Cannabinoid receptor subtype influence on neuritogenesis in human SH-SY5Y cells.

Human SH-SY5Y neuroblastoma cells stably expressing exogenous CB1 (CB1XS) or CB2 (CB2XS) receptors were developed to investigate endocannabinoid signaling in the extension of neuronal projections. Expression of cannabinoid receptors did not alter proliferation rate, viability, or apoptosis relative to parental SH-SY5Y. Transcripts for endogenous cannabinoid system enzymes (diacylglycerol lipase, monoacylglycerol lipase, α/ß-hydrolase domain containing proteins 6 and 12, N-acyl phosphatidylethanolamine-phospholipase D, and fatty acid amide hydrolase) were not altered by CB1 or CB2 expression. Endocannabinoid ligands 2-arachidonoylglycerol (2-AG) and anandamide were quantitated in SH-SY5Y cells, and diacylglycerol lipase inhibitor tetrahydrolipstatin decreased 2-AG abundance by 90% but did not alter anandamide abundance. M3 muscarinic agonist oxotremorine M, and inhibitors of monoacylglycerol lipase and α/ß hydrolase domain containing proteins 6 &12 increased 2-AG abundance. CB1 receptor expression increased lengths of short (<30 µm) and long (>30 µm) projections, and this effect was significantly reduced by tetrahydrolipstatin, indicative of stimulation by endogenously produced 2-AG. Pertussis toxin, Gßγ inhibitor gallein, and ß-arrestin inhibitor barbadin did not significantly alter long projection length in CB1XS, but significantly reduced short projections, with gallein having the greatest inhibition. The rho kinase inhibitor Y27632 increased CB1 receptor-mediated long projection extension, indicative of actin cytoskeleton involvement. CB1 receptor expression increased GAP43 and ST8SIA2 mRNA and decreased ITGA1 mRNA, whereas CB2 receptor expression increased NCAM and SYT mRNA. We propose that basal endogenous production of 2-AG provides autocrine stimulation of CB1 receptor signaling through Gi/o, Gßγ, and ß-arrestin mechanisms to promote neuritogenesis, and rho kinase influences process extension.

An inverse agonist of estrogen-related receptor γ regulates 2-arachidonoylglycerol synthesis by modulating diacylglycerol lipase expression in alcohol-intoxicated mice.

Chronic alcohol feeding increases the levels of 2-arachidonoylglycerol (2-AG) in the liver, which activates hepatic cannabinoid receptor type 1 (CB1R), leading to oxidative liver injury. 2-AG biosynthesis is catalyzed by diacylglycerol lipase (DAGL). However, the mechanisms regulating hepatic DAGL gene expression and 2-AG production are largely unknown. In this study, we show that CB1R-induced estrogen-related receptor γ (ERRγ) controls hepatic DAGL gene expression and 2-AG levels. Arachidonyl-2'-chloroethylamide (ACEA), a synthetic CB1R agonist, significantly upregulated ERRγ, DAGLα, and DAGLß, and increased 2-AG levels in the liver (10 mg/kg) and hepatocytes (10 µM) of wild-type (WT) mice. ERRγ overexpression upregulated DAGLα and DAGLß expressions and increased 2-AG levels, whereas ERRγ knockdown abolished ACEA-induced DAGLα, DAGLß, and 2-AG in vitro and in vivo. Promoter assays showed that ERRγ positively regulated DAGLα and DAGLß transcription by binding to the ERR response element in the DAGLα and DAGLß promoters. Chronic alcohol feeding (27.5% of total calories) induced hepatic steatosis and upregulated ERRγ, leading to increased DAGLα, DAGLß, or 2-AG in WT mice, whereas these alcohol-induced effects did not occur in hepatocyte-specific CB1R knockout mice or in those treated with the ERRγ inverse agonist GSK5182 (40 mg/kg in mice and 10 µM in vitro). Taken together, these results indicate that suppression of alcohol-induced DAGLα and DAGLß gene expressions and 2-AG levels by an ERRγ-specific inverse agonist may be a novel and attractive therapeutic approach for the treatment of alcoholic liver disease.

Endocannabinoid contributions to alcohol habits and motivation: Relevance to treatment.

