DEVELOPMENT OF HINDBRAIN FUNCTIONAL CONNECTION FROM MAUTHNER CELL ESCAPE NETWORKS IN ZEBRAFISH TO SEGMENTALLY HOMOLOGOUS RETICULOSPINAL NEURONS IN THE GOLDFISH HINDBRAIN Tsunehiko Kohashi, Natsuyo Nakata, Yoichi Oda 1 2 3 1 Graduate School of Science, Nagoya University, Japan Daisuke Neki , Hisako Nakayama , Takashi Fujii , Daiyu Kodama , Yoichi Oda1 A paired large reticulospinal neurons (RSNs) in matured zebrafish hindbrain, 1Department of Biological Science, Nagoya University, Japan, 2Graduate the Mauthner (M) cells, initiate fast escape away from sudden noxious school of Medicine, Osaka University, Japan, 3Graduate school of Frontier stimulus. Here, we examined the contributions of sensory inputs and the Bioscience, Osaka University, Japan M-cell firing to escape initiation in early (<70 hours postfertilization; hpf) Teleost hindbrain contains segmentally arranged reticulospinal neurons (RSNs). Paired and late (>120 hpf) zebrafish larvae. Confocal calcium imaging of RSNs large RSNs in the fourth segment (r4), Mauthner (M) cells, are known to initiate during escape in the late larva, with its rostral body embedded in agar, escape behavior. RSNs in r5 and r6 are also thought to be involved in control of the revealed that short-latency (<6 ms) escape in response to water pulse applied movement. Here, we investigated the physiological connection between M-cell and to otic vesicle (OV; immature inner ear) was always associated with single the RSNs by paired recordings in adult goldfish. The intraaxonal activation of M-cell produced inhibitory postsynaptic potentials (IPSPs) in dorsal RSNs (MiD2cm, MiD2i, spiking of the M-cell (M-escape). Head-tactile stimulus elicited delayed MiD3cm, MiD3i) on the ipsilateral side, and the contralateral MiD2cm. The IPSPs (>6 ms) escape without M-cell firing (non-M-escape) but with bursting of were strong enough to stop bursting of the postsynaptic RSNs. By contrast, in the MiD3cm, a morphological homolog of the M-cell. Elimination of OV or dorsal RSNs on the contralateral side, except MiD2cm, the M-cell firing elicited long- laser ablation of the M-cell abolished the short-latency escape in the late lasting depolarization, which enhanced postsynaptic spiking. The M-cell firing elicited larva. By contrast in the early larva, neither of the above lesioning affected strong depolarization followed by spikes in ventral RSNs (MiV2, MiV3) bilaterally. escape onset, whereas ablation of trigeminal ganglion eliminated the escape. A significant respons was never induced in the M-cell by firing of these RSNs. The connection patterns from M-cell to the RSNs seem repeated in r5 and r6, except Thus, sound-induced M-cell pathway may be added to touch-induced non- MiD2cm. The bilaterally asymmetric and symmetric projections from M-cell to RSNs M-pathway during early development of zebrafish to escape quickly from a may play a key role to control the initial turn and following swimming in the M-cell hostile sound source before it reaches the fish. initiated escape. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 189 P1PM-10-13 P1PM-10-14 OPTOKINETIC RESPONSES OF C57BL/6 MICE: I. OPTOKINETIC RESPONSES OF C57BL/6 MICE: II. RESPONSE PROPERTIES OF THE INITIAL OPEN- DEPENDENCE ON SPATIAL PROPERTIES OF VISUAL LOOP PERIOD STIMULUS 1 1 2 Hiromitsu Tabata , Naoki Shimizu , Yoshiro Wada , Kenichiro Naoki Shimizu1, Yuko Sugita1, Hiromitsu Tabata1, Yoshiro Wada2, 1 1 Miura , Kenji Kawano Toshiaki Yamanaka3, Hiroshi Hosoi3, Kenji Kawano1 1 2 Integrative Brain Science, Kyoto University, Japan, Department of 1Integrative Brain Science, Kyoto University, Japan, 2Physiology, Nara Physiology, Nara Medical University, Japan Medical University, Japan, 3Otorhinolaryngology, Nara Medical University, We studied the initial part of the optokinetic response (OKR) in C57BL/6 mice Japan with systematically manipulating the visual stimulus. The visual stimulus was In the retina of the mouse, ganglion cells are unequally distributed and the highest a drifting vertical sinusoidal grating which was presented on three computer density region is located just temporal to the optic disk. To know the role of this monitors set around the animal. The eye movements were recorded using an high density region in visually guided behavior, we studied the dependence of infra-red video-camera system. We found that the magnitude of the initial the optokinetic response (OKR) on spatial properties of the visual stimulus. The OKR monotonically increased as the stimulus contrast increased. The initial eye movements were recorded in male C57BL/6 mice using an infra-red video- OKR showed a band-pass property for spatio-temporal frequency giving oculography. We measured the initial part of the OKR evoked by horizontal motion of largest response at 0.125 cyc/deg and 1.5 Hz. The range of the spatio-temporal vertical black and white gratings (spatio-temporal frequency at 0.125 cyc/deg and 1.5 frequency to cause significant steady-state OKR was fairly limited around the Hz). The gratings were displayed on three computer monitors set around the animal peak. When the temporal frequency of the stimulus was higher than 1.5 Hz, the and could occupy the full screen (76.5 deg high) or a horizontal stripe (5 deg high) OKR was transient, i.e. no or weak sustained responses were observed. These extending the full width of the displays. The stripe was located at one of 8 vertical results suggest that the initial part of the OKR of mice has two components: first, positions (from -10 deg to 35 deg), where the 0 deg position corresponds to bregma- a transient component that diminishes in about 200 ms, and a tonic component lambda axis being horizontal. We found that a single 5 deg stripe elicited OKR in sustains more than 400 ms. The tonic component might have a role in stabilizing most cases and the maximal initial OKR was elicited when the stimulus was at around the moving visual images on the retina, and the transient component a role in 25 deg. The result was consistent with the distribution of the retinal ganglion cells, detecting the onset and/or the acceleration of the visual motion. suggesting an important role of the high density region in initiating the OKR.
P1PM-11-1 P1PM-11-2 MECHANISMS UNDERLYING SURROUND NEURAL MECHANISMS OF ORIENTATION SUPPRESSION IN THE CAT LATERAL GENICULATE SELECTIVITY IN CAT LATERAL GENICULATE NUCLEUS NUCLEUS 1 1 1 Satoshi Shimegi1, Masahiro Okamoto2, Akihiko Kimura1, Tomoyuki Tomoyuki Naito , Hironobu Osaki , Osamu Sadakane , Masahiro 2 1 1 Naito1, Hironobu Osaki1, Shin-ichiro Hara2, Ayako Ishikawa1, Okamoto , Satoshi Shimegi , Hiromichi Sato Hiromichi Sato1 1Graduate School of Medicine, Osaka University, Japan, 2Fukushima 1Graduate School of Medicine, Osaka University, Japan, 2Graduate School Medical University, Japan of Frontier Biosciences, Osaka University, Japan We investigated effects of stimulus-size, spatial frequency, and luminance- In neurons of the lateral geniculate nucleus (LGN), response to a stimulation of the contrast on the orientation selectivity of the lateral geniculate nucleus (LGN) classical center-surround receptive field (CRF) is suppressed by a stimulation of the neurons in the anesthetized cats. We found that more than 90% of LGN extraclassical receptive field (ECRF) (surround suppression, SS). In order to study neurons exhibited significant orientation selectivity in responses to large-size how GABAergic inhibition is responsible for SS, we performed exracellular single (larger than the receptive field) grating stimulus with high spatial frequency. unit recording from LGN neurons of anesthetized and paralyzed cats, and examined Then, the orientation selectivity indices (OSIs) and width of tunings were effects of a blockade of GABAergic inhibition with an administration of bicuculline measured under high (50-90%) and low contrast (7-30%) conditions. There (BIC) on stimulus-size tuning property of response to drifting sinusoidal gratings. BIC were no significant differences in the medians of OSIs and tuning widths facilitated both spontaneous firing and visual response. When stimulus was enlarged between high and low contrast conditions. These results demonstrated that beyond the optimal size for CRF response, most neurons showed a reduction of most of LGN neurons exhibit the contrast-invariant orientation selectivity response (SS) regardless of a presence or an absence of BIC. However, the optimal as well as neurons in the primary visual cortex. Furthermore, we measured stimulus size became larger under BIC administration than that under the control orientation tuning selectivity of LGN neurons under an iontophoretic condition. These results suggest that there are two types of SS; 1) GABA-mediated SS application of GABAa receptor antagonist (bicuculline). We found that the arose from near the CRF, and 2) non-GABA-mediated SS from distal area in ECRF. bicuculline had little or no effect on the sharpness of the orientation tuning Presumably, a main body of SS in LGN is inherited from retina, which is enhanced by and its contrast-invariance. We concluded that the orientation selectivity of intrathalamic inhibition. visual information processing starts from retinal ganglion cells in cat.
P1PM-11-3 P1PM-11-4 THE ROLE OF SURROUND SUPPRESSION AT HIGH SPATIAL ARRANGEMENT OF ORIENTATION SPATIAL FREQUENCY IN THE CAT V1: A POSSIBLE SELECTIVITY IN LAYER 2/3 OF MACAQUE V1 BASIS FOR SCALE-INVARIANT PROCESSING REVEALED WITH IN VIVO 2-PHOTON CALCIUM Hironobu Osaki1, Tomoyuki Naito1, Osamu Sadakane2, Masahiro IMAGING Okamoto3, Hiromichi Sato1 Koji Ikezoe1, Yoshiya Mori1, Kazuo Kitamura2, Hiroshi Tamura1, 1 1Graduate school of Medicine, Osaka University, Japan, 2National Institute Ichiro Fujita 3 for Basic Biology, Graduate School of Frontier Biosciences, Osaka 1Graduate School of Frontier Biosciences, Osaka University, Japan, University, Japan 2Graduate School of Medicine, University of Tokyo, Japan The scale-invariant visual perception refers to the phenomenon that the perceived In the monkey primary visual cortex (V1), neurons are arranged according to their feature of an object remains constant, despite a change in the retinal image size preferred orientation. Because the arrangement has been studied with recording according to the distance from the object. This property is perceptually beneficial techniques with limited spatial resolution, the organization at single-cell resolution for stabilizing the visual scene and recognition of moving objects. The retinal is still unclear. In this study, we investigated the spatial arrangement of layer 2/3 image of an object becomes larger as the distance decreases, and also, its spatial neurons based on their preferred orientation with in vivo 2-photon calcium imaging frequency (SF) becomes lower. To examine the possibility that SF selectivity of techniques. We recorded calcium signals from neurons in response to drifting square- V1 neurons changes in scale-invariant manner, we measured SF tuning curves at wave gratings in opiate-anesthetized and paralyzed monkeys. About half of the
Poster Session different stimulus size conditions in V1 neurons (N=77) in anesthetized cats, and neurons showed orientation-selective modulation in calcium signal. The tuning width found that the preferred SF of V1 neurons shifted to lower side as an increment of the orientation tuning curve based on calcium measurements was similar to that of of stimulus size. We also tested area-summation tuning curves at various stimulus previously reported spike responses. The median preferred orientation difference in SFs and found high SF stimulus strengthened surround suppression (N=55), neuron pairs separated by less than 50 μm tangential to the cortical surface was 11.3 which is the suppressive response modulation induced by a stimulation of outside degrees (1219 pairs). This value monotonically increased with separation. In neuron the receptive field. We conclude that surround suppression tuned to high SF pairs separated by 150 to 200 μm, the median difference was 36.7 degree (734 pairs). causes the shift of the preferred SF shift to lower for large stimuli, which may Thus, in macaque V1, we found that neurons are orderly arranged based on preferred contributes to the scale-invariant image processing. orientation at single-cell resolution. This research was supported by CREST, JST.
190 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-11-5 P1PM-11-6 IMAGING OF HIGHER VISUAL CORTICES BASED ON MICROSTIMULATION OF MONKEY INFERIOR DIFFERENCES IN RESPONSE PROPERTIES BETWEEN TEMPORAL CORTEX INDUCES CHANGE IN THE PRIMARY AND HIGHER VISUAL CORTICES IN PERCEPTUAL COLOR JUDGEMENT MICE Kowa Koida, Hidehiko Komatsu 1 2 1 Manavu Tohmi , Reiko Meguro , Kentaro Nagami , Ryuichi National Institute for Physiologica Sciences, Japan 1 2 3 1 Hishida , Masao Norita , Takeshi Yagi , Katsuei Shibuki We have previously identified a patchy accumulation of color selective 1Dept Neurophysiol, Brain Res Inst, Niigata Univ, Japan, 2Dept Neurobiol 3 neurons in the anterior inferior temporal cortex (area TE) of the monkey, Anat, Sch Med, Niigata Univ, Niigata, Japan, Osaka Univ, Osaka, Japan and have shown that the activities of cells in the patch correlated with the V1 is surrounded by multiple higher visual areas with distinct retinotopic maps. monkey's color judgment. To examine the causal relationship between Activities in higher visual cortices were visualized using transcranial flavoprotein neuronal activities and color judgment, we tested the effects of electrical fluorescence imaging in mice. When the speed of drifting grating patterns was changed microstimulation on color judgments. Monkeys were trained to perform a from 10 to 50 deg/s in a step, flavoprotein fluorescence signals in area LM, AL and fine color judgment task, where they had to judge whether the sample color RL were increased while those in V1 were decreased. A step-like change from 50 to was more similar to either one of the two target colors. A sample color set 10 deg/s produced an increase of the signals in V1 and a decrease in higher areas. consisted of 7 colors that slightly differed in chromaticity. Microstimulation We thought that the higher visual areas might be specialized for detecting moving (20 μA, biphasic, 200 Hz) was applied through the microelectrode inserted objects. To test this hypothesis, mice were reared under flash light, since motion of in or around the patch during a sample color presentation. In some sites, objects could not be detected under this condition. The preferred speeds of the higher visual cortices were agreed with those of V1, while those of V1 were unchanged. microstimulation induced a large shift in the monkey's color judgment These results suggest the presence of experience-dependent plasticity in the higher indicating that neural activities are causally related to color judgment. The visual cortices. We found that V1 received inputs from LGN while higher areas from sites where large effects were observed coincided with the distribution of the nuclei surrounding LGN. In mice with impaired protocadherin-α, a neurospecific color selective cells. Direction of the shift had clear tendency on chromaticity cell adhesion molecule with multiple variant forms, higher visual areas were not diagram and did not depend on the color preference of cells in stimulating clearly separated from V1, suggesting that protocadherin-α has an important role in site. These results provide important hints on the way color is represented in development of higher visual cortices. area TE.
P1PM-11-7 P1PM-11-8 CHIOCE-PREDICTIVE ACTIVITY IN MACAQUE AREA V4 SPATIALLY SELECTIVE INTEGRATION OF BINOCULAR DURING FINE DISCRIMINATION OF STEREOSCOPIC DISPARITIES IN FINE DEPTH PERCEPTION DEPTH Takahiro Doi, Takahiro Okada, Ichiro Fujita Hiroshi Shiozaki, Seiji Tanabe, Takahiro Doi, Ichiro Fujita Graduate School of Frontier Biosciences, Osaka University, Japan Graduate School of Frontier Biosciences, Osaka University, Japan The acuity for perceiving stereoscopic depth is greatly improved when Binocular disparity is represented in a number of areas in the primate visual a reference stimulus is presented at a different location, suggesting that cortex, yet the contribution of each area to stereopsis has not been fully combining different binocular disparities over space is essential for clarified. Area MT in the dorsal visual pathway contributes to coarse but not stereoacuity. Here we examined how the brain spatially weighs and integrates fine disparity discrimination, meaning other areas are responsible for fine binocular disparities in a fine depth discrimination task. To estimate depth perception. We examined whether the disparity signal in area V4, an the contribution of disparities at different locations in depth judgment, intermediate stage in the ventral visual pathway, is such an area. We recorded we introduced disparity noise to various locations within random-dot single V4 neuron activity while a Japanese monkey reported whether the stereograms, which were otherwise binocularly correlated, consisting of test center region of a random-dot stereogram was perceived as nearer or farther and reference planes. Subjects were required to report whether the test or than its adjacent surround. The disparity of the center region relative to the reference plane was perceived as nearer. We then calculated the correlation surround varied across trials. The range of disparity covered the monkey's between depth judgment and noise location. The estimated contribution or psychophysical threshold. Neuronal activities during stimulus presentation weight of binocular disparities was largest near the boundary between the were predictive of the monkey's choice on a trial-by-trial basis. One-third test and reference planes and declined monotonically with distance from of the tested cells (12/35) significantly predicted the behavioral choice, the border. At 4 deg from the boundary, the weight was 10% of the peak for which we quantified as choice probability. The sign of the predicted choice stimuli with 3.5 deg eccentricity. We suggest that binocular disparities near was consistent with the monkey's subsequent choice. We suggest that V4 the boundary contribute more to stereoacuity than those distant. The brain cells contribute to fine disparity discrimination by signaling the sign of their uses disparity signals only from near the boundary despite available signals preferred disparity with the spike count. over a wider area.
P1PM-11-9 P1PM-11-10 DIFFERENCE IN NEURAL REPRESENTATIONS OF THE EFFECTS OF GAZE DIRECTION ON THE FACE BETWEEN THE TEMPORAL CORTEX AND THE PERCEIVED DURATION OF EMOTIONAL FACE AMYGDALA IN MONKEYS Hirokazu Doi, Kazuyuki Shinohara Mikio Inagaki, Ichiro Fujita Graduate School of Biomedical Sciences, Nagasaki University, Japan Graduate School of Frontier Biosciences, Osaka University, Japan Recent research has demonstrated that the perceived duration of the Neuropsychological and brain imaging studies in human suggest that visual emotional face is influenced by the valence of the emotional expression; the processing of face is carried out by both cortical and subcortical pathways. negative expressions were perceived to last longer than the positive ones. Here we showed electrophysiological evidence of the difference in face However, to our knowledge, little is known about the effect of the gaze representations between the temporal visual cortex and the amygdala in direction, an another important facial information, on the perceived duration monkeys. We examined whether face responsive neurons in the temporal of face. To address this issue, we measured the perceived duration of the cortex and the amygdala are tuned to image-based spatial frequencies (cycles/ angry and happy expression combined with the straight or the averted gaze image), which are relative spatial frequencies referenced by the image size, direction by the use of the time-bisection task. The results revealed that or retina-based spatial frequencies (cycles/degree), which are absolute the angry expression was perceived to last longer when combined with the spatial frequencies on the retina, by assessing the effects of the image size on straight gaze than when with the averted gaze. However, there was no effect tuning. We found most neurons in the temporal cortex and a few neurons in of gaze direction on the perceived duration of the happy faces. These findings the amygdala were tuned to image-based spatial frequencies, indicating that indicate that the subjective estimation of time is elongated when the observer face representation in the temporal cortex is invariant to retinal image size. encounters a socially important survival signal, considering that an angry The results demonstrate that representations of face differed in their frame face with a direct gaze may be perceived as a threat requiring a fight-or-flight of reference for spatial frequencies between the temporal cortex and the response. On the basis of these results, together with the previous findings amygdala. on the time-perception, the possible physiological mechanism underlying the modulation of the perceived duration by the emotional stimuli is discussed. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 191 P1PM-11-11 P1PM-11-12 A COMPUTATIONAL MODEL OF TASK SWITCHING NEURAL REPRESENTATION OF VISUAL SALIENCY IN USING LEAKY INTEGRATORS THE MONKEY CEREBRAL CORTEX: AN fMRI STUDY Ryo Sasaki, Takanori Uka Naokazu Goda1, Takuya Harada1, Tadashi Ogawa2, Minami Ito1, Department of Physiology, Juntendo University, Japan Hiroshi Toyoda1, Norihiro Sadato1, Hidehiko Komatsu1 We previously reported on the responses of MT neurons while monkeys 1National Institute for Physiological Sciences, Japan, 2Graduate School of switched between discriminating either motion direction (UP or DOWN) Medicine, Kyoto University, Japan or stereoscopic depth (FAR or NEAR) in a moving random dot stereogram. We can rapidly and effortlessly detect a salient object in a cluttered visual Choice related modulation (choice probability: CP) of neurons that scene. Recent physiological experiments in monkeys and humans suggest responded strongly to “congruent” stimuli were large for both direction and that multiple areas in frontal and parietal cortices play important roles in depth discriminations, whereas CPs of neurons that responded strongly to the efficient detection of salient objects as well as competitive interaction “incongruent” stimuli were large for either task. Here, we examined the time in retinotopic areas in occipital cortex. To understand the whole picture of course of CPs in 35 incongruent neurons that had CPs larger than 0.5 in one of the two tasks. In the task with the smaller CP, CPs decreased 200ms after this distributed network, we applied functional MRI in the awake macaque stimulus onset, reaching a trough near the end of visual stimulation. monkeys. We presented monkeys an array stimulus containing a salient color We developed a computational model based on two independent integrators singleton while they were fixating at central fixation spot. The presence of the accumulating sensory evidence separately for direction and depth. Task singleton was irrelevant to the task, thus the response modulations observed switching was accomplished by leaking the accumulated evidence of the were primarily due to the bottom-up salience. We found that the presence of irrelevant task. Integration was terminated near the end of visual stimulation, the salient singleton enhanced activities in a topographic manner in V2/V3 and the final choice was determined from the sum of the activities of the and V4, as well as in MT and LIP. Interestingly, we also found a strong two integrators. The model reproduced the time course of CP. Overall, response enhancement on the ventral bank of the STS, which was anterior to our physiological results and modeling suggest that task switching is the motion-sensitive region in the STS. Our findings shed new light on the accomplished by leaking evidence of the irrelevant integrator. neural network involved in the representation of the visual salience
P1PM-11-13 P1PM-11-14 EVIDENCE THAT VISUALLY DETECTED MOTION PROPERTIES OF TOP-DOWN SIGNALS IN THE VECTOR IS PROCESSED AS TWO DISTINCT SCALARS PRIMATE FRONTAL CORTEX DURING COVERT Yoshiki Kaneoke, Tomokazu Urakawa TRACKING OF A MOVING OBJECT 1 2 Department of Integrative Physiology, National Institute for Physiological Ayano Matsushima , Masaki Tanaka Sciences, Japan 1Department of Physiology, Hokkaido university, Japan, 2Department of We investigated whether human visual system process motion information Physiology, Hokkaido university, Japan & PRESTO, JST, Japan as vectors or as scalars (direction and speed). In various motion stimuli We can track moving objects attentively without eye movements. To examine the composed of numerous red and green dots, observers perceived that top-down signals, we recorded from single neurons in the frontal eye field (FEF) peripheral dots moved similarly as central dots based on the same direction and the lateral prefrontal cortex (LPFC) when monkeys performed the covert or speed even when either one of them was different, indicating that direction tracking task. In this task, monkeys were required to continuously track one of and speed of local motion can be processed separately and their distributions moving visual stimuli that were indistinguishable from their color or shape. are analyzed independently. In MEG study, coherent motions with varied Consistent with the previous studies, the majority of neurons in both areas speeds and directions were presented using random dot kinematogram. Peak responded more to the target than to the distractor ("Attention type"). In the latency of the response to the motion onset was inversely related to the speed LPFC, we also found two additional types of neurons; "Distractor type" neurons for all the directions. The mean mutual information entropy calculated using responded solely to the distractor, whereas "Conditional type" neurons took 4 response data with different directions revealed that the value exceeded account of both the target and the distractor locations. When a second, easily more than 2 after the motion onset, indicating that the response signal can distinguishable distractor was presented simultaneously, some LPFC neurons indeed carry information of more than 4 directions. The value was related to exhibited firing modulation depending on the location of the original, but not the the coherence level but was less affected by the motion speed. The results second, distractor. indicate that speed information is represented in the peak latency and that These results suggest that, in addition to the enhancement of signals originated from direction information is represented in the properties of the response distinct the target location, the frontal cortex may also play roles in the active suppression from that for the speed information. of signals originated from the distractor location. These top-down signals might be essential for discriminating the target from the distractor during mental tracking.
P1PM-11-15 P1PM-11-16 FUNCTIONAL ORGANIZATION OF THE RODENT THE PREFRONTAL CORTEX WAS ACTIVATED IN PONTINE PARABRACHIAL NUCLEUS IN TERMS OF MOTHERS DURING THE ODOR DETECTION TASK OF INGESTIVE BEHAVIOR THE NEWBORN INFANT Takashi Yamamoto1, Naohiro Maeda2, Makoto Ohmoto2, Ichiro Shota Nishitani, Saori Kuwamoto, Asuka Takahira, Matsumoto2, Keiko Abe2 Kazuyuki Shinohara 1Health Science, Kio University, Japan, 2Graduate School of Agricultural Department of Neurobiology and Behavior, Nagasaki University, Japan and Life Sciences, The University of Tokyo, Japan In rodent species, it has been reported that the prefrontal cortex (PFC) lesions impair maternal behavior, so that PFC may play an important function in Previous studies in non-primates suggest that the parabrachial nucleus (PBN) is not maternal behavior. Likewise, a high activation of the PFC of human mothers merely a relay station but also plays an important role in ingestive behavior. To explore while listening to infant cries and viewing infant facial expressions has been more about the functional features of different subnuclei of the PBN, we employed reported. However, little information is available on while smelling infant- different techniques such as electrophysiological unit recording, immunohistochemistry associated odors. In the present study, we examined whether PFC activated in for FOS and gene expression analysis using the DNA microarray technologies in rats mothers during the infant-associated odor detection task measured with near- and mice under different experimental paradigms. FOS neurons were detected in the infrared spectroscopy (NIRS). Mothers (n=12) and non-mother female (n=12) external lateral subnucleus (els), external medial subnucleus, dorsal lateral subnucleus subjects were participated in the present study. Subjects were instructed to Poster Session (dls), central lateral subnucleus, and the central medial subnucleus (cms) depending on answer the each task whether A) infant-associated odors or B) adult male- the kind of taste and visceral stimulation. The expression pattern was different under associated odors were detected or not. We found that adult male-associated odors nutritionally sufficient and deficient conditions, novel and familiar conditions and detection task was increased the PFC activity, but no significant difference was learned and unlearned conditions. As for the possible functions, the rostral part of the found between mothers and non-mother females. However, infant-associated els is related to general visceral inputs; the caudal part of the els, aversive behavior; the odor detection task was activated only in mothers but not in Non-mother females. dls, ingestive behavior; the cms, taste of NaCl. Several genes were expressed diffusely These results suggest that olfactory inputs from the infant may also be a part of in the PBN, others were localized in specific subnuclei. Such information in the PBN the maternal behavior system to activate the complementary caregiving system is sent to the limbic and reward systems to exert appropriate ingestive behavior. of the mother in order to gain their support.
192 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-11-17 P1PM-11-18 NEW EVENT-RELATED POTENTIAL (ERP) INDUCED PREDICTION OF EVENT-RELATED POTENTIALS TO BY LOADING OF AUDITORY AND VISUAL STIMULI SPEECH SOUNDS Hiroko Toshima1, Tutomu Tukui2, Yoshimasa Kobayashi2, Satoshi Shunji Sugimoto1, Nobuaki Tsukahara1, Naoki Ikeda1, Yoko Kurihara1 Yamaguchi2, Junsei Horikawa1 1Department of Cell Physiology, Jikei University School of Medicine, Japan, 1Department of Knowledge-Based Information Engineering, Toyohashi 2All-Japan Karuta Association, Japan University of Technology, Japan, 2Laboratory for Dynamics of Emergent [Purpose] We previously reported that “Ogura Hyakunin-isshu Kyo-gi Karuta” Intelligence, Brain Science Institute, RIKEN, Japan (a Japanese traditional sport using poems) players show a characteristic ERP Speech sounds are known to elicit various event-related potentials (ERPs), (P300). The response time was significantly shorter than the P300 latency, and including components regarded as evoked potentials (EPs). However, it is little the potentials like P300 were also recorded in response to non-target stimuli in known about how time-frequency features of speech sounds are encoded in a players. Recently, we induced a new ERP by loading subjects with a Karuta-like population of cortical neurons and how they elicit ERPs. The present study is task. based on a hypothesis that the majority of ERPs to speech sounds reflects the [Method] Thirty-five players (22.5±2.0 years old) were recruited. The ERP was sum of responses of the second-order linear systems. A computational model was recorded from electrodes attached at Fz, Cz, and Pz. The task was similar to that constructed on the basis of typical ERPs elicited by a pure tone. The proposed in a Karuta game. The stimuli contained visual and auditory verbal stimuli. A model shows oscillatory activities whose frequencies are approximately 5 button was pushed when the visual and auditory stimuli had the same phonemes. and 2 Hz, corresponding to those of N1 and P2 components, respectively. The [Results] We recorded a positive potential with a latency of 430±120msec, amplitude change of speech sounds was fed to the model systems. A comparison dominantly at Cz, but when the stimuli were given in the order of visual followed between simulated and experimentally-recorded ERPs to speech sounds showed by auditory stimuli, no positive potential was recorded. predictability of the present model. These results indicate a theoretical basis [Conclusion] This positive potential recorded in this study is thought to be related of speech-sound encoding that is achieved by simulating multiple oscillatory to visual and auditory information processing. The provision of stimuli in the activities of neuronal populations as second-order linear systems. The proposed order of visual followed by auditory might be similar to that in a Karuta game, model is thought to contribute to the development of brain researches for and might be easy to process in players, so the potential was not recorded. understanding semantic and syntactic processing of spoken sentences.
P1PM-11-19 P1PM-11-20 OPTICAL IMAGING OF AZIMUTHAL ACTIVITIES IN HETEROGENEOUS NEURAL CONTRIBUTION TO MULTI-FIELDS OF THE LEFT AND RIGHT GUINEA PIG BEHAVIORAL DISCRIMINATION OF TEMPORALLY AUDITORY CORTICES ASYMMETRIC SOUNDS IN CAT PRIMARY AUDITORY Yutaka Hosokawa1, Michinori Kubota2, Junsei Horikawa3 CORTEX 1Department of Physiology, Ryukyus University, Japan, 2Med. Res. Inst., Yu Sato, JingYu Wang, Ling Qin 3 Tokyo Medical and Dental Univ., Japan Dept. of Knowlege-based Info. Department of Physiology, University of Yamanashi, Japan Engin., Toyohashi Univ. of Technology, Japan Temporal modulation of sound envelope is crucial for acoustic perception. Azimuthal sound information is crucial for animals. The left and right auditory Although previous studies have shown that amplitude modulated sounds cortices may play an important roll in processing this information. In this study we evoked both stimulus-synchronized and non-synchronized responses in examined the azimuthal sensitivity in the both guinea-pig auditory cortices using primary auditory cortex (A1) neurons, functional significance of the diverse optical imaging with a photodiode array (12x12) and voltage-sensitive dye (RH795). response modes remains unclear. This issue was addressed in this paper by Ten guinea pigs (300-450g) were anesthetized with ketamine (80mg/kg) and xylazine combining the electricphysiological and psychological experiments in cats. (40mg/kg). The auditory cortex of guinea pigs consists of the core fields: the primary We recorded single-unit activities in A1 to calculate neural discrimination and dorsocaudal fields and the surrounding fields: the posterior (P), ventroposterior (VP) fields, etc. The tone (2, 4, 8, 16 kHz) and noise stimuli were presented to the magnitudes between sounds with ramped and damped envelopes. The result animal by a loudspeaker located at contralateral 45 degrees (C45), center (CE) and was compared with the cat’s behavior performance for ramped/damped ipsilateral 45 degrees (I45). The speciotemporal neural activities of the core field to sound discrimination. We found that the behavioral discrimination could CE stimuli was a little weaker than to C45 stimuli in both cortices. However the be based on both the non-synchronized tonic-response representing the delay and level of the inhibition were different in left and right cortices. Especially in gentle amplitude modulation in the middle of sounds, and the synchronized fields P and VP, the response to CE or I45 stimuli was weaker than that to C45 stimuli phasic-response representing the abrupt amplitude change especially at the in both cortices. This tendency was strongly observed in the pure tone stimulation. sound “offset”. This finding of heterogeneous neural mechanism for sound The findings suggest that the caudal fields is concerned with processing the azimuthal envelope discrimination challenges the traditional view that auditory neurons information. represent sounds in a homogeneous phasic response mode.
P1PM-11-21 P1PM-11-22 DEVELOPMENT OF A FLEXIBLE SURFACE ORAL STRUCTURE REPRESENTATION IN HUMAN MICROELECTRODE ARRAY FOR MULTICHANNEL SOMATOSENSORY CORTEX RECORDING OF AUDITORY EVOKED POTENTIALS Yoshiyuki Shibukawa1, Yohei Tamura2, Masuro Shintani3, FROM THE RAT’S CORTEX Masakazu Tasaki1, Tatsuya Ichinohe2, Yuzuru Kaneko2 Yasuhiro X Kato, Katuhiro Maki, Shigeto Furukawa, Makio 1Department of Physiology, Tokyo Dental College, Japan, 2Department of Kashino Dental Anesthesiology, Tokyo Dental College, Japan 3Oral Health Science NTT Communication Science Laboratories, Nippon Telegraph and Center, Laboratory of Brain Research, Tokyo Dental College, Japan Telephone Corporation, Japan To clarify the topography of the areas representing whole intraoral structures and Our ongoing project explores the functional significance of across-area interactions elucidate bilateral neuronal projection to those areas in the primary somatosensory (S1) of cortical activities in auditory processing. For this purpose, we have developed a cortex, we recorded somatosensory-evoked magnetic fields (SEFs). Following tactile multichannel flexible surface microelectrode array (SMA), aiming to record auditory stimulation on the oral mucosa (inferior/superior buccal, posterior/anterior tongue, evoked potentials (AEPs) at sites widely distributed across the cortex of anesthetized and upper/lower lip mucosa), SEFs with a peak latency of 15 ms (1M) were identified or awake animals. The SMA was fabricated by using surface micromachining bilaterally. In contrast, SEF with a peak latency of 30 ms following right index technologies of Micro-Electro-Mechanical-Systems. The advantage of our method finger tactile stimulation were identified in the contralateral hemisphere. Equivalent based on photosensitive polyimide, in contrast to conventional ways using non- current dipoles (ECDs) generating 15 ms components were found along the posterior photosensitive material such as parylene, is to eliminate a complicated dry etching process and allow us to design the geometry more flexibly, depending on experimental wall of the central sulcus, bilaterally. The ECDs for oral mucosa-representing areas purposes at a lower cost with improved process yields. The fabricated SMA had were located inferiorly to those for the index finger, with the following pattern of 16-channel microelectrodes and physical properties (i.e., size and impedance) that organization from top to bottom along the central sulcus: index finger, upper or lower were satisfactory for neural recordings. Multichannel recordings of AEPs were made lip, anterior or posterior tongue and superior or inferior buccal mucosa, with a wide successfully with the SMA, which was placed epidurally on the auditory cortex of an distribution. These results indicate that sensory afferents from intraoral region project anesthetized rat. A frequency map and level-dependent activities of the rat auditory to both the contralateral and ipsilateral S1 cortices, where contralateral projection is cortex were confirmed. The SMA could be used repeatedly without any breakages and predominant. The results clarify the intraoral structure-representing areas in the S1 mechanical failures, permitting stable recording for a long time. cortex. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 193 P1PM-11-23 P1PM-11-24 OTHER’S BODY REPRESENTATION REFERRED TO HYPERALGESIA INDUCED BY CHEMOTHERAPEUTIC SELF BODY IN THE PARIETAL CORTEX OF THE AGENT PACLITAXEL MODULATES NEGATIVE MONKEY AFFECTIVE COMPONENT OF PAIN IN RAT Akira Murata, Hiroaki Ishida, Katsumi Nakajima, Masahiko Inase Kazuko Noda, Hisanao Akita, Masanori Ogata, Makoto Saji Department of Physiology, Kinki University School of Medicine, Japan Department of Physiology, School of Allied Health Sciences, Kitasato Neurons in the parietal and premotor cortices of the monkey shows visuo- University, Japan tactile bimodal properties that may contribute to not only motor control but also mapping of one’s own body parts. Furthermore, as revealed by mirror Paclitaxel is one of the chemotherapeutic agent for treatment of tumour, neurons, the brain represents others’ action in the motor control system. This which produces side-effects such as peripheral neuropathy, sensory suggests that other’s body parts are represented in the brain. We expected that abnormalities and hyperalgesia. These side-effects are left with the cessation a population of visuo-tactile neurons in the parietal cortex would concern of paclitaxel therapy but become chronic. Especially, the hyperalgesia to mapping other’s body parts onto one’s own body representation. In the severely damages quality of life. However, little is known on the mechanism current experiment, we recorded visuo-tactile bimodal neurons from area of the drug-induced hyperalgesia. In this study using in rats, we examined VIP and 7b, then examined activity while the monkey was observing visual whether the drug-induced hyperalgesia involves the pain-induced place or tactile stimuli on other’s body that was confronting the monkey. Some aversion as an aspect of the negative affective component of pain. The drug- bimodal neurons of those receptive fields anchored on the monkey’s body induced hyperalgesia was confirmed by using von Frey filament test during showed visual response on the corresponding body parts of other within 1-2 weeks after the intraperitoneal injection of paclitaxel (1mg/kg/day) for approximately 30cm from the body surface. These results were the first evidences of sensory mirror neuron. We suggest the existence of self-other 4 days. The negative affective component of pain was assessed by using the body parts matching system in the parietal bimodal area, in which self body place-conditioned paradigm, the intraplantar injection of formalin produced image is available as reference for perception of others’ body parts. These the conditioned place aversion. The formalin-induced conditioned place neurons may send information of other’s body parts to the mirror neuron aversion was abolished by the paclitaxel treatment conducted 8 days prior to system. the behavioral test.
P1PM-11-25 P1PM-12-1 EFFECT OF ELECTRICALLY STIMULATING GREATER REGULATION OF MICROGLIAL AMYLOID-β SPLANCHNIC NERVE ON INTRACELLULAR PHAGOCYTOSIS IN THE THERAPEUTIC STRATEGY POTENTIALS OF NEURONS IN ANTERIOR CINGULATE FOR ALZHEIMER'S DISEASE Kazuyuki Takata1, Yoshihisa Kitamura1, Shigehiro Morikawa2, GYRUS OF CATS 2 3 1 1 2 3 Toshiro Inubushi , Ikuo Tooyama , Takashi Taniguchi Minfan Wu , Chunfu Wu , Guoxi Teng 1 1 2 Department of Neurobiology, Kyoto Pharmaceutical University, Japan, Department of Physiology, Shenyang Medical College, China, Department 2 3 Biomedical MR Science Research Center, Shiga University of Medical of Pharmacology, Shenyang Pharmaceutical University, China, Laboratory of 3 Science, Japan, Molecular Neuroscience Research Center, Shiga Neurophysiology, Brain Research Institute, China Medical University, China University of Medical Science, Japan To explore the cerebral cortex mechanism of visceral nociceptiev sensation, In the vaccination therapeutic strategy in Alzheimer's disease (AD), microglial electrophysiological properties of 394 neurons in the bilateral anterior cingulate gyrus(ACG) amyloid-β (Aβ) phagocytosis has been proposed as one mechanism for the brain Aβ of cats responding to strongly electrically stimulating greater splanchnic nerve(GSN) were clearance, while the details are unclear. We analyzed the microglial Aβ phagocytosis investigated. According to latency of the response, the neurons could be classified into in in vitro and in vivo studies. Primary cultured rat microglia phagocytosed Aβ, and visceral nociceptive neurons(VNNs) showing long latency and visceral non-nociceptive anti-Aβ antibody increased the phagocytosis. However, deglycosylated antibody neurons (NVNNs) only having short latency(<50 ms). Spontaneous discharges of both VNNs had no effect on the microglial Aβ phagocytosis. The glycochain on the antibody is and NVNNs were not obvious distinct. But the percentage of VNNs having spontaneous critically involved in binding to Fc receptor (FcR). Thus, it is suggested that anti-Aβ biological electric activities was significantly higher than that in NVNNs(P<0.05) . The antibody enhances the microglial Aβ phagocytosis via FcR on microglia. In in vivo results indicated that the excitability of the former was higher than that of the latter. Patterns study, we transplanted rat microglia into the rat lateral ventricle just after the intra- of the evoked responses of VNNs and NVNNs were excitatory, inhibitory and mixed. hippocampal Aβ injection, and investigated its contribution to Aβ clearance. Migration Compared with the responses of NVNNs, the responses of VNNs had the specificities with of exogenous microglia on Aβ plaque was detected by MRI, and it was confirmed by higher threshold, longer latency, complex reactive patterns, and being inhibited by the action histochemical study. Furthermore, the clearance of Aβ in the brain was significantly of morphine. The results suggested that the GSN afferent fibers project to the bilateral ACG, increased by the microglial transplantation. These findings demonstrate that microglial and there exist VNNs and NVNNs in the bilateral ACG, which have obvious difference in Aβ phagocytosis could be a target for the vaccination therapeutic strategy in AD and electrophysiological properties. importantly contribute to the brain Aβ clearance.
P1PM-12-2 P1PM-12-3 DISTRIBUTION OF NEURON/GLIAL2 EXPRESSING AMPA RECEPTORS MEDIATE LTP LIKE RESPONSE IN MICROGLIA IN PARKINSON'S DISEASE RAT MODEL DENTATE GYRUS ASTROCYTE 1 2 2 Masatoshi Inden , Yoshihisa Kitamura , Kazuyuki Takata , Takashi Yuemin Ding, Chu Chen, Xiong Zhang 2 Taniguchi Department of Physiology, Zhejiang University School of Medicine, China 1 2 Ritsumeikan University, Japan, Department of Neurobiology, Kyoto Increased evidence indicated astrocytes can respond to glutamate overflowed Pharmaceutical University, Japan from neuronal synapses. We aim to astrocytes’ response to enhanced synaptic Neuron/glial2 (NG2) expressing cells are often referred to as oligodendrocyte activities during long term potentiation (LTP). With patch clamp technique, precursor cells. NG2 expressing cells are also identified as multipotent a LTP like response was invoked in dentate gyrus astrocyte after induction progenitor cells or intrinsic central nervous system adult stem cells. Recently, of the field recording LTP (fLTP) in response to tetanus stimulation at the NG2 positive macrophage-like cells markedly accumulate during a subacute perforant path in hippocampal slice from GFAP-GFP transgenic mice. The phase of ischemic events. However, NG2 expressing microglia was not fully potentiated astrocytic potential rise more slowly and the phase is reversed examined in neurodegenerative diseases such as Parkinson's disease (PD). In + + the present study, we chose a rat model of PD, such as intrastriatal injection to the potential of fLTP. Both the K channel inhibitor Cs and the glutamate of 6-hydroxydopamine (6-OHDA). At 10 days after injection of 6-OHDA, transporter inhibitors THA and DHK cannot block the potentiated astrocytic the morphology of NG2-positive cells changed to an activated form like response. By isolating the current components that composed astrocytic
Poster Session microglia. In addition, these cells expressed the immunoreactivity for ionized potential, AMPA receptor mediated currents increase significantly after calcium-binding adaptor molecule 1 (Iba1), a microglial maker. On the tetanus stimulation compared to before tetanus stimulation. These results other hand, in vehicle-injected animals, NG2- and Iba1-double positive cells suggest a LTP like response can be invoked in astrocyte accompanying were undetected in the SN. Interestingly, activated NG2-positive cells were with LTP induction in neuron. The astrocytic response is independent to the located in immediate proximity to tyrosine hydroxylase-positive neurons. increased potassium uptaking and enhanced glutamate transporter activities. These results suggest that NG2- and Iba1-double positive cells may play the It mainly attribute to the AMPA receptor mediated current may due to the protective roles for dopamine neurons, during processes of dopaminergic increased presynaptic glutamate release. neuronal cell death. Supported by National Natural Science Foundation of China (30600169)
194 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-12-4 P1PM-12-5 PURINERGIC AGONISTS INDUCE ENZYME ACTIVITIES ADP AND ATP ACTIVATE MICROGLIAL MIGRATION OF IODOTHYRONINE DEIODINASES IN CULTURED AND PHAGOCYTOSIS VIA Gi-COUPLED P2Y ASTROCYTES RECEPTOR Stanislav Pavelka Chizuru Tsuzuki, Hiroshi Hibino, Yoshihisa Kurachi Dept. of Radiometry, Institute of Physiology, Czech Acad. Sci., Prague and, Division of Molecular and Cellular Pharmacology, Department of Institute of Biochemistry, Masaryk University, Brno, Czech Republic Pharmacology, Osaka University, Japan Described are the effects of new classes of effectors - purinergic agonists - and of Brain microglial cells, a type of immune cells, migrate toward the sites of some agents controlling the intracellular Ca2+ concentration in cultured astroglial tissue damage to engulf dead cells and secrete various cytokines. These cells on the enzyme activities of iodothyronine deiodinases (ID) of the types 2 reactions can be triggered by the extracellular nucleotides released from (D2) and 3 (D3). The changes in ID activities caused by short-term incubation the injured cells. The migration is known to be driven by stimulation of Gi- of the cells in a chemically defined medium with different concentrations of coupled receptor P2Y12. Here we report that ADP and ATP regulate not only these effectors were quantified with our newly developed radiometric assays. the migration but also the phagocytosis by affecting the similar receptor in rat Physiological concentrations of ATP, ADP, AMP or adenosine caused a very microglial cells. Each of ADP and ATP enhanced the phagocytosis with EC50 marked (up to 30-fold) increase in D2 activity. The enzyme activity reached a of roughly 10 μM. The enhancement was completely blocked when the cells plateau after 4-h incubation of astrocytes with purinergic agonists. D3 activity were pretreated with PTX or exposed to a specific antagonist of i/oG -coupled was also induced, but to a much lesser extent (3 to 7-fold increase in comparison P2Y12 or P2Y13 receptor, cangrelor, suggesting the involvement of such with controls). Two representatives with direct effects on the intracellular Ca2+ receptor(s) in the process. Furthermore, the cells that had already engulfed concentration (ionophore A23187 and chelator EGTA), and another two agents zymosan as bait in the presence of ADP showed prominent impairment of manifesting indirect influences (endothelin and thrombin) were also tested. ADP-induced migration. Therefore, in the brain, microglial cells will first The results indicate multiregulation of induction of ID activities. Support from move towards the focus of injury by sensing low concentration of ADP and/ the Acad. Sci. of the Czech. Rep. (Project No. AV0Z50110509), Ministry of or ATP, and cease migration when they engulf damaged cells. The coupling Educ. of CR (Project No. MSM0021622413) and from the GA CR (Grant No. of these two distinct functions seems to be mediated by P2Y12 or P2Y13 304/08/0256) is acknowledged. receptor.
