The rostroventromedial medulla is not involved in α2-adrenoceptor-mediated antinociception in the rat

A’europhormacotog~Vol. 32, No. 12, pp. 141 I-1418, 1993 Printed in Great Britain. All rights reserved

Copyright 0

0028-3908/93 $6.00 + 0.00 1993 Pergamon Press Ltd

THE ROSTROV~~ROMED~AL MEDULLA IS NOT INVOLVED IN a,-ADRENOCEPTOR-MEDIATED ANTINOCICEPTION IN THE RAT MINNA M. HAMALAINEN and ANTTI PERTOVAARA*

Department of Physiolo~,

P.O. Box 9, 00014 University of Helsinki, Finland (Accepted

6 July 1993)

Summary-The aim of the current study was to investigate the role of the rostroventromedial medulla (RVM) in a,-adrenoceptor-mediated antinociception. Medetomidine or clonidine, selective a,-adrenoceptor agonists were microinjected into the RVM in unanesthetized rats with a chronic guide cannula. The antin~i~ptive effects were evaluated using the tail-flick and hot-plate tests. For comparison, mecleto-

midine was microinjected into the cerebellum or the periaqueductal gray (PAG). To study the role of medullospinal pathways, the tail-flick latencies were also measured in spinalized rats. The reversal of the antinociception induced by intracerebral microinjections of medetomidine was attempted by S.C.atipamezole, a selective a,-adrenoceptor antagonist. The reversal of the antinociception induced by systemic administration of medetomidine was attempted by microinjections of 5% lidocaine or atipamezole into the RVM. When administered into the RVM, medetomidine produced a dose-dependent (l-30pg) antin~iception in the tail-flick and hot-plate tests, which antin~~ptive effect was completely reversed by atipamezole (1 mg/kg, s.c.). Also clonidine produced a dosedependent (3-30 pg) antinocic~tion following microinjection into the RVM. Microinjections of medetomidine into the cerebellum or the PAG produced an identical dose-response curve in the tail-flick test as that obtained following microinjection into the RVM. In spinal&d rats the antinociceptive effect (tail-flick test) induced by medetomidine microinjected into the RVM was not less effective than in intact rats. Lidocaine (5%) or atipamezole (5 fig) microinjected into the RVM did not attenuate the antinociception induced by systemically administered medetomidine (lOO&kg, s.c.). The adapting skin temperature of the tail was increased in a nonmonotonic fashion following m~etomidine. The results indicate that the RVM is not a site which is critical for the a,adrenergic antinociception. The antinociception following intracerebral microinjections of medetomidine into the RVM, PAG or the cerebellum in the current study can be explained by a spread of the a,-adrenoceptor agonist into the spinal level to activate directly spinal a,-adrenoceptors. Also, the antinociception following systemic administration of medetomidine can be explained by spinal a,-adrenergic mechanisms. The medetomidine-induced increase of the adapting skin temperature may have attenuated the medetomidine-induced increases in the response latencies to noxious heat. Key words++adrenergic analgesia, atipamezole, m~etomidine, microinjection, nucleus raphe magnus, periaqueductal gray, a*-adrenoceptors, nociception, rostroventromedial medulla.

Activation of a,-adrenoceptors by various agonists administered systemically or intrath~aIly is known to produce antinociception in animals and in humans (Danzebrink and Gebhart, 1990; Pertovaara, 1993; Proudfit, 1988; Yaksh, 1985). There is plenty of evidence indicating that pre- and postsynaptic mechanisms in the spinal dorsal horn are important for the ot2-adrenergic antin~i~eption ~Da~eb~nk and Gebhart, 1990; Pertovaara, 1993; Proudfit, 1988; Yaksh, 1985). To a large extent these spinal CQadrenoceptors are activated by noradrenaline released from the terminals of descending axons of brainstem neurons (Carlton, Honda, Willcockson, Lacrampe, Zhang, Denoroy, Chung and Willis, 1991). There are a,-adrenoceptors also in various supraspinal sites (Unnerstall Kopjtic and Kuhar, -*To whom correspondence should be addressed. Abbreviarions: RVM, rostroventromedial medulla; PAG, periaqueductal gray; s.c., subcutaneous.

