- Email: [email protected]
Cannabinoid receptor-mediated inhibition of the rat tail-flick reflex after microinjection into the rostral ventromedial medulla
Neuroscience Letters 242 (1998) 33–36
Cannabinoid receptor-mediated inhibition of the rat tail-flick reflex after microinjection into the rostral ventromedial medulla William J. Martin1*, Kang Tsou, J. Michael Walker Departments of Psychology and Neuroscience, Brown University, Providence, RI 02912, USA Received 11 November 1997; received in revised form 22 December 1997; accepted 22 December 1997
Abstract Systemic administration of cannabinoids produce profound antinociception in rodents. The purpose of this study was to examine the contribution of the rostral ventromedial medulla (RVM) to cannabinoid-mediated inhibition of the tail-flick reflex. Rats received direct injections of two selective cannabinoid agonists, WIN55,212-2 and HU210, into the RVM. Both compounds significantly elevated tail-flick latencies by over 50%. WIN55,212-3, the inactive enantiomer, was without effect. Furthermore, coadministration of the selective cannabinoid receptor antagonist, SR141716A greatly attenuated the antinociception produced by HU210. Finally, injections of WIN55,212-2 outside the region of the RVM did not affect tail-flick latencies. These results demonstrate that the cannabinoid receptor system participates in the descending control of nociception and raise the possibility that actions of endogenous cannabinoids in the RVM may modulate nociceptive responsiveness. 1998 Elsevier Science Ireland Ltd.
Keywords: Cannabinoid receptors; Anandamide; Antinociception; Analgesia; Rat; HU-210; WIN55212-2; SR141716A
Recent evidence has suggested that one function of the central cannabinergic receptor system is to modulate pain sensitivity. Spinal administration of a cannabinoid receptor antagonist, SR141716A, evokes a significant thermal hyperalgesia in mice [16]. Systemic administration of both synthetic and endogenous cannabinoids produces profound antinociception [6,12], an effect likely due to their ability to inhibit the activity of nociceptive neurons in both the spinal cord and thalamus [8,13,19]. Nonetheless, the neural substrates through which cannabinoids modulate nociceptive processing have yet to be fully identified. Both spinal and supraspinal spinal sites contribute to cannabinoid receptor-mediated antinociception. Intrathecal administration of cannabinoids produces antinociception [20]. Moreover, spinal transection attenuates the antinociceptive effects of both systemically and intrathecally administered cannabinoids [10]. Supraspinal sites are also in* Corresponding author. Tel.: +1 415 4764311; fax: +1 415 4764845; e-mail: [email protected] 1 Present address: Department of Anatomy, University of California, Box 0452, San Francisco, CA 94143-0452, USA.
volved since intracerebroventricular [14] or direct intracerebral administration of cannabinoids into the periaqueductal gray (PAG) [15] produces antinociception. Neurons in the PAG project to the rostral ventromedial medulla (RVM) which, in turn, projects extensively to the dorsal horn of the spinal cord. Pharmacological or electrical activation of either the PAG or the RVM inhibits both behavioral responses to nociceptive stimuli as well as neuronal activity in the dorsal horn [4]. The demonstration that cannabinoids act in the PAG to inhibit nociceptive responses and that this site projects to the RVM raises the possibility that this area may be part of a cannabinergic pain modulatory circuit. Cannabinoid receptors are present in the RVM at densities that exceed those of surrounding areas [7]. In addition, the activity of the precursor enzyme for the synthesis of anandamide, an endogenous ligand for the cannabinoid receptor, is highest in the brainstem [1] lending support to the notion that cannabinoids may have antinociceptive actions in this area of the brainstem. The purpose of the current study was to test the hypothesis that cannabinoids can inhibit nociception via actions at brainstem cannabinoid receptors in the RVM.