P1PM-12-6 P1PM-12-7 ALLOSTERIC POTENTIATING LIGAND FOR CALCIUM-DEPENDENT TRANSIENT FORMATION OF NICOTINIC RECEPTOR INCREASES MICROGLIAL BLEB-LIKE STRUCTURES IN ASTROCYTES PHAGOCYTOSIS OF AMYLOID-β Remi Susuki (Tsuchiya)1, Takashi Sakurai2, Yoshihisa Kudo3, 2 Sachiko Kagitani1, kazuyuki Takata1, Yoshihisa Kitamura1, Susumu Terakawa 1 2 1Photon medical research center, Hamamatsu Univ. Sch. Med., Japan, Takashi Taniguchi , Shun Shimohama 2 1 Photon medical research center, Hamamatsu Univ. Sch. Med., JST, Department of Neurobiology, Kyoto Pharmaceutical University, Japan, 3 2 CREST Japan, Department of Life Science, Tokyo Univ. Pharmacy and Department of Neurology, Sapporo Medical University, Japan Life Sciences, Japan The deposition of amyloid-β (Aβ) is the hallmark of Alzheimer's disease (AD). The To study the distributions of neurotransmitter receptors regulating the Ca2+-dependent reduction of brain Aβ is proposed as a therapeutic approach for AD, and microglial 2+ 2+ responses in astrocytes, we measured the intracellular Ca concentration ([Ca ]i) phagocytosis is noted as an Aβ clearance system in brain. Recently, it was reported using a Ca2+ sensitive dye (Fura-2AM). In human astrocytoma cell line (U251-MG), that microglia expressed nicotinic acetylcholine receptors (nAChRs). Therefore, we we identified the expression of various neurotransmitter receptors to ATP, histamine, investigated the effect of the nAChR stimulation on microglial Aβ phagocytosis. acetylcholine, noradrenaline and so on. When the cells were stimulated with Microglia were treated with Aβ1-42 (Aβ42) in the presence of nicotine or allosteric histamine, several seconds after the initial peak of Ca2+ response, many fluorescent potentiating ligand (APL) for nAChR. The expression of mRNA of nAChR was spots transiently appeared at the periphery of the cells. These spots observed under examined by RT-PCR, and that of the protein was confirmed by confocal laser DIC microscope were bleb-like structures (BLS) of which diameter was about 1 μm microscopy and western blotting. The level of Aβ42 phagocytosed by microglia was in average. Stimulation of the cells with a Ca2+ ionophore (ionomycin) increased 2+ measured by ELISA. Rat primary cultured microglia were expressed mRNAs and both the amplitude of [Ca ]i response and the number of BLS to an extent greater proteins of nAChR. After the treatment with Aβ42, the level of Aβ42 phagocytosed by than those with histamine. Other stimulants we examined induced few number of microglia was increased by the treatment of nicotine or APL for nAChR. This effect BLS. When Ca2+ was removed from the extracellular medium, the formation of BLS was inhibited by the pre-treatment of nAChR inhibitors. These results suggest that was significantly suppressed. Therefore, it is suggested that BLS formation was the stimulation of nAChR by nicotine or APL increases microglial Aβ phagocytosis. Ca2+-dependent and histamine-specific response. It is possible that a local release of Thus, the stimulation of nAChR may effectively reduce Aβ in AD brain by increasing histamine from a synaptic terminal induces the BLS from an astrocyte to strengthen microglial phagocytosis. the synaptic structure.
P1PM-12-8 P1PM-12-9 FAST AND LOCALIZED INTRACELLULAR CALCIUM NEUROPEPTIDES AS MESSENGERS TO MICROGLIA DYNAMICS IN HIPPOCAMPAL ASTROCYTES IN RESPONSE TO PATHOLOGIC CONDITIONS Eiji Shigetomi, Khakh S Baljit Mami Noda, Yuko Okuno, Yukiko Yamakawa, Masataka Ifuku Physiology and Neurobiology, David Geffen School of Medicine, UCLA, Pathophysiology, Kyushu University, Graduate School of Pharmaceutical USA Sciences, Japan Astrocytes are known to provide passive supportive roles to neurons. Microglia, brain's immune cell, express various receptors for neurotransmitter Growing evidence also indicates that astrocytes may actively participate in or neuropeptides. In responding to these neurotransmitters or peptides, brain function through interactions with neurons. It has been proposed that microglia show chemotaxis, release different kinds of cytokines and trophic astrocyte Ca2+ elevations can trigger transmitter release from astrocytes. The factors, or attenuating excess release of cytokines induced by bacterial toxins. intracellular Ca2+ level at the plasma membrane may be an important factor Many neuropeptides are produced in the brain during trauma and stroke, and for this process. We analyzed real-time Ca2+ dynamics within ~100 nm of are regarded as mediators of inflammation. Therefore, microglial cells are the plasma membrane using total internal reflection fluorescence microscopy likely to play an important role in the brain via various neuropeptides. Using with fast acquisition (10-40 Hz) in rat hippocampal astrocytes. We found fast primary cultured of microglial cells, some neuropeptides, such as bradykinin,
(T50, 1.3 ± 0.2 s) and highly-localized (distance at 50% of the peak, 5.5 ± endothelin, vasopressin and galanin, increased microglial migration. Though 0.4 μm) Ca2+ transients occurred spontaneously (2.0 ± 0.5 events/min). The many of them are regarded as mediators of pain and inflammation and pharmacological profiles of the Ca2+ signals suggested that Ca2+-permeable are produced at the site of injury, they had rather neuroprotective effects. plasma-membrane channels mediate this fast and localized Ca2+ increases. They inhibited the release of lipopolysaccharide (LPS)-induced TNF-α or Activation of GPCRs caused large amounts of Ca2+ release from stores and interleukin-1β. These results suggest that some neuropeptides may have therefore collapsed localized Ca2+ signals. Our data suggest that physiological anti-inflammatory and neuroprotective effects through multiple functions on Ca2+ signals in astrocytes have compartmentalized near membrane features. immune cells in the brain. These observations may help to understand the These data provide a basis to understand how astrocytes may participate in paradox on the role of neuropeptides in the central nervous system and may intracellular signaling in the brain. be useful for therapeutic strategy. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 195 P1PM-12-10 P1PM-12-11 DEVELOPMENT OF A NEW IN VITRO MODEL OF INVOLVEMENT OF GLIAL P2X7 RECEPTOR IN LONG- BRAIN ISCHEMIA BY FOCAL PHOTOLYSIS OF TERM PLASTICITY IN THE SPINAL DORSAL HORN OF A CAGED GLUTAMATE IN NEURON AND GLIA THE RAT COCULTURES Yuxia Chu1,2, Ning Lu1, Yu Qiu Zhang1, Zhi Qi Zhao 1 Sadahiro Iwabuchi, Tomoharu Watanabe, Koichi Kawahara 1Institute of Neurobiology, Institute of Brain Science and State Key 2 Laboratory of Cellular Cybernetics, Graduate School of Information Laboratory of Medical Neurobiology, Fudan University, China College of Science and Technology, Hokkaido University, Japan Medicine, Jiaxing University, China In the ischemic core region (ICR), a marked increase in the concentration of It has been shown that spinal glia was involved in tetanically sciatic stimulation- extracellular glutamate (Glu) results in the activation of neuronal Glu receptors induced long-term potentiation (LTP) of C-fiber-evoked field potentials in spinal (especially NMDA-type receptors), leading to massive neuronal death. During dorsal horn and application of ATP onto spinal surface effectively induced spinal reperfusion after brain ischemia, diffusion of extracellular Glu itself and/or LTP. In the present study, the involvement and roles of the ionotropic purinergic changes in gene expressions in neurons and glias possibly influences the P2X7 receptor (P2X7R) in glia in the induction of C-fiber-evoked field potentials induction of delayed neuronal death in the region surrounding the ICR (SICR). were assessed by electrophysiological recording in rats. A detailed in vivo To prevent the delayed cell death after ischemia, it seems crucial to understand study showed that unilaterally tetanic stimuli elicited LTP of C-fiber-evoked the detailed mechanisms for the changes in cellular communications between field potentials. However, intrathecal ( i.t.) administration of a highly specific ICR and SICR. In this study, we demonstrated that focal photolysis of a caged antagonist of P2X7R Brilliant Blue G ( 20μM/10μl ,i.t.) and a less specific Glu induced acute neuronal death in the photolytic-flashed region (PR) and oxidized ATP ( 300μM/10μl, i.t.) given 0.5h prior to stimulation blocked the delayed neuronal death in the region surrounding the PR (SPR) in neuron/glia co- induction of spinal LTP. Nevertheless, both of the drugs at the same concentration cultures. In addition, treatment with an antagonist of NMDA receptors attenuated given 1h after stimulation had no significant influence on the maintenance of not only the acute neuronal death in the PR, but also the delayed neuronal death spinal LTP. Immunostaining revealed co-localization of P2X7R with OX42 in the SPR. This new in vitro brain ischemia model would contribute to the (microglia marker) and GFAP (astrocyte marker), but not with NeuN (neuronal further understanding of the cellular mechanisms responsible for the ischemia- marker). Our results suggest the pivotal role of glial P2X7R in the induction of induced brain dysfunction. LTP but not the maintenance.
P1PM-12-12 P1PM-12-13 DECREASED EXPRESSION OF SPINAL AQUAPORIN DEPENDENCY AND INDEPENDENCY OF GLIOMA 4 CONTRIBUTES TO THE DEVELOPMENT OF CELL INVASIONS ON Arf6 WHICH IS AN ESSENTIAL TOLERANCE TO MORPHINE ANALGESIA IN RODENTS FACTOR FOR BREAST CANCER CELL INVASION Hajime Yano1, Anna Smirkin1, Akihiro Inoue2, Kokeguchi Tomoki1, Meng-Ling Chen, Feng Bao, Zhi-Qi Zhao, Yu-Qiu Zhang 1 2 1 Institute of Neurobiology, Fudan University, China Hisaaki Takahashi , Ohnishi Takanori , Junya Tanaka 1Molecular and Cellular Physiology, Graduate School of Medicine, Ehime Spinal glial cells participate in the development of morphine analgesic University, Japan, 2Department of Neurosurgery, Ehime University, Japan tolerance. Aquaporin 4 (AQP4) is most strongly expressed in astrocytes throughout central nervous system, and plays an important role in some Glioma cell invasion is one of major threats of brain tumors. The small GTPase pathophysiological processes in the spinal cord. However, whether AQP4 Arf6 protein levels had been found to exhibit strong positive correlation with modulates opioid analgesia and tolerance remains unclear. The present study breast cancer cell invasive activities. Subsequently, the GTPase has been elucidated as an essential factor for breast cancer cell invasions, and either found that: (1) repeated treatment with morphine resulted in downregulation upstream or downstream signaling axis of Arf6 has been explored thereafter. of spinal AQP4 expression in mice and rats detected by Western blotting; (2) So far, specific factors namely GEP100 and AMAP1 have been identified, in the tests of tail-flick to radiant heat, the maximal possible analgesic effect respectively. Furthermore, a chemical compound that can inhibit the interaction (MPE) of morphine (10 mg/kg) was lower in AQP4 knockout (KO) mice of AMAP1 and its co-factor cortactin, indeed, suppressed in vivo metastasis of than that in wide type (WT) mice; (3) the spinal Mu opioid receptor (MOR) breast cancer cells. This “Arf6 signaling axis” scenario rendered an excellent expression in AQP4 KO mice was lower than that in WT mice; (4) the reference for exploration into mechanisms of various cancer cell invasions morphine tolerance was potentiated in AQP4 KO mice compared with WT and also clinical applications. We have assessed the correlations between Arf6 mice. It is suggested that the down-regulation of spinal expression of AQP4 protein levels and invasiveness of various glioma cells, and found that they and MOR might contribute to the development of morphine tolerance. can be classified into at least 3 groups in the aspect of Arf6 scenario, different from breast cancer cells. We would like to discuss about either physiological or pathological significance of this Arf6 dependency/independency of glioma cell invasions.
P1PM-12-14 P1PM-12-15 PREDICTING DRUGS DURATION EFFECT ON GLIAL GABA-B RECEPTORS IN THE PERIPHERAL NERVOUS CELL DAMAGES DUE TO NEUROPATHIC PAIN: SYSTEM ARE INVOLVED IN MYELINATION AND ARTIFICIAL NEURAL NETWORK APPROACH NOCICEPTION Mohammad Reza Raoufy1, Parivash Eftekhari1, Jalal Pourahmad2, Valerio Magnaghi1, Marinella Ballabio1, Bernhard Bettler2, Bijan Shafaghi2 Alessandro Faroni1, Patrizia Procacci3, Marcella Motta1 1Department of Physiology, Tarbiat Modares University, Iran, 2Faculty of 1Department of Endocrinology, University of Milan, Italy, 2Department of pharmacy, Shahid Beheshti University, M.C., Tehran, Iran Biomedicine, University of Basel, Switzerland, 3Department of Human We investigated the efficacy of Etanercept on brain glial cell reactive oxygen Morphology, University of Milan, Italy species (ROS), mitochondrial membrane potential decrease (MMPD) and GABA-B receptors are present in the nervous system where they play important lysosomal membrane damages (LMD) in a neuropathic pain model and roles in the nociceptive transmission and pain. Baclofen, a GABA-B specific agonist, compared it to those of Morphine, Aspirin and Celecoxib. Artificial neural is antinociceptive in models of acute and chronic pain. A tonic GABA-B receptor activation, therefore, contributes to the establishment of the nociceptive threshold. network (ANN) was developed to predict the effect of drugs duration on glial -/- cell damages. We found significant decrease of ROS in all days after ligation The GABA-B1 mice are hyperalgesic further supporting the role of GABA-B in Etanercept and Morphine groups in comparison with no drug group, while receptors in the central nociception. Interestingly, GABA-B receptors are expressed in the peripheral nervous system (PNS), mainly in the Schwann cells where they in Aspirin and Celecoxib groups it was seen in 2 and 6 days after ligation. In -/- control cell proliferation and myelination. Emerging evidence obtained in GABA-B1 Poster Session all of drugs groups, MMPD and LMD decreased with time increase. During mice indicates that these mice have gait alterations and reduced allodynic sensitivity. interpolation and extrapolation of designed ANN, Morphine showed remarkable GABA-B1 -/- mice also show morphological and molecular changes in peripheral ROS, MMPD and LMD increase in about 10th day, while Etanercept steadily nerves, including an increased number of small myelinated fibers and small neurons reduced those during all days in comparison with the other drugs; so that by of the lumbar DRG. These fibers are supposed to be A-delta nociceptive fibers. increasing of the days (after 14 days) Etanercept has better effect on preventing Consequently, it has been suggested that GABA-B receptors are implicated in the PNS glial cell damages. This result is a consequence of designed ANN extrapolation. myelination and nociceptive processes. The study in conditional mice specifically We suggest more long term experimental studies to confirm Etanercept effects on lacking GABA-B1 receptor in Schwann cells or motoneurons will aim at clarifying the glial cell damages due to neuropathic pain. role of GABA-B receptors in peripheral pain sensitivity.
196 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-12-16 P1PM-12-17 INVOLVEMENT OF TRPV4 IN THE PROCESS OF NMDA CURRENTS ON HIPPOCAMPAL ASTROCYTES MICROGLIAL ACTIVATION Pei-Yu SHIH, Alexey Semyanov Hisashi Shirakawa, Ikkei Matsutani, Masakazu Konno, Kusano BSI, RIKEN, Japan Ayaka, Takayuki Nakagawa, Shuji Kaneko NMDA receptors (NMDARs) play a critical role in neuronal synaptogenesis Department of Molecular Pharmacology, Graduate School of and plasticity. Recent studies indicated that functional NMDARs are also Pharmaceutical Sciences, Kyoto University, Japan expressed in certain glial cells in white matter and neocortex. Using whole- 2+ Activated microglia have been proposed to play an important role in the pathogenesis cell voltage clamp recordings and Ca imaging in rat hippocampal slices of neurodegenerative diseases in conjunction with Ca2+ signaling, however there we described currents activated by 1 mM NMDA puff application in 2+ CA1 astrocytes. These inward currents were blocked by APV, NMDARs is not enough explanation about a unique Ca mobilization. Transient receptor 2+ potential (TRP) superfamily comprises a group of non-selective cation channels that antagonist, and enhanced in zero Mg solution. They were also insensitive to + open in response to divergent stimuli. Although TRP channels are widely distributed tetrodotoxin, Na -channel blocker, and bafilomycin A1, vesicular H+-ATPase in the brain, their roles in microglia remain unknown. Using rat cultured cortical inhibitor, preventing neuronal excitability and vesicular release, respectively. microglia, we found that TRPV4, which is a non-selective cation channel sensitive to These finding is consistent with the presence of NMDARs in hippocampal extracellular hypoosmolarity or moderate temperature, is functionally expressed in the astrocytes which was not previously reported. Interestingly, NMDA induced 2+ microglia as detected by RT-PCR, immunostaining, Ca2+ imaging and whole-cell patch currents in astrocytes were not accompanied by increase in somatic Ca . This clamp experiments. Quantitative RT-PCR analysis demonstrated that LPS, a potent can be due to localization of the receptors in astrocytic processes or because 2+ stimulus for microglial activation, decreased the expression level of TRPV4 mRNA. of their low Ca permeability. Finally, MK801, use-dependent NMDARs Moreover, simultaneous application of 4αPDD, a TRPV4 selective agonist, suppressed antagonist, reduced paired-puff depression of glutamate transporter current LPS-induced increase in nitric oxide production and galectin-3/MAC-2 expression, in the astrocytes pointing to a role of NMDARs in regulation of glutamate a marker of activated microglia, and its suppression was cancelled by TRPV4 uptake. antagonists such as ruthenium red or gadolinium. Taken together, TRPV4 could play an important role in modulating microglial activation state.
P1PM-12-18 P1PM-12-19 MICROGLIA INSTRUCTS NEUROGENESIS AND ASTROCYTIC RESPONSES TO GABA SPILL-OVER GLIOGENESIS IN THE SUBVENTRICULAR ZONE BY INTERNEURON FIRINGS IN CA1 STRATUM Yukari Shigemoto-Mogami, Ken Nakazawa, Kaoru Sato LACUNOSUM-MOLECULARE (SLM) 1 1 1 2 Department of Pharmacology, National Institute of Health Sciences, Japan Kiyoshi Egawa , Yamada Junko , Furukawa Tomonori , Yuchio Yanagawa , Atuo Fukuda1 In the postnatal brain, neurogenesis occurs in the subgranular zone 1 Department of Psysiology Division of Neurophysiology, Hamamatu University (SGZ) and subventricular zone (SVZ). Increasing evidence suggests that school of medicine, Japan, 2Department of developmental and integratetive inflammation influences several steps of adult neurogenesis, but microglial Neuroscience, Gunma University Graduate scholl of Medicine, Japan contribution to the modulation of neurogenesis is still unclear. Both pro- and In CA1 astrocytes, GABA application induced inward currents and bidirectional Cl- - - anti-neurogenic effects have been reported, likely reflecting the complexity flux through GABAA receptors (Cl efflux) and GAT3 (Cl influx). To clarify glial of the microglial activation process. Here we investigated the contribution responses to GABA spill-over in the neuronal networks, we performed dual whole- of microglia to modulation of neurogenesis in the neonatal rat. We examined cell patch clamp recordings from interneuron-astrocyte pairs in SLM layer using the time dependence of microglial activation and distribution in the neonatal GAD67-GFP knock in mice. The [Cl-]i of astorcytes was adjusted to physiological period and found that the activated microglial cells concentrated in SVZ up to condition (40 mM). Trains of interneuron firing (50Hz, 2sec.) induced inward currents 10 days after birth. Intraperitoneal treatment with minocycline, an inhibitor in the adjacent astrocyte, which were completely blocked by BMI. GAT1 inhibitor of microglial activation, caused reduction of KI-67 positive neogenerative significantly increased the astrocytic inward currents and induced BMI-insensitive, cells surrounding SVZ. Moreover, experiments using differentiation markers GAT3 inhibitor-sensitive currents. Gap junction inhibitor (carbenoxolone) significantly reduced the inward currents by the distance dependent manner. Currents recorded from showed that minocycline inhibited neurogenesis and gliogenesis in SVZ. the pairs with relatively longer distance were completely abolished by carbenoxolone. Our findings suggest an essential role of microglial cells in contributing and The results indicate that spill-over of GABA induces Cl- efflux from CA1 astorocyte maintaining neurogenesis and gliogenesis. - via GABAA receptors accompanied by Cl conductance between astrocytes through gap junctions. Since GABAA conductance of postsynaptic neurons and GATs could take up Cl- from synaptic cleft, these astrocytic Cl- conductance might spatially buffer [Cl-]o change and maintain GABAergic synapse transmission.
P1PM-12-20 P1PM-12-21 DIFFERENTIAL MICROGLIAL ACTIVATION BETWEEN MECHANISMS UNDERLYING UPREGULATION OF P2Y6 ACUTE STRESS AND LIPOPOLYSACCHARIDE RECEPTORS IN MICROGLIA IN KAINATE-INDUCED INJURY MODEL Shuei Sugama 1 2 3 1 Department of Physiology, Nippon Medical School, Japan Kayoko Fujishita , Atsuhito Nakao , Kazuhide Inoue , Schuichi Koizumi 1Department of pharmacology, Interdisciplinary Graduate School of Medicine In the previous study, we demonstrated that an exposure of rats to acute and Engineering, University of Yamanashi, Japan, 2Department of Immunology, stress induces morphological microglial activation in several brain regions Interdisciplinary Graduate School of Medicine and Engineering, University including the midbrain periaqueductal gray (PAG), an area that plays of Yamanashi, Japan, 3Department of Pharmacology, Graduate School of important roles in behavioral responses to uncontrollable stress, threat, Pharmaceutical Science, Kyusyu University, Japan anxiety, and pain. To determine whether neuronal activation may be involved Extracellular nucleotides function as important neuron-to-glia mediators in the in the stress-induced microglial activation, the present study investigated physiological and pathological conditions in the CNS. When neurons are injured the correlation between neuronal activity measured as c-Fos expression and by kainate (KA), they release a pyrimidine nucleotide UDP, an endogenous agonist to P2Y receptors, and inform microglia of their crisis. Although resting microglia morphological microglial activation in the PAG. Acute stress was followed 6 express no or low levels of P P2Y6 receptors, activated microglia dramatically by morphological activation of microglia and increased c-Fos expression in upregulate the P2Y6 receptors, by which they sense UDP, and start to phagocytose the PAG but not in the surrounding midbrain. Double immunohistochemistry dead neurons (Koizumi et al., 2007). However, mechanisms underlying microglia- and topological analysis demonstrated that microglial activation occurred specific upregulation of P2Y6 receptors remain unknown. In the present study, we adjacent to responsive neurons. By contrast, lipopolysaccharide (LPS) show that transforming growth factor (TGF)-β1 is a responsible molecule for the treatment induced microglial activation even in the absence of neuronal upregulation. Exogenously applied TGF-β1 caused a significant increase in mRNAs responses in the PGA as well as in the rest of the midbrain. These findings and proteins of P2Y6 receptors in a TGF-β-receptor-dependent fashion in cultured microglia. TGF-β1 was increased in KA-treated neuron-glia co-cultures, and this suggest that the mechanism of microglial activation during stress may differ conditioned medium upregulated microglial P2Y6 receptors in a TGF-β1-dependent from those of infection or inflammation. It also indicates that the neuronal manner. The main source of TGF-β1 was astrocytes. These findings suggest that cells expressing c-Fos protein may play some roles to trigger microglial TGF-β1 is a key molecule that upregulates microglial P2Y6 receptors, and that TGF-β1 activation. mediates transformation of microglia into phagocytes. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 197 P1PM-12-22 P1PM-12-23 PHORBOL 12-MYRISTATE 13-ACETATE SUPPRESSES 2+ CELLULAR DISTRIBUTION OF ALPHA2A- Ca -DEPENDENT EXOCYTOTIC RELEASE FROM RAT ADRENOCEPTORS IN THE CEREBELLUM OF RATS ASTROGLIAL CELLS 1 1 2 1 Yue Liu, Fang Liu, Bao-Ming Li Keiichi Yasuda , Makoto Itakura , Kyota Aoyagi , Tsukiko Sugaya , Institute of Neurobiolgy, Fudan University, China Saori Yamamori1, Masami Takahashi1 1Department of Biochemistry, Kitasato University School of Medicine, Previous studies suggest that all the three subtypes of α2-adrenoceptors (α2A-, Japan, 2Department of Biochemistry, Kyorin University School of Medicine, α2B- and α2C-ARs) are present in the central nervous system. However, most Japan studies employed in situ hybridization to detect the mRNAs of these receptor Glial cells release various bioactive substances such as growth factors and subtypes and little is known about the cellular distributions of α2A-AR. neurotransmitters by exocytosis, and these functions are believed to be quite important This present study investigated the cellular localizations of α2A-AR in the for maintaining and modulating brain functions. To know the regulatory mechanisms cerebellum of Sprague-Dawley adult rats, using double immunofluorescence of exocytotic release in glial cells, we transiently transfected human growth hormone labeling and confocal laser scanning microscopy. Our results show that (hGH) in cultured rat astrocytes and in cultured rat glioma C6 cells. Enhanced almost all of the GFAP (glial fibrillary acidic protein) positive cells, primarily green fluorescent protein (EGFP)-tagged hGH was distributed in granular structures in cytoplasm of these cells. A significant amount of hGH was released in a Ca2+- Bergman cells and astrocytes, express α2A-AR, whereas few NeuN positive dependent manner after stimulating these cells with either ATP or ionomycin. Phorbol cells (mainly granule cells) and purkinje cells express it. The impressively 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), suppressed the large expression of α2A-AR in Bergmann cells may have specific functional hGH release from both astrocytes and C6 cells, and this suppression was reversed by a significance. PKC inhibitor, bisindolylmaleimide I (BIS). PMA caused a mobility shift of SNAP-23 in SDS-PAGE and this change was abolished by BIS. SNAP-23 was immunoaffinity purified from C6 cells with or without a treatment of PMA, and was analyzed by LC- MS/MS. Ser120 was revealed to be phosphorylated in a PMA-dependent manner. These results suggested that the exocytotic release from glial cells was negatively regulated by PKC, possibly through the phosphorylation of SNAP-23.
P1PM-12-24 P1PM-12-25 LIDOCAINE INHIBITS VOLTAGE-GATED PROTON GLAST IS ESSENTIAL FOR CYTOLOGICAL CHANNELS IN RAT MICROGLIA DIFFERENTIATION OF BERGMANN GLIA AND Tadashi Matsuura1, Takashi Mori1, Miyuki Kuno2, Megumi Hasaka1, Makoto CLIMBING FIBER MONOINNERVATION BY Sawada3, Kiyonobu Nishikawa1, Akira Asada1 SUPPRESSING ECTOPIC INNERVATION 1Department of Anesthesiology, Osaka City University Graduate School of 1 1 2 2 Taisuke Miyazaki , Miwako Yamasaki , Kouichi Tanaka , Masahiko Medicine, Japan, Department of Physiology, Osaka City University Graduate 1 School of Medicine, Japan, 3Department of Brain Science, Research Institute of Watanabe Environmental Medicine, Nagoya University, Japan 1Department of Anatomy, Hokkaido University, Japan, 2Department of INTRODUCTION Voltage-gated proton channels expressed abundantly on microglia Molecular Neuroscience, Tokyo Medical and Dental University, Japan are thought to be essential for phagocytosis. Local anesthetics are known to have a Glutamate transporters play a central role in shaping excitatory signaling by rapid variety of anti-inflammatory profiles which include suppression of phagocytosis in uptake of glutamate from the synaptic cleft and by restricting its spillover to adjacent monocytes. However, the effects of local anesthetics on microglial proton channels synapses. In the cerebellum, glutamate transporter GLAST is richly expressed in remain to be defined. Bergmann glial cells (BG) that enwrap Purkinje cells (PCs). In this study, GLAST- METHODS Actions of lidocaine on proton channels were studied in rat microglia deficient mice were analyzed to clarify anatomical patterns of multiple innervation. using the whole-cell recordings. The external and internal pHs were set to be 7.3 and In adult mutant mice, the distribution of climbing fiber (CF) terminals was 5.5. The pH of lidocaine (0.3 - 10 mM) test solutions was adjusted to 7.3. decreased proximally and multiple CF innervation was caused through frequent RESULTS Lidocaine decreased the current amplitude of proton channels in a dose- ectopic innervations by ascending and transverse CF branches. Particularly, ectopic dependent manner together with increase of the activation time constant. The IC50 innervations by transverse branches rarely occurred in developing mutant mice. was ~2.7 mM and the inhibition was reversible. The current-voltage relationship Furthermore, lamellate processes of mutant BG fibers were retracted progressively revealed that lidocaine shifted the reversal potential to more positive voltages. This toward the adulthood, resulting in severe impairments of glial enwrapping of PC resulting reduction of the driving force for H+ efflux, however, could explain less than dendrites and synaptic cleft. These results suggest that GLAST is essential for the 25% of total inhibition. maintenance of CF-mono innervation, through preventing local ectopic innervations DISCUSSION These results suggested that lidocaine inhibited proton channels by to nearby PCs and also through promoting cytological differentiation of BGs. GLAST modulating the channel properties. The inhibititory effects of lidocaine on proton thus appears to provide functional and structural insulations to suppress local crosstalk channels can lead to suppression of phagocytsis in microglia. between and beyond CF-to-PC units.
P1PM-12-26 P1PM-12-27 DEPENDENCE OF SYNAPTIC ACTIVITY ON MACROPHAGES EXPRESSING NG2 PROTEOGLYCAN MONOCARBOXYLATE TRANSPORT IN THE NUCLEUS IN THE ISCHEMIC AND TRAUMATIC RAT BRAIN OF THE SOLITARY TRACT OF THE RAT LESIONS ELICIT NEUROPROTECTIVE EFFECTS Masashi Nagase, Fusao Kato THROUGH MULTIPLE WAYS Department of Neuroscience, Jikei University School of Medicine, Japan Junya Tanaka, Anna Smirkin, Hisaaki Takahashi, Hajime Yano Transfer of lactate through monocarboxylate transporters (MCTs) from Department of Molecular and Cellular Physiology, Ehime University, astrocytes to neurons is one of the important mechanisms to fuel the neurons. Graduate School of Medicine, Japan In the hippocampus, a structure vulnerable to low energy supply, synaptic Macrophage-like cells accumulate in ischemic and traumatic rat brain lesions during transmission is suppressed by inhibiting MCT in the absence of glucose acute and sub-acute phases. Because such macrophage-like cells expressed NG2 (Izumi et al., 1997). Here we analyzed whether lactate transfer plays a chondroitin sulfate proteoglycan (NG2) as well as a macrophage-marker Iba1+, primary role in maintaining synaptic transmission in the structures more they were termed BINCs (Brain Iba1+/NG2+ Cells). 5-fluorouracil (5FU) was resistant to energy deprivation. To address this issue, we analyzed effects of intraperitoneally injected to ischemic or traumatic rats to kill BINCs specifically, a MCT inhibitor, alpha-cyano-4-hydroxycinnamic acid (CHCA), on neuronal because BINCs are highly proliferative. This treatment caused marked loss of BINCs activities in the nucleus of the solitary tract (NTS) in brainstem slices of in lesions and enlargement of the lesions. When BINCs were transplanted into the young rat. CHCA (1 mM, 15 min) did not markedly affect resting potential ischemic lesions that had been enlarged by 5FU-injection, the volume of the lesions Poster Session and action potential generation. In contrast, CHCA significantly decreased markedly reduced. These results indicate that BINCs play protective roles in the the amplitude of excitatory postsynaptic current without significantly ischemic events. BINCs were found to produce many kinds of neuroprotective factors affecting the paired-pulse ratio and AMPA-induced current. The amplitude such as BDNF, IGF-1, PDGF or HGF. In fact, when cocultured with embryonic of inhibitory postsynaptic currents was also decreased but to a lesser extent rat brain tissues or cells, BINCs markedly enhanced viability of the tissues or cells. than the excitatory ones. A plausible interpretation of these results is that in BINCs actively phagocytosed degenerated cell debris, a probable cause to enhance the NTS, unlike in the hippocampus, maintenance of synaptic transmission the regeneration of damaged brain tissue. Furthermore, BINCs have an ability to largely depends on the MCT-mediated energy supply even in the presence of transdifferentiate into glial cells or neurons. Through such multiple mechanisms, ambient glucose. BINCS elicit neuroprotective effects on the severely damaged brains.
198 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-12-28 P1PM-12-29 LONG LASTING POST MORTEM ACTIVITY OF METABOLIC CHARACTERISTICS OF NEURONS, MICROGLIA IN SITU IN THE MOUSE SPINAL CORD ASTROCYTES AND MICROGLIA IN PRIMARY 13 Eike D. Schomburg1, Payam Dibaj2, Heinz Steffens1, Fabien CULTURED CELLS - STUDIED BY C-NMR ANALYSIS - Nadrigny3, Clemens Neusch2, Frank Kirchhoff3 Tomoyuki Kanamatsu1, Kazuyuki Nakajima2, Akiko Koyama3, 1 1Institute of Physiology, University of Goettingen, Germany, 2Department Noriko Sawa 3 of Neurology, University of Goettingen, Germany, Department of 1Department of Environmental Engineering for Symbiosis, Faculty of Neurogenetics, Max-Planck-Institute of Experimental Medicine, Goettingen, Engineering, Soka University, Japan, 2Department of Bioinfomatics, Faculty Germany of Engineering, Soka University, Japan, 3Department of Biotechnology, Microglia (MG) shows a spontaneous high motility of its extensions. Upon local Faculty of Engineering, Soka University, Japan CNS injury processes of the surrounding microglia turn to the lesion to phagocytose We investigated the metabolic characteristics of neurons, astrocytes and microglia in degenerating axons and MG cells migrate towards the injured site. We now used primary cultured cells and metabolic trafficking between neurons and astrocytes using 2-photon laser-scanning microscopy to study the survival of MG reactions under 13C-NMR spectroscopy. Microglia, neurons, astrocytes and co-culture of neurons and ischemic conditions in the spinal cord of double-transgenic mice (CX3CR1-EGFP/ astrocytes with transwells were incubated with DMEM (glucose or glutamine free) for Thy1-EYFP). Spontaneous MG activity and the MG responsiveness to laser-evoked 12 hours and then incubated for 4 hours after an addition of [1, 6-13C] glucose or [3-13C] local injury were investigated first in situ in vivo (fully anaesthetised mice) and lactete or [4-13C] glutamine into the medium. When all types of cells were incubated then post mortem (p.m.), i.e. after cardiac and respiratory arrest in the progressively with [1, 6-13C] Glc, 13C-NMR spectra of acid extracts of neurons revealed that 13C degrading nervous tissue. Results: 1st hour p.m. spontaneous MG activity and early was incorporated into aspartate (Asp), glutamate(Glu), Lact and alanine, and those of MG reaction to injury almost unchanged; axons start developing irregular swellings. astrocytes revealed that 13C was dominantly incorporated into Gln and Lact, however 2-3 hours p.m. persisting clear reaction of MG to injury; increasing fragmentation of those of microglia showed only a peak of 13C-Glc. When three types of cells were axons. 4 and partly up to 10 hours p.m. still some MG response, but with decreasing incubated with [3-13C] Lact, spectra of acid extracts of neurons revealed that 13C was liveliness; further deterioration of axons. After 5 to 10 hours p.m. MG lost its incorporated into Glu, Gln and Asp, and those of astrocytes showed a high labeling responsiveness, but some spontaneous activity could remain for about 1 hour. The of Gln and an uptake of large amount of Lact, however those of microglia showed observations demonstrate that MG can survive long lasting anoxia and ischemia, no significant peak of 13C. These results suggest that the metabolic characteristics of obviously being able to fulfil its function even in dead nervous tissue. microglia differ from those of neurons and also those of astrocytes.
P1PM-12-30 P1PM-13-1 EXERCISE-INDUCED DEPLETION OF GLYCOGEN IN ORGANOTYPIC CULTURE OF ADULT RAT RETINA SOME BRAIN LOCI Amane Koizumi 1 1 2 Takashi Matsui , Masahiro Okamoto , Kentaro Kawanaka , Yukio National Institute for Physiological Sciences, Japan Ichitani3, Hideaki Soya1 1 Previously I and colleagues invented organotypic culture of adult rabbit Laboratory of Exercise Biochemistry, University of Tsukuba Graduate retina (PLoS One, 2007). This allowed us to transfect plasmids into adult School of Comprehensive Human Sciences, Japan, 2Department of Health 3 nervous tissue of rabbit retina to manipulate gene expression. However, and Nutrition, Niigata University of Health and Welfare, Japan, Department of Behavioral Neuroscience, Institute of Psychology, University of Tsukuba, because of limited rabbit genome resources, the culture system of adult Japan rodent retina would be much suitable for gene manipulation. The differences between rabbit and rodent retina are thickness and vascularity: rabbit retina Some brain loci utilize glucose as an energy source during exercise. On the other hand, brain glycogen is increasingly understood to be an important factor in brain is thinner and non-vascular tissue, diffusion is mainly needed to harvest it, energy metabolism. However, it is uncertain whether glycogen is utilized as an energy while rodent retina is thicker and vascular. Therefore, our organotypic culture source in the brain during exercise. This is because brain glycogen is rapidly depleted system should be improved for applying it for adult rodent retina. Here, I by activation of glycogenolysis enzymes under the hypoxic-ischemic conditions introduce organotypic culture system of adult rat retina. This is basically the that occur after animals are killed. Therefore, in our present study, by using a high- same design of our organotypic culture system of adult rabbit retina, ie an energy focused microwave irradiation (MI) method which can momentarily inactivate inter-phase chamber with high volume of Ames' medium (Sigma) agitated by glycogenolysis enzymes, we tried to accurately measure the changes in brain glycogen rotary shaker. For culturing adult rat retina, I increased the concentration of during exercise. Rats were subjected to running at 20 m/min for 2 hours. Nine brain horse serum in the medium to 10%. Rotation ratio was set at 55 rpm. These loci were collected following MI fixation immediately after cessation of exercise and were used for determination of glycogen. Glycogen levels in the cortex, hippocampus, improvements allowed me to keep adult rat retina for at least 3-4 days intact. hypothalamus, cerebellum, and medulla oblongata decreased more than 50 % with EGFP expression was detected 24 to 48 hours after gene transfection. Further exercise. On the other hand, glycogen levels in the septum, striatum, thalamus, application of this culture system of adult rat retina will be discussed. and midbrain were not significantly reduced by exercise. These results suggest that glycogen in some brain loci may serve as an energy store during prolonged exercise.