Individuals with alcohol use disorder exhibit compulsive habitual behaviors that are thought to be, in part, a consequence of chronic and persistent use of alcohol. The endocannabinoid system plays a critical role in habit learning and in ethanol self-administration, but the role of this neuromodulatory system in the expression of habitual alcohol seeking is unknown. Here, we investigated the role of the endocannabinoid system in established alcohol habits using contingency degradation in male C57BL/6 mice. We found that administration of the novel diacyl glycerol lipase inhibitor DO34, which decreases the biosynthesis of the endocannabinoid 2-arachidonoyl glycerol (2-AG), reduced habitual responding for ethanol and ethanol approach behaviors. Moreover, administration of the endocannabinoid transport inhibitor AM404 or the cannabinoid receptor type 1 antagonist AM251 produced similar reductions in habitual responding for ethanol and ethanol approach behaviors. Notably, AM404 was also able to reduce ethanol seeking and consumption in mice that were insensitive to lithium chloride-induced devaluation of ethanol. Conversely, administration of JZL184, a monoacyl glycerol lipase inhibitor that increases levels of 2-AG, increased motivation to respond for ethanol on a progressive ratio schedule of reinforcement. These results demonstrate an important role for endocannabinoid signaling in the motivation to seek ethanol, in ethanol-motivated habits, and suggest that pharmacological manipulations of endocannabinoid signaling could be effective therapeutics for treating alcohol use disorder.

Characterisation and localisation of the endocannabinoid system components in the adult human testis.

Heavy use of cannabis (marijuana) has been associated with decreased semen quality, which may reflect disruption of the endocannabinoid system (ECS) in the male reproductive tract by exogenous cannabinoids. Components of ECS have been previously described in human spermatozoa and in the rodent testis but there is little information on the ECS expression within the human testis. In this study we characterised the main components of the ECS by immunohistochemistry (IHC) on archived testis tissue samples from 15 patients, and by in silico analysis of existing transcriptome datasets from testicular cell populations. The presence of 2-arachidonoylglycerol (2-AG) in the human testis was confirmed by matrix-assisted laser desorption ionization imaging analysis. Endocannabinoid-synthesising enzymes; diacylglycerol lipase (DAGL) and N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), were detected in germ cells and somatic cells, respectively. The cannabinoid receptors, CNR1 and CNR2 were detected at a low level in post-meiotic germ cells and Leydig- and peritubular cells. Different transcripts encoding distinct receptor isoforms (CB1, CB1A, CB1B and CB2A) were also differentially distributed, mainly in germ cells. The cannabinoid-metabolising enzymes were abundantly present; the α/ß-hydrolase domain-containing protein 2 (ABHD2) in all germ cell types, except early spermatocytes, the monoacylglycerol lipase (MGLL) in Sertoli cells, and the fatty acid amide hydrolase (FAAH) in late spermatocytes and post-meiotic germ cells. Our findings are consistent with a direct involvement of the ECS in regulation of human testicular physiology, including spermatogenesis and Leydig cell function. The study provides new evidence supporting observations that recreational cannabis can have possible deleterious effects on human testicular function.

CHP1 Regulates Compartmentalized Glycerolipid Synthesis by Activating GPAT4.

Cells require a constant supply of fatty acids to survive and proliferate. Fatty acids incorporate into membrane and storage glycerolipids through a series of endoplasmic reticulum (ER) enzymes, but how these enzymes are regulated is not well understood. Here, using a combination of CRISPR-based genetic screens and unbiased lipidomics, we identified calcineurin B homologous protein 1 (CHP1) as a major regulator of ER glycerolipid synthesis. Loss of CHP1 severely reduces fatty acid incorporation and storage in mammalian cells and invertebrates. Mechanistically, CHP1 binds and activates GPAT4, which catalyzes the initial rate-limiting step in glycerolipid synthesis. GPAT4 activity requires CHP1 to be N-myristoylated, forming a key molecular interface between the two proteins. Interestingly, upon CHP1 loss, the peroxisomal enzyme, GNPAT, partially compensates for the loss of ER lipid synthesis, enabling cell proliferation. Thus, our work identifies a conserved regulator of glycerolipid metabolism and reveals plasticity in lipid synthesis of proliferating cells.

