Cannabinoid-induced Antinociception Research Articles

Sex‐Differences in Acute Cannabinoid Response and Tolerance as a Function of Mouse Strain

Cannabinoids have been increasingly used to alleviate chronic pain; however, tolerance to the antinociceptive effects of cannabinoids, including delta‐9‐tetrahydrocannabinol (Δ9‐THC), may limit their therapeutic utility. Likewise, with more women than men now using medical cannabis for pain relief, it is imperative that we understand how sex may influence cannabinoid‐mediated antinociception and subsequent tolerance. While studies in rats have consistently found female rats to be more sensitive to the acute antinociceptive effects of cannabinoids compared to male rats, work in our lab consistently finds the opposite finding that male mice are more sensitive to the acute antinociceptive effects of both Δ9‐THC and CP55,940 compared to female littermates. Studies in our lab have consistently utilized mice on a C57BL6/J (B6) background. Therefore, the purpose of the present study is to examine whether our observed sex‐differences in Δ9‐THC‐induced antinociception and tolerance are consistent across multiple mouse strains or are strain‐dependent. Male and female B6 and DBA mice were first assessed for differences in acute Δ9‐THC‐induced antinociception using the tail‐flick assay across a range of doses of (0‐100 mg/kg). After a significant washout period, these mice were subsequently assessed for sex‐differences in antinociceptive tolerance development to 30 mg/kg Δ9‐THC following once‐daily treatment for seven consecutive days. Consistent with our previous findings, male B6 mice were more sensitive to the acute antinociceptive effects of Δ9‐THC than female B6 mice. Male and female DBA, however, mice did not differ in their antinociceptive response to Δ9‐THC, suggesting that sex‐differences in cannabinoid‐induced antinociception in mice is likely strain‐specific. These studies highlight the therapeutic potential of Δ9‐THC in pain management and underscore the importance of considering sex when evaluating their clinical utility.

Neuropeptide VF Enhances Cannabinoid Agonist WIN55,212-2-Induced Antinociception in Mice.

Cannabinoids produce analgesia in several pain models, but the undesirable side effects from high doses of cannabinoid drugs limit their clinic use. Our recent results indicate that cannabinoid-induced antinociception was enhanced by neuropeptide VF (NPVF). Here, we investigate whether low-dose cannabinoid agonists combined with NPVF can produce effective antinociception with limited side effects. The in vivo properties of (R)-(-1)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN55,212-2) given alone and its combination with NPVF were evaluated in nociceptive modulation, locomotor activity, gastrointestinal transit, and tolerance development assays after intracerebroventricular administration in mice. In the radiant tail-flick test, the antinociception of combination of WIN55,212-2 and NPVF was more potent than that of cannabinoid agonist given alone, with an ED50 shift from 3.51 to 0.69 nmol; 9 nmol WIN55,212-2 alone and 3 nmol WIN55,212-2 combined with NPVF induced equivalent antinociception after supraspinal administration. The cannabinoid-potentiating effects of NPVF were reduced by both the cannabinoid receptor type 1 and the neuropeptide FF receptor antagonists. In the formalin assay, WIN55,212-2 combined with NPVF also significantly reduced pain-related behaviors. However, the combination of WIN55,212-2 with NPVF exerted significant hypoactivity in a manner similar to high doses of WIN55,212-2. It was important to note that the combination of WIN55,212-2 with NPVF produced nontolerance-forming antinociception and weaker inhibition of gastrointestinal transit compared with high dose of WIN55,212-2. These data suggest that the cannabinoid agonist combined with NPVF produces effective antinociception-lacking tolerance via both cannabinoid receptor type 1 and neuropeptide FF receptors in the brain.

Effects of neuropeptide FF and related peptides on the antinociceptive activities of VD-hemopressin(α) in naive and cannabinoid-tolerant mice