P1PM-13-2 P1PM-13-3 DEVELOPMENT OF P2X2-PURINOCEPTORS IN THE THE ENDOCOCHLEAR POTENTIAL DEPENDS ON TWO MOUSE RETINA K+ DIFFUSION POTENTIALS AND AN ELECTRICAL Makoto Kaneda1, Koichi Ito2, Yasuhide Shigematsu3, Yukio BARRIER IN THE STRIA VASCULARIS OF THE INNER Shimoda3 EAR 1Physiology, Keio University School of Medicine, Japan, 2Department of Fumiaki Nin1, Hiroshi Hibino2, Katsumi Doi3, Toshihiro Suzuki1, Yasuo Comparative Pathophysiology, Graduate School of Agricultural and Life Hisa1, Yoshihisa Kurachi1 3 Sciences, The University of Tokyo, Japan Medical Research Institute, 1Department of Otolaryngology-Head and Neck Surgery, Kyoto Prefectural Tokyo Women's Medical University, Japan University of Medicine, Japan, 2Division of Molecular and Cellular Pharmacology, Department of Pharmacology, Graduate School of Medicine, Osaka University, Previous reports have shown that signaling pathway in the retina develops Japan, 3Department of Otolaryngology, Osaka University, Japan before eye opening, and is refined by visual experience. In the present study, An endocochlear potential (EP) of 80 mV is essential for audition. We have used we examined the development of P2X2-purinoceptor signaling-pathway in + multibarreled electrodes to measure the potential, [K ], and input resistance in each the mouse retina by the immunohistochemical technique. P2X2-purinoceptor compartment of the stria vascularis. The stria faces two fluids, perilymph and endolymph, signaling-pathway starts to develop after eye opening in both ON- and and contains an extracelluar compartment, the intrastrial space (IS), surrounded by two OFF-cholinergic amacrine cells. By 5 weeks after birth, P2X2-purinoceptor epithelial layers, the marginal cell (MC) layer and that composed of intermediate and signaling-pathway is refined and became adult pattern, strong in OFF- basal cells. Fluid in the IS exhibits a low [K+] and a positive potential, called the intrastrial cholinergic amacrine cells while weak in ON-cholinergic amacrine cells. potential (ISP). We found that the input resistance of the IS was high, indicating this space is electrically isolated from the neighboring extracellular fluids. This arrangement The refinement of P2X2-purinoceptor signaling-pathway is independent of + visual experience. Our data showed that development of P2X2-purinoceptor is indispensable for maintaining positive ISP. Inhibiting the K transporters of the stria by anoxia, ouabain, or bumetanide caused the [K+] of the IS to increase and the intracellular signaling-pathway is a quite unique process in mouse retina. + [K ] of MCs to decrease, reducing both the ISP and the EP. Calculations indicate that the ISP represents the K+ diffusion potential across the apical membranes of intermediate cells through Ba2+-sensitive K+ channels. The K+ diffusion potential across the apical membranes of MCs also contributes to the EP. Interference with any of these elements in the stria vascularis can interrupt hearing. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 199 P1PM-13-4 P1PM-13-5 PUPILLARY LIGHT REFLEXES TO COLOR STIMULI RECEPTIVE FIELDS OF INTERHEMISPHERICALLY IN RATS USING A NEWLY DEVELOPED VIDEO CORRELATED NEURON PAIRS IN THE EARLY VISUAL PUPILOMETER WITH A DIFFUSE ILLUMINATION CORTEX SYSTEM 1 1,2 1,2 1 2 3 Yusuke Asada , Takahisa M Sanada , Izumi Ohzawa Atsuhiko Iijima , Munetaka Haida , Shigehisa Iida , Shigeaki 1 3 1 Graduate School of Frontier Biosciences, Osaka University, Japan, Sonoda , Isao Hasegawa 2 CREST, JST, Japan 1Division of Integrative Physiology, Graduate school of Medical and Dental Sciences, Niigata University, Japan, 2Department of Medical Education, Information in left visual hemifield is processed in the right visual cortex, and vice Tokai University, School of Medicine, Japan, 3Newopto Co., Ltd., Japan versa, in mammals with frontally located eyes. Callosal connections are thought to We analyzed pupillary light responses in rats to RGB color stimuli with a newly play an important role in seamless perception of the whole visual field. However, it is developed video pupilometer (Rap-10, Newopto, Japan). This system includes a CCD not known how the two visual hemifields are stitched together at the receptive field camera unit, an illumination unit, a stimulation unit, and an image analyzing unit. The (RF) level. How are RFs of callosally-connected neuron pairs organized? Are these doughnut-shaped illumination mounted around the camera lens put on an animal’s eye. connected neurons tuned to similar parameters such as orientations (OR) and spatial The light was transdermally input into the animal and diffused inside it. This lighting frequencies (SF) of visual stimuli? To address these questions, RFs were measured technique clearly illuminated animal’s pupil without traditional lamps so we had clear simultaneously for pairs of neurons across the two hemispheres. The presence of and precise pupil images without any lightning noise. We obtained pupil images in callosal connections was examined by cross-correlation. Pairs were found with a albino rats with this system and analyzed their pupillary RGB light responses. The small number of uni-directional neural connections as well as cases of common stimulation unit could produce red (660nm), green (525nm), and blue (470nm) light input. Preferred SFs were generally similar, but there was a significant tendency for stimuli, respectively and the luminance was controllable. The stimuli were guided with an optic fiber to the rat’s eye. We compared their pupillary responses and the blue callosally-related neurons to prefer opposite directions of stimulus movements. It is light stimulation elicited larger pupil constriction compared to red light stimulation. not clear how such tendency for opposite direction preference might be useful. One The time course of these responses was different from each other. The system may speculation is that this is a part of a mechanism for encoding optic flow information, be efficient for pupil studies, especially on responses to color stimuli in small animals where directions of visual stimuli are opposite. including non-pigmented rats. [Supported by: MEXT 18020017, CREST JST, and Global COE]
P1PM-13-6 P1PM-13-7 MANY OTHER ATTENTIONS BESIDES THE VISUAL A MODEL OF RETINAL GANGLION CELL SPIKE ATTENTION ARE EVOKED WITHIN THE DURATION OF GENERATION BASED ON STOCHASTIC ION CHANNEL THE VISUAL AXIS KEPT ON THE OBJECT KINETICS Yumi Suzuki1, Hiromi Fujii2, Tosaku Nikara3 Yoshimi Kamiyama, Yuichiro Sakuragi 1Division of Rehabilitation, Okitama Public General Hospital, Japan, 2Dept. Information Science and Technology, Aichi Prefectural University, Japan of Occupational Therapy, Yamagata Prefectural University of Health 3 The ganglion cells of the vertebrate retina form the pathway by which the Sciences, Japan, Tohoku Medical College, Japan retina communicates with the visual cortex. The ganglion cells convert the The aim of this study was to analyze the relations between visual attention graded potentials into a pattern of spikes whose characteristics is modulated by to be maintained and visual axis kept on the object. When subjects supped the synaptic and membrane currents. The ganglion cells respond with precise various amount of juice of the spoon from the plate on the desk, the speed and reliable spikes to randomly flickering light. A spiking model with a filter, and time of their movement of the spoon and the position which their visual threshold and internal noise sources has been proposed, and the model was used axis removed from the cup of the spoon (PVRC) were measured. When the to fit the spike responses of the ganglion cells (Keat et al., 2001). However, amount of juice in the cup of the spoon was decreased, the distance of the the underlying biophysical mechanisms of the spike reproducibility are not PVRC in all subjects was tending to shortness. However, the size of the well understood.Here we propose a stochastic model of spike generation in the distance of each subject was decided by individual as if based on own motor ganglion cells, based on discrete stochastic ion channels represented by Markov skill. When the visual axis was kept on the spoon, the speed of the spoon was processes. We modeled eight types of ion channels, i.e., Na, Ca(T), Ca(L), Kv, A, slower. The slowness derived by keeping the visual axis on the cup of the K(Ca), h and leakage channels. The proposed model showed precise and reliable spoon might be considered to be equal to maintaining the visual attention. spikes to randomly fluctuating current. In order to clarify the role of ion channel However, we concluded that the duration keeping the visual axis on the cup stochasticity, we evaluated the contribution of each channel in the spike timing, was composed of total of sensitive factors derived one after another such as by changing the single channel conductance in simulation. The results suggest an attentive vision, a waning confidence of decision, a fearing to failure and that the spike reproducibility is much influenced by the potassium channels, Kv so on. and A, not by the sodium channel, Na.
P1PM-13-8 P1PM-13-9 CYCLOPEAN IMAGE SIZE TUNING IN MACAQUE AREA IMMUNOHISTOCHEMICAL LOCALIZATION OF α1G V4 IS DEPENDENT ON BINOCULAR DISPARITY IN A SUBUNIT OF T-TYPE CALCIUM CHANNEL IN THE MANNER CONSISTENT WITH SIZE CONSTANCY DORSAL LATERAL GENICULATE NUCLEUS OF Shingo Tanaka, Ichiro Fujita MOUSE BRAIN Graduate School of Frontier Biosciences, Osaka University, Japan Laxmi Kumar Parajuli1, Yugo Fukazawa1, Masahiko Watanabe2, The perceived size of an object remains relatively stable despite changes in Ryuichi Shigemoto1 the retinal image size that accompany changes in distance from the observer. 1Division of Cerebral structure, National Institute for physiological sciences, This perceptual phenomenon is called size constancy. The brain uses both Japan, 2Department of Anatomy, Hokkaido University, Sapporo, Japan distance information and retinal image size to estimate the size of an object. The visual information from the retina to the visual cortex is transmitted via the To explore the neural basis for size constancy, we examined the interaction dorsal lateral geniculate nucleus (dLGN). Neural communication in the dLGN largely between binocular disparity and size information in macaque area V4, a depends on the precise subcellular distribution of ion channels, in which the T-type visual cortex known to be involved in stereopsis and size discrimination. We calcium channels plays a crucial role. The purpose of this study was to analyze the recorded cyclopean image size tunings of 96 V4 neurons in two macaque distinct subcellular localization of α1G subunit of T-type Ca2+ channel by means monkeys performing a fixation task at various binocular disparities. Random of highly specific antibody against this subunit. At the light microscopic level, the dot stereograms were to eliminate monocular cues for distance and size. thalamic region exhibited an intense immunoreactivity compared with other sub- Poster Session Eighty-five neurons were found to be selective for both binocular disparity cortical structures. The staining pattern was largely reduced in slices obtained from and cyclopean image size. In some cells, tuning curves to cyclopean image α1G knock-out animal. Electron microscopic analysis of immunogold demonstrated size were scaled depending on the binocular disparity while the preferred size that this channel is selectively distributed in the somato-dendritic surface of the dLGN became larger as the stimulus was presented nearer. This was consistent with cells. The dendritic plasma membrane exhibited 3 fold higher immunogold density psychophysical tested with the same stimuli in humans. From these results, than the somatic plasma membrane. The density of immunoparticles was negatively we suggest that V4 neurons contribute to size constancy. correlated with the dendritic cross sectional length. This selective localization of T-type calcium channels in dLGN suggests their involvement in regulating dendritic calcium dependent processes which may be important for sensory gating, sleep and arousal.
200 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-13-10 P1PM-13-11 INFORMATION DERIVED FROM NUCLEAR AND PRE- AND POSTOVULATORY AUDITORY BRAINSTEM PERIKARYON SIZE OF NEURONS RESPONSE IN NORMAL WOMEN Hui-Pin Lu, Paul W Poon Namrata Upadhayay1, Bishnu Hari Paudel1, Paras Nath Singh1, Physiology, National Cheng Kung University, Taiwan Shaligram Dhungel2, Bal Krishna Bhattarai3 For a given cell type, the nucleus usually remains rather constant in size and 1Department of Physiology, BP Koirala Institute of Health Sciences, Nepal, may vary when metabolism changes drastically. In the brain, neurons can 2Department of Anatomy, BP Koirala Institute Of Health Sciences, Nepal, appear with markedly different sizes. A relationship between nuclear and 3Department of Anesthesiology and Critical care, BP Koirala Institute Of perikaryon sizes has been reported at the human cerebral cortex and this Health Sciences, Nepal relationship could change under dysfunctional conditions like dementia. Studies with ovarian hormones on auditory brainstem response (ABR) have It remains unclear if a similar relationship holds across brain regions. contradictory reports although women have ABR shorter than men. This study If so it could be a useful index to assess functional changes based on a compared ABR between pre- and postovulatory phases of menstrual cycle in simple measurement of the nuclei. Here we determined the relationship consenting 40 healthy female volunteers (age 19±2.35 years). Ears were stimulated between nuclear and perikaryon sizes of randomly selected neurons from separately and simultaneously using standard protocol. ABR was recorded in pre- and 4 sensory and 5 motor regions in the rat brainstem from 7 um histological postovulatory phases. Ovulation estimated by measuring basal body temperature. The sections stained with Toluidine blue. Morphometric measurements using ABR wave latencies (WL) I, II, III, IV, V and inter-peak latencies (IPL) I-III, III-V, Image Proplus software showed a linear relationship between the nuclear I-V were compared between the two phases of menstrual cycle using Student paired and perikaryon sizes when plotted on the log scales over a nuclear size- t test. Postovulatory phase showed shorter right ear ABR WL II (mean ± SD: 2.77 range of 5-25 um in diameter. Results suggested the nuclear- perikaryon ±.16 vs. 2.82± .17 ms, p<.05) than preovulatory on separately stimulating the ears. On size relationship holds across brain regions and species. Preliminary data simultaneous stimulation, the postovulatory phase had shorter WL V (5.71± .18 vs. 5.81 from auditory brainstem of rats after prolonged sound exposure showed ± .19 ms, p< .01), IPL III-V (1.89± .16 vs. 1.94± .19 ms, p< .05), and I-V (3.88± .16 both enlargements in both nuclei and soma that could reflect the underlying vs. 3.95± .18 ms, p< .05) than in preovulatory. Other WL and IPL showed decreasing activity-driven plastic changes of the neurons. trend in postovulatory phase. Sensory conduction (measured by ABR) is better in postovulatory phase compared to preovulatory probably due to progesterone.
P1PM-13-12 P1PM-13-13 Ik,n CURRENTS IN ISOLATED INNER HAIR CELLS AUDITORY BRAINSTEM RESPONSE IN MICE FROM GUINEA-PIG COCHLEA Meihong Lu, Masataka Nishimura, Kazuya Saitoh, Wen-Jie Song Takashi Kimitsuki Department of Sensory and Cognitive Physiology, Kumamoto University, Department of Otorhinolaryngology, Kyushu University, Japan Japan In the mammalian cochlea, hair cells are anatomically and functionally Auditory brainstem response (ABR) is widely used for testing sensory divided into inner hair cells (IHCs) and outer hair cells (OHCs). The single transduction in the auditory pathway. Because mice can be used for studying row of IHCs receives nearly all the afferent innervations and are the primary molecular mechanisms of auditory transduction with transgenic strategies, it sensory receptors. Sound-induced vibrations onto the hair bundle are is desirable to establish a typical ABR in mice. So far few studies have been transduced into a receptor potential that controls afferent synaptic activity. done on mice ABR, and have reported inconsistent results. Here we aimed to Receptor potential is thought to be shaped by potassium conductances. In establish a typical ABR in mice. IHCs, three types of potassium currents (Ik,f, Ik,s and Ik,n) were distinguishable. We recorded responses between the left ear lobe and the vertex of the scalp Using whole-cell voltage-clamp recordings, we studied the Ik,n in acutely from eight anaesthetized adult mice, using subcutaneous wire electrodes. isolated IHCs of guinea-pig cochlea. Ik,n was blocked by the KCNQ-channel An one-cycle sine wave (0.1 msec period) was fed to a speaker for sound blockers linopirdine and XE991 but was insensitive to tetraethylammonium stimulation. We found that mice ABR typically exhibited six peaks; the first (Ik,f blocker) and 4-aminopyrdine (Ik,s blocker). Ik,n was 70 % activated five waves had fairly constant peak latencies among animals, while the sixth around the resting membrane potential of -60 mV and deactivated on wave was broader in duration and had more variable peak latencies. Similar hyperpolarization. There was no significant difference in the size of Ik,n observations were obtained at different stimulus intensity levels (50-80 dB, between the apical turn and basal turn IHCs. 10 dB steps), although peak latencies progressively became longer with decreasing intensity level. Immediately preceding the first wave, a response of small amplitude, oscillating at ~8 kHz for 5-6 cycles was also observed in all animals. We conclude that mice ABR has six peaks with a preceding oscillatory activity.
P1PM-13-14 P1PM-13-15 FLUID-MEDIATED COUPLING AND DRAG REDUCTION ANOMALOUS BROWNIAN MOTION IN ARRAYS OF CLOSELY APPOSED STEREOCILIA AND VISCOELASTICITY OF THE EAR'S Andrei Kozlov1, Johannes Baumgart2, Thomas Risler3, Corstiaen MECHANOELECTRICAL TRANSDUCER Versteegh1, Jim Hudspeth1 1 2 Andrei Kozlov, Daniel Andor, Jim Hudspeth Sensory Neuroscience, The Rockefeller University, USA, Institute for Sensory Neuroscience, The Rockefeller University, USA Aerospace Engineering, Faculty of Mechanical Engineering, Technische Universitaet Dresden, Germany, 3Laboratoire Physicochimie Curie UMR 168, The Brownian motion of a particle in a complex environment is known Institut Curie, France to display anomalous power-law scaling in which the mean squared The successful analysis of acoustical stimuli by the brain relies on high sensitivity and displacement is proportional to a fractional power of time. Using laser sharp frequency selectivity of the mechanoelectrical transducers in the ear, the hair interferometry and the analytical methods of microrheology, we examine cells. Each hair cell bears a bundle of closely apposed, cylindrical stereocilia whose nanometer-scale thermal motions of hair bundles in the internal ear and displacement controls the open probability of the mechanotransduction channels. show that these cellular organelles undergo fractional Brownian motion. Key to the hair bundle's function is a collective activity of the channels resulting Anomalous scaling is caused by viscoelasticity of the gating springs, the from the coherent motion of stereocilia. Here, we test a hypothesis that neighboring elastic elements that transmit energy in a sound to the mechanosensitive ion stereocilia move coherently because of strong hydrodynamic coupling resulting from channels. These results demonstrate a connection between rheology and an extensive overlap of their viscous boundary layers. Using laser interferometry, auditory physiology and indicate that the statistical properties of the thermal finite-element analysis, stochastic simulations, and mechanical measurements from a scaled physical model of a hair bundle, we demonstrate that―even at relatively low noise in the ear may be determined by the dynamics of a small number of key auditory frequencies―fluid coupling is both necessary and sufficient to account for molecules. the high coherence of motion. We conclude that grouping stereocilia in a hair bundle, with very small gaps between them, impedes fluid flow through the stereociliary array, forestalling separation between stereocilia, synchronizing channel activity, and reducing drag on a hair bundle―features that are essential for highly sensitive and sharply tuned hearing. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 201 P1PM-13-16 P1PM-13-17 CORTICOFUGAL MODULATION OF THE AUDITORY MODULATION OF OLFACTORY SIGNAL PROCESSING INFERIOR COLLICULUS NUCLEUS IN RAT: AN IN VIVO BY THE TRIGEMINAL NERVE INTRACELLULAR RECORDING STUDY Philipp Daiber, Raphael Steinfeld, Stephan Frings, Ying Xiong, Zhiru Zhu, Yonghai Zhang, Lei Han, Li Mi, Jing Yang Frank Moehrlen Department of Physiology, Third Military Medical University, China Department of molecular physiology, University of Heidelberg, Germany The inferior colliculus(IC) is a major auditory station that integrates In recent years, psychophysical experimentation has revealed that the trigeminal information from ascending, descending, and intrinsic sources. In this system plays a role in the modulation of olfaction. While the effects of trigeminal study, we investigated neuronal responses to acoustic and cortical stimulus activity on olfactory performance are well documented, little is known about the in the IC neurons of rats, using in vivo intracellular recording and labeling relevant interactions of olfactory and trigeminal systems on a molecular level. technique. The auditory responses of the IC neurons was examined at To demonstrate the innervation density trigeminal neurosecretory fibres, we membrane potentials at −40 to −80mV. Cortical stimulus elicited membrane stained against substance P and CGRP in the olfactory mucosa. Because potential elevation or depression of the IC neurons. This caused an increase neurosecretion is strongly affected by inflammatory processes, we also stained or a decrease in the neuronal response to sound stimulus. In the 69 neurons against various receptors of inflammatory mediators implicated in peripheral examined with auditory stimulus, 52 showed EPSP responses and 17 showed sensitization, e.g. the substance P receptor NK-1 and the bradykinin receptor IPSP responses. Of the 52 EPSP neurons examined with cortical stimulus, B2-R. 53% received potentiation and 25% hyperpolarization, 22% showed no effect. Our localization studies show a dense trigeminal innervation of the olfactory In the 17 IPSP neurons, 78% received corticofugal inhibition and 22% were epithelium and the olfactory bulb. Receptors of inflammatory mediators were not affected. The mean amplitude of the EPSPs evoked by acoustic stimulus localized prominently in the ciliary layer of the olfactory epithelium which was similar to that evoked by the cortical electrical stimulation. Thirty-six of suggests a possible role of these mediators in modulating olfactory transduction. the 63 physiological characterized neurons were anatomically identified. The To examine functional effects of neuromodulators on the sensory activity of results significantly improved our understanding of the cellular mechanism olfactory neurons, we used electro-olfactogramm (EOG) recordings. underlying corticofugal modulation in IC neurons.(Support by China NSFC This study is aimed at the systemic interactions between trigeminal and olfactory 30770681 and CSTC, 2008BB5020) signal processing and its role in odor perception.
P1PM-13-18 P1PM-13-19 THE ENDOPIRIFORM NUCLEUS FUNCTIONALLY INFLUENCES OF ORAL RINSE ON CONNECTS THE OLFACTORY AND GUSTATORY ELECTROENCEPHALOGRAM INFORMATION IN THE RAT Kotaro Fujimaki1, Shukan Okano2, Goro Fujimaki3 1 1 2 Tokio Sugai , Ryo Yamamoto , Yoshifumi Ueta , 1Dental, Pastoral Dental Office, Japan, 2Tokyo Medical and Dental Hiroshi Yoshimura3, Nobuo Kato1 University, Tokyo Metropolitan Institute for Neuroscience, Japan, 3Pastoral 1Department of Physiology, Kanazawa Medical University, Japan, 2Division Dental Office, Tokyo, Japan of Cerebral Circuitry, National Institute for Physiological Siences, Japan, 3 Oral rinse (OR) and mouth wash (MW) are becoming widely popular among busy Department od Oral and Maxillofacial Surgery, Kanazawa Medical people these days. However there is no report on the effect of OR and MW on University, Japan electroencephalogram (EEG). We analyzed the influence of OR and MW on human The endopiriform nucleus (EPN) is a large group of multipolar cells located EEG to select the moderate one. Subjects were five healthy men. Five materials (DW, in the depth of the pirifprm cortex (PC). Although many studies have MO, GU, RE, LI) were prepared as the test samples. Subjects were asked to keep one suggested that the EPN plays a role in temporal lobe epilepsy, the normal of OR or MW in the mouth for thirty seconds. EEG was recorded for two minutes function of the EPN remains unknown. In the present study, by using optical soon after the rinsing/washing of the mouth. Distribution of delta, theta, alpha and imaging of brain slices with voltage-sensitive dye, we found the signal beta waves were analyzed by the emotion spectrum analyzer. propagation from the PC or gustatory cortex (GC) to the EPN in normal MO, DW and GU had a moderate taste. On the other hand, RE and LI had a strong medium. A double pulse stimulation to either the PC or GC (inter-pulse taste. Moderate materials were favorable and alpha waves were frequently observed. interval: 20-100 ms) induced signal propagation to the EPN through the However, strong taste materials had negative effects and beta waves were mainly excitation in the agranular division (AI) of the insular cortex, with further observed. extension to the claustrum. A paired-pulse stimulation to the PC and GC OR and MW have been used every day or when brushing in some people. A pamphlet evoked the excitation in the AI, claustrum and EPN. Consistently, we found says OR/MW prevent "calmness of the dental plaque", "inflammation of gums" and c-Fos-positive cells following natural stimuli, such as feeding an apple, in "smelly breath." However some of the products had excessive stimulation. From this the PC, GC, EPN, AI and claustrum. These results thus indicate that the EPN study it is suggested to chose a moderate one if the materials have the same effect. may contribute to integration of olfactory and gustatory information.
P1PM-13-20 P1PM-13-21 TASTE-EVOKED VASODILATATION IN THE TONGUE TASTE PREFERENCES AND NEURAL RESPONSES TO STUDIED BY LASER DOPPLER FLOWMETRY SUGAR-ALCOHOLS IN C57BL/6 AND BALB/c MICE 1 1 1 Hisashi Ogawa1, Yuka Tomonaga2, Hideaki Nagami2, Yuji Orita2, Noritaka Sako , Hideo Katsukawa , Michiya Kobayashi , Kazunori Shiotsu3 Takashi Yamamoto2 1Department of Neurology, Kumamoto Kinoh Hospital, Japan, 2Department 1Department of Oral Physiology, Asahi University School of Dentistry, 2 of Research, Kumamoto Kinoh Hospital, Japan, 3Department of Pharmacy, Japan, Department of Nursing and Medical Care, Faculty of Health Kumamoto Kinoh Hospital, Japan Science, Kio University, Japan We studied possible induction of lingual vasodilatation by tastants in 15 volunteers Some sugar alcohols are widely used as anti-cariogenic sweeteners. The (20-29 years old; 9 male and 6 female). Tastants used were NaCl of various behavioral and receptor characteristics for these sweeteners, however, are concentrations from 0.39 mg to 50 mg/ 100 ml, as in the whole mouth method of not well understood. In the present study, to investigate whether there is clinical taste examination. Tastants (1 ml, 35°C) were applied in an ascending order a strain difference between C57BL/6 and BALB/c mice for receptivity of over the anterior tongue protruded from the mouth, followed by water rinse with sugar alcohols, we conducted behavioral and electrophysiological studies. As stimulation interval of 2 min. Changes in vasodilatation on the tip of the tongue was sugar alcohols, mannitol, xylitol, sorbitol and palatinit were used. In the 48 detected by laser-Doppler flowmetry with a non-contact probe (1 mm in diameter) hr two-bottle preference test, one of the above sweeteners vs distilled water (ALF21N, Advance Co. LTD., Japan). Care was taken not to infiltrate fluid into the (dw), was carried out. C57BL/6 mice preferred 0.1-0.3M xylitol, sorbitol Poster Session oral cavity. After repetitive application of water (1 ml, 35°C), vasodilatation to water and paratinit rather than dw. On the other hand, BALB/c mice did not prefer became transient. Tastants induced two phases of vasodilatation, early one with latency these sugar alcohols in all tested concentrations rather than dw. Either strain of 0~2 s as in water application, and late one with that of 2~5 s. The two phases were did not prefer 0.03-0.5M mannitol rather than dw. In the electrophysiological often fused into one. Vasodilatation began to increase around the cognitive threshold study, the chorda tympani responses to above sugar-alcohols except measured over the same tongue area in each subject, but the magnitude did not mannitol in C57BL/6 mice were suppressed by 2% pronase E, a sweet taste increase with NaCl concentration. Early phase was probably induced by the tactile suppressant. But the tongue treatment of pronase E did not suppress the component but late phase by the taste component. The present study may present a responses to sugar-alcohols in BALB/c. These results suggest that there are new method for objective evaluation of taste. strain differences for receptivity of anti-cariogenic sugar alcohols in mice.
202 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-13-22 P1PM-13-23 SPINDLE AFFERENTS DISCHARGE OF JAW-CLOSING FUNCTIONAL ROLES OF THE TONGUE IN MUSCLES DURING CHEWING DIFFERENT HARDNESS BREASTFEEDING IN DEVELOPING RATS OF FOODS IN AWAKE RABBITS Nanae Fukushima, Kumiko Yokouchi, Kenya Fujita, Tetsuhiro Zakir Hossain, Kensuke Yamamura, Masayuki Kurose, Fukuyama, Kyutaro Kawagishi, Tetsuji Moriizumi Mostafeezur Rahman, Yoshiaki Yamada Department of Anatomy, Shinshu University School of Medicine, Japan Division of Oral Physiology, Department of Oral Biological Science, Niigata Functional roles of the tongue in breastfeeding were examined in developing University, Japan rats in which the lingual or hypoglossal (XII) nerves had been injured Spindle afferents discharge of the jaw-closing muscles was recorded from the during the suckling period. The unilaterally lingual nerve-resected pups did mesencephalic trigeminal nucleus during chewing different hardness of foods in not show any suckling disturbance, whereas the bilaterally lingual nerve- awake rabbits to evaluate the role of muscle spindles in mastication. Eighteen units resected pups showed suckling disturbance with marked shortening in nipple were analyzed. Thirteen units discharged both during the jaw-closing and jaw-opening attachment time (42% of the control value) and a low survival rate (P1 pups: phases and remaining five discharged mainly during the jaw-opening phase. Spindle 33%; P11 pups: 41%). The XII nerve is made up of functionally different discharge increased significantly in the fast and slow closing phases for hard food. nerve branches: the medial branch related to protrusion of the tongue and When EMG activity increased spindle discharge increased, however the peak and the lateral branch related to its retraction. Newborn rat pups with bilateral time course of discharge were not correlated with that of EMG activity. In addition, resection of either of the XII nerve components (main trunk: XII-trunk; spindle discharge increased when the speed of closing was slow. Furthermore, spindle medial branch: XII-med; lateral branch: XII-lat) failed to suckle milk and did discharge was affected by the horizontal movement of the jaw in the fast closing, slow not survive. The unilaterally XII nerve-resected neonates showed different closing and opening phases depending on the side of chewing. In the opening phase, milk-suckling capabilities, which resulted in differences in survival rate (XII- spindle discharge increased in the late part of the phase where the velocity and length trunk: 38%; XII-med: 24%; XII-lat: 92%). The survival and growth of the of opening were high. Above findings suggest that muscle spindle afferents convey XII nerve-resected rats differed considerably depending on the injured XII sensory information about jaw movements so that CPG can conduct precise complex nerve components and the developmental ages of the rats at the time of nerve movements according to need during mastication. In addition, they act as servo- insult. We conclude that tongue sensation and tongue movement are very assistant for load compensation. important for suckling.
P1PM-13-24 P1PM-13-25 STRENGTHENING OF THE TACTILE SENSE CALLOSAL CONNECTIONS MAY MEDIATE CAPABILITY BY USE OF FINGERTIPS IPSILATERAL PROJECTION OF THE TAIL TO A MEDIAL Manabu Yoshioka PART OF THE PRIMARY SOMATOSENSARY CORTEX Division of health sciences, Kanazawa University graduate school of IN RATS medicine, Japan Tomio Hayama, Koji Hashimoto, Yuki Fujiyama, Yoko Iiboshi, The purpose of this study is to verify that using fingertips frequently makes Yuko Imamura, Misato Kanegae, Ryoko Kawashita, Mai Noda, the ability of tactile sensibility high. The subjects is a personal computer club Naomi Tashiro, Tatsuro Yasukochi student, and a general student and a visually-impaired student.In a previous Department of Physiology, Kumamoto University, Japan study, it was reported that frequency characteristics for a vibrotactile The present study electrophysiologically located projection from the tail on threshold of one point were not shown in a contactor area of less than 0.02cm2 the somatosensory map visualized using cytochrome oxidase histochemistry but frequency characteristics indicating minimum threshold of around 200Hz in a medial part of the primary somatosensory cortex (SI) and further were shown in a contactor area of more than 0.08cm2. It cannot be said that investigated callosal connections of the area in rats with a neuronal tracing vibrotactile threshold in dairy life is appropriately evaluated by one-point method using WGA-HRP. Both field potentials (FPs) evoked by electrical stimulation of the contra- and ipsi-lateral sides of the tail were located vibrotactil discrimination test because it is very influenced to the distribution in the same region, the most caudomedial portion in SI. Amplitudes of density of the receptors organ of the place where stimulation is added.In this the FPs evoked ipsilaterally (iFPs) were comparable to those of the FPs study, static two-point discrimination test, moving two-point discrimination evoked contralaterally (cFPs), while latencies of iFPs were slightly longer test and two-points vibrotactile discrimination test were used.The last test than those of cFPs. Neuronal tracing study showed that the tail region changed the vibration from one-point to two-point in the subjects in order had dense callosal connections. The results added the tail region on the SI to appropriately evaluate based on the hypothesis that fingertips which are somatosensory map and showed bilateral projections from the tail to the SI, often used for key punching and braille reading is more sensitive. Result of and further suggested that callosal connections of the tail region may mediate all experiments, we clarified that using fingertips more frequently made the ipsilaterally evoked field potentials. ability of tactile sensibility to stimulation high.
P1PM-13-26 P1PM-13-27 LONG AND SHORT TERM EFFECTS OF GLUTAMATE CALCIUM-BASED NEGATIVE FEEDBACK ON SPIDER MECHANOSENSORY NEURONS DURING ON SENSORY TRANSDUCTION IN SPIDER RANDOM STIMULATION MECHANORECEPTORS Paivi Helena Torkkeli, Keram Pfeiffer, Shannon Meisner, Andrew Andrew French, Ulli Hoeger, Shannon Meisner, Paivi H. Torkkeli S French Department of Physiology and Biophysics, Dalhousie University, Canada Department of Physiology and Biophysics, Dalhousie University, Canada The compound slit sense organ VS-3 in the patella of the spider, Cupiennius salei, consists of 7-8 cuticular slits, each innervated by a Mechanosensory neurons innervating the VS-3 slit sensilla in the spider (Cupiennius pair of bipolar mechanosensory neurons. VS-3 neurons are accessible to salei) patella receive extensive efferent innervation. The efferent fibers contain GABA, intracellular recording and mechanical stimulation in the periphery, where glutamate, octopamine and acetylcholine, and the mechanosensory neurons have mechanotransduction occurs. During mechanical stimulation, Ca2+ enters receptors to these transmitters. Activation of the structurally related ionotropic GABA 2+ - VS-3 neurons via voltage activated Ca channels when they are opened by and glutamate receptors opens Cl channels. However, while GABA depolarizes VS-3 action potentials. We used a Ca2+ sensitive fluorescent dye (Oregon Green neurons, glutamate does not have this effect. BAPTA-1) to show that intracellular [Ca2+] rises with a similar time course in When GABA was applied to VS-3 neurons while they were stimulated with all peripheral regions of VS-3 neurons following individual action potentials. pseudorandom noise signals, triphasic responses were observed, consisting of brief An antibody against Ca2+ channels showed that they are located in the same initial excitation, followed by short duration inhibition and ending in prolonged regions. Intracellular Ca2+ levels were raised experimentally by releasing excitation. Here, we applied glutamate to the VS-3 neurons during similar random intracellular caged Ca2+ (NP-EGTA) using UV illumination. Increased [Ca2+] stimulation. We observed a reduction in firing rate that returned to control level within reduced the receptor potential amplitude produced by mechanical stimulation ~100 s. During this inhibitory period the neurons’ sensitivity was reduced and their of the slits. Single-electrode voltage clamp recording showed that receptor information capacity decreased. Contrary to the GABA effects, glutamate did not current was reduced with a similar time course to receptor potential. These cause significant long term changes in neuronal excitability. These results suggest that experiments indicate that one function of the [Ca2+] increase during action in addition to Cl- channels, glutamate may also activate K+ channels and the resulting potential firing in VS-3 sensory neurons is to provide negative feedback K+ efflux may constrain depolarization and prevent excitation. control of neuronal sensitivity. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 203 P1PM-13-28 P1PM-13-29 POSTNATAL REFINEMENT OF GRAVITY-RELATED MECHANOSENSITIVE NMDA RECEPTORS SPATIAL CODING TO THE CENTRAL VESTIBULAR CONTRIBUTE TO RENAL SENSORY ACTIVATION IN CIRCUITRY RATS 1 2 3 Ka Pak Ng1, Lei Han1, Yau Pok Lau1, Chuan Li1, Chun Hong Lai1, Ming-Chieh Ma , Ho-Shiang Huang , Yih-Sharng Chen Daisy Kowk Yan Shum2, Ying Shing Chan1 1School of Medicine, Fu-Jen Catholic University, Taiwan, 2Department of 3 1Department of Physiology, The University of Hong Kong, Hong Kong, Urology, National Taiwan University Hospital, Taipei, Taiwan, Department 2Department of Biochemistry, The University of Hong Kong, Hong Kong of Cardiovascular Surgery, National Taiwan University Hospital, Taipei, Taiwan To investigate gravity-related spatial coding during postnatal development of otolith- The NMDA subtype of the ionotropic glutamate receptor is found in the periphery. related neurons in the downstream relay, inferior olivary (IO), and the upstream The present study tested whether NMDA receptors (NMDARs) are present in the ends relay, thalamus, of the vestibular circuitry, conscious rats were subjected to linear of afferent renal nerves in the renal pelvis, an area concerned mainly with transmitting translations on horizontal or vertical planes. Expression of c-fos was used as a marker sensation and the to reflex regulation of body fluid. The main NMDAR subunit, of neuronal activation. In IO, P11 was the earliest age at which 3-dimensional gravity- NMDAζ1 was found to be more abundant in the renal pelvis than the renal cortex related spatial orientation was recognized. This spatial map within IO reached adult and medulla, and was mainly colocalized with the pan-neuronal marker PGP9.5 or level by P28. In thalamus, topographic coding of 3-D spatial signals began at P28 the sensory nerve marker, the neurokinin-1 receptor. Intrapelvic administration of and reached adult level by P45. Further, immunohistochemical results demonstrated the NMDAR ligand D-serine caused a dose-dependent increase in substance P (SP) dynamic changes in the expression of glutamate receptors and GABA receptors in release and afferent renal nerve activity, but had no effect on arterial pressure. The developing IO. The nature of these excitatory and inhibitory inputs to IO neurons D-serine-induced sensory activation and SP release were abrogated by specific ion during development was also examined using whole-cell patch-clamp technique. channel blocker (+)-MK-801, the SP receptor blocker L-703,606, or dorsal rhizotomy. The amplitude of glutamatergic mEPSCs increased from P7 to P14 whereas the rise Increasing intrapelvic pressure resulted in an increase in afferent renal nerve activity time and decay time of GABAergic mIPSCs decreased from P7 to P14, indicating and a diuretic/natriuretic response. Interestingly, these effects were attenuated by prior progressive maturation of excitatory and inhibitory inputs to IO neurons. Taken administration of (+)-MK-801. These results indicate that NMDAR-positive sensory together, our result demonstrated developmental refinement of gravity-related spatial nerves are present in the renal pelvis and contribute to the renorenal reflex control of coding along the central vestibular circuitry. body fluid.
P1PM-13-30 P1PM-13-31 FORCE ENHANCEMENT OF TWITCH FORCE BY TWO DIFFERENT PATHWAYS FOR ITCH IN MAN STRETCH AND THE EFFECTS OF TEMPERATURE ON Hermann Otto Handwerker, Barbara Namer, Frauke Kosteletzky, IT IN NERVE-SKELETAL MUSCLE PREPARATION OF Clemens Forster THE FROG Department of Physiology & Pathophysiology, University of Erlangen/ Yoshiki Ishii1, Takashi Watari2, Teizo Tsuchiya3 Nuremberg, Germany 1Faculty of Health Care Sciences, Himeji Dokkyo University, Japan, Histamine is the most commonly used substance to induce itch under 2Graduate School of Medicine, Okayama University, Japan, 3Faculty of experimental conditions. Recent findings suggest that itch produced by Health, Teikyo Heisei University, Japan intradermal insertion of cowhage spicules in human is histamine independent. We aimed to investigate the different neuronal input and itch sensations evoked We investigated the mechanism of the enhancement of twitch force by stretch and the by cowhage and histamine. effects of temperature on it in nerve-skeletal muscle preparations of whole muscles of In microneurographic recordings from the peroneal nerves of healthy subjects it was iliofibularis isolated from the frog, Rana brevipoda. When a preparation was stimulated found that cowhage spicules activated all tested mechano-responsive C-units, but no indirectly and stretched, the twitch force after a stretch was enhanced remarkably in mechano-insensitive C-fibers. In contrast, histamine caused lasting activation only in comparison to that before a stretch at low temperature. The enhanced force by a stretch mechano-insensitive units. of 20% lo (resting muscle length) at low temperature was as high as the force by the Cowhage induced a more stinging, sharp and pricking itch sensation compared to direct stimulation. The phenomenon was not dependent on the velocity but on the histamine. After blind application of histamine with spicules the itch sensation rose amplitude of stretch. The enhanced force obeyed the length-force relationship when a faster than after application of cowhage spikules and the peak of the sensation was stretch was long enough. The amplitude of the action potential recorded extracellularly reached earlier. However, after scratching the itch sensation reappeared later after from muscle surface increased remarkably after a stretch, while it was same before histamine. and after a stretch when recorded from the nerve innervating muscle. These results Cowhage and histamine activate different non-overlapping populations of C-fibers in the present study suggest that the force enhancement by stretch in a nerve-muscle while inducing itch of similar magnitude, but of different quality. The two different preparation is caused by the increase in the transmission rate between nerve and forms of itch are also differently modulated by scratching and these differences muscle and the amplitude of the enhanced force is also determined by the length-force indicate that they are also differently processed in the brain. relation of muscle. Supported by the DFG, grant Ha 831-14/1&2
P1PM-13-32 P1PM-13-33 TACTILE STIMULATION AND DOPAMINE RELEASE IN ELECTRICAL CONDUCTIVITY AND TEMPERATURE OF THE RAT NUCLEUS ACCUMBENS THE SKIN IN ACUPUNCTURE POINTS Rie Shimoju1, Kimiko Maruyama2, Yuichi Mita3, Mieko Kurosawa1 Vladimir Dmitrijevich Musienko 1Center for Medical Science, International University of Health and Welfare, Department of Pathophysiology, Lugansk State Medical University, Ukraine Japan, 2Department of Rehabilitation, International University of Health 3 The mechanisms of therapeutic effect of acupuncture treatment are unclear and Wealfare Hospital, Japan, Course of Health and Welfare Sciences, today. Acupoints require detailed study because they are the main and International University Graduate School of Health and Welfare, Japan well known component of the meridian system of the traditional Chinese We investigated the effect of tactile skin stimulation on the dopamine (DA) medicine. The aim of the investigation was the study of both: electrical release in the nucleus accumbens in anesthetized rats. A coaxial microdialysis conductivity and temperature of the skin in acupoints. Clinical measurements probe was stereotaxically implanted in the left nucleus accumbens and perfused showed anomalous character of the skin electrical conductivity, contact with modified Ringer’s solution at a speed of 2 μl/min. The dialysate output from and radiant temperature in representative acupoint of the meridians. High the probe over consecutive periods of 5 minutes was directly transferred into the variability of the skin electrical conductivity in acupuncture points and HPLC by the autosampler and the amount of DA was measured with an electro- correlation their parameters with functional activity of correspondent organs chemical detector. The tactile stimulation consisted in stroking, either unilaterally and systems were determined. Irritation of acupoints by electromagnetic or bilaterally, the back of the animal for 5 min at a pressure of 80 mmH2O and microwave (54-75 GHz) during disease restores physiologic level of skin
Poster Session at a frequency of 1 Hz. Bilateral stimulation of the back significantly increased electrical conductivity. Certain interrelations between changes of electrical the DA release for 10 min after the onset of stimulation. An increase in DA conductivity and temperature indices were noted. Radiant temperature, as a release was also observed after stimulation of the back contralateral (right) to the rule, was higher within few grades than that of contact one. Some findings nucleus accumbens where release of DA was measured, but not after ipsilateral confirming existence of the meridian system and functional ties between the stimulation. The DA response to tactile stimulation applied bilaterally was meridians were noted. Original hypothesis of meridian was offered. Results abolished after electrical lesion of the left ventral tegmental area. These results of the present study have theoretical and practical value because they form show that tactile stimulation increases DA release in the nucleus accumbens, and scientific basis of acupuncture. that the effect is mediated via excitation of the ventral tegmental area.
204 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-13-34 P1PM-13-35 REGIONAL DIFFERENCES IN THERMAL COMFORT IN ESTABLISHMENT OF AN IN VITRO FORCED HUMANS EXPRESSION SYSTEM FOR VOMERONASAL Mayumi Nakamura1, Tamae Yoda2, Yuki Uchida3, Ken Tokizawa3, RECEPTORS USING A Cre-LoxP STRATEGY 3 1 Kei Nagashima , Kazuyuki Kanosue Toshiharu Namba1, Kazuyo Muramoto2, Keiko Moriya-Ito3, Masumi 1Faculty of Sport Sciences, Waseda University, Japan, 2Faculty of 3 1 3 Ichikawa , Hideto Kaba International Liberal Arts, Dokkyo University, Japan, Faculty of Human 1 2 Department of Physiology, Kochi Medical School, Japan, Division of Sciences, Waseda University, Japan 3 Physiology, School of Dentistry, Meikai University, Japan, Laboratory of Cell Sensations evoked by thermal stimulation (temperature-related sensations) Biology and Anatomy, Tokyo Metropolitan Institute for Neuroscience, Japan can be divided into two categories, temperature sensation and thermal Many mammals have a specialized chemosensory system for pheromones, the vomeronasal comfort. In this study, I examined regional differences in temperature- system. For mammals, pheromones convey various information about spices, age, related sensations with special attention to “thermal comfort”. We examined reproductive stage, social status and individual identity. They have dramatic endocrine regional differences in temperature sensation and thermal comfort by effects including the induction of estrus and pregnancy block via their receptors in the applying local temperature stimulation for the face, neck, chest, abdomen, vomeronasal sensory neurons which project their axons to the accessory olfactory bulb thigh, sole, and hand during whole-body exposure to mild heat or cold. The (AOB). Vomeronasal sensory neurons express two types of vomeronasal receptors, V1Rs thermal comfort seen in this study suggests that if given the chance, humans and V2Rs. In rodents, about 100 repertories for each receptor type have been identified. would preferentially cool the head in the heat, and maintain the warmth Moreover, V1Rs detect volatile pheromones, whereas V2Rs detect non-volatile pheromones of the trunk areas in the cold. Thermal stimulation of the hand produced like peptides and proteins. However, it is largely unknown what compound acts as a small effect for “whole-body” thermal comfort as compared with other pheromone and which receptor detects the pheromone. For elucidation of pheromone- regions. Thermal comfort of the neck was similar to that of the face for receptor pair, establishment of the system that can express only one receptor and easily detect cold stimulation and to that of the trunk for warm stimulation. Regional its response to a specific pheromone is hoped. Therefore, cultured vomeronasal cells were differences in thermal comfort investigated in this study cannot be explained infected with the adenovirus vector which has one V1R gene. After the forced expression, solely by the density or properties of the peripheral thermal receptors, and receptor expression and pheromonal response were evaluated. We found that a pheromone- consistent with the biological roles of each body part. specific response was observed only in vomeronasal cells cocultured with AOB neurons.
