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Poster Sessions ABSTRACT Free Communications (Poster Sessions) P1-1 Prostaglandin E2 modulates P1-2 Tacrine treatment-induced synaptic transmission through upregulation of VEGF-VEGFR2 system presynaptic EP1 receptors in the rat in the hippocampus spinal trigeminal subnucleus caudalis Daishu Mizuki Yuka Mizutani1,2, Yoshiaki Ohi1, Naoki Yoshida1, Inst. Natural Med., Univ. Toyama. Shunpei Fukuyama1, Satoko Kimura1, 2 2 1 We previously reported that tacrine (THA) reduced Ken Miyazawa , Shigemi Goto , Akira Haji hippocampal cells damage caused by NMDA-induced 1Lab., Neuropharmacol., Sch. Pharm., Aichigakuin Univ., 2Dept., excitotoxicity in mouse hippocampal slice cultures (OHSCs). Orthodontics, Sch. Dent., Aichigakuin Univ. Our results suggested that endogenous acetylcholine (ACh) played a rescuing role towards cell damage via hippocampal The spinal trigeminal subnucleus caudalis (Vc) receives VEGF systems. To further clarify the relationship between nociceptive afferent signals from the orofacial region. cholinergic and VEGF systems, we investigated the effects Nociceptive stimuli to the orofacial region induced of THA on the expression of genes coding VEGF-A and cyclooxygenase both peripherally and centrally, which can VEGF receptor 2 (VEGFR2) in the hippocampus. Male ddY synthesize a major prostanoid prostaglandin E2 (PGE2) that mice were treated daily with THA (2.5 mg/kg, i.p.) for 1-14 implicates in diverse physiological functions. To clarify the days. The hippocampal tissues were obtained 1 hr after the role of centrally-induced PGE2, effects of exogenous PGE2 on last treatment with THA and used for RNA extraction. The synaptic transmission in the Vc neurons were investigated expression levels of genes were analyzed by quantitative RT- in the rat brainstem slice. Spontaneously occurring PCR. THA treatment for 14 days signiÀ cantly increased the excitatory and inhibitory postsynaptic currents (sEPSCs expression levels of VEGF-A and VEGFR2 mRNAs, whereas and sIPSCs) were recorded under blockade of inhibitory THA had no effect on the expression of HIF1ǂ and nNOS and excitatory transmission, respectively, by whole-cell mRNAs. The effects of THA on VEGF-A and VEGFR2 mRNA patch-clamp mode. PGE2 exerted a concentration-dependent expression depended on the number of administrations. increase in the frequency of sIPSCs, but no discernible In in vitro experiments, treatment of OHSCs with THA effect on the amplitude. Similarly to the effects on sIPSCs, (30 ǍM) for 6 days clearly induced a morphological and PGE2 increased the sEPSC frequency without effect on the activation-related changes in astrocyte expressing VEGF-A. amplitude. These facilitatory effects of PGE2 on sIPSCs Our results suggested that ACh has an upregulatory role in and sEPSCs were blocked by EP1 antagonist, SC19220. On VEGF-VEGFR2 system in the hippocampus. the other hand, PGE2 had no effect on the trigeminal tract- evoked EPSCs in the Vc neurons. These data suggest that centrally-induced PGE2 may modify nociceptive transmission in the Vc region. P1-3 Molecular mechanisms P1-4 Enhanced dopaminergic underlying the formation of Munc13-1 neurotransmission in the nucleus nanoclusters at presynaptic terminals accumbens by the gene mutation of Tetsuro Ariyoshi, Hirokazu Sakamoto, synaptic vesicle protein 2A (SV2A) Shigeyuki Namiki, Kenzo Hirose Kentaro Tokudome1, Saki Shimizu1, Dept. Neurobiol., Grad. Sch. of Med., Tokyo Univ. Naofumi Kunisawa1, Azuki Hashimoto1, 1 1 1 At presynaptic terminals synaptic vesicles are docked at Yuki Hiraoka , Kazuki Fukuda , Ayako Ikari , Yumiko Iguchi1, Takafumi Sugahara1, a set number of release sites, where vesicles fuse with the 2 1,2 1 membrane for exocytosis. Previously we have discovered Tomoji Mashimo , Tadao Serikawa , Yukihiro Ohno that nanoscale clusters of Munc13-1 molecules at nerve 1Lab. Pharmacol., Osaka Univ. Pharm. Sci., 2Inst. Lab. Animals, Kyoto Univ. terminals are the molecular entity of the release sites. Sch. Med However, molecular mechanisms of Munc13-1 clustering have not been elucidated. Here we examined how Munc13-1 SV2A is specifically expressed in synaptic vesicles and forms nanoclusters by combining reconstitution experiments regulates neurotransmitter release. We previously reported in non-neuronal cells and super-resolution imaging. First, that SV2A-mutant rats, carrying a missense mutation to assess if Munc13-1 molecules can cluster by themselves, (L174Q) in the Sv2a gene, showed impaired hippocampal we expressed Munc13-1 in HEK-293 cells. Heterologously GABA release and enhanced seizure susceptibility. expressed Munc13-1 formed large micrometer-sized puncta. Here, we performed in vivo microdialysis and behavioral Super-resolution imaging by dSTORM revealed that studies using the SV2A-mutant rats to evaluate the role Munc13-1 molecules form nanoclusters which resemble of SV2A in accumbal dopaminergic neurotransmission. Microdialysis study showed that the Sv2a gene mutation synaptic ones, suggesting that Munc13-1 can self-assemble. + Second, to discover a critical region for self-assembling, we significantly increased both depolarization (100 mM K )- examined several mutants and found that a deletion of a and methamphetamine (MAP, 100 M)-elicited dopamine previously uncharacterized region abolishes the formation of release in the nucleus accumbens without affecting puncta in HEK-293 cells. Finally, replacement of neuronal basal release. In behavioral studies, MAP (1 mg/kg, i.p.)