Neuropeptide FF (NPFF) system has recently been reported to modulate cannabinoid-induced antinociception. The aim of the present study was to further investigate the roles of NPFF system in the antinociceptive effects induced by intracerebroventricular (i.c.v.) administration of mouse VD-hemopressin(α), a novel endogenous agonist of cannabinoid CB1 receptor, in naive and VD-hemopressin(α)-tolerant mice. The effects of NPFF system on the antinociception induced by VD-hemopressin(α) were investigated in the radiant heat tail-flick test in naive mice and VD-hemopressin(α)-tolerant mice. The cannabinoid-tolerant mice were produced by given daily injections of VD-hemopressin(α) (20nmol, i.c.v.) for 5 days and the antinociception was measured on day 6. In naive mice, intracerebroventricular injection of NPFF dose-dependently attenuated central analgesia of VD-hemopressin(α). In contrast, neuropeptide VF (NPVF) and D.NP(N-Me)AFLFQPQRF-NH2 (dNPA), two highly selective agonists for Neuropeptide FF1 and Neuropeptide FF2 receptors, enhanced VD-hemopressin(α)-induced antinociception in a dose-dependent manner. In addition, the VD-hemopressin(α)-modulating activities of NPFF and related peptides were antagonized by the Neuropeptide FF receptors selective antagonist 1-adamantanecarbonyl-RF-NH2 (RF9). In VD-hemopressin(α)-tolerant mice, NPFF failed to modify VD-hemopressin(α)-induced antinociception. However, both neuropeptide VF and dNPA dose-dependently potentiated the antinociception of VD-hemopressin(α) and these cannabinoid-potentiating effects were reduced by RF9. The present works support the cannabinoid-modulating character of NPFF system in naive and cannabinoid-tolerant mice. In addition, the data suggest that a chronic cannabinoid treatment modifies the pharmacological profiles of NPFF, but not the cannabinoid-potentiating effects of neuropeptide VF and dNPA.

Repeated morphine treatment alters cannabinoid modulation of GABAergic synaptic transmission within the rat periaqueductal grey.

Cannabinoids and opioids produce antinociception by modulating GABAergic synaptic transmission in a descending analgesic pathway from the midbrain periaqueductal grey (PAG). While chronic opioid treatment produces opioid tolerance, it has recently been shown to enhance cannabinoid-induced antinociception within the PAG. This study examined the effect of repeated opioid treatment on opioid and cannabinoid presynaptic modulation of GABAergic synaptic transmission in PAG. Midbrain PAG slices were prepared from untreated rats, and rats that had undergone repeated morphine or saline pretreatment. Whole-cell voltage-clamp recordings were made from neurons within the ventrolateral PAG. In slices from untreated animals, the cannabinoid receptor agonist WIN55212 and the μ receptor agonist DAMGO inhibited electrically evoked GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) IPSCs in PAG neurons, with IC50 s of 30 and 100 nM respectively. The inhibition of evoked IPSCs produced by WIN55212 (30 nM) and DAMGO (100 nM) was similar in PAG neurons from morphine- and saline-treated animals. The cannabinoid CB1 receptor antagonist AM251 increased the frequency of spontaneous miniature IPSCs in PAG neurons from repeated morphine-, but not saline-treated animals. DAMGO inhibition of evoked IPSCs was enhanced in the presence of AM251 in morphine-, but not saline-treated animals. These results indicate that the efficiency of agonist-induced inhibition of GABAergic synaptic transmission is enhanced by morphine treatment, although this is dampened by endocannabinoid-mediated tonic inhibition. Thus, endocannabinoid modulation of synaptic transmission could provide an alternative analgesic approach in a morphine-tolerant state. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Blockade of D1/D2 dopamine receptors within the nucleus accumbens attenuated the antinociceptive effect of cannabinoid receptor agonist in the basolateral amygdala

Previous studies showed the role of basolateral amygdala (BLA) in cannabinoid-induced antinociception. Furthermore, the nucleus accumbens (NAc) plays an important role in mediating the suppression of pain in animal models. The present study extended the role of dopamine receptors within the NAc in antinociceptive effect of cannabinoid receptor agonist, WIN55,212-2, microinjected into the BLA following the tail-flick and formalin tests in rats. In this study, 174 adult male albino Wistar rats were unilaterally implanted by two separate cannulae into the BLA and NAc. In two separated groups, rats received intra-NAc infusions of the D1 receptor antagonist, SCH-23390 (0.25, 1 and 4μg/0.5μl saline) or D2 receptor antagonist, sulpiride (0.25, 1 and 4μg/0.5 DMSO), and just 2min later, WIN55,212-2 (15μg/rat) was microinjected into the BLA. In the tail-flick test, antinociceptive responses of drugs represented as maximal possible effect (%MPE) in 5, 15, 30, 45 and 60min after their administrations. Moreover, in the formalin test, pain related behaviors were monitored in 5-min blocks for 60min test period. Our findings showed that intra-accumbal SCH-233909 dose-dependently prevented antinociception induced by intra-BLA administration of WIN55,212-2 (15μg/rat) in time set intervals in formalin, but not tail-flick test. Besides, administration of sulpiride in the NAc could affect WIN-induced analgesia in both models of pain. In conclusion, it seems that D2 receptors located in the NAc, in part, mediate the antinociceptive responses of cannabinoid within the BLA, while D1 receptors only are involved in modulation of persistent inflammatory model of pain.

