The endocannabinoid signaling system regulates diverse physiologic processes and has attracted considerable attention as a potential pharmaceutical target for treating diseases, such as pain, anxiety/depression, and metabolic disorders. The principal ligands of the endocannabinoid system are the lipid transmitters N-arachidonoylethanolamine (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), which activate the two major cannabinoid receptors, CB1 and CB<em>2</em>. Anandamide and <em>2</em>-AG signaling pathways in the nervous system are terminated by enzymatic hydrolysis mediated primarily by the serine hydrolases fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. In this review, we will discuss the development of FAAH and MAGL inhibitors and their pharmacological application to investigate the function of anandamide and <em>2</em>-AG signaling pathways in preclinical models of neurobehavioral processes, such as pain, anxiety, and addiction. We will place emphasis on how these studies are beginning to discern the different roles played by anandamide and <em>2</em>-AG in the nervous system and the resulting implications for advancing endocannabinoid hydrolase inhibitors as next-generation therapeutics.

OBJECTIVE

The link between excess intra-abdominal adiposity (IAA) and metabolic complications leading to type <em>2</em> diabetes and cardiovascular disease is well recognized. Blockade of endocannabinoid action at cannabinoid CB(1) receptors was shown to reduce these complications. Here, we investigated the relationship between IAA, circulating endocannabinoid levels and markers of cardiometabolic risk in male obese subjects.

METHODS

Fasting plasma levels of the endocannabinoids, anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), were measured by liquid chromatography-mass spectrometry in a study sample of 6<em>2</em> untreated asymptomatic men with body mass index (BMI) from 18.7 to 35.<em>2</em> kg/m(<em>2</em>).

RESULTS

Plasma <em>2</em>-AG, but not AEA, levels correlated positively with BMI, waist girth, IAA measured by computed tomography, and fasting plasma triglyceride and insulin levels, and negatively with high-density lipoprotein cholesterol and adiponectin levels. Obese men with similar BMI values >> or =30 kg/m(<em>2</em>)) but who markedly differed in their amount of IAA (< vs>> or = 130 cm(<em>2</em>), n=17) exhibited higher <em>2</em>-AG levels in the presence of high IAA. No difference in <em>2</em>-AG concentrations was observed between obese men with low levels of IAA vs nonobese controls.

CONCLUSIONS

These results provide evidence for a relationship in men between a key endocannabinoid, <em>2</em>-AG, and cardiometabolic risk factors, including IAA.

It is now well established that the psychoactive effects of Cannabis sativa are primarily mediated through neuronal CB1 receptors, while its therapeutic immune properties are primarily mediated through CB<em>2</em> receptors. Two endocannabinoids, arachidonoylethanolamide and <em>2</em>-<em>arachidonoylglycerol</em>, have been identified, their action on CB1 and CB<em>2</em> thoroughly characterized, and their production and inactivation elucidated. However, many significant exceptions to these rules exist. Here we review the evidence suggesting that cannabinoids can modulate synaptic transmission, the cardiovascular system, and the immune system through receptors distinct from CB1 and CB<em>2</em>, and that an additional "independent" endocannabinoid signaling system that involves palmitoylethanolamide may exist.

Cancer cells couple heightened lipogenesis with lipolysis to produce fatty acid networks that support malignancy. Monoacylglycerol lipase (MAGL) plays a principal role in this process by converting monoglycerides, including the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), to free fatty acids. Here, we show that MAGL is elevated in androgen-independent versus androgen-dependent human prostate cancer cell lines, and that pharmacological or RNA-interference disruption of this enzyme impairs prostate cancer aggressiveness. These effects were partially reversed by treatment with fatty acids or a cannabinoid receptor-1 (CB1) antagonist, and fully reversed by cotreatment with both agents. We further show that MAGL is part of a gene signature correlated with epithelial-to-mesenchymal transition and the stem-like properties of cancer cells, supporting a role for this enzyme in protumorigenic metabolism that, for prostate cancer, involves the dual control of endocannabinoid and fatty acid pathways.

