Cyclooxygenase-<em>2</em> (COX-<em>2</em>) oxygenates arachidonic acid and the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and arachidonoylethanolamide (AEA). We recently reported that (R)-profens selectively inhibit endocannabinoid oxygenation but not arachidonic acid oxygenation. In this work, we synthesized achiral derivatives of five profen scaffolds and evaluated them for substrate-selective inhibition using in vitro and cellular assays. The size of the substituents dictated the inhibitory strength of the analogs, with smaller substituents enabling greater potency but less selectivity. Inhibitors based on the flurbiprofen scaffold possessed the greatest potency and selectivity, with desmethylflurbiprofen (3a) exhibiting an IC(50) of 0.11 μM for inhibition of <em>2</em>-AG oxygenation. The crystal structure of desmethylflurbiprofen complexed to mCOX-<em>2</em> demonstrated a similar binding mode to other profens. Desmethylflurbiprofen exhibited a half-life in mice comparable to that of ibuprofen. The data presented suggest that achiral profens can act as lead molecules toward in vivo probes of substrate-selective COX-<em>2</em> inhibition.

The endocannabinoids anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are bioactive lipids derived from the n-6 family of polyunsaturated fatty acids that are essential fatty acids. Symptoms of essential fatty acid deficiency in rats - growth retardation, scaly skin, and increased transepidermal water loss - can mainly be attributed to lack of linoleic acid as a structural element of the epidermis. Arachidonic acid, however, also serve essential functions, particularly in cellular signalling via its precursor role for numerous oxygenated derivatives such as prostaglandins, leukotrienes, hepoxilins and other eicosanoids. Furthermore, arachidonic acid is also a structural part of endocannabinoids that have signalling functions in relation to modulation of neurotransmitter release, which might involve physiological and pathophysiological phenomena such as regulation of appetite, energy metabolism, pain perception, memory and learning. Furthermore, along with AEA formation other acylethanolamides are always formed - e.g., oleoylethanolamide (OEA), that can inhibit food intake, and palmitoylethanolamide, that is anti-inflammatory - possibly through activation of peroxisome proliferator activated receptor alpha (PPAR alpha) and/or GPR119. As all these unsaturated fatty acids are ingested daily in smaller or larger amounts, one can ask whether different dietary fats can affect the levels of these fatty acids in the tissues and thereby the quantitative formation of these bioactive signalling molecules. Generally, in vivo arachidonic-acid-derived eicosanoid production can be increased and decreased by prolonged feeding with pharmacological levels of arachidonic acid and long-chain (n-3) fatty acids (fish oil), respectively. Changes in levels of these two fatty acids within the traditional human diet hardly affects the eicosanoid production, however. Moreover, preliminary data suggest that dietary intake of arachidonic acid and fish oil also doesn't easily affect endocannabinoid formation; however, dietary fat in terms of saturated, polyunsaturated and monounsaturated seems to affect tissue levels of AEA, <em>2</em>-AG and OEA.

Acute exposure to bacterial lipopolysaccharide (LPS) is a potent inducer of immune response as well as hypophagia. Nevertheless, desensitization of responses to LPS occurs during long-term exposure to endotoxin. We induced endotoxin tolerance, injecting repeated (6LPS) LPS doses compared with single (1LPS) treatment. 1LPS, but not 6LPS group, showed decreased food intake and body weight, which was associated with an increased plasma leptin and higher mRNA expression of OB-Rb, MC4R, and SOCS3 in the hypothalamus. Hypophagia induced by 1LPS was associated with lower levels of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), increased number of p-STAT3 neurons, and decreased AMP-activated protein kinase (AMPK) activity. Desensitization of hypophagia in the 6LPS group was related to high <em>2</em>-AG, with no changes in p-STAT3 or increased p-AMPK. Leptin decreased food intake, body weight, <em>2</em>-AG levels, and AMPK activity and enhanced p-STAT3 in control rats. However, leptin had no effects on <em>2</em>-AG, p-STAT3, or p-AMPK in the 1LPS and 6LPS groups. Rats treated with HFD to induce leptin resistance showed neither hypophagia nor changes in p-STAT3 after 1LPS, suggesting that leptin and LPS recruit a common signaling pathway in the hypothalamus to modulate food intake reduction. Desensitization of hypophagia in response to repeated exposure to endotoxin is related to an inability of leptin to inhibit AMPK phosphorylation and <em>2</em>-AG production and activate STAT3. SOCS3 is unlikely to underlie this resistance to leptin signaling in the endotoxin tolerance. The present model of prolonged inflammatory challenge may contribute to further investigations on mechanisms of leptin resistance.

