Cannabinoid-induced Analgesia Research Articles

Identification of the TRPA1 cannabinoid-binding site

Chronic pain accounts for nearly two-thirds of conditions eligible for medical cannabis licenses, yet the mechanisms underlying cannabis-induced analgesia remain poorly understood. The principal phytocannabinoids, the psychoactive Δ9-tetrahydrocannabinol (THC) and non-psychoactive cannabidiol (CBD), exhibit comparable efficacy in pain management. Notably, THC functions as an agonist of cannabinoid receptor 1 (CB1), whereas CBD shows minimal activity on CB1 and CB2 receptors. Elucidating the molecular targets through which phytocannabinoids modulate the pain system is required for advancing our understanding of the pain pathway and optimizing medical cannabis therapies. Transient receptor potential ankyrin 1 (TRPA1), a pivotal chemosensor in the pain pathway, has been identified as a phytocannabinoid target. Unlike most TRPA1 activators, phytocannabinoid activation is not mediated through the electrophilic binding site, suggesting an alternative mechanism. Here, we identified the human TRPA1 channel cannabinoid-binding site (CBS) and demonstrated that mutations at residue Y840 abolished responses to both THC and CBD at saturating concentrations, indicating a shared primary binding site. Molecular modeling revealed distinct interactions of THC and CBD with the Y840 residue within the CBS. Additionally, CBD binds to the adjacent general anesthetic binding site at oversaturating concentrations. Our findings define the CBS of TRPA1 as overlapping with and adjacent to binding sites for other allosteric activators, suggesting that TRPA1 possesses a highly adaptable domain for binding non-electrophilic activators. This underscores its unique role as a chemosensor in the pain pathway. Furthermore, our results provide new insights into the molecular mechanisms of cannabinoid-induced analgesia and identify novel targets for pain management therapies.

Transcriptomic Profiling in Mice With CB1 receptor Deletion in Primary Sensory Neurons Suggests New Analgesic Targets for Neuropathic Pain.

Type 1 and type 2 cannabinoid receptors (CB1 and CB2, respectively) mediate cannabinoid-induced analgesia. Loss of endogenous CB1 is associated with hyperalgesia. However, the downstream targets affected by ablation of CB1 in primary sensory neurons remain unknown. In the present study, we hypothesized that conditional knockout of CB1 in primary sensory neurons (CB1cKO) alters downstream gene expression in the dorsal root ganglion (DRG) and that targeting these pathways alleviates neuropathic pain. We found that CB1cKO in primary sensory neurons induced by tamoxifen in adult Advillin-Cre:CB1-floxed mice showed persistent hyperalgesia. Transcriptome/RNA sequencing analysis of the DRG indicated that differentially expressed genes were enriched in energy regulation and complement and coagulation cascades at the early phase of CB1cKO, whereas pain regulation and nerve conduction pathways were affected at the late phase of CB1cKO. Chronic constriction injury in mice induced neuropathic pain and changed transcriptome expression in the DRG of CB1cKO mice, and differentially expressed genes were mainly associated with inflammatory and immune-related pathways. Nerve injury caused a much larger increase in CB2 expression in the DRG in CB1cKO than in wildtype mice. Interfering with downstream target genes of CB1, such as antagonizing CB2, inhibited activation of astrocytes, reduced neuroinflammation, and alleviated neuropathic pain. Our results demonstrate that CB1 in primary sensory neurons functions as an endogenous analgesic mediator. CB2 expression is regulated by CB1 and may be targeted for the treatment of neuropathic pain.

Systematic review and meta-analysis of cannabinoids, cannabis-based medicines, and endocannabinoid system modulators tested for antinociceptive effects in animal models of injury-related or pathological persistent pain.