Forsythiaside prevents ß-amyloid-induced hippocampal slice injury by upregulating 2-arachidonoylglycerol via cannabinoid receptor 1-dependent NF-κB pathway.

In the study, the neuroprotectivities of forsythiaside, a main constituent of Forsythia suspensa (Thunb.) Vahl (F. suspensa, Lianqiao in Chinese), were investigated in the hippocampal slices. Forsythiaside suppressed the overexpression of cyclooxygenase-2 (COX-2) and monoacylglycerol lipase (MAGL) proteins induced by ß-amyloid (Aß25-35) to upregulate the levels of 2-arachidonoylglycerol (2-AG), an endogenous endocannabinoids. Then the inhibition of forsythiaside on COX-2 was deeply studied by the molecular docking. Forsythiaside prevented neuroinflammation and apoptosis from Aß25-35 insults, and this action appeared to be mediated via cannabinoid receptor 1 (CB1R)-dependent nuclear factor-κB (NF-κB) signaling pathways. More importantly, forsythiaside functionally improved Aß25-35-induced learning and memory deficits, which was indicated by long term potentiation (LTP). Taken together, forsythiaside may have therapeutic potential for Alzheimer's diseases (AD) by increasing the levels of 2-AG.

Thioesterase superfamily member 2 promotes hepatic insulin resistance in the setting of glycerol-3-phosphate acyltransferase 1-induced steatosis.

Hepatic insulin resistance in the setting of steatosis is attributable at least in part to the accumulation of bioactive lipids that suppress insulin signaling. The mitochondria-associated glycerol-3-phosphate acyltransferase 1 (GPAT1) catalyzes the first committed step in glycerolipid synthesis, and its activity diverts fatty acids from mitochondrial ß-oxidation. GPAT1 overexpression in mouse liver leads to hepatic steatosis even in the absence of overnutrition. The mice develop insulin resistance owing to the generation of saturated diacylglycerol and phosphatidic acid molecular species that reduce insulin signaling by activating PKCϵ and by suppressing mTORC2, respectively. Them2, a mitochondria-associated acyl-CoA thioesterase, also participates in the trafficking of fatty acids into oxidative versus glycerolipid biosynthetic pathways. Them2-/- mice are protected against diet-induced hepatic steatosis and insulin resistance. To determine whether Them2 contributes to hepatic insulin resistance due to hepatic overexpression of GPAT1, recombinant adenovirus was used to overexpress GPAT1 in livers of chow-fed Them2+/+ and Them2-/- mice. Hepatic GPAT1 overexpression led to steatosis in both genotypes. In the setting of GPAT1 overexpression, glucose tolerance was reduced in Them2+/+ but not Them2-/- mice, without influencing whole-body insulin sensitivity or basal hepatic glucose production. Improved glucose tolerance in Them2-/- mice was associated with reduced PKCϵ translocation. Preserved insulin receptor activity was supported by Thr-308 phosphorylation of Akt following GPAT1 overexpression in Them2-/- hepatocytes. These findings suggest a pathogenic role of Them2 in the biosynthesis of glycerolipid metabolites that promote hepatic insulin resistance.

Lipase-catalyzed glycerolysis of anchovy oil in a solvent-free system: Simultaneous optimization of monoacylglycerol synthesis and end-product oxidative stability.

The production of mono- and diacylglycerols rich in polyunsaturated fatty acids is achieved in this study, by solvent-free glycerolysis of anchovy oil with lipase PS-DI from Burkholderia cepacia. Attention is focused on the oxidative stability of the reaction products, determined in terms of induction time (It). The effects of glycerol/triacylglycerol molar ratio, enzyme concentration, and reaction temperature on mono- and diacylglycerol production and It are all assessed. The operating conditions that optimized monoacylglycerol yields and oxidative stability were a glycerol/triacylglycerol ratio of 3/1, 9.0% (w/w) Lipase PS-DI, a stirring rate of 200 rpm, and a reaction time of 4 h, at 45.8 °C, producing a content of 24.8% and 51.9% of mono- and diacylglycerols, respectively, over an It of 1.41 h. The glycerolysis conditions determined by simultaneous optimization strategy increased the oxidative stability of the glycerolysis products by 68%, which rose from 0.84 h (individual optimization) to 1.41 h.