Hepatic ischemia-reperfusion (I/R) injury continues to be a fatal complication that can follow liver surgery or transplantation. We have investigated the involvement of the endocannabinoid system in hepatic I/R injury using an in vivo mouse model. Here we report that I/R triggers several-fold increases in the hepatic levels of the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, which originate from hepatocytes, Kupffer, and endothelial cells. The I/R-induced increased tissue endocannabinoid levels positively correlate with the degree of hepatic damage and serum TNF-alpha, MIP-1alpha, and MIP-<em>2</em> levels. Furthermore, a brief exposure of hepatocytes to various oxidants (H<em>2</em>O<em>2</em> and peroxynitrite) or inflammatory stimuli (endotoxin and TNF-alpha) also increases endocannabinoid levels. Activation of CB<em>2</em> cannabinoid receptors by JWH133 protects against I/R damage by decreasing inflammatory cell infiltration, tissue and serum TNF-alpha, MIP-1alpha and MIP-<em>2</em> levels, tissue lipid peroxidation, and expression of adhesion molecule ICAM-1 in vivo. JWH133 also attenuates the TNF-alpha-induced ICAM-1 and VCAM-1 expression in human liver sinusoidal endothelial cells (HLSECs) and the adhesion of human neutrophils to HLSECs in vitro. Consistent with the protective role of CB<em>2</em> receptor activation, CB<em>2</em>-/- mice develop increased I/R-induced tissue damage and proinflammatory phenotype. These findings suggest that oxidative/nitrosative stress and inflammatory stimuli may trigger endocannabinoid production, and indicate that targeting CB<em>2</em> cannabinoid receptors may represent a novel protective strategy against I/R injury. We also demonstrate that CB<em>2</em>-/- mice have a normal hemodynamic profile.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is a unique molecular species of monoacylglycerol isolated in 1995 from rat brain and canine gut as an endogenous ligand for the cannabinoid receptors. <em>2</em>-AG is rapidly formed from arachidonic acid-containing phospholipids through increased phospholipid metabolism, such as enhanced inositol phospholipid turnover, in various tissues and cells upon stimulation. <em>2</em>-AG binds to the cannabinoid receptors (CB1 and CB<em>2</em>) and exhibits a variety of cannabimimetic activities in vitro and in vivo. Notably, anandamide, another endogenous ligand for the cannabinoid receptors, often acts as a partial agonist at these cannabinoid receptors, whereas <em>2</em>-AG acts as a full agonist in most cases. The results of structure-activity relationship studies suggested that <em>2</em>-AG rather than anandamide is the true natural ligand for both the CB1 and the CB<em>2</em> receptors. Evidence is gradually accumulating which shows that <em>2</em>-AG plays physiologically essential roles in diverse biological systems. For example, several lines of evidence indicate that <em>2</em>-AG plays an important role as a retrograde messenger molecule in the regulation of synaptic transmission. <em>2</em>-AG has also been shown to be involved in the regulation of various types of inflammatory reactions and immune responses. In this review, we focused on <em>2</em>-AG, and summarized information concerning its biosynthesis, metabolism, bioactions and physiological significance, including our latest experimental results.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is a naturally occurring monoglyceride that activates cannabinoid receptors and meets several key requisites of an endogenous cannabinoid substance. It is present in the brain (where its levels are 170-folds higher than those of anandamide), is produced by neurons in an activity- and calcium-dependent manner, and is rapidly eliminated. The mechanism of <em>2</em>-AG inactivation is not completely understood, but is thought to involve carrier-mediated transport into cells followed by enzymatic hydrolysis. We examined the possible role of the serine hydrolase, monoglyceride lipase (MGL), in brain <em>2</em>-AG inactivation. We identified by homology screening a cDNA sequence encoding for a 303-amino acid protein, which conferred MGL activity upon transfection to COS-7 cells. Northern blot and in situ hybridization analyses revealed that MGL mRNA is unevenly present in the rat brain, with highest levels in regions where CB1 cannabinoid receptors are also expressed (hippocampus, cortex, anterior thalamus and cerebellum). Immunohistochemical studies in the hippocampus showed that MGL distribution has striking laminar specificity, suggesting a presynaptic localization of the enzyme. Adenovirus-mediated transfer of MGL cDNA into rat cortical neurons increased the degradation of endogenously produced <em>2</em>-AG in these cells, whereas no such effect was observed on anandamide degradation. These results indicate that hydrolysis via MGL may be a primary route of <em>2</em>-AG inactivation in intact neuronal cells.