Intra-accumbal NMDA but not AMPA/kainate receptor antagonist attenuates WIN55,212-2 cannabinoid receptor agonist-induced antinociception in the basolateral amygdala in a rat model of acute pain

Previous studies showed the role of basolateral amygdala (BLA) in cannabinoid-induced antinociception. Several lines of evidence indicated that the nucleus accumbens (NAc) receives excitatory glutamatergic inputs primarily from limbic-related structures, including the hippocampus, BLA, and various thalamic nuclei. Additionally, it has been shown that the NAc plays an important role in mediating the suppression of animal models of pain. In the present study, we examined the role of NMDA and AMPA/kainate receptors within the NAc in antinociception induced by intra-BLA cannabinoid receptor agonist WIN55,212-2 in rats. 126 adult male albino Wistar rats weighing 230–280g were unilaterally implanted by two separate cannulae into the BLA and NAc. Dose–response antinociceptive effects of different doses of intra-BLA WIN55,212-2 (5, 10 and 15μg/0.3μl/rat) were evaluated in this study. Moreover, animals received intra-accumbal infusions of either NMDA receptor antagonist, AP5 (0.5, 2.5 and 5μg/0.5μl saline) or AMPA/kainate receptor antagonist, CNQX (0.1, 0.5 and 2.5μg/0.5μl DMSO), just 2min before microinjection of WIN55,212-2 into the BLA. Antinociceptive responses of drugs were obtained by tail-flick analgesiometer and represented as maximal possible effect (%MPE) at 5, 15, 30, 45 and 60min after their administrations. Results showed that intra-accumbal AP5 dose-dependently prevented antinociception induced by intra-BLA administration of WIN55,212-2 (15μg/rat) in time set intervals. Nonetheless, administration of AMPA/kainate receptor antagonist, CNQX, could not affect WIN-induced analgesia. Additionally, solely administration of intra-accumbal injection of CNQX (2.5μg/0.5μl DMSO), but not AP5 (5μg/0.5μl saline), could significantly change the baseline tail-flick latencies in the rats. It seems that NMDA receptors located in the NAc, in part, mediate the antinociceptive responses of cannabinoid within the BLA in acute model of pain.

Inhibitory CB1 and activating/desensitizing TRPV1-mediated cannabinoid actions on CGRP release in rodent skin

Cannabinoid-induced antinociception relies on activation of inhibitory cannabinoid receptors (CB1) in the peripheral and central nervous system. However, most cannabinoids at higher concentration also activate excitatory ionotropic transient receptor potential (TRP) channels coexpressed with CB1 in primary nociceptive neurons that contain and release calcitonin gene-related peptide (CGRP) upon activation. Over a wide concentration range (0.01-100μM) we investigated the molecular action principles of the endocannabinoid anandamide and of the plant-derived Δ(9)-THC that can be prescribed for analgesia. Isolated rat and mouse skin preparations were used to measure CGRP release induced by noxious heat (47°C) and capsaicin (0.5μM), stimuli known to activate the capsaicin receptor TRPV1. At low concentration (0.1μM) both cannabinoids inhibited stimulated CGRP release by 34-65%, which effects were absent under CB1 block by AM 251 and in global CB1 but not TRPV1 knockout mice. At high concentration (100μM) both cannabinoids evoked CGRP release by themselves and desensitized subsequent heat responses, which effects were absent under TRPV1 block by BCTC and in global TRPV1 but not CB1 knockouts. A lower (0.01μM) and the intermediate concentrations (1 and 10μM) of cannabinoids were ineffective. Excitatory and desensitizing effects were not more expressed (disinhibited) in CB1(-/-), inhibitory effects not stronger in TRPV1(-/-). CGRP release induced by unspecific depolarization (KCl) was not modulated by cannabinoids. An incidental finding was that global CB1(-/-) showed reduced heat sensitivity, almost as low as TRPV1(-/-) and in accord with their behavioral phenotype. In conclusion, the antinociceptive potency of peripherally acting CB1 agonists is not restrained by opposing irritant effects through TRPV1 but by their own limited efficacy and narrow concentration-response relationship.

