Activation of Serotonin 1A Receptors in Ventrolateral Orbital Cortex Depresses Persistent Nociception: a Presynaptic Inhibition Mechanism

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Neurochemistry International 57 (2010) 749–755

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Neurochemistry International

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Activation of serotonin 1A receptors in ventrolateral orbital cortex depresses persistent nociception: A presynaptic inhibition mechanism

Fu-Quan Huo a, Fen-Sheng Huang a, Bo-Chang Lv b, Tao Chen b, Jie Feng a, Chao-Ling Qu a, Jing-Shi Tang a,*, Yun-Qing Li b,** a Department of Physiology and Pathophysiology, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an Jiaotong University School of Medicine, Xi’an 710061, China b Department of Anatomy, Histology and Embryology and K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi’an 710032, China

ARTICLE INFO ABSTRACT

Article history: The present study examined the effect of serotonin 1A (5-HT1A) receptor activation in the ventrolateral Received 26 May 2010 orbital cortex (VLO) upon formalin-evoked ﬂinching behavior and spinal Fos expression, and further Received in revised form 24 July 2010 determined whether activation of 5-HT1A receptors affected the spontaneous GABAergic miniature Accepted 11 August 2010 inhibitory postsynaptic currents (mIPSCs) in rat VLO slice by pharmacologically separated neurons to Available online 8 September 2010 understand the possible mechanism underlying this effect. Microinjection of the 5-HT1A receptors agonist 8-OH-DPAT (8-hydro-2-(di-n-propylamino) tetralin) into the VLO depressed the formalin- Keywords: evoked nociceptive behavior ﬂinching response and the Fos expression in the lumbar spinal cord dorsal, 5-HT1A receptor which was antagonized by pre-treatment with 5-HT receptors antagonist NAN-190 (1-(2- Fos 1A mIPSCs methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide). Furthermore, application of Ventrolateral orbital cortex 8-OH-DPAT into VLO slice inhibited GABAergic mIPSC frequency in a dose-dependent manner without Antinociception effects on amplitude of the GABAergic mIPSCs, this effect was blocked by NAN-190. These results provide

Formalin test evidence for the involvement of 5-HT1A receptors in VLO in the modulation of persistent inﬂammatory

1. Introduction Zhang et al., 1995, 1998b, 1999). However, electrically or chemically induced activation of the VLO depresses tail flick and jaw-opening Previous studies in our laboratory have demonstrated that reflexes. These antinociceptive effects are eliminated by lesion or electrolytic lesions or microinjection of g-aminobutyric acid (GABA) functional blocking of the periaqueductal gray (PAG) (Y.Q. Zhang into the ventrolateral orbital cortex (VLO) eliminates antinocicep- et al., 1997; S. Zhang et al., 1997; Zhang et al., 1998a). These data tive effects induced by peripheral electrical stimulation, or by suggest that the VLO is involved in an endogenous analgesic system activation of the thalamic nucleus submedius (Sm) (Lu et al., 1996; consisting of a spinal/medulla cord-Sm-VLO-PAG-spinal/medulla cord loop (Tang et al., 2009). Morphological studies have indicated that the VLO receives the ascending projections of serotonin (5-hydroxytryptamine, 5- Abbreviations: 5-HT, 5-hydroxytryptamine, serotonin; 8-OH-DPAT, 8-hydro-2-(di- HT)-ergic terminals from the dorsal raphe nucleus (Huo et al., n-propylamino) tetralin; ABC, avidin–biotin-peroxidase; ACSF, artificial cerebro- 2009; Li et al., 1993; Matsuzaki et al., 1993), and 5-HT receptors spinal fluid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DAB, 3,3-diaminoben- are distributed in the VLO (Barnes and Sharp, 1999; Hossein et zidine tetrahydrochloride; DL-AP5, DL-2-amino-5-phosphovaleric acid; F 13640, (3-chloro-4-fluoro-phenyl)-[4-fluoro-4-[[(5-methyl-pyridin-2-ylmethyl)-amino]- al., 1996). Behavioral studies also demonstrated that the 5-HT1A methyl]piperidin-1-yl]-methadone; GABA, g-aminobutyric acid; mIPSCs, minia- receptors are implicated in mediating the VLO-evoked descend- ture inhibitory postsynaptic currents; NAN-190, 1-(2-methoxyphenyl)-4-[4-(2- ing antinociception in the physiological pain model (tail flick phthalimido)butyl]piperazine hydrobromide; NGS, normal goat serum; PAG, test) (Qu et al., 2008; Huo et al., 2008). However, it is unknown, periaqueductal gray; PB, phosphate buffer; PBS, phosphate-buffered saline; Sm, thalamic nucleus submedius; TTX, tetrodotoxin; VLO, ventrolateral orbital cortex; under persistent inflammatory pain state, whether the 5-HT1A WAY-100635, {N-2-[4-(2-methoxyphenyl-1-piperazinyl]ethyl]-N-2-pyridinylcy- receptors also are involved. The formalin injected into rat clohexane carboxamide}. hindpaw evoked nociceptive behavior and spinal Fos expression * Corresponding author. Tel.: +86 29 82655172; fax: +86 29 82656364. has been widely used in studying the persistent inflammatory ** Corresponding author. Tel.: +86 29 84774501; fax: +86 29 83283229. painprocessesanditsmodulation(Abbadie et al., 1997; E-mail addresses: [email protected] (J.-S. Tang), [email protected] (Y.-Q. Li). Buritova et al., 2005; Chapman and Besson, 1997; Harris,