Endocannabinoids and N-acylethanolamines are lipid mediators regulating a wide range of biological functions including food intake. We investigated short-term effects of feeding rats five different dietary fats (palm oil (PO), olive oil (OA), safflower oil (LA), fish oil (FO) and arachidonic acid (AA)) on tissue levels of <em>2</em>-<em>arachidonoylglycerol</em>, anandamide, oleoylethanolamide, palmitoylethanolamide, stearoylethanolamide, linoleoylethanolamide, eicosapentaenoylethanolamide, docosahexaenoylethanolamide and tissue fatty acid composition. The LA-diet increased linoleoylethanolamide and linoleic acid in brain, jejunum and liver. The OA-diet increased brain levels of anandamide and oleoylethanolamide (not <em>2</em>-<em>arachidonoylglycerol</em>) without changing tissue fatty acid composition. The same diet increased oleoylethanolamide in liver. All five dietary fats decreased oleoylethanolamide in jejunum without changing levels of anandamide, suggesting that dietary fat may have an orexigenic effect. The AA-diet increased anandamide and <em>2</em>-<em>arachidonoylglycerol</em> in jejunum without effect on liver. The FO-diet decreased liver levels of all N-acylethanolamines (except eicosapentaenoylethanolamide and docosahexaenoylethanolamide) with similar changes in precursor lipids. The AA-diet and FO-diet had no effect on N-acylethanolamines, endocannabinoids or precursor lipids in brain. All N-acylethanolamines activated PPAR-alpha. In conclusion, short-term feeding of diets resembling human diets (Mediterranean diet high in monounsaturated fat, diet high in saturated fat, or diet high in polyunsaturated fat) can affect tissue levels of endocannabinoids and N-acylethanolamines.

Ethanol and opiate self-administration are sensitive to manipulations of cannabinoid CB1 receptor function and, from this, a role for the endogenous cannabinoid system in the modulation of drug reward has been hypothesized. However, direct in vivo evidence of drug-induced alterations in brain endocannabinoid (eCB) formation has been lacking. To address this issue, we explored the effect of drug self-administration on interstitial eCB levels in the nucleus accumbens (NAc) shell using in vivo microdialysis. Ethanol, heroin, and cocaine were compared because the rewarding properties of ethanol and heroin are reduced by CB1 receptor inactivation, whereas cocaine reward is less sensitive to these manipulations. Ethanol self-administration significantly increased dialysate <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) levels with no concomitant change in dialysate anandamide (AEA) concentrations. Conversely, heroin self-administration significantly increased dialysate AEA levels, and induced a subtle but significant decrease in dialysate <em>2</em>-AG levels. In each case, the relative change in dialysate eCB content was significantly correlated with the amount of drug consumed. In contrast, cocaine self-administration did not alter dialysate levels of either AEA or <em>2</em>-AG. Local infusion of the CB1 antagonist SR 141716A into the NAc significantly reduced ethanol, but not cocaine, self-administration. Together with our previous observation that intra-NAc SR 141716A reduces heroin self-administration, these data provide novel in vivo support for an eCB involvement in the motivational properties of ethanol and heroin but not cocaine. Furthermore, the selective effects of ethanol and heroin on interstitial <em>2</em>-AG and AEA provide new insight into the distinct neurochemical profiles produced by these two abused substances.