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00044- 5
34
W.J. Martin et al. / Neuroscience Letters 242 (1998) 33–36
Guide cannulae (24 gauge thinwall stainless steel tubing, Small Parts, Miami, FL, USA) were implanted above the nucleus raphe magnus in male Sprague–Dawley rats weighing 275–325 g under pentobarbital (60 mg/kg) anesthesia using the following coordinates: AP = −2.6, LM = 0 from lambda, DV = −10.1 from the surface of the skull. Microinjections were made with a 31 ga stainless steel injection needle that extended 5.0 mm beyond the tip of the guide. Control off-site injections of WIN55,212-2 (5 mg/0.5 ml) were also made into the caudal aspect of the pontine reticular field. Two high affinity cannabinoid receptor agonists [3], WIN55,212-2 mesylate (RBI, Natick, MA, USA) and HU210 (a gift from Dr. Raphael Mechoulam), and the inactive enantiomer of WIN55,212-2, WIN55,212-3 (a gift from Dr. Dean Haycock), were dissolved in 60% dimethyl sulfoxide (DMSO) and delivered at a dose of 5 mg/0.5 ml over 72 s. SR141716A (a gift from Sanofi Research), a potent cannabinoid receptor antagonist [17], was dissolved in 100% DMSO and delivered either alone, at a dose of 50 mg/0.5 ml, or in combination with HU210 at an equivalent dose. Three to seven days after surgery, the antinociceptive effects of WIN55,212-2 and HU210 were examined using the tail-flick test. The latency to withdraw the tail from a radiant heat source was recorded every 3 min. A 10 s cut-off was used and baseline tail-flick latencies (2.5–4.5 s) were recorded for 15 min. WIN55,212-2, HU210 or a vehicle solution of 60% DMSO was microinjected into the RVM and tail-flick latencies were recorded for at least 30 min. Receptor specificity was tested by injecting either the inactive enantiomer of WIN55,212-2, WIN55,212-3, or by coadministration of SR171614A with HU210. Each animal received only one injection, was tested only once and the number of animals in each group was kept to a minimum. All experiments conformed to the guidelines on the study of pain in awake animals established by the International Association for the Study of Pain and were approved by the Brown University Institutional Animal Care and Use Committee. For verification of the microinjections, animals were injected with pentobarbital (100 mg/kg) and perfused transcardially with 0.9% saline and 10% formalin. Brains were removed, stored overnight in a 30% sucrose-formalin solution. Forty-micrometer frozen sections were cut on a cryostat and stained with cresyl violet. The location of each injection was determined, and only animals with verified injection sites were included in the data analysis. Antinociception was calculated according to the following equation: % maximum possible effect (MPE) = 100 × (test latency − control latency)/(10 − control latency), where 10 represents the cut-off latency and the control latency equals the average of three baseline tests prior to injection. A repeated measures analysis of variance (ANOVA) was used to compare the %MPE of animals from the five groups over time. Where appropriate, Dun-
nett’s test was used to make post-hoc comparisons between treatment groups. A total of 45 rats were included in this study. Placement of microinjections in the RVM was confirmed for 40 animals. Five additional animals served as off-site controls. Injections of vehicle or WIN55,212-3, the inactive enantiomer of WIN55,212-2, had no effect on tail-flick latencies (Fig. 1). By contrast, microinjections of both WIN55,212-2 and HU210 into the RVM led to a greater than 50% decrease in nociceptive responding. ANOVA revealed a significant effect of treatment among these groups (F(5,34) = 4.83; P = 0.0019). SR141716A had no effect on tail-flick latencies when given alone. However, co-administration of SR141716A significantly attenuated the HU210-induced antinociception over the course of the 30 min test session to a level not statistically different from vehicle-treated animals. Fig. 2 illustrates the onset and duration of cannabinoid receptor-mediated antinociception. Microinjections of both WIN55,212-2 and HU210 into the RVM produced a suppression of the tail-flick reflex within 3 min after injection. WIN55,212-2 produced its peak effect of 56 ± 13% at 3 min and a mean antinociceptive effect of 35% over the 30 min. HU210 also produced a rapid elevation of tail-flick latencies, with peak antinociception occurring 15 min post injection (70 ± 18%). For HU210, we recorded a mean antinociception of 52 ± 5.5% over the 30 min test session. Although the effects of HU210 appeared to be more potent than those of WIN55212-2, the magnitude and duration of antinociception produced by these two agonists were not statistically different from one another. When WIN55, 212-2 was microinjected dorsal to the RVM, there was no
Fig. 1. Summary of the mean ± SEM antinociception produced over the 30 min after microinjection of WIN55,212-2 (n = 10) and HU210 (n = 6) into the RVM. The antinociceptive effects of both compounds were significantly different from vehicle (n = 8). In addition, coadministration of HU210 with the cannabinoid receptor antagonist, SR141716A, greatly attenuated the HU210-induced elevation in tailflick latencies (n = 5). WIN55,212-3 (inactive enantiomer) and SR141716A (administered alone) were not statistically different from vehicle. ***P , 0.0001.