P1PM-14-1 P1PM-14-2 SIGNALLING MECHANISM INVOLVED IN MUSCARINIC THE INVOLVEMENT OF RHO-ASSOCIATED RESPONSES OF BOVINE CILIARY MUSCLE STUDIED KINASE IN RABBIT URETHRAL SMOOTH MUSCLE USING YM-254890, AN INHIBITOR OF THE Gq/11- CONTRACTION PROTEIN Michael Walsh1, Keith Thornbury2, William Cole1, Gerard 2 2 2 Akira Takai, Motoi Miyazu, Fuminori Yasui, Yoshiko Takai Sergeant , Mark Hollywood , Noel McHale 1 Department of Physiology, Asahikawa Medical College, Japan Department of Biochemistry & Molecular Biology, University of Calgary, Canada, 2Smooth Muscle Research Centre, Dundalk Institute of In the ciliary muscle, tonic contraction produced by cholinergic agonists requires Technology, Ireland continuous Ca2+ influx through non-selective cation channels (NSCCs) opened upon Rho-associated kinase (ROK) involvement in rabbit urethral smooth muscle stimulation of M3-muscarinic receptors (M3R). We examined effects of YM-254890 2+ contraction was investigated by examining the effects of Y27632 and H1152 on the (YM), a specific inhibitor of the G -protein, on contraction, NSCC currents and [Ca ] q/11 i contractile responses to electric field stimulation, membrane depolarization, and elevation induced by carbachol (CCh). alpha1-adrenoceptor activation. Both ROK inhibitors attenuated contractions elicited Isometric tension was recorded from ciliary muscle bundles excised from bovine by all three stimuli. Phosphorylation of three direct or indirect substrates of ROK was eyes. In 2-5 day cultured ciliary myocytes, whole-cell currents were recorded by 2+ analysed: myosin regulatory light chain (LC20), myosin targeting subunit (MYPT1) voltage clamp and the [Ca ]i was monitored using Fluo-4 fluorophore. Existence and of myosin light chain phosphatase (MLCP), and cofilin. Results: (i) at resting tension, localization of M3R and Gq/11 were examined by immunofluorescence microscopy. LC20 was phosphorylated at S19 (~0.5 mol Pi / mol) and phosphorylation did not Both phasic and tonic components of contractions evoked by 2 μM CCh were dose- change in response to KCl or phenylephrine; (ii) ROK inhibition had no effect on dependently inhibited by YM (10-1000 nM). In the cultured cells, CCh (2 μM) evoked 2+ LC20 phosphorylation at rest or in response to KCl or phenylephrine; (iii) under an NSCC current as well as an elevation of the [Ca ]i. Both initial and sustained resting conditions, MYPT1 was partially phosphorylated at T697 and T855 and cofilin phases of these responses were abrogated by YM (1-10 μM). Immunostaining of at S3, and phosphorylation was unaffected by KCl or phenylephrine. Conclusions: the cytoplasmic side of the cell membrane of ciliary myocytes revealed a dense ROK plays an important role in urethral smooth muscle contraction, but not via distribution of M3R and Gq/11. inhibition of MLCP or phosphorylation of LIM kinase (which phosphorylates cofilin). The tonic as well as phasic component of the ciliary muscle contraction is likely to be The high level of LC20 phosphorylation at resting tension suggests that activation of controlled by a Gq/11-coupled mechanism. ROK leads to alleviation of inhibition of cross-bridge cycling.
P1PM-14-3 P1PM-14-4 ERK AND P38MAPK REGULATE MYOSIN LIGHT CHAIN PKG TYPE I NEGATIVELY REGULATES TRPC6 BY PHOSPHATASE AND CONTRIBUTE TO CARBACHOL- PHOSPHORYLATION THROUGH INTERACTION WITH INDUCED CONTRACTION IN RAT INTESTINAL TRPC6 N TERMINAL DOMAIN SMOOTH MUSCLE Akira Honda, Zhong Jian, Lin Hai, Shinichi Takahashi, Ryuji Inoue Eikichi Ihara, Queena Yu, Lori Moffat, Mona Chappellaz, Justin Department of Physiology, Faculty of Medicine, Fukuoka University, Japan MacDonald cGMP/cGMP-dependent protein kinase (PKG) signaling pathway plays an Department of biochemistry and Molecular Biogoly, University of Calgary, important role in vasorelaxation. In this pathway, PKG is a key enzyme, Canada which lowers the intracellular Ca2+ level by protein phosphorylation. Both ERK and p38MAPK contribute to smooth muscle contraction. The objective of However, the downstream of PKG is poorly elucidated. Here, we report the present study was to examine whether MAPKs are involved in MLCP regulation that one of the transient receptor potential (TRP) non-voltage-gated cation and contribute to intestinal smooth muscle contraction. Contractile responses to channel super family, TRPC6, is a novel PKG substrate and is negatively carbachol (CCh) and effects of MAPK inhibitors on CCh-induced contraction were regulated by PKG phoshorylation. In TRPC6 over-expressed HEK cells, assessed in rat ileal longitudinal smooth muscle strips. Pretreatment with PD98059 TRPC6 channel activities were suppressed by SNAP, NOR-3, ANP and 8Br- (10 μM; ERK inhibitor) or SB203580 (10 μM; p38MAPK inhibitor), but not with cGMP. These inhibitory effects were strongly attenuated by the presence of Y27632 (10 μM; ROK inhibitor) significantly inhibited CCh-induced contractile force. inhibitors of guanylyl cyclase and PKG such as ODQ, KT5823 and DT-3. Decreased MLCP activity was observed during CCh-induced sustained contraction; Site directed mutagenesis for the PKG phosphorylation candidate sites this inhibition of MLCP was completely abolished by treatment with PD98059 or SB203580. However, the phosphorylation of MYPT1 (Thr-697 and Thr-855) and revealed that T69 is a putative PKG phosphorylation site, responsible for the CPI-17 (Thr-38) were not affected by addition of MAPK inhibitors. Application of negative regulation of TRPC6 channel. Furthermore, yeast two-hybrid system ML-7 (MLCK inhibitor) during CCh-induced sustained contraction elicits a relaxation was used to investigate the PKG-TRPC6 interaction. In the combination of that is dependent on MLCP. This relaxation rate was accelerated by PD98059 and PKG Iα or Iβ as a bait and TRPC6 N-terminal domain as a pray, α-Gal blue- SB203580 with proportional changes in LC20 phosphorylation levels. In conclusion, white colony assay showed positive in cGMP-independent manner. These both ERK and p38MAPK inhibited MLCP activity possibly by a novel mechanism results suggest that physical interaction of PKG may be required for the and contributed to CCh-induced sustained contraction of ileal smooth muscle. negative regulation of TRPC6 channel via phosphorylation on T69. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 205 P1PM-14-5 P1PM-14-6 ROLE OF PI3 KINASE/AKT PATHWAY IN CD38 SPONTANEOUS ELECTRICAL AND CALCIUM SIGNALS EXPRESSION IN AIRWAY SMOOTH MUSCLE IN ATYPICAL SMOOTH MUSCLES CELLS OF THE Mathur Srinivasan Kannan1, Joseph A Jude1, Krishnaswamy G MOUSE PYELOURETERIC SYSTEM 1 2 1 Tirumurugaan1, Reynold A Panettieri2, Timothy F Walseth1 Richard John Lang , Hikaru Hashitani , Mary A Tonta , Helena C 1 2 1Veterinary and Biomedical Sciences, College of Veterinary Medicine, Parkington , Hikaru Suzuki 1 2 University of Minnesota, USA, 2Department of Medicine, University of Department of Physiology, Monash University, Australia, Department Pennsylvania, Philadelphia, USA of Cell Physiology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan The ADP-ribosyl cyclase activity of CD38, a membrane protein, generates cyclic ADP-ribose, a calcium mobilizing agent. TNF-α induces CD38 expression through The pyeloureteric system, consisting of the renal pelvis and ureter, is unique in mitogen activated protein kinases and transcription factors NFκB and AP-1. We physiology in that morphological, electrophysiological and Ca imaging evidence determined PI3 Kinase role in CD38 expression in HASM cells. The inhibitors of suggests that ureteric peristalsis is driven by a population of spindle-shaped atypical PI3K, LY294002, decreased while, wortmannin increased CD38 expression and smooth muscle cells (atypical SMCs) situated in the proximal renal pelvis. Atypical SMCs in situ display high frequency spontaneous transient depolarizations (STDs) ADP-ribosyl cyclase activity in HASM cells following TNF-α (40ng/ml). Transient and Ca waves that are prevented upon blockade of IP3-dependent release of stored transfection of HASM cells with the catalytically active PI3K (p110) increased Ca and only reduced by nifedipine or ryanodine. After enzymatic dispersal, typical CD38 expression while the phosphatase and tensin homologue (PTEN) had no SMCs under perforated-patch voltage clamp are electrically quiescent. However, effect. Nuclear extracts from TNF-α-treated HASM cells in the presence of the PI3K a subgroup of SMCs display a distinctive cation-selective tail current as well as inhibitors showed no significant difference in NF-κB and AP-1 activation. LY294002 spontaneous transient inward currents (STICs) and large inward currents (LICs) that and wortmannin increased the basal level of ERK (p42/44) activation which was could well be responsible for STD generation. We conclude that Ca entry through ‘L augmented by TNF-α. Inhibition of ERK activation increased activated Akt. p110 type’ Ca channels and Ca-induced release of Ca from internal stores is involved in the transient transfection increased Akt activation while PTEN reduced it. The results synchronization (‘entrainment’) of STDs before they can provide a pacemaker drive indicate cross talk between PI3K/Akt and ERK in regulating CD38 expression. The to the SMC wall. However, the molecular identification of these pacemaker channels decrease in CD38 expression by LY294002 suggests the involvement of additional and the site(s) or mechanisms of entrainment that trigger propagating contractions pathways in regulating the CD38 expression in HASM cells. have yet to be established.
P1PM-14-7 P1PM-14-8 ROLE OF K+ CHANNELS IN REGULATING PROTEIN TRANSLATION INITIATION IS ACTIVATED BY SPONTANEOUS ACTIVITY IN DETRUSOR SMOOTH MECHANICAL STRETCH IN C2C12 MYOBLAST MUSCLE IN SITU OF THE MOUSE URINARY Naoya Nakai1, Fuminori Kawano1, Yoshihiko Oke1, Sachiko BLADDER Nomura1, Masahiro Terada2, Takashi Ohira2, Yoshinobu Ohira3 1 2 2 1 1Department of Health and Sports Sciences, Osaka University, Graduate Hikaru Hashitani , Masa Hayase , Kenjiro Kohri , Hikaru Suzuki 2 1Department of Cell Physiology, Nagoya City University, Japan, School of Medicine, Japan, Department of Health and Sports Sciences, 2 Graduate School of Frontier Bioscience, Osaka University, Japan, Department of Nephro-Urology, Nagoya City University, Japan 3 + Department of Health and Sports Sciences, Osaka University, Graduate The functional role of K channels in regulating spontaneous activity in detrusor School of Medicine and Graduate School of Frontier Bioscience, Japan smooth muscle (DSM) of the mouse urinary bladder was investigated using 2+ It has been proposed that mechanically-induced tension was the critical event for intracellular recording techniques and Ca imaging with fluo-4 fluorescence. initiating muscle hypertrophy. However, the molecular mechanisms involved in this Iberiotoxin or charybdotoxin (CTX) increased the amplitude of spontaneous process are still under investigation. In the present study, effect of mechanical stretch action potentials and prolonged their repolarizing phase without inhibiting after- on the protein translation initiation is studied in C2C12 myoblast. C2C12 cells were hyperpolarizations (AHPs). TEA (10 mM) suppressed AHPs, and further increased 2+ plated on fibronectin-coated silicone elastomer chamber in DMEM containing 10% the amplitude and half-duration of action potentials. Spontaneous Ca transients were fetal bovine serum. Cells were grown in CO2 incubator and subjected to 30 min of generated in individual DSM cells and seldom propagated to neighbouring cells to cyclic 15% biaxial stretch at a frequency of 1Hz. 30 min of cyclic stretch increased form intercellular Ca2+ waves. Transmural nerve stimulations initiated synchronous 2+ the phosphorylation levels of p38 mitogen activated protein kinase (MAPK) and Ca transients both within and across muscle bundles. CTX increased the amplitude extracellular-regulated kinase 1 and 2 (ERK1/2). p70 S6 kinase and eukaryotic 2+ of Ca transients, while the subsequent application of TEA (10 mM) increased their elongation factor 2, a marker for translation initiation and peptide chain elongation, 2+ half-duration. In addition, TEA increased the synchronicity of Ca transients within respectively, were also activated by cyclic stretch. These results suggest that p38 muscle bundles. These results suggest that BK and IK channels may contribute to the MAPK and/or ERK1/2 pathway may be involved in stretch-induced activation of action potential repolarization and restrict the excitability of DSM. In addition, the protein translation initiation process. This study is supported by Grant-in-Aid for activation of TEA-sensitive Kv channels may be involved in both repolarization and Scientific Research C (20500577 to N.N.) from the Ministry of Education, Culture, AHPs, and play a fundamental role in stabilizing DSM excitability. Sports, Science and Technology, Japan.
P1PM-14-9 P1PM-14-10 MECHANICAL STRETCH STIMULATES GLUCOSE RAC-SIGNALLING IS NECESSARY FOR INSULIN-, UPTAKE THROUGH A MECHANISM DISTINCT FROM CONTRACTION- AND AICAR-STIMULATED GLUCOSE THAT OF INSULIN-LIKE GROWTH FACTOR 1 IN UPTAKE IN INCUBATED MOUSE MUSCLES SKELETAL MUSCLE CELLS Thomas Elbenhardt Jensen, Erik A. Richter Masahiro Iwata1, Shigeyuki Suzuki2, Kimihide Hayakawa3, Takayuki Inoue2, Department of Exercise and Sport Sciences, University of Copenhagen, Keiji Naruse4 Denmark 1Department of Rehabilitation, Nihon Fukushi University, Japan, 2Program Cortical actin-turnover is critical to insulin-stimulated translocation of GLUT4 in Physical and Occupational Therapy, Nagoya University Graduate School and the small Rho-GTPase, Rac1, has been proposed as an essential regulator of of Medicine, Japan, 3ICORP/SORST Cell Mechanosensing, Japan Science 4 actin-turnover in L6 myotubes. Furthermore, a recent study in C2C12 myoblasts and Technology Agency, Japan Department of Cardiovascular Physiology, reported that AMPK may regulate the activation of Rac1. Here, we tested the Okayama University Graduate School of Medicine, Japan effect of the actin-depolymerising agent, latrunculin B, and a novel Rac1- Objective: Both insulin-like growth factor 1 (IGF-1), an autocrine/paracrine factor potentiated inhibitor, EHT1864, on glucose uptake into fully differentiated mouse soleus myogenesis, and mechanical stimuli such as stretch increase glucose transport in skeletal muscle. (SOL) and EDL muscles ex vivo in unstimulated basal condition or in response Some reports suggest that IGF-1 is secreted from skeletal muscle cells in response to stretch. Therefore, we examined whether IGF-1 is involved in the mechanism by which mechanical stretch to insulin, electrically induced contraction or the AMPK-activator, AICAR. regulates glucose transport using cultured muscle cells. Methods: C2C12 myotubes were treated Depending on the muscle, latrunculin B significantly inhibited insulin (60% Poster Session with various combinations of IGF-1 (maximum 10 nM), cyclic stretch (110% of original length, at in SOL, 20% in EDL), contraction- (50% in SOL, 80% in EDL) and AICAR 1 Hz, for 30 min), dantrolene (an inhibitor of Ca2+ release from sarcoplasmic reticulum) (25 μM), (~50% in EDL)-stimulated 2-deoxyglucose (2DG) uptake (p < 0.01). EHT1864 and/or phosphatidylinositol 3-kinase inhibitor wortmannin (1 μM), after which 2-deoxy-D-glucose reduced non-stimulated uptake by 60-80% and nearly prevented stimulation of (2-DG) uptake was measured. Results: IGF-1 increased 2-DG uptake, and this created an additive 2DG uptake in both muscles by all 3 stimuli (p < 0.001). These data suggest that effect with cyclic stretch. IGF-1-stimulated 2-DG uptake was not blocked by dantrolene, whereas the stretch-stimulated effect was abolished. Conversely, the IGF-1-stimulated 2-DG uptake was the actin-cytoskeleton is partly required for GLUT4 translocation both insulin prevented by wortmannin, which did not prevent the stretch-stimulated 2-DG uptake. Conclusions: and non-insulin stimuli. Rac1 is proposed to provide a major essential signal to These results suggest that mechanical stretch stimulates glucose uptake in skeletal muscle through a basal and stimulated glucose uptake and, by comparison with the effect of actin mechanism distinct from that of IGF-1. depolymerisation, likely with actions beyond cytoskeletal turnover.
206 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-15-1 P1PM-15-2
PGE2-INDUCED ICAM-1 EXPRESSION IS MEDIATED BY A RAP1 SMALL GTPase ENHANCES VE-CADHERIN- PI3K/NF-κB SIGNALING PATHWAY DEPENDENT CELL ADHESION BY INDUCING ACTIN Tae-Yeoup Park, Yi-Sook Jung, Eun Joo Baik, Chang-Hyun Moon, BUNDLING AT CELL-CELL CONTACTS Soo Hwan Lee Kazuomi Noda, Jianghui Zhang, Shigetomo Fukuhara, Department of Physiology, Ajou University, School of Medicine, Korea Atsuko Sakurai, Naoki Mochizuki Induction of ICAM-1 expression is implicated in the various brain pathologies. Department of Structural Analysis, National Cardiovascular Center Research Institute, Japan In the present study, we investigated the roles of prostaglandin E2(PGE2) in the Vascular endothelial-cadherin (VE-cad) is a cell-cell adhesion molecule involved in expression of ICAM-1 in bEnd.3. We demontrated that PGE2 induced ICAM-1 expression at the levels of protein and mRNA in the time and dose dependent the formation of endothelial adherens junctions. Previously, we have reported that cyclic AMP (cAMP) enhances VE-cad-dependent cell adhesion through an Epac-Rap1 manner. Sulprostone, an EP1/3 agonist, and 1-OH-PGE1, an EP3/4 agonist signaling pathway. Here, we further scrutinized the molecular mechanism underlying mimicked the effect of PGE2. However, butaprost, an EP2 agonist, failed to the stabilization of cell-cell adhesion initiated by cAMP. show significant effect. PGE2 induced the phosphorylation of IκB and the Forskolin (FSK; an adenylyl cyclase activator) induced actin bundling close and translocation of NF-κB into nucleus. NF-κB inhibitors such as BAY-11 and parallel to the cell-cell borders and accumulation of VE-cad at the cell-cell contacts. MG-132 diminished the PGE2-induced ICAM-1 expression. While dbcAMP and Fluorescence recovery after photobleaching analysis using VE-cad carboxy-terminally forskolin stimulated ICAM-1 expression, PI3K inhibitor LY294002 attenuated fused with GFP (VEC-GFP) revealed that FSK stabilized VEC-GFP at the cell-cell contacts. The stabilizing effect of FSK was prevented by an actin depolymerization PGE2-induced ICAM-1 expression. Though PGE2 increased the phosphorylation of ERK and JNK, inhibitors of MAPKs did not show any significant effect agent, latrunculin A. Furthermore, a VEC-GFP mutant lacking β-catenin (ctn) binding on PGE -induced ICAM-1 expression. Taken together, these data suggest that site and an another mutant VEC-GFP in which cytoplasmic domain of VE-cad was 2 replaced with α-ctn exhibited less and more stabilization at the cell-cell contacts, PGE2 induces ICAM-1 expression through ligation with the specific EP receptor respectively. sutype(s) followed by the activation of NF-κB and PI3K signaling pathways. Collectively, these results suggest that a cAMP-Epac-Rap1 pathway initially induces This study was supported by grant from Ajou University Medical Center, actin bundling close to cell-cell contacts, resulting in recruitment of VE-cad through α/ Gyunggi-do through CCRB-GRRC and Brain Korea 21 for Medical Sciences. β-ctn and stabilization of cell-cell adhesion.
P1PM-15-3 P1PM-16-1 REGULATION OF ISOPRENYL TRANSFERASE TRANSCRIPTIONAL REGULATION OF THE RAPID ACTIVITY BY SIGNALS FROM HETEROTRIMERIC G ANTI-SECRETORY RESPONSE TO ESTROGEN IN PROTEIN-COUPLED RECEPTORS FEMALE RAT DISTAL COLONIC CRYPTS Isao Matsuoka, Masaaki Ito, Yomogida Shinichi Fiona O'Mahony, Rodrigo R Alzamora, Brian J Harvey Laboratory of Pharmacology, Faculty of Pharmacy, Takasaki University of Molecular Medicine, Royal College of Surgeons in Ireland, Ireland Health and Welfare, Japan In the distal colon, basolateral KCNQ1 channels carry out the K+ recycling Isoprenylation of small G-proteins at carboxyterminal cysteine is critical for their required for Cl- secretion. 17 β-estradiol (E2) inhibits KCNQ1 via PKC δ membrane localization and molecular switch functions. HMG-CoA reductase (1). This inhibition occurs only in the female distal colon, which has a higher inhibitor is known to decrease cellular pool of isoprenoids, such as farnesyl expression level of PKC δ compared to the male tissue. We investigated pyrophosphate and geranylgeranyl pyrophosphate, thereby inhibiting the lipid whether the anti-secretory response was regulated throughout the estrous modification of various small G proteins. However, regulation of isoprenyl cycle. Distal colonic epithelia from Sprague-Dawley rats were used for short transferase activity has yet to be investigated. In this study, we investigated circuit current measurements (Cl- secretion stimulated by a CAMP agonist). effects of activation of heterotrimeric G protein-coupled receptor (GPCR) E2 (10 nM) inhibited Cl- secretion at the estrus, metaestrus and diestrus on protein farnesyl transferase (FTase) activity in human aortic endothelial stages of the cycle. A weak inhibition of Cl- secretion by E2 was observed cells (HAECs). FTase activity was determined by measuring farnesylation of in proestrus. An increase in PKC δ levels throughout the cycle correlated fluorescent dansylated peptide substrate. HAEC lysates contained FTase activity. with an enhanced anti-secretory effect of E2. PKC δ was up-regulated at a Stimulation of HAECs with sphingsine-1-phosphate and lysophosphatidic transcriptional level via the transcription factor CREB. Confocal microscopy acid increased FTase activity in a pertussis toxin-sensitive manner. In contrast, analysis revealed a rapid phosphorylation of CREB at Ser133 in the nuclei of fluvastatin did not affect FTase activity. Addition of GTPγS and tyrosine colonic crypt cells following E2 treatment. This study demonstrates estrogen phosphatase inhibitor to HAEC lysate increased FTase activity. Such modulation regulation of fluid secretion in the rat distal colon both at non-genomic and by GTPγS or tyrosine phosphatase inhibiter did not observed with recombinant genomic levels. FTase enzyme. These results suggest that FTase activity is regulated by GPCR (1) O Mahony F et al., J. Biol. Chem. 2007; 282(34):24563-73. signal in a tyrosine phosphorylation-dependent manner. Supported by the HEA-PRTLI Programme and the Wellcome Trust.
P1PM-16-2 P1PM-16-3 DIFFERENT FUNCTION OF CELL-SPECIFIC CLCA MULTI PATHWAYS OF ATP-RELEASE IN MAMMARY ISOFORMS IN THE RAT SUBMANDIBULAR GLAND EPITHELIAL CELLS REVEALED BY ATP IMAGING 1 2 3 Jun Yamazaki , Kazuhiko Okamura , Kazunari Ishibashi , Kishio Furuya1, Kyoko Harada1, Masahiro Sokabe2 1 Kenji Kitamura 1Cell Mechano-sensing Project, JST, Japan, 2Department of Physiology, 1Department of Physiological Science & Molecular Biology, Fukuoka Dental Nagoya University Graduate School of Medicine, Japan 2 College, Japan, Department of Morphological Biology, Fukuoka Dental Mammary epithelial cells are highly sensitive to mechanical stimulations and College, Japan, 3Department of Functional Bioscience, Fukuoka Dental College, Japan release ATP. The ATP surrounds mammary alveoli and acts as autocrine and 2+ - paracrine mediators on the secretory and myo- epithelial cells. This mechano- Ca -dependent Cl transport is known to be important to modulation of the purinergic coupling regulates or modulates milk ejection. To investigate the composition of saliva. In the present study, we found that a member of the CLCA family, rCLCA-f, is strongly expressed in the striated ducts of the submandibular mechanism of ATP release from the cells, we have developed a real-time ATP gland (SMG) of the rat. Transient transfection of HEK293 cells with rCLCA-f imaging system under microscope. Bioluminescence emitted by the reaction cDNA increased the marked Ca2+-dependent Cl- conductance evoked by of Luciferin-Luciferase and ATP is detected using a high quantum-yield ionomycin. Retrograde injection of rCLCA-f siRNA into a submandibular duct of image intensifier and a high performance cooled-CCD camera. The system a rat significantly increased the Cl- concentration of the saliva collected after i.p. enables the ATP imaging with 100 ms time-resolution under an upright - administration of pilocarpine, suggesting a physiological role of rCLCA-f in ductal Cl microscope with x20 water immersion objective. The system also enables transport. We also discovered expression of a truncated isoform, rCLCA-t (60 kDa), - simultaneous imaging of Nomarski during ATP luminescence imaging. Using in the SMG. Although rCLCA-t failed to increase Cl conductance in the transfected this system we have found mechanically (touch and stretch) induced ATP- HEK293 cells, it reduced surface expression of mature β -integrin and cell adherence. 1 release, spontaneous ATP-release especially in Calcium-free solution, and Immunohistochemistry of the rat SMG revealed rCLCA-t to be located only in the basal, undifferentiated cells of the excretory ducts, where integrins were observed to be ligand gated ATP-release in mammary epithelial cells and mammary organs. localized in the perinuclear region. The striking differences between the two isoforms These pathways possess different kinetics and pharmacological properties. suggest that cell-specific splicing of rCLCA mRNA affords epithelial cells different Multi pathways may work concomitantly to achieve mechano-purinergic functions. coupling in mammary glands. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 207 P1PM-16-4 P1PM-16-5 MKP1-DEPENDENT ERK DEPHOSPHORYLATION PARACELLULAR MAGNESIUM TRANSPORT IS UP- STIMULATES Na+ REABSORPTION THROUGH BETA- REGULATED BY PHOSPHORYLATION OF CLAUDIN-16 AND GAMMA-ENaC EXPRESSION IN HYPOTONIC Akira Ikari, Midori Ito, Yohei Sasaki, Yasuhiro Yamazaki, STRESS Masao Miwa, Junko Sugatani Naomi Niisato, Mariko Ohta, Yoshinori Marunaka Department of Pharmaco-Biochemistry, University of Shizuoka, Japan Department of Molecular Cell Physiology, Kyoto Prefectural University of Renal magnesium filtrated in the glomeruli is predominantly reabsorbed Medicine, Japan through the paracellular pathway in the thick ascending limb TAL of Henle. We have indicated that hypotonic stress increases Na+ reabsorption in renal epithelial Claudin-16 appears to function as a paracellular pore for Mg2+. We found A6 cells by stimulating both translocation of ENaC to the apical membrane through that claudin-16 expression leads to an increase of transepithelial electrical an EGFR-JNK-dependent pathway in the early phase and induction of ENaC resistance (TER) and the paracellular transport of divalent cations. The mRNA expression via a p38-dependent pathway in the late phase. Although ERK tight junctional localization of claudin-16 was regulated by a cAMP/PKA- is known to suppress ENaC expression, the role of ERK in hypotonic regulation of + dependent phosphorylation. The de-phosphorylated claudin-16 was Na reabsorption via ENaC expression is still unclear. In this study, we found that translocated into lysosome. The interaction between claudin-16 and PKA hypotonic stress caused dephosphorylation of ERK by elevating MAPK phosphatase was promoted by dibutyryl-cAMP (DBcAMP) in a concentration-dependent 1 (MKP1) mRNA expression via a p38-dependent pathway. We next clarified the manner, indicating that PKA directly interacts with claudin-16. S217A relationship between the ERK dephosphorylation and ENaC mRNA expression. Application of an MKP1 inhibitor, which caused activation of ERK, suppressed mutant of claudin-16 was not phosphorylated by PKA. This mutant was the ENaC expression, whereas inhibition of ERK by application of MEK inhibitor distributed in lysosome and did not increase TER and magnesium transport. enhanced the ENaC expression in hypotonic stress. Further, a p38 inhibitor rescued These results indicate that the PKA-dependent phosphorylation of Ser217 ERK from its inactivated form caused by MKP1. Based on these observations, it is is essential for its localization in the tight junction. The activation of a suggested that hypotonic stress inactivates ERK through a p38-dependent induction of polyvalent cation-sensing receptor (CaSR) decreased PKA activity, TER and MKP1, releasing expression of ENaC mRNA from ERK-dependent suppression. This magnesium transport, which were recovered by co-treatment with DBcAMP. novel mechanism contributes to hypotonic stimulation of Na+ reabsorption in the late We suggest that the activation of CaSR might prevent excess reabsorption of phase. divalent cations and back-flow from peritubular space to tubule lumen.
P1PM-16-6 P1PM-16-7 EGF AND NEUROTENSIN MEDIATED PROLIFERATION REGULATION OF EPIDERMAL TIGHT JUNCTION IN HT-29 COLON CELLS; IS NHERF-1 THE KEY? PERMEABILITY BY AN INTESTINAL ABSORPTION Wade Andrew Kruger1, Gregory R Monteith2, Philip Poronnik1 ENHANCER SODIUM CAPRATE 1 1 1 1School of Medical Sciences, RMIT University, Australia, 2School of Masumi Kurasawa , Shohei Kuroda , Naoko Kida , Pharmacy, The University of Queensland, Australia Takuya Yamamoto2, Ai Oba1, Hiroyuki Sasaki3 Epidermal growth factor (EGF) and neurotensin (NT) are important 1Cutaneous Drug Research Department, POLA Chamical Industries, INC., synergistic factors implicated in epithelial cell cancer progression. Our Japan, 2Intellectual Properties Strategy, R&D Planning Department, POLA research focuses on the roles of PSD-95/Dlg/Zo-1 (PDZ) scaffold in Chamical Industries, INC., Japan, 3Department of Molecular Cell Biology, mediating cell signalling in epithelia. The Na+/H+ exchanger regulatory Institute of DNA Medicine, The Jikei University School of Medicine, Japan factor 1 (NHERF-1) a PDZ scaffold has been shown to interact with Tight junction (TJ) in epithelia and endothelia restricts paracellular flux of water and solutes. the EGF receptor. This study was undertaken to investigate the role of TJs exist also in epidermis, houever, physiological significance of epidermal TJ is largely NHERF-1 in EGF and NT mediated proliferation of HT-29 colorectal cancer unknown on account of its structural complexity. Sodium caprate (C10) is a well-known cells. Infection of HT-29 cells with a lentivirus expressing siRNA against absorption enhancer that elicits dilatations of TJ and increase paracellular permeability NHERF-1 reduced endogenous levels of NHERF-1 by 80±5%. MTT-assays in the intestine. To help understanding epidermal TJ functions, we applied 1mM C10 on demonstrated that silencing NHERF-1 reduced basal cell proliferation cultured human epidermal keratinocytes (HEKs) for 24hrs. By C10 treatment, transepithelial by 57±12%.Treatment of the cells with EGF or NT increased the rate of electrical resistance decreased and paracellular permeability increased although expression of TJ-related molecules did not decrease. Immunofluorescence microscopy and immuno-replica proliferation by 30±2% (EGF) and 17±5% (NT). No increases over these electron microscopy showed that localization of the TJ-related molecules was dispersed levels were observed with EGF and NT combined, suggesting signalling and TJ strands altered into poor networks on the cell surface. The fragmented strands were via similar endpoints. In cells silenced for NHERF-1 these growth effects observed also in C10 treated reconstructed epidermis. All functional and structural alterations were abolished. The peak Ca2+ response to NT was reduced by 70±5% of 2+ were restored within a few hours after C10 removal. Our results suggested that C10 induced the control in these cells, as determined using the Ca sensitive dye Fluo-3. reversible physical TJ disruption in the HEKs and the reconstructed epidermis and showed However, co-immunoprecipitation of a GFP-tagged C-terminus of NTR-1 importance of proper localization of TJ-related molecules on the cell membrane for the TJ showed no detectable interaction to NHERF-1 in HT-29 cells. barrier function.
P1PM-16-8 P1PM-17-1 EPIDERMAL TIGHT JUNCTION MAINTAINS CALCIUM THE PI3K/AKT/FOXO-3A/P27KIP1 PATHWAY DISTRIBUTION IN HUMAN RECONSTRUCTED CONTRIBUTE TO ANTI-INFLAMMATORY DRUG EPIDERMIS AND REGULATES THE EPIDERMAL CAUSED SUPPRESSION OF PROLIFERATION IN DIFFERENTIATION HUMAN OSTEOBLASTS Tetsuo Maeda1, Shohei Kuroda1, Masumi Kurasawa1, Ai Oba1, 2 3 Ching-Ju Li, Je-Ken Chang, Chia-Hsuan Chou, Gwo-Jaw Wang, Takuya Yamamoto , Hiroyuki Sasaki Mei-Ling Ho 1Cutaneous Drug Research Department, POLA Chemical Industries, INC., Japan, 2Intellectual Properties Strategy, R&D Planning Department, POLA Orthopaedic Research Center, Kaohsiung Medical University, Taiwan 3 Chemical Industries, INC., Japan, Department of Molecular Cell Biology, Akt has been indicated to suppress p27kip1 promoter activity through Forkhead box Institute of DNA Medicine, The Jikei University School of Medicine, Japan O (FoxO) in various cells. We previously found that anti-inflammatory drugs (AIDs) Tight Junction (TJ) among adjacent epithelial or endothelial cells control paracellular up-regulated p27kip1 and this effect may play an important role in AID-caused cell permeability of solutes. In this study, we applied sodium caprate (C10) which elicits cycle arrest in hOBs. Accordingly, we hypothesized that AIDs may enhance p27kip1 dilations of TJ on human reconstructed epidermis, and investigated the role of TJs for calcium ion (Ca2+)distribution in the epidermis. The localization of Ca2+ was observed by expression through altering Akt/FoxO pathway in hOBs. In this study, we found that ion-capture electron microscopic cytochemistry and electron energy-loss spectroscopy. By the three classes of AIDs suppressed canonical level of p-Akt accompanied with C10 treatment, the epidermal Ca2+ localization was largely altered compared to untreated elevating FoxO-3a, and enhancing promoter activity, mRNA and protein expressions 2+ kip1 Poster Session epidermis. Ca -containing deposits appeared in the cells and extra-cellular spaces of the of p27 . Furthermore, AIDs suppressed EGF-enhanced phosphorylation and nucleus stratum corneum, and large clusters of Ca2+-containing deposits were occasionally observed translocation of Akt, and was accompanied with the suppression of proliferation. AIDs in the stratum corneum and granulosum. Additionally, abnormal differentiation (e.g. also attenuated EGF-decreased FoxO-3a accumulation in nucleus, and p27kip1 mRNA parakeratosis) was observed in the stratum granulosum. To confirm that these changes were expression in hOBs. Most importantly, FoxO silencing significantly attenuated AIDs- caused by TJ disruption, we observed the structure of TJ strands by freeze fracture electron kip1 2+ 2+ induced elevation of p27 and suppression of proliferation in hOBs. This study firstly microscopy, and measured trans-epidermal Ca permeability by quantifying diffused Ca kip1 through the epidermis. As a result, the TJ strands were fragmented and the Ca2+ permeability showed that Akt/FoxO3a/p27 pathway contributes to AID-suppressed proliferation increased. These data suggest that the epidermal TJs maintain Ca2+ under the stratum of hOBs. We suggest that AIDs suppressed hOBs proliferation, at least partly, through corneum, and regulates the epidermal differentiation. inactivating Akt, activating FoxO-3a, and eventually up-regulating p27kip1 expression.
208 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-17-2 P1PM-18-1 REGULATION OF PAI-1 EXPRESSION IN FUNCTION OF MEMBRANE RAFTS AS A PLATFORM OSTEOARTHRITIC CHONDROCYTES BY SHEAR OF CELL SIGNALING ON ABNORMAL VASCULAR STRESS: ROLE OF PROTEIN KINASE C SMOOTH MUSCLE CONTRACTION Cheng-Nan Chen, Wan-Tin Tsai, Hsin-I Chang Katsuko Kajiya, Hiroko Kishi, Hozumi Kawamichi, Yuichi Takada, Department of Biochemical Science and Technology, National Chiayi Daisuke Tokumori, Sei Kobayashi University, Taiwan Department of Molecular Physiology and Medical Bioregulation, In the present study, we used fluid flow system to investigate the influence of Yamaguchi University Graduate School of Medicine, Japan shear stress on plasminogen activator inhibitor-1 (PAI-1) expression of human A Rho-kinase-mediated Ca2+-sensitization of vascular smooth muscle (VSM) plays a SW-1353 chondrocytes and human osteoarthritic articualr chondroctyes. critical role in abnormal VSM contraction such as vasospasm. We previously identified Chondrocytes were cultured on a type-II collagen coated glass and connected sphingosylphosphorylcholine (SPC) as an upstream mediator of the abnormal with the flow camber. Chondrocytes were exposed to different intensity of shear contraction, and found that SPC activates this pathway, dependently on cholesterol stress and then using real-time PCR to analyze the PAI-1 gene expression. By the in the VSM tissues and cells, and through the activation of Fyn. These results taken addition of protease inhibitors or siRNAs, the regulation of shear stress induced together with the localization of Fyn in cholesterol-enriched membrane domains, signal transduction cascades were investigated. lipid rafts, suggest the essential roles of lipid rafts in abnormal contraction. Although Results showed the significant reduction of PAI-1 expression in lesional articular several signaling molecules accumulate lipid rafts, the membrane-permeabilized chondrocytes compared with non-lesional sites. Two-hour flow shear stress VSM revealed that the activation of G-proteins or GPCRs was not required for the studies indicated 5 and 10 dyne/cm2 shear stress generated large PAI-1 mRNA SPC-induced contraction. Therefore, we investigated the direct interaction of SPC expression in SW-1353 chondrocytes and was regulated through phosphorylation with lipid rafts. For this purpose, we successfully and originally developed the model of protein kinase C-alpha (PKC-alpha) and Sp1 activation. In addition, shear lipid rafts, which contain high concentrations of cholesterol and sphingomyelin, and stress can also increased PAI-1 expression in non-lesional articular chondrocytes examined their direct interaction with SPC. Here we provide the first direct evidence through the same signal transduction cascades but no effect on lesional site. that SPC has very high affinity for the model lipid rafts. In addition, the surface of the These results explain OA chondrocytes from different sites of cartilages may model lipid rafts, interacted with and without SPC was observed by scanning electron have the diverse responses and hence induce various gene expressions. microscope and atomic force microscope.
P1PM-18-2 P1PM-18-3 NANO-SCALE ANALYSIS OF PHOSPHATIDYLINOSITOL DETECTION AND CLONING OF PLCζ FROM TESTES 4,5-BISPHOSPHATE (PI(4,5)P2) DISTRIBUTION BY OF RATTUS ARGENTIVENTER AND APPLICATION OF ELECTRON MICROSCOPY siRNA AGAINST PLCζ EXPRESSION IN-VITRO Akikazu Fujita, Jinglei Cheng, Toyoshi Fujimoto Fadzlina Amir Shapuddin, Sabrina Sukardi, Kqueen Yoke Cheah, Department of Anatomy and Molecular Cell Biology, Nagoya University Han Learn Lee Graduate School of Medicine, Japan Biomedical Sciences, University Putra Malaysia, Malaysia Live imaging using GFP-tagged phosphoinositide (PI)-binding domains Phospholipase C-zeta (PLC ζ) is a sperma specific enzyme in sperm that advanced our knowledge greatly by showing the real-time distribution of PIs triggers calcium oscillation and leads to eggs activation during fertilization. in situ. However, several drawbacks of the method, such as disturbance of in mammalian egg. It sparks a services of surges in calcium levels in the the intracellular signaling by GFP probes, and insufficient space resolution to eggs that occurs during fertilization. Rattus argentiventer (Rice Field detect the two-dimensional heterogeneity in a membrane, have been pointed Rat) is often responsible for depredations on paddy fields and palm oil. out. Labeling of ultrathin cryosections by the probes have been done for Reproductive potential of Rattus argentiventer may causing destruction of electron microscopy, but the method also has a potential problem because PIs agricultural crops. This study is to identify PLC-ζ in local rodent and to are not likely be immobilized by chemical fixation. We developed a method to silence expression of PLC ζ via siRNA approach. Three phases involved analyze the nanoscale distribution of PI(4,5)P2 by electron microscopy using in this study which is RT-PCR amplification and cloning of PLC-ζ, siRNA cell membranes that were physically stabilized by rapid freezing and platinum/ delivery and validation of siRNA effects. RNA interference (RNAi) is the carbon vacuum evaporation. By this method, we observed that PI(4,5)P2 shows pathway by which short interfering RNA (siRNA) or short hairpin RNA only a low level clustering in the flat membrane areas, whereas it is densely (shRNA) are used to inactivate the expression of target genes in the rodent. concentrated at the caveolar orifice. The present method does not require The result will indicate PLC ζ is present in the species and siRNA delivery expression of artificial probes, can be applied to any cell in vivo and in vitro, and is successfully in vitro thus validation of knockdown efficiency by RealTime gives a superb spacio-temporal resolution to define the PI(4,5)P2 distribution. PCR will show that PLC-ζ siRNA will reduce PLC-ζ mRNA levels by more We are now examining the effects by agonist stimulation, intracellular Ca2+ than 70%. It would be useful to silence PLC ζ as an approach in controlling mobilization and other agents which can affect the cellular PI(4,5)P2 level. and preventing the population of Rattus argentiventer.