1998; Jinks et al., 2002; Okuda et al., 2001; Watanabe et al., were transcardially perfused with 100 ml of 0.01 M phosphate-buffered saline 2003). The aim of the present study was to examine whether (PBS, pH 7.4), followed by 500 ml of 4% (w/v) paraformaldehyde and 15% (v/v) saturated picric acid in 0.1 M phosphate buffer (PB, pH 7.4). The brain and spinal microinjection of 5-HT1A receptor agonist into the VLO could cord were immediately removed and placed into the fresh fixative. Subsequently, depress the formalin-evoked nociceptive behavior (paw flinch- the brain and lumbar spinal cord were placed in 30% sucrose solution/0.1 M PB (pH ing response) and the Fos expression in the spinal dorsal horn. 7.4) as a cryprotectant overnight at 4 8C. The brain was serially cut into 50-mm thick coronal sections on a freezing microtome. The sections were mounted and Since 5-HT1A receptors are coupled to inhibitory G-proteins and stained with Cresyl Violet. The injection sites were plotted on photographs of the their activation results in hyperpolarization of cell membrane coronal sections modified from the Paxinos and Watson’s atlas (1986).The and inhibits neuronal activity (Aghajanian, 1995; Albert et al., locations of drug injection sites were verified histologically to be within the VLO. 1997), it has been suggested that the excitatory effect of 5-HT1A An example is shown in Fig. 1. receptors on neurons may be produced by inhibiting an The lumbar spinal cords were cut in 30-mm serial sections on a freezing inhibitory action of the GABAergic interneuron on the projection microtome (Kryostat 1720, Leitz, Mannheim, Germany). The L4-5 spinal cord was isolated. Lumbar spinal cord sections were serially collected into three separate neurons (Koyama et al., 1999, 2002; Susana et al., 2003). To dishes containing 0.01 M PBS (pH 7.4), according to numerical order. All sections provide evidence for this suggestion, this study also examined were carefully washed with 0.01 M PBS and immunohistochemically processed as the influence of 5-HT1A receptor agonist application into the VLO free-floating sections. slice on spontaneous GABAergic miniature inhibitory postsyn- The sections in the first dish were used for immunohistochemistry staining of Fos using the avidin–biotin-peroxidase (ABC) method. Briefly, the sections were aptic currents (mIPSCs) by using patch clamp technique. washed in 0.01 M PBS (pH 7.4) and incubated sequentially with: (1) rabbit antiserum against Fos polyclonal antibody (ab7963, 1:500 dilution; Abcam, 2. Materials and methods Cambridge, MA) in 0.01 M PBS containing 5% normal goat serum (NGS), 0.3% Triton