W.J. Martin et al. / Neuroscience Letters 242 (1998) 33–36
Fig. 2. Time course of antinociceptive actions of 5 mg injections of (A) WIN55,212-2 and (B) HU210 directly into the RVM (mean ± SEM). This illustrates the rapid onset and duration of antinociception produced by these compounds. Vehicle injections were without effect. Receptor specificity of this effect is suggested by the lack of effect of the inactive enantiomer and the attenuation of the HU210-induced antinociception when co-administered with 50 mg of SR141716A, a cannabinoid receptor antagonist.
significant change in tail-flick latencies over the 30 min test session (6.7 ± 3.8%, n = 5). The purpose of this study was to examine the contribution of brainstem modulatory circuits to supraspinal cannabinoid receptor-mediated antinociception. Microinjections of WIN55,212-2 and HU210 into the RVM produced potent, long-lasting antinociception in the rat tail-flick reflex. WIN55,212-3, the inactive enantiomer of WIN55,212-2, had no effect on tail-flick latencies. SR141716A, the cannabinoid receptor antagonist, significantly attenuated the increase in tail-flick latency produced by HU210. Together, these results suggest that the observed antinociceptive effects of cannabinoids after microinjection into the RVM were mediated by central cannabinoid receptors. Since both WIN55,212-2 and HU210 exhibited a rapid (within 3 min) onset of the elevation of tail-flick latencies, it is likely that actions within, or close to, the RVM account for the observed analgesia. The delay in peak antinocicep-
35
tion, however, suggests that some degree of diffusion may be required to enable the agonists to act on a sufficient number of brainstem cannabinoid receptors. Nonetheless, the finding that injections in the brainstem, but outside the RVM, had no significant effect on tail-flick latencies illustrates the limited anatomical substrate of cannabinoidinduced antinociception in the brainstem. The mechanism by which cannabinoids activate neurons in the RVM to produce antinociception is not currently known. The activity of two types of neurons in the RVM is believed critical for pain modulation [5]. A thermal stimulus sufficient to evoke a tail-flick withdrawal reflex activates ‘on-cells’ and produces a pause in ‘off-cell’ firing just prior to a tail-flick. Systemic opioid administration inhibits the tail-flick during which time on-cell firing is inhibited and off-cell activity is increased. Since the effects of opioids are inhibitory, opioid-induced antinociception is believed to derive, in part, from a disinhibition of off-cell activity via actions at on-cells. The direct cellular actions of cannabinoids are also generally inhibitory. Cannabinoid agonists inhibit adenylyl cyclase activity via an inhibitory G-protein [9]. Furthermore, WIN55,212-2 inhibits voltage-gated Ca2 + channels and activates K + channels [2,11], actions consistent with presynaptic inhibition. Shen et al. [18] demonstrated cannabinoid receptor-mediated inhibition of glutamate release from hippocampal cultures. One possibility is that cannabinoids directly inhibit on-cell firing to prevent a facilitating action of these neurons on nociceptive transmission. Inhibition of on-cells should also lead to a disinhibition of offcells and a subsequent reduction in nociceptive transmission [5]. The RVM represents one of the most important brain areas for the descending control of spinal cord activity and, ultimately, the modulation of nociceptive transmission. The finding that cannabinoid actions in the RVM are sufficient to inhibit nociceptive responses suggests that one mechanism by which systemically administered cannabinoids produce analgesia is by activating descending pain inhibitory pathways. This work was supported by National Institute on Drug Abuse (DA10043). W.J.M. was supported by graduate fellowships from the National Science Foundation and the National Institute on Drug Abuse (F31DA05617). J.M.W. is grateful for the financial support provided by the Public Health Service/National Institutes of Health (KO2MH01083, NS33247). [1] Cadas, H., di Tomaso, E. and Piomelli, D., Occurrence and biosynthesis of endogenous cannabinoid precursor, N-arachidonoyl phosphatidylethanolamine in rat brain, J. Neurosci., 17 (1997) 1226–1242. [2] Deadwyler, S.A., Hampson, R.E., Mu, J., Whyte, A. and Childers, S., Cannabinoids modulate voltage sensitive potassium A-current in hippocampal neurons via a cAMP-dependent process, J. Pharmacol. Exp. Ther., 273 (1995) 734–743.