P1PM-18-4 P1PM-18-5 CROSS-TALK BETWEEN PI3K/AKT AND MEK/ ROLES OF ERK, PI3 KINASE, PLC-γ PATHWAYS ERK SIGNAL PATHWAYS MEDIATED BY ER WAS INDUCED BY TRANSLATIONALLY CONTROLLED ACTIVATED BY CAVOLIN-1 DOWN-REGULATION IN TUMOR PROTEIN OVEREXPRESSION IN HeLa CELLS MAMMARY EPITHELIAL CELL Moon-Hee Kim, Jae-Hoon Jung, Kyung-Lim Lee Shuang Feng1, Wei Zou1, Yang Wang1, Zhao Yi Wang2, Xi Wang3 College of Pharmacy, Center for Cell Signaling & Drug Discovery 1College of Life and Science, Liaoning Normal University, China, 2 3 Research, Ewha Womans University, Korea Cancer Center, Creighton University, USA Division of Neurobiology and Physiology, School of Medicine, Zhejiang University, China We reported previously that Translationally Controlled Tumor Protein (TCTP) Caveolin-1 may function as a tumor suppressor to inhibit many growth-promoting is a cytoplasmic repressor of Na,K-ATPase in HeLa cell. In the current signaling pathways. Multiple evidences support that Caveolin-1 play an important role study, we showed that TCTP overexpression using adenovirus as vehicle, in E2 induced signal transduction. Recent report shows that the existence of a plasma induced partial inhibition of Na,K-ATPase; phosphorylation of EGFR membrane ERαand ERα-Caveolin-1 are localization. However, the mechanism whereby tyrosine residues 845, 992, 1068, and 1148; activation of Ras/Raf/ERK ERαis constitutively activated during transformation of normal mammary cells has not pathway; activation of PI3K/Akt pathway; and phosphorylation of PLC-γ in been well established. Here, we compared MCF-10A and MCF10A-ST1-7SD cells with ERαand proteins associated with PI3K/AKT, MEK/ERK signal pathways. LY294002 and HeLa cells. Specific inhibition of PI3K/Akt pathway significantly decreased Wortmainn pharmacological inhibitor of PI3K/AKT transduction pathway treatment could TCTP overexpression-induced survival signal, but inhibition of ERK had inhibit the activation of PI3K/AKT and MEK/ERK pathways obviously in MCF10A-ST1- little or no impact. Inhibition of PLC-γ pathway significantly decreased 7SD but not in MCF10A. Our data shows that E2 can induce growth-promoting, LY294002 TCTP overexpression-induced cell migration but inhibition of ERK had less and Wortmainn could induce growth inhibiting effects via the binding of ERα-Caveolin-1. effect. These results suggest that TCTP plays a key physiological role in cell This loss of Caveolin-1 in MCF10A-ST1-7SD cell could activate the signal cascade that survival through Akt pathway and migration through PLC-γ pathway. trigger cell proliferation and increase the sensitivity of various pharmacological inhibitors. Caveolin-1 may be a potential target for mediating membrane-initiated estrogen-signaling pathway, it played an important role in mammary tumorigensis and could effect on cell metastasis in vitro. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 209 P1PM-18-6 P1PM-19-1 THE GIANT MAMMALIAN CADHERIN FAT1 ACTS AS A NOX4 MODULATES ACTIVATION AND INACTIVATION REGULATOR OF TRANSCRIPTION OF THE VOLUME-SENSITIVE TAURINE LEAK Werner H. Hofer, Dietmar Schreiner, Katja Fiedler, PATHWAY IN NIH3T3 FIBROBLASTS Kerstin Rosenberger Ian Henry Lambert, Martin Barfred Friis Department of Biology, University of Konstanz, Germany Department of Biology, University of Copenhagen, Denmark The influence of the mammalian cadherin-superfamily protein FAT1 on Release of the organic osmolyte taurine in NIH3T3 fibroblasts is increased transcriptional regulation was investigated by a luciferase reporter system by hypotonic exposure. The production of reactive oxygen species (ROS) is in H1299 cells. The intracellular domain of FAT1 strongly stimulated the concomitantly potentiated and the potentiation increases with the magnitude expression of the Serum Response Element-dependent reporter and slightly of the osmotic challenge. NIH3T3 cells express the NADPH oxidase stimulated AP1-, CRE and NFAT-dependent expression. E2F-, p53-, Rb-, components: p22phox, a NOX4 isotype, p47phox, and p67phox. H2O2 Myc-, and HSE-dependent reporters remained unaffected. Stimulation potentiates the swelling induced taurine release and delays the inactivation of SRE- and CRE-dependent expression was 4 fold enhanced when a of the volume sensitive efflux pathway. NOX4 knock down (siRNA) impairs transmembrane form of FAT1 was expressed instead of the intracellular the increase in the ROS production and taurine release following hypotonic domain. The stimulation by FAT1 was strongly inhibited by the MEK1/2 exposure. Thus, a NOX4 isotype plus p22phox account for the swelling- inhibitor PD98059 but not by inhibitors of JNK or p38, suggesting that FAT1 induced increase in ROS production and potentiation of volume-sensitive activates the classical MAPK/ERK pathway. The stimulatory effect on the taurine release. The protein tyrosine phosphatase (PTP) inhibitor vanadate expression of the SRE-reporter was almost eliminated by the deletion of a potentiates the ROS production and the concomitant taurine release under putative C-terminal PDZ-binding domain of FAT1. Although the stimulation hypotonic conditions. Exposure to vanadate also delays inactivation of the of the SRE-reporter by FAT1 was strongly enhanced by co-expression volume sensitive taurine efflux in the absence but not in the presence of the of PDZ-RhoGEF, this activation was possibly not mediated by a direct flavoprotein inhibitor diphenylene iodonium chloride. It is suggested that an interaction, since it was independent on the presence of the PDZ-binding increased tyrosine phosphorylation of the volume sensitive NOX4/p22phox motif in FAT1. We conclude that FAT1 activates SRE-regulated transcription system leads to an increased ROS production and subsequent delays the in a dual manner by MAPK/ERK- as well as by Rho-mediated pathways. inactivation of the volume-sensitive taurine efflux pathway.
P1PM-19-2 P1PM-19-3 RELEASE OF INTRACELLULAR CALCIUM INCREASE GHRELIN ANTAGONIZED1-METHYL-4- PRODUCTION OF MITOCHONDRIAL REACTIVE PHENYLPYRIDINIUM (MPP+)-INDUCED APOPTOSIS IN OXYGEN SPECIES IN RENAL DISTAL EPITHELIAL MES23.5 CELLS CELLS Juanjuan Dong, Ning Song, Junxia Xie, Hong Jiang Henning F. Bjerregaard State Key Discipline: Physiology (in incubation); Department of Physiology, Science, Systems and Models, Roskilde University, Denmark Medical College of Qingdao University, China Reactive oxygen species (ROS) like, hydrogen peroxide (H2O2) has traditionally Ghrelin is an endogenous ligand for the growth hormone secretagogue been regarded as toxic by-products of aerobic metabolism. However, recent receptor (GHSR) acting to stimulate growth hormone release. In the previous findings indicate that 2H O2 act as a signalling molecule The aim of the present study, we have observed the neuroprotective effect of ghrelin in MPTP- study was to monitor, in real time, the rates of ROS generation in order to treated mice. In order to illustrate the mechanism, in the present study, directly determine their production dynamics in living cells in response to we investigated the neuroprotective effects of ghrelin in MPP+-treated hormonal signal events in the A6 cell culture. A6 cells have a divalent cation- dopaminergic cells. MPP+ treatment could cause an decreased mitochondrial sensing receptor (the extracellular calcium receptor) that can be stimulated with transmembrane potential, an elevated level of ROS production and activation cadmium (Cd) and thereby induce a fast and transient liberation of calcium from intracellular stores, due to G-protein stimulation of phospholipase C and release of caspase-3. These cells showed the apoptotic morphorlogical changes. of inositol -3 phosphate. Cd (0.4 mM) treatment of A6 cells enhanced the ROS Ghrelin(10-12 -10-7M) could fully abolish the MPP+-induced apoptotic production after one minutes incubation. The production rate was constant for changes in a dose-dependent manner. The results suggest that ghrelin can at least 10 to 20 min. Experiments showed that the Cd induced increase in ROS antagonize MPP+-induced apoptosis in MES23.5 cells. The protective effect production was inhibited by buffering of intracellular calcium with BAPTA, by of ghrelin involves the restoration of mitochondria function. the antioxidant N-acetylcysteine and by uncoupling of mitochondrial oxidative phosphorylation from respiration with CCCP. These results indicate that Cd generate a prompt initiation of ROS production from mitochondria due to an increase in the intracellular calcium concentration.
P1PM-19-4 P1PM-19-5 THE ROLES OF CRAG AND CacyBP/SIP IN STRESS- OXIDATIVE MODIFICATION OF SERUM ALBUMIN MEDIATED SIGNALLING THROUGH PARACELLULAR PATHWAY OF RAT Liwei Yang, Toshifumi Fukuda, Shun Nagashima, Ryoko Inatome, SALIVARY GLAND 1 2 3 Shigeru Yanagi Tomoya Hayashi , Masataka Murakami , Yukie Matsuyama , 3 School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Seiichi Era 1 Japan Department of Physiology, Meiji University of Integrated Medicine, Japan, 2Department of Nano-Structure Physiology, National Institute for Reactive oxygen species (ROS) is involved in the regulation of a variety of stress 3 Natural Sciences, Japan, Department of Physiology and Biophysics, Gifu responses. Previously, we determined that CRAM-associated GTPase (CRAG) is one University Graduate School of Medicine, Japan of the binding proteins of CRAM (CRMP5), which controls the axonal retraction in the downstream of neuronal repulsion factor Semaphorin-3A. Until now, we reported In order to clarify the oxidative modification of sulfhydryl group in serum albumin in that in response to ROS stress CRAG changes its localization from cytoplasm to paracellular pathway, we examined redox state of the albumin in saliva secreted from nucleus and then forms nuclear inclusions called PML body with the expanded the isolated rat submandibular gland which perfused with the perfusate containing commercial human albumin. The collected samples were analyzed by an ion-exchange polyglutamine proteins (polyQs), a responsible proteins that induce neurogenerative HPLC. The findings indicated that: 1) small amount of the human albumin could be diseases. detected in the rat saliva at 1.0 μM carbachol stimulation and its concentration was Here, we identified calcyclin-binding protein/Siah-interacting protein (CacyBP/SIP) approximately 0.05-0.7% of the albumin in the perfusate; 2) secretion-perfusate ratio Poster Session as a novel binding partner of CRAG by the yeast two-hybrid system. CacyBP/SIP, a showed a tendency to increase exponentially according to decline of flow rate at 0.5 target protein of S100, has been specified as a component of a novel ubiquitinylation μM carbachol-0.5 μM isoproterenol stimulation; 3) the S-nitrosoalbumin fraction was complex leading to β-catenin degradation. However, the relationship between CRAG detected in saliva only, and an inhibitor on synthesis of nitric oxide (l-NAME) hardly and CacyBP/SIP remain unclear. affected secretion volume of saliva; 4) in the salivary gland, the intercellular passage In the present study, we have investigated the physiological relevance of the interaction tended to increase an irreversible albumin fraction which was directly oxidized by between CRAG and CacyBP/SIP. Here, we report that the CRAG nuclear localization reactive oxygen species. These facts suggested that the human albumin in saliva might with CacyBP/SIP may synergistically contributes to cell survival and accelerates cell be passed through the paracellular pathway such as tight junction in rat submandibular differentiation, which triggered by ROS stress or Wnt pathway. gland and oxidative modification of the albumin might be occurred in this pathway.
210 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-19-6 P1PM-19-7 MRSA CAUSES DNA DAMAGE IN ALVEOLAR IN VIVO EFFECTS OF SS-DNA-SWCNT ON OXIDANT/ EPITHELIAL CELLS BY PERINUCLEAR FORMATION ANTIOXIDANT BALANCE OF REACTIVE NITROGEN SPECIES Simona Valeria Clichici1, Teodora Mocan1, Adriana G Filip1, Alexandru 2 2 2 1 1 Kamna Bansal, Rhykka L Connelly, Daniel L Traber, Lillian L Biris , Dan Lupu , Stefania Simon , Nicoleta Decea , Adriana V Muresan 1Department of Physiology, University of Medicine and Pharmacy Iuliu Traber, Perenlei Enkhbaatar Hatieganu Cluj Napoca, Roumania, 2National R&D Institute of Isotopic and Department of Anesthesiology, University of Texas Medical Branch and Molecular Technologies INCDTIM Cluj Napoca, Roumania Shriners Hospitals for Children, Galveston, Texas, USA Although single walled carbon nanotubes (SWCNT) have been proposed for various medical Rationale: MRSA (Methicillin-resistant Staphylococcus aureus)commonly referred applications, reports on SWCNT in vivo cytotoxicity are still controversial. to as a ‘super bug’ because of its resistance to antibiotic therapy. We have previously Our aim was to evaluate the effects of intraperitoneal injection of ss-DNA-SWCNT on shown that MRSA causes vascular hyperpermeability, multi-organ system failure, oxidant/antioxidant balance. and death in our ovine model of MRSA-induced sepsis and smoke inhalation. We Material and Method: 70mg/ml ss-DNA-SWCNT water solution was obtained through hypothesized that RNS may play a pivotal role in MRSA-induced DNA damage and 7 hr sonication. NaCl 0,9% was added up to the final concentration of 0.9%. 0.5 ml of obtained solution was i.p. administered to 30 male Wistar rats. Controls were similarly consequent cellular death. Methods: Pulmonary epithelial cell (A549) were challenged injected with 0.5 ml NaCl. 20 rats (10 controls and 10 test) were sacrificed at each at with 105 cfu MRSA over a time course of 4 hrs then probed for markers of RNS the following intervals after administration: 3hr, 6hr and 24 hrs. Malondialdehyde, (2,7-Dichlorodihydrofluorescein [DCF] and 3-Nitrotyrosine {3-NT}), as well as poly carbonylated proteins, hydrogen donor ability (HD), sulfhydryl groups and gluthation ADP-ribose (PAR), a marker of DNA damage, by confocal imaging and western blot blood levels were assessed. Results were compared by means of Mann-Whitney U analyses. Neuronal nitric oxide synthase (nNOS) and NADPH oxidase-4 (NOX4) test. were localized by confocal microscopy. Results: MRSA caused nNOS and NOX4 Results: We obtained a significant decrease of HD in test group compared to controls translocation to a perinuclear location, resulting in a 7-fold increase in RNS (measured at 24 hr interval (22.3(3.8) vs 16.2(4.5), p=0.009) and an increase of oxidative stress by DCF) at the perinuclear compartment compared to normal cells. MRSA also caused biomarkers. Difference between groups showed marginal significance when compared PAR (3.4 fold) and 3-NT (4.8 fold) formation within 1h of MRSA exposure observed to difference recorded for 6 hr interval. by both confocal and western blotting analyses. Conclusions: MRSA causes DNA Conclusion: Our results support the lack of oxidant/antioxidant balance after i. p. damage in alveolar epithelial cells as result of perinuclear formation of RNS. administration of ss-DNA-SWCNT.
P1PM-19-8 P1PM-19-9 HETEROGENEITY OF POST-TRANSLATIONAL ROLE OF NEURONAL NOS IN VASCULAR MODIFICATION OF COMMERCIAL HUMAN SERUM SUPEROXIDE LEVEL AND MITOGEN-ACTIVATED ALBUMIN PRODUCTS: THIOL OXIDATION AND PROTEIN KINASE PHOSPHORYLATION PROTEIN CARBONYLATION Guo-Xing Zhang, Juichiro Shimizu, Hiroko Matsuyoshi, Miyako Seiichi Era, Yukie Matsuyama, Tomoyoshi Terada, Takeshi Minami Takaki Department of Physiology and Biophysics, Gifu University Graduate Department of Physiology II, Nara Medical University, Japan School of Medicine, Japan Role of nNOS in the regulation of vascular MAPK activity under basal and Albumin has been widely served as nutrients for cell cultures in laboratory field and as Ang II-stimulated conditions was investigated. nNOS inhibitor (L-VNIO) plasma expander for blood transfusion in clinical field. Human serum albumin (HSA) significantly increased 2O - production and MAPK phosphorylation in aorta and is a mixture of HMA (reduced form) and HNA (oxidized form) (HNA-1, mixed VSMC from wild type mice, which were inhibited by Tempol. O2- production disulfide with cysteine; HNA-2, more higher oxidation product). By using an HPLC and phosphorylation of MAPK were higher in aorta from nNOS-/- mice than system for the separation from HSA to HMA, HNA-1 and HNA-2, we examined the that from wild mice, which were suppressed by tempol and oxypurinal. In wild heterogeneity of HSA products. HSA products were obtained mainly from Sigma Co. type VSMC or aorta, stimulation with Ang II for 30 min markedly increased (product No. A1653, A9511, A1887, A8763 and A3782). The A1653 is the product O2- production and MAPK phosphorylation. L-VNIO markedly reduced Ang corresponding to Cohn Fraction V, which a starting material from pooled sera. In the II-induced increases of O - production and MAPK phosphorylation. Apocynin case of A1653 (lot No. 86K7540), values (%) for HMA, HNA-1 and HNA-2 were 2 37.5, 51.9 and 10.6%, respectively. In contrast, corresponding values for the final further inhibited Ang II-induced O2- production and MAPK phosphorylation product (A3782; lot No. 78H7603), which prepared from A1653 through lyophilized compared with L-VNIO treated group. In contrast to wild type, Ang II failed to and defatted processes, were 13.0, 38.7 and 48.3%, respectively, and it was contained increase O2- production, phosphorylated MAPK in aorta or VSMC from nNOS- dimer remarkably. With respect to carbonyl modification of protein, carbonylated /-mice.These results suggested that under basal condition, nNOS-derived NO value for A3782 was higher than that for A1653. These results suggested that the acting as antioxidant reduces O2- accumulation and suppresses vascular MAPK heterogeneity of amino acid modification of various kinds of commercial HSA phosphorylation. Under Ang II-stimulated condition, NAD(P)H oxidase-derived products appears to occur during manufacturing process of HSA from large-scale O2- inducing nNOS uncoupling potentiates Ang II-induced increase of O2- pooled blood. generation and participate in Ang II-induced activation of vascular MAPK.
P1PM-19-10 P1PM-19-11 THE RELATIONSHIP BETWEEN OXIDATION OF SUBSTRATE ACTIVATION MECHANISM OF SERUM ALBUMIN AND SERUM PROTEIN CARBONYL BRANCHED-CHAIN ACYL-CoA DEHYDROGENASES: A CONTENT IN REGULAR HEMODIALYSIS SESSIONS STUDY USING ARTIFICIAL FLAVIN Yukie Matsuyama1, Hiroyuki Terawaki2, Tomoyoshi Terada1, Yoichi Yasuzo Nishina1, Kyosuke Sato2, Chiaki Setoyama3, Haruhiko Tamaoki2, 3 1 Yuka Tominaga1, Maiko Horai1, Hiromi Maeda1, Kiyoshi Shiga4 Funakoshi , Seiichi Era 1 1 Department of Physiology, School of Health Sciences, Kumamoto University, Department of Physiology and Biophysics, Gifu University, Japan, 2 2 Japan, Department of Molecular Physiology, Graduate School of Medical Department of Kidney and Hypertension, The Jikei University School of 3 3 Sciences, Kumamoto University, Japan, Department of Molecular Enzymology, Medicine, Japan, Funakoshi Clinic, Japan Graduate School of Medical Sciences, Kumamoto University, Japan, 4 Oxidative stress is enhanced in regular hemodialysis (HD) patients. Oxidized Department of Nursing, Kyushu University of Nursing and Social Welfare, albumin is a reliable marker of oxidative stress. Depending on redox state, there Japan are three major fractions of human serum albumin (HSA); HMA (reduced form) Iso(2)valeryl-CoA (i2VD) and iso(3)valeryl-CoA (i3VD) dehydrogenases are members with a free thiol group on cysteine-34, HNA-1 (reversible oxidation product) of acyl-CoA dehydrogenase (ACD) family of flavoproteins. ACDs catalyze the oxidation of acyl-CoA thioesters to the corresponding 2-enoyl-CoA products. The substrate with cystine, HNA-2 (irreversible oxidation product) with more oxidized state. activation mechanism in i2VD and i3VD was compared to that in medium-chain acyl-CoA We have studied the albumin thiol oxidation and the serum protein carbonyl dehydrogenase (MCAD). The mechanism was investigated using reconstituted i2VD with content (CC) in 83 HD patients. The mean values for f (HMA) for pre- and post- artificial FADs, i.e., 8-CN-. 8-Cl-, 8-H-, 8-OCH3-, 8-NH2-, and ribityl-2’-deoxy-FADs. The HD session were 42.4 % and 64.9 % respectively. In contrast, f (HNA-1) was pKa value of substrate analog 3-thiabutyryl-CoA at αC-H was drastically lowered from 16 significantly decreased the course of the HD session (52.2 % at pre-, 31.4 % at in the free state to 6.7 on binding to i2VD. This phenomenon reflects substrate activation post-HD), and f (HNA-2) was also decreased (5.4 % at pre-, 3.7 % at post-HD). during the enzymatic process. The pKa lowering was more extensive when the 8-CH3 group CC was also decreased (0.8 nM/mg protein at pre-, 0.6 nM/mg protein at post- of FAD was substituted by a strong electron-withdrawing group such as CN, indicating that the flavin ring itself directly affects the Kp a of the ligand, hence flavin ring activates HD). In relationship between the thiol oxidation and the carbonyl formation, the substrate in i2VD reaction by lowering the pKa value. The destruction of the hydrogen value for CC was not correlated with that for HNA-1 but significantly with that bond of the ligand C(1)=O with ribityl-2’-OH of FAD raised the pKa. Similar results were for HNA-2. We found the close relationship between irreversible oxidation of obtained for i3VD and they are also similar to the previous results for MCAD, suggesting HSA and serum protein carbonyl formation in HD patients. that the substrate is activated in common mechanism among ACD family. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 211 P1PM-19-12 P1PM-19-13 EXERCISE, OXIDATIVE STRESS AND BRAIN-DERIVED MELATONIN PREVENTS RENOVASCULAR NEUROTROPHIC FACTOR GENE EXPRESSION UP- HYPERTENSION-INDUCED OXIDATIVE DAMAGE AND REGULATION IN HIPPOCAMPUS OF DIABETIC RATS IMPROVES CARDIAC FUNCTION IN RATS Iraj Salehi1, Mohamadi Mostafa2, Farajnia Safar3 Goksel Sener1, Ozer Sehirli1, Mehmet Ersahin2, Hale Toklu1, Selami 3 4 1Department of Physiology, Hamadan University of Medical Sciences, Iran, Suleymanoglu , Berrak Yegen 2 3 Department of Physiology, Tabriz University of Medical Sciences, Iran, Drug 1Department of Pharmacology, Marmara University, Turkey, 2Department of Applied Research Center Tabriz University of Medical Sciences, Iran Neurosurgery, Haydarpasa Numune Education and Research Hospital, Istanbul, Turkey, 3Department of Pediatric Cardiology, Gulhane Military Medical Academy, Oxidative stress, as an outcome of diabetes, plays a role in both pathogenesis and 4 numerous pathophysiological mechanisms that trigger diabetic complications. Brain- Istanbul, Turkey, Department of Physiology, Marmara University, School of Derived Neurotrophic Factor (BDNF) promotes plasticity and survival of synapses Medicine, Istanbul, Turkey and neurons in CNS and protects it against insults. The effect of melatonin was investigated in an angiotensin II-dependent renovascular 36 male wistar rats were divided into three groups (control, diabetic and diabetes hypertension model in Wistar albino rats by placing a renal artery clip (two-kidney, one- exercise). Diabetes was induced by injection of single dose of Streptozotocin (50mg/ clip; 2K1C), while sham rats did not have clip placement. Starting either on the operation Kg, i.p). Exercise was performed for 8 weeks and one hour every day. The antioxidant day or 3 weeks after the operation, the rats received melatonin (10 mg/kg/day) or vehicle for enzymes (SOD, GPX, CAT and GR) and oxidant indexes with BDNF protein and its the following 6 weeks. Blood pressure (BP) and echocardiographic recordings were made before, at the 3rd and 9th weeks of surgery. At the end of the 9th week, heart and kidney mRNA were measured in hippocampus of rats. tissues were obtained to determine malondialdehyde (MDA) and glutathione (GSH) levels, A significant decrease in antioxidant enzymes activities, GSH level and increase MDA myeloperoxidase (MPO) and Na+-K+ ATPase activities. 2K1C led to increases in BP, left level were observed in diabetic rats (P=0.004). In response to exercise antioxidant ventricular (LV) posterior wall thickness, LV end-diastolic and end-systolic dimensions, enzymes activities and GSH level increased (P<0.004). In contrast, MDA level while % fractional shortening and % ejection fraction were significantly decreased. Cardiac decreased in diabetic rats (P = 0.004). Induction of diabetes caused an increase of and renal MDA levels, as well as MPO activity, were increased with concomitant decreases BDNF protein and its mRNA expression. In response to exercise, BDNF protein and in GSH and Na+-K+ ATPase activity. Both melatonin regimens significantly reduced BP, its mRNA expression reduced in hippocampus in diabetic rats. alleviated oxidative injury and improved LV function. In conclusion, melatonin protected Exercise ameliorates oxidative stress induced BDNF gene expression up-regulation against renovascular hypertension-induced tissue damage and improved cardiac function due and protects of hippocampus against oxidative stress damage in diabetic rat. to its direct antioxidant and receptor-dependent actions.
P1PM-19-14 P1PM-19-15 ALPHA LIPOIC ACID ALLEVIATES OXIDATIVE STRESS THE EFFECT OF CUCUMIS MELO EXTRACT (JUICE) AND PRESERVES BLOOD BRAIN PERMEABILITY IN ON RESPIRATORY BURST OF MICE PERITONEAL RATS WITH SUBARACHNOID HEMORRHAGE MACROPHAGES Goksel Sener1, Mehmet Ersahin2, Hale Toklu1, Can Erzik3, Sule Cetinel4, Parivash Eftekhari1, Mohammad Reza Raoufy1, Nariman Mosaffa2 5 6 Meral Yuksel , Berrak Yegen 1Department of Physiology, Tarbiat Modares University, Iran, 2Department 1Department of Pharmacology, Marmara University, Turkey, 2 Department of Immunology, Shahid Beheshti University of Medical Sciences, Tehran, of Neurosurgery; Haydarpasa Numune Education and Research Hospital, Iran Istanbul, Turkey, 3Department of Medical Biology; Marmara University, School of Medicine, Istanbul, Turkey, 4Department of Histology & Embryology, Marmara Nowadays uses of plants and food products for purpose of diseases treatment, University, School of Medicine,; Istanbul, Turkey, 5Vocational School of Health begins. Cucumis melo is one of these fruits which is cultivates in Iran. Our Related Professions; Marmara University, Istanbul, Turkey, 6Department of purpose from this study was investigating the effect of Cucumis melo on Physiology; Marmara University, School of Medicine, Istanbul, Turkey oxidative stress and viability of macrophages harvested from peritonea of mice. Oxidative stress has detrimental effects in several models of neurodegenerative diseases, including 40 balb/c mices (20-25g) divided into 4 groups. Control group had been fed subarachnoid hemorrhage (SAH). In order to investigate the putative neuroprotective effect of alpha- with solvent of extract and 3 groups had been fed with 0.1 , 1 , 2 mg/ml of lipoic acid (ALA), Wistar albino rats were divided as control, SAH, and ALA-treated (100 mg/kg, Cucumis melo total extract, twice a day. After a week animals were sacrificed intraperitoneally) SAH groups, where SAH was induced by injection of blood into cisterna magna. At the 48th h of SAH induction, neurological examination scores were recorded and brain tissue and macrophages of peritonea from 4 groups were harvested. MTT was used to samples were taken for histological analysis and for the determination of blood brain barrier (BBB) show activation of respiratory burst of macrophages. At last results were red with permeability, malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) elisa reader. 1 and 2 mg/ml of total extract of Cucumis melo was significantly and Na+-K+ ATPase activities, oxidant-induced DNA fragmentation and formation of reactive increased respiratory burst and oxidative stress of peritoneal macrophages. 0.1 oxygen species (ROS). Increased neurological scores in the SAH group were accompanied with mg/ml of extract had no effect on these parameters. significant decreases in GSH content and Na+-K+ ATPase activity, and with significant increases with note to ability of the extract of Cucumis melo on oxidative stress of in MDA levels, MPO activity, DNA fragmentation and ROS generation. On the other hand, ALA treatment reversed impaired BBB and the alterations in biochemical and histopathological analyses. peritoneal macrophages, application of this plant in treatment of immune system In conclusion, ALA, which can easily cross BBB, exerts neuroprotection in SAH by alleviating deficiencies is suggested. Fractioning of this extract can show precise effect of oxidative stress and preserving BBB permeability. the extract.
P1PM-20-1 P1PM-20-2 NITRIC OXIDE SYNTHESIS IN CHRONIC A ROLE OF NITRIC OXIDE (NO) AND ITS CELL PERIODONTITIS SIGNALLING AT FERTILIZATION IN SEA URCHIN Alina Elena Parvu1, Sandu F Alb2, Simona Clichici3, Alexandra Craciun4, EGGS Constantin Craciun5, Marian-Aurel Taulescu6, Grigore Baciut7, Mihaela 1 2 3 7 1 8 Tatsuma Mohri , Masahiro Sokabe , Keiichiro Kyozuka Baciut , Adriana Bulboaca , Angelo Bulboaca 1Division of Intracellular Metabolism, Department of Molecular Physiology, 1Pathophysiology, University of Medicine and Pharmacy Iuliu Hatieganu, Roumania, National Institute for Physiological Sciences, Japan, 2Department of Physiology, 2Periodontology, University of Medicine and Pharmacy Iuliu Hatieganu, Romania, 3Physiology, 3 4 Graduate School of Medicine, Nagoya University, Nagoya, Japan, Research University of Medicine and Pharmacy Iuliu Hatieganu, Romania, Biochemistry, University of Center for Marine Biology, Asamushi, Graduate School of Life Science, Tohoku Medicine and Pharmacy Iuliu Hatieganu, Romania, 5Center of Molecular and Cellular, Biology 6 University, Asamushi, Japan Babes Bolyai University, Romania, Pathology, University of Agricultural and Veterinary Sciences, Romania, 7Oromaxillofacial Surgery, University of Medicine and Pharmacy Iuliu Little is known about complex cell signaling at fertilization in sea urchin eggs 8 Hatieganu, Romania, Neurology, University of Medicine and Pharmacy Iuliu Hatieganu, although it has been studied for more than 120 years. Previous research indicates that Roumania the nitric oxide (NO) increase at fertilization in sea urchin eggs is Ca2+-dependent and In periodontitis iNOS expression plays beneficial effects as antimicrobial activity, immune attributed to the late Ca2+ rise. However, its role in fertilization still remains unclear. modulation, inhibition of microvascular thrombosis, as well as increased tissue perfusion, and Simultaneous measurements of the activation current, by a single electrode voltage detrimental effects as cytotoxic action towards the host tissues. Therefore, in the present study we clamp, and NO, using the NO indicator DAF-FM, showed that the NO increase aimed to evaluate nitric oxide (NO) synthesis in patients with chronic periodontitis. 2+ occurred at the time of peak current (tp) which corresponds to peak [Ca ]i, suggesting Blood samples and periodontal biopsies were harvested from 76 patients with periodontitis and 20 2+
Poster Session that NO is not related to any other ionic changes besides [Ca ]i. To find the role of NO healthy volunteers. NO synthesis was evaluated systemically through its serum metabolites nitrites/ increase, we measured O consumption by a polarographic method, redox changes nitrates (Griess reaction) and locally immunohistochemical localization of iNOS. Results: systemic 2 nitrite/nitrate concentration increased significantly and there was a marked increase in the number by detecting the egg’s autofluorescence of NAD(P)H, and 2H O2 using Amplex Red in of iNOS-immunoreactive gingivomucosal immune and epithelial cells compared to the control. control and when NO was eliminated by a NO scavenger, PTIO. Surprisingly, PTIO NO synthesis and iNOS expression was positively correlated with disease severity. Serum nitrites/ decreased O2 consumption, the rate of the fluorescence change and the late phase of nitrates were positively correlated with iNOS expression only in severe forms of periodontitis. increase in NAD(P)H was eliminated. PTIO also suppressed the production of H2O2, Conclusions: 1. In periodontitis there is a significant increase of induced NO synthesis, which is the activity of ovoperoxidase, and caused weak and high fertilization envelope (FE). correlated with disease severity. 2. Systemic effects of NO excess seem to be significant only in Our results suggest that NO increase upregulates NAD(P)H and H2O2 production and severe forms of periodontitis. consolidates FE hardening by H2O2.
212 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-21-1 P1PM-21-2 COMPREHENSIVE ANALYSIS OF LEUCINE- STIMULATION OF AMINO ACIDS TO GROWTH SIGNAL STIMULATION DEPENDENT PHOSPHORYLATED IN HEK293T PROTEINS Ryuichi Ohgaki1, Narakorn Khunweeraphong1, Yoshikatsu Kanai1, Kazuaki Takafuji, Pattama Wiriyasermkul, Kanyarat Promchan, Shushi Nagamori1, Taku Hirata2 Shushi Nagamori, Yoshikatsu Kanai 1Division of Bio-system Pharmacology, Dept. of Pharmacology 2 Division of Bio-system Pharmacology, Department of Pharmacology, Graduate School of Medicine, Osaka University, Japan, Department Graduate School of Medicine, Osaka University, Japan of Pharmacology and Toxicology, Kyorin University School of Medicine, Amino acids are important substrate for a lot of biological reactions. Recently, it has Japan been indicated that amino acids are not only substrates for protein biosynthesis, but Nutrients are essential for cellular processes to regulate their fundamental also serve as signaling molecules controlling signal transduction pathways of protein functions and the growth. Amino acids are not only essential as nutrients biosynthesis. Especially, among all amino acids, it is reported that leucine stimulates for cellular mechanism, but also play an important role in protein synthesis, protein biosynthesis remarkably. mainly via a serine/threonine kinase multiprotein complex called mTOR Many evidences show that the stimulation with leucine promotes activation of mTOR (mammalian target of Rapamycin) complex by achievement through the signal transduction pathway. This pathway is related to various biological phenomena dominant functions of two regulatory downstream regulators of mRNA of cell growth, cell cycle and autophagy inhibition besides the promotion of protein translation, p70S6K and 4EBP1. However, the mechanisms of sensing biosynthesis. However, the activation mechanism is yet poorly understood. and responding to amino acids are still unclear. In this study, HEK 293T Here we have examined the leucine-stimulation to the mTOR pathway by using phosphoproteomics approaches. Cells were cultured in the absence of leucine and cell is used as a model system to elucidate the amino acids stimulation to harvested after leucine-stimulation or no stimulation. Then, total proteins were cells. Among several amino acids tested such as phenylalanine, alanine and separated by strong cation-exchange, reverse-phased, and titanium oxide columns. The leucine, L-Leucine stimulated the phosphorylation of p70S6K strikingly and procedures allow us to analyze expression and phosphorylation of proteins by LC-MS promoted cell growth. The phosphorylation of p70S6K and the cell growth comprehensively and quantitatively. Finally, we have identified proteins which show increased in a concentration-dependent manner. Moreover, D-leucine does different profiles of expression and/or phosphorylation in the absence or presence of not stimulate as well as L-Leucine. leucine-stimulation.
P1PM-21-3 P1PM-21-4 CHANGES OF ErbB-RELATED SURVIVAL SIGNALINGS FRUCTOSE-1,6-DIPHOSPHATE MODULATES TLR4 BETWEEN ANDROGEN-DEPENDENT AND SIGNALING PATHWAY IN BRAIN MICROVASCULAR -INDEPENDENT TRANSITION OF PROSTATE CANCER ENDOTHELIAL CELLS CELLS Sun Mi Seok, Tae Gu Lee, Yi-Sook Jung, Eun Joo Baik, Chang- Fu-Ning Hsu, Min-Shiou Yang, Ho Lin Hyun Moon, Soo Hwan Lee Department of Life Sciences, National Chung Hsing University, Taiwan Department of Physiology, Ajou University School of Medicine, Korea Androgen ablation therapy is the most common strategy to suppress prostate Toll-like receptors (TLRs) play a significant role in perception of different microbial tumor progression, however, cancer cells eventually escape androgen pathogens like LPS and induction of innate immune responses. The activation of TLRs requirement and progress into androgen-independent phase. In order to triggers two downstream signals through MyD88- and TRIF-dependent pathways answer this transition mystery in prostate cancer, we established an androgen- leading to activation of NF-kappa B and IFN-beta respectively. independent prostate cancer cell line (LNCaPDCC) by long-term screening Fructose 1,6-diphosphate (F1,6DP), a glycolytic intermediate, is reported to have LNCaP cells in androgen-deprived condition to investigate changes of neuroprotective effects, of which underlying mechanisms remain largely unknown. molecular mechanism before and after androgen withdrawal. LNCaPDCC cells In the present study, we explored the molecular targets of F1,6DP in TLR4 signaling displayed the morphology in neuroendocrine differentiation, lower androgen pathways leading to the induction of inflammatory mediators in brain microvascular sensitivity, and less aggressive growth although the cell cycle distribution endothelial cells (bEnd.3). was similar to parental LNCaP cells. It is interesting that higher expression F1,6DP reduced NF-kappa B activation through ectopic expression of MyD88, of ErbB receptors, AR and PSA proteins in LNCaPDCC cells were observed. TRIF, and TRAF6. However, F1,6DP did not affect the IKK-beta-induced NF-kappa Unexpectedly, growth effects of estrogen on LNCaPDCC and LNCaP cells B activation. These data suggest that molecular targets of F1,6DP exist in between were dramatic different. Besides, these two cell lines also exhibited distinct downstream of TRAF6 and upstream of IKK-beta. And also, F1,6DP significantly responses to ErbB activators and inhibitors. Taken together, we demonstrate diminished IRF3 activity, in which TBK1 appeared to be the target molecule. Our the changes of ErbB-dependent survival signalings between androgen- results suggest that F1,6DP can modulate TLR4 signaling pathway in bEnd.3 cells. dependent and -independent prostate cancer cells and hope these findings This study was supported, in part, by Gyunggi-do through CCRB-GRRC and Brain will provide detail mechanisms in the transition under androgen deprivation. Korea 21 for Medical Sciences.
P1PM-21-5 P1PM-21-6 INHIBITION OF AUTOPHAGY AT THE LATE STAGE OF PROGRESS IN INTRACELLULAR SITE-DIRECTED POLYMICROBIAL SEPSIS IN KIDNEY AND LIVER pH MEASUREMENTS UTILIZING CONFOCAL Hsu Chin1, Hsiu-Wen Hsiao1, Wei-Shan Chien1, Shu-Mien FLUORESCENCE CORRELATION SPECTROSCOPY Chuang2, Su-Hwa Jin1, Yen-Hsu Chen3 (CFCS) 1Department of Physiology, Kaohsiung Medical University, Taiwan, Norbert Opitz, Stephan Gude, Antje Berken 2 Graduate Institute of Medicine, Kaohsiung Medical University, Taiwan, Structural Biology, Max-Planck-Institute for molecular Physiology, Germany 3Division of Infectious Diseases, Department of Internal Medicine, In view of CFCS-based intracellular site-directed pH measurements we Kaohsiung Medical University, Taiwan succeeded to suitably mutate the Green Fluorescent Protein (GFP) in order to The changes of timing and location of autophagy in kidney and liver during the yield ecliptic GFP (EcGFP : S147D/A206T/Q204T/S202F/N149Q/T161I) which progression of rat septic model induced by cecal ligation and puncture (CLP) is particularly well-suited for intracellular pH measurements. Here we report were investigated. Level of autophagy was quantified by Western blot analysis of first in vitro and in vivo investigations using this mutant in conjunction with LC3-II, a marker for autophagy. The localization of autophagy was identified by advanced microscopical techniques such as confocal laser scanning microscopy immunohistochemistry and TEM. BUN, creatinine, bilirubin, and alkaline phosphate and confocal fluorescence correlation spectroscopy. The pH associated flickering (ALP) were measured as indicators of renal and hepatic dysfunction. In kidney, our results showed that level of LC3-II declined at 9h till 18h after CLP. More LC3 of EcGFP molecules generates a typical shoulder within the short time range aggregation was located in the cortex, especially in ACE (a marker of proximal tubule) (<1ms) of the autocorrelation function. From these autocorrelation functions pH positive cells than in D28K (a marker of distal tubule) positive cells. Renal dysfunction calibration curves are determined, e.g. degree of protonation (Fp) and average began at 6h and became worse at 18 h after CLP. In liver, elevation of LC3-II occurred count rate (CR) are plotted in dependence on the pH value. These curves at 3h, peaked at 6h and then declined until 18h. Ratio of LC3 aggregation positive can be well approximated by sigmoidal sensor characteristics with highest cells in hepatocytes was significantly higher than that in kupffer cells. A significant pH sensitivities around the pKa value (7.1+- 0.5). Since we are interested in hepatic dysfunction was observed at 18h after CLP. These results indicated that, during intracellular site-directed pH measurements, we fused EcGFP to the membrane polymicrobial sepsis, autophagy occurred in high-energy demanding cells and the loss targeting sequence of the small GTP-binding protein Rac1 and expressed of recycle function of autophagy at late stage was associated with the organ failure this protein in mammalian cells, e.g. HEK 293T cells, in order to measure both in kidney and liver. intracellular pH values adjacent to the inner leaflet of the plasma membrane. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 213 P1PM-21-7 P1PM-22-1 AMPK FAMILY MEMBER ARK5 IS ESSENTIAL DIVERSITY AND COMPARATIVE ANATOMICAL BASIS FOR WHITE ADIPOSE TISSUE HYPERTROPHY IN OF THE CENTRAL NERVOUS SYSTEM OF FOUR RESPONSE TO HIGH-FAT DIET FEEDING SALMONID SPECIES Atsushi Suzuki, Suni Lee, Shiki Okamoto, Tetsuya Shiuchi, Tamaki Ohya Yasuhiko Minokoshi Laboratory of Endocrinology, Graduate School of Integrated Science, Department of Developmental Physiology, National Institute for Yokohama City University, Japan Physiological Sciences, Japan Although brain studies began in ancient Egypt, speculations on vertebrate ARK5 (AMPK-related kinase #5) is a class of AMPK (AMP-activated protein kinase) brain evolution occurred only much later, after the publication of Darwin's catalytic subunit family. While ARK5 function in non-tumor cells remains unclear, Origin of Species in 1859. Subsequently, views of brain evolution have been we have reported that ARK5 promotes tumor progression by enhancing tumor cell shaped by a complex interplay of theory and technique. With the development survival against metabolic stress and inflammation. In the current study, we found of histological techniques, research shifted to descriptions of cellular structure, that ARK5 expression increases in adipocyte both of obese human as well as mouse. cellular aggregates and their putative interconnections. In spite of these technical To investigate the phathophysiological role of ARK5 in adipocyte, we generated advances, brain evolution continued to be viewed within the context of scala transgenic mouse expressing dominant negative-ARK5 (dnARK5) in adipocyte naturae. Following the publication of The Comparative Anatomy of the Nervous (aP2/dnARK5 mouse). Although high-fat diet feeding increased body weight, white System of Vertebrates by Ariens Kappers, Huber, and Crosby in 1936, there adipose tissue (WAT) organ weight and plasma glucose level in wild-type mice, aP2/ followed a period of stasis, after which biological views of evolution were dnARK5 mice did not show any increase. Interestingly, we found that adipocytes of radically altered by the confluence of genetics, paleontology, and systematics, aP2/dnARK5 mice underwent caspase-dependent apoptotic cell death followed by termed the Evolutionary Synthesis. Against this background, the development chromosomal DNA fragmentation and caspase 3 activation. Furthermore, in vitro of new experimental techniques for establishing neural connections resulted in experiments with 3T3-L1/adipocyte also showed that ARK5 promotes cell survival of a new flowering of comparative neuroanatomy. Present studies were diversity adipocyte against metabolic stress and inflammatory cytokine TNF (tumor necrosis and comparative aspect of four salmonid fish, rainbow trout, chum salmon, masu factor). These results suggest that tumor progression factor ARK5 promotes WAT salmon, and sockeye salmon, referred the work of Rudolf Nieuwenhuys entitled hypertrophy in response to high-fat diet by enhancing cell survival of adipocyte. The Central Nervous System of Vertebrates, published 1998.