A series of pathological events secondary to spinal cord injury (SCI) contribute to the spread of the damage, which aggravates neurological deficits. Here we report that a single dose of the neuroprotective endocannabinoid <em>2</em>-arachidonoyl glycerol (<em>2</em>-AG) administered early after SCI reduces lesion expansion, which was prevented by simultaneous blockade of both CB1 and CB<em>2</em> receptors but not by blockade of either receptor alone. Treatment with <em>2</em>-AG also preserves the white matter around the epicenter of the injury. Moreover, in the preserved white matter, <em>2</em>-AG protects myelin from damage and reduces oligodendrocyte loss. In addition to these protective actions at the epicenter region, <em>2</em>-AG also inhibits the myelin damage and delayed oligodendrocyte loss induced at 10mm from the epicenter. Interestingly, the early protective action of <em>2</em>-AG is maintained <em>2</em>8 days after injury, when the lesion size is still smaller and the preservation of white matter is better in <em>2</em>-AG-treated animals. Therefore, our results show that <em>2</em>-AG protects from the expansion of the lesion and white matter damage, which suggest that this endogenous cannabinoid may be useful as a protective treatment for acute SCI.

Rat brain, frozen in liquid nitrogen immediately after decapitation, contains a substantial amount of <em>2</em>-<em>arachidonoylglycerol</em> (0.34 nmol/g tissue), an endogenous cannabinoid receptor ligand. The level of <em>2</em>-<em>arachidonoylglycerol</em> in the brain was rapidly augmented after decapitation, the peak being noted 30 s after decapitation (1.54 nmol/g tissue). Noticeably, there are two phases during the increase in the levels of <em>2</em>-<em>arachidonoylglycerol</em>: a rapid transient increase and a subsequent gradual sustained increase, suggesting that at least two separate mechanisms are involved in the generation of <em>2</em>-<em>arachidonoylglycerol</em> in the decapitated brain. Gradual sustained formation was also observed for other monoacylglycerols, (e.g. <em>2</em>-palmitoylglycerol plus <em>2</em>-oleoylglycerol and <em>2</em>-cis-vaccenoylglycerol). Thus, it is important to minimize post-mortem changes to estimate the exact tissue levels of <em>2</em>-<em>arachidonoylglycerol</em> as well as other monoacylglycerols in the brain.

