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BackgroundMicroglia provide continuous immune surveillance of the CNS and upon activation rapidly change phenotype to express receptors that respond to chemoattractants during CNS damage or infection. These activated microglia undergo directed migration towards affected tissue. Importantly, the molecular species of chemoattractant encountered determines if microglia respond with pro- or anti-inflammatory behaviour, yet the signaling molecules that trigger migration remain poorly understood. The endogenous cannabinoid system regulates microglial migration via CB2 receptors and an as yet unidentified GPCR termed the 'abnormal cannabidiol' (Abn-CBD) receptor. Abn-CBD is a synthetic isomer of the phytocannabinoid cannabidiol (CBD) and is inactive at CB1 or CB2 receptors, but functions as a selective agonist at this Gi/o-coupled GPCR. N-arachidonoyl glycine (NAGly) is an endogenous metabolite of the endocannabinoid anandamide and acts as an efficacious agonist at GPR18. Here, we investigate the relationship between NAGly, Abn-CBD, the unidentified 'Abn-CBD' receptor, GPR18, and BV-2 microglial migration.ResultsUsing Boyden chamber migration experiments, yellow tetrazolium (MTT) conversion, In-cell Western, qPCR and immunocytochemistry we show that NAGly, at sub-nanomolar concentrations, and Abn-CBD potently drive cellular migration in both BV-2 microglia and HEK293-GPR18 transfected cells, but neither induce migration in HEK-GPR55 or non-transfected HEK293 wildtype cells. Migration effects are blocked or attenuated in both systems by the 'Abn-CBD' receptor antagonist O-1918, and low efficacy agonists N-arachidonoyl-serine and cannabidiol. NAGly promotes proliferation and activation of MAP kinases in BV-2 microglia and HEK293-GPR18 cells at low nanomolar concentrations - cellular responses correlated with microglial migration. Additionally, BV-2 cells show GPR18 immunocytochemical staining and abundant GPR18 mRNA. qPCR demonstrates that primary microglia, likewise, express abundant amounts of GPR18 mRNA.ConclusionsNAGly is the most effective lipid recruiter of BV-2 microglia currently reported and its effects mimic those of Abn-CBD. The data generated from this study supports the hypothesis that GPR18 is the previously unidentified 'Abn-CBD' receptor. The marked potency of NAGly acting on GPR18 to elicit directed migration, proliferation and perhaps other MAPK-dependent phenomena advances our understanding of the lipid-based signaling mechanisms employed by the CNS to actively recruit microglia to sites of interest. It offers a novel research avenue for developing therapeutics to elicit a self-renewing population of neuroregenerative microglia, or alternatively, to prevent the accumulation of misdirected, pro-inflammatory microglia which contribute to and exacerbate neurodegenerative disease.

Cannabinoids have been shown to exert anti-inflammatory activities in various in vivo and in vitro experimental models as well as ameliorate various inflammatory degenerative diseases. However, the mechanisms of these effects are not completely understood. Using the BV-2 mouse microglial cell line and lipopolysaccharide (LPS) to induce an inflammatory response, we studied the signaling pathways engaged in the anti-inflammatory effects of cannabinoids as well as their influence on the expression of several genes known to be involved in inflammation. We found that the two major cannabinoids present in marijuana, ⌬ 9 -Tetrahydrocannabinol (THC) 3 is a major constituent of Cannabis and serves as an agonist of the cannabinoid receptors CB1 (located mainly in neural cells) and CB2 (located mainly on immune cells). The second major constituent of Cannabis extract is cannabidiol (CBD), which is virtually inactive at the CB1 and CB2 receptors (1). Thus, because of its negligible activity at the CB1 receptor, CBD lacks the psychoactive effects that accompany the use of THC. Moreover, CBD was demonstrated to antagonize some undesirable effects of THC, including intoxication, sedation, and tachycardia, while sharing neuroprotective, anti-oxidative, anti-emetic, and anti-carcinogenic properties (2-4). Both THC and CBD have been shown to exert anti-inflammatory properties and to modulate the function of immune cells, including suppression of humoral response, immune cell proliferation, maturation, and migration, and antigen presentation (5-9). Despite increasing amounts of such observations, the molecular mechanisms involved in these cannabinoid-mediated effects are not yet fully understood.Microglial cells are resident macrophages of the central nervous system and serve as early host defense against pathogens. Activation of microglial cells leads to the release of proinflammatory and neurotoxic factors and serves as part of the neuroinflammatory process (10). The BV-2 murine microglial cell line is known to retain morphological, phenotypic, and functional properties associated with freshly isolated microglia such as expression of nonspecific esterase activity, phagocytic ability, and the absence of peroxidase activity (11,12). Furthermore, these cells release lysozyme and, when stimulated, interleukin (IL)-1 and tumor necrosis factor ␣ (11, 12). Close similarities between BV-2 and primary microglia in mechanisms mediating microglial stimulations, e.g. by lipopolysaccharide (LPS), S100B, or ␤-amyloid, were reported (13). These properties make BV-2 cells an appropriate model for studying the activation of microglia in vitro. It has recently been shown that BV-2 cells express elements of the cannabinoid signaling systems, including the presence of endocannabinoids, i.e. anandamide and 2-arachidonoylglycerol, and cannabinoid or cannabinoid- 3 The abbreviations used are: THC, ⌬ 9 -tetrahydrocannabinol; CBD, cannabidiol; abn-CBD, abnormal cannabidiol; STAT, signal transducers and activators of transcription; IL, interleukin; IFN, ...

