105
Pain, 47 (1991) 105-113 0 1991 Elsevier Science Publishers B.V. All rights reserved 0304-3959/91/$03.50 ADONIS 0304395991002038
PAIN 01868
GABA-mediated inhibition in rostra1 ventromedial medulla: role in nociceptive modulation in the lightly anesthetized rat Mary M. Heinricher
and Hilary J. Kaplan
Department of Neurology, Uniuersity of California at San Francisco, San Francisco, CA 94143 (U.S.A.)
(Received 1 October 1990, revision received 21 February 1991, accepted 28 February 1991)
Local microinjection of GABA, receptor agonists and antagonists was used to characterize the Summary role of GABA-mediated inhibitory processes in the nociceptive modulatory functions of the rostra1 ventromedial medulla (RVM) in the lightly anesthetized rat. Microinjection of selective GABA, receptor antagonists bicuculline methiodide and SR95531 produced a significant increase in tail-flick (TF) latency. This antinociception was dose related, showed recovery and was attenuated by prior injection of the GABA, receptor agonist THIP at the same site. Microinjection of saline or the glycine receptor antagonist strychnine did not significantly affect TF latency. In contrast, administration of GABA, receptor agonists THIP and muscimol resulted in a significant decrease in TF latency. Microinjections at sites surrounding the RVM did not significantly affect TF latency. These results demonstrate that a GABA-mediated process within the RVM is crucial in permitting execution of the TF and, presumably, other spinal nociceptive reflexes. Key words:
Rostra1 ventromedial modulation
medulla;
Nucleus
Introduction
An extensive body of evidence has implicated brain-stem systems in descending control of nociceptive input and in opiate analgesia [3,47,52]. Microinjection of opiates into, or electrical stimulation of, several brain-stem structures inhibits both nocifensor reflexes and neuronal responses to noxious stimuli. One region that has attracted intense interest is the rostra1 ventromedial medulla (RVM), which includes the nucleus raphe magnus, and the laterally adjacent nucleus reticularis gigantocellularis pars alpha and nucleus reticularis paragigantocellularis lateralis medial to the facial nucleus. Stimulation of the RVM electrically or by glutamate microinjection has antinociceptive effects [23,34,41,53] and also inhibits dorsal horn neurons [14,16,43,48]. Cell bodies and terminals showing opioid-like immunoreactivity are found in this region
Correspondence to: Mary M. Heinricher, Department of Neurology, Box 0114, University of California at San Francisco, San Francisco, CA 94143, U.S.A.
raphe
magnus; Tail flick; Antinociception;
GABA;
Pain
[5,29,46], and microinjection of morphine or opioid peptides into the RVM is also antinociceptive [1,10,22,23,25]. In addition, lesions of the RVM or local microinjection of naloxone can significantly reduce the antinociceptive effect of systemically administered morphine [1,39,511. These studies provide compelling evidence that the RVM has a key role in the modulation of nociception and, specifically, in mediating opiate analgesia. Although the functional importance of the RVM in modulation of nociception is well established, the local circuitry involved in control of RVM neurons is only now being elucidated. A substantial body of evidence suggests a significant role for GABA in modulation of nociception. Systemic administration of GABA, receptor agonists such as THIP and muscimol has been shown by several groups to inhibit responses to noxious stimulation [8,45]. However, GABA is distributed throughout the nervous system and participates in a wide variety of neuronal circuits [12,33], presumably including systems crucial to both transmission and modulation of nociception. Thus, direct application of GABA agonists and antagonists at discrete CNS sites
that arc involved in nociccptive tr~~~~srn~ssi~~~7 of its rn~~dul~~tio~~ is required. and ~nvestigat~~rs taking this ~~pprc~ach have demonstrated that microjnjection of GABA, receptor antagonists at several sites involved in n~~ciceptive modulation can inhibit spinal r~ocif~nsive reflexes and dorsal horn nociceptive neurons [9,11,X, 31.441. Moreover, unlike systemic administrations, microinjection of GABA, receptor agonists into brainstem sites involved in nociceptive modulation has no effect on or may even facilitate responses to noxious stimuli. Thus, THIP was reported to have no consistent dose-related effect when microinjected into the PAG I401 and resulted in a decrease in TF latency when applied in the RVM [ 1i]. We have recently shown that iontophoretic application of GABA, receptor antagonists eliminates a synaptically mediated inhibition of RVM neurons that is associated with occurrence of nocifensor reflexes [20]. An understanding of the functional role of GABA in this preparation would thus complement our knowledge of the physiology of RVM neurons obtained under essentially identical conditions and greatly strengthen the conclusions to be drawn from both classes of experiments. The current experjmental strategy involved identi~ing changes in nociceptive responsiveness produced by local injection of severaf GABA,% receptor antagonists (bicucul~ine methiodide (EM11 and SR955.11 ~~-tcarboxy-3’“pr~~pyl)-3-amin~~-4-~~~~ff-mcthoxy-phenyl”pyridazinium bromide)~ and of the GABA, receptor agonists muscimof and THIP (4,S,h,7-tetrahydroisoxazolo(S,4-c)pyridin-3-(~1~ into the RVM of the lightly anesthetized rat. The results demonstrate that GARA plays an important role in controlling nociceptive m~~du~ation within this region. A preiimina~ report of these findings has been presented [211.
Methods Animals Male Sprague-Dawiey rats weighing 250-275 g were initially anesthetized with pentobarbitai (60 mg/kg, i.p.) and a catheter placed in an external jugular vein. Each animal was then placed in a stereotaxic frame and a small craniectomy was performed to allow placement of an injection can&a within the RVM. .A 25-gauge stainless steel guide cannula was aimed towards the RVM and lowered slowly to a position 2 mm dorsal to the intended injection site. After cornp~~tio~ of these procedures, the animal was alIowed to recover from the initial anesthesia to the point at which the TF response could be evoked by application of noxious heat To the tail. The animal was subsequently maintained in a lightly anesthetized state by continuous infusion of methohexitai (1.5-30 mg/kg/h). The animals were monitored continuously throughout the
course of the experiments and showed no signs cti discomfort; they did not move spontane[~usly, vocatize or show vigorous withdrawal responses to intense pinch. Nocicepti~~e testing was initiated 35-45 min after stnrting the methohexit~l infusion. Body temperature was maintained at appr~~xini~~te~y37 o C using a circulating water blanket.