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[3] Felder, C.C., Joyce, K.E., Briley, E.M., Mansouri, J., Mackie, K., Blond, O., Lai, Y., Ma, A.L. and Mitchell, R.L., Comparison of the pharmacology and signal transduction of the human cannabinoid CB1 and CB2 receptors, Mol. Pharmacol., 48 (1995) 443–450. [4] Fields, H.L., Basbaum, A.I., Clanton, C.H. and Anderson, S.D., Nucleus raphe magnus inhibition of spinal cord dorsal horn neurons, Brain Res., 126 (1977) 441–453. [5] Fields, H.L., Heinricher, M.M. and Mason, P., Neurotransmitters in nociceptive modulatory circuits, Annu. Rev. Neurosci., 14 (1991) 219–245. [6] Fride, E. and Mechoulam, R., Pharmacological activity of the cannabinoid receptor agonist, anandamide, a brain constituent, Eur. J. Pharmacol., 231 (1993) 313–314. [7] Herkenham, M., Lynn, A.B., Johnson, M.R., Melvin, L.S., de Costa, B.R. and Rice, K.C., Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study, J. Neurosci., 11 (1991) 563–583. [8] Hohmann, A.G., Martin, W.J., Tsou, K. and Walker, J.M., Inhibition of noxious stimulus-evoked activity of spinal cord dorsal horn neurons by the cannabinoid WIN 55,212–2, Life Sci., 56 (1995) 2111–2118. [9] Howlett, A.C. and Fleming, R.M., Cannabinoid inhibition of adenylate cyclase. Pharmacology of the response in neuroblastoma cell membranes, Mol. Pharmacol., 26 (1984) 532–538. [10] Lichtman, A.H. and Martin, B.R., Spinal and supraspinal components of cannabinoid-induced antinociception, J. Pharmacol. Exp. Ther., 258 (1991) 517–523. [11] Mackie, K. and Hille, B., Cannabinoids inhibit N-type calcium channels in neuroblastoma-glioma cells, Proc. Natl. Acad. Sci. USA, 89 (1992) 3825–3829. [12] Martin, B.R., Cellular effects of cannabinoids, Pharmacol. Rev., 38 (1986) 45–74.
[13] Martin, W.J., Hohmann, A.G. and Walker, J.M., Suppression of noxious stimulus-evoked activity in the ventral posterolateral nucleus of the thalamus by a cannabinoid agonist: correlation between electrophysiological and antinociceptive effects, J. Neurosci., 16 (1996) 6601–6611. [14] Martin, W.J., Lai, N.K., Patrick, S.L., Tsou, K. and Walker, J.M., Antinociceptive actions of cannabinoids following intraventricular administration in rats, Brain Res., 629 (1993) 300–304. [15] Martin, W.J., Patrick, S.L., Coffin, P.O., Tsou, K. and Walker, J.M., An examination of the central sites of action of cannabinoid-induced antinociception in the rat, Life Sci., 56 (1995) 2103–2109. [16] Richardson, J.D., Aanonsen, L. and Hargreaves, K.M., SR 141716A, a cannabinoid receptor antagonist, produces hyperalgesia in untreated mice, Eur. J. Pharmacol., 319 (1997) R3– R4. [17] Rinaldi-Carmona, M., Barth, F., Heaulme, M., Shire, D., Calandra, B., Congy, C., Martinez, S., Marvani, J., Neliat, G., Caput, D., Ferrara, P., Sovbrie, P., Breliere, J.C. and Le Fur, G., SR141716A, a potent and selective antagonist of the brain cannabinoid receptor, FEBS Lett., 350 (1994) 240–244. [18] Shen, M., Piser, T.M., Seybold, V.S. and Thayer, S.A., Cannabinoid receptor agonists inhibit glutamatergic synaptic transmission in rat hippocampal cultures, J. Neurosci., 16 (1996) 4322– 4334. [19] Tsou, K., Lowitz, K.A., Hohmann, A.G., Martin, W.J., Hathaway, C.B., Bereiter, D.A. and Walker, J.M., Supression of noxious stimulus-evoked expression of fos protein-like immunoreactivity in rat spinal cord by a selective cannabinoid agonist, Neuroscience, 70 (1996) 791–798. [20] Yaksh, T.L., The antinociceptive effects of intrathecally administered levonantradol and desacetyllevonantradol in the rat, J. Clin. Pharmacol., 21 (1981) 334S–340S.