X-100, 0.05% NaN3, and 0.25% carrageenan (PBS-NGS, pH 7.4) for 48–72 h at 4 8C; 2.1. Part I: animal experiment (2) biotinylated goat anti-rabbit IgG (1:200 dilution; Vector, Burlingame, CA) in 2.1.1. Animal preparation PBS-NGS overnight at 4 8C; and (3) ABC Elite complex (Vector: 1:100) in 0.01 M Male Sprague–Dawley rats (220–250 g) were provided by the Experimental PBS (pH 7.4) containing 0.3% Triton X-100 (PBS-X) for 2 h at room temperature. Animal Center of the Fourth Military Medical University (Xi’an, China). The Bound peroxidase was visualized by incubation with 0.05% 3, 3-diaminobenzidine experimental protocol was approved by the Institutional Animal Care Committee tetrahydrochloride (DAB; Dojin, Kumamoto, Japan) and 0.003% H2O2 in 0.05 M of the University. According to the ethical guidelines of the International Association Tris–HCl buffer (pH 7.6) for 20–30 min. The sections were rinsed at least three for the Study of Pain (Zimmermann, 1983), all efforts were made to minimize the times in 0.01 M PBS (pH 7.4), for at least 10 min, after all incubations. The sections number of animals used, as well as to minimize distress to the animals. The animals were then mounted onto gelatin-coated glass slides, air-dried, dehydrated and were anesthetized with sodium pentobarbital (50 mg kgÀ1, intraperitoneally) and the cleaned, coverslipped with DPX, and observed under light microscope. Micro- head was immobilized in a stereotaxic frame. A small craniotomy was performed just photographs were taken with a digital camera (DP-70; Olympus) attached to a above the VLO. A stainless steel guide cannula (0.8 mm in diameter) was microscope. stereotaxically inserted, with its tip 2.0 mm dorsal to the VLO, at the following Sections in the second dish were mounted onto gelatin-coated glass slides and coordinates: 3.2–3.7 mm anterior to bregma, 2.0–2.5 mm lateral, 4.0–5.0 mm below processed for Nissl staining. Sections in the third dish were used for control tests. In cortical surface (Paxinos and Watson, 1986), and attached to the skull with three the control experiments, primary antibodies were omitted or replaced with normal microscrews and dental cement. Once the animals had recovered from anesthesia, rabbit serum; no positive staining for the omitted or replaced antibodies was they received sodium penicillin (0.2 million units per day for 4 days, intraperitoneally) detected. Sections from control and treated animals were processed in parallel to prevent wound and intracerebral infections. The animals were carefully nursed, under identical experimental conditions. then housed and fed in clear cages. [(Fig._1)TD$IG]

2.1.2. Intracerebral microinjection and drug preparation On the day of testing, the rats were acclimated to the experimental arena for 30 min prior to testing. The rats were then lightly anesthetized with enﬂurane (Baxter Caribe, Guayama, Puerto Rico, USA), a very short-acting inhalational anesthetic, and a 1.0-ml microsyringe, with the tip extending 2 mm beyond the end of the guide cannula, was inserted to the VLO through the guide cannula. The drugs were dissolved in saline (0.5 ml) and slowly infused through the microsyringe at a constant speed over a 30-s period.

Drugs used in this study, including the selective 5-HT1A receptor agonist 8-OH-

DPAT (8-hydro-2-(di-n-propylamino) tetralin, 5.0 mg, in 0.5 ml) and 5-HT1A receptor antagonist NAN-190 (1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine hydrobromide, 20 mg, in 0.5 ml), were purchased from RBI/Sigma, St. Louis, MO, USA. The drugs were freshly prepared in saline. 8-OH-DPAT was injected into the VLO contralateral to formalin-injected paw 10 min prior to formalin injection, and NAN-190 was administered 5 min prior to 8-OH-DPAT injection. The doses of both drugs were chosen according to previous studies (Huo et al., 2008; Nogueira and Graeff, 1995), where the dose of agonist was reported to be most effective in dose-dependent tests and the dose of antagonist did not inﬂuence the nociceptive response when it was injected alone. Saline of equal volume was injected into the VLO as vehicle control.

2.1.3. Formalin test The formalin test was performed as previously described (Xie et al., 2004). Brieﬂy, rat was placed in the plastic chamber with a mirror positioned below the chamber at a 458 angle to allow unobstructed observation of the rat’s injected paw. Ten minutes after intracerebral injection, the rat received a 50-ml subcutaneous injection of diluted (5%) formalin into the hindpaw pad, contralateral to the intracerebral injection. The rat was then immediately returned to the chamber. Formalin-induced nociceptive behaviors were observed, and the number of times the injected paw ﬂinched was counted every 5 min during a 60 min observation period, including early phase (0– 10 min) and late phase (20–60 min) (Abbott et al., 1999). Saline, injected into rat hind paw, was used as vehicle control. The observer was blind to treatment conditions.

2.1.4. Histology and immunohistochemistry Fig. 1. Photomicrograph showing an example of location of injection site within the At the end of the experiment, the drug injection site was marked by injection of VLO. Arrow points to the injection site. Scale bar = 1000 mm. AI, agranular insular Pontamine Sky Blue dye (0.5 ml; 2% in 0.5 M sodium acetate acid). One hour after cortex; CI, claustrum; fmi, forceps minor of the corpus callosum; LO, lateral orbital behavior observation (i.e., 2 h after formalin injection) under deep anesthesia, rats cortex; VLO, ventrolateral orbital cortex. F.-Q. Huo et al. / Neurochemistry International 57 (2010) 749–755 751