We report a systematic review and meta-analysis of studies that assessed the antinociceptive efficacy of cannabinoids, cannabis-based medicines, and endocannabinoid system modulators on pain-associated behavioural outcomes in animal models of pathological or injury-related persistent pain. In April 2019, we systematically searched 3 online databases and used crowd science and machine learning to identify studies for inclusion. We calculated a standardised mean difference effect size for each comparison and performed a random-effects meta-analysis. We assessed the impact of study design characteristics and reporting of mitigations to reduce the risk of bias. We meta-analysed 374 studies in which 171 interventions were assessed for antinociceptive efficacy in rodent models of pathological or injury-related pain. Most experiments were conducted in male animals (86%). Antinociceptive efficacy was most frequently measured by attenuation of hypersensitivity to evoked limb withdrawal. Selective cannabinoid type 1, cannabinoid type 2, nonselective cannabinoid receptor agonists (including delta-9-tetrahydrocannabinol) and peroxisome proliferator-activated receptor-alpha agonists (predominantly palmitoylethanolamide) significantly attenuated pain-associated behaviours in a broad range of inflammatory and neuropathic pain models. Fatty acid amide hydrolase inhibitors, monoacylglycerol lipase inhibitors, and cannabidiol significantly attenuated pain-associated behaviours in neuropathic pain models but yielded mixed results in inflammatory pain models. The reporting of criteria to reduce the risk of bias was low; therefore, the studies have an unclear risk of bias. The value of future studies could be enhanced by improving the reporting of methodological criteria, the clinical relevance of the models, and behavioural assessments. Notwithstanding, the evidence supports the hypothesis of cannabinoid-induced analgesia.

Cholesterol regulates cannabinoid analgesia through glycine receptors

Cholesterol plays vital roles in many central physiological and pathological processes. As a key component in the cell membrane, cholesterol can regulate a variety of ion channels, including ligand-gated ion channels (LGICs). However, relatively little is known about the molecular detail and in vivo consequence of cholesterol-LGIC interaction. Here, we reveal that membrane cholesterol depletion significantly inhibits the potentiating effects of dehydroxylcannabidiol (DH-CBD) on glycine-activated currents (IGly) in HEK 293T cells expressing α1/α3 glycine receptors (GlyRs). Simvastatin considerably decreases cholesterol levels and DH-CBD-induced potentiation of IGly in the spinal cord of mice. Simvastatin also significantly decreases DH-CBD analgesia in acute and chronic pain of mice. The cholesterol levels in the dorsal horn of spinal cord, measured by mass spectrometry imaging, are specifically correlated with cannabinoid potentiation of spinal GlyRs and cannabinoid-induced analgesia. These findings suggest that spinal cholesterol is critical for the efficacy of glycinergic cannabinoid-induced analgesia.

Cannabinoids and Pain: New Insights From Old Molecules.

Cannabis has been used for medicinal purposes for thousands of years. The prohibition of cannabis in the middle of the 20th century has arrested cannabis research. In recent years there is a growing debate about the use of cannabis for medical purposes. The term ‘medical cannabis’ refers to physician-recommended use of the cannabis plant and its components, called cannabinoids, to treat disease or improve symptoms. Chronic pain is the most commonly cited reason for using medical cannabis. Cannabinoids act via cannabinoid receptors, but they also affect the activities of many other receptors, ion channels and enzymes. Preclinical studies in animals using both pharmacological and genetic approaches have increased our understanding of the mechanisms of cannabinoid-induced analgesia and provided therapeutical strategies for treating pain in humans. The mechanisms of the analgesic effect of cannabinoids include inhibition of the release of neurotransmitters and neuropeptides from presynaptic nerve endings, modulation of postsynaptic neuron excitability, activation of descending inhibitory pain pathways, and reduction of neural inflammation. Recent meta-analyses of clinical trials that have examined the use of medical cannabis in chronic pain present a moderate amount of evidence that cannabis/cannabinoids exhibit analgesic activity, especially in neuropathic pain. The main limitations of these studies are short treatment duration, small numbers of patients, heterogeneous patient populations, examination of different cannabinoids, different doses, the use of different efficacy endpoints, as well as modest observable effects. Adverse effects in the short-term medical use of cannabis are generally mild to moderate, well tolerated and transient. However, there are scant data regarding the long-term safety of medical cannabis use. Larger well-designed studies of longer duration are mandatory to determine the long-term efficacy and long-term safety of cannabis/cannabinoids and to provide definitive answers to physicians and patients regarding the risk and benefits of its use in the treatment of pain. In conclusion, the evidence from current research supports the use of medical cannabis in the treatment of chronic pain in adults. Careful follow-up and monitoring of patients using cannabis/cannabinoids are mandatory.

