Involvement of supraspinal GABA-ergic systems in clonidine-induced antinociception in the tail-pinch test in mice

life Sciaw, Vd 61,No 11,pi 1097~ll@+, 1997 capyti@.1997EbvierSdccrclac RialedkthcUSA Auli@ltsmemd cIow32mp7 sl7.00 + oo PII soo24.3205(97)00618-8 INVOLVEMENT OF SUPRASPINAL INDUCED ANTINOCICEPTION GABA-ERGIC SYSTEMS IN CLONIDINEIN THE TAIL-PINCH TEST IN MICE Nguyen Thi Thu Huong, Kinzo Matsumoto and Hiroshi Watanabe* Department of Pharmacology, Research Institute for Wakan-Yaku (Oriental Medicines), Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan zyxwvutsrqponm (Received in final form June 6,19!27) Summary W e investigated the involvement of supraspinal GARAergic systems in the antinociceptive effect of clonidine using the tail-pinch test in mice Muscimol (31.2-250 rig/moose,, i.c.v.) and R(t)-baclofen (lo-100 q/mouse, i.c.v.), selective agonists for the GABA, and GABA, receptors, respectively, significantly attenuated the antinociceptive effect of subcutaneously (s.c.) administered clonidine (1 mg/kg) in a dose-dependent manner The attenuating effect of muscimol (62.5 @mouse, i.c.v.) on the clonidine-induced antinociception was significantly blocked by the GABA, antagonists bicuculline (100-400 @mouse, i.c.v.) and picrotoxin (250 rig/moose,, i.c.v.) but not by the GABA, antagonist 2hydroxysaclofen (10 pg/mouse, i.c.v.) On the other hand, the attenuating effect of R(+)-baclofen (50 r&mouse, i.c.v.) was blocked by the coadministration with 2hydroxysaclofen (20 l&/mouse), but was not affected by the coadministration with bicuculline (400 r&mouse) These results indicate that both supraspinal GABA, and GABA, receptors play inhibitory roles in the antinociception caused by systemically administered clonidine w U-ix&: imthO&e#o~ GABAA receptor, GABA, receptor, clonidine The descending noradrenergic system is one of the major pain-modulating systems originating from the brainstem; it participates in opioid-induced antinociception in rodents (l-3) This system exerts tonic inhibitory influence on the transmission of nociceptive information through a,-adrenoceptors located at pre- and postsynaptic sites of the primary afferent fibers (35) In fact, several reports have shown that the a,-adrenoceptor agonist clonidine exerts a more potent antinociceptive activity in various nociceptive tests than does morphine (5-7) Corresponding Author: Hiroshi Watanabe, Ph.D., Department of Pharmacology, Research Institute for Wakan-Yaku (Oriental Medicines), Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan, FAX: &l-764-34-5056 cJlo&be Antinociccption & zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM GABA zyxwvutsrqponmlkjihgfedcbaZYXWVUTS Systems VoL 61, No ll, 1997 The amino acid neurotransmitter, GABA, exerts a potent inhibitory activity in the central nervous system, through the GABA, receptor coupled with a chloride channel, and the GABA, receptor, the stimulation of which decreases Ca’+ conductance and increases K’ conductance via adenylate cyclase-coupled G-protein (8, 9) Some of a,-adrenoceptor-mediated pharmacological actions of clonidine appear to be exhibited through GABAergic systems in the brain, since clonidine facilitates the release of endogenous GABA via a2- and a,- presynaptic adrenoceptors in the brain (10) Recent findings have demonstrated that a descending antinociceptive system involving the spinal GABA receptors plays an important role in the regulation of the nociceptive response (11, 12), and that the spinal GABA, and GABA, receptors mediate 6-opioid agonistinduced antinociception in the tail-flick test in mice (13) Several reports have elucidated the role of GABAergic systems in the a,-adrenoceptor agonist-induced antinociception using a thermal noxious stimulation Przesmycki et al showed that neither GABA, agonists nor GABA, antagonists affected the clonidine-induced antinociception in the tail-withdrawal test (14) Nabeshima et al reported that the a,-adrenoceptor-mediated suppression of the nociceptive response in the tail-flick test was not modified by selective GABA, receptor antagonists (15) However, no information is available on whether GABAergic systems can modulate the a2adrenoceptor-mediated inhibition of the nociceptive response caused by a mechanical noxious stimulation Thus, in the present study, we investigated the effect of supraspinally administered GABAergic agonists and antagonists on the