Role of spinal phosphatases in cannabinoid-induced antinociception in a rodent neuropathic pain model

A cannabinoid receptor type 2 (CBR2) agonist administered intrathecally reduces peripheral nerve injury-induced mechanical hypersensitivity without exhibiting neurological side effects. Using in vitro rat cortical primary microglial cells, we previously observed that a CBR2 agonist induces the expression of MAP kinase phosphatase (MKP)-1 and 3 and reduces p-ERK, TNF expression and microglial migration. Herein, we investigated the hypothesis that CBR2 agonists produce analgesia by inducing spinal MKP expression and ERK dephosphorylation. We tested our hypothesis in vivo using male Sprague-Dawley rats and the L5 spinal nerve transection (L5NT) neuropathic pain model. Sham surgery was used as the control group. JWH015 (50 μg), a CBR2 agonist, was administered intrathecally by lumbar puncture four days after surgery. Mechanical withdrawal thresholds were determined with von Frey filaments, before surgery, four days after surgery and two hours after treatment. MKP-1 and pCREB expression was assessed using immunohistochemistry. Peripheral nerve injury-induced mechanical hypersensitivity, increased spinal pCREB and reduced spinal MKP-1 expression. JWH015 reversed nerve injury-induced pCREB expression and restored MKP-1 expression levels in association with its antinociceptive effects. To further investigate the functional association of MKPs with the antinociceptive effects of CBR2 agonists, we challenged the antinociceptive effects of JWH015 with triptolide (a MKP-1 and MKP-3 inhibitor). We observed that the intrathecal co-administration of triptolide (20 μg) with JWH015 blocked the antinociceptive effects of JWH015. These results suggest that CBR2 agonists may induce antinociception in neuropathic pain conditions via MKP induction. In conclusion, our results demonstrate that MKP modulation is part of the mechanism of action of CBR2 agonists. To further investigate the role of MKP in neuropathic pain and CBR2 agonist mechanisms of action we will determine the expression of spinal MKP-3 and the cellular localization of MKP-1 and MKP-3. (Supported by APS 2009 future leaders in pain research award and NIH 1R01DA025211.) This abstract will also be presented as an Oral Paper Presentation on May 8. Refer to the daily Schedule-At-A-Glance for presentation time and location.

Antinociceptive effect of the cannabinoid agonist, WIN 55,212-2, in the orofacial and temporomandibular formalin tests

Orofacial pain disorders are frequent in the general population and their pharmacological treatment is not always adequately resolved. Cannabinoids have demonstrated their analgesic effect in several pain conditions, both in animal models and in clinical situations. The aim of the present study was to evaluate the cannabinoid-mediated antinociception in two inflammatory models of orofacial pain (orofacial and temporomandibular joint (TMJ) formalin test) and to compare it with a spinal inflammatory model (paw formalin test). WIN 55,212-2 (0.5, 1 mg/kg), a synthetic cannabinoid agonist, was intraperitoneally (i.p.) administered prior to formalin and significantly reduced the nociceptive behavioural responses in these inflammatory tests. To elucidate which subtype of receptor could be involved in such effect, two selective cannabinoid antagonists were administered prior to WIN. SR141716A (1 mg/kg i.p.), the CB1 receptor-selective antagonist, was able to prevent the cannabinoid-induced analgesia in all three models, whereas SR144528 (1 mg/kg i.p.), the CB2 receptor-selective antagonist, only prevented it in the paw formalin test. A comparison with the antinociceptive effects of morphine (2.5, 5, 10 mg/kg, i.p.), indomethacin (2.5, 5 mg/kg, i.p.) and ketamine (25, 50 mg/kg, i.p.) was also performed. Morphine displayed a dose-dependent reduction of acute and inflammatory pain in all three models, whereas indomethacin and ketamine only attenuated inflammatory pain at the highest tested doses. These results indicate that the cannabinoid-induced antinociception in the orofacial region is mediated by activation of CB1 cannabinoid receptor. Moreover WIN was as effective as morphine and more effective than indomethacin and ketamine, in oral inflammatory pain.

The cannabinoid anticonvulsant effect on pentylenetetrazole-induced seizure is potentiated by ultra-low dose naltrexone in mice