Involvement of glycine receptor α1 subunits in cannabinoid-induced analgesia

Some cannabinoids have been shown to suppress chronic pain by targeting glycine receptors (GlyRs). Although cannabinoid potentiation of α3 GlyRs is thought to contribute to cannabinoid-induced analgesia, the role of cannabinoid potentiation of α1 GlyRs in cannabinoid suppression of chronic pain remains unclear. Here we report that dehydroxylcannabidiol (DH-CBD), a nonpsychoactive cannabinoid, significantly suppresses chronic inflammatory pain caused by noxious heat stimulation. This effect may involve spinal α1 GlyRs since the expression level of α1 subunits in the spinal cord is positively correlated with CFA-induced inflammatory pain and the GlyRs antagonist strychnine blocks the DH-CBD-induced analgesia. A point-mutation of S296A in TM3 of α1 GlyRs significantly inhibits DH-CBD potentiation of glycine currents (IGly) in HEK-293 cells and neurons in lamina I-II of spinal cord slices. To explore the in vivo consequence of DH-CBD potentiation of α1 GlyRs, we generated a GlyRα1S296A knock-in mouse line. We observed that DH-CBD-induced potentiation of IGly and analgesia for inflammatory pain was absent in GlyRα1S296A knock-in mice. These findings suggest that spinal α1 GlyR is a potential target for cannabinoid analgesia in chronic inflammatory pain.

The critical role of spinal 5-HT7 receptors in opioid and non-opioid type stress-induced analgesia

The opioid and non-opioid types of stress-induced analgesia have been well defined. One of the non-opioid type involve the endocannabinoid system. We previously reported that the spinal serotonin 7 receptor (5-HT7) blockers inhibit both morphine and cannabinoid-induced analgesia, thus we hypothesized that descending serotonergic pathways-spinal 5-HT7 receptor loop might contribute to stress-induced analgesia.Stress-induced analgesia was induced with warm (32°C) or cold (20°C) water swim stress in male Balb-C mice. The effects of intrathecal injection of a selective 5-HT7 receptor antagonist, SB 269970, of the denervation of serotonergic neurons by intrathecal administration of 5,7-dihydroxytryptamine (5,7-DHT) and of lesions of the dorsolateral funiculus on opioid and non-opioid type stress-induced analgesia were evaluated with the tail-flick and hot plate tests. The expression of 5-HT7 receptors mRNA in the dorsal lumbar region of spinal cord were analyzed by RT-PCR following spinal serotonin depletion or dorsolateral funiculus lesion. The effects of the selective 5-HT7 receptor agonists LP 44 and AS 19 were tested on nociception.Intrathecal SB 269970 blocked both opioid and non-opioid type stress-induced analgesia. Dorsolateral funiculus lesion or denervation of the spinal serotonergic neurons resulted in a marked decrease in 5-HT7 receptor expression in the dorsal lumbar spinal cord, accompanied by inhibition of opioid and non-opioid type stress-induced analgesia. However, the systemic or intrathecal LP 44 and AS 19 alone did not produce analgesia in unstressed mice.These results indicate that descending serotonergic pathways and the spinal 5-HT7 receptor loop play a crucial role in mediating both opioid and non-opioid type stress-induced analgesia.

Positive Modulators of Glycine Receptors with Analgesic Potential Identified by Virtual Screening

Human glycine receptors (hGlyRs) are important targets for neuroactive drugs, including for analgesic therapies. A previous study has identified the crucial role of residue S296 in hGlyR-α1 potentiation by Δ9-tetrahydrocannabinol (THC) and in cannabinoid-induced analgesia. The recently determined NMR structure of the hGlyR-α1 transmembrane domain (TMD) provides a basis for structure-based virtual screening to discover new analgesic drugs. Using a large ensemble of hGlyR-α1 structures generated from molecular dynamics simulations based on the NMR structure, we screened 1549 FDA approved compounds from the DrugBank database targeted to a cannabinoid binding site near residue S296. Drugs were ranked based on their predicted binding affinities across the ensemble of hGlyR-α1 TMD structures. Four leading compounds were selected for experimental validation in Xenopus laevis oocytes expressing hGlyR-α1. At a low concentration (1 μM), all four leading compounds potentiate hGlyR-α1 currents more than two times, which was greater than that of THC. The hit rate is remarkable. The study provides strong evidence that these leading compounds will be at least as effective as THC for analgesia by acting on hGlyR-α1, but without psychoactive effects. The protocol developed here can easily be applied to the discovery of novel analgesic compounds of even higher efficacy on hGlyRs. Research supported by grants from the NIH and XSEDE.