clonidine-induced antinociception by means of the tail-pinch test in mice Methods Animals Male ddY mice (Japan SIC, Shizuoka, Japan) were obtained at the age of weeks The animals were housed in groups of 20-25 per cage for at least week before starting the experiments, with free access to food and water Housing conditions were thermostatically maintained at 24 * “C and a relative humidity of 55 -c 5% with a 12 h lightdark cycle (lights on: 08.00-20.00) Total number of animals used were 326 Each animal was used only once The present studies were conducted in accordance with the standards established by the Guide for the Care and Use of Laboratory Animals of Toyama Medical and Pharmaceutical University The following drugs were used: Clonidine HCl, muscimol and picrotoxin (Sigma Chem Co St Louis, MO), R(+)-baclofen hydrochloride and 2-hydroxysaclofen (Research Biochemical International, Natick, MA), bicuculline (Calbiochem., La Jolla, CA) Muscimol, R(+)-baclofen, picrotoxin and clonidine were dissolved in saline Bicuculline and 2-hydroxysaclofen were dissolved in saline by adding a few drops of O.lN HCl Clonidine HCl (1 mg/kg) was administered subcutaneously (s.c.) in a constant volume of 10 ml/kg Other test drugs were injected intracerebroventricularly (i.c.v.) just before the S.C administration of clonidine The i.c.v injection of test drugs was performed in a constant total volume of @/mouse according to the methods of Haley and McCormick (16) (2 mm lateral, mm caudal to bregma and mm deep) The i.c.v doses were expressed as ng or pg/mouse When testing antagonism at the supraspinal level, test drugs were coadministered i.c.v All drug solutions were prepared immediately before the start of the experiments Measurement of the nociceptive response in the tail-pinch test The nociceptive response in the tail-pinch test was measured according to Haffner’s method as previously reported (17-19) Briefly, hemostatic forceps (3 mm width, 500 g constant pressure) Clonidine Antinoeieeption & GABA Vol 61, No 11, 1997 Systems 1099 were applied to the root of the tail, and only the mice that showed the nociceptive responses such as biting the forceps within s were used for the experiments To prevent tissue damage, a cut-off time of s was selected zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB Statistical analy sis Data are expressed as the mean percent maximum possible effect (%MPE according to Dewey et al (20) The %MPE was calculated by the following equation %MPE = (post-drug latency - pre-drug latency) / (cut-off time - Here: pre-drug and post-drug latencies represent the latencies administration, respectively The cut-off time is s SEM) pre-drug latency) x 100 measured before and after drug The %MPE was analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s test or by two-way ANOVA followed by Tukey’s test for multiple comparison among groups Differences of PcO.05 were considered statistically significant Results Effect of muscimol and baclofen on clonidine- induced antinociception The systemic administration of clonidine (1 mg/kg, s.c.) significantly prolonged the latency of nociceptive responses in the tail-pinch test in mice We chose this dose of clonidine in the present experiments because, in our previous study, we found that clonidine, at mg/kg (s.c.), exhibited a submaximal antinociceptive activity in the tail-pinch test in mice As shown in Fig 1, this antinociceptive action of clonidine peaked at 15 after administration, and then gradually decreased Clonidine, at the same dose, also produced a decrease in spontaneous motor activity in m- ml (B) r -l Muscimol so- + &oa9 (10) 31.2 (IO) -o- 62.5 (IO) o (10) -+10 (10) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHG -f t- 125 (IO) u 250 (10) 50 (12) 20- 4/ ;s ilt Time after clonidine P administration & (min) 4/ is io Time after clonidine is administration io (min) Fig Effects of muscimol and baclofen on the systemic clonidine-induced antinociception in the tail-pinch test After the basal nociceptive responses were recorded, mgikg clonidine was injected S.C at time Thirty before clonidine injection, muscimol (A: 31.2-250 nglmouse) or baclofen (B: lo-100 rig/moose)) was injected i.c.v The latency of the nociceptive response was measured every 15 over a 60-min observation period Each point represents the mean %MPE S.E.M The number of animals used is shown in parentheses *PcO.O5, **P