Molecular Sites for the Positive Allosteric Modulation of Glycine Receptors by Endocannabinoids

Glycine receptors (GlyRs) are transmitter-gated anion channels of the Cys-loop superfamily which mediate synaptic inhibition at spinal and selected supraspinal sites. Although they serve pivotal functions in motor control and sensory processing, they have yet to be exploited as drug targets partly because of hitherto limited possibilities for allosteric control. Endocannabinoids (ECs) have recently been characterized as direct allosteric GlyR modulators, but the underlying molecular sites have remained unknown. Here, we show that chemically neutral ECs (e.g. anandamide, AEA) are positive modulators of α1, α2 and α3 GlyRs, whereas acidic ECs (e.g. N-arachidonoyl-glycine; NA-Gly) potentiate α1 GlyRs but inhibit α2 and α3. This subunit-specificity allowed us to identify the underlying molecular sites through analysis of chimeric and mutant receptors. We found that alanine 52 in extracellular loop 2, glycine 254 in transmembrane (TM) region 2 and intracellular lysine 385 determine the positive modulation of α1 GlyRs by NA-Gly. Successive substitution of non-conserved extracellular and TM residues in α2 converted NA-Gly-mediated inhibition into potentiation. Conversely, mutation of the conserved lysine within the intracellular loop between TM3 and TM4 attenuated NA-Gly-mediated potentiation of α1 GlyRs, without affecting inhibition of α2 and α3. Notably, this mutation reduced modulation by AEA of all three GlyRs. These results define molecular sites for allosteric control of GlyRs by ECs and reveal an unrecognized function for the TM3-4 intracellular loop in the allosteric modulation of Cys-loop ion channels. The identification of these sites may help to understand the physiological role of this modulation and facilitate the development of novel therapeutic approaches to diseases such as spasticity, startle disease and possibly chronic pain

Interaction between cannabis consumption and childhood abuse in psychotic disorders:preliminary findings on the role of different patterns of cannabis use

AIM: Several studies have suggested that lifetime cannabis consumption and childhood abuse synergistically contribute to the risk for psychotic disorders. This study aimed to extend existing findings regarding an additive interaction between childhood abuse and lifetime cannabis use by investigating the moderating role of type and frequency of cannabis use. METHODS: Up to 231 individuals presenting for the first time to mental health services with psychotic disorders and 214 unaffected population controls from South London, United Kingdom, were recruited as part of the Genetics and Psychosis study. Information about history of cannabis use was collected using the Cannabis Experiences Questionnaire. Childhood physical and sexual abuse was assessed using the Childhood Experience of Care and Abuse Questionnaire. RESULTS: Neither lifetime cannabis use nor reported exposure to childhood abuse was associated with psychotic disorder when the other environmental variable was taken into account. Although the combination of the two risk factors raised the odds for psychosis by nearly three times (adjusted OR\u2009=\u20092.94, 95% CI: 1.44-6.02, P\u2009=\u20090.003), no evidence of interaction was found (adjusted OR\u2009=\u20091.46, 95% CI: -0.54 to 3.46, P\u2009=\u20090.152). Furthermore, the association of high-potency cannabis and daily consumption with psychosis was at least partially independent of the effect of childhood abuse. CONCLUSIONS: The heavy use of high-potency cannabis increases the risk of psychosis but, in addition, smoking of traditional resin (hash) and less than daily cannabis use may increase the risk for psychosis when combined with exposure to severe childhood abuse

An endocannabinoid mechanism for stress-induced analgesia

Acute stress suppresses pain by activating brain pathways that engage opioid or non-opioid mechanisms. Here we show that an opioid-independent form of this phenomenon, termed stress-induced analgesia, is mediated by the release of endogenous marijuana-like (cannabinoid) compounds in the brain. Blockade of cannabinoid CB(1) receptors in the periaqueductal grey matter of the midbrain prevents non-opioid stress-induced analgesia. In this region, stress elicits the rapid formation of two endogenous cannabinoids, the lipids 2-arachidonoylglycerol (2-AG) and anandamide. A newly developed inhibitor of the 2-AG-deactivating enzyme, monoacylglycerol lipase, selectively increases 2-AG concentrations and, when injected into the periaqueductal grey matter, enhances stress-induced analgesia in a CB1-dependent manner. Inhibitors of the anandamide-deactivating enzyme fatty-acid amide hydrolase, which selectively elevate anandamide concentrations, exert similar effects. Our results indicate that the coordinated release of 2-AG and anandamide in the periaqueductal grey matter might mediate opioid-independent stress-induced analgesia. These studies also identify monoacylglycerol lipase as a previously unrecognized therapeutic target

Endocannabinoids at the spinal level regulate, but do not mediate, nonopioid stress-induced analgesia.

Recent work in our laboratories has demonstrated that an opioid-independent form of stress-induced analgesia (SIA) is mediated by endogenous cannabinoids [Hohmann et al., 2005. Nature 435, 1108]. Non-opioid SIA, induced by a 3-min continuous foot shock, is characterized by the mobilization of two endocannabinoid lipids--2-arachidonoylglycerol (2-AG) and anandamide--in the midbrain periaqueductal gray (PAG). The present studies were conducted to examine the contributions of spinal endocannabinoids to nonopioid SIA. Time-dependent increases in levels of 2-AG, but not anandamide, were observed in lumbar spinal cord extracts derived from shocked relative to non-shocked rats. Notably, 2-AG accumulation was of smaller magnitude than that observed previously in the dorsal midbrain following foot shock. 2-AG is preferentially degraded by monoacylglycerol lipase (MGL), whereas anandamide is hydrolyzed primarily by fatty-acid amide hydrolase (FAAH). This metabolic segregation enabled us to manipulate endocannabinoid tone at the spinal level to further evaluate the roles of 2-AG and anandamide in nonopioid SIA. Intrathecal administration of the competitive CB1 antagonist SR141716A (rimonabant) failed to suppress nonopioid SIA, suggesting that supraspinal rather than spinal CB1 receptor activation plays a pivotal role in endocannabinoid-mediated SIA. By contrast, spinal inhibition of MGL using URB602, which selectively inhibits 2-AG hydrolysis in the PAG, enhanced SIA through a CB1-selective mechanism. Spinal inhibition of FAAH, with either URB597 or arachidonoyl serotonin (AA-5-HT), also enhanced SIA through a CB1-mediated mechanism, presumably by increasing accumulation of tonically released anandamide. Our results suggest that endocannabinoids in the spinal cord regulate, but do not mediate, nonopioid SIA

2 - Arachidonoylglycerol and anandamide are endocannabinoid mediators of stress - induced analgesia

Convegno: Society for Neuroscience's 35th Annual Meeting, Program No. 982.16 Abstract (Poster

Inhibitors of monoacylglycerol lipase as novel analgesics

2-Arachidonoylglycerol (2-AG) is an endogenous cannabinoid (endocannabinoid) lipid whose functions remain poorly understood. Guindon and colleagues report the novel finding that exogenous application of 2-AG induces peripheral antinociceptive effects that are mediated, at least in part, by actions at peripheral cannabinoid CB2 receptors. URB602, a recently described inhibitor of monoacylglycerol lipase, an enzyme that catalyzes 2-AG hydrolysis in vivo, also induced peripheral antinociceptive effects and enhanced the actions of 2-AG. Peripheral analgesic mechanisms represent promising therapeutic targets for suppressing pain in the absence of unwanted central nervous system side-effects (e.g. psychoactivity) associated with activation of central CB1 receptors. The therapeutic potential of inhibitors of 2-AG deactivation for the treatment of inflammatory pain is discussed