Cannabis constituents for chronic neuropathic pain; reconciling the clinical and animal evidence.

Chronic neuropathic pain is a debilitating pain syndrome caused by damage to the nervous system that is poorly served by current medications. Given these problems, clinical studies have pursued extracts of the plant Cannabis sativa as alternative treatments for this condition. The vast majority of these studies have examined cannabinoids which contain the psychoactive constituent delta-9-tetrahydrocannabinol (THC). While there have been some positive findings, meta-analyses of this clinical work indicates that this effectiveness is limited and hampered by side-effects. This review focuses on how recent preclinical studies have predicted the clinical limitations of THC-containing cannabis extracts, and importantly, point to how they might be improved. This work highlights the importance of targeting channels and receptors other than cannabinoid CB1 receptors which mediate many of the side-effects of cannabis.

Cannabinoids and Opioids Differentially Target Extrinsic and Intrinsic GABAergic Inputs onto the Periaqueductal Grey Descending Pathway.

The midbrain periaqueductal gray (PAG) plays a central role in pain modulation via descending pathways. Opioids and cannabinoids are thought to activate these descending pathways by relieving intrinsic GABAergic inhibition of PAG neurons which project to the rostroventromedial medulla (RVM), a process known as disinhibition. However, the PAG also receives descending extrinsic GABAergic inputs from the central nucleus of the amygdala (CeA) which are thought to inhibit PAG GABAergic interneurons. It remains unclear how opioids and cannabinoids act at these different synapses to control descending analgesic pathways. We used optogenetics, tract tracing and electrophysiology to identify the circuitry underlying opioid and cannabinoid actions within the PAG of male and female rats. It was observed that both RVM-projection and nonprojection PAG neurons received intrinsic-PAG and extrinsic-CeA synaptic inputs, which were predominantly GABAergic. Opioids acted via presynaptic µ-receptors to suppress both intrinsic and extrinsic GABAergic inputs onto all PAG neurons, although this inhibition was greater in RVM-projection neurons. By contrast, cannabinoids acted via presynaptic CB1 receptors to exclusively suppress the direct descending GABAergic input from the CeA onto RVM-projection PAG neurons. These findings indicate the CeA controls PAG output neurons which project to the RVM via parallel direct and indirect GABAergic pathways. While µ-opioids indiscriminately inhibit GABAergic inputs onto all PAG neurons, cannabinoids selectively inhibit a direct extrinsic GABAergic input from the amygdala onto PAG projection neurons. These differential actions of opioids and cannabinoids provide a flexible system to gate the descending control of analgesia from the PAG.SIGNIFICANCE STATEMENT The disinhibition hypothesis of analgesia states that opioids activate the midbrain periaqueductal gray (PAG) descending pathway by relieving the tonic inhibition of projection neurons from GABAergic interneurons. However, the PAG also receives extrinsic GABAergic inputs and is the locus of action of cannabinoid analgesics. Here, we show the relative sensitivity of GABAergic synapses to opioids and cannabinoids within the PAG depends on both the origin of presynaptic inputs and their postsynaptic targets. While opioids indiscriminately inhibit all GABAergic inputs onto all PAG neurons, cannabinoids selectively inhibit a direct extrinsic GABAergic input from the amygdala onto PAG descending projection neurons. These differential actions of opioids and cannabinoids provide a flexible system to gate PAG descending outputs.

Intrathecal Actions of the Cannabis Constituents Δ(9)-Tetrahydrocannabinol and Cannabidiol in a Mouse Neuropathic Pain Model.