2.1.5. Quantification of Fos immunoreactive neurons obtained from Hebei Province Marine Science and Technology, China. Drugs were Considerable evidence indicates that the number of Fos-labeled neurons evoked at applied by a custom ‘‘Y-tube system’’ for rapid and complete solution exchange 2 h after hindpaw formalin injection is maximal at superficial (I–II) and deep (V–VI) within 20 ms. dorsal horn laminae of the L4–L5 segment levels (Buritova et al., 2005), which contain neurons responsive to noxious stimuli, corresponding to segmental innervation of the 2.2.4. Data analysis rat plantar hindpaw (Jinks et al., 2002). Therefore, the number of formalin-induced Analysis was performed using pclampfit (Axon) and MiniAnalysis Program Fos-expressing cells was quantified at the L4–L5 segment levels. The sections were (Synaptosoft Inc., Leonia, NJ, USA). All data are presented as mean Æ SEM. Differences first examined using lightfield microscopy at 4Â magnification to determine in amplitude and frequency distributions of miniature synaptic events were compared by segmental level, according to the cytoarchitectonic organization of the rat spinal non-parametric analysis (Kolmogorov–Smirnov test). Group means amplitude and cord reported by Molander et al. (1984). The sections were then examined under frequency of mIPSCs were compared using paired student’s t-test. P < 0.05 was lightfield microscopy at 10Â magnification to localize Fos-labeled nuclei, and considered to be statistically significant. photomicrographs were taken with a digital camera (DP-70; Olympus) attached to a microscope. Fos-labeled nuclei were quantified from collected images by an 3. Results investigator who was blind to treatment conditions of each animal. For analysis of Fos-labeled nuclei location, 10 sections with the greatest degree of Fos expression were selected from L4 to L5 segments of each animal. For each 3.1. Effects of 8-OH-DPAT on formalin-induced nociceptive behavior animal, two counts were made in the gray matter of the 10 sections: (1) the mean number of Fos-labeled nuclei in the entire spinal dorsal horn, and (2) the mean Subcutaneous injection of 5% formalin into the rat hindpaw pad- number of Fos-labeled nuclei in laminae I–II and V–VI. induced typical biphasic nociceptive flinching behavior of the injected paw. The early phase began immediately after injection and 2.1.6. Statistical analysis lasted for approximately 5–10 min. Subsequently, after a short All data were expressed as mean Æ SEM. Behavioral data were analyzed by two- way repeated measures of analysis of variance (two-way RM ANOVA) with a post hoc quiescent period (10–15 min), a prolonged tonic response persisted multiple comparison (Bonferroni t-test). The data obtained from the early and late for over 45 min (late phase). The response peak appeared at phases of the formalin-induced flinching behavior and Fos expression were analyzed approximately 35 min after formalin injection. Microinjection of by one-way ANOVA with a post hoc multiple comparison (Bonferroni t-test). P < 0.05 saline into the VLO did not influence the formalin-evoked flinching was considered to be statistically significant. The data were analyzed by using the response (Fig. 2A). SigmaStat 2.03 software. However, microinjection of 8-OH-DPAT (5.0 mg), a selective 5-

2.2. Part II: brain slice patch clamp experiment HT1A receptor agonist, into the VLO, 10-min prior to formalin injection significantly decreased the number of flinches. This 2.2.1. Brain slice preparation inhibitory effect was blocked by pre-treatment with the 5-HT1A VLO brain slices were prepared from Sprague–Dawley rats aged 13–15 postnatal receptor antagonist NAN-190 (20 g). As shown in Fig. 2A, the time- days (Kishimoto et al., 2001) provided by the Medical Experimental Animal Center m of Shaanxi Province, China. The experimental protocols were approved by the course curves (i.e., saline plus saline, saline plus 8-OH-DPAT, and Institutional Animal Care Committee of the University. The rats were decapitated [(Fig._2)TD$IG]NAN-190 plus 8-OH-DPAT treated groups) were significantly under pentobarbital anesthesia (50 mg/kg i.p.). The brain was removed and placed in an ice-cold artificial cerebrospinal fluid (ACSF) composed of (mM):124 NaCl, 3.0

KCl, 2 CaCl2, 6 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose, saturated with 95%

O2 and 5% CO2. Transverse brain slices (400 mm) were cut using a vibrating tissue slicer (Campden Instruments, London, UK), and transferred to a holding chamber containing ACSF saturated with 95% O2 and 5% CO2, and stored at room temperature (about 22–25 8C) at least one and half hour.