Drugs were delivered into the RVM in a volume of 11.5,ul using a 31”gauge cannula having a hevefled tip. This was connected to a l-p1 Hamilton syringe via a length of polyethylene tubing (PE 201, The injection cannula, which extended 3 mm beyond the end of the guide cannuia, was inserted into the RVM just prior to initiation of the behavioral testing protocol and was left in place for a minimum of 5 min after the injection was completed. Progress of the injection was monitored by observing the movement of a small air bubble through the tubing. The following drugs were injected: BMI (RI%, pH 5.3-5.51, SR95531 (RI%, pH 5.5-5.91, THIP HCI (RBI, pH 5.0-5.31, mnscimoi (Sigma, pH 565.91, strychnine HCI (Sigma, pH 5.91 and 0.9% saline vehicle (0.5 ylf. With the exception of experiments that involved pretreatment with agonist prior to antagonist microinjections. only one injection was made per animal.
The tail flick response was used as an index of nociceptive responsiveness [7f. Noxious heat was appiied to the blackened ventral surface of the tail using a feedback-controlled projection lamp. Three sites. 3, 5 and 7 cm from the distal tip of the tail, were used in rotation. A miniature thermistor probe placed in contact with the heated surface of the tai1 altowed precise measurement of tail temperature. The tail surface temperature at the stimulation site was maintained at 35 o C between TF trials which were initiated at 5 min intervals. The noxious heat stimulus consisted of a linear 10 set temperature ramp from the 35 o C holding temperature to a maximum of 53” C at 10 sec. The heat was automatically returned to 35’ C upon occurrence of the TF or if 10 set etapsed in the absence of a TF. Following determination of baseline TF iatency (mean of 3 TF triais), an injection was made into the RVM over a period of 3 min. TF latencies were determined at 5 min intervats for the period from 2 to 27 min after completing drug injection. In experiments involving pretreatment with THIP prior to administratioI1 of BMI, THIP (25-100 ngf was injected into the RVM immediately following determination of baseline latency. BMT (100 ng) was injected 10 min later (immediate& after the fifth TF trial) and TF latency monitored for the ensuing 27 min.
107
Histological uerification
At completion of the experiments, animals were sacrificed with an overdose of methohexital and perfused intracardially with physiological saline followed by 10% formalin. Microinjection sites were histologically verified and plotted on standardized sections derived from the atlas of Paxinos and Watson 1371.
3 :J ,
Statistical analysis
TF latencies following administration of GABA. receptor antagonists, strychnine and saline were compared to baseline latency using Friedman’s analysis of variance by ranks. A non-parametric test was required because many animals failed to perform to TF response within the 10 set cut-off time following administration of BMI or SR95531. In experiments involving pretreatment with THIP prior to administration of BMI, TF latencies following microinjection of BMI were compared to baseline latencies using a t test for correlated means. TF latencies following administration of GABA, receptor agonists THIP and muscimol were analyzed using analysis of variance with repeated measures within each dose group. In this case, Tukey’s test was employed for post hoc comparisons. A probability level of 0.05 was considered significant.
Results The mean baseline TF latency in these experiments was 4.8 rt 0.1 sec. Prior to drug injection, there were no significant differences in baseline latencies among the groups (ANOVA; P > 0.05; n = 128). Microinjection of GABA,
receptor antagonists
Microinjection of BMI (25, 50, 300 or 200 ng; 6-9 animals/group) into the RVM resuhed in a dose-reloo-
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Fig. 1. Time course and dose dependence of effects of BMI (25-200 ng) on TF latency. Each point represents the mean of determinations made in 6-9 rats. Mean and S.E.M. are shown for baseline latencies. Following microinjection, the number of animals in which TF was completely inhibited (10 set cut-off) is shown next to each point. Effects of saline microinjection are shown for comparison. A saline: n 25 ng BMI; 0 50 ng BMI; l 100 ng I3MI; 0 200 ng BMI.
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Fig. 2. Time coume of effects of microinjection of SR95531 (0.5-10 ng) on TF latency. Each line represents the mean of determinations made in 3 rats. Mean and S.E.M. are shown for baseline latencies. Following microinjection. the number of animals in which TF was completely inhibited (IO set cut-off) is shown next to each point. A 0.5 ng: W 1.0 ng; 0 2.5 ng; l 5.0 ng; o 10.0 ng SR95531.
lated antinociception (Fig. 1). The percentage of animals failing to respond within 10 set ranged from 28% following administration of 2.5 ng to 100% following 100 and 200 ng BMI (7 min after drug injection). Onset of the effect was quite rapid; at all doses tested, the TF latency was significantly increased within 2 min of completing the microinjection (i.e., the first test), with a peak antinociceptive effect attained by 7 min post injection. The duration of BMI-induced antinociception was also dose dependent. The increase in TF latency produced by microinjection of 25 ng BMI was reversed by 12 min after the microinjection, whereas recovery required 17 and 22 min following administration of 100 and 200 ng, respectively (Friedman’s analysis of variance by ranks). Microinjection of a second GABA, receptor antagonist, SR95531CO.5, 1,2.5,5 or 10 ng; 3 animals/group) also produced a profound antinociception when microinjected in RVM (Fig. 2). The percent of animals failing to respond within 10 set ranged from 33% following administration of 0.5 ng SR95531 to 100% following 2.5-10 ng SR95531 (7 min after drug injection). As with microinjection of BMI, the increase in TF latency following SR95531 administration was evident within 2 min of completing the microinjection. However, the antinociceptive effect of SR95531 was generally more prolonged than that of BMI, with TF suppression (10 set cut-off) often lasting throughout the 27 min post-injection observation period. Unlike the GABA, receptor antagonists, microinjection of the glycine receptor antagonist strychnine (75 ng) had no effect on TF latency when injected into the RVM (Friedman’s test; P < 0.20; n = 3). Similarly, microinjection of 0.5 ~1 saline vehicIe had no effect on TF latency (Friedman’s test; P < 0.20; n = 6). The TF inhibition produced by microinjection of BMI into the RVM was attenuated by prior injection
Fig. 4. Representative coronal sections of the medulla showing histologically verified sites at which SR95531 was microinjected. A 0.5 ng; U 1.Ong: 0 2.5 ng; A 5.1)ng; n 10.0 ng. VII, facial nerve and nucleus; P. pyramids. Fig. 3. Representative coronal sections of the medulla showing histologically verified sites at which BMI was microinjected, 28 sites within the RVM and 9 sites dorsolateral or caudal to the RVM. RVM (dotted outline) includes nucleus raphe magnus and the adjacent nucleus reticularis gigantocellularis pars alpha, as well as the rostra1 portion of nucleus reticularis paragigantocellularis lateralis, extending from the caudat poIe of the facial nucleus rostraliy to the level of the trapezoid body. o saline; CI 25 ng BMI; l 50 ng BMI; A 100 ng BMI; W 200 ng BMI. VII, facial nerve and nucleus: P, pyramids: AMB, nucleus ambiguus, IO, inferior olive.