Cannabinoid compounds are anticonvulsant since they have inhibitory effects at micromolar doses, which are mediated by activated receptors coupling to G(i/o) proteins. Surprisingly, both the analgesic and anticonvulsant effects of opioids are enhanced by ultra-low doses (nanomolar to picomolar) of the opioid antagonist naltrexone and as opioid and cannabinoid systems interact, it has been shown that ultra-low dose naltrexone also enhances cannabinoid-induced antinociception. Thus, concerning the seizure modulating properties of both classes of receptors this study investigated whether the ultra-low dose opioid antagonist naltrexone influences cannabinoid anticonvulsant effects. The clonic seizure threshold was tested in separate groups of male NMRI mice following injection of vehicle, the cannabinoid selective agonist arachidonyl-2-chloroethylamide (ACEA) and ultra-low doses of the opioid receptor antagonist naltrexone and a combination of ACEA and naltrexone doses in a model of clonic seizure induced by pentylenetetrazole (PTZ). Systemic injection of ultra-low doses of naltrexone (1pg/kg to 1ng/kg, i.p.) significantly potentiated the anticonvulsant effect of ACEA (1mg/kg, i.p.). Moreover, the very low dose of naltrexone (500pg/kg) unmasked a strong anticonvulsant effect for very low doses of ACEA (10 and 100microg/kg). A similar potentiation by naltrexone (500pg/kg) of anticonvulsant effects of non-effective dose of ACEA (1mg/kg) was also observed in the generalized tonic-clonic model of seizure. The present data indicate that the interaction between opioid and cannabinoid systems extends to ultra-low dose levels and ultra-low doses of opioid receptor antagonist in conjunction with very low doses of cannabinoids may provide a potent strategy to modulate seizure susceptibility.

Ultra-low dose cannabinoid antagonist AM251 enhances cannabinoid anticonvulsant effects in the pentylenetetrazole-induced seizure in mice

Several lines of evidence suggest that cannabinoid compounds are anticonvulsant since they have inhibitory effects at micromolar doses, which are mediated by activated receptors coupling to G i/o proteins. Surprisingly, both the analgesic and anticonvulsant effects of opioids are enhanced by ultra-low doses (nanomolar to picomolar) of the opioid antagonist naltrexone and as opioid and cannabinoid systems interact, it has been shown that ultra-low dose naltrexone also enhances cannabinoid-induced antinociception. However, regarding the seizure modulating properties of both classes of receptors this study investigated whether ultra-low dose cannabinoid antagonist AM251 influences cannabinoid anticonvulsant effects. The clonic seizure threshold (CST) was tested in separate groups of male NMRI mice following injection of vehicle, the cannabinoid selective agonist arachidonyl-2-chloroethylamide (ACEA) and ultra-low doses of the cannabinoid CB 1 antagonist AM251 and a combination of ACEA and AM251 doses in a model of clonic seizure induced by pentylenetetrazole (PTZ). Systemic administration of ultra-low doses of AM251 (10 fg/kg–100 ng/kg) significantly potentiated the anticonvulsant effect of ACEA at 0.5 and 1 mg/kg. Moreover, inhibition of cannabinoid induced excitatory signaling by AM251 (100 pg/kg) unmasked a strong anticonvulsant effect for very low doses of ACEA (100 ng/kg–100 μg/kg), suggesting that a presumed inhibitory component of cannabinoid receptor signaling can exert strong seizure-protective effects even at very low levels of cannabinoid receptor activation. A similar potentiation by AM251 (100 pg/kg and 1 ng/kg) of anticonvulsant effects of non-effective dose of ACEA (0.5 and 1 mg/kg) was also observed in the generalized tonic–clonic model of seizure. The present data suggest that ultra-low doses of cannabinoid receptor antagonists may provide a potent strategy to modulate seizure susceptibility, especially in conjunction with very low doses of cannabinoids.

Ultra-low dose naltrexone enhances cannabinoid-induced antinociception

Both opioids and cannabinoids have inhibitory effects at micromolar doses, which are mediated by activated receptors coupling to Gi/o-proteins. Surprisingly, the analgesic effects of opioids are enhanced by ultra-low doses (nanomolar to picomolar) of the opioid antagonist, naltrexone. As opioid and cannabinoid systems interact, this study investigated whether ultra-low dose naltrexone also influences cannabinoid-induced antinociception. Separate groups of Long-Evans rats were tested for antinociception following an injection of vehicle, a sub-maximal dose of the cannabinoid agonist WIN 55 212-2, naltrexone (an ultra-low or a high dose) or a combination of WIN 55 212-2 and naltrexone doses. Tail-flick latencies were recorded for 3 h, at 10-min intervals for the first hour, and at 15-min intervals thereafter. Ultra-low dose naltrexone elevated WIN 55 212-2-induced tail flick thresholds without extending its duration of action. This enhancement was replicated in animals receiving intraperitoneal or intravenous injections. A high dose of naltrexone had no effect on WIN 55 212-2-induced tail flick latencies, but a high dose of the cannabinoid 1 receptor antagonist SR 141716 blocked the elevated tail-flick thresholds produced by WIN 55 212-2+ultra-low dose naltrexone. These data suggest a mechanism of cannabinoid-opioid interaction whereby activated opioid receptors that couple to Gs-proteins may attenuate cannabinoid-induced antinociception and/or motor functioning.