(1) Background: The psychoactive and non-psychoactive constituents of cannabis, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), synergistically reduce allodynia in various animal models of neuropathic pain. Unfortunately, THC-containing drugs also produce substantial side-effects when administered systemically. We examined the effectiveness of targeted spinal delivery of these cannabis constituents, alone and in combination. (2) Methods: The effect of acute intrathecal drug delivery on allodynia and common cannabinoid-like side-effects was examined in a mouse chronic constriction injury (CCI) model of neuropathic pain. (3) Results: intrathecal THC and CBD produced dose-dependent reductions in mechanical and cold allodynia. In a 1:1 combination, they synergistically reduced mechanical and cold allodynia, with a two-fold increase in potency compared to their predicted additive effect. Neither THC, CBD nor combination THC:CBD produced any cannabis-like side-effects at equivalent doses. The anti-allodynic effects of THC were abolished and partly reduced by cannabinoid CB1 and CB2 receptor antagonists AM281 and AM630, respectively. The anti-allodynic effects of CBD were partly reduced by AM630. (4) Conclusions: these findings indicate that intrathecal THC and CBD, individually and in combination, could provide a safe and effective treatment for nerve injury induced neuropathic pain.

Oral efficacy of Δ(9)-tetrahydrocannabinol and cannabidiol in a mouse neuropathic pain model.

The psychoactive and non-psychoactive constituents of cannabis, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), have synergistic analgesic efficacy in animal models of neuropathic pain when injected systemically. However, the relevance of this preclinical synergy to clinical neuropathic pain studies is unclear because many of the latter use oral administration. We therefore examined the oral effectiveness of these phytocannabinoids and their interactions in a mouse chronic constriction injury (CCI) model of neuropathic pain. THC produced a dose-dependent reduction in mechanical and cold allodynia, but also induced side-effects with similar potency. CBD also reduced allodynia, albeit with lower potency than THC, but did not produce cannabinoid-like side-effects at any dose tested. Combination THC:CBD produced a dose-dependent reduction in allodynia, however, it displayed little to no synergy. Combination THC:CBD produced substantial, synergistic side-effects which increased with the proportion of CBD. These findings demonstrate that oral THC and CBD, alone and in combination, have analgesic efficacy in an animal neuropathic pain model. Unlike prior systemic injection studies, combination THC:CBD lacks analgesic synergy when delivered orally. Furthermore, both THC and combination THC:CBD display a relatively poor therapeutic window when delivered orally. This suggests that CBD provides a safer, albeit lower efficacy, oral treatment for nerve injury induced neuropathic pain than THC-containing preparations. This article is part of the special issue on 'Cannabinoids'.

The anticonvulsant zonisamide positively modulates recombinant and native glycine receptors at clinically relevant concentrations.

GABAA and glycine receptors mediate fast synaptic inhibitory neurotransmission. Despite studies showing that activation of cerebral glycine receptors could be a potential strategy in the treatment of epilepsy, few studies have assessed the effects of existing anticonvulsant therapies on recombinant or native glycine receptors. We, therefore, evaluated the actions of a series of anticonvulsants at recombinant human homo-oligomeric glycine receptor α1, α2 and α3 subtypes expressed in Xenopus oocytes using two-electrode voltage-clamp methods, and then assessed the most effective drug at native glycine receptors from entorhinal cortex neurons using whole-cell voltage-clamp recordings. Ganaxolone, tiagabine and zonisamide positively modulated glycine induced currents at recombinant homomeric glycine receptors. Of these, zonisamide was the most efficacious and exhibited an EC50 value ranging between 450 and 560 µM at α1, α2 and α3 subtypes. These values were not significantly different indicating a non-selective modulation of glycine receptors. Using a therapeutic concentration of zonisamide (100 µM), the potency of glycine was significantly shifted from 106 to 56 µM at α1, 185 to 112 µM at α2, and 245 to 91 µM at α3 receptors. Furthermore, zonisamide (100 µM) potentiated exogenous homomeric and heteromeric glycine mediated currents from layer II pyramidal cells of the lateral or medial entorhinal cortex. As therapeutic concentrations of zonisamide positively modulate recombinant and native glycine receptors, we propose that the anticonvulsant effects of zonisamide may, at least in part, be mediated via this action.