2.2.2. Electrical measurement The whole-cell voltage patch clamp recordings were similar as our previous method (Huang et al., 2007). For recordings, slice was transferred to a recording chamber, where it was perfused (2–2.5 ml/min) at 30–32 8C. The extracellular solution contained (mM): 124 NaCl, 3.0 KCl, 2.5 CaCl2, 1.3 MgCl2, 1.25 NaH2PO4,26

NaHCO3, 10 glucose, saturated with 95% O2 and 5% CO2. The slices were placed on an upright infrared video microscope with differential interference contrast (DIC) optics (Olympus BX51WI). Slices were observed through a 40Â waterimmersion objective using an infrared-sensitive camera (DAGE-MTI, IR-1000). Whole-cell voltage clamp recordings from visually identiﬁed VLO cells were performed with an Axopatch 700B ampliﬁer (Axon Instruments Inc., Burlingame, CA, USA) at holding potential of À70 mV, and digitized using a data-acquisition board (Digital data 1322A) operated by pCLAMP 9.2 software (Axon Instruments). Patch pipettes (1.5 mm/0.86 mm) were pulled using a horizontal puller (Model P-97; Sutter Instruments Co., Novato, CA, USA). They had a resistance of 3–5 MV and were neither polished nor coated. The pipette solution contained (in mM) 130 cesium methanesulfonate, 5 NaCl, 2 MgCl2, 0.1 CaCl2, 1 EGTA, 10 HEPES, 2 Na2ATP, and 0.25

Na3GTP (pH 7.3–7.4, adjusted with CsOH). In order to isolate mIPSCs, external solutions routinely contained 0.5 mM tetrodotoxin (TTX) to block voltage- dependent Na+ channels. In addition, 10 mM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50 mM DL-2-amino-5-phosphovaleric acid (DL-AP5) were present to block glutamatergic synaptic currents. Under these conditions the spontaneous mIPSCs could be stably recorded and continuously monitored and stored in a computer for off-line analysis. After getting gigaseal, we rupture the membrane patch by suction. Normally we waited about 5–10 min until the series resistance was stable. The slow capacitances were compensated about 70–80%. The series resistance, which was continuously monitored during the experiments using a À10 mV depolarizing pulse, and lower than 30 MV (10–30 MV) recordings were accepted. Recordings included for analysis were collected during periods of stable Fig. 2. (A) Time-course plots showing the effects of 8-OH-DPAT microinjected into series resistance (changes less than 5%). Responses were filtered at 2 kHz and the VLO on formalin-evoked flinching behavior. *P < 0.05, compared with saline sampled at 10 kHz. group and #P < 0.05, NAN-190 plus 8-OH-DPAT group compared with saline plus 8- OH-DPAT group at those time points, respectively. (B) Bar graph showing mean 2.2.3. Drugs number of flinches per 5 min during early phase and late phase. *P < 0.05 and # Drugs used in the present study were CNQX, AP5, EGTA, HEPES, Mg-ATP, Na3GTP, **P < 0.001, compared with saline group and P < 0.05, compared with saline plus bicuculline and 8-OH-DPAT, NAN-190, from RBI/Sigma, St. Louis, MO, USA. TTX was 8-OH-DPAT group, respectively. 752[(Fig._3)TD$IG] F.-Q. Huo et al. / Neurochemistry International 57 (2010) 749–755

Fig. 3. Photomicrographs showing examples of altered c-fos expression in the spinal dorsal horn under various treatment conditions. (A) Saline injected into hindpaw did not induce Fos expression; (B and C) formalin injected into hindpaw-induced signiﬁcant Fos expression in the ipsilateral (B), but not contralateral dorsal horn (C); (D) 8-OH-DPAT applied to VLO depressed formalin-evoked Fos expression; (E) NAN-190 applied to VLO antagonized 8-OH-DPAT-evoked inhibition of Fos expression; (F) NAN-190 alone applied to VLO did not inﬂuence formalin-evoked Fos expression. Scale bar = 200 mm.