Secretion of glucocorticoid hormones during stress produces an array of physiological changes that are adaptive and beneficial in the short term. In the face of repeated stress exposure, however, habituation of the glucocorticoid response is essential as prolonged glucocorticoid secretion can produce deleterious effects on metabolic, immune, cardiovascular, and neurobiological function. Endocannabinoid signaling responds to and regulates the activity of the hypothalamic-pituitary-adrenal (HPA) axis that governs the secretion of glucocorticoids; however, the role this system plays in adaptation of the neuroendocrine response to repeated stress is not well characterized. Herein, we demonstrate a divergent regulation of the two endocannabinoid ligands, N-arachidonylethanolamine (anandamide; AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), following repeated stress such that AEA content is persistently decreased throughout the corticolimbic stress circuit, whereas <em>2</em>-AG is exclusively elevated within the amygdala in a stress-dependent manner. Pharmacological studies demonstrate that this divergent regulation of AEA and <em>2</em>-AG contribute to distinct forms of HPA axis habituation. Inhibition of AEA hydrolysis prevented the development of basal hypersecretion of corticosterone following repeated stress. In contrast, systemic or intra-amygdalar administration of a CB(1) receptor antagonist before the final stress exposure prevented the repeated stress-induced decline in corticosterone responses. The present findings demonstrate an important role for endocannabinoid signaling in the process of stress HPA habituation, and suggest that AEA and <em>2</em>-AG modulate different components of the adrenocortical response to repeated stressor exposure.

Food, drugs and brain stimulation can serve as strong rewarding stimuli and are all believed to activate common brain circuits that evolved in mammals to favour fitness and survival. For decades, endogenous dopaminergic and opioid systems have been considered the most important systems in mediating brain reward processes. Recent evidence suggests that the endogenous cannabinoid (endocannabinoid) system also has an important role in signalling of rewarding events. First, CB(1) receptors are found in brain areas involved in reward processes, such as the dopaminergic mesolimbic system. Second, activation of CB(1) receptors by plant-derived, synthetic or endogenous CB(1) receptor agonists stimulates dopaminergic neurotransmission, produces rewarding effects and increases rewarding effects of abused drugs and food. Third, pharmacological or genetic blockade of CB(1) receptors prevents activation of dopaminergic neurotransmission by several addictive drugs and reduces rewarding effects of food and these drugs. Fourth, brain levels of the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> are altered by activation of reward processes. However, the intrinsic activity of the endocannabinoid system does not appear to play a facilitatory role in brain stimulation reward and some evidence suggests it may even oppose it. The influence of the endocannabinoid system on brain reward processes may depend on the degree of activation of the different brain areas involved and might represent a mechanism for fine-tuning dopaminergic activity. Although involvement of the various components of the endocannabinoid system may differ depending on the type of rewarding event investigated, this system appears to play a major role in modulating reward processes.

The endogenous phospholipid l-alpha-lysophosphatidylinositol (LPI) was recently identified as a novel ligand for the orphan G protein-coupled receptor 55 (GPR55). In this study we define the downstream signaling pathways activated by LPI in a human embryonic kidney (HEK) <em>2</em>93 cell line engineered to stably express recombinant human GPR55. We find that treatment with LPI induces marked GPR55 internalization and stimulates a sustained, oscillatory Ca(<em>2</em>+) release pathway, which is dependent on Galpha13 and requires RhoA activation. We then establish that this signaling cascade leads to the efficient activation of NFAT (nuclear factor of activated T cells) family transcription factors and their nuclear translocation. Analysis of cannabinoid ligand activity at GPR55 revealed no clear effect of the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em>; however, the classical CB(1) antagonist AM<em>2</em>51 evoked GPR55-mediated Ca(<em>2</em>+) signaling. Thus, LPI is a potent and efficacious ligand at GPR55, which is likely to be a key plasma membrane mediator of LPI-mediated signaling events and changes in gene expression.