Inflammation induces developmentally regulated sumatriptan inhibition of spinal synaptic transmission.

BACKGROUND AND PURPOSE: While triptans are used to treat migraine, there is evidence that they also reduce inflammation-induced pain at the spinal level. The cellular mechanisms underlying this spinal enhancement are unknown. We examined whether inflammation alters sumatriptan modulation of synaptic transmission in the rat spinal dorsal horn. EXPERIMENTAL APPROACH: Three to four days following intraplantar injection of complete Freund's adjuvant (CFA) or saline, whole cell recordings of evoked glutamatergic EPSCs were made from lumbar lamina I-II dorsal horn neurons in rat spinal slices KEY RESULTS: In 2- to 3-week-old animals, sumatriptan reduced the amplitude of evoked EPSCs and this was greater in slices from CFA, compared to saline-injected rats. In CFA-injected animals, sumatriptan increased the paired pulse ratio of evoked EPSCs and reduced the rate of spontaneous miniature EPSCs. The 5-HT1B and 5-HT1D agonists CP9 3129 and PNU109291 both inhibited evoked EPSCs in CFA but not saline-injected rats. By contrast, the 5-HT1A agonist R(+)-8-OH-DPAT inhibited evoked EPSCs in saline but not CFA-injected rats. In CFA-injected rats, the sumatriptan-induced inhibition of evoked EPSCs was reduced by the 5-HT1B and 5-HT1D antagonists NAS181 and BRL-15572. Intriguingly, the difference in sumatriptan inhibition between CFA and saline-injected animals was only observed in animals less than 4 weeks old. CONCLUSION AND IMPLICATIONS: These findings indicate that inflammation induces a developmentally regulated 5-HT1B/1D presynaptic inhibition of excitatory transmission into the rat superficial dorsal horn. Thus, triptans could potentially act as spinal analgesic agents for inflammatory pain in the juvenile setting.

A new mouse line with reduced GluA2 Q/R site RNA editing exhibits loss of dendritic spines, hippocampal CA1-neuron loss, learning and memory impairments and NMDA receptor-independent seizure vulnerability.

Calcium (Ca2+)-permeable AMPA receptors may, in certain circumstances, contribute to normal synaptic plasticity or to neurodegeneration. AMPA receptors are Ca2+-permeable if they lack the GluA2 subunit or if GluA2 is unedited at a single nucleic acid, known as the Q/R site. In this study, we examined mice engineered with a point mutation in the intronic editing complementary sequence (ECS) of the GluA2 gene, Gria2. Mice heterozygous for the ECS mutation (named GluA2+/ECS(G)) had a ~ 20% reduction in GluA2 RNA editing at the Q/R site. We conducted an initial phenotypic analysis of these mice, finding altered current-voltage relations (confirming expression of Ca2+-permeable AMPA receptors at the synapse). Anatomically, we observed a loss of hippocampal CA1 neurons, altered dendritic morphology and reductions in CA1 pyramidal cell spine density. Behaviourally, GluA2+/ECS(G) mice exhibited reduced motor coordination, and learning and memory impairments. Notably, the mice also exhibited both NMDA receptor-independent long-term potentiation (LTP) and vulnerability to NMDA receptor-independent seizures. These NMDA receptor-independent seizures were rescued by the Ca2+-permeable AMPA receptor antagonist IEM-1460. In summary, unedited GluA2(Q) may have the potential to drive NMDA receptor-independent processes in brain function and disease. Our study provides an initial characterisation of a new mouse model for studying the role of unedited GluA2(Q) in synaptic and dendritic spine plasticity in disorders where unedited GluA2(Q), synapse loss, neurodegeneration, behavioural impairments and/or seizures are observed, such as ischemia, seizures and epilepsy, Huntington's disease, amyotrophic lateral sclerosis, astrocytoma, cocaine seeking behaviour and Alzheimer's disease.