of the GABA, agonist TI-IIP at the same site. Thus, following pretreatment with 100 ng THIP in RVM, microinjection of 100 ng BMI resulted in only a small increase in TF latency, from 4.0 i 0.2 set prior to the injections to 6.2 + 1.3 set measured 7 min after microinjection of BMI (t test for correlated means; P > 0.05;n = 6). This attenuation of the antinociceptive effect of BMI was not likely due to non-specific effects of the double injection procedure, as pretreatment with the lowest dose of THIP (25 ng) did not prevent the antinociception produced by BMI microinjection (n = 3). Microinjection of BMI at sites dorsal (n = 6) or caudal (n = 3) to the RVM did not significantly alter the TF response (Friedman’s analysis of variance by ranks; P > 0.05in each case). Fig. 3 illustrates the sites in the medulla where BMI was injected, 28 within and 9 outside of the RVM; Fig. 4 shows the distribution of 15 RVM sites where SR95.531 injections were carried out. Sites for injections of strychnine and pretreatment with THIP were comparable.
~icro~njection of GABA. receptor agonists Microinjection of the GABA, receptor agonists THIP and muscimol in the RVM resulted in significant decreases in TF latency. THIP was microinjected at 30 sites within RVM (12.5, 25, 50, 100, 300 or 1000 ng; 5 animals/group). Following drug administration, all groups except that receiving 300 ng THIP showed a significant decrease in TF latency (repeated measures ANOVA; Fig. 5). DecreaSed TF latencies were manifest within 2-7 min of the microinjection, with a maxi-
DOSE
/
SAL
THIP
(ng) MUSciMoL
I
Fig. 5. Effects of THIP and muscimol microinjections on TF latency. Results, expressed as percent of pretreatment .control (mean* S.E.M.) are shown for values determined 12 min after microinjection of saline, THIP or muscimol in RVM. (* P < 0.05; * * P < 0.01; repeated measures ANOVA).
109 -1.80
mum effect at 12 min post injection. This hyperalgesia was maintained throughout the 27 min post-injection observation period. The magnitude of hyperalgesia produced by THIP was not significantly related to the dose administered, although there was a tendency for increasing hyperalgesia with increasing dose to a maximum at 100 ng THIP. Administration of the highest dose WI00 ng) was associated with no greater, or possibly even a smaller, decrease in TF latency (TF latencies at 12 min post injection, Fig. 5). Muscimol was microinjected at 20 sites within the RVM (0.1, 1,5, 10 or 50 ng; 4 animals/group) and, like THIP, produced a significant decrease in TF latency (repeated measures ANOVA, Fig. 5). As with THIP microinjection, hyperalgesia produced by muscimol was evident within 2-7 min of completing the microinjection, was maximal at I2 min post injection and lasted throughout the 27 min post-injection period. Sites at which THIP and muscimol were microinjetted into the medulla are shown in Figs. 6 and 7, respectively. Microinjection of 100 ng THIP at 8 sites
Fig. 7. Representative coronal sections of the medulla showing histologicalIy verified sites at which muscimol was injected. A 0.1 ng; 0 1.0 ng; l 5.0 ng; A 10 ng; n 50 ng. VII, facial nerve and nucleus; P, pyramid.
dorsal or caudal to the RVM did not result in significant changes in TF latency (ANOVA, P > 0.05).
Discussion
Fig. 6. Representative coronal sections of the medulla showing histologically verified sites at which THIP was microinjected, 30 sites within RVM (dotted outline), and 8 sites dorsal or caudal to RVM. o 12.5 ng; A 25 ng; 0 50 ng; l 100 ng; A 300 ng; n 1000 ng. VII, facial nerve and nucleus; P, pyramids; AMB, nucleus ambiguus; IO, inferior olive.
The present studies demonstrate that a GABAmediated process plays a key role in the nociceptive modulating functions within the RVM; IocaI application of GABA, receptor antagonists in RVM induced a profound antinociception, whereas GABA, receptor agonists produced an enhancement of nociceptive responsiveness. Microinjection of BMI, a selective GABA, receptor antagonist, suppressed the TF response in a dose-related manner. Although it is impossible to conclude unequivocally that this effect was mediated by an antagonism of GABA-mediated neurotransmission, the observations that the antinociception was dose related, showed recovery and was attenuated by prior treatment with the GABA, agonist THIP injected at the same site provide evidence that this effect was not a non-specific effect of the microinjection procedure. Moreover, microinjection of SR9.5531, a recently developed pyridazinyl-GABA derivative having GABA, receptor antagonist properties [17,18,27], produced a similar dose-dependent suppression of the TF response, Finally, microinjection of the glycine receptor antago-
1 to
nist strychnine or saline vehicle was without effect. Taken together, these findings strongly suggest that BMI-induced antinociception involved blockade of GABA-mediated inhibition within the RVM. Administration of the GABA, receptor agonists THIP and muscimol resulted in a decrease in TF latency. This hyperalgesia was likely mediated by activation of a GABA, receptor, since muscimol is only a weak agonist and THIP is largely inactive at the GABA, receptor [4]. Moreover, microinjection of baclofen, the prototypical GABA, agonist, in the RVM is reported to have no effect on TF latency [25]. Micr~injections made into areas outside the RVM did not significantly affect TF latency. Thus, the low doses required and the rapid onset of the drug effect argue against the possibility that the antagonists diffused to a site of action outside of the RVM. As in the case of antagonist microinjections, localization of THIP and muscimol effects to the RVM is supported by the observation that microinjections at sites surrounding the RVM were ineffective. The present findings extend previous observations implicating GABA-mediated inhibitory processes in the control of nociceptive modulatory circuitry within the RVM [I 11. BMI microinjected into nucleus raphe magnus or nucleus reticularis gigantocellularis pars alpha in awake rats produced only small increases in TF latency that were not related to the dose administered. These authors therefore concluded that GABA neurotransmission did not make a substantial tonic contribution to control of nociceptive modulating neurons in the RVM. Their animals were, however, first tested 15 min after the microinjection, well after the time of peak effect of BMI injections seen in our experiments and reported by others [26,31,44]. Thus, although it is possible that GABA plays a less significant role in awake than in barbiturate-anesthetized animals (see below), it may be that more profound effects would have been evident if the awake animals had been tested within 2-10 min of the microinjection. The same authors also reported that microinjection of 300 or 1000 ng THIP, comparable to the highest doses used in the present experiments in anesthetized animals, enhanced nociceptive responsiveness as measured in the paw pinch and TF but not in hot-plate tests. Given our present observation that the highest doses of both THIP and muscimol did not produce the greatest decreases in TF latency, it would be interesting to determine whether lower doses of THIP affect hot-plate latencies in awake animals. As in the barbiturateanesthetized animals, in which a maximal decrease in TF latency developed by I2 min after the injection, the peak effect was manifest within 15 min of the agonist injection in awake animals. Thus, the time course of the THIP-induced hyperalgesia was similar in the 2 preparations.