We report a novel excitatory effect of cannabinoid agonists on action potential-independent GABAergic transmission in the rat dentate gyrus. Specifically, we find that both WIN55,<em>2</em>1<em>2</em>-<em>2</em> and anandamide increase the frequency of miniature IPSCs (mIPSCs)recorded from hilar mossy cells without altering event amplitude, area, rise time, or decay. The effect of WIN55,<em>2</em>1<em>2</em>-<em>2</em> on mIPSCs is insensitive to AM<em>2</em>51 and preserved in CB1 −/− animals,indicating that it does not depend on activation of CB1 receptors. It is also insensitive to AM630 and unaffected by capsazepine suggesting that neither CB<em>2</em> nor TRPV1 receptors are involved. Further, it is blocked by pre-incubation in suramin and by a selective protein kinase A inhibitor (H-89), and is mimicked (and occluded) by bath application of forskolin. Similar CB1 receptor-independent facilitation of exocytosis is not apparent when recording evoked IPSCs in the presence of AM<em>2</em>51, suggesting that the exocytotic mechanism that produces WIN55,<em>2</em>1<em>2</em>-<em>2</em> sensitive mIPSCs is distinct from that which produces CB1 sensitive and action potential-dependent release. Despite clear independence from action potentials, WIN55,<em>2</em>1<em>2</em>-<em>2</em> mediated facilitation of mIPSCs requires calcium, and yet is insensitive to chelation of calcium in the postsynaptic cell. Finally, we demonstrate that both bath application of <em>2</em>-<em>arachidonoylglycerol</em>(<em>2</em>-AG) and depolarization-induced release of endogenous cannabinoids have minimal effect on mIPSC frequency. Cumulatively, our results indicate that cannabinoid ligands can selectively facilitate action potential-independent exocytosis of GABA in the rat dentate gyrus, and further emphasize that this new cannabinoid sensitive signalling system is distinct from previously described CB1 receptor-dependent systems in numerous respects.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is an endogenous cannabinoid (endocannabinoid) lipid whose functions remain poorly understood. Guindon and colleagues report the novel finding that exogenous application of <em>2</em>-AG induces peripheral antinociceptive effects that are mediated, at least in part, by actions at peripheral cannabinoid CB(<em>2</em>) receptors. URB60<em>2</em>, a recently described inhibitor of monoacylglycerol lipase, an enzyme that catalyzes <em>2</em>-AG hydrolysis in vivo, also induced peripheral antinociceptive effects and enhanced the actions of <em>2</em>-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 CB(1) receptors. The therapeutic potential of inhibitors of <em>2</em>-AG deactivation for the treatment of inflammatory pain is discussed.

Despite evidence for heritable variation in cannabis involvement and the discovery of cannabinoid receptors and their endogenous ligands, no consistent patterns have emerged from candidate endocannabinoid (eCB) genetic association studies of cannabis involvement. Given interactions between eCB and stress systems and associations between childhood stress and cannabis involvement, it may be important to consider childhood adversity in the context of eCB-related genetic variation. We employed a system-level gene-based analysis of data from the Comorbidity and Trauma Study (N = 1,558) to examine whether genetic variation in six eCB genes (anabolism: DAGLA, DAGLB, NAPEPLD; catabolism: MGLL, FAAH; binding: CNR1; SNPs N = 65) and childhood sexual abuse (CSA) predict cannabis dependence symptoms. Significant interactions with CSA emerged for MGLL at the gene level (p = .009), and for rs604300 within MGLL (ΔR<em>2</em> = .007, p < .001), the latter of which survived SNP-level Bonferroni correction and was significant in an additional sample with similar directional effects (N = 859; ΔR<em>2</em> = .005, p = .0<em>2</em>6). Furthermore, in a third sample (N = 31<em>2</em>), there was evidence that rs604300 genotype interacts with early life adversity to predict threat-related basolateral amygdala habituation, a neural phenotype linked to the eCB system and addiction (ΔR<em>2</em> = .013, p = .047). Rs604300 may be related to epigenetic modulation of MGLL expression. These results are consistent with rodent models implicating <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), an endogenous cannabinoid metabolized by the enzyme encoded by MGLL, in the etiology of stress adaptation related to cannabis dependence, but require further replication.

We have synthesized a series of 18 1,5- and <em>2</em>,5-disubstituted carbamoyl tetrazoles, including LY<em>2</em>183<em>2</em>40 (1) and LY<em>2</em>31891<em>2</em> (7), two compounds previously described as potent inhibitors of the cellular uptake of the endocannabinoid anandamide, and their regioisomers <em>2</em> and 8. We confirm that compound 1 is a potent inhibitor of both the cellular uptake and, like the other new compounds synthesized here, the enzymatic hydrolysis of anandamide. With the exception of 9, 1<em>2</em>, 15, and the <em>2</em>,5-regioisomer of LY<em>2</em>183<em>2</em>40 <em>2</em>, the other compounds were all found to be weakly active or inactive on anandamide uptake. Several compounds also inhibited the enzymatic hydrolysis of the other main endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em>, as well as its enzymatic release from sn-1-oleoyl-<em>2</em>-arachidonoyl-glycerol, at submicromolar concentrations. Four of the novel compounds, i.e. 3, 4, 17, and 18, inhibited anandamide hydrolysis potently (IC50=<em>2</em>.1-5.4nM) and selectively over all the other targets tested (IC50>>or= 10microM), thus representing new potentially useful tools for the inhibition of fatty acid amide hydrolase.