We examined the effect of <em>2</em>-<em>arachidonoylglycerol</em>, an endogenous cannabinoid receptor ligand, on the intracellular free Ca(<em>2</em>+) concentrations in HL-60 cells that express the cannabinoid CB<em>2</em> receptor. We found that <em>2</em>-<em>arachidonoylglycerol</em> induces a rapid transient increase in intracellular free Ca(<em>2</em>+) concentrations in HL-60 cells. The response was affected by neither cyclooxygenase inhibitors nor lipoxygenase inhibitors, suggesting that arachidonic acid metabolites are not involved. Consistent with this notion, free arachidonic acid was devoid of any agonistic activity. Importantly, the Ca(<em>2</em>+) transient induced by <em>2</em>-<em>arachidonoylglycerol</em> was blocked by pretreatment of the cells with SR1445<em>2</em>8, a CB<em>2</em> receptor-specific antagonist, but not with SR141716A, a CB1 receptor-specific antagonist, indicating the involvement of the CB<em>2</em> receptor but not the CB1 receptor in this cellular response. G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the cells abolished the response. We further examined the structure-activity relationship. We found that <em>2</em>-<em>arachidonoylglycerol</em> is the most potent compound among a number of naturally occurring cannabimimetic molecules. Interestingly, anandamide and N-palmitoylethanolamine, other putative endogenous ligands, were found to be a weak partial agonist and an inactive ligand, respectively. These results strongly suggest that the CB<em>2</em> receptor is originally a <em>2</em>-<em>arachidonoylglycerol</em> receptor, and <em>2</em>-<em>arachidonoylglycerol</em> is the intrinsic natural ligand for the CB<em>2</em> receptor that is abundant in the immune system.

Endocannabinoids are lipid molecules that serve as natural ligands for the cannabinoid receptors CB1 and CB<em>2</em>. They modulate a diverse set of physiological processes such as pain, cognition, appetite, and emotional states, and their levels and functions are tightly regulated by enzymatic biosynthesis and degradation. <em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is the most abundant endocannabinoid in the brain and is believed to be hydrolyzed primarily by the serine hydrolase monoacylglycerol lipase (MAGL). Although <em>2</em>-AG binds and activates cannabinoid receptors in vitro, when administered in vivo, it induces only transient cannabimimetic effects as a result of its rapid catabolism. Here we show using a mouse model with a targeted disruption of the MAGL gene that MAGL is the major modulator of <em>2</em>-AG hydrolysis in vivo. Mice lacking MAGL exhibit dramatically reduced <em>2</em>-AG hydrolase activity and highly elevated <em>2</em>-AG levels in the nervous system. A lack of MAGL activity and subsequent long-term elevation of <em>2</em>-AG levels lead to desensitization of brain CB1 receptors with a significant reduction of cannabimimetic effects of CB1 agonists. Also consistent with CB1 desensitization, MAGL-deficient mice do not show alterations in neuropathic and inflammatory pain sensitivity. These findings provide the first genetic in vivo evidence that MAGL is the major regulator of <em>2</em>-AG levels and signaling and reveal a pivotal role for <em>2</em>-AG in modulating CB1 receptor sensitization and endocannabinoid tone.

OBJECTIVE

Endocannabinoids are defined as endogenous agonists of cannabinoid receptors, that is, of the two G-protein-coupled receptors for the Cannabis psychoactive principle Delta-tetra-hydrocannabinol. Two such endogenous mediators have been most thoroughly studied so far: anandamide and <em>2</em>-<em>arachidonoylglycerol</em>. Here we review the mechanisms for the regulation of their levels under physiological and pathological conditions, and recent findings on their role in disease.

RESULTS

It is becoming increasingly clear that, although both anandamide and <em>2</em>-arachidonoyl-glycerol are produced and degraded 'on demand', the levels of these two compounds appear to be regulated in different, and sometimes even opposing, ways, often using redundant molecular mechanisms. Alterations of endocannabinoid levels have been found in both animal models of pain, neurological and neurodegenerative states, gastrointestinal disorders and inflammatory conditions, and in blood, cerebrospinal fluid and bioptic samples from patients with various diseases.

CONCLUSIONS

Endocannabinoid levels appear to be transiently elevated as an adaptive reaction to re-establish normal homeostasis when this is acutely and pathologically perturbed. In some chronic conditions, however, this system also contributes to the progress or symptoms of the disorder. As a consequence, new therapeutic drugs are being designed from both stimulants and blockers of endocannabinoid action.