Opioid presynaptic disinhibition of the midbrain periaqueductal grey descending analgesic pathway.

BACKGROUND AND PURPOSE: The midbrain periaqueductal grey (PAG) plays a central role in modulating pain through a descending pathway that projects indirectly to the spinal cord via the rostroventral medial medulla (RVM). While opioids are potent analgesics that target the PAG, their cellular actions on descending projection neurons are unclear. EXPERIMENTAL APPROACH: Patch clamp recordings in voltage- and current-clamp mode were made from acutely prepared PAG slices from animals that received retrograde tracer injections into the RVM. KEY RESULTS: The µ-agonist DAMGO reduced GABAergic evoked inhibitory postsynaptic currents (IPSCs) in retro-labelled, RVM-projecting neurons to a greater extent than in unlabelled neurons. The κ-opioid agonist U69593 reduced evoked IPSCs to a similar extent in both neuronal groups, while the δ-opioid agonist deltorphin-II was without effect. DAMGO and U69593 both produced a reduction in the rate, but not amplitude of spontaneous miniature IPSCs and asynchronous evoked IPSCs in retro-labelled neurons. DAMGO and U69593 also suppressed glutamatergic EPSCs in retro-labelled and unlabelled neurons. The DAMGO inhibition of evoked EPSCs, however, was less than that for evoked IPSCs in retro-labelled, but not unlabelled neurons. In current clamp, DAMGO produced a depolarizing increase in evoked postsynaptic potentials in retro-labelled neurons, but directly inhibited unlabelled neurons. CONCLUSION AND IMPLICATIONS: These findings suggest that µ-opioids activate the descending analgesic pathway from the midbrain PAG by a combination of presynaptic disinhibition of RVM-projecting neurons and postsynaptic inhibition of presumptive interneurons.

Endogenous cannabinoid modulation of restraint stress-induced analgesia in thermal nociception.

It is thought that endogenous cannabinoids have a role in the analgesia induced by specific forms of stress. We examined if the role of endogenous cannabinoids is also dependent upon the mode of nociception, and whether this could be altered by drugs which block their enzymatic degradation. In C57BL/6 mice, restraint stress produced analgesia in the hot-plate and plantar tests, two thermal pain assays that engage distinct supraspinal and spinal nociceptive pathways. Stress-induced analgesia in the hot-plate test was abolished by pre-treatment with the opioid receptor antagonist naltrexone but was unaffected by the cannabinoid receptor antagonist 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281). By contrast, stress-induced analgesia in the plantar test was abolished by pre-treatment with naltrexone plus AM281, but not by either antagonist individually. Remarkably, inhibiting the breakdown of endocannabinoids, with the dual fatty acid amide hydrolase and monoacylglycerol lipase inhibitor JZL195, rescued stress-induced analgesia in the hotplate test when endogenous opioid signalling was blocked by naltrexone. Furthermore, JZL195 recruited analgesia induced by sub-threshold restraint stress in both thermal pain assays. These findings indicate the role of endocannabinoids in stress-induced analgesia differs with the type of thermal pain behaviour. However, by inhibiting their breakdown, endocannabinoids can be recruited to substitute for endogenous opioid signalling when their activity is blocked, indicating a degree of redundancy between opioid and cannabinoid systems. Together these data suggest targeting endocannabinoid breakdown could provide an alternative, or adjuvant to mainstream analgesics such as opioids.

Correction to: Electrophysiological Actions of N/OFQ.

In the last paragraph of Sect. 2.1.2 on line 3 the word 'off-cells' is misspelt. It should be 'on-cells'.

Electrophysiological Actions of N/OFQ.