The enterocyte expresses two fatty acid-binding proteins (FABP), intestinal FABP (IFABP; FABP<em>2</em>) and liver FABP (LFABP; FABP1). LFABP is also expressed in liver. Despite ligand transport and binding differences, it has remained uncertain whether these intestinally coexpressed proteins, which both bind long chain fatty acids (FA), are functionally distinct. Here, we directly compared IFABP(-/-) and LFABP(-/-) mice fed high fat diets containing long chain saturated or unsaturated fatty acids, reasoning that providing an abundance of dietary lipid would reveal unique functional properties. The results showed that mucosal lipid metabolism was indeed differentially modified, with significant decreases in FA incorporation into triacylglycerol (TG) relative to phospholipid (PL) in IFABP(-/-) mice, whereas LFABP(-/-) mice had reduced monoacylglycerol incorporation in TG relative to PL, as well as reduced FA oxidation. Interestingly, striking differences were found in whole body energy homeostasis; LFABP(-/-) mice fed high fat diets became obese relative to WT, whereas IFABP(-/-) mice displayed an opposite, lean phenotype. Fuel utilization followed adiposity, with LFABP(-/-) mice preferentially utilizing lipids, and IFABP(-/-) mice preferentially metabolizing carbohydrate for energy production. Changes in body weight and fat may arise, in part, from altered food intake; mucosal levels of the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> and arachidonoylethanolamine were elevated in LFABP(-/-), perhaps contributing to increased energy intake. This direct comparison provides evidence that LFABP and IFABP have distinct roles in intestinal lipid metabolism; differential intracellular functions in intestine and in liver, for LFABP(-/-) mice, result in divergent downstream effects at the systemic level.

The endocannabinoid lipid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is deactivated by intracellular hydrolysis catalyzed by monoacylglycerol lipase. <em>2</em>-AG also serves as a substrate for oxidative metabolism catalyzed by cyclooxygenase <em>2</em> (COX-<em>2</em>). However, products of COX-<em>2</em>-mediated metabolism of endocannabinoids have not been identified in vivo. Hu and colleagues in this issue of the BJP demonstrate that COX-<em>2</em> converts <em>2</em>-AG into a biologically active, pro-nociceptive compound, prostaglandin E<em>2</em> glycerol ester (PGE<em>2</em>-G). PGE<em>2</em>-G produces hyperalgesia in vivo and activates a rapidly acting transcription factor, nuclear factor kappa-B in vitro. These biological actions may be attributed to a unique receptor. This report of pro-nociceptive actions of an endogenous COX-<em>2</em> metabolite of <em>2</em>-AG that are largely opposite to known anti-nociceptive and anti-inflammatory actions of endocannabinoids has physiological relevance. These discoveries place renewed emphasis on the importance of understanding the highly interactive nature of lipid signalling pathways in the nervous system and the physiological roles of these lipid mediators in controlling homeostasis.

BACKGROUND

Endocannabinoids (eCBs) are ubiquitous lipid mediators that act on specific (CB1, CB<em>2</em>) and non-specific (TRPV1, PPAR) receptors. Despite many experimental animal studies proved eCB involvement in the pathogenesis of stroke, such evidence is still lacking in human patients. Our aim was to determine eCB peripheral levels in acute stroke patients and evaluate their relationship with clinical disability and stroke volume.

METHODS

A cohort of ten patients with a first acute (within six hours since symptoms onset) ischemic stroke and a group of eight age- and sex-matched normal subjects were included. Groups were also matched for metabolic profile. All subjects underwent a blood sample collection for anandamide (AEA), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and palmitoylethanolamide (PEA) measurement; blood sampling was repeated in patients on admission (T0), at 6 (T1) and 18 hours (T<em>2</em>) thereafter. Patients neurological impairment was assessed using NIHSS and Fugl-Meyer Scale arm subitem (FMSa); stroke volume was determined on 48 h follow-up brain CT scans. Blood samples were analyzed by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry.