- Munc13-1 to the deletion mutant partially disrupted the induced hyperlocomotion was significantly augmented formation of synaptic nanoclusters. These results show that by the SV2A mutation. In addition, development of Munc13-1 can self-assemble into nanoclusters at synapses. locomotor sensitization (reverse tolerance) to repeated MAP treatments (0.3 mg/kg/day, 12 days) was significant enhanced in SV2A-mutant rats as compared to control (F344) rats. The present results suggest that dysfunction of SV2A by the missense mutation (L174Q) enhances synaptic dopamine release in the nucleus accumbens, which may be linked to vulnerability to psychotic disorders. S152 P1-5 Type-1 metabotropic P1-6 Establishment of a versatile glutamate receptor regulates adenosine BDNF gene modulating system A1 receptor signaling Fumiya Izumiseki1, Junichi Nabekura2, 2 1 Hakushun Sakairi, Yuji Kamikubo, Takashi Sakurai Akiko Miyamoto , Kenji Tanaka 1 2 Dept. Pharmacol., Juntendo Univ. Dept. Neuropsychiatry, Keio Univ. Sch. Med., NIPS Adenosine A1 receptor (A1R) is a G protein-coupled receptor Gain-of-function and loss-of-function studies are commonly (GPCR) for the ubiquitous neuromodulator adenosine and used to examine the function of genes in vivo. To examine is known to play roles in regulation of neuronal excitability, the function of BDNF gene in vivo, we have developed a arousal level, and pain sensitivity. In previous presentation, gene targeting mouse that allows us to manipulate BDNF we reported that A1R activation leads to the attenuation of gene expression levels. The system, called STOP-tetO- type-1 metabotropic glutamate receptor (mGluR1) signaling BDNF, enables us to rapidly produce 1) a gene knock down, 2) Cre-mediated rescue, and 3) tTA-mediated rescue. in cerebellar Purkinje cells. mGluR1 is a Gq/11 protein- coupled GPCR and this signaling is essential to induction Moreover, when crossed with Flpe mice, the STOP cassette of long-term depression of cerebellar Purkinje cells. Here can be removed, producing tetO-BDNF knock-in mice. tetO- we explore in more detail signal crosstalk between A1R BDNF knock-in mice have wild-type expression levels, but and mGluR1 using heterologous expression systems. Our permit 4) tTA-mediated over/ectopic expression and 5) tTS- immunocytochemical, biochemical, and FRET analysis mediated conditional knock-down. These manipulations revealed that A1R and mGluR1 form complexes even in the are effected by crossing STOP-tetO or tetO knock-in mice cell lines. We used cAMP production assay to evaluate the with established Cre, tTA, or tTS lines. We exemplified activity of the signaling cascade coupled to A1R and found tTA-mediated overexpression by crossing dopamine that mGluR1 activation regulate this cascade. Our À ndings neuron specific tTA line (dopamine transporter (DAT)- demonstrate that distinct GPCRs, each of which plays a tTA) with tetO-BDNF knock-in line. We found the tTA- distinct role by itself, may form complex and bidirectional dependent BDNF mRNA induction in the ventral tegmental functional interplay and thereby cooperatively regulate dopamine neurons and BDNF protein overexpression in signal cascades. their terminal, the nucleus accumbens. Cell-type specific, time controllable BDNF gene modulating system will be a promising tool for understanding the roles of BDNF in various neuropsychiatric disease models. P1-7 Effects of sigma receptor P1-8 Role of cerebellar D3 ligands on the uptake activity of receptors in modulating dopamine D1 serotonin transporter receptor-mediated oral dyskinesia in Masaya Asano1, Naoto Usuki1, Hikaru Yamamoto2, rats Toshihiko Shirafuji1, Izumi Hide1, Shigeru Tanaka1, 1 Takahiro Mukai, Saki Shimizu, Hitomi Yoshimura, Norio Sakai Aya Yamauchi, Yoshihiro Suzuki, Yumi Irie, 1Dept. Mol. Pharmacol. Neurosci., Grad. Sch. Biomed. Health Sci., Hiroshima Taiki Kubo, Fumiya Yokokawa, Yoshiko Kawai, Univ., 2Biosig. Res. Ctr., Kobe Univ. Yukihiro Ohno The function of serotonin transporter (SERT), a membrane Lab. Pharmacol., Osaka Univ. Pharm. Sci. protein that terminates serotonergic neurotransmission, is
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