Whilst the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) has similar intracellular coupling mechanisms to opioid receptors, it has distinct modulatory effects on physiological functions such as pain. These actions range from agonistic to antagonistic interactions with classical opioids within the spinal cord and brain, respectively. Understanding the electrophysiological actions of N/OFQ has been crucial in ascertaining the mechanisms by which these agonistic and antagonistic interactions occur. These similarities and differences between N/OFQ and opioids are due to the relative location of NOP versus opioid receptors on specific neuronal elements within these CNS regions. These mechanisms result in varied cellular actions including postsynaptic modulation of ion channels and presynaptic regulation of neurotransmitter release.

THC and gabapentin interactions in a mouse neuropathic pain model.

Clinical studies have shown that the major psychoactive ingredient of Cannabis sativa Δ9-tetrahydrocannabinol (THC) has some analgesic efficacy in neuropathic pain states. However, THC has a significant side effect profile. We examined whether the profile of THC could be improved by co-administering it with the first-line neuropathic pain medication gabapentin. This was done using the chronic constriction injury (CCI) model of neuropathic pain in C57BL6 mice. At 8 days post-CCI nerve injury, acute systemic administration of gabapentin produced a dose-dependent decrease in CCI-induced mechanical and cold allodynia, and increased motor incoordination. Coadministration of THC and gabapentin in a fixed-ratio dose-dependently reduced mechanical and cold allodynia, and produced all the side-effects observed for THC, including motor incoordination, catalepsy and sedation. Isobolographic analysis indicated that the ED50 for the THC:gabapentin induced reduction in allodynia was 1.7 times less than that predicted for an additive interaction. The therapeutic window of combination THC:gabapentin was greater than that for THC alone. These findings indicate that gabapentin synergistically enhances the anti-allodynic actions of THC and improves its therapeutic window. Thus, THC may represent a potential adjuvant for neuropathic pain medications such as gabapentin.

Plant-Based Cannabinoids for the Treatment of Chronic Neuropathic Pain.

Chronic neuropathic pain is a prevalent condition that places a heavy burden on individuals and the healthcare system. Current medications have limitations and new approaches are needed, particularly given the current opioid crisis. There is some clinical evidence that the plant Cannabis sativa produces relief from neuropathic pain. However, current meta-analyses suggest that this efficacy is limited and there are problems with side effects. Most of this clinical research has examined whole cannabis, the psychoactive phytocannabinoid 9-tetrahydrocannabinol (THC), and nabiximols, which are a mixture of THC and the non-psychoactive phytocannabinoid cannabidiol. In the past, there has been little evidence based, preclinical animal research to guide clinical studies on phytocannabinoids. Recent animal studies indicate that while THC and high dose nabiximols are effective in animal neuropathic pain models, significant pain relief is only achieved at doses that produce substantial side effects. By contrast, cannabidiol and low dose nabiximols have moderate pain relieving efficacy, but are devoid of cannabinoid-like side effects. This animal data suggests that cannabidiol and low dose nabiximols warrant consideration for clinical studies, at least as adjuvants to current drugs. Preclinical research is also required to identify other phytocannabinoids that have therapeutic potential.

Cannabis constituent synergy in a mouse neuropathic pain model.

Cannabis and its psychoactive constituent Δ9-tetrahydrocannabinol (THC) have efficacy against neuropathic pain, however, this is hampered by their side effects. It has been suggested that co-administration with another major constituent cannabidiol (CBD) might enhance the analgesic actions of THC and minimise its deleterious side effects. We examined the basis for this phytocannabinoid interaction in a mouse chronic constriction injury (CCI) model of neuropathic pain. Acute systemic administration of THC dose-dependently reduced CCI-induced mechanical and cold allodynia, but also produced motor incoordination, catalepsy, and sedation. Cannabidiol produced a lesser dose-dependent reduction in allodynia, but did not produce the cannabinoid side effects. When co-administered in a fixed ratio, THC and CBD produced a biphasic dose-dependent reduction in allodynia. At low doses, the THC:CBD combination displayed a 200-fold increase in anti-allodynic potency, but had lower efficacy compared with that predicted for an additive drug interaction. By contrast, high THC:CBD doses had lower potency, but greater anti-allodynic efficacy compared with that predicted for an additive interaction. Only the high dose THC:CBD anti-allodynia was associated with cannabinoid side effects and these were similar to those of THC alone. Unlike THC, the low dose THC:CBD anti-allodynia was not cannabinoid receptor mediated. These findings demonstrate that CBD synergistically enhances the pain-relieving actions of THC in an animal neuropathic pain model, but has little impact on the THC-induced side effects. This suggests that low dose THC:CBD combination treatment has potential in the treatment of neuropathic pain.