different between treatments (F(2, 165) = 12.123; P < 0.001), across contralateral dorsal horn (Fig. 3C). Saline injection into the times (F(11, 165) =71.405;P < 0.001), and for their interactions (F(22, hindpaw did not induce spinal Fos expression (Fig. 3A). 165) =3.312;P < 0.001). Further analyses indicated that the mean Microinjection of 8-OH-DPAT (5.0 mg) into the VLO significant- number of flinches in the saline plus 8-OH-DPAT treated group was ly reduced the formalin-evoked Fos expression (Fig. 3D), while pre- significantly less than the saline-treated group at 9 of 12 time points treatment with NAN-190 (20 mg) reversed this effect (Fig. 3E). In (P < 0.001), as well as the NAN-190 plus 8-OH-DPAT group at 6 of 12 the entire spinal dorsal horn, or laminae I–II and laminae V–VI, the time points (P < 0.05). However, no significant difference (P > 0.05) number of Fos-positive neurons was significantly less in the saline was measured between the NAN-190 plus 8-OH-DPAT group and plus 8-OH-DPAT treated group compared with the saline (P < 0.05) the saline-treated group in all but one time point (P > 0.05), as and NAN-190 plus 8-OH-DPAT (P < 0.05) treated groups, with no shown in Fig. 2A. Significant differences between various groups in significant difference between the NAN-190 plus 8-OH-DPAT either the early phase or the late phase are shown in Fig. 2B. group and saline-treated group (P > 0.05), as shown in Fig. 4. NAN- NAN-190 (20 mg) injected alone into the VLO did not influence 190 microinjection alone into the VLO (i.e., NAN-190 plus saline- the nociceptive behavior, the number of flinches in the NAN-190 treated group) did not alter the formalin-evoked increase in the plus saline group was not significantly different from the saline- number of Fos-labeled neurons in the spinal dorsal horn, as shown treated group, in entire observation period or at any time point in Figs. 3F and 4. (Fig. 2A and B). 3.3. Effects of 8-OH-DPAT on GABAergic mIPSCs 3.2. Effects of 8-OH-DPAT on formalin-induced spinal Fos expression Pharmacologically separated spontaneous mIPSCs of the Formalin injection significantly induced spinal Fos expression neurons in the VLO slice were stably recorded lasting for over ipsilaterally to the formalin-injected hindpaw (Fig. 3B), and the 60–120 min in presence of TTX, CNQX and AP5 (Fig. 5A1).

Fos-labeled neurons were mainly located in the superﬁcial dorsal Application of bicuculline, a selective GABAA receptor antagonist horn (laminae I–II) and in the deep dorsal horn (laminae V–VI) (10 mM), into the extracellular solution completely and reversibly [(Fig._4)TD$IG]Fig. 3). The Fos-labeled neurons were virtually absent in the blocked the mIPSCs (Fig. 5A) which indicated that the mIPSCs were mediated by GABAA receptors, namely as GABAergic mIPSCs. Application of the 8-OH-DPAT, a selective 5-HT1A receptor agonist (0.1, 0.3, 1.0, 3.0 and 10.0 mM) into the VLO slice inhibited the GABAergic mIPSCs frequency in a concentration- dependent manner, without affecting the amplitude of the mIPSCs. A sample of the 8-OH-DPAT-evoked decrease of the GABAergic mIPSC frequency is shown in Fig. 5B1 and B2, without effect on the mIPSC amplitude (Fig. 5b1, b2 and b4). From Fig. 5G, it can be seen that the mean mIPSC frequency was decreased following the increase of the 8-OH-DPAT concentration. In 24 neurons tested, the mean mIPSC frequency induced by 1.0 mM of 8-OH-DPAT decreased to 68.4 Æ 18.9% of the control (P < 0.05, Fig. 5C and G, also see Fig. 5D). However, the amplitude of the mIPSCs was not affected by applying same doses of 8-OH- DPAT, as shown in Fig. 5EandF.

Application of the NAN-190, a 5-HT1A receptor antagonist Fig. 4. Bar graph showing inhibitory effect of 8-OH-DPAT microinjected into VLO on (1.0 mM), into the VLO slice, 5.0 min prior to the 8-OH-DPAT formalin-induced Fos expression, as well as the blocking effect of NAN-190 on 8- application, completely blocked the 8-OH-DPAT-induced decrease OH-DPAT-induced decreased Fos expression in the laminae I–II, laminae V–VI, and of the GABAergic mIPSC frequency, without effect on its amplitude entire spinal dorsal horn ipsilateral to formalin-injected paw. The mean Æ SEM of the each group (n = 6) was counted according to the mean number of 10 sections in (Fig. 5B3, b3, C, E and F). In addition, NAN-190 alone applied to the each animal. *P < 0.05 compared with saline group, #P < 0.05 compared with 8-OH- VLO slice had no any effect on either GABAergic mIPSC frequency DPAT plus saline group. or its amplitude (Fig. 5H). [(Fig._5)TD$IG] F.-Q. Huo et al. / Neurochemistry International 57 (2010) 749–755 753

Fig. 5. Picture showing the effect induced by 8-OH-DPAT application into VLO slice on the GABAergic mIPSC frequency, and the inﬂuence of NAN-190 on this effect. (A1–3) Spontaneous mIPSCs of the neurons in the VLO slice which was blocked by bicuculline; (B1–3) a sample showing that 8-OH-DPAT (1.0 mM) decreased the GABAergic mIPSC frequency, this effect was blocked by NAN-190 (1.0 mM). (b1–4) 8-OH-DPAT did not affect the amplitude of the GABAergic mIPSCs; (C and D) changes (%) of the mean GABAergic mIPSC frequency and the cumulative probability plots of the frequency distribution of mIPSCs from control, application of 8-OH-DPAT and 8-OH-DPAT plus NAN- 190, respectively; (E and F) changes (%) of the mean GABAergic mIPSC amplitude and the cumulative probability plots of the amplitude distribution of mIPSCs from control, application of 8-OH-DPAT and 8-OH-DPAT plus NAN-190, respectively; (G) GABAergic mIPSC frequency was decreased following the increase of the 8-OH-DPAT concentration; (H) NAN-190 alone application did not affect the GABAergic mIPSC frequency or amplitude. *P < 0.05, 8-OH-DPAT application compared with the control or NAN-190 plus 8-OH-DPAT application.