P1PM-22-2 P1PM-22-3 ULTRASTRUCTURAL DIFFERENCES AND THE IMPORTANCE OF THE CROCODILIAN HEPATIC HISTOCHEMICAL CHARACTERISTICS IN SWIMMING PISTON PUMP TO VENTILATION DURING ALTERED MUSCLES BETWEEN WILD AND REARED ATLANTIC RESPIRATORY DEMAND SALMON Suzanne Munns1, Tomasz Owerkowicz2, Sarah Andrewartha3, Peter Frappell4 Katja Anttila, Satu Manttari 1 Veterinary and Biomedical Sciences, James Cook University, Australia, Department of Biology, University of Oulu, Finland 2Department of Ecology and Evolutionary Biology, University of California, USA, 3 4 The swimming capacity of wild and reared fish differs. Whether the difference is Department of Zoology, LaTrobe University, Australia, School of Zoology, associated with variations in swimming musculature is unknown. In order to compare University of Tasmania, Australia the musculature of wild and reared salmon, several morphological and enzymatic Crocodilians ventilate through a combination of the intercostal muscles, the abdominal parameters were measured. muscles and the diaphragmaticus muscle. Previous studies report that the caudal- The density and size of the mitochondria was higher in the muscles of wild fish than in cranial movement of the liver during the ventilatory cycle by the diaphragmaticus reared ones. Similar variability was noted in the density of triads. Conversely, the size muscle, termed the hepatic piston pump, is solely responsible for ventilation in floating and density of lipid droplets was lower in the muscles of wild versus reared salmon. caimans. However, the importance of the hepatic-piston pump to ventilation in crocodilians under altered conditions of ventilatory demand is unknown. The hepatic- The densities of two muscle contraction components, dihydropyridine and ryanodine piston pump made only a limited contribution to ventilation while crocodiles rested receptor, were higher in wild salmon. Similar difference was observed in the activities at 30oC, following a decrease in temperature (20oC, reduced ventilatory demand), of aerobic enzymes. Phosphorylase activity was, on the other hand, lower in the and during hypercapnia (5% CO , increased ventilatory drive). The diaphragmatic muscles of wild fish. 2 2+ muscle was important for facilitating ventilation during exercise (increased To conclude, there are significant differences in morphology, Ca regulating capacity, ventilatory demand) as loss of the hepatic piston pump, following inactivation of the and enzyme activities in swimming muscles between wild and reared salmon. diaphragmaticus muscle, compromised exercise induced increases in tidal volume and These results provide evidence that the prerequisites for efficient contraction of the minute ventilation. A relative hyperventilation was induced by exercise (both with and swimming muscles are better met in wild as compared to reared salmon. Importantly, without a functional hepatic piston pump) and, as a result, the alterations in ventilation the results also suggest that the observed variation is a major contributing factor to the following inactivation of the diaphragmaticus muscle did not significantly alter arterial difference in the swimming capacity between wild and reared salmon. oxygenation.
P1PM-22-4 P1PM-22-5 UTILITY OF THE TRANSTHYRETIN GENE (TTR) GENOMIC RESPONSES TO HEAT STRESS IN MARINE INTRON ONE FOR ELUCIDATING CROCODYLIAN FISHES PHYLOGENETIC RELATIONSHIPS Bradley A Buckley Ray E Willis Biology, Portland State University, USA Cell Physiology and Molecular Biophysics, Texas Tech University Health The increasing availability of genomic resources for a variety of fish Sciences Center, USA species have made these organisms ideal model systems in which to study transcriptional responses to environmental stresses. Comparing eurythermal The gene encoding transthyretin (TTR) is an attractive candidate for use in and stenothermal species allows for inferences to made as those genotypic phylogenetic analysis because it is a short, single-copy nuclear gene with and phenotypic responses to heat stress that confer thermotolerance. Gene regions that are highly conserved across evolutionarily-divergent organisms. expression profiling via cDNA microarray was used to determine the extent This makes the first intron of the TTR gene well-suited for sequencing across to which one species of cold adapted Antarctic notothenioid, Trematomus diverse species. TTR intron 1 (787-bp) was sequenced in 22 crocodylian bernacchii, has retained the ability to alter gene expression in response to species and analyzed using maximum likelihood, maximum parsimony and heat stress. Hundreds of genes, associated with a broad range of cellular Bayesian analyses. Uncorrected pair-wise divergence values ranged from less processes, were responsive to heat. Many of these genes are associated with than 0.3% when comparing species within Crocodylus to a high of 6.85% central aspects of the evolutionarily conserved cellular stress response (CSR), Poster Session between members of Crocodylus and the caimans. Detailed comparisons which plays a pivotal role in responding to physical and chemical stresses. The inability of T. bernacchii to mount a heat shock response underscores of TTR intron 1 resolved the three expected lineages, Alligatoroidea, the potential susceptibility of this species to the effects of global warming. In Gavialoidea, and Crocodyloidea with high support at most nodes. It offered addition, comparison of gene expression profiles from the Antarctic species additional evidence for the utility of synapomorphic indels in elucidating to those of temperate species revealed a novel gene candidate that may higher-level phylogenetic relationships. When used in conjunction with other provide a link between heat stress and cell cycle arrest in heat-stressed fishes. nuclear and mitochondrial markers, TTR intron 1 should be a valuable tool in the investigation of other closely-related groups.
214 IUPS 2009 July 27 - August 1, 2009 in Kyoto P1PM-22-6 P2AM-1-1 SYSTEMIC PATHOLOGICAL ALTERNATIONS IN IMPAIRED MYOFIBRILLAR FUNCTION IN SOLEUS THORNFISH TERAPON JARBUA BY HEAT POLLUTION MUSCLE OF MICE WITH COLLAGEN-INDUCED Chau-Heng Chien, Eileen Jea Chien ARTHRITIS 1 1 2 1 Department of Physiology, National Yang-Ming University, Taiwan Takashi Yamada , Joseph D Bruton , Nicolas Place , Shi-jin Zhang , Natalia Kosterina3, Helena E Harris4, Therese Ostberg4, Cecilia Grundtman4, Thornfish are usually present in the India-Pacific Ocean. These have been Birgitta Glenmark5, Hakan Westerblad1 many incidents of vertebral deformed thornfish found near the second nuclear 1 Department of Physiology and Pharmacology, Karolinska Institutet, Sweden, power plant in Northern Taiwan since 1992. The deformed fish were X-rayed 2Faculty of Medecine ISMMS-EEPS, University of Geneve, Switzerland 3KTH and fixed in formalin or Bouin’s solution for histopathological studies. A Mechanics, Royal Institute of Technology, Sweden 4Department of Medecine, high temperature (36ºC) in the sea water was found to be the major cause Rheumatology Unit, Karolinska University Hospital, Solna, Karolinska Instiutet, 5 of the deformation to lordosis and scoliosis in thornfish. According to the Sweden Nacka Narsjukhus Proxima AB, Sweden histopathological studies, the symptom could be classified into 3 aspects. (1) Patients with rheumatoid arthritis (RA) suffer from a progressive muscle weakness, Hyperthermia: heat stroke, brain cells edema, spinal cord degeneration and which undoubtedly impairs daily life activities. However, there is no data as to whether the muscle fibres’ intrinsic contractile properties are affected in RA. We necrosis. (2) Hypovolemic shock: hypoxic necrosis in liver, nephritis and 2+ 2+ tubular ectasia (flatting of the epithelial cells,luminal dilation, and proteins investigated muscle contractility and myoplasmic free Ca concentration ([Ca ]i) in in urine), and dehydrated kidney failure. (3) Disseminated intravascular slow-twitch soleus muscle of mice with collagen-induced arthritis (CIA). The results show a markedly decreased tetanic force per cross-sectional area, which was not due coagulation: anemia and the blood coagulation. The nuclear power plant 2+ to decreased [Ca ]i in CIA soleus muscles. Immunoblot analyses showed specific seems to emit vast amounts of thermal cooling water into bay area during changes in protein tyrosine nitration, nitrosylation, malondialdehyde-adducts and the summer. This abnormal environment places the thornfish at the limit of carbonyl content in myofibrillar proteins from CIA muscles. These changes were their thermal tolerance. The deformation of the vertebrae can be attributed accompanied by a significant increase in neuronal nitric oxide synthase expression. to circulatory shock. The results indicated global warming might expedite We conclude that arthritis causes intrinsic contractile defects in slow-twitch muscle the subtropics marine organism crisis to due to possible thermal and oxygen primarily reflecting myofibrillar dysfunction, which is related to post-translational limitation disasters. modifications mediated by nitric oxide-derived radicals.
P2AM-1-2 P2AM-1-3 ISCHEMIA-REPERFUSION CHANGES IN SKELETAL AUTOLOGOUS BONE MARROW MONONUCLEAR MUSCLE MORPHOLOGY CELLS TRANSPLANTATION COMBINED WITH Motoharu Itoh1, Noriaki Shimokawa1, Yuki Tajika2, Tohru CHINESE MEDICINE TO TREAT LOWER LIMB Murakami2, Hiroshi Yorifuji2, Noriyuki Koibuchi1 ISCHEMIA 1 2 3 2 1Department of Integrative Physiology, Gunma University, Japan, Nianhe Rong , Jianxun Dong , Guanglin Lu , Yudong Kang , 2Department of Anatomy, Gunma University Graduate School of Medicine, Xiaohui Niu2, Xuying Xu2 Gunma, Japan 1Department of Medical Humanities, Peiking University, China, 2Clinical The purpose of this study is to elucidate the regeneration mechanism of Medical School of Chinese Medicine, Capital University of Medical 3 skeletal muscle following ischemia. Male Wistar rats were anesthetized Sciences, China Beijing University of Chinese Medicine, China with intraperitoneal ketamine/xylasine. Ischemia of the right anterior tibial Objective To evaluate the therapeutic effect of autologous bone marrow mononuclear muscles was induced using clamping the anterior tibial artery. After 2 h cells transplantation combining with Chinese medicine for the treatment of limb ischemia, the rats underwent reperfusion. Muscle injuries were evaluated ischemia. Methods Forty two patients with limb ischemia were treated and by measurement of the plasma creatine phosphokinase (CPK) activity. Two, Granulocyte Colony-Stimulating Factor (CSF) was used to stimulate the bone marrow. 5, 14, 21, and 28 days after ischemia, animals were sacrificed under ether The mononuclear cells, amount more than 1×109, were separated from the aspirated anesthesia and anterior tibial muscles were removed. Cross section of the bone marrow fluid in the stem cell studio. The transplantation was performed by muscle was cut and stained with HE. Ischemia for 2 h produced a transient intramuscular multi-injection. Chinese medicine was prescribed from the first day after operation. The pain evaluation, poikilothermia evaluation, the ulcer or necrosis and increase of activity of CPK compared with the value before ischemia. After ankle/brachial index (ABI) of the ischemic limb were contrasted before and after the 2 days of ischemia, the muscular area in ischemic side was significantly treatment. Results The pain score and poikilothermia score decreased one week after decreased by 30% compared with the control (sham-ischemia) side, and the transplantation, and increased a little two weeks after the treatment, and decreased recovered after 28 days. Centronuclei were appeared after 5 days, reached a steadily one month after the treatment. The patients with limb necrosis undertook peak after 14 days and disappeared after 28 days. The number of interstitial amputation one month after the treatment, and the incision healed well. Conclusion nuclei significantly increased after 2 to 14 days of ischemia. Our results Autologous bone marrow mononuclear cells transplantation combined with Chinese indicate that reperfusion promotes muscle regeneration from ischemia. medicine improve the symptom and sign of severe lower limb ischemia efficaciously.
P2AM-1-4 P2AM-1-5 REPETITIVE STRETCHING SUPPRESSES MUSCLE DOES PASSIVE STRETCHING INFLUENCE MUSCLE ATROPHY OF DENERVATED SOLEUS MUSCLE VIA INJURY AND HSPs EXPRESSION DURING RELOADING Akt/mTOR PATHWAYS AFTER CAST IMMOBILIZATION IN RATS? 1 2 3 Nobuhide Agata , Nobuaki Sasai , Masumi Inoue-Miyadu , Keisuke Takayuki Inoue1, Shigeyuki Suzuki1, Ryusuke Hagiwara1, Masahiro Iwata2, 1 4 5 6 Kawakami , Kimihide Hayakawa , Kunihiko Kobayashi , Masahiro Sokabe Yasuhiro Banno3, Minoru Okita4 1Physical and Occupational Therapy Program, Nagoya University Graduate 1 2 Program in Physical and Occupational Therapy, Graduate School of Medicine, School of Medicine, Japan, Department of Physical Therapy, Himeji Dokkyo 2 3 Nagoya University, Japan, Department of Rehabilitation, Faculty of Health University, Himeji, Japan, Department of Rehabilitation Science Therapy, Sciences, Nihon Fukushi University, Japan, 3Faculty of Care and Rehabilitation, Aichi Medical College for Physical and Occupational Therapy, Kiyosu, Japan, Seijoh University, Japan, 4Unit of Physical and Occupational Therapy, Nagasaki 4ICORP/SORST, "Cell mechan-sencing Project", Japan Science and Technology 5 University Graduate School of Biomedical Sciences, Japan Agency, Nagoya, Japan, Department of Biomedical Sciences, Chubu University College of Life and Health Sciences, Kasugai, Japan, 6Department of Objective: We examined whether 1) passive stretching protects against/ameliorates rat Physiology, Nagoya University Graduate School of Medicine, Nagoya, Japan. soleus muscle injury induced by hindlimb cast immobilization and subsequent reloading ICORP/SORST, "Cell mechan-sencing Project", Japan Science and Technology and 2) passive stretching affects expression of heat shock proteins (HSPs) during reloading Agency, Nagoya, Japan following cast immobilization. Methods: Rat hindlimbs were immobilized for four weeks, It is well known that denervation induces atrophy of the muscle. We investigated whether passive followed by reloading through normal ambulation with or without stretching exercise (30 mechanical loading on skeletal muscle can suppress denervation-induced muscle atrophy. minutes each day). Muscle fibers infiltrating nuclei and HSPs expression were measured. Denervated soleus muscle was subjected to a 15 min repetitive stretching daily for 2 weeks. Results: The rate of invading muscle fibers increased drastically during the 1st and 2nd Histochemical analysis showed that the cross-sectional area of denervated soleus muscle fibers days of reloading. Compared with reloading alone, stretching exercise reduced invading receiving repetitive stretching was significantly larger than that of control denervated muscle muscle fibers at most time points following cast removal. Additionally, HSPs expression (p<0.05). We also examined the involvement of the Akt/mammalian target of the rapamycin (mTOR) increased progressively with time during reloading. Levels of HSPs expression following cascade in the suppressive effects of repetitive stretching, and found that levels of phosphorylated Akt, p70S6K, 4E-BP1 were significantly increased in denervated soleus muscle subjected to the application of stretching exercise were low throughout the experimental period relative to mechanical load compared to controls (p<0.05). Furthermore, suppressive effect of repetitive reloading alone; however, no significant differences were evident, respectively. Conclusions: stretching on muscle atrophy was fully inhibited by rapamycin, a potent inhibitor of mTOR. These Following immobilization, passive stretching exercise protected against/ameliorated rat results indicate that denervation-induced muscle atrophy is considerably suppressed by passively soleus muscle injury induced by reloading. Meanwhile, expression of HSPs increased during applied repetitive stretching through the upregulation of the Akt/mTOR signal pathway. reloading with or without affecting stretching exercise. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 215 P2AM-1-6 P2AM-1-7 miR-23a ATTENUATES SKELETAL MUSCLE ATROPHY EFFECTS OF THE SYNTHESIZED GLUCOCORTICOID, BY TARGETING MAFbx /ATROGIN-1 AND MuRF1 DEXAMETHASONE ON BETA1-3-ADRENOCEPTOR Shogo Wada1, Yoshio Kato2, Mitsuharu Okutsu3, Shigeru Miyaki4, mRNA EXPRESSIONS OF SKELETAL AND LEFT Katsuhiko Suzuki5, Hiroshi Asahara4, Takashi Ushida1, Takayuki Akimoto1 VENTRICLE MUSCLES 1Center for Disease Biology and Integrative Medicine, The University of Tokyo, Fuuun Kawano1, Jun Tanihata1, Shogo Sato1, Kaoru Tachiyashiki2, Japan, 2Research Institute for Cell Engineering, National Institute of Advanced 1 3 Kazuhiko Imaizumi Industrial Science and Technology (AIST), Japan, Duke-NUS Graduate Medical 1 2 School, Japan, 4Department of Regenerative Biology and Medicine, National Faculty of Human Sciences, Waseda University, Japan, Department of Research Institute for Child Health and Development, Japan, 5Faculty of Sport Living and Health Sciences, Joetsu University of Education, Japan Sciences, Waseda University, Japan Glucocorticoids are known to increase the density and mRNA levels of beta-adrenoceptors MicroRNAs (miRNAs) are small non-coding RNAs that interact with 3’ UTR of (beta-AR) in many tissues. However, little information is available on these increased effects specific target mRNAs to down-regulate translation. Recently, miRNA have been in the skeletal and cardiac muscles. Therefore, the effects of the synthetic glucocorticoid shown to play an important role in skeletal muscle development but their roles in dexamethasone (DexM, 1.0 mg/kg BW/day for 10 days, s.c.) on beta1-3-AR mRNA expressions of fast-twitch fiber-rich extensor digitorum longus (EDL), slow-twitch fiber- muscle plasticity remain to be determined. We searched miRNAs that regulate the rich soleus (SOL) and left ventricle (LV) muscles were investigated. Male Sprague Dawley expression of Atrogin-1 and MuRF1, which are E3 ubiquitin ligases and key molecules rats were divided into the DexM group and control (CON) group. The weight, RNA of skeletal muscle atrophy. Through the bioinformatics search, we nominated miR- concentration and total RNA content of EDL were 0.76-0.84 times significantly lower in 23a as a potential candidate, which is also known as one of stress responsive miRNAs DexM group than in CON group. The weight and RNA concentration of SOL were 0.92-0.87 in cardiac muscle remodeling. Luciferase reporters with 3’ UTRs of the two ubiquitin times significantly lower in DexM group than in CON group. However, the weight/BW and ligases revealed that miR-23a could down-regulate both of them. Forced expression total RNA content/BW of LV were about 1.40 times higher in DexM group than in CON of miR-23a in muscle brought significant resistance against glucocorticoid-induced group. No effects of DexM on beta1-3-AR mRNA expressions of EDL were observed. muscle atrophy and down-regulation of over-expressed miR-23a with anti sense LNA However, the DexM decreased significantly beta2-AR mRNA expression of SOL, and oligonucleotids led to cancel the resistance. These findings suggest that miR-23a increased significantly beta1-AR mRNA expression of LV. These results suggest that the targets the major two ubiquitin pathways in skeletal muscle atrophy to protect skeletal effects of DexM on beta1- and beta2-AR mRNA expressions and muscle atrophy depend on muscle from atrophy. muscle fiber types.
P2AM-1-8 P2AM-1-9 DOWNSTREAM UTROPHIN ENHANCER IS REQUIRED HYPOXIA INDUCIBLE FACTOR-1 ALPHA IS INVOLVED FOR EXPRESSION OF UTROPHIN IN SKELETAL IN MYOGENIC DIFFERENTIATION MUSCLE Akira Wagatsuma, Ryouichi Matsuda 1 2 1 Jun Tanihata , Yuko Suzuki-Miyagoe , Kazuhiko Imaizumi , Department of Life Sciences, The University of Tokyo, Japan Shin'ichi Takeda2 1 2 Hypoxia inducible factor-1 (HIF-1) transactivates numerous genes related Faculty of Human Science, Waseda University, Japan, Department of to cell proliferation, survival, and glucose/iron metabolism. The biological Molecular Therapy, National Institute of Neuroscience, Japan activity of HIF-1 is determined by the expression and activity of the HIF-1 Duchenne muscular dystrophy is caused by the absence of the muscle cytoskeletal alpha subunit. The HIF-1 alpha gene is required for somite segmentation protein dystrophin (DYS). Utrophin (UTR) is an autosomal homologue of DYS, during mouse embryogenesis. To elucidate the possible role of HIF-1 alpha and overexpression of UTR is expected to compensate for the DYS deficit. Recent study showed that the UTR gene containing the UTR core promoter did not drive in the process of myogenic differentiation, we have investigated the effect transgene expression in skeletal muscles. To clarify the regulatory mechanism of UTR of pharmacologically disruption of the HSP90-HIF-1 alpha interaction on expression, we generated a nuclear localization signal-tagged LacZ transgenic (Tg) myogenic differentiation in C2C12 cells. HIF-1 alpha mRNA was constantly mouse, in which the LacZ gene was driven by the downstream UTR enhancer (DUE) expressed during myogenesis. HIF-1 alpha protein was observed in both and the UTR promoter. The transgene mRNA expression levels were examined by the nucleus and the cytoplasm of myoblasts and myotubes. Geldanamycin RT-PCR and quantitative RT-PCR. Several tissues were stained with H&E and X-gal. induced degradation of HIF-1 alpha protein and inhibited myotube formation The transgene expression patterns were consistent with endogenous UTR in several with concomitant decreases in myogenic regulatory factors, MyoD and tissues including skeletal muscles. Transgene expression was up-regulated more in myogenin proteins. Finally, HIF-1 alpha protein was highly expressed in regenerating muscle than in non-regenerating muscle. We also established primary cultures of myogenic cells from this Tg mice and found that UTR up-regulation the regenerating muscle fibers from mice were subjected to freeze injury or during muscle differentiation depends on the DUE motif. In conclusion, DUE is received an intramuscular injection of bupivacaine and from dystrophin- indispensable for UTR expression in skeletal muscle and primary myogenic cells from deficient mdx mice. These results suggest that HIF-1 alpha may play a role in this Tg mice provide a high through-put screening system for drugs for up-regulate myogenic differentiation. UTR expression.
P2AM-1-10 P2AM-2-1 ANALYSIS OF CHANNELOPATHIES ON THE HODGKIN- REGION-SPECIFIC RESPONSES OF ADDUCTOR HUXLEY EQUATIONS FOR MUSCLE LONGUS MUSCLE TO UNLOADING AND RELOADING IN WISTAR HANNOVER RATS Kazuko Terada 1 1 2 2 Department of Medical Informatics, Toho University, Japan Takashi Ohira , Masahiro Terada , Fuminori Kawano , Naoya Nakai , Toshimasa Ochiai3, Jun-ichiro Gyotoku4, Masamichi Sudoh5, Yoshinobu Repetitive firings and depolarization of the resting potential on muscle membranes are Ohira6 the main electrophysiological features of myotonia and periodic paralysis, respectively. 1Graduate School of Frontier Biosciences, Osaka University, Japan, 2Graduate In order to reveal the mechanism behind these channelopathies, some mathematical School of Medicine, Osaka University, Japan, 3Mitsubishi Heavy Industries, bifurcation phenomena in the space-clamped Hodgkin-Huxley equations for muscles Kobe, Japan, 4Tsurui Chemical Company, Hyogo, Japan, 5School of Medicine, 6 (HHM) have been studied. Some functional defects of ion channel correspond to some Jikei University, Japan, Graduate School of Medicine and Frontier Biosciences, change of parameter value of HHM. Osaka University, Japan Two parameters on HHM are chosen as bifurcation parameters. One is the leakage Response of adductor longus (AL) muscle to unloading and reloading was studied. Male conductance, 70% of which consists of chloride conductance which decreases in Wistar Hannover rats (5 wk old) were hindlimb-suspended (HS) for 16 days with or without 16-day recovery. The integrated electromyogram (EMG) activity level during quadrupedal myotonia congenita. The other is the parameter concerning with sodium channel resting position on the floor was greater in caudal than rostral region. The EMG level in functions, which is thought to vary under the effect of drugs or in paramyotonia and both regions decreased following acute HS, but that in rostral region was even elevated hyperkalemic periodic paralysis. during continuous HS. The EMG levels in caudal region gradually increased up to 1 week, Poster Session As results, good correspondences of the real muscle and the model are found: but decreased again. All fibers in caudal region were pure type I, although ~40% were type normal action potential and normal behavior, myotonia and periodic firing, paralysis I and I+II in rostral region. De novo appearance of type I+II and II was noted in caudal or depolarized block and depolarized potential which appear in increased ionic region after HS. After HS, atrophy of type I and/or type I+II, not pure II, fibers and shift of permeability. fiber phenotype toward fast-twitch type were seen in both regions. However, adaptability of fibers to reloading was different between the regions. Fiber phenotype was recovered only The positions of point defects of sodium channel gene in paramyotonia and in caudal region after 16 days of reloading. These results suggest that different responses hyperkalemic periodic paralysis mingled on the muscle sodium channel gene. The of fibers to loading levels in AL are closely related to region-specific activity patterns. This results suggest the functional difference between those diseases might be small. study was supported by Grant-in-Aid for Scientific Research S (19100009) from Japan Society for the Promotion of Sciences.
216 IUPS 2009 July 27 - August 1, 2009 in Kyoto P2AM-2-2 P2AM-2-3 EFFECTS OF SWIMMING EXERCISE TIME ON TRANSCRIPTIONAL PROFILING IN HUMAN SKELETAL SKELETAL MUSCLE AMPKα2 EXPRESSION MUSCLE FOLLOWING A SINGLE BOUT OF HYPOXIC Ying Zhang, Yang Wang EXERCISE 1 2 3 1 Exercise Biochemistry, Sports Science, Beijing Sport University, China Nao Ohiwa , Taisuke Enoki , Toru Okuwaki , Akiko Honda , 1 4 3 The purpose of this study was to determine the effects of the different Hideyuki Takahashi , Motohiko Tanino , Takashi Kawahara 1 exercise (swimming) time on AMPKα2 gene and protein expression, and Department of Sports Sciences, Japan Institute of Sports Sciences, 2 the relationships between the expression of AMPK and the blood glucose, Japan, Department of Health Science, Osaka Kyoiku University, Japan, 3Depatment of Sports Medicine, Japan Institute of Sports Sciences, Japan, muscle glycogen and blood insulin. 4 30 C57 mice were divided into three groups: exhausted swimming exercise, Clinical Buisiness Group, DNA Chip Research Inc., Japan 90-minute swimming exercise and no exercise control. The AMPKα2 gene The hypoxic training is known to be effective methods that improve athletic performance. and protein expression in the quadriceps muscle were measured separately However, little is known about the molecular mechanisms underlying hypoxic training. To by real-time quantitative PCR and western immunoblot after the swimming search for transcriptional profiles in skeletal muscle after hypoxic training, we used an oligo exercise .Blood glucose, muscle glycogen and blood insulin were also DNA microarray, whole human genome 4*44K (Agilent Technology), to measure global measured at the same time. mRNA expression after a single bout of moderate intensity of cycling. Healthy sedentary The results showed that the 90-minute and the exhausted swimming exercise males performed the cycling bout in hypoxic or normoxic condition, and muscle biopsies were taken from the vastus lateralis before (pre), immediately after (post 1), and 4-6h after induced a significant increase in the skeletal muscle AMPKα2 gene and exercise (post2). Blood samples were collected at the same time points of muscle biopsies protein expression compared with the control group, and the AMPKα2 for measuring erythropoietin, cortisol, and ACTH. Heart rate (HR), oxygen saturation (SPO2) expression was higher after exhausted swimming than 90-minute swimming and rating of perceived exertion (RPE) were measured during sixty minutes of cycling. exercise . In the meantime, there was a significant negative correlation HR and RPE during exercise were not significantly different in both conditions. SPO2 between the AMPKα2 expression and the blood glucose level. The high significantly decreased in hypoxic exercise. Exercise-induced erythropoietin and cortisol AMPKα2 gene and protein expression induced by exercise may be one of the concentrations were significantly increased in hypoxic exercise. From our microarray data, main reasons in the blood glucose reduction. we found that 26 genes which were expressed higher after the hypoxic condition than that of normoxic by using a K-means method.
P2AM-2-4 P2AM-2-5 BONE BENDING STIFFNESS AND MINERAL DENSITY EFFECTS OF EXERCISE TRAINING AND AGING ON IN TRAINED CYCLISTS, RUNNERS AND UNTRAINED MACROAUTOPHAGY IN RAT SKELETAL MUSCLES ADULTS Yuji Ogura1, Hisashi Naito2, Ryo Kakigi2, Mitsutoshi Kurosaka2, 1 Michael T.C. Liang1, Kevin M. Lewis1, Thomas W. Spalding1, Tatsuo Akema 2 1Department of Physiology, St. Marianna University School of Medicine, Sara B. Arnaud 2 1 Japan, Department of Exercise Physiology, Juntendo University, Japan Kinesiology and Health Promotion, California State Polytechnic University, Pomona, USA, 2NASA Ames Research Center, Moffett Field, California, Macroautophagy is a major pathway of autophagy in cells, which breaks the USA included proteins using lysosomal proteases. This study investigated the effects The purpose of this study was to examine the effects of weight bearing and non-weight of chronic exercise training on macroautophagy in skeletal muscles of both adult bearing endurance training adaptation on bone mineral density (BMD) and bone and older rats. Adult (18 months) and old (28 months) male Fischer 344 rats strength. Mechanical Response Tissue Analyzer (MRTA) was used to non-invasively were assigned to either a sedentary control or an endurance training group (n = measure bone strength (EI, N.m2) of the left ulnar (EI-U) and left tibia (EI-T). A DXA 6 per group). Animals in the training groups ran on a treadmill for 8 weeks. The (PIXI GE Lunar) was used to scan the left heel (hBMD) and left wrist (wBMD). We training intensity was adjusted to be identical between adult and old groups. After recruited 29 male subjects (23.8± 4.8y) for this study: 12 trained cyclists (CYC), 10 completing the training program, the soleus and plantaris muscles were taken trained runners (RUN), and 7 untrained controls (CON). Univariate ANOVAs show for subsequent analyses. As an index of macroautophagy, the relative levels of significant (p<0.05) differences between CYC and CON for hBMD (0.63±0.02 vs. microtubule-associated protein 1 light chain (LC3)-I and LC3-II were determined 0.56±0.03 g/cm2), EI-T (304±29 vs. 246±39 N.m2), and EI-U (63.3±5.7 vs. 43.1± through immunoblotting. LC3-I was significantly increased in older animals in 7.7 N.m2) and between CYC and RUN for wBMD (0.57±0.02 vs. 0.52±0.02 g/cm2). comparison to adult animals in both soleus (P = 0.05) and plantaris (P < 0.05) When RUN was compared to CON significant differences were found for hBMD muscles. LC3-II was significantly increased in older animals in comparison to (0.66±0.03 vs. 0.56±0.03 g/cm2), EI-T (333±32 vs. 246±39 N.m2), and EI-U (59.2± adult animals in plantaris (P < 0.05) muscles. However, the exercise training 6.3 vs. 43.1±7.7 N.m2). The trained subjects show greater hBMD, EI-U and EI-T than did not modulate LC3-I and LC3-II. These results indicate that the basal state the CON. Low statistical power may prohibit us from detecting significant differences of macroautophagy is influenced by aging rather than exercise training in rat between the trained groups. skeletal muscles.
P2AM-2-6 P2AM-2-7 EXPOSURE TO PRESSURE STIMULUS SUPPRESSES ASTAXANTHIN IMPROVES OXIDANT STATUS MYOGENIN EXPRESSION IN DIFFERENTIATING WITHOUT ALTERING THE DYNAMIC PATTERN OF MYOBLASTS OXIDATIVE STRESS UNDER STRENUOUS EXERCISE Noriteru Morita1, Kenji Iizuka2, Takuji Machida2, Masahiro Horiuchi3, Nova Sylviana, Ieva Baniasih Akbar, Ambrosius Purba Masahiko Hirafuji2, Koichi Okita3 Department of Physiology and Sport Medicine, Faculty of Medicine 1Sports Education, Hokkaido University of Education, Japan, 2Department of Padjadjaran University, Indonesia Pharmacological Sciences, Health Sciences University of Hokkaido, Japan, 3 The dynamic changes of oxidative stress after strenuous exercise are still Hokusho University, Japan elusive. This study has been conducted to clarify the pattern of oxidative stress When skeletal muscle contracts during exercise, elevated intramuscular pressure is generated under supplementation of astaxanthin in trained and untrained subject. Thirty in the contracted muscle. We previously reported that artificial pressure enhanced glucose subjects, aged 17-25 years old were divided into trained and untrained groups, uptake and energy metabolism associated with protein synthesis. We attempted to relate the with or without astaxanthin supplementation. Astaxanthin was given for 1 week pressure-induced metabolic activation to myogenetic responses of the pressurized myoblasts. followed with strenuous physical test. MDA measurement was done at pre test, AIM: To test the effects of elevated pressure on myogenetic responses in myoblasts. immediately post test, 6th and 24th hours post test. The data were analyzed METHODS: L6 myoblasts were used. After change to differentiation media, atmospheric with ANOVA test. On trained subjects, the results showed significant difference pressure at 160 mmHg to the cells was applied for 3 h daily for 3 days. between astaxanthin and placebo effects on MDA level at immediate (11,55± RESULTS: Compared to controls kept under normal pressure, the number of fused 2,34 vs 16,33±7,09mmol/ml)( p= 0,01), 6-hour (5,58±3,32vs7,73±3,06mmol/l) myoblasts by morphological measure of Giemsa-stained cells reduced after 3 days pressurization. Myogenin proteins in the pressurized cells were lowered at day 1 (30%, (p= 0,021), and 24-hour post test (4,25±1,86 vs 5,14±1,07mmol/ml( p= 0,027). p<0.05) and day 3 (54%, p=0.09) compared to the controls. Myogenin mRNA decreased The same results were observed in untrained male at immediate (18,98 vs at day 1 (72%, p<0.05) and day 3 (62%, p<0.05) compared to the controls. Pressurization 20,09mmol/ml( p= 0,047), 6-hour (9,31 vs 12,74mmol/ml( p= 0,048), and increased phosphorylated ERK (2.7-fold, p<0.01) and JNK (1.9-fold, p<0.01), but not at 24-hour post test (5,02 vs 8,14mmol/ml( p= 0,048). MDA level in trained affected p38. subjects with astaxanthin showed better results. These findings support similar CONCLUSIONS: These results suggest that elevated intramuscular pressure may suppress dynamic changes between trained and untrained subjects. Astaxanthin can myogenic differentiation from myoblasts to myotubes in contracted skeletal muscles. improve oxidant status in both subjects after strenuous exercise. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 217 P2AM-2-8 P2AM-2-9 ORAL ADMINISTRATION OF LUFFA CYLINDRICA EFFECTS OF CITRIC ACID SUPPLEMENTATION ON EXTRACT INCREASES PHYSICAL STAMINA IN MICE GAS EXCHANGE DYNAMICS DURING EXERCISE 1 2 2 2 Ji-Hong Kang, Byung-Il Min, Hyun-Myung Choi Tadashi Saitoh , Noriko Taya , Saori Nakayama , Miyuki Suto , 1 Department of East-West Medicine, Graduate School, Kyung Hee Kyuichi Niizeki University, Seoul, Korea 1Department of Bio-System Engineering, Graduate School of Science and 2 The aim of the present study was to investigate the effects of Luffa cylindrica Engineering, Yamagata University, Japan, Department of Bio-System Extract (LCE) on fatigue or physical stamina. LCE (0.1 and 1 g/kg/day) Engineering, Faculty of Engineering, Yamagata University, Japan was orally administered to mice for 10 days. The control group of mice The tricarboxylic acid (TCA) cycle plays a key role in oxidative metabolism was given distilled water (0.1 ml/day). In the Forced swimming test, the and leads to the production of large amounts of adenosine triphosphate. immobility time decreased in the 0.1 g/kg and 1 g/kg LCE treated-groups (197 Citric acid is a main TCA cycle intermediate that contributes substantially ±5.2 s, and 202±8.9 s, respectively), in comparison with the control group to the TCA cycle flux. In recent years, many nutritional products containing (214±5.3 s). In addition, the contents of creatine kinase(CK), and lactic citric acid are being sold in markets. In addition, some effects of citric dehydrogenase(LDH) in the blood serum decreased by oral administration of acid supplementation are expected during exercise. However, the effects LCE. These results suggest that LCE could enhance physical stamina in mice of citric acid on the dynamics or kinetics of gas exchange during exercise by reducing muscle damage through down regulation of CK and LDH levels. remain unclear. In this study, we investigated the relationship between It may thus seem that LCE has a potential benefit for anti-fatigue. citric acid supplementation before exercise and the dynamics and kinetics of gas exchange during exercise. However, we did not observe a significant effect of citric acid supplementation on gas exchange. Thus, citric acid supplementation does not accelerate TCA cycle flux during exercise.
P2AM-2-10 P2AM-2-11 REDOX STATUS AND FITNESS AFTER GREEN TEA SUMMATION EFFECT OF GLUCOSE AND CAFFEINE EXTRACT SUPPLEMENTATION ON AEROBIC PREDOMINANT SPORT PERFORMANCE Christina Karatzaferi1, Giorgos K Sakkas2, Michalis Nikolaidis2, Vassilis IN TRAINED SUBJECTS Paschalis1, Georgia Tsiotra2, Ahanassios Jamurtas1, Dimitrios Kouretas3, 1 2 2 4 1 Iwan Setiawan , Noni Zakiah , Gaga Irawan Nugraha Ioannis Stefanidis , Yiannis Koutedakis 1 1 Physiology, Faculty of Medicine Universitas Padjadjaran, Indonesia, Department of P.E. and Sport Sciences, University of Thessaly, Greece, 2 2 Department of Nutrition, Universitas Padjadjaran, Indonesia Institute for Human Performance and Rehabilitation, CERETETH, Greece, 3Department of Biochemistry and Biotechnology, University of Thessaly, Greece, Use of glucose and caffeine alone and combination is still controversial in 4 Department of Nephrology, University of Thessaly, Greece enhancing sport performance. Therefore, the study has been done to clarify We examined, in a randomized, double blind, placebo-controlled study, whether 3 this issue in particular to aerobic predominant sport. months supplementation with caffeine-free green tea extract (GTE) would: a) affect An experimental study with pre-post-test design was performed in 27 male blood redox status, and b) improve fitness. Twenty healthy volunteers were assigned to trained subjects aged 18-21 years, divided into group with glucose, caffeine, receive placebo or 300 mg GTE (5 male and 5 female in each group). Subjects run on and the combination of glucose and caffeine. Lap time of 3000-m run before a treadmill, for 45 min at 75% VO2max and thereafter to exhaustion at 90% VO2max, and after treatment was measured. pre and post 3 month supplementation. Blood was sampled at rest and exhaustion The results showed that the administration of glucose alone in 3000-m for determination of GSH, GSSG (and GSH/GSSG), TBARS, protein carbonyls, run improved lap-time by 7.5% (990.33 ± 19.81 seconds vs. 916 ± 94.28 catalase, and total antioxidant capacity (TAC). Pre and post supplementation, subjects’ seconds), caffeine alone by 7.8% (989.22 ± 129.39 seconds vs. 912.11 ± body composition was assessed by DEXA. Possible differences over time & between groups were examined at p<0.05. Exercise caused significant oxidative stress to all 120.85 seconds) and the combination of glucose and caffeine by 13% (993.56 subjects on both occasions. With GTE, GSH concentration at rest was decreased ±125.88 seconds vs. 864.78 ± 94.25 seconds). Combination of glucose and and the exercise-induced increase in GSSG was lower vs placebo (p<0.05). GTE caffeine was the most effective compared to glucose and caffeine alone caused higher increases in exercise-induced protein carbonyls vs placebo (p<0.05). (864.78 ± 94.25 seconds vs. 912.11 ± 120.85 seconds vs. 916 ± 94.28 Subjects on GTE reduced their body fat (p<0.05). There was no gender effect: GTE seconds, respectively). supplementation resulted in: a) altered blood redox status at rest & post exercise, b) an This study clarifies summation effect of glucose and caffeine on aerobic improvement of body composition predominant sport performance in trained subjects.
P2AM-2-12 P2AM-2-13 EFFECTS OF β2-AGONIST, CLENBUTEROL ON TESTOSTERONE STIMULATES MYOGLOBIN THE mRNA EXPRESSION OF mRNA BINDING/ EXPRESSION IN DIFFERENT MUSCLES OF THE DEGRADATION FACTORS OF β-ADRENOCEPTOR IN MOUSE RAT MUSCLES Satu Manttari, Katja Anttila 1 1 1 2 Shogo Sato , Jun Tanihata , Fuuun Kawano , Kaoru Tachiyashiki , Department of Biology, University of Oulu, Finland 1 Kazuhiko Imaizumi The regulation of energy metabolism is one of the major functions of steroid 1 2 Faculty of Human Sciences, Waseda University, Japan, Department of hormones. This study was performed to explore whether testosterone Living and Health Sciences, Waseda University, Japan can regulate the aerobic capacity of skeletal muscles via myoglobin
β2-agonist, clenbuterol (CLE) is used as a non-steroidal anabolic drug for sports (Mb) expression. Changes in testosterone level were quantified, and the doping. Recently, we reported that the effects of CLE on β1- and β2-adrenoceptor level of Mb in mice subjected to six weeks of training or testosterone (AR) mRNA expressions in skeletal and cardiac muscles depend on muscle fiber administration was analyzed. Both treatments significantly increased the types (Sato S et al. J Pharmacol Sci, 107:393-400, 2008). However, little is known plasma testosterone level. Training induced a significant increase in the Mb about CLE-induced changes of the β-AR transcriptional efficiency and/or mRNA content in gastrocnemius and plantaris muscles (287 and 83%, respectively). stability. Therefore, the effects of CLE on the mRNA expressions of mRNA binding/ Testosterone administration increased Mb concentration in plantaris (183%) degradation factors such as hnRNP A1 and HuR (construct β-ARBP), and hnRNP D0 but not in gastrocnemius. In extensor digitorum longus the protein content (AUF-1) in fast-twitch fiber rich extensor digitorum longus (EDL), slow-twitch fiber Poster Session decreased slightly after exercise, but increased 78% after testosterone rich soleus (SOL) and left ventricle (LV) were studied by real-time RT-PCR. Adult male rats were divided into CLE-administered (dose=1.0 mg/kg BW/day, for 10 days, administration. In soleus and rectus femoris the Mb content was unchanged s.c.) and the control groups. CLE significantly decreased hnRNP A1 and HuR mRNA after both treatments. The data show that testosterone and training have expressions in EDL. CLE significantly also decreased hnRNP D0 mRNA expression differential effects on the concentration of Mb in some, but not all muscles. in EDL. However, no effects of CLE on the mRNA expressions of these factors in This may have an influence on the aerobic capacity in mouse skeletal SOL and LV were observed. These results suggest that CLE-induced down-regulation muscles. The data demonstrated that both testosterone administration and of β2-AR mRNA expressions in fast-twitch muscle is weakly associated with β-ARBP training induced an increase in plasma testosterone level. However, the and/or AUF-1 and mainly with transcriptional factors (e.g. CREB). effects of the treatments on the Mb concentration differ.
218 IUPS 2009 July 27 - August 1, 2009 in Kyoto P2AM-2-14 P2AM-2-15 EFFECTS OF REACTIVE OXYGEN SPECIES ON NEURONAL NITRIC OXIDE SYNTHASE IS AN SKELETAL MUSCLE METABORECEPTORS ACTIVITIES ESSENTIAL MEDIATOR FOR MUSCLE HYPERTROPHY FROM GROUP IV AFFERENTS IN RATS Naoki Ito1, Beryl Nyamekye Ampong2, Akira Kudo1, Yuko Miyagoe- 3 3 Stephane Delliaux, Christelle Brerro-Saby, Yves Jammes Suzuki , Shin'ichi Takeda 1Department of Biological Information, Graduate School of Bioscience and Physiology - UMR MD2, P2COE, IFR Jean Roche, Faculte de Medecine 2 de Marseille, Aix-Marseille Universite, France Biotechnology, Tokyo Institute of Technology, Japan, Children's National Medical Center, Washington DC, USA, 3Department of Molecular Therapy, The group IV muscle afferents (>50% of skeletal muscle afferents) are activated National Institute of Neuroscience, NCNP, Japan by biochemicals as lactic acid, potassium, and inflammatory mediators. Muscle In skeletal muscle, neuronal nitric oxide synthase (nNOS) is mainly localized at the contraction produces also reactive oxygen species (ROS), which exert facilitatory sarcolemma, as a member of the dystrophin-glycoprotein complex. We previously action on muscle spindles (Delliaux S. et al., Pflugers Arch. 2008). We tested the demonstrated that unloading activates nNOS and promotes muscle atrophy by hypothesis that ROS had similar effects on the group IV afferents. activating FoxO3a (J Clin Invest. 117:2468-76, 2007). In addition, we observed In adult rats, we tested the effects of intra-muscular (IM) injection of H2O2 that re-growth of atrophied muscle after reloading is impaired in nNOS knock- and of superoxide dismutase (SOD) on the frequency discharge of group IV out mice (nNOS-/-), suggesting that nNOS activity is dispensable in muscle growth afferents from the tibialis anterior muscle, at rest and after electrically-induced process. To investigate the function of nNOS in muscle hypertrophy, we surgically ablated gastrocnemius and soleus muscles of nNOS+/+ and nNOS-/- mice to induce muscle fatigue. Afferents were identified using measurement of nerve conduction -/- velocity, determination of field receptor and activation by lactic acid. compensatory hypertrophy of plantaris muscle. Interestingly, nNOS plantaris muscle In resting muscle, H2O2 markedly activated the group IV fibres (+ 570 ± 169 %) showed delayed muscle hypertrophy. Immunohistochemistry revealed that nNOS levels at the sarcolemma were largely decreased after surgery. Western blotting while SOD significantly lowered their spontaneous discharge rate (- 36 ± 13 %). confirmed that the level of nNOSmu (major nNOS isoform in skeletal muscle) greatly Moreover, the early post-fatigue-activation of group IV fibres was suppressed by decreased, but instead, shorter nNOS isoforms lacking N-terminal nNOS PDZ domain SOD pre-treatment. increased in overloaded muscle. These data suggest that nNOS is sensitive to the We concluded that metaboreceptors activities of the group IV muscle afferents mechanical load on muscle and nNOS isoforms not associated with the sarcolemma depend on ROS, both at rest and during exercise or fatigue. are involved in muscle hypertrophy.