In the ventrolateral periaqueductal gray (PAG), activation of excitatory output neurons projecting monosynaptically to OFF cells in the rostral ventromedial medulla (RVM) causes antinociceptive responses and is under the control of cannabinoid receptor type-1 (CB1) and vanilloid transient receptor potential vanilloid type 1 (TRPV1) receptors. We studied in healthy rats the effect of elevation of PAG endocannabinoid [anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG)] levels produced by intra-PAG injections of the inhibitor of fatty acid amide hydrolase URB597 [cyclohexylcarbamic acid-3'-carbamoyl-biphenyl-3-yl ester] on 1) nociception in the "plantar test" and <em>2</em>) spontaneous and tail-flick-related activities of RVM neurons. Depending on the dose or time elapsed since administration, URB597 (0.5-<em>2</em>.5 nmol/rat) either suppressed or increased thermal nociception via TRPV1 or CB1 receptors, respectively. TRPV1 or cannabinoid receptor agonists capsaicin (6 nmol) and (R)-(+)-[<em>2</em>,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,<em>2</em>,3,-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate [WIN55,<em>2</em>1<em>2</em>-<em>2</em> (4 nmol)] also suppressed or enhanced nociception, respectively. URB597 dose dependently enhanced PAG anandamide and <em>2</em>-AG levels, with probable subsequent activation of TRPV1/CB1 receptors and only CB1 receptors, respectively. The TRPV1-mediated antinociception and CB1-mediated nociception caused by URB597 correlated with enhanced or reduced activity of RVM OFF cells, suggesting that these effects occur via stimulation or inhibition of excitatory PAG output neurons, respectively. Accordingly, several ventrolateral PAG neurons were found by immunohistochemistry to coexpress TRPV1 and CB1 receptors. Finally, at the highest doses tested, URB597 (4 nmol/rat) and, as previously reported, WIN55,<em>2</em>1<em>2</em>-<em>2</em> (<em>2</em>5-100 nmol) also caused CB(1)-mediated analgesia, correlating with stimulation (possibly disinhibition) of RVM OFF cells. Thus, endocannabinoids affect the descending pathways of pain control by acting at either CB1 or TRPV1 receptors in healthy rats.

Endocannabinoids were defined in 1995 as endogenous agonists of cannabinoid receptors, i.e. of the G protein-coupled receptors for cannabis's psychoactive principle, Delta9-tetrahydrocannabinol. Although there appear to be several endocannabinoids, only two of such endogenous mediators have been thoroughly studied so far: anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). A general strategy seems to apply to the biosynthesis and degradation of anandamide and <em>2</em>-AG, although the levels of these two compounds appear to be regulated in different, and sometimes even opposing, ways. "Endocannabinoid enzymes", that is to say enzymes that catalyse endocannabinoid biosynthesis or degradation, have been identified and in some cases cloned, and will be described in this review together with their possible pharmacological targeting for therapeutic purposes. The cellular and subcellular localization and the modes for the regulation of the expression and activity of these enzymes play an important role in the functions played by the endocannabinoids under physiological and pathological conditions.

Two endogenous ligands for cannabinoid receptors, anandamide (arachidonylethanolamide) and <em>2</em>-<em>arachidonoylglycerol</em>, lose their biological activities by enzymatic hydrolysis. A cDNA for a rat liver enzyme hydrolyzing anandamide as well as oleamide was overexpressed in COS-7 cells. When the particulate fraction was allowed to react with <em>2</em>-<em>arachidonoylglycerol</em>, arachidonic acid was produced. In contrast, this hydrolytic reaction did not occur with the control cells. The hydrolysis of <em>2</em>-<em>arachidonoylglycerol</em> proceeded about 4-fold faster than the anandamide hydrolysis with a Km value as low as 6 microM and an optimal pH of 10. Phenylmethylsulfonyl fluoride and methyl arachidonyl fluorophosphonate inhibited the hydrolysis of both anandamide and <em>2</em>-<em>arachidonoylglycerol</em> in parallel. Furthermore, the hydrolysis of [14C]<em>2</em>-<em>arachidonoylglycerol</em> was inhibited by anandamide dose-dependently. These results suggest that anandamide and <em>2</em>-<em>arachidonoylglycerol</em> can be inactivated by the same enzyme.