Peripheral Pain-Sensing Neurons: from Molecular Diversity to Functional Specialization

Recent work in Cell Reports by Minett et al. and Yang et al. highlights the complexity of pain pathways and points to new ways of thinking about chronic and disease-related pain.

Cannabinoid potentiation of glycine receptors contributes to cannabis-induced analgesia

Cannabinoids enhance the function of glycine receptors (GlyRs). However, little is known about the mechanisms and behavioral implication of cannabinoid-GlyR interaction. Using mutagenesis and NMR analysis, we have identified a serine at 296 in the GlyR protein critical for the potentiation of I(Gly) by Δ(9)-tetrahydrocannabinol (THC), a major psychoactive component of marijuana. The polarity of the amino acid residue at 296 and the hydroxyl groups of THC are critical for THC potentiation. Removal of the hydroxyl groups of THC results in a compound that does not affect I(Gly) when applied alone but selectively antagonizes cannabinoid-induced potentiating effect on I(Gly) and analgesic effect in a tail-flick test in mice. The cannabinoid-induced analgesia is absent in mice lacking α3GlyRs but not in those lacking CB1 and CB2 receptors. These findings reveal a new mechanism underlying cannabinoid potentiation of GlyRs, which could contribute to some of the cannabis-induced analgesic and therapeutic effects.

Desensitization of Transient Receptor Potential Ankyrin 1 (TRPA1) by the TRP Vanilloid 1-Selective Cannabinoid Arachidonoyl-2 Chloroethanolamine

Recent studies on cannabinoid-induced analgesia implicate certain transient receptor potential (TRP) channels as a therapeutic target along with metabotropic cannabinoid receptors. Although TRP ankyrin 1 (TRPA1)-selective cannabinoids, such as (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone (WIN55,212), are effective at desensitizing TRPA1 and TRP vanilloid 1 (TRPV1), there is a gap in knowledge in understanding the opposite situation, namely whether TRPV1-selective cannabinoids desensitize TRPA1. We selected the TRPV1-specific synthetic cannabinoid, arachidonoyl-2 chloroethanolamine (ACEA), to study peripheral antihyperalgesic properties because ACEA is known to activate TRPV1. Hence, we used in vitro as well as in vivo assays to evaluate the following: 1) the effects of ACEA on the TRPA1-selective agonist, mustard oil (MO), for calcitonin gene-related peptide (CGRP) release from rat hindpaw skin in vitro; 2) the effects of a peripherally selective dose of ACEA on MO-induced nocifensive behavior in vivo; and 3) the effects of five ACEA-insensitive TRPV1 mutations on ACEA-inhibition of MO-evoked calcium accumulation using a Chinese hamster ovary cell expression system. Our results demonstrate that 1) ACEA significantly attenuated (∼40%) MO-evoked CGRP release from rat hindpaw skin, and this effect was not antagonized by the TRPV1 antagonist, capsazepine; 2) ACEA significantly inhibited (∼40%) MO-induced nocifensive behavior in wild-type mice but not in TRPV1 knockout mice; and 3) all TRPV1 mutations insensitive to ACEA lacked the ability to inhibit MO-evoked calcium accumulation in Chinese hamster ovary cells transfected with TRPV1 and TRPA1. Taken together, the results indicate that a TRPV1-selective cannabinoid, ACEA, inhibits MO-evoked responses via a TRPV1-dependent mechanism. This study strengthens the hypothesis that cannabinoids mediate their peripheral analgesic properties, at least in part, via the TRP channels.

