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

Auli@ltsmemd cIow32mp7 sl7.00 + .oo PII soo24.32059700618-8 INVOLVEMENT OF SUPRASPINAL GABA-ERGIC SYSTEMS IN CLONIDINE- INDUCED ANTINOCICEPTION IN THE TAIL-PINCH TEST IN MICE Nguyen

capyti@.1997EbvierSdccrclac

RialedkthcUSA Auli@ltsmemd

cIow32mp7 sl7.00 + oo

PII soo24.3205(97)00618-8

INVOLVEMENT OF SUPRASPINAL GABA-ERGIC SYSTEMS IN CLONIDINE-

INDUCED ANTINOCICEPTION IN 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 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

(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 2-

hydroxysaclofen (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 2-

hydroxysaclofen (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~ GA BA A 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 (3-

5) 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 & zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAGABA zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBASystems 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 agonist-

induced 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 a2-

adrenoceptor-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 4 weeks The

animals were housed in groups of 20-25 per cage for at least 1 week before starting the

experiments, with free access to food and water Housing conditions were thermostatically

maintained at 24 * 1 “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 5 @/mouse according to the

methods of Haley and McCormick (16) (2 mm lateral, 2 mm caudal to bregma and 3 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 & GA BA 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 2 s were used for the experiments To prevent tissue damage, a cut-off

time of 6 s was selected zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Statistical analy sis

Data are expressed as the mean percent maximum possible effect (%MPE 2 SEM)

according to Dewey et al (20) The %MPE was calculated by the following equation

%MPE = (post-drug latency - pre-drug latency) / (cut-off time - pre-drug latency) x 100

Here: pre-drug and post-drug latencies represent the latencies measured before and after drug

administration, respectively The cut-off time is 6 s

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 1 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 min after administration, and then gradually

decreased Clonidine, at the same dose, also produced a decrease in spontaneous motor activity in

m-

so-

&o-

a9 -

20-

Muscimol

0 (10)

+ 62.5 (IO) t- 125 (IO)

u 250 (10)

ml (B)

-o- 0 (10) -+- 10 (10) o 50 (12)

Time after clonidine administration (min) Time after clonidine administration (min)

Fig 1 Effects of muscimol and baclofen on the systemic clonidine-induced antinociception

in the tail-pinch test After the basal nociceptive responses were recorded, 1 mgikg

clonidine was injected S.C at time 0 Thirty min 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 min over a 60-min

observation period Each point represents the mean %MPE 2 S.E.M The number of

animals used is shown in parentheses *PcO.O5, **P<O.Ol vs vehicle groups

(Dunnett’s test)

1100 Clonidine Autinoeiception & GABA Systems Vol 61, No ll, 1997

mice and the clonidine-induced hypolocomotion lasted over a 60-min period (data not shown)

The intracerebroventricular (i.c.v.) administration of muscimol (31.2-250 q/mouse), a selective

GABA, agonist, significantly prevented the antinociceptive effect of clonidine in a dose-

dependent manner (Fig 1A) The i.c.v administration of baclofen (lo-100 ng/mouse), a selective

GABA, agonist, also produced a dose-dependent inhibition of the clonidine-induced antinocicep-

tion in the tail-pinch test (Fig 1B) However, when given alone, muscimol and baclofen had no

effect on the nociceptive response at the doses tested in the same test (data not shown)

Effects of GABA, and GABA, receptor antagonists on the suppression of the clonidine-induced

antinociception caused by muscimol

As shown in Fig 2, i.c.v administered picrotoxin (250 @mouse) and bicuculline (100-400

q/mouse) significantly attenuated the suppressing effect of muscimol (62.5 @mouse, i.c.v.) on

the systemic clonidine-induced antinociception [Fig 24: F,- x piaotmio (1.32)= 4.935, PcO.05;

Fig 2B: Fe, x ,,iarcullinc 4w ng (1.35)=11.265, P< 0.01 at 15 min after clonidine administration]

