Ibuprofen and mefenamic acid are weak, competitive inhibitors of cyclooxygenase-<em>2</em> (COX-<em>2</em>) oxygenation of arachidonic acid (AA) but potent, noncompetitive inhibitors of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) oxygenation. The slow, tight-binding inhibitor, indomethacin, is a potent inhibitor of <em>2</em>-AG and AA oxygenation whereas the rapidly reversible inhibitor, <em>2</em>'-des-methylindomethacin, is a potent inhibitor of <em>2</em>-AG oxygenation but a poor inhibitor of AA oxygenation. These observations are consistent with a model in which inhibitors bind in one subunit of COX-<em>2</em> and inhibit <em>2</em>-AG binding in the other subunit of the homodimeric protein. In contrast, ibuprofen and mefenamate must bind in both subunits to inhibit AA binding.

The endocannabinoid system consists of the cannabinoid (CB) receptors, CB(1) and CB(<em>2</em>), the endogenous ligands anandamide (AEA, arachidonoylethanolamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and their synthetic and metabolic machinery. The use of cannabis has been described in classical and recent literature for the treatment of pain, but the potential for psychotropic effects as a result of the activation of central CB(1) receptors places a limitation upon its use. There are, however, a number of modern approaches being undertaken to circumvent this problem, and this review represents a concise summary of these approaches, with a particular emphasis upon CB(<em>2</em>) receptor agonists. Selective CB(<em>2</em>) agonists and peripherally restricted CB(1) or CB(1)/CB(<em>2</em>) dual agonists are being developed for the treatment of inflammatory and neuropathic pain, as they demonstrate efficacy in a range of pain models. CB(<em>2</em>) receptors were originally described as being restricted to cells of immune origin, but there is evidence for their expression in human primary sensory neurons, and increased levels of CB(<em>2</em>) receptors reported in human peripheral nerves have been seen after injury, particularly in painful neuromas. CB(<em>2</em>) receptor agonists produce antinociceptive effects in models of inflammatory and nociceptive pain, and in some cases these effects involve activation of the opioid system. In addition, CB receptor agonists enhance the effect of mu-opioid receptor agonists in a variety of models of analgesia, and combinations of cannabinoids and opioids may produce synergistic effects. Antinociceptive effects of compounds blocking the metabolism of anandamide have been reported, particularly in models of inflammatory pain. There is also evidence that such compounds increase the analgesic effect of non-steroidal anti-inflammatory drugs (NSAIDs), raising the possibility that a combination of suitable agents could, by reducing the NSAID dose needed, provide an efficacious treatment strategy, while minimizing the potential for NSAID-induced gastrointestinal and cardiovascular disturbances. Other potential "partners" for endocannabinoid modulatory agents include alpha(<em>2</em>)-adrenoceptor modulators, peroxisome proliferator-activated receptor alpha agonists and TRPV1 antagonists. An extension of the polypharmacological approach is to combine the desired pharmacological properties of the treatment within a single molecule. Hopefully, these approaches will yield novel analgesics that do not produce the psychotropic effects that limit the medicinal use of cannabis.

Palmitoylethanolamide (PEA) and oleamide are two fatty acid amides which 1) share some cannabimimetic actions with delta9-tetrahydrocannabinol, anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, and <em>2</em>) may interact with proteins involved in the biosynthesis, action and inactivation of endocannabinoids. Due to its pharmacological actions and its accumulation in damaged cells, PEA may have a physio-pathological role as an analgesic, anti-oxidant and anti-inflammatory mediator. However, its mechanism of action is puzzling. In fact, PEA does not bind to CB1 and CB<em>2</em> receptors transfected into host cells, but might be a ligand for a putative CBn receptor present in the RBL-<em>2</em>H3 cell line. On the other hand, the analgesic effect of PEA is reversed by SR1445<em>2</em>8, a CB<em>2</em> antagonist. PEA may act as an entourage compound for endocannabinoids, i.e. it may enhance their action for example by inhibiting their inactivation. Oleamide is a sleep inducing lipid whose mechanism of action is far from being understood. Although it does not bind with high affinity to CB1 or CB<em>2</em> receptors, it exhibits some cannabimimetic actions which could be explained at least in part by entourage effects. It is likely that oleamide and anandamide have common as well as distinct pathways of action. The 5-HT<em>2</em>A receptor appears to be a target for oleamide but the possibility of the existence of specific receptors for this compound is open. The biosynthesis and tissue distribution of oleamide remain to be assessed in order to both substantiate its role as a sleep-inducing factor and investigate its participation in other physiopathological situations.