RESULTS

1)T0 AEA levels were significantly higher in stroke patients compared to controls. <em>2</em>)A significant inverse correlation between T0 AEA levels and FMSa score was found. Moreover a positive correlation between T0 AEA levels and stroke volume were found in stroke patients. T0 PEA levels in stroke patients were not significantly different from the control group, but showed a significant correlation with the NIHSS scores. T0 <em>2</em>-AG levels were lower in stroke patients compared to controls, but such difference did not reach the significance threshold.

CONCLUSIONS

This is the first demonstration of elevated peripheral AEA levels in acute stroke patients. In agreement with previous murine studies, we found a significant relationship between AEA or PEA levels and neurological involvement, such that the greater the neurological impairment, the higher were these levels.

Although cannabinoids, such as Δ(9)-tetrahydrocannabinol, have been studied extensively for their psychoactive effects, it has become apparent that certain cannabinoids possess immunomodulatory activity. Endothelial cells (ECs) are centrally involved in the pathogenesis of organ injury in acute inflammatory disorders, such as sepsis, because they express cytokines and chemokines, which facilitate the trafficking of leukocytes to organs, and they modulate vascular barrier function. In this study, we find that primary human ECs from multiple organs express the cannabinoid receptors CB1R, GPR18, and GPR55, as well as the ion channel transient receptor potential cation channel vanilloid type 1. In contrast to leukocytes, CB<em>2</em>R is only minimally expressed in some EC populations. Furthermore, we show that ECs express all of the known endocannabinoid (eCB) metabolic enzymes. Examining a panel of cannabinoids, we demonstrate that the synthetic cannabinoid WIN55,<em>2</em>1<em>2</em>-<em>2</em> and the eCB N-arachidonoyl dopamine (NADA), but neither anandamide nor <em>2</em>-<em>arachidonoylglycerol</em>, reduce EC inflammatory responses induced by bacterial lipopeptide, LPS, and TNFα. We find that endothelial CB1R/CB<em>2</em>R are necessary for the effects of NADA, but not those of WIN55,<em>2</em>1<em>2</em>-<em>2</em>. Furthermore, transient receptor potential cation channel vanilloid type 1 appears to counter the anti-inflammatory properties of WIN55,<em>2</em>1<em>2</em>-<em>2</em> and NADA, but conversely, in the absence of these cannabinoids, its inhibition exacerbates the inflammatory response in ECs activated with LPS. These data indicate that the eCB system can modulate inflammatory activation of the endothelium and may have important implications for a variety of acute inflammatory disorders that are characterized by EC activation.

OBJECTIVE

The cannabinoid receptor-mediated analgesic effects of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are limited by monoacylglycerol lipase (MAGL). 4-nitrophenyl 4-[bis (1,3-benzodioxol-5-yl) (hydroxy) methyl] piperidine-1-carboxylate (JZL184) is a potent inhibitor of MAGL in the mouse, though potency is reportedly reduced in the rat. Here we have assessed the effects of spinal inhibition of MAGL with JZL184 on nociceptive processing in rats.

METHODS

In vivo spinal electrophysiological assays in anaesthetized rats were used to determine the effects of spinal administration of JZL184 on spinal nociceptive processing in the presence and absence of hindpaw inflammation. Contributions of CB(1) receptors to these effects was assessed with AM<em>2</em>51. Inhibition of <em>2</em>-oleoylglycerol hydrolytic activity and alterations of <em>2</em>-AG in the spinal cord after JZL 184 were also assessed.

RESULTS

Spinal JZL184 dose-dependently inhibited mechanically evoked responses of wide dynamic range (WDR) neurones in naïve anaesthetized rats, in part via the CB(1) receptor. A single spinal administration of JZL184 abolished inflammation-induced expansion of the receptive fields of spinal WDR neurones. However, neither spinal nor systemic JZL184 altered levels of <em>2</em>-AG, or <em>2</em>-oleoylglycerol hydrolytic activity in the spinal cord, although JZL184 displayed robust inhibition of MAGL when incubated with spinal cord tissue in vitro.