Endocannabinoids mediate retrograde signaling and modulate synaptic transmission in various regions of the CNS. Depolarization-induced elevation of intracellular Ca<em>2</em>+ concentration causes endocannabinoid-mediated suppression of excitatory/inhibitory synaptic transmission. Activation of G(q/11)-coupled receptors including group I metabotropic glutamate receptors (mGluRs) also causes endocannabinoid-mediated suppression of synaptic transmission. However, precise mechanisms of endocannabinoid production initiated by physiologically relevant synaptic activity remain to be determined. To address this problem, we made whole-cell recordings from Purkinje cells (PCs) in mouse cerebellar slices and examined their excitatory synapses arising from climbing fibers (CFs) and parallel fibers (PFs). We first characterized three distinct modes to induce endocannabinoid release by analyzing CF to PC synapses. The first mode is strong activation of mGluR subtype 1 (mGluR1)-phospholipase C (PLC) beta4 cascade without detectable Ca<em>2</em>+ elevation. The second mode is Ca<em>2</em>+ elevation to a micromolar range without activation of the mGluR1-PLCbeta4 cascade. The third mode is the Ca<em>2</em>+-assisted mGluR1-PLCbeta4 cascade that requires weak mGluR1 activation and Ca<em>2</em>+ elevation to a submicromolar range. By analyzing PF to PC synapses, we show that the third mode is essential for effective endocannabinoid release from PCs by excitatory synaptic activity. Furthermore, our biochemical analysis demonstrates that combined weak mGluR1 activation and mild depolarization in PCs effectively produces <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), a candidate of endocannabinoid, whereas either stimulus alone did not produce detectable <em>2</em>-AG. Our results strongly suggest that under physiological conditions, excitatory synaptic inputs to PCs activate the Ca<em>2</em>+-assisted mGluR1-PLCbeta4 cascade, and thereby produce <em>2</em>-AG, which retrogradely modulates synaptic transmission to PCs.

In 1964, the psychoactive ingredient of Cannabis sativa, Δ(9)-tetrahydrocannabinol (THC), was isolated. Nearly 30 years later the endogenous counterparts of THC, collectively termed endocannabinoids (eCBs), were discovered: N-arachidonoylethanolamine (anandamide) (AEA) in 199<em>2</em> and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in 1995. Since then, considerable research has shed light on the impact of eCBs on human health and disease, identifying an ensemble of proteins that bind, synthesize, and degrade them and that together form the eCB system (ECS). eCBs control basic biological processes including cell choice between survival and death and progenitor/stem cell proliferation and differentiation. Unsurprisingly, in the past two decades eCBs have been recognized as key mediators of several aspects of human pathophysiology and thus have emerged to be among the most widespread and versatile signaling molecules ever discovered. Here some of the pioneers of this research field review the state of the art of critical eCB functions in peripheral organs. Our community effort is aimed at establishing consensus views on the relevance of the peripheral ECS for human health and disease pathogenesis, as well as highlighting emerging challenges and therapeutic hopes.

BACKGROUND

Cannabinoid agonists are potential therapeutic agents because of their antinociceptive and anxiolytic-like effects, although an important caveat to their use is the possible adverse responses related to memory impairment. An alternative approach to circumvent this limitation consists of enhancing the concentration of the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em>.

METHODS

Using low doses of the specific inhibitors of the endocannabinoid metabolizing enzymes fatty acid amide hydrolase, URB597, and monoacylglycerol lipase, JZL184, we analyzed their acute and chronic effects on memory consolidation, anxiolytic-like effects, and nociception in mice (n = 6-1<em>2</em> per experimental group).

RESULTS

We show that anandamide is a central component in the modulation of memory consolidation, whereas <em>2</em>-<em>arachidonoylglycerol</em> is not involved in this process. Interestingly, both URB597 and JZL184 induce anxiolytic-like effects through different cannabinoid receptors. In addition, the results show that the antinociceptive and anxiolytic-like responses of both inhibitors, as well as their acute effects on memory consolidation, are maintained after chronic treatment.

CONCLUSIONS

These results dissociate the role of anandamide and <em>2</em>-<em>arachidonoylglycerol</em> in memory consolidation and anxiety and reveal the interest of cannabinoid receptor <em>2</em> as a novel target for the treatment of anxiety-related disorders.