Chronic morphine reduces the readily releasable pool of GABA, a presynaptic mechanism of opioid tolerance.

KEY POINTS: Chronic treatment with opioids, such as morphine, leads to analgesic tolerance. While postsynaptic opioid tolerance is well documented, the involvement of presynaptic mechanisms remains unclear. We show that chronic morphine reduces the ability of periaqueductal grey (PAG) neurons to maintain GABAergic transmission. This depression of GABAergic transmission was due to a reduction in the effective size of the readily releasable pool. This also led to a reduction in opioid presynaptic inhibition; these presynaptic adaptations need to be considered in the development of strategies to reduce opioid tolerance. ABSTRACT: The midbrain periaqueductal grey (PAG) plays a critical role in tolerance to the analgesic actions of opioids such as morphine. While numerous studies have identified the postsynaptic adaptations induced by chronic morphine treatment in this and other brain regions, the presence of presynaptic adaptations remains uncertain. We examined GABAergic synaptic transmission within rat PAG brain slices from animals which underwent a low dose morphine treatment protocol which produces tolerance, but not withdrawal. Evoked GABAergic IPSCs (inhibitory postsynaptic currents) were less in morphine compared to control saline treated animals. Postsynaptic GABAA receptor mediated currents and desensitization, presynaptic release probability (Pr ), and inhibition by endogenous neurotransmitters were similar in morphine and saline treated animals. By contrast, the effective size of the readily releasable pool (RRP) was smaller in morphine treated animals. While the µ-opioid agonist DAMGO produced a reduction in Pr and RRP size in saline treated animals, it only reduced Pr in morphine treated animals. Consequently, DAMGO-induced inhibition of evoked IPSCs during short burst stimulation was less in morphine, compared to saline treated animals. These results indicate that low dose chronic morphine treatment reduces presynaptic µ-opioid inhibition by reducing the size of the pool of vesicles available for action potential dependent release. This novel presynaptic adaptation may provide important insights into the development of efficacious pain therapies that can circumvent the development of opioid tolerance.

The tarantula toxin ß/δ-TRTX-Pre1a highlights the importance of the S1-S2 voltage-sensor region for sodium channel subtype selectivity.

Voltage-gated sodium (NaV) channels are essential for the transmission of pain signals in humans making them prime targets for the development of new analgesics. Spider venoms are a rich source of peptide modulators useful to study ion channel structure and function. Here we describe ß/δ-TRTX-Pre1a, a 35-residue tarantula peptide that selectively interacts with neuronal NaV channels inhibiting peak current of hNaV1.1, rNaV1.2, hNaV1.6, and hNaV1.7 while concurrently inhibiting fast inactivation of hNaV1.1 and rNaV1.3. The DII and DIV S3-S4 loops of NaV channel voltage sensors are important for the interaction of Pre1a with NaV channels but cannot account for its unique subtype selectivity. Through analysis of the binding regions we ascertained that the variability of the S1-S2 loops between NaV channels contributes substantially to the selectivity profile observed for Pre1a, particularly with regards to fast inactivation. A serine residue on the DIV S2 helix was found to be sufficient to explain Pre1a's potent and selective inhibitory effect on the fast inactivation process of NaV1.1 and 1.3. This work highlights that interactions with both S1-S2 and S3-S4 of NaV channels may be necessary for functional modulation, and that targeting the diverse S1-S2 region within voltage-sensing domains provides an avenue to develop subtype selective tools.