The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is a lipid mediator involved in various physiological processes. In response to neural activity, <em>2</em>-AG is synthesized post-synaptically, then activates pre-synaptic cannabinoid CB1 receptors (CB1Rs) in a retrograde manner, resulting in transient and long-lasting reduction of neurotransmitter release. The signalling competence of <em>2</em>-AG is tightly regulated by the balanced action between 'on demand' biosynthesis and degradation. We review recent research on monoacylglycerol lipase (MAGL), ABHD6 and ABHD1<em>2</em>, three serine hydrolases that together account for approx. 99% of brain <em>2</em>-AG hydrolase activity. MAGL is responsible for approx. 85% of <em>2</em>-AG hydrolysis and colocalizes with CB1R in axon terminals. It is therefore ideally positioned to terminate <em>2</em>-AG-CB1R signalling regardless of the source of this endocannabinoid. Its acute pharmacological inhibition leads to <em>2</em>-AG accumulation and CB1R-mediated behavioural responses. Chronic MAGL inactivation results in <em>2</em>-AG overload, desensitization of CB1R signalling and behavioural tolerance. ABHD6 accounts for approx. 4% of brain <em>2</em>-AG hydrolase activity but in neurones it rivals MAGL in efficacy. Neuronal ABHD6 resides post-synaptically, often juxtaposed with CB1Rs, and its acute inhibition leads to activity-dependent accumulation of <em>2</em>-AG. In cortical slices, selective ABHD6 blockade facilitates CB1R-dependent long-term synaptic depression. ABHD6 is therefore positioned to guard intracellular pools of <em>2</em>-AG at the site of generation. ABHD1<em>2</em> is highly expressed in microglia and accounts for approx. 9% of total brain <em>2</em>-AG hydrolysis. Mutations in ABHD1<em>2</em> gene are causally linked to a neurodegenerative disease called PHARC. Whether ABHD1<em>2</em> qualifies as a bona fide member to the endocannabinoid system remains to be established.

The reason why neurons synthesize more than one endocannabinoid (eCB) and how this is involved in the regulation of synaptic plasticity in a single neuron is not known. We found that <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide mediate different forms of plasticity in the extended amygdala of rats. Dendritic L-type Ca(<em>2</em>+) channels and the subsequent release of <em>2</em>-AG acting on presynaptic CB1 receptors triggered retrograde short-term depression. Long-term depression was mediated by postsynaptic mGluR5-dependent release of anandamide acting on postsynaptic TRPV1 receptors. In contrast, <em>2</em>-AG/CB1R-mediated retrograde signaling mediated both forms of plasticity in the striatum. These data illustrate how the eCB system can function as a polymodal signal integrator to allow the diversification of synaptic plasticity in a single neuron.

One of the two major endocannabinoids, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), serves as a retrograde messenger at various types of synapses throughout the brain. Upon postsynaptic activation, <em>2</em>-AG is released immediately after de novo synthesis, activates presynaptic CB1 cannabinoid receptors, and transiently suppresses neurotransmitter release. When CB1 receptor activation is combined with some other factors such as presynaptic activity, the suppression is converted to a long-lasting form. Whereas <em>2</em>-AG primarily transmits a rapid, transient, point-to-point retrograde signal, the other major endocannabinoid, anandamide, may function as a relatively slow retrograde or non-retrograde signal or as an agonist of the vanilloid receptor. The endocannabinoid system can be up- or down-regulated by a variety of physiological and environmental factors including stress, which might be clinically important.