Cannabinoid receptor-mediated inhibition of the rat tail-flick reflex after microinjection into the rostral ventromedial medulla William J. Martin1*, Kang Tsou, J. Michael Walker Departments of Psychology and Neuroscience, Brown University, Providence, RI 02912, USA Received 11 November 1997; received in revised form 22 December 1997; accepted 22 December 1997
Abstract Systemic administration of cannabinoids produce profound antinociception in rodents. The purpose of this study was to examine the contribution of the rostral ventromedial medulla (RVM) to cannabinoid-mediated inhibition of the tail-flick reflex. Rats received direct injections of two selective cannabinoid agonists, WIN55,212-2 and HU210, into the RVM. Both compounds significantly elevated tail-flick latencies by over 50%. WIN55,212-3, the inactive enantiomer, was without effect. Furthermore, coadministration of the selective cannabinoid receptor antagonist, SR141716A greatly attenuated the antinociception produced by HU210. Finally, injections of WIN55,212-2 outside the region of the RVM did not affect tail-flick latencies. These results demonstrate that the cannabinoid receptor system participates in the descending control of nociception and raise the possibility that actions of endogenous cannabinoids in the RVM may modulate nociceptive responsiveness. 1998 Elsevier Science Ireland Ltd.
Keywords: Cannabinoid receptors; Anandamide; Antinociception; Analgesia; Rat; HU-210; WIN55212-2; SR141716A
Recent evidence has suggested that one function of the central cannabinergic receptor system is to modulate pain sensitivity. Spinal administration of a cannabinoid receptor antagonist, SR141716A, evokes a significant thermal hyperalgesia in mice [16]. Systemic administration of both synthetic and endogenous cannabinoids produces profound antinociception [6,12], an effect likely due to their ability to inhibit the activity of nociceptive neurons in both the spinal cord and thalamus [8,13,19]. Nonetheless, the neural substrates through which cannabinoids modulate nociceptive processing have yet to be fully identified. Both spinal and supraspinal spinal sites contribute to cannabinoid receptor-mediated antinociception. Intrathecal administration of cannabinoids produces antinociception [20]. Moreover, spinal transection attenuates the antinociceptive effects of both systemically and intrathecally administered cannabinoids [10]. Supraspinal sites are also in* Corresponding author. Tel.: +1 415 4764311; fax: +1 415 4764845; e-mail: [email protected] 1 Present address: Department of Anatomy, University of California, Box 0452, San Francisco, CA 94143-0452, USA.
volved since intracerebroventricular [14] or direct intracerebral administration of cannabinoids into the periaqueductal gray (PAG) [15] produces antinociception. Neurons in the PAG project to the rostral ventromedial medulla (RVM) which, in turn, projects extensively to the dorsal horn of the spinal cord. Pharmacological or electrical activation of either the PAG or the RVM inhibits both behavioral responses to nociceptive stimuli as well as neuronal activity in the dorsal horn [4]. The demonstration that cannabinoids act in the PAG to inhibit nociceptive responses and that this site projects to the RVM raises the possibility that this area may be part of a cannabinergic pain modulatory circuit. Cannabinoid receptors are present in the RVM at densities that exceed those of surrounding areas [7]. In addition, the activity of the precursor enzyme for the synthesis of anandamide, an endogenous ligand for the cannabinoid receptor, is highest in the brainstem [1] lending support to the notion that cannabinoids may have antinociceptive actions in this area of the brainstem. The purpose of the current study was to test the hypothesis that cannabinoids can inhibit nociception via actions at brainstem cannabinoid receptors in the RVM.