1984), and some of these sites (e.g. the rostroventromedial medulla, locus coerufeus and ~riaqueductal grey) are known to be involved in the modulation of nociceptive signals as verified by electrical or chemical stimulation of these structures (Basbaum and Fields, 1984; Jensen and Yaksh, 1989; Proudfit, 1988). The antinociceptive role of a,-adrenoceptors located in various supraspinal structures is, however, only studied to a limited extent. It has been proposed that the antinociceptive effect of systemically administered clonidine, the prototype cr,-adrenoceptor agonist, is entirely due to spinal mechanisms as indicated by the lack of effect of spinalization on the clonidineinduced increase in tail-flick late&es (Spaulding, Venafro, Ma and Fielding, 1979). Similarly, systemically administered medetomidine, also an cr,-adrenoceptor agonist, had an equal effect on the tail-flick response in spinalized and intact rats (Pertovaara, Kauppila, Jyvgsjarvi and Kalso, 1991). Furthermore, microinjections of clonidine have had no antinocicep-

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MINNAM. H~~M~~L~~INEN and ANTII PERT~VAARA

tive effects when microinjected into the periaqueductal gray (PAG; Ossipov and Gebhart, 1983), hyperalgesic effects when microinjected into the lateral reticular nucleus (Ossipov and Gebhart, 1986), and the central antinociception induced by etorphine has been attenuated following microinjection of clonidine into the locus coeruleus (Ossipov, Malseed, Eisenman and Goldstein, 1984). On the other hand, a number of studies have indicated that noradrenaline may modulate serotonergic raphe-spinal neurons and consequently, nociceptive signals in the spinal dorsal horn (Fields, Heinricher and Mason, 1991; Proudfit, 1988). Concerning the effect of selective a,-adrenergic agents, there are studies which have indicated that clonidine microinjected into the rostroventromedial medulla (RVM) produces increased tail-flick latencies in awake (Sagen and Proudfit, 1985) and pentobarbitone-anaesthetized rats (Haws, Heinricher and Fields, 1990), whereas microinjection of cc,-adrenoceptor antagonists into the RVM has produced either decreased tail-flick latencies (Sagen and Proudfit, 1985) or no effect (Haws et al., 1990). Thus, conflicting results exist concerning the antinociceptive role of a,-adrenoceptors located in supraspinal structures, including the RVM. In the current investigation we tried to re-evaluate the role of the RVM in cc,-adrenergic antinociception using a highly selective a,-adrenoceptor agonist medetomidine (MacDonald, Scheinin and Scheinin, 1988; MacDonald, Scheinin, Scheinin and Virtanen, 1991, Virtanen, Salvola, Saano and Nyman, 1988). Medetomidine was microinjected into the RVM, or for comparison into the PAG or cerebellum, in unaneasthetized rats and its effect was determined on the nocifensive response latencies in the tail-flick and hot-plate tests. Atipamezole, a highly selective c(~adrenoceptor antagonist (Scheinin, MacDonald and Scheinin, 1988; Virtanen, Savola and Saano, 1989) was used to reverse the medetomidine-induced effects. The tests were also performed in spinalized rats to determine the role of medullospinal pathways in the observed effects. Systemic administration of medetomidine is known to produce antinociception (Kauppila, Kemppainen, Tanila and Pertovaara, 1991; Pertovaara, Kauppila and Tukeva, 1990; Pertovaara, Bravo and Herdegen, 1993a) and it is possible that a,-adrenoceptors in the RVM might contribute to this antinociception. To investigate this possibility, we microinjected lidocaine or atipamezole into the RVM to reverse the antinociception induced by systemic administration of medetomidine.