Endocannabinoids acting on CB(1) cannabinoid receptors are involved in short- and long-term depression of synaptic transmission. The aim of the present study was to determine which endocannabinoid, anandamide or <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), is involved in depolarization-induced suppression of inhibition (DSI) in the cerebellar cortex, which is the most widely studied form of short-term depression. Depolarization of Purkinje cells in the mouse cerebellum led to an increase in intracellular calcium concentration and to suppression of the inhibitory input to these neurons (i.e. DSI occurred). Orlistat and RHC80<em>2</em>67, two blockers of sn-1-diacylglycerol lipase, the enzyme catalysing <em>2</em>-AG formation, abolished DSI by acting downstream of calcium influx. In contrast, DSI occurred also in the presence of a phospholipase C inhibitor. Intact operation of the calcium-dependent messengers calmodulin and Ca(<em>2</em>+)-calmodulin-dependent protein kinase II were necessary for DSI. DSI was potentiated by an inhibitor of the main <em>2</em>-AG-degrading enzyme, monoacylglycerol lipase. Interference with the anandamide metabolizing enzyme, fatty acid amide hydrolase, did not modify DSI. Thus, three kinds of observations identified <em>2</em>-AG as the endocannabinoid involved in DSI in the mouse cerebellum: DSI was abolished by diacylglycerol lipase inhibitors; DSI was potentiated by a monoglyceride lipase inhibitor; and DSI was not changed by an inhibitor of fatty acid amide hydrolase. Further experiments indicated that <em>2</em>-AG is the endocannabinoid mediating short-term retrograde signalling also at other synapses: orlistat abolished DSI in the rat cerebellum, DSI in the mouse substantia nigra pars reticulata and depolarization-induced suppression of excitation in the mouse cerebellum.

Increasing evidence indicates that endocannabinoid (EC) signalling is dysregulated during hyperglycemia and obesity, particularly at the level of anandamide (AEA) and/or <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) concentrations in tissues involved in the control of energy intake and processing, such as the liver, white adipose tissue and pancreas. Here we review this previous evidence and provide new data on the possible dysregulation of EC levels in organs with endocrine function (adrenal glands and thyroid), involved in energy expenditure (brown adipose tissue and skeletal muscle), or affected by the consequences of metabolic disorders (heart and kidney), obtained from mice fed for 3, 8 and 14 weeks with two different high fat diets (HFDs), with different fatty acid compositions and impact on fasting glucose levels. Statistically significant elevations (in the skeletal muscle, heart and kidney) or reductions (in the thyroid) of the levels of either AEA or <em>2</em>-AG, or both, were found. Depending on the diet, these changes preceded or accompanied the development of overt obesity and/or hyperglycemia. In the adrenal gland, first a reduction and then an elevation of EC levels were observed. In the brown fat, a very early elevation of both AEA and <em>2</em>-AG normalized levels was observed with one of the diets, whereas delayed decreases were explained by an increase of the amount of fat tissue weight induced by the HFDs. The potential implications of these and previous findings in the general framework of the proposed roles of the EC system in the control of metabolic, endocrine and cardiovascular and renal functions are discussed.

We reported earlier that closed head injury (CHI) in mice causes a sharp elevation of brain <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) levels, and that exogenous <em>2</em>-AG reduces brain edema, infarct volume and hippocampal death and improved clinical recovery after CHI. The beneficial effect of <em>2</em>-AG was attenuated by SR141716A, a CB1 cannabinoid receptor antagonist, albeit at relatively high doses. In the present study, we further explored the role of CB1 receptors in mediating <em>2</em>-AG neuroprotection. CB1 receptor knockout mice (CB1-/-) showed minor spontaneous recovery at <em>2</em>4 h after CHI, in contrast to the significant improvement in neurobehavioral function seen in wild-type (WT) mice. Moreover, administration of <em>2</em>-AG did not improve neurological performance and edema formation in the CB1-/- mice. In addition, <em>2</em>-AG abolished the three- to four-fold increase of nuclear factor kappaB (NF-kappa B) transactivation, at <em>2</em>4 h after CHI in the WT mice, while it had no effect on NF-kappaB in the CB1-/- mice, which was as high as in the WT vehicle-treated mice. We thus propose that <em>2</em>-AG exerts its neuroprotection after CHI, at least in part, via CB1 receptor-mediated mechanisms that involve inhibition of intracellular inflammatory signaling pathways.

Cannabinoids are antinociceptive in animal models of acute, tissue injury-, and nerve injury-induced nociception. This review examines the biology of endogenous cannabinoids (endocannabinoids) and behavioral, neurophysiological, and neuroanatomical evidence supporting the notion that cannabinoids play a role in pain modulation. Behavioral pharmacological approaches, in conjunction with the identification and quantification of endocannabinoids through the use of liquid and gas chromatography mass spectrometry, have provided insight into the functional roles of endocannabinoids in pain modulation. Here we examine the distribution of cannabinoid receptors and endocannabinoid-hydrolyzing enzymes within pain modulatory circuits together with behavioral, neurochemical, and neurophysiological studies that suggest a role for endocannabinoid signaling in pain modulation. This review will provide a comprehensive evaluation of the roles of the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> and anandamide in stress-induced analgesia. These findings provide a functional framework with which to understand the roles of endocannabinoids in nociceptive processing at the supraspinal level.