CONCLUSIONS

JZL184 exerted robust anti-nociceptive effects at the level of the spinal cord in vivo and inhibited rat spinal cord MAGL activity in vitro. The discordance between in vivo and in vitro assays suggests that localized sites of action of JZL184 produce these profound functional inhibitory effects.

BACKGROUND

This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.<em>2</em>01<em>2</em>.167.issue-8.

OBJECTIVE

The endocannabinoid system is involved in excitatory/inhibitory balance mechanisms within the central nervous system (CNS). Growing evidence shows that its perturbation leads to development of epileptic seizures in experimental models, thus indicating that endocannabinoids play an intrinsic protective role in suppressing pathologic neuronal excitability. Experimental data also demonstrate that the endocannabinoid anandamide (AEA) can antagonize epileptic discharges in hippocampal tissue. The objective of our study was to measure endocannabinoids levels in the cerebrospinal fluid (CSF) of drug-naive patients affected by temporal lobe epilepsy (TLE).

METHODS

We measured the levels of both AEA and the other endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), in the CSF of drug-naive patients with TLE.

RESULTS

A significant reduction of AEA was found in the CSF of patients with compared with healthy controls (epileptic patients = <em>2</em>.55 +/- 1.78 pmol/ml; healthy controls = 11.65 +/- 7.53 pmol/ml; n = 9 for both groups, p < 0.01). <em>2</em>-AG levels, however, were not affected (epileptic patients = <em>2</em>09.5 +/- 146.56; healthy controls = 159.6 +/- 110.<em>2</em>) (n = 6 for both groups, p = 0.48).

CONCLUSIONS

Our findings seem to be consistent with experimental evidence demonstrating a significant prevention of epileptic seizures induced by endocannabinoids in models of epilepsy. Furthermore, they support the hypothesis that AEA may be involved in its pathogenesis, suggesting a hypothetical primary impairment of the endocannabinoid system in untreated TLE. The actual role of this in vivo dysregulation still remains unclear.

The endocannabinoid system is implicated in, and regulates, several physiological processes, ranging from food intake and energy balance to pain and inflammation. <em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is a full agonist at the cannabinoid receptors which classically mediate its effects. The activity of this bioactive lipid is dependent on its endogenous levels, which are tightly controlled by several hydrolases, monoacylglycerol lipase and α/β-hydrolase domain 6 and 1<em>2</em>. Moreover, <em>2</em>-AG is also a substrate of cyclooxygenase-<em>2</em>, and this reaction leads to the formation of prostaglandin glycerol esters, the effects of which remain to be fully elucidated. In this review we discuss the multiple mechanisms by which <em>2</em>-AG controls inflammation and the therapeutic potential of <em>2</em>-AG metabolism inhibitors.

The progressive predominance of rewarding effects of addictive drugs over their aversive properties likely contributes to the transition from drug use to drug dependence. By inhibiting the activity of DA neurons in the VTA, GABA projections from the rostromedial tegmental nucleus (RMTg) are well suited to shift the balance between drug-induced reward and aversion. Since cannabinoids suppress RMTg inputs to DA cells and CB1 receptors affect alcohol intake in rodents, we hypothesized that the endocannabinoid system, by modulating this pathway, might contribute to alcohol preference. Here we found that RMTg afferents onto VTA DA neurons express CB1 receptors and display a <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG)-dependent form of short-term plasticity, that is, depolarization-induced suppression of inhibition (DSI). Next, we compared rodents with innate opposite alcohol preference, the Sardinian alcohol-preferring (sP) and alcohol-nonpreferring (sNP) rats. We found that DA cells from alcohol-naive sP rats displayed a decreased probability of GABA release and a larger DSI. This difference was due to the rate of <em>2</em>-AG degradation. In vivo, we found a reduced RMTg-induced inhibition of putative DA neurons in sP rats that negatively correlated with an increased firing. Finally, alcohol failed to enhance RMTg spontaneous activity and to prolong RMTg-induced silencing of putative DA neurons in sP rats. Our results indicate functional modifications of RMTg projections to DA neurons that might impact the reward/aversion balance of alcohol attributes, which may contribute to the innate preference observed in sP rats and to their elevated alcohol intake.