GABA-mediated inhibition may also play a role in nociceptive modulation in other medullary regions, In rats anesthetized with alphaxalone/alphadolone, bilateral but not unilateral injections of 200 or 400 ng (total dose of 400 or 800 ng) BMI in the retrofaciai portion of nucleus reticularis paragigantocellularis generally cauda1 and lateral to the RVM as defined here, produced complete inhibition of the TF response beginning within I-10 min of the injection [26]. These results suggest an important role for GABA neurotransmission within the caudal ventrolateral medulla in nociceptive modulation. Although use of the lightly anesthetized rat has the advantage that it allows a direct comparison with electrophysiological observations made in the same preparation, the possibility that the present findings were influenced by barbiturate anesthesia must be considered. It is well known that barbiturates, including methohexitai, facilitate GABA-mediated inhibition [24,28.35,49]. Thus, a barbiturate-induced potentiation of endogeneous GABA transmission could have influenced the present results. That is. the effects of blocking GABA transmission by microinjection of antagonists might be more readily apparent in barbiturateanesthetized animals because there could be an clevation in basal levels of GABA-mediated inhibition within the RVM. Acting against this, however, could be a reduction in the effectiveness of the microinjected antagonists, since antagonist binding is reduced in the presence of barbiturates [27,51]. In any case, the relative significance of these two factors, increased inhibitory ‘tone’ and reduced antagonist binding, is not clear. Because blockade of GABAergic synaptic transmission by local application of competitive receptor antagonists within the RVM completely suppresses the TF response in lightly anesthetized animals, it is fikely that a GABA-mediated inhibition of some population of RVM neurons is crucial in permitting the execution of the TF and, presumably, other spinal nocifensive rcflexes. The observation that microinjection of GABA, receptor agonists, which would be expected to mimic such an obligatory GABAergic inhibitory process, results in a decrease in TF latency adds further weight to this inference. Although 3 populations of neurons have been identified in the RVM using electrophysiological techniques [13], only 1 class shows unequivocal evidence of a powerful inhibitory input that is temporally correlated with the TF response. These cells, called ‘off-cells,’ are characterized by an abrupt cessation in firing that occurs just prior to the execution of the TF. Several lines of evidence suggest that activation of off-cells inhibits responses to noxious stimulation and, conversely, that silence of cells of this class would permit such responses to occur. Thus, manipulations known to prevent the off-cell from pausing, for exam-
ple morphine given systemically or microinjected into the PAG [6,15], also suppress the TF response. In addition, periods of spontaneously occurring off-cell silence are associated with TF latencies that are more rapid than those obtained during a period in which off-cells are active [191. Cells of the other 2 physiologically characterized populations of RVM neurons respond very differently to nociceptive inputs and to manipulations producing antinociception. ‘on-cells’ display a sudden increase in activity just prior to the occurrence of the TF. This increase in on-cell activity presumably reflects a prepotent TF-related excitatory input to this cell class. Administration of morphine suppresses on-cell activity [2,6], and periods of on-cell silence are associated with relatively long TF latencies [19]. Cells of the third class, ‘neutral cells,’ are unaffected by either noxious stimulation or opiate administration [2,13]. These considerations would suggest that it is unlikely that GABAmediated inhibition of either on-cell or neutral cell activity would be required in order for the TF response to occur. However, the possibility that a fourth, as yet uncharacterized, neuron is involved cannot be excluded. Thus, the simplest explanation for the results of the present study would be that the GABA, receptor antagonists block a phasic, GABA-mediated inhibitory input responsible for the off-cell pause. Consistent with this, we have provided evidence that the TF-related off-cell pause can be blocked by iontophoretic application of BMI [20]. Blockade of additional, possibly tonic, inhibitor inputs to the off-cell could also contribute to the antinoci~eption produced by GABA antagonists. Conversely, microinje~tion of GABA, receptor agonists would silence the off-cell, permitting the TF to occur without hindrance from descending brain-stem modulatory circuitry. This is consistent with the fact that elimination of ull descending modulatory influences originating in the RVM (by means of electrolytic lesions or local anesthetic injection) can, under some conditions, produce a shortening of TF latency [38,39,421. Although GABA, receptor agonist microinjections could potentially suppress the activity of all cell classes within the RVM, it is unlikely that inhibition of on-cells would contribute to the decrease in TF latency, since administration of morphine, which suppresses the activity of on-cells [2,6], produces an increase, not a decrease, in TF latency, The source of the GABA input to the off-cell has not been identified. However, it may be intrinsic to the region; GABA-containing cell bodies are found in RVM [30,32] and, in the medullary slice preparation, local electrical stimulation evoked GABA-mediated synaptic potentials in RVM neurons [36]. If local GABA-containing neurons are responsible for the offcell pause, they would by definition have the properties
of on-cells in that they would show a burst of activity temporally correlated with the TF response. In summary, the results of the present study demonstrate that in the intact, lightly anesthetized rat, GABA-mediated inhibitory input to RVM neurons, most likely off-cells, is required for expression of spinally organized responses to noxious stimulation.
This work was supported by a grant from NIDA (DA05608). We wish to thank Beth Budra for histology and artwork, and Howard L. Fields, Michael M. Morgan and Kathleen Gogas for invaluable criticism of the manuscript.