The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is biosynthesized by diacylglycerol lipases DAGLα and DAGLβ. Chemical probes to perturb DAGLs are needed to characterize endocannabinoid function in biological processes. Here we report a series of 1,<em>2</em>,3-triazole urea inhibitors, along with paired negative-control and activity-based probes, for the functional analysis of DAGLβ in living systems. Optimized inhibitors showed high selectivity for DAGLβ over other serine hydrolases, including DAGLα (∼60-fold selectivity), and the limited off-targets, such as ABHD6, were also inhibited by the negative-control probe. Using these agents and Daglb(-/-) mice, we show that DAGLβ inactivation lowers <em>2</em>-AG, as well as arachidonic acid and eicosanoids, in mouse peritoneal macrophages in a manner that is distinct and complementary to disruption of cytosolic phospholipase-A<em>2</em>. We observed a corresponding reduction in lipopolysaccharide-induced tumor necrosis factor-α release. These findings indicate that DAGLβ is a key metabolic hub within a lipid network that regulates proinflammatory responses in macrophages.

OBJECTIVE

Recombinant cyclooxygenase-<em>2</em> (COX-<em>2</em>) oxygenates <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in vitro. We examined whether prostaglandin E<em>2</em> glycerol ester (PGE<em>2</em>-G), a COX-<em>2</em> metabolite of <em>2</em>-AG, occurs endogenously and affects nociception and immune responses.

METHODS

Using mass spectrometric techniques, we examined whether PGE<em>2</em>-G occurs in vivo and if its levels are altered by inhibition of COX-<em>2</em>, monoacylglycerol (MAG) lipase or inflammation induced by carrageenan. We also examined the effects of PGE<em>2</em>-G on nociception in rats and NFkappaB activity in RAW<em>2</em>64.7 cells.

RESULTS

PGE<em>2</em>-G occurs endogenously in rat. Its levels were decreased by inhibition of COX-<em>2</em> and MAG lipase but were unaffected by carrageenan. Intraplantar administration of PGE<em>2</em>-G induced mechanical allodynia and thermal hyperalgesia. In RAW<em>2</em>64.7 cells, PGE<em>2</em>-G and PGE<em>2</em> produced similar, dose-related changes in NFkappaB activity. PGE<em>2</em>-G was quickly metabolized into PGE<em>2</em>. While the effects of PGE<em>2</em> on thermal hyperalgesia and NFkappaB activity were completely blocked by a cocktail of antagonists for prostanoid receptors, the same cocktail of antagonists only partially antagonized the actions of PGE<em>2</em>-G.

CONCLUSIONS

Thermal hyperalgesia and immunomodulation induced by PGE<em>2</em>-G were only partially mediated by PGE<em>2</em>, which is formed by metabolism of PGE<em>2</em>-G. PGE<em>2</em>-G may function through a unique receptor previously postulated to mediate its effects. Taken together, these findings demonstrate that <em>2</em>-AG is oxygenated in vivo by COX-<em>2</em> producing PGE<em>2</em>-G, which plays a role in pain and immunomodulation. COX-<em>2</em> could act as an enzymatic switch by converting <em>2</em>-AG from an antinociceptive mediator to a pro-nociceptive prostanoid.

Inflammatory bowel diseases (IBDs) are chronic inflammatory conditions for which new therapeutic approaches are needed. Genetic and pharmacological data point to a protective role of CB(1) and CB(<em>2</em>) cannabinoid receptor activation in IBD experimental models. Therefore, increasing the endogenous levels of <em>2</em>-<em>arachidonoylglycerol</em>, the main full agonist of these receptors, should have beneficial effects on colitis. <em>2</em>-<em>Arachidonoylglycerol</em> levels were raised in the trinitrobenzene sulfonic acid (TNBS)-induced colitis mouse model by inhibiting monoacylglycerol lipase (MAGL), the primary enzyme responsible for hydrolysis of <em>2</em>-<em>arachidonoylglycerol</em>, using the selective inhibitor JZL184. MAGL inhibition in diseased mice increased <em>2</em>-<em>arachidonoylglycerol</em> levels, leading to a reduction of macroscopic and histological colon alterations, as well as of colonic expression of proinflammatory cytokines. The restored integrity of the intestinal barrier function after MAGL inhibition resulted in reduced endotoxemia as well as reduced peripheral and brain inflammation. Coadministration of either CB(1) (SR141716A) or CB(<em>2</em>) (AM630) selective antagonists with JZL184 completely abolished the protective effect of MAGL inhibition on TNBS-induced colon alterations, thus demonstrating the involvement of both cannabinoid receptors. In conclusion, increasing <em>2</em>-<em>arachidonoylglycerol</em> levels resulted in a dramatic reduction of colitis and of the related systemic and central inflammation. This could offer a novel pharmacological approach for the treatment of IBD based on the new protective role of <em>2</em>-<em>arachidonoylglycerol</em> described here.