Conditioned gaping produced by high dose Δ9-tetrahydracannabinol: Dysregulation of the hypothalamic endocannabinoid system.

Δ9-tetrahydracannabinol (THC) is recognized as an effective treatment for nausea and vomiting via its action on the cannabinoid 1 (CB1) receptor. Paradoxically, there is evidence that THC can also produce nausea and vomiting. Using the conditioned gaping model of nausea in rats, we evaluated the ability of several doses of THC (0.0, 0.5, 5 and 10 mg/kg, i.p.) to produced conditioned gaping reactions. We then investigated the ability of the CB1 receptor antagonist, rimonabant, to block the establishment of THC-induced conditioned gaping. Real-time polymerase chain reaction (RT-PCR) was then used to investigate changes in endocannabinoid related genes in various brain regions in rats chronically treated with vehicle (VEH), 0.5 or 10 mg/kg THC. THC produced dose-dependent gaping, with 5 and 10 mg/kg producing significantly more gaping reactions than VEH or 0.5 mg/kg THC, a dose known to have anti-emetic properties. Pre-treatment with rimonabant reversed this effect, indicating that THC-induced conditioned gaping was CB1 receptor mediated. The RT-PCR analysis revealed an upregulation of genes for the degrading enzyme, monoacylglycerol lipase (MAGL), of the endocannabinoid, 2-arachidolyl glycerol (2-AG), in the hypothalamus of rats treated with 10 mg/kg THC. No changes in the expression of relevant genes were found in nausea (interoceptive insular cortex) or vomiting (dorsal vagal complex) related brain regions. These findings support the hypothesis that THC-induced nausea is a result of a dysregulated hypothalamic-pituitary-adrenal axis leading to an overactive stress response.

The endocannabinoid 2-arachidonoylglycerol regulates oligodendrocyte progenitor cell migration.

While the endocannabinoid 2-arachidonoylglycerol (2-AG) is thought to enhance the proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) in vitro, less is known about how endogenous 2-AG may influence the migration of these cells. When we assessed this in Agarose drop and Boyden chemotaxis chamber assays, inhibiting the sn-1-diacylglycerol lipases α and ß (DAGLs) that are responsible for 2-AG synthesis significantly reduced the migration of OPCs stimulated by platelet-derived growth factor-AA (PDGF) and basic fibroblast growth factor (FGF). Likewise, antagonists of the CB1 and CB2 cannabinoid receptors (AM281 and AM630, respectively) produced a similar inhibition of OPC migration. By contrast, increasing the levels of endogenous 2-AG by blocking its degradation (impairing monoacylglycerol lipase activity with JZL-184) significantly increased OPC migration, as did agonists of the CB1, CB2 or CB1/CB2 cannabinoid receptors. This latter effect was abolished by selective CB1 or CB2 antagonists, strongly suggesting that cannabinoid receptor activation specifically potentiates OPC chemotaxis and chemokinesis in response to PDGF/FGF. Furthermore, the chemoattractive activity of these cannabinoid receptor agonists on OPCs was even evident in the absence of PDGF/FGF. In cultured brain slices prepared from the corpus callosum of postnatal rat brains, DAGL or cannabinoid receptor inhibition substantially diminished the in situ migration of Sox10+ OPCs. Overall, these results reveal a novel function of endogenous 2-AG in PDGF and FGF induced OPC migration, highlighting the importance of the endocannabinoid system in regulating essential steps in oligodendrocyte development.

The expression of cannabinoid type 1 receptor and 2-arachidonoyl glycerol synthesizing/degrading enzymes is altered in basal ganglia during the active phase of levodopa-induced dyskinesia.