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Pain, 47 (1991) 105-113 0 1991 Elsevier Science Publishers B.V. All rights reserved 0304-3959/91/$03.50 ADONIS 0304395991002038
PAIN 01868
GABA-mediated inhibition in rostra1 ventromedial medulla: role in nociceptive modulation in the lightly anesthetized rat Mary M. Heinricher
and Hilary J. Kaplan
Department of Neurology, Uniuersity of California at San Francisco, San Francisco, CA 94143 (U.S.A.)
(Received 1 October 1990, revision received 21 February 1991, accepted 28 February 1991)
Local microinjection of GABA, receptor agonists and antagonists was used to characterize the Summary role of GABA-mediated inhibitory processes in the nociceptive modulatory functions of the rostra1 ventromedial medulla (RVM) in the lightly anesthetized rat. Microinjection of selective GABA, receptor antagonists bicuculline methiodide and SR95531 produced a significant increase in tail-flick (TF) latency. This antinociception was dose related, showed recovery and was attenuated by prior injection of the GABA, receptor agonist THIP at the same site. Microinjection of saline or the glycine receptor antagonist strychnine did not significantly affect TF latency. In contrast, administration of GABA, receptor agonists THIP and muscimol resulted in a significant decrease in TF latency. Microinjections at sites surrounding the RVM did not significantly affect TF latency. These results demonstrate that a GABA-mediated process within the RVM is crucial in permitting execution of the TF and, presumably, other spinal nociceptive reflexes. Key words:
Rostra1 ventromedial modulation
medulla;
Nucleus
Introduction
An extensive body of evidence has implicated brain-stem systems in descending control of nociceptive input and in opiate analgesia [3,47,52]. Microinjection of opiates into, or electrical stimulation of, several brain-stem structures inhibits both nocifensor reflexes and neuronal responses to noxious stimuli. One region that has attracted intense interest is the rostra1 ventromedial medulla (RVM), which includes the nucleus raphe magnus, and the laterally adjacent nucleus reticularis gigantocellularis pars alpha and nucleus reticularis paragigantocellularis lateralis medial to the facial nucleus. Stimulation of the RVM electrically or by glutamate microinjection has antinociceptive effects [23,34,41,53] and also inhibits dorsal horn neurons [14,16,43,48]. Cell bodies and terminals showing opioid-like immunoreactivity are found in this region
Correspondence to: Mary M. Heinricher, Department of Neurology, Box 0114, University of California at San Francisco, San Francisco, CA 94143, U.S.A.
raphe
magnus; Tail flick; Antinociception;
GABA;
Pain
[5,29,46], and microinjection of morphine or opioid peptides into the RVM is also antinociceptive [1,10,22,23,25]. In addition, lesions of the RVM or local microinjection of naloxone can significantly reduce the antinociceptive effect of systemically administered morphine [1,39,511. These studies provide compelling evidence that the RVM has a key role in the modulation of nociception and, specifically, in mediating opiate analgesia. Although the functional importance of the RVM in modulation of nociception is well established, the local circuitry involved in control of RVM neurons is only now being elucidated. A substantial body of evidence suggests a significant role for GABA in modulation of nociception. Systemic administration of GABA, receptor agonists such as THIP and muscimol has been shown by several groups to inhibit responses to noxious stimulation [8,45]. However, GABA is distributed throughout the nervous system and participates in a wide variety of neuronal circuits [12,33], presumably including systems crucial to both transmission and modulation of nociception. Thus, direct application of GABA agonists and antagonists at discrete CNS sites
that arc involved in nociccptive tr~~~~srn~ssi~~~7 of its rn~~dul~~tio~~ is required. and ~nvestigat~~rs taking this ~~pprc~ach have demonstrated that microjnjection of GABA, receptor antagonists at several sites involved in n~~ciceptive modulation can inhibit spinal r~ocif~nsive reflexes and dorsal horn nociceptive neurons [9,11,X, 31.441. Moreover, unlike systemic administrations, microinjection of GABA, receptor agonists into brainstem sites involved in nociceptive modulation has no effect on or may even facilitate responses to noxious stimuli. Thus, THIP was reported to have no consistent dose-related effect when microinjected into the PAG I401 and resulted in a decrease in TF latency when applied in the RVM [ 1i]. We have recently shown that iontophoretic application of GABA, receptor antagonists eliminates a synaptically mediated inhibition of RVM neurons that is associated with occurrence of nocifensor reflexes [20]. An understanding of the functional role of GABA in this preparation would thus complement our knowledge of the physiology of RVM neurons obtained under essentially identical conditions and greatly strengthen the conclusions to be drawn from both classes of experiments. The current experjmental strategy involved identi~ing changes in nociceptive responsiveness produced by local injection of severaf GABA,% receptor antagonists (bicucul~ine methiodide (EM11 and SR955.11 ~~-tcarboxy-3’“pr~~pyl)-3-amin~~-4-~~~~ff-mcthoxy-phenyl”pyridazinium bromide)~ and of the GABA, receptor agonists muscimof and THIP (4,S,h,7-tetrahydroisoxazolo(S,4-c)pyridin-3-(~1~ into the RVM of the lightly anesthetized rat. The results demonstrate that GARA plays an important role in controlling nociceptive m~~du~ation within this region. A preiimina~ report of these findings has been presented [211.
Methods Animals Male Sprague-Dawiey rats weighing 250-275 g were initially anesthetized with pentobarbitai (60 mg/kg, i.p.) and a catheter placed in an external jugular vein. Each animal was then placed in a stereotaxic frame and a small craniectomy was performed to allow placement of an injection can&a within the RVM. .A 25-gauge stainless steel guide cannula was aimed towards the RVM and lowered slowly to a position 2 mm dorsal to the intended injection site. After cornp~~tio~ of these procedures, the animal was alIowed to recover from the initial anesthesia to the point at which the TF response could be evoked by application of noxious heat To the tail. The animal was subsequently maintained in a lightly anesthetized state by continuous infusion of methohexitai (1.5-30 mg/kg/h). The animals were monitored continuously throughout the
course of the experiments and showed no signs cti discomfort; they did not move spontane[~usly, vocatize or show vigorous withdrawal responses to intense pinch. Nocicepti~~e testing was initiated 35-45 min after stnrting the methohexit~l infusion. Body temperature was maintained at appr~~xini~~te~y37 o C using a circulating water blanket.