Endocannabinoid signaling is a key regulator of synaptic neurotransmission throughout the brain. Compelling evidence shows that its perturbation leads to development of epileptic seizures, thus indicating that endocannabinoids play an intrinsic protective role in suppressing pathologic neuronal excitability. To elucidate whether long-term reorganization of endocannabinoid signaling occurs in epileptic patients, we performed comparative expression profiling along with quantitative electron microscopic analysis in control (postmortem samples from subjects with no signs of neurological disorders) and epileptic (surgically removed from patients with intractable temporal lobe epilepsy) hippocampal tissue. Quantitative PCR measurements revealed that CB(1) cannabinoid receptor mRNA was downregulated to one-third of its control value in epileptic hippocampus. Likewise, the cannabinoid receptor-interacting protein-1a mRNA was decreased, whereas 1b isoform levels were unaltered. Expression of diacylglycerol lipase-alpha, an enzyme responsible for <em>2</em>-<em>arachidonoylglycerol</em> synthesis, was also reduced by approximately 60%, whereas its related beta isoform levels were unchanged. Expression level of N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D and fatty acid amide hydrolase, metabolic enzymes of anandamide, and <em>2</em>-<em>arachidonoylglycerol</em>'s degrading enzyme monoacylglycerol lipase did not change. The density of CB(1) immunolabeling was also decreased in epileptic hippocampus, predominantly in the dentate gyrus, where quantitative electron microscopic analysis did not reveal changes in the ratio of CB(1)-positive GABAergic boutons, but uncovered robust reduction in the fraction of CB(1)-positive glutamatergic axon terminals. These findings show that a neuroprotective machinery involving endocannabinoids is impaired in epileptic human hippocampus and imply that downregulation of CB(1) receptors and related molecular components of the endocannabinoid system may facilitate the deleterious effects of increased network excitability.

Cyclooxygenase-<em>2</em> (COX-<em>2</em>) catalyzes the oxygenation of arachidonic acid and the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> and arachidonoylethanolamide. Evaluation of a series of COX-<em>2</em> inhibitors revealed that many weak competitive inhibitors of arachidonic acid oxygenation are potent inhibitors of endocannabinoid oxygenation. (R) enantiomers of ibuprofen, naproxen and flurbiprofen, which are considered to be inactive as COX-<em>2</em> inhibitors, are potent 'substrate-selective inhibitors' of endocannabinoid oxygenation. Crystal structures of the COX-<em>2</em>–(R)-naproxen and COX-<em>2</em>–(R)-flurbiprofen complexes verified this unexpected binding and defined the orientation of the (R) enantiomers relative to (S) enantiomers. (R)-Profens selectively inhibited endocannabinoid oxygenation by lipopolysaccharide-stimulated dorsal root ganglion (DRG) cells. Substrate-selective inhibition provides new tools for investigating the role of COX-<em>2</em> in endocannabinoid oxygenation and a possible explanation for the ability of (R)-profens to maintain endocannabinoid tone in models of neuropathic pain.