Potentiation by WIN 55,212‐2 of GABA‐activated currents in rat trigeminal ganglion neurones

Although both natural and synthetic cannabinoid compounds have been shown to exert an antinociceptive effect on acute and persistent pain, the anatomical locus of the target of cannabinoid-induced analgesia has not been fully elucidated. Here, we investigated the effects of the cannabinoid agonist WIN 55,212-2 on GABA-activated currents (I(GABA)) in rat primary sensory neurones. In the present study, experiments were performed on neurones freshly isolated from rat trigeminal ganglion (TG) by using whole-cell patch clamp and repatch techniques. GABA-evoked inward currents were potentiated by pretreatment with WIN 55,212-2 in a concentration-dependent manner (10(-10)-10(-8) M). WIN 55,212-2 shifted the GABA concentration-response curve upwards, with an increase of 30.3 +/- 3.7% in the maximal current response but with no significant change in the EC(50) (agonist concentration producing a half-maximal response) value. WIN 55,212-2 potentiated the responses to GABA in a manner independent of holding potential and in the absence of any change in the reversal potential of the current. This potentiation of I(GABA) induced by WIN 55,212-2 was almost completely blocked by AM 251 (3 x 10(-8) M), a CB(1) receptor antagonist, and, using the repatch technique, was found to be abolished after intracellular dialysis with the protein kinase A (PKA) activator cAMP or the PKA inhibitor H89. The potentiation by WIN 55,212-2 of I(GABA) in primary sensory neurones may help to elucidate the mechanism underlying the modulation of analgesia by cannabinoids in the spinal dorsal horn.

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.

Involvement of female sex hormones in the cannabinoid-induced catalepsy and analgesia in ovarectomized mice

Materials and methods Female swiss mice (weighing 25-30 g) were divided into 3 main groups: unoperated, sham-operated and OVX. Both the catalepsy and analgesia induced by different doses of synthetic cannabinoid WIN 55,212-2 (2 and 4 mg/kg, i.p.) were examined in three groups in the presence or absence of the CB1 antagonist AM251. We also evaluated effects of estradiol and progesterone on catalepsy and analgesia induced by WIN 55,212-2 in OVX group.

Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors

Although endocannabinoids constitute one of the first lines of defense against pain, the anatomical locus and the precise receptor mechanisms underlying cannabinergic modulation of pain are uncertain. Clinical exploitation of the system is severely hindered by the cognitive deficits, memory impairment, motor disturbances and psychotropic effects resulting from the central actions of cannabinoids. We deleted the type 1 cannabinoid receptor (CB1) specifically in nociceptive neurons localized in the peripheral nervous system of mice, preserving its expression in the CNS, and analyzed these genetically modified mice in preclinical models of inflammatory and neuropathic pain. The nociceptor-specific loss of CB1 substantially reduced the analgesia produced by local and systemic, but not intrathecal, delivery of cannabinoids. We conclude that the contribution of CB1-type receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia is paramount, which should enable the development of peripherally acting CB1 analgesic agonists without any central side effects.

A Molecular Basis of Analgesic Tolerance to Cannabinoids

Clinical usage of cannabinoids in chronic pain states is limited by their central side effects and the pharmacodynamic tolerance that sets in after repeated dosage. Analgesic tolerance to cannabinoids in vivo could be caused by agonist-induced downregulation and intracellular trafficking of cannabinoid receptors, but little is known about the molecular mechanisms involved. We show here that the type 1 cannabinoid receptor (CB1) interacts physically with G-protein-associated sorting protein 1 (GASP1), a protein that sorts receptors in lysosomal compartments destined for degradation. CB1-GASP1 interaction was observed to be required for agonist-induced downregulation of CB1 in spinal neurons ex vivo as well as in vivo. Importantly, uncoupling CB1 from GASP1 in mice in vivo abrogated tolerance toward cannabinoid-induced analgesia. These results suggest that GASP1 is a key regulator of the fate of CB1 after agonist exposure in the nervous system and critically determines analgesic tolerance to cannabinoids.