Drugs were delivered into the RVM in a volume of 11.5,ul using a 31”gauge cannula having a hevefled tip. This was connected to a l-p1 Hamilton syringe via a length of polyethylene tubing (PE 201, The injection cannula, which extended 3 mm beyond the end of the guide cannuia, was inserted into the RVM just prior to initiation of the behavioral testing protocol and was left in place for a minimum of 5 min after the injection was completed. Progress of the injection was monitored by observing the movement of a small air bubble through the tubing. The following drugs were injected: BMI (RI%, pH 5.3-5.51, SR95531 (RI%, pH 5.5-5.91, THIP HCI (RBI, pH 5.0-5.31, mnscimoi (Sigma, pH 565.91, strychnine HCI (Sigma, pH 5.91 and 0.9% saline vehicle (0.5 ylf. With the exception of experiments that involved pretreatment with agonist prior to antagonist microinjections. only one injection was made per animal.
The tail flick response was used as an index of nociceptive responsiveness [7f. Noxious heat was appiied to the blackened ventral surface of the tail using a feedback-controlled projection lamp. Three sites. 3, 5 and 7 cm from the distal tip of the tail, were used in rotation. A miniature thermistor probe placed in contact with the heated surface of the tai1 altowed precise measurement of tail temperature. The tail surface temperature at the stimulation site was maintained at 35 o C between TF trials which were initiated at 5 min intervals. The noxious heat stimulus consisted of a linear 10 set temperature ramp from the 35 o C holding temperature to a maximum of 53” C at 10 sec. The heat was automatically returned to 35’ C upon occurrence of the TF or if 10 set etapsed in the absence of a TF. Following determination of baseline TF iatency (mean of 3 TF triais), an injection was made into the RVM over a period of 3 min. TF latencies were determined at 5 min intervats for the period from 2 to 27 min after completing drug injection. In experiments involving pretreatment with THIP prior to administratioI1 of BMI, THIP (25-100 ngf was injected into the RVM immediately following determination of baseline latency. BMT (100 ng) was injected 10 min later (immediate& after the fifth TF trial) and TF latency monitored for the ensuing 27 min.
107
Histological uerification
At completion of the experiments, animals were sacrificed with an overdose of methohexital and perfused intracardially with physiological saline followed by 10% formalin. Microinjection sites were histologically verified and plotted on standardized sections derived from the atlas of Paxinos and Watson 1371.
3 :J ,
Statistical analysis
TF latencies following administration of GABA. receptor antagonists, strychnine and saline were compared to baseline latency using Friedman’s analysis of variance by ranks. A non-parametric test was required because many animals failed to perform to TF response within the 10 set cut-off time following administration of BMI or SR95531. In experiments involving pretreatment with THIP prior to administration of BMI, TF latencies following microinjection of BMI were compared to baseline latencies using a t test for correlated means. TF latencies following administration of GABA, receptor agonists THIP and muscimol were analyzed using analysis of variance with repeated measures within each dose group. In this case, Tukey’s test was employed for post hoc comparisons. A probability level of 0.05 was considered significant.
Results The mean baseline TF latency in these experiments was 4.8 rt 0.1 sec. Prior to drug injection, there were no significant differences in baseline latencies among the groups (ANOVA; P > 0.05; n = 128). Microinjection of GABA,
receptor antagonists
Microinjection of BMI (25, 50, 300 or 200 ng; 6-9 animals/group) into the RVM resuhed in a dose-reloo-
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Fig. 1. Time course and dose dependence of effects of BMI (25-200 ng) on TF latency. Each point represents the mean of determinations made in 6-9 rats. Mean and S.E.M. are shown for baseline latencies. Following microinjection, the number of animals in which TF was completely inhibited (10 set cut-off) is shown next to each point. Effects of saline microinjection are shown for comparison. A saline: n 25 ng BMI; 0 50 ng BMI; l 100 ng I3MI; 0 200 ng BMI.
s
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2
7
12
17
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TIME
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Fig. 2. Time coume of effects of microinjection of SR95531 (0.5-10 ng) on TF latency. Each line represents the mean of determinations made in 3 rats. Mean and S.E.M. are shown for baseline latencies. Following microinjection. the number of animals in which TF was completely inhibited (IO set cut-off) is shown next to each point. A 0.5 ng: W 1.0 ng; 0 2.5 ng; l 5.0 ng; o 10.0 ng SR95531.
lated antinociception (Fig. 1). The percentage of animals failing to respond within 10 set ranged from 28% following administration of 2.5 ng to 100% following 100 and 200 ng BMI (7 min after drug injection). Onset of the effect was quite rapid; at all doses tested, the TF latency was significantly increased within 2 min of completing the microinjection (i.e., the first test), with a peak antinociceptive effect attained by 7 min post injection. The duration of BMI-induced antinociception was also dose dependent. The increase in TF latency produced by microinjection of 25 ng BMI was reversed by 12 min after the microinjection, whereas recovery required 17 and 22 min following administration of 100 and 200 ng, respectively (Friedman’s analysis of variance by ranks). Microinjection of a second GABA, receptor antagonist, SR95531CO.5, 1,2.5,5 or 10 ng; 3 animals/group) also produced a profound antinociception when microinjected in RVM (Fig. 2). The percent of animals failing to respond within 10 set ranged from 33% following administration of 0.5 ng SR95531 to 100% following 2.5-10 ng SR95531 (7 min after drug injection). As with microinjection of BMI, the increase in TF latency following SR95531 administration was evident within 2 min of completing the microinjection. However, the antinociceptive effect of SR95531 was generally more prolonged than that of BMI, with TF suppression (10 set cut-off) often lasting throughout the 27 min post-injection observation period. Unlike the GABA, receptor antagonists, microinjection of the glycine receptor antagonist strychnine (75 ng) had no effect on TF latency when injected into the RVM (Friedman’s test; P < 0.20; n = 3). Similarly, microinjection of 0.5 ~1 saline vehicIe had no effect on TF latency (Friedman’s test; P < 0.20; n = 6). The TF inhibition produced by microinjection of BMI into the RVM was attenuated by prior injection
Fig. 4. Representative coronal sections of the medulla showing histologically verified sites at which SR95531 was microinjected. A 0.5 ng; U 1.Ong: 0 2.5 ng; A 5.1)ng; n 10.0 ng. VII, facial nerve and nucleus; P. pyramids. Fig. 3. Representative coronal sections of the medulla showing histologically verified sites at which BMI was microinjected, 28 sites within the RVM and 9 sites dorsolateral or caudal to the RVM. RVM (dotted outline) includes nucleus raphe magnus and the adjacent nucleus reticularis gigantocellularis pars alpha, as well as the rostra1 portion of nucleus reticularis paragigantocellularis lateralis, extending from the caudat poIe of the facial nucleus rostraliy to the level of the trapezoid body. o saline; CI 25 ng BMI; l 50 ng BMI; A 100 ng BMI; W 200 ng BMI. VII, facial nerve and nucleus: P, pyramids: AMB, nucleus ambiguus, IO, inferior olive.