Adolescence is a critical phase of active brain development often characterized by the initiation of marijuana (Cannabis sativa) use. Limited information is known regarding the endogenous cannabinoid system of the adolescent brain as well as related neurotransmitters that appear sensitive to cannabis exposure. We recently observed that adult rats pre-exposed to Delta-9-tetrahydrocannabinol (THC) during adolescence self-administered higher amounts of heroin and had selective impairments of the enkephalin opioid system within the nucleus accumbens (NAc) implicated in reward-related behavior. To explore the ontogeny of the cannabinoid and opioid neuronal systems in association with adolescence THC exposure, rats were examined at different adolescent stages during an intermittent THC paradigm (1.5 mg/kg i.p. every third day) from postnatal days (PNDs) <em>2</em>8-49. Rat brains were examined <em>2</em>4 h after injection at PND <em>2</em>9 (early adolescence), PND 38 (mid adolescence) and PND 50 (late adolescence) and analyzed for endocannabinoids (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>), Met-enkephalin, cannabinoid CB(1) receptors and micro opioid receptors (microOR) in the NAc, caudate-putamen and prefrontal cortex (PFC). Of the markers studied, the endocannabinoid levels had the most robust alterations throughout adolescence and were specific to the PFC and NAc. Normal correlations between anandamide and <em>2</em>-<em>arachidonoylglycerol</em> concentrations in the NAc (positive) and PFC (negative) were reversed by THC. Other significant THC-induced effects were confined to the NAc - increased anandamide, decreased Met-enkephalin and decreased microORs. These findings emphasize the dynamic nature of the mesocorticolimbic endocannabinoid system during adolescence and the selective mesocorticolimbic disturbance as a consequence of adolescent cannabis exposure.

Intact endogenous cannabinoid signaling is involved in several aspects of drug addiction. Most importantly, endocannabinoids exert pronounced influence on primary rewarding effects of abused drugs, including exogenous cannabis itself, through the regulation of drug-induced increase in bursting activity of dopaminergic neurons in the ventral tegmental area (VTA). Previous electrophysiological studies have proposed that these dopaminergic neurons may release endocannabinoids in an activity-dependent manner to regulate their various synaptic inputs; however, the underlying molecular and anatomical substrates have so far been elusive. To facilitate understanding of the neurobiological mechanisms involving endocannabinoid signaling in drug addiction, we carried out detailed analysis of the molecular architecture of the endocannabinoid system in the VTA. In situ hybridization for sn-1-diacylglycerol lipase-alpha (DGL-alpha), the biosynthetic enzyme of the most abundant endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), revealed that DGL-alpha was expressed at moderate to high levels by most neurons of the VTA. Immunostaining for DGL-alpha resulted in a widespread punctate pattern at the light microscopic level, whereas high-resolution electron microscopic analysis demonstrated that this pattern is due to accumulation of the enzyme adjacent to postsynaptic specializations of several distinct morphological types of glutamatergic and GABAergic synapses. These axon terminal types carried presynaptic CB(1) cannabinoid receptors on the opposite side of DGL-alpha-containing synapses and double immunostaining confirmed that DGL-alpha is present on the plasma membrane of both tyrosine hydroxylase (TH)-positive (dopaminergic) and TH-negative dendrites. These findings indicate that retrograde synaptic signaling mediated by <em>2</em>-AG via CB(1) may influence the drug-reward circuitry at multiple types of synapses in the VTA.

Psychosocial stress is a risk factor for development and exacerbation of neuropsychiatric illness. Repeated stress causes biochemical adaptations in endocannabinoid (eCB) signaling that contribute to stress-response habituation, however, the synaptic correlates of these adaptations have not been examined. Here, we show that the synthetic enzyme for the eCB <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), diacylglycerol (DAG) lipase alpha, is heterogeneously expressed in the amygdala, and that levels of <em>2</em>-AG and precursor DAGs are increased in the basolateral amygdala (BLA) after 10 days, but not 1 day, of restraint stress. In contrast, arachidonic acid was decreased after both 1 and 10 days of restraint stress. To examine the synaptic correlates of these alterations in <em>2</em>-AG metabolism, we used whole-cell electrophysiology to determine the effects of restraint stress on depolarization-induced suppression of inhibition (DSI) in the BLA. A single restraint stress exposure did not alter DSI compared with control mice. However, after 10 days of restraint stress, DSI duration, but not magnitude, was significantly prolonged. Inhibition of <em>2</em>-AG degradation with MAFP also prolonged DSI duration; the effects of repeated restraint stress and MAFP were mutually occlusive. These data indicate that exposure to repeated, but not acute, stress produces neuroadaptations that confer BLA neurons with an enhanced capacity to elevate <em>2</em>-AG content and engage in <em>2</em>-AG-mediated short-term retrograde synaptic signaling. We suggest stress-induced enhancement of eCB-mediated suppression of inhibitory transmission in the BLA could contribute to affective dysregulation associated with chronic stress.

OBJECTIVE

We previously reported that the plasma levels of the endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), in a cohort of viscerally obese men are directly correlated with visceral adipose tissue (VAT) accumulation and metabolic risk factors including low HDL-cholesterol and high triacylglycerol. It is not known, however, if such correlations persist after vigorous lifestyle interventions that reduce metabolic risk factors. We analysed the changes in endocannabinoid levels in a subsample from the same cohort following a 1 year lifestyle modification programme, and correlated them with changes in VAT and metabolic risk factors.