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00044- 5
34
W.J. Martin et al. / Neuroscience Letters 242 (1998) 33–36
Guide cannulae (24 gauge thinwall stainless steel tubing, Small Parts, Miami, FL, USA) were implanted above the nucleus raphe magnus in male Sprague–Dawley rats weighing 275–325 g under pentobarbital (60 mg/kg) anesthesia using the following coordinates: AP = −2.6, LM = 0 from lambda, DV = −10.1 from the surface of the skull. Microinjections were made with a 31 ga stainless steel injection needle that extended 5.0 mm beyond the tip of the guide. Control off-site injections of WIN55,212-2 (5 mg/0.5 ml) were also made into the caudal aspect of the pontine reticular field. Two high affinity cannabinoid receptor agonists [3], WIN55,212-2 mesylate (RBI, Natick, MA, USA) and HU210 (a gift from Dr. Raphael Mechoulam), and the inactive enantiomer of WIN55,212-2, WIN55,212-3 (a gift from Dr. Dean Haycock), were dissolved in 60% dimethyl sulfoxide (DMSO) and delivered at a dose of 5 mg/0.5 ml over 72 s. SR141716A (a gift from Sanofi Research), a potent cannabinoid receptor antagonist [17], was dissolved in 100% DMSO and delivered either alone, at a dose of 50 mg/0.5 ml, or in combination with HU210 at an equivalent dose. Three to seven days after surgery, the antinociceptive effects of WIN55,212-2 and HU210 were examined using the tail-flick test. The latency to withdraw the tail from a radiant heat source was recorded every 3 min. A 10 s cut-off was used and baseline tail-flick latencies (2.5–4.5 s) were recorded for 15 min. WIN55,212-2, HU210 or a vehicle solution of 60% DMSO was microinjected into the RVM and tail-flick latencies were recorded for at least 30 min. Receptor specificity was tested by injecting either the inactive enantiomer of WIN55,212-2, WIN55,212-3, or by coadministration of SR171614A with HU210. Each animal received only one injection, was tested only once and the number of animals in each group was kept to a minimum. All experiments conformed to the guidelines on the study of pain in awake animals established by the International Association for the Study of Pain and were approved by the Brown University Institutional Animal Care and Use Committee. For verification of the microinjections, animals were injected with pentobarbital (100 mg/kg) and perfused transcardially with 0.9% saline and 10% formalin. Brains were removed, stored overnight in a 30% sucrose-formalin solution. Forty-micrometer frozen sections were cut on a cryostat and stained with cresyl violet. The location of each injection was determined, and only animals with verified injection sites were included in the data analysis. Antinociception was calculated according to the following equation: % maximum possible effect (MPE) = 100 × (test latency − control latency)/(10 − control latency), where 10 represents the cut-off latency and the control latency equals the average of three baseline tests prior to injection. A repeated measures analysis of variance (ANOVA) was used to compare the %MPE of animals from the five groups over time. Where appropriate, Dun-
nett’s test was used to make post-hoc comparisons between treatment groups. A total of 45 rats were included in this study. Placement of microinjections in the RVM was confirmed for 40 animals. Five additional animals served as off-site controls. Injections of vehicle or WIN55,212-3, the inactive enantiomer of WIN55,212-2, had no effect on tail-flick latencies (Fig. 1). By contrast, microinjections of both WIN55,212-2 and HU210 into the RVM led to a greater than 50% decrease in nociceptive responding. ANOVA revealed a significant effect of treatment among these groups (F(5,34) = 4.83; P = 0.0019). SR141716A had no effect on tail-flick latencies when given alone. However, co-administration of SR141716A significantly attenuated the HU210-induced antinociception over the course of the 30 min test session to a level not statistically different from vehicle-treated animals. Fig. 2 illustrates the onset and duration of cannabinoid receptor-mediated antinociception. Microinjections of both WIN55,212-2 and HU210 into the RVM produced a suppression of the tail-flick reflex within 3 min after injection. WIN55,212-2 produced its peak effect of 56 ± 13% at 3 min and a mean antinociceptive effect of 35% over the 30 min. HU210 also produced a rapid elevation of tail-flick latencies, with peak antinociception occurring 15 min post injection (70 ± 18%). For HU210, we recorded a mean antinociception of 52 ± 5.5% over the 30 min test session. Although the effects of HU210 appeared to be more potent than those of WIN55212-2, the magnitude and duration of antinociception produced by these two agonists were not statistically different from one another. When WIN55, 212-2 was microinjected dorsal to the RVM, there was no
Fig. 1. Summary of the mean ± SEM antinociception produced over the 30 min after microinjection of WIN55,212-2 (n = 10) and HU210 (n = 6) into the RVM. The antinociceptive effects of both compounds were significantly different from vehicle (n = 8). In addition, coadministration of HU210 with the cannabinoid receptor antagonist, SR141716A, greatly attenuated the HU210-induced elevation in tailflick latencies (n = 5). WIN55,212-3 (inactive enantiomer) and SR141716A (administered alone) were not statistically different from vehicle. ***P , 0.0001.