Management of levodopa-induced dyskinesias (LID) is one of the main challenges in the treatment of Parkinson's disease patients. Mechanisms involved in the appearance of these involuntary movements are not well known but modifications in the activity of different neurotransmitter pathways seem to play an important role. The objective of this study was to determine differences in the expression levels of the endocannabinoid system (ECS) elements that would support a role in LID. The basal ganglia nuclei, putamen, external segment of the globus pallidus (GPe), internal segment of the globus pallidus (GPi), subthalamic nucleus (STN) and substantia nigra (SN) were dissected out from cryostat sections obtained from two groups of parkinsonian monkeys treated with levodopa to induce dyskinesias. One group of dyskinetic animals was sacrificed under the effect of levodopa, during the active phase of LID, and the other group 24 h after the last levodopa dose (OFF levodopa). Biochemical analysis by real-time PCR for ECS elements was performed. CB1 receptor expression was upregulated in the putamen, GPe and STN during the active phase of dyskinesia and downregulated in the same nuclei and in the SN when dyskinetic animals were OFF levodopa. Changes in the 2-arachidonoyl glycerol (2-AG) synthesizing/degrading enzymes affecting the pallidal-subthalamic projections in dyskinetic animals OFF levodopa would suggest that 2-AG may play a role in LID. Anandamide (AEA) synthesizing/degrading enzymes were altered specifically in the GPe of untreated parkinsonian monkeys, suggesting that increased AEA levels may be a compensatory mechanism. These results indicate that the expression of the ECS elements is influenced by alterations in dopaminergic neurotransmission. On one hand, changes in CB1 receptor expression and in the 2-AG synthesizing/degrading enzymes suggest that they could be a therapeutic target for the active phase of LID. On the other hand, AEA metabolism could provide a non-dopaminergic target for symptomatic relief. However, further research is needed to unravel the mechanism of action of the ECS and how they could be modulated for a therapeutic purpose.

Enhanced synthesis of feruloylated acylglycerols by the lipase-catalyzed transesterification of glyceryl monoferulate with different acyl donors using ionic liquids as reaction solvents.

Feruloylated acylglycerols (FAG) can be used as antioxidants and UV absorbing ingredients in food and cosmetics. In this work, FAG was prepared by the lipase-catalyzed transesterification of glyceryl monoferulate (GMF) with different acyl donors using ionic liquids (ILs) as reaction solvents. The effect of different imidazolium ILs (BF4-, PF6- and TF2N-) and acyl donors (monoacylglycerols and diacylglycerols) on the transesterification and lipase selectivity for FAG formation were compared. The effect of reaction parameters (temperature, enzyme concentration, substrates ratio and time) on the reaction were also studied. The results showed that FAG preparation can be enhanced using monoolein (MO) and distearin (DS) as acyl donors. High transesterification activity and excellent lipase selectivity for lipophilic FAG formation were achieved using [C18MIM]PF6 as reaction solvent. The activation energy to form the lipophilic FAG by transesterification using MO as an acyl donor was 37.2 kJ/mol, which was lower than that of DS (92.9 kJ/mol). The activation energy to form the hydrophilic glyceryl diferulate by the esterification of GMF with feruloyl formed by the hydrolysis of another GMF (21.5 kJ/mol) using MO as an acyl donor was lower than that of DS (61.9 kJ/mol).

Chemical tools to modulate 2-arachidonoylglycerol biosynthesis.

2-Arachidonoylglycerol (2-AG) is an important endogenous signaling lipid that activates the cannabinoid receptors (CB1 R and CB2 R), thereby regulating a diverse range of physiological processes including anxiety, appetite, inflammation, memory, pain sensation, and nociception. Diacylglycerol lipases (DAGLs) are the principle enzymes responsible for 2-AG biosynthesis. Recently, the (patho)physiological functions of DAGLs have been explored by both genetic methods and chemical tools. This review will focus on the recent efforts to develop highly selective and in vivo active DAGLs inhibitors using activity-based protein profiling.

Formation of OX-1R/CB1R heteromeric complexes in embryonic mouse hypothalamic cells: Effect on intracellular calcium, 2-arachidonoyl-glycerol biosynthesis and ERK phosphorylation.