METHODS These experiments were approved by the Institutional Ethics Committee of the University of Helsinki, and by the Regional Government of Uusimaa.

Surgical preparation

Male Hannover-Wistar rats (250-450 g) were anaesthetized with pentobarbital(60 mg/kg, i.p.). The rat was placed into a stereotaxic frame according to Paxinos and Watson (1986). A stainless steel microinjection guide tube (22-gauge) was implanted such that the tip lay 2mm dorsal to the injection site. The stereotaxic coordinates of the injection sites were as follows (reference: the ear bar). The RVM: posterior, 2.3 mm; horizontal, -0.5 mm; lateral, 0.0 mm. The PAG: posterior, 0.7 mm; horizontal, 4.0 mm, lateral, 0.7 mm. The cerebellum: posterior, 2.3 mm; horizontal, 7.0 mm; lateral, 0.0 mm. The guide cannula was held in place with dental acrylic which was anchored to the skull with stainless steel screws. After the last experiment Fast Green was injected through the cannula to help in histological localization of the injection sites. The animals were individually housed and allowed free access to food and water. In one experiment the rat was additionally spinalized at midthoracic level with a knife under pentobarbital anaesthesia (60 mg/kg, i.p.). The effectiveness of spinalization was macroscopically verified. Microinjection procedures

Drugs were microinjected through a 27-gauge stainless steel injection cannula inserted into the guide cannula. The tip of the injection cannula extended 2 mm beyond the end of the guide cannula. The injection cannula was connected to a 20 cm length of polyethylene tubing (PE 20) filled with drug solution. The volume of each injection was 1.O~1 and was delivered over a period of 30 set by a 10 ~1 Hamilton microsyringe. The efficacy of the injection was monitored by watching the movement of a small airbubble through the tubing. The injection cannula was left in place for 30 set after the injection to minimize flow of the drug solution up the cannula track. Testing of nocifensive reflexes

Two tests were used to evaluate the possible antinociceptive effects: the tail-flick test and the hot-plate test. The tail-flick response was elicited by applying radiant heat to the dorsal surface of the rat’s tail using a commercially available device (Socrel Model DS-20). The time interval between the onset of the radiant heat stimulus and the tail-flick response was measured electronically and the stimulus was terminated after 12 set in the absence of response. Three separate spots were marked in the tail and these spots were consecutively stimulated in order to avoid skin damage. Each threshold (at various time points before and after the drug administration) was based on three measurements performed at one min intervals. During the testing of the tail-flick response the rat was in a standard restraint tube made of plexiglass. A calibrated thermocouple was glued to the skin of the tail 5 cm proximally to the stimulus site to

Descending a,-adrenergic antinociception measure the adapting skin temperature during the experiment. In the hot-plate test the rat was placed on a metal plate (maintained at 54°C) and the latency to the lick of the hind paws was measured. A cut-off time of 30 set was used. One measurement before and one IO min after the drug administration was made in each rat. Before the first hot-plate test the rats were allowed to habituate to the hot-plate device for one min. During habituation the temperature of the hot-plate was 30°C. Furthermore, all the rats had spent at least 1 day before the first experiment in the laboratory in order to habituate to the laboratory environment. Drugs Medetodimine and atipamezole were obtained from the Farmos Group, Orion Pharmaceuticals Corp., Turku, Finland, clonidine from Sigma, St Louis, Missouri, U.S.A., and lidocaine from Astra, Sweden. The drugs were dissolved in physiological saline. Dexmedetomidine is the pharmacologically active d-isomer of medetomidine. The effect of 1 pg of dexmedetomidine equals 2pg of medetomidine (MacDonald et al., 1991). Experimental protocol Following recovery from surgery (3 days) baseline latencies were determined and the rats were given an intracerebral microinjection of medetomidine (l-3Opg, 1 PI), clonidine (3-3Opg, 1 pl) or physiological saline ( = control, 1 ~1). Unless otherwise specified, the post-drug taif-flick latencies were measured 5 and 15 min after the intracerebral microinjection (the maximal effect reported), and the post-drug hot-plate latency 10 min after the intracerebra1 injection. The reversal of the effect induced by intracerebrally injected medetomidine was attempted by S.C.atipamezole (1 mg/kg, s.c.). When the reversal of the antinociception induced by systemic administration of medetomidine was attempted, medetomidine (l~~g~kg, s.c.) was administered after determining the base line tail-flick latency. Then, 10 min later the effect of systematically administered medetomidine on the tail-flick latency was measured. Lidocaine (S%, 1~1) or atipamezole (5 pg, 1 ,ul) was microinjected into the RVM after the antinociceptive effect of systemically administered medetomidine had been verified. The effect of intracerebral lidocaine/ atipamezole on the medetomidine-induct antinociception (tail-flick test) was determined 5 and 15 min following the central injection. It should be noted that due to the first-pass metabolism in the liver considerably lower medetomidine doses are needed to produce antinociception following subcutaneous than intaperitoneal injections (Kalso, PSyhia and Rosenberg, 1991). Each unspinalized rat was used in 3-5 experiments. Each drug dose/saline control or drug combination was tested on a separate day, and