4. Discussion mediated at the spinal level, while the lifting/licking response is believed to be a more complex reflex mediated at the supraspinal Results of the present study have indicated that formalin injected level (Okuda et al., 2001; Watanabe et al., 2003). Therefore, subcutaneously into the hindpaw pad of rats induces a distinct measuring the Fos expression in the spinal dorsal horn and the spatial pattern of Fos expression in the lumbar spinal cord dorsal paw flinching responses induced by formalin injection can horn ipsilateral to the injected hindpaw and a significant paw investigate the descending modulation actions of the cerebral flinching response. The Fos-like immunoreactive neurons were higher centers on spinal nociception. densely distributed in the layers I–II and in layers V–VI in the L4–5 The present study has demonstrated that microinjection of 8- spinal dorsal horn. These regions have been suggested to contain OH-DPAT, a selective 5-HT1A receptor agonist, into the VLO numerous nociceptive primary afferents and nociceptive neurons significantly reduced both formalin-induced flinching behavior (Jinks et al., 2002; Willis and Coggeshall, 2004). These results are in and Fos expression in the lumbar spinal dorsal horn and in a NAN- agreement with previous results reporting Fos expression (Jinks et 190 reversible manner. These results extend previous studies that al., 2002) and in behavioral tests (Abbott et al., 1999; Bardin et al., found 5-HT1A agonist microinjected into the VLO depressed the tail 2001; Okuda et al., 2001; Shannon and Lutz, 2000). There is strong flick reflex (Huo et al., 2008; Qu et al., 2008). Furthermore, this evidence that the expression of Fos-like immunoreactive neurons in study provided novel evidence for the involvement of 5-HT1A the spinal cord is a function of the intensity and duration of the receptors in the VLO in the modulation of persistent inflammatory noxious stimulus (Abbadie et al., 1997). It has been demonstrated nociception. that Fos expression represents nociceptive transmission as well as Many studies have indicated that the central 5-HT1A receptor antinociceptive modulation occurring in the spinal cord following system participates in the modulation of nociception in various noxious stimuli such as subcutaneous formalin or bee venom animal models. Fasmer et al. (1986) demonstrated that intracer- injections (Luo et al., 1998). On the other hand, formalin injections ebroventricular injections of 8-OH-DPAT significantly reduced early into the rat hindpaw-induced spontaneous nociceptive behaviors phase formalin-induced nociceptive behavior in mice. Other studies including flinching and lifting/licking responses in the injected paw also reported that intraperitoneal or intrathecal injection of 8-OH- for over 1 h. The flinching response is considered to be a flexor reflex DPAT, or PAG injection, produced dose-dependent inhibition of 754 F.-Q. Huo et al. / Neurochemistry International 57 (2010) 749–755 licking and flinching responses induced by formalin injection in attenuates this effect (Huo et al., 2008). These results provide rodents (Bardin et al., 2001; Millan et al., 1996; Shannon and Lutz, support for involvement of GABAergic modulation in the VLO, 5-

2000). Furthermore, Oyama et al. (1996) reported that, in rats HT1A receptor-mediated descending antinociception, i.e.,activa- treated intrathecally with the neurotoxin 5,7-DHT to deplete the tion of the 5-HT1A receptors inhibits the inhibitory action of lumbar spinal cord of 5-HT, intrathecal administration of both 5-HT GABAergic interneuron on the VLO neurons projecting to the PAG and 8-OH-DPAT could produce antinociception in the formalin test, (disinhibition), leading to activation of the brainstem descending and this effect was blocked by the 5-HT1A antagonist NAN-190. The inhibitory system and depression of the nociceptive transmission present findings demonstrated that 8-OH-DPAT microinjection into atthespinaldorsalhorn. the VLO depressed formalin-induced nociceptive behavior, which In addition, there is evidence showing that a number of 5-HT provides novel data indicating the involvement of 5-HT1A receptors, receptor ligands displayed affinity to a-adrenoceptor binding sites at the cerebral cortex level, in the modulation of persistent and were used in experimental research to interact with a- inflammatory nociception. Indeed, 5-HT1A receptor agonists, such adrenoceptors in the rat (Yoshio et al., 2001; Centurio´ netal., as (3-chloro-4-fluoro-phenyl)-[4-fluoro-4-[[(5-methyl-pyridin-2- 2006; Foong and Bornstein, 2009). For example, the widely used ylmethyl)-amino]-methyl]piperidin-1-yl]-methadone (F 13640), a specific 5-HT1A receptor antagonist NAN-190 could block a2- newly discovered high-efficacy 5-HT1A receptor agonist, are adrenoceptor, and hence depress inhibitory synaptic potentials and attracting increasing interest for the management of chronic pain hyperpolarization evoked by noradrenalin in submucosal noncho- conditions (Bardin et al., 2003; Buritova et al., 2005; Colpaert, 2006; linergic secretomotor neurons of guinea pig (Foong and Bornstein,