W.J. Martin et al. / Neuroscience Letters 242 (1998) 33–36
Fig. 2. Time course of antinociceptive actions of 5 mg injections of (A) WIN55,212-2 and (B) HU210 directly into the RVM (mean ± SEM). This illustrates the rapid onset and duration of antinociception produced by these compounds. Vehicle injections were without effect. Receptor specificity of this effect is suggested by the lack of effect of the inactive enantiomer and the attenuation of the HU210-induced antinociception when co-administered with 50 mg of SR141716A, a cannabinoid receptor antagonist.
significant change in tail-flick latencies over the 30 min test session (6.7 ± 3.8%, n = 5). The purpose of this study was to examine the contribution of brainstem modulatory circuits to supraspinal cannabinoid receptor-mediated antinociception. Microinjections of WIN55,212-2 and HU210 into the RVM produced potent, long-lasting antinociception in the rat tail-flick reflex. WIN55,212-3, the inactive enantiomer of WIN55,212-2, had no effect on tail-flick latencies. SR141716A, the cannabinoid receptor antagonist, significantly attenuated the increase in tail-flick latency produced by HU210. Together, these results suggest that the observed antinociceptive effects of cannabinoids after microinjection into the RVM were mediated by central cannabinoid receptors. Since both WIN55,212-2 and HU210 exhibited a rapid (within 3 min) onset of the elevation of tail-flick latencies, it is likely that actions within, or close to, the RVM account for the observed analgesia. The delay in peak antinocicep-
35
tion, however, suggests that some degree of diffusion may be required to enable the agonists to act on a sufficient number of brainstem cannabinoid receptors. Nonetheless, the finding that injections in the brainstem, but outside the RVM, had no significant effect on tail-flick latencies illustrates the limited anatomical substrate of cannabinoidinduced antinociception in the brainstem. The mechanism by which cannabinoids activate neurons in the RVM to produce antinociception is not currently known. The activity of two types of neurons in the RVM is believed critical for pain modulation [5]. A thermal stimulus sufficient to evoke a tail-flick withdrawal reflex activates ‘on-cells’ and produces a pause in ‘off-cell’ firing just prior to a tail-flick. Systemic opioid administration inhibits the tail-flick during which time on-cell firing is inhibited and off-cell activity is increased. Since the effects of opioids are inhibitory, opioid-induced antinociception is believed to derive, in part, from a disinhibition of off-cell activity via actions at on-cells. The direct cellular actions of cannabinoids are also generally inhibitory. Cannabinoid agonists inhibit adenylyl cyclase activity via an inhibitory G-protein [9]. Furthermore, WIN55,212-2 inhibits voltage-gated Ca2 + channels and activates K + channels [2,11], actions consistent with presynaptic inhibition. Shen et al. [18] demonstrated cannabinoid receptor-mediated inhibition of glutamate release from hippocampal cultures. One possibility is that cannabinoids directly inhibit on-cell firing to prevent a facilitating action of these neurons on nociceptive transmission. Inhibition of on-cells should also lead to a disinhibition of offcells and a subsequent reduction in nociceptive transmission [5]. The RVM represents one of the most important brain areas for the descending control of spinal cord activity and, ultimately, the modulation of nociceptive transmission. The finding that cannabinoid actions in the RVM are sufficient to inhibit nociceptive responses suggests that one mechanism by which systemically administered cannabinoids produce analgesia is by activating descending pain inhibitory pathways. This work was supported by National Institute on Drug Abuse (DA10043). W.J.M. was supported by graduate fellowships from the National Science Foundation and the National Institute on Drug Abuse (F31DA05617). J.M.W. is grateful for the financial support provided by the Public Health Service/National Institutes of Health (KO2MH01083, NS33247). [1] Cadas, H., di Tomaso, E. and Piomelli, D., Occurrence and biosynthesis of endogenous cannabinoid precursor, N-arachidonoyl phosphatidylethanolamine in rat brain, J. Neurosci., 17 (1997) 1226–1242. [2] Deadwyler, S.A., Hampson, R.E., Mu, J., Whyte, A. and Childers, S., Cannabinoids modulate voltage sensitive potassium A-current in hippocampal neurons via a cAMP-dependent process, J. Pharmacol. Exp. Ther., 273 (1995) 734–743.
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