1413

the order of testing varying drug doses/saiine control was counterbalanced between the animals. When spinalized rats were studied, the experiments were performed 3-5 hr after the spinalization, when the rats had recovered from the surgery and anaesthesia. Chronic spinalized rats were not studied due to ethical reasons. To keep the number of spinal rats in minimum (n = 4) only one dose of medetomidine was tested in spinalized rats. Decerebration was not performed, since the purpose was to study the role of a supraspinal structure in antinociception. In spinalized rats the baseline tail-flick latency was first determined, then medetomidine was microinjected into the RVM at a dose which had proved effective in intact rats (10 fig, 1~1). Post-drug tail-flick latency was measured 5 min following the intracerebral microinjection. Then atipamezole was administered (1 mg/kg, s.c.) and the effect of atipamezole on the tail-flick latency measured 10 min later. Immediately after the last latency determination an overdose of pentobarbital was given to kill the rat. Statistics For the dose-response analysis, the data of hotplate and tail-&k latencies were converted to% MPE, where% MPE = [(Latency with drug Baseline latency)/(Cut-off time - Baseline latency)] x 100. The dose producing a 50 % MPE was calculated by linear regression analysis of the doseresponse curve. Analysis of variance (ANOVA, one-way, unless otherwise specified) followed by Newman-Keuls test was used to evaluate statistically the results. P < 0.05 was considered to represent a significant difference. RESULTS

Figure 1 shows the histologically verified injection sites in the RVM and the PAG. The cerebellar injection sites (not shown) were all located in the mediodorsal part of the cerebellum. The mean baseline latencies over all experimental conditions in the tail-flick and hot-plate tests were 3.1 f 0.1 and 10.4 f 0.9 set, respectively (+SEM, n = 104 and 28, respectively). The baseline latencies in various experimental groups were in the same range. ~icroinj~tion of medetomidine into the RVM produced a significant dose-dependent (l-30 pg) increase of the tail-flick latency [F(4, 18) = 24.8, P < 0.001, ANOVA; Fig. 2(a)] and of the hot-plate latency [F(4, 18) = 13.6, P < 0.001, ANOVA; Fig. 2(b)]. The 50% MPEs in the tail-flick test and the hot-plate test were obtained following microinjection of medetomidine into the RVM at a dose of 16.0 and 17.Opg, respectively. The peak antinociceptive effect induced by injection of medetomidine into the RVM was obtained within 5 min as indicated by the tail-flick test, and ;he antinociception remained at the same level at least for the duration of 30min [Fig. 2(c)]. The antinociceptive effect in-