Cannabinoids Ameliorate Pain and Reduce Disease Pathology in Cerulein-Induced Acute Pancreatitis

The functional involvement of the endocannabinoid system in modulation of pancreatic inflammation, such as acute pancreatitis, has not been studied to date. Moreover, the therapeutic potential of cannabinoids in pancreatitis has not been addressed. We quantified endocannabinoid levels and expression of cannabinoid receptors 1 and 2 (CB1 and CB2) in pancreas biopsies from patients and mice with acute pancreatitis. Functional studies were performed in mice using pharmacological interventions. Histological examination, serological, and molecular analyses (lipase, myeloperoxidase, cytokines, and chemokines) were performed to assess disease pathology and inflammation. Pain resulting from pancreatitis was studied as abdominal hypersensitivity to punctate von Frey stimuli. Behavioral analyses in the open-field, light-dark, and catalepsy tests were performed to judge cannabinoid-induced central side effects. Patients with acute pancreatitis showed an up-regulation of cannabinoid receptors and elevated levels of endocannabinoids in the pancreas. HU210, a synthetic agonist at CB1 and CB2, abolished abdominal pain associated with pancreatitis and also reduced inflammation and decreased tissue pathology in mice without producing central, adverse effects. Antagonists at CB1- and CB2-receptors were effective in reversing HU210-induced antinociception, whereas a combination of CB1- and CB2-antagonists was required to block the anti-inflammatory effects of HU210 in pancreatitis. In humans, acute pancreatitis is associated with up-regulation of ligands as well as receptors of the endocannabinoid system in the pancreas. Furthermore, our results suggest a therapeutic potential for cannabinoids in abolishing pain associated with acute pancreatitis and in partially reducing inflammation and disease pathology in the absence of adverse side effects.

Regional differences in naloxone modulation of Δ 9-THC induced Fos expression in rat brain

Recent behavioral and pharmacological research shows extensive interplay between cannabinoid and opioid neurochemical systems. Here we examined the neuroanatomical basis of this interaction using c- fos immunohistochemistry. We compared Fos immunoreactivity in groups of male albino Wistar rats treated with vehicle, Δ 9-tetrahydrocannabinol (THC, 10 mg/kg, i.p.), naloxone (10 mg/kg, i.p.) or THC and naloxone in combination. Locomotor activity was depressed in both THC treatment groups and moderately inhibited in rats given naloxone alone. Results showed that naloxone inhibited THC-induced Fos immunoreactivity in several key brain regions including the ventral tegmental area, ventromedial and dorsomedial hypothalamus, central caudate-putamen and ventrolateral periaqueductal grey. Conversely, naloxone and THC had an additive effect on Fos immunoreactivity in the central nucleus of the amygdala, the bed nucleus of the stria terminalis (lateral division), the insular cortex, and the paraventricular nucleus of the thalamus. These findings complement earlier pharmacological results showing potent modulation of cannabinoid-induced analgesia, appetite and reward by opioids. The inhibitory effects of naloxone on THC-induced ventral tegmentum, hypothalamic and periaqueductal grey Fos expression point to these structures as key sites involved in cannabinoid-opioid interactions.

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.

Recent developments in the pathophysiology of acute pain

Tissue damage induces a state of hyperalgesia. Traditionally, sensory physiology was elucidated by examining responses to ephemeral transient stimuli. However, in order to study acute clinical pain, it is necessary to re-examine nociceptive processing using models which include a significant hyperalgesic component. Primary hyperalgesia results from sensitisation of primary afferent nociceptive neurons by various components of the ‘inflammatory soup’, in a complex interaction between the immune and nervous systems. It is now possible to identify key molecules which participate in this process and exploitation of these may result in the development of novel peripherally acting analgesics: Nerve growth factor: In the developing animal this target-derived growth factor is vital to the survival of the small diameter neurons associated with nociception. However, in the developed animal there is now considerable evidence that this neurotrophin takes on a different role in that it occupies a pivotal position in the development of hyperalgesia. A ligand trap molecule which sequesters nerve growth factor attenuates inflammatory hyperalgesia in several animal models. Bradykinin: Although the potent algogenic effects of bradykinin have been appreciated for some time, the recent discovery of bradykinin receptor sub-types has renewed interest in this molecule. By the use of selective antagonists at the B1 and B2 receptors it has been ascertained that they play disparate roles in nociception and hyperalgesia, thus giving scope for further development. Cannabinoids: Although a component of cannabinoid-induced analgesia is mediated through central (CB1) receptors, the discovery of a second immune cell associated cannabinoid receptor (CB2) has allowed further investigation of the cannabinoids. It appears that putative endogenous CB2 ligands are active during inflammation and attenuate inflammatory hyperalgesia. Exploitation of CB2 receptor agonists may allow the analgesic actions of cannabinoids to be divorced from their CNS side effects.