However, neither picrotoxin nor bicuculline had effect on the nociceptive response (data not

shown) or on the antinociceptive effect of clonidine in the tail-pinch test In contrast, the GABA,

antagonist 2-hydroxysaclofen (10 p&mouse, i.c.v.) failed to attenuate the effect of muscimol

(62.5 rig/moose,, i.c.v.) on the clonidine-induced antinociception (Fig 2C) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

BlC(ng) 0

MUS (ng) 0

WI

SL

Fig 2 Effects of picrotoxin, bicuculline and 2-hydroxysaclofen on the suppression of the

clonidine-induced antinociception by the GABA, agonist muscimol in the tail-pinch

test After the basal nociceptive responses were recorded, clonidine (1 mgkg) was

administered S.C at time 0 Either vehicle or muscimol (MUS, 62.5 q/mouse) was

injected i.c.v with (A) picrotoxin (PTX: 250 rig/moose,,, i.c.v.), (B) bicuculline (BIG:

100-400 q/mouse, i.c.v.) and (C) 2-hydroxysaclofen (ZOH-SAC: 10 pg./mouse,

i.c.v.) just before the clonidine administration (1 mgkg, s.c.) Each column represents

the mean %MPE * S.E.M 15 min after the clonidine administration The number of

anhnals used is shown in parentheses **P<O.Ol vs vehicle groups; #P<O.O5,

##P<O.Ol vs muscimol alone (Tukey’s test)

Vol 61, No zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA11, 1997 Clonidine Antinocieeption & GABA Systems 1101 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

Effects of GABA, and GABA, receptor antagonists on the suppression of the cloniiiine- induced

antinociception caused by baclofen

When coadministered i.c.v with 2-hydroxysaclofen (lo-20 @mouse) or bicuculline (400

q/mouse), a significant interaction between 2-hydroxysaclofen and baclofen treatments [ Fbadofcn x

Zhydmxysadofcn (2,73)=3.155, P-zO.05 at 15 min after clonidine administration] was observed 2-

Hydroxysaclofen (20 &mouse) significantly reversed the suppressing effect of baclofen (50

ngmouse, i.c.v.) on the clonidine-induced antinociception Neither did the GABA, antagonist

bicuculline (400 @mouse, i.c.v.) affect the suppressing effect of baclofen on the clonidine-

induced antinociception at the dose (400 q/mouse, i.c.v.) which significantly antagonized the

suppressing effect of muscimol on the clonidine-induced antinociception in the same test

80 (4

1

s zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

20

0 IL (10)

2-OH-SAC (pg) 0

BAC (ng) 0

I

(10)

-

10

0

I

(10)

-

20

0

_I

a0

60

E

Ln z40

E

3

8 20

**

El

6

‘(10) (10)

0

1 ** 1 T

**

J !!!!I (10) lm (9 (10)

Fig 3 Effects of 2-hydroxysaclofen (A) and bicuculline (B) on the suppression of the

clonidine-induced antinociception by the GABA, agonist baclofen in the tail-pinch

test After the basal nociceptive responses were recorded, clonidine (1 mg/kg) was

administered S.C at time 0 Either vehicle or baclofen (BAC, 50 rig/moose)) was

injected i.c.v with 2-hydroxysaclofen (ZOH-SAC: 10 and 20 pg/mouse, i.c.v.) or

bicuculline (BIC: 400 q/mouse, i.c.v.) just before the clonidine administration (1

mg/kg, s.c.) Each column represents the mean %MPE f S.E.M 15 min after the

clonidine administration The number of animals used is shown in parentheses

*P<O.O5, **P<O.Ol vs vehicle groups; #PcO.O5 vs baclofen alone (Tukey’s test)

Discussion The present results demonstrate that supraspinally administered muscimol and R(t)-

baclofen can inhibit the suppressive effect of clonidine on the mechanical stimulation-induced

nociceptive response through GABA, and GABA, receptors, respectively, and provide evidence

that supraspinal GABAergic systems play an important role in the a,-adrenoceptor-mediated

regulation of nociceptive responses in mice

Kawabata et al reported that L-three-3,4_dihydrophenylserine (L-three-DOPS), a

noradrenaline precursor, produced opioid-independent antinociception in the tail-flick test in

mice, and they found that the effect of L-three-DOPS was mediated by the noradrenaline

stimulation of supraspinal a,-adrenoceptors and spinal a,-adrenoceptors (21) Spaulding et al