MINNA M. Hk~jili;fm

and ANTI-I PERTOVAARA

Fig. I. The centres of the histologically verified injection sites (m) in the rostroventromedial medulla and in the mesencephalon. DPGi = dorsal ~ra~ganto~IIu1~ nucleus, Gi = gigantoc&uIar nucleus, RMg = nucleus raphe magnus, PAG = ~~aqu~u~t~ gray. The scale bar represents 1 mm.

medetomidine (100 pg/kg). The microinjection of duced by microinjection of medetomidine into the lidocaine [Fig. 4(b)] or atipamezole alone (n = 4, RVM was completely reversed by S.C.atipamezole at data not shown) at these doses into the RVM had a dose (1 mg/kg) which itself produced no effects on no effects on the tail-flick latency. the tail-flick latency [Fig. Z(dd)]. Also microinjection Since a change in the adapting skin temperature of clonidine into the RVM produced a significant can influence the response latency to heat stimuli dose-dependent (3-30 pg) increase of the tail-flick (Berge, Garcia-Cabrera and Hole, 1988), we also latency [F(3, 16) = 22.8, P < 0.001, ANOVA; studied the effect of medetomidine microinjections on Fig. 3(a)]. Clonidine microinjected into the RVM the adapting skin temperature of the tail. The mean produced a 50% MPE in the tail-flick test at a dose adapting skin temperature over all experimental conof 24.2 fig. ditions before the intra~rebral injections was The microinj~ons of medeto~dine into the PAG 25.6 f 0.16”C (n = 30), and there were no significant or into the cerebellum also produced significant dosedifferences in the pre-drug skin temperatures between dependent increases in the tail flick test FAG: F(3, 18) = 7.35, P = 0.002; Cerebellum: F(3, 11) = 38.8, the various experimental groups (ANOVA). Medetomidine microinjected into the RVM produced a P < 0.001, ANOVA]. The doses producing 50% significant non-monotonous increase in the tail skin MPEs in the tail-flick test following microinjection of temperature [F(4, 19) = 3.63, P < 0.05, ANOVA; m~etomidine into the PAG or the cerebellum were Fig. 51. 19.9 and 15.5 pig, respectively. There were no significant differences in the dose-response curves following medetomidine microinjections into the RVM, PAG DISCUSSION and cerebellum as indicated by 2-way ANOVA [Fig. 3@)1. In the current study microinjection of medetoIn order to study the role of descending medulmidine, a highly selective aradrenoceptor agonist, lospinal pathways in the antinociception, medetointo the RVM produced a dose-dependent increase in midine was microinjected into the RVM at an the tail-flick and hot-plate latencies, and this antiantinociceptive dose (10 bg) also in spinalized rats. nociceptive effect was reversed by atipamezole, a Medetomidine microinjected into the RVM produced highly selective a,-adrenoceptor antagonist. Also a highly significant increase in the tail-flick latencies clonidine, a less selective a,-adrenoceptor agonist, also in spinalized rats, and this increase was commicroinj~t~ into the RVM produced a dose-depenpletely reversed by S.C. atipamezole fl mg/kg; dent antinociceptive effect. These results are in line Fig. 4(ab)]. with the previously described increase in tail-flick We also studied the role of medullary a,-adrenolatencies following microinjections of clonidine into ceptors in the antinoception induced by systemic the RVM in unanaesthetized (Sagen and Proudfit, administration of medetomidine. This was done by 1985) and anaesthetized (Haws er al., 1990) rats. attempting to attenuate the increase in tail-flick However, in the current study an equipotent effect on latency induced by systemically administered medetothe tail-flick latency was obtained by microinjecting midine by microinjecting lidocaine (S??, 1.~1) or medetomidine into the cerebellum or the PAG. Furatipamezole (5 pg, 1 ~1) into the RVM. Neither thermore, also in spinalized rats the tail-flick latency lidocaine [Fig. 4(b)] nor atipamezole (n = 4, data was at least as effectively increased by microinjection not shown) microinjected into the RVM attenuof medetomidine into the RVM as in intact rats, and ated the antinoceptive effect induced by S.C. the antinociception in spinal&d rats as well as in