P2AM-2-16 P2AM-2-17 POSSIBLE ROLE OF HEAT SHOCK FACTOR 1 IN SOME ASPECTS OF NF-KAPPAB-RELATED LOADING-ASSOCIATED MUSCLE HYPERTROPHY OF SIGNALS IN HEAT STRESS-ASSOCIATED MUSCLE MICE HYPERTROPHY IN RATS Katsumasa Goto1, Yoshitaka Ohno1, Akira Nakai2, Takao Sugiura3, Yoshitaka Ohno1, Katsumasa Goto1, Sumio Yamada2, Takao Yoshinobu Ohira4, Toshitada Yoshioka5 Sugiura3, Yoshinobu Ohira4, Toshitada Yoshioka5 1 2 Laboratory of Physiology, Toyohashi SOZO University, Japan, Department 1Laboratory of Physiology, Toyohashi SOZO University, Japan, 2School of Molecular Biology, Yamaguchi University School of Medicine, Japan, of Health Sciences Nagoya University, Japan, 3Faculty of Education, 3Department of Exercise and Health Sciences, Yamaguchi University, Japan, 4 4 5 Yamaguchi University, Japan, Graduate School of Medicine, Osaka Graduate School of Medicine, Osaka University, Japan, Hirosaki Gakuin University, Japan, 5Hirosaki Gakuin University, Japan University, Japan Molecular mechanism responsible for muscle hypertrophy in response to heat stress Hypertrophic stimuli, such as mechanical as well as heat stress, for skeletal muscles cause an is still unclear. The purpose of this study was to investigate the contribution of nuclear increase in the expression of stress proteins, so-called heat shock proteins (HSPs) in skeletal muscles. The precise mechanism, as well as the physiological roles, for HSPs induction in factor-κB (NF-κB)-related intracellular signals for heat-stress-associated muscle hypertrophy. Male Wistar rats (7-week-old) were randomly divided into two groups; (1) skeletal muscles is still not fully understood. The induction of HSPs expression is regulated o by heat shock factor 1 (HSF1), which binds to heat shock elements located on the upstream control and (2) heat-stressed groups that were exposed to heat stress (42 C for 60 min) region of all Hsp genes. In the present study, using the transgenic mice expressing the active in an incubator without anesthesia. Significant decrease in the expression of activated form of HSF1 (Tg-HSF1), we examined the role of HSF1 in the overloading-associated NF-κB was observed 1 day after the application of heat stress. There were significant hypertrophy of skeletal muscles. Functional overloading on soleus was induced by tenotomy increases in the number of satellite cells and protein content of soleus muscle 3 and of synergistic muscles, and was maintained for 4 weeks. Increase in overloading-induced 7 days after heat stress, respectively. It is suggested that NF-κB signaling would protein content of soleus muscles was observed compared with that of wild type animals. be, in part, involved in heat-stress-associated skeletal muscle hypertrophy. These Results from this study indicated that active HSF1 has strong facilitated effect on the protein observations suggest that the application of heat stress to skeletal muscle is a useful synthesis under the hypertrophic condition. This study was supported, in part, by Grant-in- tool for an increase in muscle mass and force generation in human subjects. This Aid for Scientific Research (B, 20300218, KG; A, 18200042, TY; S, 19100009, YO) from study was supported, in part, by Grants-in-Aid for Young Scientists (B, 19700451, Japan Society for the Promotion of Science, and Research Grant from KAO Health Science YO), Grants-in-Aid for Scientific Research (B, 20300218, KG; A, 18200042, TY; S, Research (KG). 19100009, YO) from Japan Society for the Promotion of Sciences.
P2AM-2-18 P2AM-2-19 THE EFFECTS OF COENZYME Q10 INTRAMUSCULAR MUSCULAR METABOLIC LOAD SUPPLEMENTATION ON PERFORMANCE DURING IN MULTIPLE SETS LOW-INTENSITY RESISTANCE REPEATED BOUTS OF SUPRAMAXIMAL EXERCISE IN EXERCISE WITH DIFFERENT PATTERS OF BLOOD SEDENTARY MEN FLOW RESTRICTION 1 2 2 2 Shingo Takada1, Koichi Okita1, Tadashi Suga2, Noriteru Morita3, Hakki Gokbel , Ibrahim Gul , Muaz Belviranli , Nilsel Okudan 1 1 2 1 2 Masashi Omokawa , Masahiro Horiuchi , Takashi Yokota , Department of Physiology, Selcuk University, Turkey, Department of 2 2 2 Physiology, Meram Faculty of Medicine, Selcuk University, Turkey Kagami Hirabayashi , Shintaro Kinugawa , Hiroyuki Tsutsui 1Graduate School, Hokusho University, Japan, 2Department of Cardiovascular The objective of this study was to determine the effects of oral coenzyme Q10 (CoQ10) Medicine, Hokkaido University Graduate School of Medicine, Japan, 3Hokkaido supplementation on performance during repeated bouts of supramaximal exercise. University of Education, Japan This randomized, double blind, crossover study on 15 healthy and sedentary men was Background: Resistance exercise with blood flow restriction (BFR) effectively increases approved by the Ethics Committee of Meram Faculty of Medicine, Selcuk University. muscle bulk and strength despite using smaller, whereas we reported that its usual protocol The study composed of two 8-week periods (separated by a 4-week washout period) of a single set could not result in energetically sufficient stimulus. The purpose of this study -1 of supplementation with CoQ10 (100 mg.day ) and placebo. Five Wingate tests (WT) was to examine the effective low-intensity resistance exercise with BFR protocol composed with 75 g.kg body weight-1 load with 2 min intervals between tests were performed of multiple sets. Method: Twelve healthy subjects (22+-1 yr, mean+-SE) performed 3 sets three times at baseline and after CoQ10 and supplementation. Peak power, mean power of 1-min unilateral plantar-flexion (30 repetitions) with 1-min intervals. Protocols were and fatigue index were calculated. During Wingate tests peak power and mean power randomly performed with high-intensity resistance exercise (65%1RM) without BFR (H), tended to decrease and fatigue index tended to increase in all groups. Peak power low-intensity exercise (20%1RM) combined with intermittent (only during exercise) BFR decreased with CoQ (WT1 and WT2) and placebo supplementation (for WT2). Mean (I-BFR) and continuous BFR (C-BFR). Intramuscular metabolic load was measured using 10 31P-Magnetic resonance spectroscopy. Result: Integrated phosphocreatine depletion through power increased only with CoQ10 supplementation during the WT5. Fatigue index the whole exercises (I-BFR: 54.4+-3.9, C-BFR: 90.0+-4.5, H: 91.4+-4.9 mM×min) and decreased with CoQ10 supplementation but these decreases did not different from that intramuscular pH decrease at the end of final set (I-BFR: 6.96+-0.02, C-BHR: 6.84+-0.02, seen with placebo supplementation. In conclusion, CoQ10 may show performance H: 6.87+-0.02) in C-BFR were greater than those in I-BFR (p<0.05) and comparable in H. enhancing effects during the repeated bouts of supramaximal exercises and CoQ10 Conclusion: Intramuscular metabolic load in low-intensity resistance exercise with multiple might be used as ergogenic aid. sets might be effectively enhanced by continuous BFR. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 219 P2AM-2-20 P2AM-2-21 EXAMINING A PROTOCOL OF LOW INTENSITY EFFECTS OF REPEATED SUPRAMAXIMAL EXERCISES RESISTANCE TRAINING WITH BLOOD FLOW ON PLASMA ADIPONECTIN, IL-6 AND TNF-α LEVELS RESTRICTION Hasan Serdar Gergerlioglu1, Hakki Gokbel1, Nilsel Okudan1, 1 1 2 Masashi Omokawa , Koichi Okita , Tadashi Suga , Noriteru Ibrahim Gul1, Muaz Belviranli1, Mustafa Kemal Basarali2 3 1 1 2 Morita , Shingo Takada , Masahiro Horiuchi , Takashi Yokota , 1 2 2 2 Department of Physiology, Selcuk University Meram Faculty of Medicine, Kagami Hirabayashi , Shintaro Kinugawa , Hiroyuki Tsutsui Turkey, 2Departments Meram Faculty of Medicine, Selcuk University, 1Graduate School, Hokusho University, Japan, 2Department of Konya, Turkey Cardiovascular Medicine, Hokkaido University Graduate School, Japan, 3 Introduction: Adiponectin is a protein hormone that modulates a number of metabolic Hokkaido University of Education, Japan processes, including glucose regulation and fatty acid catabolism. Exercise causes to Background: Conventional resistance training needs to load muscles intensively and is hard increase insulin sensitivity as adiponectin does. Tumor necrosis factor α (TNF-α) is for cardiac and elder subjects. Resistance training with blood flow restriction (BFR) is a believed to modulate the release of adipokines including adiponectin and interleukin new method to effectively increase muscle bulk and strength despite using small workload, 6 (IL-6). IL-6 concentration also increases in exercise. We aimed to study the possible whereas we reported that usual Kaatsu training protocol couldn’t result in energetically effects of repeated supramaximal exercise on plasma adiponectin, IL-6 and TNF-α sufficient stimulus. The purpose of this study was to examine the optimal Kaatsu training levels. protocol in terms of muscle energy metabolism. Method: Fourteen healthy subjects (22+-6 yr) performed unilateral plantar-flexion for 2-min with 30 repetitions/min. Protocols were Material and Method: The study included 14 healthy sedentary adults. Wingate test randomly performed with larger workload (W: 30%1RM+usual BFR) and higher cuff was performed 5 times with 75 g.kg-1 body weight load with 2 min intervals. Blood pressure (P: 20%1RM+BFR with 200mmHg) compared with usual procedure (20%1RM samples were collected at rest, immediately after, 15 min and 60 min after the fifth with BFR by 1.3 times systolic pressure). Phosphocreatine and intramuscular pH were Wingate test. Plasma adiponectin, TNF α and IL 6 levels were measured. measured every 30 sec by 31P-Magnetic resonance spectroscopy. Result: Phosphocreatine Results: Plasma adiponectin level 60 min after 5th Wingate test was lower than resting depletion (W: 55.3+-12.8 vs P: 47.8+-12.7%, p<0.05) and intramuscular pH decrease (W: level. IL-6 levels immediately after and 60 min after exercise) were lower than the 6.88+-0.05 vs P: 6.91+-0.04, p<0.05) were greater in W than in P. Conclusion: Low intensity resting level TNF-α levels after 5th Wingate test were not different from resting level. resistance training with BFR could be effectively performed by increasing workload rather Conclusion: Plasma adiponectin level decreases, IL-6 level increases and TNF-α level than by increasing cuff pressure to restrict blood flow. does not change after repeated supramaximal exercises.
P2AM-2-22 P2AM-2-23 SEROTONIN CONTENT IN PREOPTIC AREA IS ENHANCED SKIN BLOOD FLOW RESPONSE TO RELATED TO CHOLINERGIC SYSTEM ACTIVATION OF HYPERTHERMIA AFTER ENDURANCE TRAINING IS HEAT LOSS IN RUNNING RATS MAINLY CAUSED BY PLASMA VOLUME EXPANSION Candido Celso Coimbra1, Alex G Rodrigues2, Danusa S Soares1, IN HUMANS Umeko Marubayashi1 Shigeki Ikegawa, Yoshi-Ichiro Kamijo, Kazunobu Okazaki, Shizue 1Department of Physiology & Biophysics, ICB - Federal University of Minas Masuki, Yoshiyuki Okada, Hiroshi Nose Gerais, Brazil, 2IBHS - Pontificia Universidade Catolica de Minas Gerais, Department of Sports Medical Sciences, Shinshu University Graduate Brazil School of Medicine, Japan To investigate the influence of central cholinergic system on thermoregulation and We tested the hypothesis that enhanced skin blood flow (SkBF) response to brain serotonin (5-HT) concentration during exercise, 2 uL of physostigmine (PHY, hyperthermia after endurance training (ET) was mainly caused by plasma volume 5x10 mM) or saline (SAL, 0.15 M) solution was injected into the lateral cerebral (PV) expansion in humans. Seven young men performed ET at 70% peak oxygen ventricle of running rats. At fatigue, brains were quickly removed and serotonin (5-HT) consumption rate (VO2 peak) at 30ºC, 30 min/day, 5 days, while monitoring daily food intake. Before and after ET, we measured esophageal temperature (T ) and forearm and 5-HIAA were measured in preoptic area (POA), hypothalamus, frontal cortex, es vascular conductance (FVC) during 30-min exercise at pre-training 65% V and hippocampus. PHY injection attenuated the exercise-induced hyperthermia and O2 peak in the same environment as in ET in 2 conditions; normovolemia (Nor) and acute heat storage that was closely related to the 5-HT (r=0.787, p<0.01) and to 5-HIAA hypovolemia (Hypo) with a diuretic. ET lowered Tes threshold for an increase in FVC (r=0.894, p<0.01) content in POA. Exercise promoted an increase in core temperature (TH ) by 0.28ºC and elevated the sensitivity of an increase in FVC at a given rise in both groups, however, changes in body temperature at fatigue point were lower in FVC in Tes (ΔFVC/ΔTes) by 32% with ~10% PV expansion. In Hypo before ET, THFVC Phy-treated than Sal-treated rats (0.92 ±0.030 C PHY vs 1.17±0.090 C; p<0.05). Phy increased by 0.13ºC and ΔFVC/ΔTes decreased by 5% compared with those in Nor treatment also increases heat dissipation by decreasing core temperature threshold for before ET with ~10% PV loss. Similarly, in Hypo after ET, TH increased by 0.22ºC 0 FVC vasodilation in 0.48 C (p<0.05). In conclusion, our data indicated that stimulation of and ΔFVC/ΔTes decreased by 38 % compared with those in Nor after ET with ~20% central cholinergic system promotes heat dissipation in running rats that is related to PV loss. We found that the effects of a given change in PV on THFVC and ΔFVC/ΔTes decreased serotonin content in preoptic area. after ET were not different from those by Hypo (P>0.05). Thus, enhanced SkBF Support: FAPEMIG, CNPq, CAPES response after ET might be mainly caused by PV expansion.
P2AM-2-24 P2AM-2-25 RAPID PLASMA VOLUME RESTORATION ENHANCES SYMPATHETIC VASOCONSTRICTOR SKIN VASODILATION BUT NOT SWEAT RATE IN RESPONSIVENESS IS REDUCED BY 5-DAY HYPOVOLEMIC AND PASSIVELY WARMED MEN ENDURANCE TRAINING WITH ENHANCED Yoshi-ichiro Kamijo, Kazunobu Okazaki, Shigeki Ikegawa, BAROREFLEX SENSITIVITY IN MEN Kazunobu Okazaki1, Yoshi-ichiro Kamijo1, Shizue Masuki1, Shigeki Yoshiyuki Okada, Hiroshi Nose 1 1 1 2 2 Department of Sports Medical Sciences, Shinshu University Graduate Ikegawa , Yoshiyuki Okada , Daisuke Yazawa , Takeki Hata , Yuji Shiba , Masafumi Takahashi2, Uichi Ikeda2, Hiroshi Nose1 School of Medicine, Japan 1Department of Sports Medical Sciences, Shinshu University Graduate School of We examined whether a rapid plasma volume (PV) restoration enhanced skin Medicine, Japan, 2Department of Cardiovascular Medicine, Shinshu University vasodilation while not sweat rate in hypovolemic hyperthermia. Sixteen young men, Graduate School of Medicine, Japan who underwent ~12% acute hypovolemia with a diuretic, were divided into 2 groups; We tested the hypothesis that sympathetic vasoconstrictor responsiveness (VCR) in the control (n=7, C) and rapid infusion (n=9, RI). Subjects in both groups were warmed trained muscles was reduced while baroreflex sensitivity (BRS) was enhanced after o for 80min with a perfused suit until core temperature rose by 0.7 C. After 50min of endurance training (ET). Ten young men (age, 21±2 (SD) yr) performed cycling exercise for o warming, in RI, saline at 37 C was infused at 0.7ml/min/kg for 30min so that PV was 5 days (70%Vo2peak, 30 min/day). Before and after ET, VCR in the forearm was determined restored to the baseline by the end of infusion while in C at 0.1ml/min/kg. After the from vascular conductance response (plethysmography) to α1 and α2 agonists, phenylephrine start of warming, cutaneous vascular conductance [CVC = skin blood flow (laser- and dexmedetomidine, respectively, locally injected through a catheter in brachial artery. Poster Session Doppler flowmetry) on the dorsal foot/mean arterial pressure] gradually increased and VCR in the calf was determined from vascular conductance response (plethysmography) at the 50th min of warming, the increase was 200% compared with the baseline in both to an increase in total muscle sympathetic nerve activity (MSNA, microneurography) groups (P<0.01). However, after infusion, CVC rose by 115% compared with before evoked by rhythmic handgrip. We also determined sympathetic (total MSNA/diastolic blood pressure (BP)) and cardiac (heart rate/systolic BP) BRS by the modified Oxford method. We infusion in RI (P<0.01) while remained unchanged in C. Sweat rate (SR; hygrometry) found that VCR in the calf decreased (-0.029 vs -0.012 AUxmin/SAU, P<0.05) while not in and burst frequency of skin sympathetic nerve activity (microneurography) from the forearm, and that both sympathetic (-0.98 vs -1.53 SAU/mmHg) and cardiac BRS (-0.59 the peroneal nerve rose during warming similarly in both groups (P<0.01) but no vs -0.73 beats/min/mmHg, both P<0.02) were enhanced after training. Thus, ET by using significant differences occurred by infusion. Thus, a rapid PV restoration enhanced lower extremities reduced VCR in the calf while not in the forearm with enhanced BRS, skin vasodilation but not SR in hypovolemic hyperthermia. suggesting a close association between VCR and BRS.
220 IUPS 2009 July 27 - August 1, 2009 in Kyoto P2AM-2-26 P2AM-2-27 INCREASE IN RESPIRATORY QUOTIENT AT NIGHT EFFECT OF MUSCLE METABOREFREX ACTIVATION IS BLUNTED IN VASOPRESSIN V1A RECEPTOR ON PUPIL DIAMETER IN HUMANS KNOCKOUT MICE 1 2 3 3 Naoyuki Hayashi, Nami Someya Shizue Masuki , Taka-aki Koshimizu , Jinze Qian , Keiichi Higuchi , Gozoh Institute of Health Science, Kyushu University, Japan Tsujimoto2, Hiroshi Nose1 1Department of Sports Medical Sciences, Shinshu University Graduate School The pupil diameter is increased by sympathetic activation. However, it of Medicine, Japan, 2Department of Genomic Drug Discovery Science, Graduate remains unclear whether activation by metabolically sensitive skeletal muscle 3 School of Pharmaceutical Sciences, Kyoto University, Japan, Department of afferents (i.e., the muscle metaboreflex) dilates the pupil. We investigated Aging Biology, Shinshu University Graduate School of Medicine, Japan the influence of muscle metaboreflex activation on the pupil diameter. Eight Vasopressin is known to modulate glucose and lipid metabolisms. We recently healthy subjects performed 2 min of isometric hand-grip exercise at 30 found that vasopressin V1a receptor polymorphism altered body mass index and % maximal voluntary contraction, which was followed by either 2 min of blood glucose in humans. In this study, we assessed glucose and lipid metabolisms during the day (inactive phase) and night (active phase) in vasopressin V1a receptor postexercise muscle ischemia (PEMI) in the forearm (experimental trial) or no PEMI (control trial). The pupil diameter and blood pressure were knockout mice (KO). We continuously measured oxygen consumption rate (VO2, mass spectrometry), carbohydrate production rate (VCO2), and activity in free-moving continuously recorded. The mean blood pressure increased significantly from KO (n=7) and wild type mice (WT, n=7) with L (600-1800) / D (1800-600) cycle rest during exercise in both trials, and this increase was maintained during the for 24 h. Activity, VO2, and VCO2 were higher at night than during the day (P<0.05) PEMI period in the experimental trial but not during the recovery period in with no differences between WT and KO (P>0.1). Respiratory quotient (RQ) also the control trial. Hand-grip exercise significantly increased the pupil diameter increased at night compared with during the day (P<0.05) in both groups. However, by 7 ± 1 % (mean ± SE ) and 5 ± 1 % from the resting baseline in the control we found that RQ tended to be lower in KO than WT during the day (0.83±0.02 vs 0.87 and experimental trials, respectively. This increase was maintained during the ±0.01, P=0.1), and it was more prominent at night (0.87±0.02 vs 0.96±0.01, P<0.01), suggesting that catabolic rate of glucose was lower while that of lipid was higher in PEMI period in the experimental trial while it decreased by half during the KO than WT, more markedly at night. Thus, V1a receptor modulates glucose and lipid recovery period in the control trial. These findings suggest that the muscle metabolisms during active phase more than inactive phase, suggesting the mechanisms metaboreflex contributes to dilation of the pupil. for the different phenotype by the polymorphism in humans.
P2AM-2-28 P2AM-2-29 MYOCARDIAL INFARCTION DECREASES NERVE REAL TIME IMAGING OF MICROVASCULAR VOLUME GROWTH FACTOR IN PRIMARY AFFERENT NEURONS IN HUMAN AND RAT SKELETAL MUSCLE DURING OF RATS INSULIN STIMULATION AND MUSCLE CONTRACTION 1 2 3 1 Jianhua Li, Jian Lu, Jihong Xing, Lawrence Sinoway Kim Anker Sjoberg , Stephen Rattigan , Natalie Hiscock , Erik A Richter , Bente Kiens1 Heart & Vascular Institute and Department of Medicine, Penn State 1 University College of Medicine, USA Molecular Physiology group, Dep. of Sport and Exercise Science, University of Copenhagen, Denmark, 2Menzies Research Institute, University of Tasmania, Nerve growth factor (NGF) plays a critical role in the maintenance and Hobart, Australia., 3Unilever Discover, Shambrook, Bedfordshire, UK survival of both sympathetic and sensory neurons. Also, NGF can alter receptor The purpose of the present study was to measure contraction and insulin induced expression and neuronal activity in the sensory neurons. Abnormalities in NGF changes in microvascular blood flow (MBF) in the forearm and in the vastus lateralis regulation are seen in patients and animals with heart failure (HF). For example, muscle in humans and in rat hindleg skeletal muscle. For this a low mechanical index plasma concentrations of NGF are almost undetectable in HF patients. In this (MI) technique and a constant infusion of Definity microbubbles were applied which experiment, the ELISA method was used to assess NGF levels in the dorsal root gives a detailed live picture of the microvascular refilling compared to the retrospective ganglion (DRG) neurons of sham-control rats and rats with myocardial infarction pulsing interval technique that has previously been used and allows assessment of (MI). The MI was induced by ligation of the coronary artery. The levels of NGF capillary blood flow (Zhang et al 2004, Diabetes: 53(2):447-53). were significantly decreased in the DRG neurons 6-10 weeks after the ligation. In humans, handgrip contractions increased MBF by 170% in forearm muscle, and The NGF was 18±3 pg/mg wet weight (P<0.05, vs. control) in eight MI rats insulin stimulation increased MBF by 30% in forearm muscle and by 70% in vastus lateralis, compared to basal conditions. In the rat hindleg muscle, electrical stimulation and 32±5 pg/mg in six sham-control rats. In addition, a close relation was seen (1Hz) increased MBF by 320% (n=7) and insulin stimulation increased MBF by 98% between the NGF levels and the left ventricular function. Chemically sensitive (n=4) compared to the basal conditions. afferents are engaged in the sympathetic response to muscle activity via a reflex In conclusion these data indicate that the low MI technique is an effective tool in the mechanism, and this response is attenuated in HF. Taken together with the assessment of microvascular blood flow. Furthermore, the technique gives a detailed previous findings, our data support the notion that NGF is an important regulator live picture of the vascular bed that may provide new insights into regulation of blood responsible for the development of abnormal sympathetic responses seen in HF. flow and allows the time course of capillary recruitment to be visualized.
P2AM-2-30 P2AM-2-31 FUNCTIONAL ANALYSIS OF THE PGC-1α PROMOTER DECREASED MUSCLE SENSORY FEEDBACK IN RESPONSE TO EXERCISE BY IN VIVO IMAGING INFLUENCES THE PROTEIN EXPRESSION IN RAT Takayuki Akimoto1, Ping Li2, Zhen Yan2 BRAIN 1 2 1 1Center for Disease Biology and Integrative Medicine, The University of Fuminori Kawano , Yoshie Nakajima , Naoya Nakai , Masahiro Tokyo, Japan, 2Department of Medicine, Duke University Medical Center, Terada3, Takashi Ohira3, Yoshihiko Oke1, Sachiko Nomura1, USA Yoshinobu Ohira1 Real-time optical bioluminescence imaging is a powerful tool for studies 1Graduate School of Medicine, Osaka University, Japan, 2School of gene regulation in living animals. To elucidate exercise-induced of Science, Osaka University, Japan, 3Graduate School of Frontier signaling/transcriptional control of the peroxisome proliferator-activated Biosciences, Osaka University, Japan receptor γ coactivator-1α (Pgc-1α) gene in skeletal muscle, we combined Effects of deafferentation on the protein expression in the brain of adult rats were this technology with electric pulse-mediated gene transfer to co-transfect studied. The dorsal roots at the L4-6 segmental levels of the spinal cord were Pgc-1α reporter gene with plasmid DNA encoding mutant/deletion forms bilaterally transected in order to inhibit the afferent neural input from the hindlimb of putative regulatory factors to assess the responsiveness of the promoter muscles. The ambulation recovery was allowed for 3 weeks after the surgery. The to skeletal muscle contraction. We show here that each of the MEF2 sites thalamus including hypothalamus region was sampled, frozen in liquid nitrogen and on the Pgc-1α promoter is required for contractile activity-induced Pgc- powdered in a liquid nitrogen-filled mortar. The powdered samples were dissolved 1α transcription. The responsiveness of the Pgc-1α promoter to contractile in lysis buffer and two-dimensional electrophoresis was performed using the soluble fraction. Subsequently, the spots with different expression were picked up and the activity could be completely blocked by overexpression of dominant negative protein was identified using a peptide mass fingerprinting method. As the results, 11 form of activating transcription factor 2 (ATF2), signaling-resistant form of proteins with lower expression level were found in the deafferentated compared with histone deacetylase 5 (HDAC5) or protein kinase D (PKD), but not by that of sham-operated rats. According to the previous studies, the decreased levels of these HDAC4. These findings provide in vivo evidence for functional interactions proteins were closely associated with the degeneration and/or dysfunction of neurons. between PKD/HDAC5 and ATF2 regulatory factors and the Pgc-1α gene in Therefore, it was suggested that the loss of afferent feedback from muscle affected the adult skeletal muscle. thalamic function. This study was supported by Grant-in-Aid for Scientific Research S (19100009) from Japan Society for the Promotion of Sciences. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 221 P2AM-2-32 P2AM-2-33 EFFECT OF HINDLIMB SUSPENSION ON LEARNING HINDLIMB SUSPENSION IMPAIRS HIPPOCAMPAL CAPACITY IN JUVENILE RATS NEUROGENESIS IN WEANED RATS Yoshihiko Oke1, Fuminori Kawano1, Takashi Ohira2, Sachiko Sachiko Nomura1, Katsuya Kami1, Fuminori Kawano1, Yoshihiko Nomura1, Masahiro Terada2, Naoya Nakai1, Yoshinobu Ohira3 Oke1, Naoya Nakai1, Masahiro Terada2, Takashi Ohira2, Kazuhiko 3 4 1Graduate School of Medicine, Osaka University, Japan, 2Graduate School Imaizumi , Yoshinobu Ohira 3 of Frontier Biosciences, Osaka University, Japan, Graduate School of 1Graduate School of Medicine, Osaka University, Japan, 2Graduate School Medicine and Frontier Biosciences, Osaka University, Japan of Frontier Biosciences, Osaka University, Japan, 3Faculty of Human 4 Effects of inhibition of muscle activity on brain function were investigated Sciences, Waseda University, Japan, Graduate School of Medicine and in juvenile male Wistar Hannover rats. Rats (3-wk old) were hindlimb- Frontier Biosciences, Osaka University, Japan suspended (HS) for 14 days and ambulation recovery was allowed in some Effects of decreased muscular activity by 2-wk hindlimb suspension (HS) on rats for 12 days. Water maize tests were conducted immediately after the neurogenesis were studied in weaned rats. Effects of ambulation recovery were also termination of HS and 12-day recovery. The maize had 3 branch points studied. Brain was fixed by infusion of phosphate buffer with 4% paraformaldehyde and 3 areas including starting point, platform, or blind end. In HS rats, in anesthetized rat and cross-sectional samples, around hippocampal area, were swim path length and the frequency of re-selecting incorrect areas were analyzed immunohistochemically. Cellular proliferation was determined by counting not improved during 3 trials, although the arrival time to the platform was the cells labeled with anti-proliferating cell nuclear antigen (PCNA) antibody. The spatial distribution of new neurons was examined by marking doublecortin (DCX)- shortened. These parameters were improved to the level of non-suspended positive cells. In all rats, DCX-positive cells were localized only in subgranular zone rats after 12-day ambulation. In addition, the patterns of protein expression (SGZ) of dentate gyrus within hippocampal formation. PCNA-positive cells in SGZ in the hippocampus were different between HS and cage-control rats. It is of unsuspended control rats were decreased gradually following growth. Decrease suggested that restriction of hindlimb activity impairs the function of brain in of PCNA-positive cells was further stimulated by HS. After 2 weeks of recovery, growing rats. But such an inhibition was reversible in response to ambulation however, the number of PCNA-positive cells of HS rats returned to the age-matched recovery. This study was supported by Grant-in-Aid for Scientific Research S control level. These results suggest that hippocampal neurogenesis is influenced by the (19100009) from Japan Society for the Promotion of Sciences. activity of hindlimb muscles. This study was supported by Grant-in-Aid for Scientific Research S (19100009) from Japan Society for the Promotion of Sciences.
P2AM-2-34 P2AM-3-2 EFFECT OF CONDITIONED FEAR ON THE ROLE OF TRPV1 AND P2X RECEPTORS IN THE PREFRONTAL DOPAMINE TRANSMISSION DURING STIMULATION OF CAPSAICIN-SENSITIVE LUNG EXPLORATION VAGAL AFFERENT FIBERS BY INHALED CIGARETTE Masatoshi Takita1, Hiroshi Yokoi2 SMOKE IN RATS 1Cognition and Action Research Group, National Institute of Advanced You Shuei Lin1, Hsu-Ting Weng2, Chun-Chun Hsu1, Yu Ru Kou2 2 Industrial Science and Technology (AIST), Japan, Department of Precision 1Department of Physiology, Taipei Medical University, Taiwan, 2Department Engineering, The University of Tokyo, Japan of Physiology, National Yang-Ming University, Taiwan Either the exploration of a novel environment or the recall of conditioned fear can Inhaled cigarette smoke triggers airway reflexes that are thought to result from stimulate dopamine (DA) transmission in the rat medial prefrontal cortex (mPFC). stimulation of capsaicin-sensitive lung vagal afferent fibers (CSLVAFs) via a hydroxyl We investigated the mutual interaction between exploration-derived and recall- radical (.OH) mechanism in rats. We investigated the role of transient receptor derived DA through simultaneous measurements of exploratory locomotion in an potential vanilloid 1 (TRPV1) and P2X receptors in this event. Activities of CSLVAFs open-field cage (45 cm square) and mPFC DA using a high-performance liquid were recorded in anesthetized and artificially ventilated rats. Airway challenge chromatography with electrochemical detection (HPLC-ECD) microdialysis of cigarette smoke produced dose-dependant fiber stimulation. Pretreatment with method, twice, 24 hr apart, one week after fear conditioning to an auditory tone dimethylthiourea (DMTU; a .OH scavenger), capsazepine (CPZ; a TRPV1 receptor with footshock (0 or 1.0 mA) in another room. The rats were kept in their home antagonist) and iso-PPADS (a P2X receptor antagonist) separately reduced the fiber cage, except during measurement periods. The effect of the conditioned tone was responses by 60, 40 and 40%, respectively, whereas pretreatment with hexamethonium tested at the first measurement and the DA release was higher in the conditioned (a nicotinic receptor antagonist) failed to alter the response. A combination of CPZ group than in the control. The basal DA level before the second exploration was and iso-PPADS exerted a greater inhibitory effect compared with the effect of either lower than that before the first exploration in the groups exposed to a tone, as single pretreatment. However, a combination of DMTU, CPZ and iso-PPADS did not compared to the groups not exposed to a tone. We performed an autocorrelation further reduce the fiber response compared with the combined effect of CPZ and iso- analysis of either DA or locomotion and a cross-correlation analysis between DA PPADS. We conclude that both TRPV1 and P2X receptors, but not nicotinic receptors, and locomotion, and discuss the fundamental roles of DA transmission in the participate in the stimulation of CSLVAFs by inhaled cigarette smoke possibly through prefrontal cortex with respect to navigation control. the action of .OH. (Supported by NSC95-2320-B-010-025-MY3)
P2AM-3-3 P2AM-3-4 ADMINISTRATION OF POLY(ADP-RIBOSE) PROTECTIVE EFFECT OF INTRATRACHEALLY POLYMERASE INHIBITOR INTO BRONCHIAL ARTERY ADMINISTERED ANTIFLAMMIN-1 ON BLEOMYCIN- IMPROVES PULMONARY DYSFUNCTION AFTER INDUCED LUNG FIBROSIS SMOKE INHALATION INJURY Wei Liu, Zi Qiang Luo, Chen Li, Jian Zhong Han, Wen Li Liu, Atsumori Hamahata1, Perenlei Enkhbaatar2, Hiroyuki Sakurai1, Motohiro Le Wang, Dan Dan Feng, Jing Wan 1 2 2 2 2 Nozaki , Traber Lillian , Yusuke Yamamoto , Csaba Szabo , Daniel Traber Department of Physiology, Xiangya Medical School, Central South 1 Department of Plastic and Reconstructive Surgery, Tokyo Womens Medical University, China University, Japan, 2Department of Anestesiology, Univarsity of Texas Medical Branch, USA Antiflammin-1(AF-1,MQMKKVLDS) is a nonapeptide which has a powerful anti- inflammatory effect . It acts as its original full length protein-Clara Cell Secretory Introduction: There is ~20-fold increase in bronchial blood flow associated with excessive Protein (CCSP) and may display potent clinical application value. Our previous studies production of poly(ADP-ribose) polymerase (PARP) following combined smoke inhalation indicated that Clara Cells on the terminal bronchioles significantly inhibited pulmonary and cutaneous burn. We hypostasized that direct delivery of low dose PJ-34 (PARP inhibitor) fibrosis induced by bleomycin. In present study, we observed firstly the protective into the bronchial artery would attenuate smoke/burn-induced acute lung injury. effect of AF-1 against pulmonary fibrosis. Mice were subjected to intratracheal Methods: Bronchial artery was cannulated preserving the blood flow. Acute lung injury was induced 7-10 days later by 40% total body surface area 3rd ° cutaneous burn and smoke administration of bleomycin (5mg/kg) to establish the fibrotic model and received inhalation. Ewes were divided into three groups. 1) Treatment 1: 1 h post-injury, PJ-34 AF-1(15mg/kg) by intratracheal administration 3 days later. The mRNA expression Poster Session (0.002 mg/kg/h, 2ml/h) was continuously injected into bronchial artery, n=4. 2) Treatment level of precollagen III and precollagen I in lung tissue were quantified by RT-PCR at 2: 1 h post-injury, PJ-34 (0.01 mg/kg/h, 2ml/h) was continuously injected into bronchial 14th day and 28th day respectively. The bleomycin-induced increase of precollagen artery, n=4. 3) Control group: 1h post-injury, equal amounts of saline was injected into III and precollagen I mRNA expression were significantly down-regulated by AF-1. bronchial artery, n=6. Results: Pulmonary function evaluated by measurement of blood gas The collagen deposition detected by Masson staining and hydroxyproline content was analysis and pulmonary transvascular fluid flux was severely deteriorated in control group. decreased by AF-1. All together, our results showed that AF-1 could reduce the degree However, the above changes were markedly attenuated by PJ-34 infusion into bronchial of lung damage and fibrosis. In conclusion, AF-1 has a protective effect on bleomycin- artery. Conclusions: Local airway production of PARP contributes to pulmonary dysfunction induced lung fibrosis and it might prove useful as an add-on therapy for the treatment following smoke inhalation and burn. of lung fibrotic disorders.
222 IUPS 2009 July 27 - August 1, 2009 in Kyoto P2AM-3-5 P2AM-3-6 A NOVEL OVINE MODEL OF CUTANEOUS BURN AND AIRWAY BIOSENSOR ACTIVATION IS MEDIATED BY SMOKE INHALATION INJURY WITH EARLY INCISION OXYGEN FREE RADICALS DURING LUNG ISCHEMIA- AND SKIN GRAFTING REPERFUSION INJURY Yusuke Yamamoto, Perenlei Enkhbaatar, Sebastian Rehberg, Huafeng Li, Lei Du, Mary Proctor, Juan Guardiolar, Rodney Folz, Lillian D Traber, David N Herndon, Daniel L Traber Jerry Yu Department of Anesthesiology, University of Texas Medical Branch, and Department of Medicine, Pulmonary Division, University of Louisville, USA Shriners Hospital for Children, Galveston, USA Despite intensive research, our understanding of the mechanisms of lung A long-term pulmonary function, especially the lung tissue healing following ischemia-reperfusion injury (LIRI) is still limited, partly due to lack of an burn and smoke inhalation is not fully investigated in the animal model. We have effective monitoring system. We propose that airway nociceptors can serve as developed a new ovine model of burn and smoke inhalation injury with early biosensors to monitor LIRI by detecting oxygen free radicals (OFRs). Airway incision and skin grafting to investigate chronically in lung tissue. Materials and nociceptors were recorded from the cervical vagus nerve (ischemic side) in Methods: Four adult female sheep (skin grafting group) were subjected to 20% anesthetized, open-chest, and mechanically ventilated rabbits. Their responses to total body surface (TBS) third-degree burn and cotton smoke inhalation. At 24 LIRI were examined by closing (ischemia, 30 min) and releasing (reperfusion, hour after injury, early excision was carried out to fascia to (20%TBS) and skin 15 min) a snare around the pulmonary artery. Nociceptor discharge increased grafting with meshed autografts was performed. Sheep were monitored for 2 or briefly and then subsided during ischemia. Their discharge (21±10 imp/min) 3 weeks. At this time survived sheep were sacrificed and aliquots of lung tissue increased sharply to 56±36 imp/min (n=9; P=0.022) upon reperfusion. Such an was taken for various assays. Second group of sheep (smoke inhalation alone increase disappeared when superoxide dismutase was infused intravenously for group) were given only smoke inhalation and handled similarly for comparison. 20 min to reduce OFR production before reperfusion. In addition, increasing Result: 3 out of 4 and 4 out of 5 sheep were survived over 2 weeks in skin OFRs by injecting a mixture of xanthine/xanthine oxidase into the receptive grafting and smoke inhalation alone group, respectively. Survival rate, pulmonary field stimulated airway nociceptors. The sensory activity increased sharply after function and hemodynamic were similar in both groups. Conclusion: This model the injection, peaked within 10 sec, and recovered in about a minute. Our data is closely resembles clinical setting and allows to monitor long-term reaction in demonstrate that airway nociceptors are sensitive to increased OFRs, and can act pulmonary function following burn and smoke inhalation injury. as biosensors to monitor LIRI by detecting increased OFRs.
P2AM-3-7 P2AM-3-8 HEMORRHAGE INDUCES MICROTHROMBUS EFFECT OF ALVEOLAR MACROPHAGE ON FORMATION AND FIBRINOGEN DEPLETION IN THE LYMPHOCYTE PROLIFERATION AND REGULATION OF LUNGS OF RATS VIP IN RATS WITH ALLERGIC ASTHMA Robert Conhaim1, Kal E Watson1, William F Dovi1, Martin J Jing Wu1, Chaxiang Guan2, Guoying Sun2, Jia He1, Yuxian Li1, Bo Mangino2, Bruce A Harms1 Wu1, Hua Yang1, Ming Ji2, Chunyan Tang2 1Surgery, University of Wisconsin School of Medicine, USA, 2Virginia 1Department of Clinical Medicine,Grade 2005, Xiangya School of Commonwealth University, Medical College of Virginia, Richmond, VA, Medicine,Central South University, China, 2Department of Physiology, USA Xiangya School of Medicine, Central South University, China Hemorrhage is associated with coagulopathy, lung injury, and increased risk of Bronchial asthma is related with plenty of inflammatory cells.Proliferator-activated pulmonary embolus formation. We showed previously that hemorrhage also causes T lymphocytes and then released lymphokine is important during the development marked perfusion mal-distribution in lung capillaries within 45 minutes (Shock of asthma. Vasoactive intestinal peptide (VIP) has the effect of anti-inflammation and 29:410-16, 2008). Is this mal-distribution due to early microthrombus formation in local immunoregulation.Objective:To study effects of alveolar macrophage(AM) on the low-pressure pulmonary circulation following blood loss? To test this, we rapidly lymphocyte Proliferation and regulation of VIP in Rats with allergic asthma. Methods: removed 30% of rats' blood volume, then removed their lungs after 45 min. or 24 hrs. Rats were divided into two groups. the breathing indexs were detected with Buxco We also prepared non-hemorrhaged controls. We measured microthrombus formation system. AM were obtained by bronchoalveolar lavage, and the peripheral blood in lung samples using immunofluorescence, and measured fibrinogen concentrations lymphocytes of normal rats were obtained by lymphocyte separation medium.The in lung homogenates using western blot. We found that microthrombi increased by 2.4- activity of lymphocyte proliferation was tested with MTT colorimetry.Results:(1) and 6.3-fold respectively at 45 minutes and 24 hours, while fibrinogen concentrations The model of asthmatic rats has been established. (2) The stimulating index(SI) fell to 24% and 58% of control at these times. Our results suggest that hemorrhage of normal lymphocyte+ashmatic AM group increased obviously.Stimulated by caused immediate lung microthrombus formation, which depleted circulating dermatophagoides pteronyssinus, the SI of two groups both increased (P<0.05). The fibrinogen stores. The microthrombi appeared to grow and multiply over 24 hours, as effect of asthmatic AM and dermatophagoides pteronyssinus on lymphocyte can liver fibrinogen synthesis replaced that lost immediately following hemorrhage. This be reversed by the pretreatment of VIP (P<0.05).Conclusion:AM of asthmatic rats could explain hemorrhage coagulopathy, and also explain why hemorrhage patients can promote lymphocyte proliferation, and then accelerate asthma's occurrence and are at increased risk for subsequent pulmonary embolus. development; VIP could reverse this effect partly.