of the GABA, agonist TI-IIP at the same site. Thus, following pretreatment with 100 ng THIP in RVM, microinjection of 100 ng BMI resulted in only a small increase in TF latency, from 4.0 i 0.2 set prior to the injections to 6.2 + 1.3 set measured 7 min after microinjection of BMI (t test for correlated means; P > 0.05;n = 6). This attenuation of the antinociceptive effect of BMI was not likely due to non-specific effects of the double injection procedure, as pretreatment with the lowest dose of THIP (25 ng) did not prevent the antinociception produced by BMI microinjection (n = 3). Microinjection of BMI at sites dorsal (n = 6) or caudal (n = 3) to the RVM did not significantly alter the TF response (Friedman’s analysis of variance by ranks; P > 0.05in each case). Fig. 3 illustrates the sites in the medulla where BMI was injected, 28 within and 9 outside of the RVM; Fig. 4 shows the distribution of 15 RVM sites where SR95.531 injections were carried out. Sites for injections of strychnine and pretreatment with THIP were comparable.
~icro~njection of GABA. receptor agonists Microinjection of the GABA, receptor agonists THIP and muscimol in the RVM resulted in significant decreases in TF latency. THIP was microinjected at 30 sites within RVM (12.5, 25, 50, 100, 300 or 1000 ng; 5 animals/group). Following drug administration, all groups except that receiving 300 ng THIP showed a significant decrease in TF latency (repeated measures ANOVA; Fig. 5). DecreaSed TF latencies were manifest within 2-7 min of the microinjection, with a maxi-
DOSE
/
SAL
THIP
(ng) MUSciMoL
I
Fig. 5. Effects of THIP and muscimol microinjections on TF latency. Results, expressed as percent of pretreatment .control (mean* S.E.M.) are shown for values determined 12 min after microinjection of saline, THIP or muscimol in RVM. (* P < 0.05; * * P < 0.01; repeated measures ANOVA).
109 -1.80
mum effect at 12 min post injection. This hyperalgesia was maintained throughout the 27 min post-injection observation period. The magnitude of hyperalgesia produced by THIP was not significantly related to the dose administered, although there was a tendency for increasing hyperalgesia with increasing dose to a maximum at 100 ng THIP. Administration of the highest dose WI00 ng) was associated with no greater, or possibly even a smaller, decrease in TF latency (TF latencies at 12 min post injection, Fig. 5). Muscimol was microinjected at 20 sites within the RVM (0.1, 1,5, 10 or 50 ng; 4 animals/group) and, like THIP, produced a significant decrease in TF latency (repeated measures ANOVA, Fig. 5). As with THIP microinjection, hyperalgesia produced by muscimol was evident within 2-7 min of completing the microinjection, was maximal at I2 min post injection and lasted throughout the 27 min post-injection period. Sites at which THIP and muscimol were microinjetted into the medulla are shown in Figs. 6 and 7, respectively. Microinjection of 100 ng THIP at 8 sites
Fig. 7. Representative coronal sections of the medulla showing histologicalIy verified sites at which muscimol was injected. A 0.1 ng; 0 1.0 ng; l 5.0 ng; A 10 ng; n 50 ng. VII, facial nerve and nucleus; P, pyramid.
dorsal or caudal to the RVM did not result in significant changes in TF latency (ANOVA, P > 0.05).
Discussion
Fig. 6. Representative coronal sections of the medulla showing histologically verified sites at which THIP was microinjected, 30 sites within RVM (dotted outline), and 8 sites dorsal or caudal to RVM. o 12.5 ng; A 25 ng; 0 50 ng; l 100 ng; A 300 ng; n 1000 ng. VII, facial nerve and nucleus; P, pyramids; AMB, nucleus ambiguus; IO, inferior olive.
The present studies demonstrate that a GABAmediated process plays a key role in the nociceptive modulating functions within the RVM; IocaI application of GABA, receptor antagonists in RVM induced a profound antinociception, whereas GABA, receptor agonists produced an enhancement of nociceptive responsiveness. Microinjection of BMI, a selective GABA, receptor antagonist, suppressed the TF response in a dose-related manner. Although it is impossible to conclude unequivocally that this effect was mediated by an antagonism of GABA-mediated neurotransmission, the observations that the antinociception was dose related, showed recovery and was attenuated by prior treatment with the GABA, agonist THIP injected at the same site provide evidence that this effect was not a non-specific effect of the microinjection procedure. Moreover, microinjection of SR9.5531, a recently developed pyridazinyl-GABA derivative having GABA, receptor antagonist properties [17,18,27], produced a similar dose-dependent suppression of the TF response, Finally, microinjection of the glycine receptor antago-
1 to
nist strychnine or saline vehicle was without effect. Taken together, these findings strongly suggest that BMI-induced antinociception involved blockade of GABA-mediated inhibition within the RVM. Administration of the GABA, receptor agonists THIP and muscimol resulted in a decrease in TF latency. This hyperalgesia was likely mediated by activation of a GABA, receptor, since muscimol is only a weak agonist and THIP is largely inactive at the GABA, receptor [4]. Moreover, microinjection of baclofen, the prototypical GABA, agonist, in the RVM is reported to have no effect on TF latency [25]. Micr~injections made into areas outside the RVM did not significantly affect TF latency. Thus, the low doses required and the rapid onset of the drug effect argue against the possibility that the antagonists diffused to a site of action outside of the RVM. As in the case of antagonist microinjections, localization of THIP and muscimol effects to the RVM is supported by the observation that microinjections at sites surrounding the RVM were ineffective. The present findings extend previous observations implicating GABA-mediated inhibitory processes in the control of nociceptive modulatory circuitry within the RVM [I 11. BMI microinjected into nucleus raphe magnus or nucleus reticularis gigantocellularis pars alpha in awake rats produced only small increases in TF latency that were not related to the dose administered. These authors therefore concluded that GABA neurotransmission did not make a substantial tonic contribution to control of nociceptive modulating neurons in the RVM. Their animals were, however, first tested 15 min after the microinjection, well after the time of peak effect of BMI injections seen in our experiments and reported by others [26,31,44]. Thus, although it is possible that GABA plays a less significant role in awake than in barbiturate-anesthetized animals (see below), it may be that more profound effects would have been evident if the awake animals had been tested within 2-10 min of the microinjection. The same authors also reported that microinjection of 300 or 1000 ng THIP, comparable to the highest doses used in the present experiments in anesthetized animals, enhanced nociceptive responsiveness as measured in the paw pinch and TF but not in hot-plate tests. Given our present observation that the highest doses of both THIP and muscimol did not produce the greatest decreases in TF latency, it would be interesting to determine whether lower doses of THIP affect hot-plate latencies in awake animals. As in the barbiturateanesthetized animals, in which a maximal decrease in TF latency developed by I2 min after the injection, the peak effect was manifest within 15 min of the agonist injection in awake animals. Thus, the time course of the THIP-induced hyperalgesia was similar in the 2 preparations.