Colpaert et al., 2006; Deseure et al., 2007; Kiss et al., 2005). 2009). Similarly, the high-efficacy 5-HT1A receptor antagonist WAY- Studies in our laboratory have provided evidence that the VLO 100635, {N-2-[4-(2-methoxyphenyl-1-piperazinyl]ethyl]-N-2-pyr- is part of an endogenous analgesic system consisting of an idinylcyclohexane carboxamide} also displayed affinity to a1- ascending pathway from the spinal cord to VLO via the Sm and a adrenoceptor binding sites (Centurio´ netal.,2006), and dose- descending pathway to the spinal cord relaying in the PAG (Tang dependently decreased diastolic blood pressure and blocked et al., 2009). Therefore, it is reasonable to propose that the vasopressor responses to phenylephrine in rats (Villalobos-Molina inhibitory effects of administration of 5-HT1A receptor agonist et al., 2002). Therefore, in study on the effects of the 5-HT1A into the VLO on formalin-induced flinching behavior and Fos receptors, choice of the antagonists needs for considerable caution. expression in the spinal dorsal horn are mediated by the VLO-PAG Nevertheless, in the present study, NAN-190 applied to the VLO did brainstem descending inhibitory system. However, it has been not increase the Fos expression and the paw flinching behavior as known that the 5-HT1A receptor is a negative G-protein coupled well as mIPSC, but completely blocked the 8-OH-DPAT-evoked receptor and its activation produces an inhibitory effect on target inhibition. This suggests that the NAN-190 really produces its neurons via a hyperpolarization of cell membrane (Aghajanian, antagonizing effect on the 5-HT1A receptors, but unlikely on the a- 1995; Albert et al., 1997). It has been suggested that the excitatory adrenoceptors in VLO. Meanwhile, the 5-HT1A receptors in the VLO effect of activation of 5-HT1A receptor may result from inhibition may lack tonic activity, and the blocking effect of NAN-190 on the 8- of an inhibitory action of the GABAergic interneuron on the OH-DPAT-evoked inhibition is not a result of either compound projection neurons (Koyama et al., 1999, 2002; Susana et al., facilitating nociceptive responses.

2003). The results of this study showed that 8-OH-DPAT In conclusion, the results of this study suggested that 5-HT1A application into rat VLO slice significantly reduced the GABAergic receptors in the VLO are involved in mediating the descending mIPSC frequency, but not affect its amplitude, and this effect is antinociceptive effect of persistent inflammatory nociception. The blocked by NAN-190, which is consistent with previous studies in possible mechanism underlying this effect is that 5-HT1A receptor other brain areas, such as basolateral amygdala (Koyama et al., activation presynaptically inhibits the GABA neurotransmitter 1999, 2002; Kishimoto et al., 2000)andPAG(Kishimoto et al., release, leading to activation of VLO neurons projecting to the PAG. 2001; Jeong et al., 2008). Since mIPSC frequency depends on the This could activate the PAG brainstem descending inhibitory GABA release probability in the presynaptic terminals and mIPSC system and depression of nociceptive information transmission at amplitude depends on the postsynaptic receptor sensitivity or the spinal cord level. conductance (Thompson et al., 1993; Seamans et al., 2001), this result suggests a presynaptic effect of 5-HT1A receptor activation. Conflict of interest Although the electrophysiological experiments were performed on postnatal 13- to 15-day-old rat pups, this result suggests that There were no conflicts of interest in relation to this article. 5-HT1A receptor activation presynaptically inhibits the GABA neurotransmitter release, which may lead to a disinhibitory effect Acknowledgment on the projection neurons to enhance the activity of the descending antinociceptive pathway in the adult rat, as suggested This project was supported by grants from the National Natural in PAG by Kishimoto et al. (2001). Morphological studies have Science Foundation of China (No. 30700222, 30800334). indicated that GABAergic neurons and terminals are distributed in the VLO and make symmetric (assumed inhibitory) synapse with References 5-HTergic terminals from the dorsal raphe nucleus and express 5-

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