P2AM-3-9 P2AM-3-10 THE EFFECT OF INTRAPULMONARY REGULATORY EFFECT OF VASOACTIVE INTESTINAL PEPTIDE PEPTIDE ON TREM-1 EXPRESSION OF LPS-INDUCED ON TGF-β EXPRESSION IN LPS-INDUCED MACROPHAGES MACROPHAGES Yunchao Li1, Chaxiang Guan2, Guoying Sun3, Yusi Chen1, He Huang1, 3 3 Guoying Sun, Chaxiang Guan, Chunyan Tang, Yong Zhou, Yanru Chunyan Tang , Yong Zhou Cui 1Department of Clinical Medicine; Grade 2006, Xiangya School of 2 Department of Physiology, Xiangya School of Medicine, Central South Medicine,Central South University, China, Department of Physiology, Xiangya School of Medicine,Central South University, China, 3Department of Physiology; University, China Xiangya School of Medicine,Central South University, China Vasoactive intestinal peptide (VIP) plays a vital role in organism’s physiological Triggering receptor expressed on myeloid cells-1(TREM-1),which is highly expressed on processes such as reducing the release of inflammation media and enhancing the the surface of mature monocytes,can magnify inflammatory response.Vasoactive intestinal repairing of wounded airway epithelium.Transforming growth factor beta(TGF-β) peptide (VIP) regulates the inflammatory response;Calcitonin gene-related peptide (CGRP) can block the activation of endothelial cell and induce the fibroblasts to generate the can promote inflammation response.Objective:To study the effects of VIP and CGRP on connective protein.Objective:To investigate the effects of VIP on the TGF-β expressed TREM-1 expression of LPS-induced macrophages.Methods:The TREM-1 mRNA expression by the LPS-induced macrophages.Methods: the TGF-β secretion was measured by of LPS-induced macrophages after pretreating with VIP or CGRP was measured by RT- ELISA ,the TGF-β mRNA expression of LPS-induced macrophages at five time points PCR,and the effects of the inhibitors of PKC,PKA,CaM and MAPK signal transduction was measured by RT-PCR; and the effects of the inhibitors of signal transduction pathway(H-7,H-89,W-7,PD98059). Results:(1)The TREM-1 mRNA expression of LPS- pathway(PKC and MAPK)were observed. Results:(1)The TGF-β mRNA expression induced macrophage increased,pretreatment of VIP could down-regulate mRNA expression increased in LPS-induced Macrophages and VIP pretreated could enhance the of TREM-1,while pretreatment of CGRP could up-regulate it (P<0.01);(2)Compared with group vs LPS+VIP, the TREM-1 mRNA expression of the group which used H-89 effects in the prophase (2h and 6h),and down-regulate it in the midanaphase (12h and PD98059 increased(P<0.05);(3)Compared with group LPS+CGRP,the TREM-1 and 24h)(P<0.05);(2) VIP promoted the TGF-β protein secretion at the early stage mRNA expression of the group which used H-7,W-7 and PD98059 decreased(P<0.05). (P<0.05);(3 )The effect of VIP could be partially cut-off by the inhibitors of PKC and Conclusion:VIP could protect organism and alleviate injury through down-regulating the MAPK signaling pathway (P<0.05).Conclusion:VIP has a protective effect in the acute TREM-1 expression of macrophage.While CGRP up-regulated the TREM-1 expression of injury phase and fibrosis stage of LPS-induced lung injury by up-regulation of TGF-β macrophage which showed an antagonistic effect to VIP. expression. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 223 P2AM-3-11 P2AM-3-12 A NEW CONCEPT ON LUNG'S PARTICIPATION IN MULTI-SCALE COMPUTATIONAL ANALYSIS OF HORMONAL CHANGES DURING ANY ADAPTIVE PULMONARY PERFUSION GRADIENTS 1 2 REACTIONS OF ORGAMISMS Kelly Suzanne Burrowes , Merryn H Tawhai 1Computing Laboratory, University of Oxford, UK, 2Auckland Bioengineering MIKHAIL I. ZALMANOV, LEONID TELL, SERGEY LISENKOV Institute, University of Auckland, New Zealand Kazakh Medical Academy, Kazakhstan Purpose: Physiological role of lungs includes not only gaseous exchange, but also active We have developed a computational modeling framework for simulation of perfusion metabolic adjustments to changing environmental circumstanses. We were in detailed anatomically-based geometric models of the pulmonary circulation. The aim of this work is to understand structure-function relationships, and the relative the first to uncovered the ability of lungs to regulate the level of hormones in influence of various integrated factors - spanning across multiple scales from micro- to arterial bloodstream of humans and animals, as well as the connection of this macro-circulatory vessels. regulation to a frontal structures of hypothalamus and medulla oblongata. All Methods: hormones produced and excreted from endocrine organs first have to pass Perfusion is simulated within the full vascular circuit via solution of Poiseuille flow through enormous pulmonary circulation. Depending on the state of organism equations in elastic vessels and sheet flow equations to represent flow through the capillary network. The flow model has been coupled to a model of parenchymal tissue and environment lungs reveal the ability to store and/or to release multiplicity mechanics to incorporate vascular branch deformation due to tissue volume change of hormons (aldosteron, insulin, angiotensin, vasopressin. ACTH, thyroxine, and tissue recoil pressure acting to expand the vessels. The relative influence of cortisol, triiodothyronine, renin) and, perhaps, influence their bioactivity in various model components was investigated. efferent pulmonary blood flow. Therefore, lungs could be the first and the Results and Conclusions: fastest organ to provide qualitative and quantitative composition of hormones in This modeling approach allows us to investigate structure-function relationships, and arterial bloodstream demanded by given physiological or pathological condition. the relative influence of various model parameters, within a model of the integrated pulmonary circulatory system. One of the main outcomes of this work has been Undoubtedly, external breathing is closely connected with hormone regulation to highlight the large amount of perfusion heterogeneity and the importance of the and it is probable to achieve desirable hormonal changes by affecting mechanical relationship between vascular branching structure and the subsequent distribution of component of breathing. flow in the lung.
P2AM-3-13 P2AM-4-1 ASSESSING THE RELATIVE INFLUENCE OF LESSONS LEARNED FROM WASHINGTON (1968) TO VASCULAR BRANCHING STRUCTURE ON KYOTO (2009) PULMONARY BLOOD FLOW DISTRIBUTION VIA Julio Cesar Cruz COMPUTATIONAL MODELS Department of Physiology, Universidad Nacional de Piura, Peru 1 1 1 2 Tom Doel , Kelly S Burrowes , Jonathan P Whiteley , Vicente Grau , David The argon (Ar) inhaled mixed with the alveolar gas and later exhaled, at 1 1 J Gavaghan atmosphere (atm) and 7 atm (Cruz et al. Proc.IUPS.7:79,1968). Sikand et 1Oxford University Computing Laboratory, University of Oxford, UK, 2Department al. (J.Appl.Physiol.21:1331,1966) explained the effect of apneas at 1 atm, of Engineering Science, University of Oxford, UK but not the Ar expirogram. Thus, I tried to explain it (Cruz, Respir. Physiol. Rationale: 86:1,1991). At IUPS-2005, two posters attempted to explain the data at 1 and The distribution of pulmonary blood flow plays a critical role in respiration, so understanding 7 atm. It was concluded that the Ar inhaled at 7 atm penetrated deeper into the factors influencing this is essential for understanding the functioning of the lung. The relative the residual volume (RV). The lessons learned through the years are: 1. Ar influence of various factors is not fully understood and is currently a hot topic of debate. We aim to show how models of the pulmonary circulation can incorporate two important factors- branch does not reach RV during inspiration. 2. Ar diffuses to RV during expiration angles and pressure discontinuities across vessel bifurcations- and how they affect flow predictions. and takes more than 20s to reach equilibration. 3.There is no Phase 4 in the Methods: Ar expirogram in spite of exhaling to RV. 4. All gases inhaled are diluted or We applied conservation laws to model blood flow through a system of elastic bifurcating vessels. concentrated in the alveolar gas, thus, when O2 and CO2 are exhaled, they Finite element geometric models of several vessel bifurcations typical of the pulmonary vascular do not reflect the alveolar gas exchange processes that take place at RV. 5. system were generated and flow predicted by solution of a reduced 1D form of the Navier-Stokes The expirograms of any gas are the weighted average of uneven emptying of equations. Branch angles and pressure discontinuities were modeled using conservation equations at regional flows that are arranged in parallel, apex-to-base of the lung. These vessel bifurcations. Flow solutions were compared to assess the importance of several factors. lessons led us to new physiological respiratory concepts, such as the mean Results: Predictions suggest that branch angles have a significant influence on blood flow distribution in the alveolar partial pressure of O2, which our peers need to re-evaluate with lung, comparable to that of other model parameters. Results agree with experimental hypotheses future experimental research. indicating a major influence of vascular branching structure on pulmonary perfusion distribution. Supported in part by CEIS
P2AM-4-2 P2AM-4-3 THE EFFECT OF NIGELLA SATIVA ON TRACHEAL EFFECTS OF DEPTH AND DIVE TYPE ON RECOVERY RESPONSIVENESS OF OVALBUMIN SENSITIZED OF ARTERIAL OXYGEN SATURATION AFTER DEEP GUINEA PIGS COMPETITION APNEA DIVES 1 2 Erika KA Schagatay1, Angelica Lodin-Sundstrom1, Fanny Z Schagatay1, Rana Keyhanmanesh , Mohammad Hossein Boskabady , Mohammad Ali 2 3 Ebrahimi Sadatloo3, Saeed Khamneh4, Fariba Mirzaee Bavil5 Johan PA Andersson , Mats H Liner 1 1Department of Engineering and Sustainable Development; Mid Sweden Department of Physiology, Tabriz University of Medical Sciences, Iran, 2 2 University, Sweden, Department of Cell- and Organism Biology, Lund University, Department of Physiology and Pharmacological Research Center of Medical 3 Plants, Mashhad University of Medical Sciences, Mashhad, Iran, 3Department Lund, Sweden, Department of Clinical Sciences, Section of Anesthesiology and of Basic Sciences, Islamic Azad University, Tabriz Branch, Iran, 4Tuberclosis Intensive Care, Lund University, Lund, Sweden and Lung Research Center, Tabriz University of Medical Sciences, Tabriz, Iran, We studied recovery of SaO2 after deep apnea dives in the competitive disciplines: “constant 5Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, weight” (CWT) where the diver uses fins to swim down and up, and “free immersion” (FIM) Iran where the diver pulls down and up on a line. A delay of recovery beyond a few minutes In the present study, the prophylactic effect of hydro-ethanolic extract of Nigella sativa (N sativa) could indicate pulmonary edema caused by lung squeeze at depth (Liner and Andersson and thymoquinone on tracheal responsiveness and white blood cell count of sensitized guinea pigs 2008). A total of 37 divers were tested at two international competitions in Egypt 2008. The was examined. Three groups of sensitized guinea pigs to ovalbumin (OA), were given drinking SaO2 after 32 CWT and 24 FIM competition dives to 25-82 m was recorded using pulse water alone (group S), drinking water containing low concentration of N sativa extract (S+LNS oximetry. Mean depth and duration were 52.6 m and 112 s for CWT and 52.5 m and 122 s group) and high concentration of N sativa extract (S+HNS group). Tracheal responses (TR) for FIM dives. There was a negative correlation between depth and SaO2 at 2 min post dive Poster Session of control animals (group C) and three groups of sensitized guinea pigs (n=7, for all groups) to (r=0.63; P<0.001). When pooled for depth, mean SaO2 at 2 min post dive was 96.9% for methacholine as effective concentration causing fifty percent of maximum response (EC50 M) dives to <50 m and 91.9% for dives to >50 m (P<0.01). SaO reached 97% at 3 min 30 s were measured. Tracheal responses to 0.1% OA, relative to contraction obtained by 10 micromol 2 after shallower dives, and at 9 min after deeper dives. Comparing CWT and FIM dives, SaO methacholine, were also examined. The measurement of TR to methacholine and OA were repeated 2 in incubated tissues with N sativa extract. The TR to both methacholine and OA of sensitized guinea recovery was the same for dives to <50 m, but slower after FIM dives to >50 m (P<0.01). pigs were significantly higher than those of controls (p<0.001 for both cases). The TR in S+LNS Nine cases (5 CWT and 4 FIM) showed a delay of SaO2 recovery beyond 10 min and/or and S+HNS groups to both methacholine and OA were significantly decreased compared to S cough with hemoptysis (3 cases). Of the 9 cases, 8 had been to >50 m depth. We conclude group (p<0.05 to p<0.001). These results showed a preventive effect of N sativa extract on tracheal that diving to depth may be associated with pulmonary edema and that both depth and dive responses and lung inflammation of sensitized guinea pigs. type may affect SaO2 recovery.
224 IUPS 2009 July 27 - August 1, 2009 in Kyoto P2AM-4-4 P2AM-4-5
ESTIMATION OF ALVEOLAR PCO2 FROM INTERMITTENT HYPOXIA IMPAIRS PHARYNGEAL REBREATHING AT REST AND DURING EXERCISE DILATOR MUSCLE FUNCTION IN MALE BUT NOT 1 2 FEMALE RATS Katsuo Uchida , Kazuaki Ito 1 2 1 1Department of Physical Therapy, Yamagata Prefectural University James Richard Skelly , Aidan Bradford , Ken D O'Halloran 2 1School of Medicine and Medical Science, University College Dublin, of Health Sciences, Japan, Graduate School, Yamagata Prefectural 2 University of Health Sciences, Japan Ireland, Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Ireland Rebreathing is a closed respiration to inspire exhaled breath in a bag. Because Upper airway muscle dysfunction is implicated in obstructive sleep apnoea. of the closed respiration oxygen decreases and carbon dioxide increases in a Intermittent hypoxia (IH) is a central feature of sleep apnoea causing oxidative bag. Using a rebreathing bag containing air, we can observe a linear fall of O2 stress. We wished to characterise the effect of IH on pharyngeal dilator muscle concentration and an exponential rise of CO2 concentration, which are caused function in male and female rats. by the differences in partial pressures between alveolar gas and mixed venous Adult Wistar rats were exposed to IH (90s air/90s N2; 5%O2 at nadir) or sham blood. Based on these concentration changes O2 uptake is constant, while CO2 treatment for 8 hours/day for 9 days. Following treatments, isometric contractile output is reduced during rebreathing, and then respiratory exchange ratio (RER) properties of the sternohyoid (SH) muscle were examined at 35OC under control declines linearly with increasing Pco2 in a bag. The slope of the linear regression (95%O2/5%CO2) or hypoxic (95%N2/5%CO2) conditions in vitro. Stress- of RER on Pco2 at rest is larger than those during exercise. We found that the frequency relationship was measured at stimulus frequencies ranging 10-100Hz. slope of the regression line was reduced with increasing exercise intensity and SH peak force was significantly decreased in IH-treated male rats [23±1 vs. 16±1 2 the regression lines intersected at a point. In our study male students participated N/cm , sham (n=8) vs. IH (n=8), P<0.001 ANOVA]. Chronic antioxidant treatment in the experiments with informed consent. They were asked to breathe in both with Tempol (superoxide scavenger) ameliorated IH-induced muscle impairment. Conversely, IH treatment had no effect on SH force in female rats [21±1 vs. 20±1 open (normal respiration) and closed (rebreathing) circuits at rest and during N/cm2, sham (n=8) vs. IH (n=8), P>0.05 ANOVA]. Under hypoxic conditions in vitro, running on a treadmill at 5.0, 7.5 and 10.0 km/h. Alveolar Pco was estimated 2 female SH muscle had greater tolerance to low PO2 compared to male SH muscle. from the crossing point of the regression lines. The estimated alveolar Pco2 was Our results are suggestive of increased antioxidant capacity in females that protects compared with end-tidal Pco2 observed in the open circuit just before rebreathing. them from the deleterious effects of IH on pharyngeal dilator muscle function.
P2AM-4-6 P2AM-4-7 IDENTIFICATION OF NUCLEAR TRANSCRIPTIONAL INTERLEUKIN-13 ENHANCES THE FREQUENCY OF FACTORS REGULATING CTNNAL1 EXPRESSION IN LTD4-INDUCED Ca OSCILLATION IN HUMAN AIRWAY HUMAN BRONCHIAL EPITHELIAL CELLS SMOOTH MUSCLE CELLS Yang Xiang, Xiao-Qun Qin, Yu-Rong Tan, Chi Liu, Fei Qu, Yutaka Hirata1, Hisako Matsumoto2, Kojiro Otsuka2, Isao Ito2, Emiko 2 2 3 2 2 Hui-Jun Liu Ogawa , Shigeo Muro , Hiroaki Sakai , Akio Niimi , Michiaki Mishima , Yoshitaka Oku1 Department of Physiology, Xiangya School of Medicine, Central South 1 University, China Division of Physiome, Department of Physiology, Hyogo College of Medicine, Japan, 2Departments of Respiratory Medicine, Kyoto University, Japan, Adhesion molecules play vital roles in airway hyperresponsiveness or airway 3Departments of Thoracic Surgery, Kyoto University, Japan inflammation. Our previous study indicated an alpha-catenin-related protein, catenin alpha-like 1 (CTNNAL1) was downregulated in asthma patients and animal Interleukin-13 (IL-13) plays a pivotal role in the development of airway hyperresponsiveness model. CTNNAL1 contribute to the wound repair and proliferation of human in asthma. However, physiological mechanisms of hyperresponsiveness have been poorly bronchial epithelial cells (BECs). In the present study, we determined molecular understood. Here we show that IL-13 enhances the frequency of LTD4-induced Ca mechanisms of CTNNAL1 regulation in human BECs. 8 oligonucleotide probes oscillation by increasing the number of CysLT receptors (CysLTRs) on airway smooth corresponding to various regions of the CTNNAL1 promoter were used in EMSA muscle cells (ASMCs), which would cause abnormal airway responsiveness in asthmatic (electrophoretic mobilityshift assays). 5 were found to have an enhanced mobility patients. We exposed cultured human ASMC to 10 ng/ml of IL-13 for 24 h. Levels of shift with extracts from BECs. On the basis of the assay of mutated probes and CysLTRs expression were determined by real-time PCR, and LTD4-induced Ca oscillation antibody supershift, they were verified as LEF-1, AP-2α and CREB. Next, ChIP were imaged with a Ca indicator Fluo-4. In IL-13-treated ASMCs, CysLTRs expression and (chromatin immunoprecipitation) assay was used to observe the interaction between frequency of LTD4-induced Ca oscillation were significantly enhanced when compared with these transcription factors and CTNNAL1 promoter. Only AP-2α and LEF-1 show non-treated ASMC. IP3R inhibitor, 2-aminoethoxydiphenylborate, completely abolished the binding on CTNNAL1 promoter. By site-directed mutagenesis of putative the Ca oscillation, whereas inhibition of RyRs by ruthenium red partially blocked the Ca transcription-factor-binding sites within pGL3/FR/luc, we observed a reduction in oscillation. Further, disruption of caveolar function with a cholesterol-depleting agent, human CTNNAL1 promoter activity of mutants of both AP-2α and LEF-1 sites. Our methyl-beta-cyclodextrin reduced the frequency of Ca oscillation. These results indicate that data suggest that AP-2α and LEF-1 may be involved in regulation of CTNNAL1 IL-13 increases CysLTRs, enhances IP3R-dependent Ca oscillation, and thereby induces expression. ASMC hypercontraction. Caveolae is involved in the frequency regulation of Ca oscillation.
P2AM-4-8 P2AM-5-1 Sp1 REGULATE CFTR EXPRESSION IN OZONE- INTRACELLULAR TRAFFICKING OF THE GENERAL STRESSED BRONCHIAL EPITHELIAL CELLS COACTIVATOR SYT Fei Qu1, Xiao-Qun Qin1, Yan-Ru Cui2, Yang Xiang1, Yu-Rong Tan1, Marina Bernarda Londono, Toshiharu Iwasaki, Kingsley Xiao-Yan Zhou1, Hui-Jun Liu1, Xiao-Lin Zhu1, Chi Liu1 Ibhazehiebo, Noriaki Shimokawa, Noriyuki Koibuchi 1Department of Physiology, Central South University Xiangya Medical Department of Integrative Physiology, Gunma University, Graduate School School, China, 2Department of Physiology, Jiangxi University of Traditional of Medicine, Japan Chinese medicine,China Nuclear hormone receptors (NR) regulate transcription by interacting with coactivators Previously, we found that cystic fibrosis transmembrane conductance regulator (CFTR) and corepressors. We previously characterized the synovial sarcoma translocation significantly decreased in an ozone-stressed bronchial epithelial cell (BEC) model and (SYT) as a general coactivator. Although in vitro assays showed its capability to modify NR-activated transcription, its physiological roles remain unknown. We resulted in CF-like symptoms in a variety of inflammatory airway diseases. In this analyzed the spatio-temporal subcellular distribution of GFP-fused-SYT in response to study, we determined molecular mechanisms of CFTR regulation in BEC in response glucocorticoid receptor (GR) and thyroid hormone receptor (TR) activation by using to ozone stress. Six oligonucleotide probes corresponding to various regions of the time-lapse imaging of living cells. CFTR promoter were used in EMSA studies. Three were found to have an enhanced We found progressive and significant ligand-dependent increase in the nuclear mobility shift with extracts from ozone-stressed cells. Based on the assay of mutated fluorescence intensity in a punctate pattern when GFP-SYT and GR or TR expression probes binding with extracts and antibody supershift, they were verified as Sp1, vectors were cotransfected. It started at 2 to 5 min of dexametasone or triiodothyronine GCF and ERα. Next, ChIP assay, site-directed mutagenesis technology and antisense (T3) treatment (10-7 M) and peaked at 15 and 40 min of exposure, with GR and TR oligonucleotide technology were used to observe these transcription factors associated respectively. Additionally, direct binding of GFP-SYT to both receptors regardless with the CFTR promoter. Only Sp1 increased the ozone-inducible DNA binding on of the presence of ligands was confirmed by immunoprecipitation. Coactivation the CFTR promoter and CFTR expression. The translocation of Sp1 was observed by efficiency of GFP-SYT was evaluated with luciferase reporter assays. immunofluorescence assay, which showed that Sp1 nuclear translocation increased This is the first report on the intracellular trafficking of SYT in living cells. It shows a after ozone exposure. Our data suggest that Sp1 may be involved specially in this dynamic change of the subcellular localization of NR coactivators according to the NR ozone-inducible down-regulation mechanism of CFTR expression. state of activation. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 225 P2AM-5-2 P2AM-5-3 DEXAMETHASONE SUPPRESSES THE NON-GENOMIC RAPID INHIBITION CYSTATHIONINE GAMMA-LYASE EXPRESSION ON Na+/H+-EXCHANGE 1 AND APOPTOTIC IN RAW264.7MACROPHAGES STIMULATED WITH IMMUNOSUPPRESSION BY GLUCORTICOID IN LIPOPOLYSACCHARIDE HUMAN T CELLS 1 1 2 2 1 Xiao-Yan Zhu , Shu-Juan Liu , Yu-Jian Liu , Jian Lu , Xin Ni Eileen Jea Chien1, Chau-Heng Chien1, Ching-Pang Chang1, Shyi-Wu 1Department of Physiology, the Second Military Medical University, China, Wang2, Zih-Ling Huang1, Der-Cherng Tarng1, Shyee-Yhee Chen1 2 Department of Pathophysiology, Second Military Medical University, China 1Department of Physiology, National Yang-Ming University, Taiwan, 2Department
Hydrogen sulfide (H2S) is recognized to be involved in inflammatory processes. of Physiology and Pharmacology, Chang-Gung University, Taiwan Glucocorticoids (GCs) are well known to be able to inhibit the production of various Glucorticoid (GCs) has been employed as immunosuppressive agents for many inflammatory mediators. We hypothesized that GCs may regulate 2H S formation during years. However, it is still unclear how GCs instantly uncouple cells from an acute some inflammatory conditions. We investigate the effects of GCs on the expression stressful responses such as anaphylactic shock. By time scale, the genomic activities of H2S-forming enzyme, cystathionine γ-lyase (CSE), in murine macrophage cell line of the classic GC receptor can not fulfill this role under crisis; but a rapid non- RAW264.7 stimulated with lipopolysaccharide (LPS). It showed that LPS stimulated genomic response can. In a previous study, intracellular acidification was found CSE expression and the transcriptional activity of CSE promoter, suggesting that LPS to be due to a rapid non-genomic inhibition of Na+/H+-exchange 1 (NHE1) and induced CSE expression is dependent on transcriptional mechanisms. H2S precursor leads to the immunosuppression of T cell proliferation by progesterone. This study (L-cysteine) reduced, whereas CSE inhibitor (DL-propargylglycine,PAG) enhanced aimed to exaime whether the rapid responses of acidification are due to an inhibition NO production in LPS-treated macrophages, suggesting that LPS induced H2S of NHE1 activity by hydrocortisone and dexamethasone. Co-stimulation of GCs formtion and H2S inhibited NO production in RAW264.7. Dexamethasone decreased with phytohemagglutinin (PHA) is able to inhibit PHA-activated IL-2 secretion, LPS-induced CSE expression as well as CSE promoter transcription. GCs receptor IL-4 secretion and T cell proliferation. Furthermore, apoptosis was induced by (GR) antagonist RU486 reversed the effects of dexamethasone, indicating that GR dexamethasone, and not by hydrocortisone in PHA-activated T cells. Moreover, the mediates dexamethasone suppression of CSE expression. Our data suggest that LPS apoptosis by dexamethasone-BSA suggests that the immunosuppression is caused by induce H2S production through CSE expression. Suppression of CSE expression a non-genomic mode via plasma membrane sites. Thus, the rapid inhibitory responses might be one of the mechanisms by which GCs modulate LPS-induced inflammatory triggered by GCs might release T cells instantly when an acute stressful response is processes. needed, but only dexamethasone later caused harm to the T cells via by apoptosis.
P2AM-5-4 P2AM-5-5 MECHANICAL STRETCH-INDUCED REDUCTION IN MULTIFACTORIAL REGULATION OF CORTISOL 11 BETA-HYDROXYSTEROID DEHYDROGENASE 2 SECRETION IN PERFUSED GUINEA PIG ADRENALS EXPRESSION IN RAT VASCULAR SMOOTH MUSCLE Toshio Shimada, Itsuro Matsumoto, Tadaomi Aikawa CELLS Department of Physiology, Nagasaki University, Japan Akira Nishiyama1, Hirofumi Hitomi1, Yoshihide Fujisawa2, Daisuke Nakano1, 1 2 The cortisol (F) synthesis in adrenal fasciculate cells is in part via a calcium/ Shoji Kimura , Yukiko Nagai calmodulin-dependent process, we studied the cross-regulation of F secretion 1Department of Pharmacology, Kagawa University Medical School, Japan, - 2 among platelet-activating factor (PAF), ACTH, AlF4 , and calcium ionophore Health Sciences Research Center, Kagawa University Medical School, Japan A23187 in perfused guinea pig adrenal gland. 1) The calmodulin inhibitor The activity of the glucocorticoid metabolism enzyme, 11 beta-hydroxysteroid - (W-7) inhibited F secretion to AlF4 (5mM NaF + 20 μM AlCl3) but not to 10 dehydrogenase (HSD), is significantly reduced in aortic tissues of spontaneously nM PAF and 100 pg/ml ACTH. 2) The secretory responses of F to repeated hypertensive rats. We tested the hypothesis that blood pressure-induced mechanical - infusion of 10 μM A23187 and AlF4 were not reproducible and second force at the vascular wall participates in a reduction in 11 beta-HSD 2 expression in infusions only slightly increased. 3) The cortisol response to PAF perfused vascular smooth muscle cells (VSMCs). Significant mRNA expression of 11 beta- for 5 min from 45 min after A23187 infusion was potently suppressed. By HSD1, 11 beta-HSD2, mineralocorticoid receptor (MR) and glucocorticoid receptor contrast, the cortisol response to A23187 after PAF was unaffected. 4) The (GR) was observed in control VSMCs. Mechanical stretch (15% surface elongation cortisol responses to ACTH after A23187 infusion and A23187 after ACTH with 60 cycles/min) resulted in biphasic increases in mRNA levels of 11 beta-HSD1. - infusion were unaffected. 5) The cortisol response to AlF4 after A23187 was Continuous mechanical stretch significantly decreased 11 beta-HSD2 mRNA levels. - Similar changes in protein expression of 11 beta-HSD2 were also observed. On the potently suppressed. By contrast, the cortisol response to A23187 after AlF4 was unaffected. other hand, mRNA levels of MR and GR were not changed during the observation - period. The present study demonstrates that continuous mechanical stretch decreases These results implicate that the cortisol response to AlF4 was calcium/ expression of 11 beta-HSD2 in VSMCs. It is possible that during the development calmodulin-dependent process and cross-regulation among processes of hypertension, high blood pressure-induced mechanical force at the vascular wall activated by PAF receptor-PKC, ACTH receptor-PKA, and calcium/ participates in a reduction in 11 beta-HSD2 expression in VSMCs that may contribute calmodulin might function in the multifactorial regulation of adrenal to the pathogenesis of hypertension and vascular remodeling. steroidogenesis.
P2AM-5-6 P2AM-5-7 EFFECTS OF EXERCISE AND LACTATE ON DISRUPTED GLUCOSE HOMEOSTASIS MAY BE CORTICOSTERONE SECRETION IN RATS RESPONSIBLE FOR THE TRANSFORMATION Ru-Lian Hsu1, Wan-Yun Chen1, Chih-Yung Lin1, Yu-Chang OF GASTROPROTECTIVE ACTION OF Chang1, Paulus S. Wang1,2 GLUCOCORTICOIDS TO ULCEROGENIC ONE 1Department of Physiology, National Yang-Ming University, Taiwan, Ludmila Filaretova, Tatiana Podvigina, Tatiana Bagaeva, Olga 2Department of Medical Research and Education, Taipei City Hospital, Morozova Taiwan Laboratory of Experimental Endocrinology, Pavlov Institute of Physiology, Russia The effects of exercise and lactate on the secretion of corticosterone (C) were Our previous results suggest that glucocorticoids released during acute stress- examined in rats. In an in vivo study, ovariectomized (Ovx) rats were catheterized induced activation of hypothalamic-pituitary-adrenocortical axis are important via the right jugular vein (RJV), and the blood samples were collected at several gastroprotective factors, allowing us to re-evaluate the traditional view about their time intervals before and after 10 min of swimming. In addition, blood samples were ulcerogenic role. This beneficial action of glucocorticoids is opposite well-known collected from RJV before and after 10 min lactate infusion (26 mg/kg/min) through ulcerogenic action of exogenous glucocorticoids used at pharmacological doses. the RJV in male rats. In an in vitro experiment male rat zona fasiculata-reticlaris Therefore, glucocorticoids have dual action on the stomach: gastroprotective and (ZFR) cells were challenged with lactate (2.5-20 mM) in the presence or absence of ulcerogenic one. The present study was designed to understand why and how the -9 -4 gastroprotective action of glucocorticoids can be transformed to ulcerogenic effect. Poster Session adrencorticotropin (ACTH, 10 M) and 8-Br-cAMP (10 M) for 60 min. The levels of C in plasma and media were measured by radioimmunoassay. The postexercise levels We hypothesized that glucocorticoid-induced disturbance of carbohydrate regulation of plasma glucose, lactate and C were significantly higher than the corresponding may be responsible for the transformation. The results obtained support the hypothesis and suggest that short-term maintenance of blood glucose levels may be responsible basal levels. An elevation of plasma lactate and C was found subsequent to 10 min of for the gastroprotective action of glucocorticoids, while disturbance of carbohydrate lactate infusion. Lactate ranging from 5 to 10 mM with or without ACTH and 8-Br- regulation during glucocorticoid-induced long-term maintenance of blood glucose cAMP significantly increased C production in a dose dependant manner. The results level or hyperglycemia (accompanied with the signs of catabolic effects) may account suggested that lactate increased C secretion by a direct action on rat ZFR cells via at least partly for the ulcerogenic action of glucocorticoid hormones. Supported by cAMP pathway. BSciM RAS-2008, RFBR-07-04-00622, Sci School RAS-1434.2008.4.
226 IUPS 2009 July 27 - August 1, 2009 in Kyoto P2AM-5-8 P2AM-6-1 ENDOCRINE-IMMUNE INTERACTION UNDER ESTROGENS POSSESS ANTI-INFLAMMATORY STRESS CONDITIONS ON HYPERTENSIVE AND EFFECT IN ULCER AND COLITIS VIA BOTH NORMOTENSIVE RATS ESTROGEN RECEPTOR (ER)-ALPHA AND ER-BETA Vadim E Tseylikman1, Olga B Tseylikman2, Denis A Kozochkin1, Anton A Berrak Caglayan Yegen1, Zarife Nigar Ozdemir1, Gulsun Memi1, Sinitsky1, Sergey V Popov1, Eugene S Katashinsky1, Alexandre M Mironov1 Feriha Ercan2 1Department of Biochemistry, Chelyabinsk State Medical Academy, Russia, 1 2 Department of Physiology, Marmara University School of Medicine, South-Ural State University, Russia Turkey, 2Department of Histology and Embryology, Marmara University We used two models of preliminary stress exposure. The first model is designed School of Medicine, Turkey to induce the tolerant strategy of adaptation. The animals sustained a repeated Estrogen receptor (ER) agonists have been demonstrated to possess anti-inflammatory immobilization stress exposure (RIS1); 60 minutes daily for three days. The second properties in various inflammatory disease models, but their role in gastrointestinal mode (RIS2) is designed to induce the resistant strategy of adaptation. The animals inflammatory processes is not clear yet. In Sprague-Dawley rats with acetic acid- sustained four episodes of immobilization stress; 60 minutes each, 72 hours apart. induced ulcer or colitis, either ERα (PPT; propylpyrazole triol; 1 mg/kg/day) or ERβ Male ISIAH (inherited stress-induced arterial hypertension) rats and male Wistar agonist (DPN; diarylpropionitrile; 1 mg/kg/day) or oil was administered for 3 days rats were used in our investigation. No significant differences were observed in serum following the induction of ulcer or colitis. At the end of the 3rd day, the rats were levels of IL-1, IL-6, in either strain of rat. Corticosterone levels in stressed Wistar decapitated and the colonic and gastric tissues were taken to measure the levels of rats were estimably higher than those of ISIAH rats. RIS2 caused an elevation of malondialdeyde (MDA), glutathione (GSH) and myeloperoxidase (MPO) activity corticosterone levels in ISIAH rats measured 24 hours post cessation. In Wistar and to assess the extent of oxidant damage histologically. Extensive damage in the rats exposed to RIS1 conditions we observed the up-regulation of delayed-type stomach and colon of oil-treated rats was accompanied with significant increases in hypersensitivity reactions (DHR). No significant differences in DHR were found MDA and MPO activity along with decreases in the GSH content of both tissues. Both between stressed and unstressed ISIAH rats. RIS2 did not modify the intensity of DHR ER agonists potently reversed the indicators of oxidative damage and ameliorated on Wistar rats. However, we observed down-regulation of DHR in ISIAH rats under the extent of gastric or colonic injury. In conclusion, the anti-inflammatory action of RIS2 conditions. estrogens in ulcer and colitis may be due to their direct antioxidant effects as well as Supported by grant of President of R.F. for young Doctors of Science (MD their inhibitory role on tissue neutrophil infiltration, which may be accomplished by -2063.2008.7) and RFBR (08-04-01-526-a) the activation of either ER isoforms.
P2AM-6-2 P2AM-6-3 GENOMIC ORGANIZATION AND STRUCTURE OF THE DIFFERENTIAL PROTEOMIC ANALYSIS REVEALS 5’-FLANCKING REGION OF THE RAT ESTROGEN THE ACTION MECHANISM OF ESTROGEN RECEPTOR RECEPTOR ALPHA GENE β-MEDIATED ANTIOXIDANT ACTIVITIES IN A549 Hirotaka Ishii, Momoko Kobayashi, Yasuo Sakuma CELLS 1 1 2 1 Department of Physiology, Nippon Medical School, Japan Hui-Ping Lee , Yi-Min Hsaio , Shu-Hui Chen , Mei-Ling Tsai 1Department of Physiology, National Cheng-Kung University, Taiwan, The estrogen receptor α (ERα) gene is transcribed from multiple promoters. 2 The alternative promoter usage and splicing generate multiple ERα mRNA Department of Chemistry, National Cheng-Kung University, Taiwan transcripts with distinct 5’-ends in the untranslated region. To understand the Estrogen (E2) elicits various biological functions through ERα and ERβ. ERβ elicits regulatory mechanism of rat ERα gene expression, the genomic organization antioxidant effects on LPS-stimulated superoxide production in microglial cells when of rat ERα gene must be investigated. We, therefore, analyzed the structure ERα exists. Oxidative stress induces the expression of ERβ but not ERα in human of 5’-flancking region of rat ERα mRNA using the rapid amplification of breast cancer cells. Overexpression of ERβ increases the antioxidant gene expression 5’-cDNA end (5’-RACE) and RT-PCR techniques. The sequence analysis in MDA-MB-231. Taken together, ERβ plays an antioxidant role when ERα exists but it is not clear if ERβ alone elicits antioxidant effects. Our preliminary study had shown revealed that the presence of multiple novel mRNA variants containing the the induction of ROS by ultrafine carbon black (ufCB) in A549 which expressed unique 5’-untranslated regions. We mapped the cDNA sequences on the rat ERβ only. Accordingly, the purpose of this study was to investigate the antioxidant genome, and identified novel untranslated leader and intermediate exons effect of ERβ in ufCB-induced ROS in A549. First, immunoblotting showed the (exons 0U, and I1-10) in rat ERα gene. We further analyzed the distribution expression of ERβ. Secondly, MTT and flow cytometery showed the influence of of rat ERα mRNA variants in uterus, ovary, testis, liver, and kidney using RT- E2on cell proliferation in MCF-7 but not in A549. Although E2 alone did not influence PCR. The analysis revealed a tissue-specific pattern of all mRNA variants. ROS production, E2 reduced ufCB-induced ROS. Differential proteomics showed the The 0S, 0N, and 0/B mRNA isoforms are widely distributed. The expression reduction of mitochondrial proteins by E2 in the presence of ufCB. Immunoblotting of 0U mRNA isoform is restricted to testis. Our results showed that the rat confirmed the reduction of PON2 and HO-1 by E2 under ufCB exposure. Taken ERα gene is a complex genomic unit that exhibits altenative promoter usage together, activation of ERβ by E2 may reduce ROS production by modulating and splicing which are regulated in a tissue-specific manner. mitochondrial activities.
P2AM-6-4 P2AM-6-5 UP-REGULATION OF ESTROGEN RECEPTOR BETA IN STIMULATORY EFFECT OF ARECOLINE ON THE THE MENOPAUSAL MONKEY HIPPOCAMPUS IS NOT PROGESTERONE PRODUCTION BY RAT GRANULOSA A DIRECT RESPONSE TO A DEFICIT IN GONADAL CELLS ESTROGEN Cai-Yun Jian1, Han-Wei Lin1, Chia-Jung Chan1, Yun-Wan Chen1, Paulus S. 2 Sayuri Higaki1, Ken Takumi1, Keiko Shimizu2, Takao Oishi1, Motoharu Wang Hayashi1 1Department of Physiology, National Yang-Ming University, Taiwan, 2Department 1Department of Cellular and Molecular Biology, Primate Research Institute, of Physiology, National Yang-Ming University, Taiwan, ROC; Department of Kyoto University, Japan, 2Department of Zoology, Okayama University of Medical Research and Education, Taipei City Hospital, Taiwan Science, Japan Arecoline, an alkaloid of betel nut of Areca catechu , might cause abnormalities of The higher prevalence of Alzheimer’s disease or depression in old women relative to old men reproductive endocrine system. However, its mechanism is not clear. The present study has led to the suggestion that the disruption of cyclical hormonal levels following menopause was to investigate the directive effects of arecoline on the production of progesterone (P) may increase the risk for these diseases. Japanese macaques enter natural menopause around in rat granulosa cells (GCs). The immature female rats (23-25 days) were injected with 25 years of age, characterized by lower estrogen and elevated gonadotropin levels. Estrogen pregnant mare serum gonadotropin at 48 hours before decapitation and prepared for the plays neurotrophic and neuroprotective roles via estrogen receptor (ER) α and β in the GCs. Rat GCs were incubated with follicle-stimulating hormone (pFSH), human chorionic central nervous system. Using immunohistochemistry, we have observed the significantly gonadotropin (hCG), 8-bromo-adenosine-3’,5’-cyclic monophosphate (8-Br-cAMP), higher ERβ expressions in the hippocampal formation of peri-menopausal (peri-: 25-26 yr) A23187, and nifedipine (a L-type Ca 2+ channel blocker) in the presence or absence of and post-menopausal (post-: 29-31 yr) Japanese macaques than control pre-menopausal (pre-: arecoline in an incubator at 37 ˚C for 2 hours. The media were collected and measured for P 9-13 yr) monkeys. Ovariectomized (ovx: 9-18 yr) monkeys, however, still showed similar by radioimmunoassay. P level in rat GCs were significantly enhanced by FSH, hCG, 8-Br- ERβ immunoreactive levels to those of pre-monkeys. In contrast, the immunoreactivity of cAMP and A23187. Arecoline at the 10-8 - 10-4 M resulted a dose-dependent increase of aromatase cytochrome P450, the enzyme responsible for local estrogen biosynthesis in the basal level of P and the secretion of P in response to 8-Br-cAMP and A23187. The arecoline hippocampus, declined in peri- and post- monkeys, while it increased in ovx monkeys. These stimulated P production was decreased by administration of nifedipine. These results findings suggest that the reduction of autocrined/paracrined estrogen rather than endocrined suggested that arecoline stimulated P production in rat Leydig cells through mechanisms estrogen may cause the up-regulation of ERβ within the menopausal monkey hippocampus. involving the increased activities of adenylyl cyclase, and L-type Ca 2+ channel. Poster Session
IUPS 2009 July 27 - August 1, 2009 in Kyoto 227 Volume 59 · Supplement 1 · 2009
Volume 59 · Supplement 1 · 2009
The XXXVI International Congress of Volume 59 · Supplement 1 · 2009 · pp 1–XX · pp 1 · 2009 Volume 59 · Supplement Physiological Sciences (IUPS2009) International Scientific Program Committees (ISPC) ISPC Chair Yoshihisa Kurachi Vice Chair Ole Petersen ISPC from IUPS Council Akimichi Kaneko (IUPS President) Irene Schulz (IUPS Vice President) Pierre Magistretti (IUPS Vice President) Malcolm Gordon (IUPS Treasurer)
ISPC IUPS2009 Members and Associated Members Proceedings of the XXXVI International Congress of Physiological Sciences (IUPS2009) Commission I Locomotion Commission VII Comparative Physiology: Hans Hoppeler, Masato Konishi, Hiroshi Nose Evolution, Adaptation & Environment Function of Life: Elements and Integration Commission II Circulation/Respiration Malcolm Gordon, Ken-ichi Honma, July 27–August 1, 2009, Kyoto, Japan Yung Earm, Makoto Suematsu, Itsuo Kodama Kazuyuki Kanosue Commission III Endocrine, Reproduction & Commission VIII Genomics & Biodiversity Development David Cook, Hideyuki Okano, Gozoh Tsujimoto Caroline McMillen, Yasuo Sakuma, Toshihiko Yada Commission IX Others Commission IV Neurobiology Ann Sefton, Peter Hunter, Osamu Matsuo, Quentin Pittman, Harunori Ohmori, Fumihiko Kajiya, Tadashi Isa, Tadaharu Tsumoto, Megumu Yoshimura Jun Tanji Commission V Secretion & Absorption Local Executives Irene Schulz, Miyako Takaki, Yoshikatsu Kanai Yasuo Mori, Ryuji Inoue Commission VI Molecular & Cellular Biology Cecilia Hidalgo, Yoshihiro Kubo, Katsuhiko Mikoshiba, Masahiro Sokabe, Yukiko Gotoh