GABA-mediated inhibition may also play a role in nociceptive modulation in other medullary regions, In rats anesthetized with alphaxalone/alphadolone, bilateral but not unilateral injections of 200 or 400 ng (total dose of 400 or 800 ng) BMI in the retrofaciai portion of nucleus reticularis paragigantocellularis generally cauda1 and lateral to the RVM as defined here, produced complete inhibition of the TF response beginning within I-10 min of the injection [26]. These results suggest an important role for GABA neurotransmission within the caudal ventrolateral medulla in nociceptive modulation. Although use of the lightly anesthetized rat has the advantage that it allows a direct comparison with electrophysiological observations made in the same preparation, the possibility that the present findings were influenced by barbiturate anesthesia must be considered. It is well known that barbiturates, including methohexitai, facilitate GABA-mediated inhibition [24,28.35,49]. Thus, a barbiturate-induced potentiation of endogeneous GABA transmission could have influenced the present results. That is. the effects of blocking GABA transmission by microinjection of antagonists might be more readily apparent in barbiturateanesthetized animals because there could be an clevation in basal levels of GABA-mediated inhibition within the RVM. Acting against this, however, could be a reduction in the effectiveness of the microinjected antagonists, since antagonist binding is reduced in the presence of barbiturates [27,51]. In any case, the relative significance of these two factors, increased inhibitory ‘tone’ and reduced antagonist binding, is not clear. Because blockade of GABAergic synaptic transmission by local application of competitive receptor antagonists within the RVM completely suppresses the TF response in lightly anesthetized animals, it is fikely that a GABA-mediated inhibition of some population of RVM neurons is crucial in permitting the execution of the TF and, presumably, other spinal nocifensive rcflexes. The observation that microinjection of GABA, receptor agonists, which would be expected to mimic such an obligatory GABAergic inhibitory process, results in a decrease in TF latency adds further weight to this inference. Although 3 populations of neurons have been identified in the RVM using electrophysiological techniques [13], only 1 class shows unequivocal evidence of a powerful inhibitory input that is temporally correlated with the TF response. These cells, called ‘off-cells,’ are characterized by an abrupt cessation in firing that occurs just prior to the execution of the TF. Several lines of evidence suggest that activation of off-cells inhibits responses to noxious stimulation and, conversely, that silence of cells of this class would permit such responses to occur. Thus, manipulations known to prevent the off-cell from pausing, for exam-
ple morphine given systemically or microinjected into the PAG [6,15], also suppress the TF response. In addition, periods of spontaneously occurring off-cell silence are associated with TF latencies that are more rapid than those obtained during a period in which off-cells are active [191. Cells of the other 2 physiologically characterized populations of RVM neurons respond very differently to nociceptive inputs and to manipulations producing antinociception. ‘on-cells’ display a sudden increase in activity just prior to the occurrence of the TF. This increase in on-cell activity presumably reflects a prepotent TF-related excitatory input to this cell class. Administration of morphine suppresses on-cell activity [2,6], and periods of on-cell silence are associated with relatively long TF latencies [19]. Cells of the third class, ‘neutral cells,’ are unaffected by either noxious stimulation or opiate administration [2,13]. These considerations would suggest that it is unlikely that GABAmediated inhibition of either on-cell or neutral cell activity would be required in order for the TF response to occur. However, the possibility that a fourth, as yet uncharacterized, neuron is involved cannot be excluded. Thus, the simplest explanation for the results of the present study would be that the GABA, receptor antagonists block a phasic, GABA-mediated inhibitory input responsible for the off-cell pause. Consistent with this, we have provided evidence that the TF-related off-cell pause can be blocked by iontophoretic application of BMI [20]. Blockade of additional, possibly tonic, inhibitor inputs to the off-cell could also contribute to the antinoci~eption produced by GABA antagonists. Conversely, microinje~tion of GABA, receptor agonists would silence the off-cell, permitting the TF to occur without hindrance from descending brain-stem modulatory circuitry. This is consistent with the fact that elimination of ull descending modulatory influences originating in the RVM (by means of electrolytic lesions or local anesthetic injection) can, under some conditions, produce a shortening of TF latency [38,39,421. Although GABA, receptor agonist microinjections could potentially suppress the activity of all cell classes within the RVM, it is unlikely that inhibition of on-cells would contribute to the decrease in TF latency, since administration of morphine, which suppresses the activity of on-cells [2,6], produces an increase, not a decrease, in TF latency, The source of the GABA input to the off-cell has not been identified. However, it may be intrinsic to the region; GABA-containing cell bodies are found in RVM [30,32] and, in the medullary slice preparation, local electrical stimulation evoked GABA-mediated synaptic potentials in RVM neurons [36]. If local GABA-containing neurons are responsible for the offcell pause, they would by definition have the properties
of on-cells in that they would show a burst of activity temporally correlated with the TF response. In summary, the results of the present study demonstrate that in the intact, lightly anesthetized rat, GABA-mediated inhibitory input to RVM neurons, most likely off-cells, is required for expression of spinally organized responses to noxious stimulation.
This work was supported by a grant from NIDA (DA05608). We wish to thank Beth Budra for histology and artwork, and Howard L. Fields, Michael M. Morgan and Kathleen Gogas for invaluable criticism of the manuscript.
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