Endocannabinoids control vesicle release mode at midbrain periaqueductal grey inhibitory synapses.

KEY POINTS: The midbrain periaqueductal grey (PAG) forms part of an endogenous analgesic system which is tightly regulated by the neurotransmitter GABA. The role of endocannabinoids in regulating GABAergic control of this system was examined in rat PAG slices. Under basal conditions GABAergic neurotransmission onto PAG output neurons was multivesicular. Activation of the endocannabinoid system reduced GABAergic inhibition by reducing the probability of release and by shifting release to a univesicular mode. Blockade of endocannabinoid system unmasked a tonic control over the probability and mode of GABA release. These findings provides a mechanistic foundation for the control of the PAG analgesic system by disinhibition. ABSTRACT: The midbrain periaqueductal grey (PAG) has a crucial role in coordinating endogenous analgesic responses to physiological and psychological stressors. Endocannabinoids are thought to mediate a form of stress-induced analgesia within the PAG by relieving GABAergic inhibition of output neurons, a process known as disinhibition. This disinhibition is thought to be achieved by a presynaptic reduction in GABA release probability. We examined whether other mechanisms have a role in endocannabinoid modulation of GABAergic synaptic transmission within the rat PAG. The group I mGluR agonist DHPG ((R,S)-3,5-dihydroxyphenylglycine) inhibited evoked IPSCs and increased their paired pulse ratio in normal external Ca2+ , and when release probability was reduced by lowering Ca2+ . However, the effect of DHPG on the coefficient of variation and kinetics of evoked IPSCs differed between normal and low Ca2+ . Lowering external Ca2+ had a similar effect on evoked IPSCs to that observed for DHPG in normal external Ca2+ . The low affinity GABAA receptor antagonist TPMPA ((1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid) inhibited evoked IPSCs to a greater extent in low than in normal Ca2+ . Together these findings indicate that the normal mode of GABA release is multivesicular within the PAG, and that DHPG and lowering external Ca2+ switch this to a univesicular mode. The effects of DHPG were mediated by mGlu5 receptor engagement of the retrograde endocannabinoid system. Blockade of endocannabinoid breakdown produced a similar shift in the mode of release. We conclude that endocannabinoids control both the mode and the probability of GABA release within the PAG.

Opioid and cannabinoid synergy in a mouse neuropathic pain model.

BACKGROUND AND PURPOSE: Clinical studies have reported that pan-cannabinoid receptor agonists may have efficacy in neuropathic pain states and that this might be enhanced by co-administration with opioids. While cannabinoid-opioid analgesic synergy has been demonstrated in animal models of acute pain, it has not been examined in neuropathic pain models. We examined the effect of combination treatment with cannabinoid and opioid receptor agonists on allodynia and side effects in a nerve injury-induced neuropathic pain model. EXPERIMENTAL APPROACH: C57BL/6 mice were subjected to chronic constriction injury (CCI) of the sciatic nerve. The effects of systemic administration of morphine and the pan-cannabinoid receptor agonist, WIN55212, on allodynia and side effects were examined at 7-10 days post-CCI surgery. Isobolographic analysis was used to determine whether the effects of the combination were synergistic. KEY RESULTS: The opioid agonist morphine reduced CCI-induced mechanical and cold allodynia and produced motor incoordination, in a dose-dependent manner. WIN55212 reduced CCI-induced allodynia and produced motor incoordination, catalepsy and sedation, in a dose-dependent manner, as we have observed previously. When administered together, WIN55212 and morphine reduced allodynia in a synergistic manner but had only an additive effect on motor incoordination. CONCLUSIONS AND IMPLICATIONS: These findings indicate that administration of a combination of a non-selective opioid and cannabinoid receptor agonist synergistically reduces nerve injury-induced allodynia, while producing side effects in an additive manner. This suggests that this combination treatment has an improved anti-allodynic potency and therapeutic index in a neuropathic pain model.