1102 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAClonicline Antinociceptlon & GABA Systems Vol 61, No ll,l!I97

(22) showed that spinalization at T6-T8 failed to change the antinociceptive activity of clonidine

in mice These findings suggest that antinociception caused by systemic clonidine is predominantly mediated by spinal a,-adrenoceptors In our preliminary study, idazoxan, an a2- adrenoceptor antagonist, attenuated the systemic clonidine-induced antinociception in the tail- pinch test when administered i.c.v or i.t (Huong et al., unpublished data) Thus, it is presumed that the supraspinal a,-adrenoceptors are partly involved in the suppressive effect of systemic clonidine on the nociceptive response caused by tail-pinch in mice

The GARA, receptor agonist muscimol and the GABA, receptor agonist R(t)-baclofen dose-dependently prevented the clonidine-induced antinociception in the tail-pinch test at the doses which exhibited no effect on the nociceptive response in the same test These findings disagree with the data reported by Przesmycki et al (14) Those authors showed that the systemic clonidine-induced antinociception in the tail-withdrawal test of rats was potentiated by systemically administered baclofen, and that, compared with the effect of baclofen, systemic muscimol produced a weak potentiating effect on the clonidine-induced antinociception even at a large dose (14) The exact reason for this discrepancy between our and their findings remains unclear but it may be due to differences in the route of administration of GABAergic drugs, the noxious stimulation applied to the animals, the spticies of animals used, and differences in processing mechanisms of thermal and mechanical information in the central nervous system GABA, receptor agonists are also known to produce opposite effects on opioid-induced antinociception depending on the route administration; i.e., they enhance opioid-induced antinociception when administered systemically, and counteract it when administered intracerebrally (8, 23)

The reversing effect of i.c.v muscimol on the a9inociception caused by clonidine was significantly antagonized by the GABA, receptor antagonists bicuculline and the GABA,-gated chloride channel blocker picrotoxin but not by the GABA, receptor antagonist 2-hydroxysaclofen

On the other hand, the reversing effect of i.c.v R(t)-baclofen on the clonidine-induced antinociception was antagonized by 2-hydroxysaclofen but not by bicuculline These results strongly indicate that i.c.v muscimol and i.c.v R(+)-baclofen attenuate the clonidine-induced antinociception by stimulating the supraspinal GABA, and GABA, receptors, respectively, and suggest that both supraspinal GABA, and GABA, receptors exhibit modulatory roles in the a2- adrenoceptor-mediated antinociception

A descending antinociceptive pathway involving GABAergic intemeurons in the spinal cord dorsal horn has been reported to play an important role in the regulation of the nociceptive response (11, 12, 24), but the GABAergic pathways implicated in the reversal of antinociception caused by the a,-adrenoceptor stimulation remain unclear Taking into account the results that when administered i.c.v., neither the GABA, antagonists, bicuculline and picrotoxin, nor the GABA, antagonist, 2-hydroxysaclofen, failed to enhance the clonidine-induced antinociception, it seems unlikely that supraspinal GABAergic systems tonically regulate the a,-adrenoceptor- mediated influence on the nociceptive response Both directly acting and indirectly acting GABAergic drugs reportedly produce antinociception through supraspinal and spinal GABA, and GABA, receptors in a variety of animal tests (8, 25-28) In the present study, the changes in the nociceptive response were not observed following the i.c.v injection of muscimol or R(t)- baclofen at the doses that exhibited a significant antagonism against the clonidine-induced antinociception Taken together, these findings suggest that supraspinal GABAergic systems may have reciprocal regulatory effects on the nociceptive response in mice; a suppressive effect on the a,-adrenoceptor-mediated antinociception and an antinociceptive effect through supraspinal GABA, and GABA, receptors

Vol 61, No 11, 1997 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAClonidme Antinoeieeption & zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAGABA Systems 1103

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