Orexin 1 (OX-1R) and cannabinoid receptor (CB1R) belong to the superfamily of G-protein-coupled receptors (GPCRs) and are mostly coupled to Gq and Gi/o proteins, respectively. In vitro studies in host cells over-expressing OX-1R and CB1R revealed a functional interaction between these receptors, through either their ability to form heteromers or the property for OX-1R to trigger the biosynthesis of 2-arachidonoylglycerol (2-AG), an endogenous CB1R ligand. Since: i) OX-1R and CB1R co-espression has been described at postsynaptc sites in hypothalamic circuits involved the regulation of energy homeostasis, and ii) increased orexin-A (OX-A) and 2-AG levels occur in hypothalamic neurons during obesity, we sought here to investigate the OX-1R/CB1R interaction in embryonic mouse hypothalamic NPY/AgRP mHypoE-N41 neurons which express, constitutively, both receptors. Treatment of mHypoE-N41 cells with OX-A (0.1-0.3µM), but not with the selective CB1R agonist, arachidonyl-2-chloroethylamide (ACEA; 0.1-0.3µM), transiently elevated [Ca(2+)]i. Incubation with a subeffective dose of OX-A (0.1µM)+ACEA (0.1µM) led to stronger and longer lasting elevation of [Ca(2+)]i, antagonized by OX-1R or CB1R antagonism with SB-334867 or AM251, respectively. FRET and co-immunoprecipitation experiments showed the formation of OX-1R/CB1R heteromers after incubation with OX-A (0.2µM), or OX-A (0.1µM)+ACEA (0.1µM), but not after ACEA (0.2µM), in a manner antagonized by SB-334867 or AM251. OX-A (0.2µM) or OX-A (0.1µM)+ACEA (0.1µM) also led to 2-AG biosynthesis. Finally, a stronger activation of ERK1/2(Thr202/185) phosphorylation in comparison to basal or each agonist alone (0.1-0.2µM), was induced by incubation with OX-A (0.1µM)+ACEA (0.1µM), again in a manner prevented by OX-1R or CB1R antagonism. We suggest that OX-A, alone at effective concentrations on [Ca(2+)]i, or in combination with ACEA, at subeffective concentrations, triggers intracellular signaling events via the formation of OX-1R/CB1R heteromers and an autocrine loop mediated by 2-AG.

Assay of DAGLα/ß Activity.

The endocannabinoid 2-arachidonoylglycerol (2-AG) exerts its physiological action by binding to and functionally activating type-1 (CB1) and type-2 (CB2) cannabinoid receptors. It is thought to be produced through the action of sn-1 selective diacylglycerol lipase (DAGL) that catalyzes 2-AG biosynthesis from sn-2-arachidonate-containing diacylglycerols. Since 2-AG biosynthetic enzymes have been identified only recently, little information on methodological approaches for measuring DAGL activity is as yet available. Here, a highly sensitive radiometric assay to measure DAGL activity by using 1-oleoyl[1-(14)C]-2-arachidonoylglycerol as the substrate is reported. All the steps needed to perform lipid extraction, fractionation by thin-layer chromatography (TLC), and quantification of radiolabeled [(14)C]-oleic acid via scintillation counting are described in detail.

Selective production of 1-monocaprin by porcine liver carboxylesterase-catalyzed esterification: Its enzyme kinetics and catalytic performance.

Porcine liver carboxylesterase (PLE) belongs to carboxylesterase family (EC 3.1.1.1) as a serine-type esterase. The PLE-catalyzed esterification of capric acid with glycerol in reverse micelles was investigated on the catalytic performance and enzyme kinetics. The most suitable structure of reverse micelles was comprised of isooctane (reaction medium) and bis(2-ethylhexyl) sodium sulfosuccinate (AOT, anionic surfactant) with 0.1 of R-value ([water]/[surfactant]) and 3.0 of G/F-value ([glycerol]/[fatty acid]) for the PLE-catalyzed esterification. In the aspect of regio-selectivity, the PLE mainly produced 1-monocaprin without any other products (di- and/or tricaprins of subsequent reactions). Furthermore, the degree of esterification at equilibrium state (after 4 h from the initiation) was 62.7% under the optimum conditions at pH 7.0 and 60 °C. Based on Hanes-Woolf plot, the apparent Km and Vmax values were calculated to be 16.44 mM and 38.91 µM/min/mg protein, respectively.

Cocaine-Induced Endocannabinoid Mobilization in the Ventral Tegmental Area.