METHODS

Forty-nine viscerally obese men (average age 49 years, BMI 30.9 kg/m(<em>2</em>), waist 107.3 cm) underwent a 1 year lifestyle modification programme including healthy eating and physical activity. Plasma levels of <em>2</em>-AG and the other most studied endocannabinoid, anandamide, were measured by liquid chromatography-mass spectrometry. Anthropometric and metabolic risk factors, including VAT, insulin resistance and glucose intolerance, HDL-cholesterol and triacylglycerol, were measured.

RESULTS

Most risk factors were improved by the intervention, which led to a significant decrease in body weight (-6.4 kg, p < 0.0001), waist circumference (-8.0 cm, p < 0.0001) and VAT (-30%, p < 0.0001), and in plasma <em>2</em>-AG (-6<em>2</em>.3%, p < 0.0001) and anandamide (-7.1%, p = 0.005) levels. The decrease in levels of <em>2</em>-AG but not those of anandamide correlated with decreases in VAT and triacylglycerol levels, and with the increase in HDL(3)-cholesterol levels. Multivariate analyses suggested that decreases in <em>2</em>-AG and VAT were both independently associated with decreases in triacylglycerol.

CONCLUSIONS

This study shows that a strong correlation exists between <em>2</em>-AG levels and high plasma triacylglycerol and low HDL(3)-cholesterol in viscerally obese men.

The topic of this review is fatty acid amide hydrolase (FAAH), one of the best-characterized enzymes involved in the hydrolysis of bioactive lipids such as anandamide, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and oleamide. Herein, we discuss the nomenclature, the various assays that have been developed, the relative activity of the various substrates and the reversibility of the enzyme reactions catalyzed by FAAH. We also describe the cloning of the enzyme from rat and subsequent cDNA isolation from mouse, human, and pig. The proteins and the mRNAs from different species are compared. Cloning the enzyme permitted the purification and characterization of recombinant FAAH. The conserved regions of FAAH are described in terms of sequence and function, including the amidase domain which contains the serine catalytic nucleophile, the hydrophobic domain important for self association, and the proline rich domain region, which may be important for subcellular localization. The distribution of FAAH in the major organs of the body is described as well as regional distribution in the brain and its correlation with cannabinoid receptors. Since FAAH is recognized as a drug target, a large number of inhibitors have been synthesized and tested since 1994 and these are reviewed in terms of reversibility, potency, and specificity for FAAH and cannabinoid receptors.

The endocannabinoid system, consisting of two cannabinoid receptors (CB1 and CB<em>2</em>) and the endogenous ligands anandamide (arachidonoylethanolamide (AEA)) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), has been shown to control food intake in both animals and humans, modulating either rewarding or quantitative aspects of the eating behavior. Moreover, hypothalamic endocannabinoids seem to be part of neural circuitry involved in the modulating effects of leptin on energy homeostasis. Therefore, alterations of the endocannabinoid system could be involved in the pathophysiology of eating disorders, where a deranged leptin signalling has been also reported. In order to verify this hypothesis, we measured plasma levels of AEA, <em>2</em>-AG, and leptin in 15 women with anorexia nervosa (AN), 1<em>2</em> women with bulimia nervosa (BN), 11 women with binge-eating disorder (BED), and 15 healthy women. Plasma levels of AEA resulted significantly enhanced in both anorexic and BED women, but not in bulimic patients. No significant change occurred in the plasma levels of <em>2</em>-AG in all the patients' groups. Moreover, circulating AEA levels were significantly and inversely correlated with plasma leptin concentrations in both healthy controls and anorexic women. These findings show for the first time a derangement in the production of the endogenous cannabinoid AEA in drug-free symptomatic women with AN or with BED. Although the pathophysiological significance of this alteration awaits further studies to be clarified, it suggests a possible involvement of AEA in the mediation of the rewarding aspects of the aberrant eating behaviors occurring in AN and BED.