Participation of the opioid system in cannabinoid-induced antinociception and emotional-like responses

Several anatomical, biochemical and pharmacological evidence support the existence of bidirectional interactions between cannabinoid and opioid systems. The present review is focused on the participation of the endogenous opioid system in the antinociceptive and emotional-like responses induced by cannabinoids, and the development of tolerance to cannabinoid pharmacological effects. Cannabinoid and opioid agonists produce antinociception by acting on similar structures within the central nervous system, and a peripheral mechanism has been also proposed for both compounds. Pharmacological studies have suggested that the endogenous opioid system could be involved in cannabinoid antinociception and the development of cannabinoid tolerance. Recent studies using knockout mice have also demonstrated the role of the opioid system in cannabinoid antinociception and tolerance, although some discrepancies with the previous pharmacological results have been reported when using knockout mice. On the other hand, cannabinoid administration can induce anxiolytic-like responses that are mediated at least in part by an endogenous opioid activity on μ- and δ-opioid receptors.

Pharmacologic interaction between cannabinoid and either clonidine or neostigmine in the rat formalin test.

Although spinal cannabinoid receptor agonist (WIN 55,212-2) has been shown to encounter various models of pain, the role of two subtypes of cannabinoid receptor for the antinociceptive effect of cannabinoids has not been investigated at the spinal level. Spinal alpha 2 receptor agonist (clonidine) and cholinesterase inhibitor (neostigmine) are also active in the modulation of nociception. The authors examined the properties of drug interaction after coadministration of WIN 55,212-2-clonidine, and intrathecal WIN 55,212-2-neostigmine, and further clarified the role of cannabinoid 1 and 2 receptors in cannabinoid-induced antinociception at the spinal level. Catheters were inserted into the intrathecal space of male Sprague-Dawley rats, and 50 microl of 5% formalin solution was injected into the hind paw to evoke the pain. Isobolographic analysis was used for evaluation of pharmacologic interaction. Intrathecal 55,212-2, clonidine, and neostigmine dose-dependently suppressed the flinching observed during phase 1 and 2 in the formalin test. Isobolographic analysis revealed a synergistic interaction after intrathecal delivery of WIN 55,212-2-clonidine or WIN 55,212-2-neostigmine mixture in both phases. The antinociceptive effect of WIN 55,212-2 was antagonized by cannabinoid 1 receptor antagonist (AM 251) but not by cannabinoid 2 receptor antagonist (AM 630). No antinociceptive effect was seen after intrathecal administration of cannabinoid 2 receptor agonist (JWH 133). Intrathecal 55,212-2, clonidine, and neostigmine attenuate the facilitated state and acute pain. WIN 55,212-2 interacts synergistically with either clonidine or neostigmine. The antinociception of WIN 55,212-2 is mediated through the cannabinoid 1 receptor, but not the cannabinoid 2 receptor, at the spinal level.

The rodent amygdala contributes to the production of cannabinoid-induced antinociception

The amygdala is a temporal lobe region that is implicated in emotional information processing. The amygdala also is associated with the processing and modulation of pain sensation. Recently, we demonstrated that in nonhuman primates, the amygdala is necessary for the full expression of cannabinoid-induced antinociception [J Neurosci 21 (2001) 8238]. The antinociceptive effect of the cannabinoid receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo(1,2,3-de)-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2) was significantly reduced in rhesus monkeys with large bilateral lesions of the amygdaloid complex. In the present study, we investigated the contribution of the amygdala to cannabinoid-induced antinociception in the rat. Using bilateral local microinjections of the GABAA receptor agonist muscimol, we inactivated neurons originating from the central nucleus of the amygdala (CeA) or basolateral nucleus of the amygdala (BLA). In rats injected with intra-CeA saline, the cannabinoid receptor agonist WIN55,212-2 produced dose-dependent antinociception on the noxious heat-evoked tail flick assay. In rats treated with intra-CeA muscimol, however, the antinociceptive effect of WIN55,212-2 was significantly reduced. Rats treated with intra-BLA muscimol showed no deficit in WIN55,212-2-induced antinociception. The effect of CeA inactivation on WIN55,212-2-induced suppression of prolonged pain in the formalin test also was tested. In rats treated with intra-CeA saline, WIN55,212-2 reduced the incidence of formalin-induced nociceptive behaviors and also reduced formalin-evoked c-fos expression in both superficial and deep laminae of the spinal cord dorsal horn. In rats treated with intra-CeA muscimol, however, these effects of WIN55,212-2 were significantly reduced.The results constitute the first causal data demonstrating the necessity of descending pain-modulatory circuitry (of which the CeA is a component) for the full expression of cannabinoid-induced antinociception in the rat. Furthermore, the results complement previous findings suggesting an overlap in neural circuitry activated by opioids and cannabinoids.