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Descending ar-adrenergic antinociception

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Fig. 2. (a) Tail-flick latencies following microinjection of medetomidine at various doses or saline (SAL) control into the RVM. (b) Paw-lick late&es in the hot-plate test following microinjection of medetomidine at various doses or saline control into the RVM. (c) The time-course of the increase in the tail-flick latency following medetomidine microinjection into the RVM at a dose of 10 pg. (d) Atipamezole (ATIP, 1 mg/kg, s.c.) reverses the tail-flick increase induced by medetomidine (MED) microinjected into the RVM at the dose of 10 pg. 0% = The baseline latency before the intracerebral injection; 100% = the maximal antinociceptive effect. The stars indicate a significant difference from the SAL-group. lP c 0.05, **P < 0.01 (Newman-Keuls test). The error bars represent SEM; in each group n = 4-g.

intact These cated were duced

rats was completely reversed by atipnmezole. findings suggest that the a,-adrenoceptors loin the RVM, or in other supraspinal structures, not mediating the antinociceptive effects inby intracerebral injections of medetomidine in

this study. A plausible explanation for the current findings is the spread of medetomidine (e.g. via circulation) into the spinal dorsal horn, and direct activation of a*-adrenoceptors at the spinal level. It is well established, that activation of a,-adrenoceptors in the spinal dorsal horn by various a,-adrenoceptor agonists (Danzebrink and Gebhart, 1990; Yaksh, (dex) medetomidine (Fisher, 1985), including Zornow, Yaksh and Peterson, 1991; Kalso, Payhi-

hnd Rosenberg, 1991; Pertovaara, 1991, Pertovaara Kauppila, Mecke, et al., 1993a; Pertovaara, Hiimlhiinen and Carlson, 1993a; Sullivan, Kalso, McQuay and Dickenson 1992; Takano and Yaksh, 1992), produces antinociception. The lowest antinocicaptive doses of intrathecally administered (dex) medetomidine are considerably lower than the dose needed to produce antinociception in the RVM, PAG or cerebellum in this study or in the locus coeruleus in our recent study (Pertovaara et al., 1993b). Consistent with the current observations, the results of earlier studies using systemic administrations of clonidine (Spaulding et al., 1979) or medetomidine (Pertovaara et al., 1993a) have suggested that the

MINNA

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M. H&XL&EN and ANTII F'ERTOVAARA

a,-adrenergic antinociception is predominantly due to spinal a,-adrenoceptors as indicated by the lack of effect of spinalization on the qadrenoceptor-induced antinociception. The antinociception induced by systemic administration of medetomidine was not attenuated by lidocaine or by atipamezole (5 pg) applied into the RVM. These observations suggest that the RVM, or raphe-spinal neurons, are not markedly involved in the antinociception induced by systemic administration of medetomidine. This findings also supports our conclusion based on the above microinjection experiments that cr,-adrenoceptors in the RVM are not critical for the cc,-adrenoceptor-mediated antinociception. Similarly, it has been reported earlier that lidocaine microinjected into the RVM did not attenuate antinociception induced by systemically administered morphine (Proudfit, 1980). In some previous investigations decreased tail-flick

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Fig. 4. (a) Tail-flick latencies in spinalized rats following microinjection of medetomidine (MED) into the RVM at the dose of lOpg, and the reversal of the medetomidineinduced effect by atipamezole (ATIP, 1 mg/kg, s.c.). (b) Systemic administration of medetomidine (100 fig/kg, s.c.) induces a significant increase in the tail-flick latency, which effect is not attenuated by lidocaine (LIDO, 5%, 1~1) microinjected into the RVM. Lidocaine alone has no effect. In both graphs the stars indicate a significant difference from the pre-drug (= 0%) latency. *P ~0.05, l*P co.01 (Newman-Keuls test). The error bars represent SEM; n = 4.