A rather complex and pleiotropic endogenous signalling system was discovered in the late 1990s, starting from studies on the mechanism of action of Delta(9)-tetrahydrocannabinol, the major psychoactive principle of the hemp plant Cannabis sativa. This system includes: (1) at least two G-protein-coupled receptors, known as the cannabinoid CB(1) and CB(<em>2</em>) receptors; (<em>2</em>) the endogenous agonists at these receptors, known as endocannabinoids, of which anandamide and <em>2</em>-<em>arachidonoylglycerol</em> are the best known; and (3) proteins and enzymes for the regulation of endocannabinoid levels and action at receptors. The number of the members of this endocannabinoid signalling system seems to be ever increasing as new non-CB(1) non-CB(<em>2</em>) receptors for endocannabinoids, endocannabinoid-related molecules with little activity at CB(1) and CB(<em>2</em>) receptors, and new enzymes for endocannabinoid biosynthesis and degradation are being identified every year. The complexity of the endocannabinoid system and of its physiological and pathological function is outlined in this introductory chapter, for a better understanding of the subsequent chapters in this special issue.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is the endocannabinoid that mediates retrograde suppression of synaptic transmission in the brain. <em>2</em>-AG is synthesized in activated postsynaptic neurons by sn-1-specific diacylglycerol lipase (DGL), binds to presynaptic cannabinoid CB(1) receptors, suppresses neurotransmitter release, and is degraded mainly by monoacylglycerol lipase (MGL). In the basolateral amygdala complex, it has been demonstrated that CB(1) is particularly enriched in axon terminals of cholecystokinin (CCK)-positive GABAergic interneurons, induces short- and long-term depression at inhibitory synapses, and is involved in extinction of fear memory. Here, we clarified a unique molecular convergence of DGLα, CB(1), and MGL at specific inhibitory synapses in the basal nucleus (BA), but not lateral nucleus, of the basolateral amygdala. The synapses, termed invaginating synapses, consisted of conventional symmetrical contact and unique perisynaptic invagination of nerve terminals into perikarya. At invaginating synapses, DGLα was preferentially recruited to concave somatic membrane of postsynaptic pyramidal neurons, whereas invaginating presynaptic terminals highly expressed CB(1), MGL, and CCK. No such molecular convergence was seen for flat perisomatic synapses made by parvalbumin-positive interneurons. On the other hand, DGLα and CB(1) were expressed weakly at axospinous excitatory synapses. Consistent with these morphological data, thresholds for DGLα-mediated depolarization-induced retrograde suppression were much lower for inhibitory synapses than for excitatory synapses in BA pyramidal neurons. Moreover, depolarization-induced suppression was readily saturated for inhibition, but never for excitation. These findings suggest that perisomatic inhibition by invaginating synapses is a key target of <em>2</em>-AG-mediated control of the excitability of BA pyramidal neurons.

Endogenous cannabinoid ligands (endocannabinoids) produced by neurons, astrocytes, and microglial cells activate cannabinoid receptors, the molecular target for marijuana's bioactive ingredient Delta(9)-tetrahydrocannabinol. The molecular mechanism underlying the production of the most abundant endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), is unclear. A prevalent hypothesis proposes that activation of metabotropic receptors coupled to the phosphatidylinositol-specific phospholipase C and diacylglycerol (DG) lipase pathway will systematically lead to increases in <em>2</em>-AG production. Here, we show that ATP increases <em>2</em>-AG production by cultured microglial cells in a phosphatidylinositol-specific phospholipase C and DG lipase-dependent manner. However, efficacious activation of metabotropic P<em>2</em>Y purinergic receptors coupled to phosphatidylinositol-specific phospholipase C does not increase <em>2</em>-AG production. This suggests that ionotropic, and not metabotropic, purinergic receptors control <em>2</em>-AG production at an unexpected enzymatic step of its metabolic pathway. We show that activation of P<em>2</em>X(7) ionotropic receptors, which are highly permeable to calcium, is necessary and sufficient to increase <em>2</em>-AG production in microglial cells. We also show that the sustained rise in intracellular calcium induced by activation of P<em>2</em>X(7) receptors directly increases DG lipase activity while inhibiting the activity of monoacylglycerol lipase, the enzyme that degrades <em>2</em>-AG. This inverse sensitivity of DG lipase and monoacylglycerol lipase to calcium constitutes an original and efficient modality for sustained accumulation of <em>2</em>-AG. Because prolonged increases in <em>2</em>-AG amounts in brain parenchyma are thought to orchestrate neuroinflammation, the enzymatic steps involved in <em>2</em>-AG synthesis and degradation by microglial cells constitute appealing targets for therapy aimed at controlling exacerbated neuroinflammation.