N-methyl-D-aspartate antagonists and WIN 55212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1-i,j]quinolin-6-one], a cannabinoid agonist, interact to produce synergistic hypothermia.

CB(1) cannabinoid receptors mediate profound hypothermia when cannabinoid agonists are administered to rats. Glutamate, the principal excitatory neurotransmitter in the central nervous system (CNS), is thought to tonically increase body temperature by activating N-methyl-D-aspartate (NMDA) receptors. Because NMDA antagonists block cannabinoid-induced antinociception and catalepsy, intimate glutamatergic-cannabinoid interactions may exist in the CNS. The present study investigated the effect of two NMDA antagonists on the hypothermic response to WIN 55212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1-i,j]quinolin-6-one], a selective cannabinoid agonist, in rats. WIN 55212-2 (1-10 mg/kg i.m.) produced dose-dependent hypothermia that peaked 60 to 180 min postinjection. Dextromethorphan (5-75 mg/kg i.m.), a noncompetitive NMDA antagonist, or LY 235959 [(-)-6-[phosphonomethyl-1,2,3,4,4a,5,6,7,8,8a-decahydro-isoquinoline-2-carboxylate]](1-4 mg/kg i.m.), a competitive and highly selective NMDA antagonist, evoked hypothermia in a dose-sensitive manner, suggesting that endogenous glutamate exerts a hyperthermic tone on body temperature. A dose of dextromethorphan (10 mg/kg) that did not affect body temperature by itself potentiated the hypothermic response to WIN 55212-2 (1, 2.5, or 5 mg/kg). The enhancement was strongly synergistic, indicated by a 2.7-fold increase in the relative potency of WIN 55212-2. Similarly, a dose of LY 235959 (1 mg/kg) that did not affect body temperature augmented the hypothermia associated with a single dose of WIN 55212-2 (2.5 mg/kg), thus confirming that NMDA receptors mediated the synergy. We have demonstrated previously that CB(1) receptors mediate WIN 55212-2-evoked hypothermia in rats. The present data are the first evidence that NMDA antagonists exert a potentiating effect on cannabinoid-induced hypothermia. Taken together, these data suggest that interactions between NMDA and CB(1) receptors produce synergistic hypothermia.

Reduction in Opioid- and Cannabinoid-Induced Antinociception in Rhesus Monkeys after Bilateral Lesions of the Amygdaloid Complex

The amygdaloid complex is a prominent temporal lobe region that is associated with "emotional" information processing. Studies in the rodent have also recently implicated the amygdala in the processing and modulation of pain sensation, the experience of which involves a considerable emotional component in humans. In the present study, we sought to establish the relevance of the amygdala to pain modulation in humans by investigating the contribution of this region to antinociceptive processes in nonhuman primates. Using magnetic resonance imaging guidance, the amygdaloid complex was lesioned bilaterally in six rhesus monkeys (Macaca mulatta) through microinjection of the neurotoxin ibotenic acid. This procedure resulted in substantial neuronal cell loss in all nuclear subdivisions of this structure. In awake unoperated control monkeys, systemic administration of the prototypical opioid morphine or the cannabinoid receptor agonist WIN55,212-2 produced dose-dependent antinociception on a warm-water tail-withdrawal assay. The antinociceptive effects of each drug were reversible with an appropriate antagonist. In monkeys with bilateral amygdala lesions, however, the antinociceptive effects of each drug were significantly reduced. These results constitute the first causal data demonstrating the necessity of neurons in a specific brain region for the full expression of opioid- and cannabinoid-induced antinociception in the primate. Because our amygdala-lesioned monkeys exhibited both a reduction in antinociception and a reduction in behavioral indices of fear (Emery et al., 2001), the possibility should be considered that, in the primate, "antinociceptive circuitry" and "fear circuitry" overlap at the level of the amygdala.