Cocaine is a highly addictive drug that acts upon the brain's reward circuitry via the inhibition of monoamine uptake. Endogenous cannabinoids (eCB) are lipid molecules released from midbrain dopamine (DA) neurons that modulate cocaine's effects through poorly understood mechanisms. We find that cocaine stimulates release of the eCB, 2-arachidonoylglycerol (2-AG), in the rat ventral midbrain to suppress GABAergic inhibition of DA neurons, through activation of presynaptic cannabinoid CB1 receptors. Cocaine mobilizes 2-AG via inhibition of norepinephrine uptake and promotion of a cooperative interaction between Gq/11-coupled type-1 metabotropic glutamate and α1-adrenergic receptors to stimulate internal calcium stores and activate phospholipase C. The disinhibition of DA neurons by cocaine-mobilized 2-AG is also functionally relevant because it augments DA release in the nucleus accumbens in vivo. Our results identify a mechanism through which the eCB system can regulate the rewarding and addictive properties of cocaine.

Biosynthesis and Fate of Endocannabinoids.

Since the discovery of the two cannabinoid receptors, CB(1) and CB(2), several molecules, commonly defined as endocannabinoids, able to bind to and functionally activate these receptors, have been discovered and characterized. Although the general thought was that the endocannabinoids were mainly derivatives of the n-6 fatty acid arachidonic acid, recent data have shown that also derivatives (ethanolamides) of n-3 fatty acids may be classified as endocannabinoids. Whether the n-3 endocannabinoids follow the same biosynthetic and metabolic routes of the n-6 endocannabinoids is not yet clear and so warrants further investigation. In this review, we describe the primary biosynthetic and metabolic pathways for the two well-established endocannabinoids, anandamide and 2-arachidonoylglycerol.

Fasting stimulates 2-AG biosynthesis in the small intestine: role of cholinergic pathways.

The endocannabinoids are lipid-derived signaling molecules that control feeding and energy balance by activating CB1-type cannabinoid receptors in the brain and peripheral tissues. Previous studies have shown that oral exposure to dietary fat stimulates endocannabinoid signaling in the rat small intestine, which provides positive feedback that drives further food intake and preference for fat-rich foods. We now describe an unexpectedly broader role for cholinergic signaling of the vagus nerve in the production of the endocannabinoid, 2-arachidonoyl-sn-glycerol (2-AG), in the small intestine. We show that food deprivation increases levels of 2-AG and its lipid precursor, 1,2-diacylglycerol, in rat jejunum mucosa in a time-dependent manner. This response is abrogated by surgical resection of the vagus nerve or pharmacological blockade of small intestinal subtype-3 muscarinic acetylcholine (m3 mAch) receptors, but not inhibition of subtype-1 muscarinic acetylcholine (m1 mAch). We further show that blockade of peripheral CB1 receptors or intestinal m3 mAch receptors inhibits refeeding in fasted rats. The results suggest that food deprivation stimulates 2-AG-dependent CB1 receptor activation through a mechanism that requires efferent vagal activation of m3 mAch receptors in the jejunum, which, in turn, may promote feeding after a fast.

ß-Neurexins Control Neural Circuits by Regulating Synaptic Endocannabinoid Signaling.

α- and ß-neurexins are presynaptic cell-adhesion molecules implicated in autism and schizophrenia. We find that, although ß-neurexins are expressed at much lower levels than α-neurexins, conditional knockout of ß-neurexins with continued expression of α-neurexins dramatically decreased neurotransmitter release at excitatory synapses in cultured cortical neurons. The ß-neurexin knockout phenotype was attenuated by CB1-receptor inhibition, which blocks presynaptic endocannabinoid signaling, or by 2-arachidonoylglycerol synthesis inhibition, which impairs postsynaptic endocannabinoid release. In synapses formed by CA1-region pyramidal neurons onto burst-firing subiculum neurons, presynaptic in vivo knockout of ß-neurexins aggravated endocannabinoid-mediated inhibition of synaptic transmission and blocked LTP; presynaptic CB1-receptor antagonists or postsynaptic 2-arachidonoylglycerol synthesis inhibition again reversed this block. Moreover, conditional knockout of ß-neurexins in CA1-region neurons impaired contextual fear memories. Thus, our data suggest that presynaptic ß-neurexins control synaptic strength in excitatory synapses by regulating postsynaptic 2-arachidonoylglycerol synthesis, revealing an unexpected role for ß-neurexins in the endocannabinoid-dependent regulation of neural circuits.