Actions of the dual FAAH/MAGL inhibitor JZL195 in a murine neuropathic pain model.

BACKGROUND AND PURPOSE: While cannabinoids have been proposed as a potential treatment for neuropathic pain, they have limitations. Cannabinoid receptor agonists have good efficacy in animal models of neuropathic pain; they have a poor therapeutic window. Conversely, selective fatty acid amide hydrolase (FAAH) inhibitors that enhance the endocannabinoid system have a better therapeutic window, but lesser efficacy. We examined whether JZL195, a dual inhibitor of FAAH and monacylglycerol lipase (MAGL), could overcome these limitations. EXPERIMENTAL APPROACH: C57BL/6 mice underwent the chronic constriction injury (CCI) model of neuropathic pain. Mechanical and cold allodynia, plus cannabinoid side effects, were assessed in response to systemic drug application. KEY RESULTS: JZL195 and the cannabinoid receptor agonist WIN55212 produced dose-dependent reductions in CCI-induced mechanical and cold allodynia, plus side effects including motor incoordination, catalepsy and sedation. JZL195 reduced allodynia with an ED50 at least four times less than that at which it produced side effects. By contrast, WIN55212 reduced allodynia and produce side effects with similar ED50s. The maximal anti-allodynic effect of JZL195 was greater than that produced by selective FAAH, or MAGL inhibitors. The JZL195-induced anti-allodynia was maintained during repeated treatment. CONCLUSIONS AND IMPLICATIONS: These findings suggest that JZL195 has greater anti-allodynic efficacy than selective FAAH, or MAGL inhibitors, plus a greater therapeutic window than a cannabinoid receptor agonist. Thus, dual FAAH/MAGL inhibition may have greater potential in alleviating neuropathic pain, compared with selective FAAH and MAGL inhibitors, or cannabinoid receptor agonists.

Endocannabinoid modulation by FAAH and monoacylglycerol lipase within the analgesic circuitry of the periaqueductal grey.

BACKGROUND AND PURPOSE: Endogenous cannabinoids (endocannabinoids) in the periaqueductal grey (PAG) play a vital role in mediating stress-induced analgesia. This analgesic effect of endocannabinoids is enhanced by pharmacological inhibition of their degradative enzymes. However, the specific effects of endocannabinoids and the inhibitors of their degradation are largely unknown within this pain-modulating region. EXPERIMENTAL APPROACH: In vitro electrophysiological recordings were conducted from PAG neurons in rat midbrain slices. The effects of the major endocannabinoids and their degradation inhibitors on inhibitory GABAergic synaptic transmission were examined. KEY RESULTS: Exogenous application of the endocannabinoid, anandamide (AEA), but not 2-arachidonoylglycerol (2-AG), produced a reduction in inhibitory GABAergic transmission in PAG neurons. This AEA-induced suppression of inhibition was enhanced by the fatty acid amide hydrolase (FAAH) inhibitor, URB597, whereas a 2-AG-induced suppression of inhibition was unmasked by the monoacylglycerol lipase (MGL) inhibitor, JZL184. In addition, application of the CB1 receptor antagonist, AM251, facilitated the basal GABAergic transmission in the presence of URB597 and JZL184, which was further enhanced by the dual FAAH/MGL inhibitor, JZL195. CONCLUSIONS AND IMPLICATIONS: Our results indicate that AEA and 2-AG act via disinhibition within the PAG, a cellular action consistent with analgesia. These actions of AEA and 2-AG are tightly regulated by their respective degradative enzymes, FAAH and MGL. Furthermore, individual or combined inhibition of FAAH and/or MGL enhanced tonic disinhibition within the PAG. Therefore, the current findings support the therapeutic potential of FAAH and MGL inhibitors as a novel pharmacotherapy for pain.