. Sex and hormonal cycle differences in rat brain levels of pain-related cannabimimetic lipid mediators. Am J Physiol Regul Integr Comp Physiol 291: R349 -R358, 2006. First published March 23, 2006 doi:10.1152/ajpregu.00933.2005.-One important function of endocannabinoids and related lipid mediators in mammalian central nervous system is modulation of pain. Evidence obtained during the last decade shows that altered levels of these compounds in the brain accompany decreases in pain sensitivity. Such changes, if sexually dimorphic, could account for sex differences in pain and differences that occur during different phases of the hormonal cycle in females. To examine this possibility, we measured the levels of the pain-modulatory lipids anandamide, 2-arachidonoyl glycerol, N-arachidonoyl glycine, N-arachidonoyl gamma amino butyric acid, and N-arachidonoyl dopamine in seven different brain areas (pituitary, hypothalamus, thalamus, striatum, midbrain, hippocampus, and cerebellum) in male rats, and in female rats at five different points in the estrous cycle. The cerebellum did not demonstrate a change in endocannabinoid production across the estrous cycle, whereas all other areas tested showed significant differences in at least one of the compounds measured. These changes in levels occurred predominantly within the 36-h time period surrounding ovulation and behavioral estrus. Differences between males and females were measured as either estrous cycle-independent (all estrous cycles combined) or cycle-dependent (comparisons of males to each estrous cycle). In cycle-independent analyses, small sex differences were observed in the pituitary, hypothalamus, cerebellum, and striatum, whereas no differences were observed in the thalamus, midbrain, and hippocampus. In cycle-dependent analyses, the hypothalamus and pituitary showed largest sex differences followed by the striatum, midbrain, and hippocampus, whereas no sex differences were measured in thalamus and cerebellum. These data provide a basis for investigations into how differences in sex and hormonal status play a role in mechanisms regulating endocannabinoid production and pain. estrous; anandamide; 2-arachidonoyl glycerol; N-arachidonoyl glycine ENDOGENOUS CANNABINOIDS are involved in a myriad of physiological processes, including immune function, feeding regulation, vascular tone, memory, mood, reproduction, and pain (for reviews, see Refs. 29,31,34,35,[44][45][46]. Sex differences in the cannabinoid signaling system have been reported, these being limited mainly to examinations of sex-and cycle-related differences in the efficacy of the exogenous cannabinoid delta-9 tetrahydrocannabinol (THC; reviewed in Ref. 6). The antinoceptive effects of THC were significantly greater in female rats vs. male rats in a model of acute peripheral pain (39). Prenatal THC exposure produced a sex-dependent effect on proenkephalin mRNA levels in several brain regions in which females had significantly higher expression than males (28). Also, the cannabinoid receptor agonist CP...

BACKGROUND AND PURPOSECannabis extracts and several cannabinoids have been shown to exert broad anti-inflammatory activities in experimental models of inflammatory CNS degenerative diseases. Clinical use of many cannabinoids is limited by their psychotropic effects. However, phytocannabinoids like cannabidiol (CBD), devoid of psychoactive activity, are, potentially, safe and effective alternatives for alleviating neuroinflammation and neurodegeneration. EXPERIMENTAL APPROACHWe used experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) in C57BL/6 mice, as a model of multiple sclerosis. Using immunocytochemistry and cell proliferation assays we evaluated the effects of CBD on microglial activation in MOG-immunized animals and on MOG-specific T-cell proliferation. KEY RESULTSTreatment with CBD during disease onset ameliorated the severity of the clinical signs of EAE. This effect of CBD was accompanied by diminished axonal damage and inflammation as well as microglial activation and T-cell recruitment in the spinal cord of MOG-injected mice. Moreover, CBD inhibited MOG-induced T-cell proliferation in vitro at both low and high concentrations of the myelin antigen. This effect was not mediated via the known cannabinoid CB1 and CB2 receptors. CONCLUSIONS AND IMPLICATIONSCBD, a non-psychoactive cannabinoid, ameliorates clinical signs of EAE in mice, immunized against MOG. Suppression of microglial activity and T-cell proliferation by CBD appeared to contribute to these beneficial effects.