Metabotropic and NMDA glutamate receptors participate in the cannabinoid-induced antinociception

The purpose of this study was to evaluate the possible contribution of metabotropic glutamate receptors (mGluRs) to cannabinoid-induced antinociception in the periaqueductal grey (PAG) matter of rats. Intra-PAG microinjection of WIN 55,212-2, a cannabinoid receptor agonist, increased the latency of the nociceptive reaction (NR) in a dose-dependent fashion in the plantar test. This effect was prevented by pretreatment with SR141716A, a selective antagonist of CB1 receptors. When injected alone, SR141716A produced, with the highest dosage used, a significant reduction in the latency of the NR. CPCCOEt, a selective mGlu1 receptor antagonist, was unable to prevent the analgesia produced by WIN 55,212-2. On the contrary, MPEP, a selective mGlu5 receptor antagonist, completely antagonized the effect of WIN 55,212-2. However, the analgesia induced by CHPG, a selective mGlu5 receptor agonist, was blocked by MPEP but not by SR141716A. When injected alone, CPCOOEt produced no effect, whereas MPEP produced, with the highest dosage used, a significant reduction in the latency of the NR. These data emphasize that mGlu5 receptors, but not mGluR1, may modulate nociception in the PAG. Similarly, a pretreatment with either 2-( S)-α-EGlu or ( RS)-α-MSOP, selective antagonists for group II and III mGluRs, respectively, prevented the WIN 55,212-2-induced analgesia. When the higher dosage of ( RS)-α-MSOP was used a decrease in the latency of the NR was observed. This was not the case for 2-( S)-α-EGlu. Pretreatment with DL-AP5, a selective antagonist of N-methyl- d-aspartate (NMDA) receptors, blocked the effect of WIN 55,212-2, and by increasing the dosage strongly reduced per se the latency of the NR. This study suggests that endogenous glutamate could tonically modulate nociception through mGlu and NMDA receptors in the PAG matter. In particular, the physiological stimulation of these receptors seems to be required for the cannabinoid-induced analgesia in this midbrain area.

Dynorphin B and spinal analgesia: induction of antinociception by the cannabinoids CP55,940, Δ 9-THC and anandamide

The endogenous opioid dynorphin B was evaluated for its role in cannabinoid-induced antinociception. Previous work in our laboratory has shown that the synthetic, bicyclic cannabinoid, CP55,940, induces the release of dynorphin B whilst the naturally occurring cannabinoid, Δ 9-tetrahydrocannabinol (Δ 9-THC), releases dynorphin A. The dynorphins contribute in part to the antinociceptive effects of both cannabinoids at the level of the spinal cord. The present study compares dynorphin B released from perfused rat spinal cord in response to acute administration of anandamide (AEA), Δ 9-THC and CP55,940 at two time points, 10 min and 30 min post administration, and attempts to correlate such release with antinociceptive effects of the drugs. Dynorphin B was collected from spinal perfusates of rats pretreated with Δ 9-THC, CP55,940 or AEA. The supernatant was lyophilized and the concentrations of dynorphin B were measured via radioimmunoassay. At a peak time of antinociception (10 min), CP55,940 and Δ 9-THC induced significant two-fold increases in the release of dynorphin B. AEA did not significantly release dynorphin B. Upon a 30-min pretreatment with the drugs, no significant dynorphin B release was observed, although antinociceptive effects persisted for CP55,940 and Δ 9-THC. Previous work indicates that Δ 9-THC releases dynorphin A while AEA releases no dynorphin A. This study confirms that although all three test drugs produced significant antinociception at 10 min, the endocannabinoid, AEA, does not induce antinociception via dynorphin release. Thus, our data indicate a distinct mechanism which underlies AEA-induced antinociception.

Cannabinoid modulation of dynorphin A: correlation to cannabinoid-induced antinociception

Intrathecal administration of anandamide, Δ 9-tetrahydrocannabinol (THC) and (−)-3-[2-hydroxy-4-(1,1-dimethyheptyl)ptyl)phenyl]-4-(3-hydroxypropyl)-cicloexan-1-ol (CP55,940) induced spinal antinociception accompanied by differential κ-opioid receptor involvement and dynorphin A peptide release. Antinociception using the tail-flick test was induced by the classical cannabinoid THC and was blocked totally by 17,17′-bis(cyclopropylmethyl)-6′,6,7,7′-tetrahydro-4,5,4′5′-diepoxy-6,6′-(imino)[7,7′-bimorphinan]-3,3′,14,14′-tetrol (norbinaltorphimine) indicating a significant and critical κ-opioid receptor component. The endogenous cannabinoid, anandamide and the non-classical bicyclic cannabinoid, CP55,940, induced non-nor-BNI-sensitive effects. The N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716A)-mediated attenuation of spinal antinociception imparted by the various cannabinoids indicates cannabinoid CB 1 receptor involvement. THC-induced an enhancement of immunoreactive dynorphin A release which coincided with the onset, but not duration antinociception. The release of dynorphin A was also attenuated by SR141716A suggesting it is cannabinoid CB 1 receptor-mediated. These data indicate a critical role for dynorphin A release in the initiation of the antinociceptive effects of the cannabinoids at the spinal level.