The mechanisms subserving the ability of glucocorticoid signaling within the medial prefrontal cortex (mPFC) to terminate stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis are not well understood. We report that antagonism of the cannabinoid CB(1) receptor locally within the mPFC prolonged corticosterone secretion following cessation of stress in rats. Mice lacking the CB(1) receptor exhibited a similar prolonged response to stress. Exposure of rats to stress produced an elevation in the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> within the mPFC that was reversed by pretreatment with the glucocorticoid receptor antagonist RU-486 (<em>2</em>0 mg/kg). Electron microscopic and electrophysiological data demonstrated the presence of CB(1) receptors in inhibitory-type terminals impinging upon principal neurons within layer V of the prelimbic region of the mPFC. Bath application of corticosterone (100 nm) to prefrontal cortical slices suppressed GABA release onto principal neurons in layer V of the prelimbic region, when examined 1 h later, which was prevented by application of a CB(1) receptor antagonist. Collectively, these data demonstrate that the ability of stress-induced glucocorticoid signaling within mPFC to terminate HPA axis activity is mediated by a local recruitment of endocannabinoid signaling. Endocannabinoid activation of CB(1) receptors decreases GABA release within the mPFC, likely increasing the outflow of the principal neurons of the prelimbic region to contribute to termination of the stress response. These data support a model in which endocannabinoid signaling links glucocorticoid receptor engagement to activation of corticolimbic relays that inhibit corticosterone secretion.

Activation of group I metabotropic glutamate (mGlu) receptors drives the endocannabinoid system to cause both short- and long-term changes of synaptic strength in the striatum, hippocampus, and other brain areas. Although there is strong electrophysiological evidence for a role of endocannabinoid release in mGlu receptor-dependent plasticity, the identity of the endocannabinoid transmitter mediating this phenomenon remains undefined. In this study, we show that activation of group I mGlu receptors triggers the biosynthesis of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), but not anandamide, in primary cultures of corticostriatal and hippocampal slices prepared from early postnatal rat brain. Pharmacological studies suggest that <em>2</em>-AG biosynthesis is initiated by activation of mGlu5 receptors, is catalyzed by phospholipase C (PLC) and 1,<em>2</em>-diacylglycerol lipase (DGL) activities, and is dependent on intracellular Ca<em>2</em>+ ions. Realtime polymerase chain reaction and immunostaining analyses indicate that DGL-beta is the predominant DGL isoform expressed in corticostriatal and hippocampal slices and that this enzyme is highly expressed in striatal neurons, where it is colocalized with PLC-beta1. The results suggest that <em>2</em>-AG is a primary endocannabinoid mediator of mGlu receptor-dependent neuronal plasticity.

Endocannabinoid (eCB) signaling mediates depolarization-induced suppression of excitation (DSE) and inhibition (DSI), two prominent forms of retrograde synaptic depression. N-Arachidonoylethanolamine (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), two known eCBs, are degraded by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. Selective blockade of FAAH and MAGL is critical for determining the roles of the eCBs in DSE/DSI and understanding how their action is regulated. 4-Nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) is a recently developed, highly selective, and potent MAGL inhibitor that increases <em>2</em>-AG but not AEA concentrations in mouse brain. Here, we report that JZL184 prolongs DSE in Purkinje neurons in cerebellar slices and DSI in CA1 pyramidal neurons in hippocampal slices. The effect of JZL184 on DSE/DSI is mimicked by the nonselective MAGL inhibitor methyl arachidonyl fluorophosphonate. In contrast, neither the selective FAAH inhibitor cyclohexylcarbamic acid 3'-carbomoylbiphenyl-3-yl ester (URB597) nor FAAH knockout has a significant effect on DSE/DSI. JZL184 produces greater enhancement of DSE/DSI in mouse neurons than that in rat neurons. The latter finding is consistent with biochemical studies showing that JZL184 is more potent in inhibiting mouse MAGL than rat MAGL. These results indicate that the degradation of <em>2</em>-AG by MAGL is the rate-limiting step that determines the time course of DSE/DSI and that JZL184 is a useful tool for the study of <em>2</em>-AG-mediated signaling.