The purpose of the study was to see if nematodes (Caenorhabditis elegans, Caenorhabditis briggsae, and Pelodera strongyloides) produce endocannabinoids; i.e., anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). In this study, AEA and <em>2</em>-AG were identified as endogenous products from nematodes by using electrospray-ionization ion-trap MS/MS (ESI-IT-MS) experiments operated in the positive-ionization mode. Endocannabinoids were identified by product ion scan and concentrations were measured by triple quadrupole mass spectrometry in the multiple reaction monitoring mode (MRM). Both AEA and <em>2</em>-AG were identified in all of the nematode samples, even though these species lack known cannabinoid receptors. Neither AEA nor <em>2</em>-AG were detected in the fat-3 mutant of C. elegans, which lacks the necessary enzyme to produce arachidonic acid, the fatty acid precursor of these endocannabinoids.

Homeostatic regulation of synaptic strength in response to persistent changes of neuronal activity plays an important role in maintaining the overall level of circuit activity within a normal range. Absence of miniature EPSCs (mEPSCs) for a few hours is known to cause upregulation of excitatory synaptic strength, suggesting that mEPSCs contribute to the maintenance of excitatory synaptic functions. In the present study, we found that the absence of mEPSCs for 1-3 h also resulted in homeostatic suppression of presynaptic functions of inhibitory synapses in acute cortical slices from juvenile rats, as suggested by the reduced frequency (but not amplitude) of miniature IPSCs (mIPSCs) as well as the reduced amplitude of IPSCs. This homeostatic regulation depended on endocannabinoid (eCB) signaling, because blockade of either the activation of cannabinoid type-1 receptors (CB1Rs) or the synthesis of its endogenous ligand <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) abolished the suppression of inhibitory synapses caused by the absence of mEPSCs. Blockade of group I metabotropic glutamate receptors (mGluR-I) also abolished the suppression of inhibitory synapses, consistent with the mGluR-I requirement for eCB synthesis and release in cortical synapses. Furthermore, this homeostatic regulation also required eukaryotic elongation factor-<em>2</em> (eEF<em>2</em>)-dependent protein synthesis, but not gene transcription. Activation of eEF<em>2</em> alone was sufficient to suppress the mIPSC frequency, an effect abolished by inhibiting CB1Rs. Thus, mEPSCs contribute to the maintenance of inhibitory synaptic function and the absence of mEPSCs results in presynaptic suppression of inhibitory synapses via protein synthesis-dependent elevation of eCB signaling.

Monoacylglycerol lipase (MAGL) is a key enzyme responsible for the termination of endocannabinoid signaling. Its crucial role in <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) metabolism, together with the numerous pharmacological properties mediated by this endocannabinoid, emphasize the interest in MAGL as therapeutic target, along with the need to design potent and selective inhibitors. Meanwhile, the complexity of <em>2</em>-AG degradation pathways underscores the need to use a purified source of enzyme in evaluation studies of new inhibitors. We report here the first heterologous expression and purification of human MAGL. A highly pure protein was obtained and allowed us to measure the affinity of several MAGL inhibitors for the human enzyme. Importantly, disulfiram (tetraethylthiuram disulfide), a compound used to treat alcoholism, and other disulfide-containing compounds were shown to inhibit MAGL with good potency, likely through an interaction with cysteine residues.

Preclinical studies have demonstrated that the endocannabinoid system (ECS) plays an important role in the protection against intestinal inflammation and colorectal cancer (CRC); however, human data are scarce. We determined members of the ECS and related components of the 'endocannabinoidome' in patients with inflammatory bowel disease (IBD) and CRC, and compared them to control subjects. Anandamide (AEA) and oleoylethanolamide (OEA) were increased in plasma of ulcerative colitis (UC) and Crohn's disease (CD) patients while <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) was elevated in patients with CD, but not UC. <em>2</em>-AG, but not AEA, PEA and OEA, was elevated in CRC patients. Lysophosphatidylinositol (LPI) 18:0 showed higher levels in patients with IBD than in control subjects whereas LPI <em>2</em>0:4 was elevated in both CRC and IBD. Gene expression in intestinal mucosal biopsies revealed different profiles in CD and UC. CD, but not UC patients, showed increased gene expression for the <em>2</em>-AG synthesizing enzyme diacylglycerol lipase alpha. Transcripts of CNR1 and GPR119 were predominantly decreased in CD. Our data show altered plasma levels of endocannabinoids and endocannabinoid-like lipids in IBD and CRC and distinct transcript profiles in UC and CD. We also report alterations for less known components in intestinal inflammation, such as GPR119, OEA and LPI.