Cerebellum

60

Medetomidine (pg) Fig. 3. (a) Comparison of the effect of clonidine (clan) with medetomidine (med) on the tail-flick latency following microinjection into the RVM. (b) Comparison of the effect of medetomidine on tail-flick latencies following microinjection at various doses into the RVM (O), PAG (V) or cerebellum (a); n = 4-7. For further details see the legend for Fig. 2.

responses have been observed following microinjections of lidocaine (Proudfit, 1980) or yohimbine, an a,-adrenoceptor antagonist, into the RVM (Sagen and Proudfit, 1985; Haws et al., 1990). In the current study no significant decrease of tail-flick latencies was observed following microinjections of atipamezole or lidocaine into the RVM. This difference in results may be due to a difference in experimental conditions. Interestingly, it has been previously reported that application of morphine or etorphine into the brainstem in cats may also produce antinociceptive action at the spinal cord level due to a direct spinal action and not to activation of descending inhibition (Clark and Ryall, 1983; Clark, Edeson and Ryall, 1983). The volume of intracerebral injections in this study was 1 ~1. According to previous studies it spreads in

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Descending a,-adrenergic antinociception 5.0

RVM

r

SAL

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following microinjections into the RVM. The medetomidine-induced antinociception was completely reversed by a specific qadrenoceptor antagonist both in unspinalized and spinalized rats. The antinociception produced by systemic administration of medetomidine was not attenuated by lidocaine microinjected into the RVM. These results support the hypothesis that the RVM, or some other supraspinal site, is not critical for the antinociception produced by u,-adrenoceptor agonists. A plausible explanation for the antinociception observed following a supraspinal microinjection of medetomidine, is the spread of the drug to activate directly the spinal a,-adrenoceptors. 3

Medetomidine

10

30

(pg)

Fig. 5. The increase in the adapting skin temperature of the tail induced by medetomidine microinjected at various doses into the RVM. 0°C = The adapting skin temperature before the intracerebral injection. The error bars represent f SEM; ?l=4-6.

cerebral tissue at the requested concentration about 1.9 mm (Myers, 1966). This should cover a major part of the RVM. Additionally, in lower concentrations

the drug can spread to more distant sites, even into the spinal cord level as indicated by the increases in tail-flick latencies following microinjections of medetomidine into the RVM in spinal&d animals. The volume of 5% lidocaine microinjected into the RVM should effectively suppress neuronal activity of brainstem neurons within a radius of 1.6-1.7 mm as indicated by previous studies (Martin, 1991; Sandkuhler, Maisch and Zimmerman, 1987). This should be enough to inhibit most of the RVM neurons. Medetomidine as well as clonidine is an imidazoletype qadrenoceptor agonist. It has been shown that part of the effects produced by clonidine can be explained due to an action on imidazoline-receptors (Ernsberger, Meeley, Mann and Reis, 1987). Also medetomidine can bind to imidazoline receptors of the cerebral cortex (Wikberg, Uhlen and Chajlani, 1991). However, binding of atipamezole is highly selective to a,-adrenoceptors of the cerebral cortex (Sjiiholm, Voutilainen, Luomala, Savola and Scheinin, 1992). The complete reversal of the medetomidine-induced antinoception by atipamezole indicates that the present medetomidine-induced effects were due to an action on a*-adrenoceptors rather than on an action on imidazoiine-receptors. Conclusions

The antinociceptive effects of medetomidine, a highly selective a,-adrenoceptor agonist, were studied following microinjections into the RVM, PAG or cerebellum. All these intracerebral microinjections produced identical dose-response curves. Furthermore, medetomidine was as potent in producing antinoception in spinalized as in unspinalized rats

Acknowledgements-We wish to thank Dr E. Mecke for his skillful1 assistance in the histological verification of the microinjection sites, and Dr R. Virtanen, Farmos Group, for generously providing us with medetomidine and atipamezole. This study was supported by grants from the Sigrid Juselius Foundation, Helsinki, and the Academy of Finland.

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