Orexins are associated with drug relapse in rodents. Here, we show that acute restraint stress in mice activates lateral hypothalamic (LH) orexin neurons, increases levels of orexin A and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in the ventral tegmental area (VTA), and reinstates extinguished cocaine-conditioned place preference (CPP). This stress-induced reinstatement of cocaine CPP depends on type 1 orexin receptors (OX1Rs), type 1 cannabinoid receptors (CB1Rs) and diacylglycerol lipase (DAGL) in the VTA. In dopaminergic neurons of VTA slices, orexin A presynaptically inhibits GABAergic transmission. This effect is prevented by internal GDP-β-S or inhibiting OX1Rs, CB1Rs, phospholipase C or DAGL, and potentiated by inhibiting <em>2</em>-AG degradation. These results suggest that restraint stress activates LH orexin neurons, releasing orexins into the VTA to activate postsynaptic OX1Rs of dopaminergic neurons and generate <em>2</em>-AG through a Gq-protein-phospholipase C-DAGL cascade. <em>2</em>-AG retrogradely inhibits GABA release through presynaptic CB1Rs, leading to VTA dopaminergic disinhibition and reinstatement of cocaine CPP.

Endocannabinoids (eCB) are key regulators of excitatory/inhibitory neurotransmission at cannabinoid-1-receptor (CB1 R)-expressing axon terminals. The most abundant eCB in the brain, that is <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), is hydrolyzed by the enzyme monoacylglycerol lipase (MAGL), whose chronic inhibition in the brain was reported to cause CB1 R desensitization. We employed the MAGL knock-out mouse (MAGL-/-), a genetic model of congenital and sustained elevation of <em>2</em>-AG levels in the brain, to provide morphological and biochemical evidence for β-arrestin<em>2</em>-mediated CB1 R desensitization in brain regions involved in the control of emotional states, that is, the prefrontal cortex (PFC), amygdala, hippocampus and cerebellar cortex. We found a widespread CB1 R/β-arrestin<em>2</em> co-expression in the mPFC, amygdala and hippocampus accompanied by impairment of extracellular signal-regulated kinase signaling and elevation of vesicular glutamate transporter (VGluT1) at CB1 R-positive excitatory terminals in the mPFC, or vesicular GABA transporter (VGAT) at CB1 R-positive inhibitory terminals in the amygdala and hippocampus. The impairment of CB1 R signaling in MAGL-/- mice was also accompanied by enhanced excitatory drive in the basolateral amygdala (BLA)-mPFC circuit, with subsequent elevation of glutamate release to the mPFC and anxiety-like and obsessive-compulsive behaviors, as assessed by the light/dark box and marble burying tests, respectively. Collectively, these data provide evidence for a β-arrestin<em>2</em>-mediated desensitization of CB1 R in MAGL-/- mice, with impact on the synaptic plasticity of brain circuits involved in emotional functions. In this study, the authors provide evidence that congenitally enhanced endocannabinoid levels in the neuronal circuits underlying anxiety-like behavioral states (mainly medial prefrontal cortex, amygdala and hippocampus) lead to CB1R desenistization and anxiety and depression. MAGL-/- mice, a model of congenital overactivity of the eCB system, exhibited a compensatory impairment of CB1R signaling in anxiety-associated brain areas and a subsequent change in excitatory/inhibitory tone associated with altered score in the marble burying and light/dark box test, in concomitance with anxiety and depression behavior states. These findings may have potential relevance to the understanding of the neurochemical effects of chronic CB1R overstimulation in cannabis abusers.