The endocannabinoid system plays a role in obesity, primarily by its role in food reward. Activity, also involved in obesity, seems to be at least partially controlled by the endocannabinoid system, but the relevant behavioral and neurochemical mechanisms have not been well established. This study represents an attempt to begin elucidating these mechanisms by examining the effects of an endogenous cannabinoid ligand, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), on the reinforcing properties of exercise reinforcement in lean and obese Zucker rats. Ten obese and 10 lean Zucker rats pressed a locked door under a progressive ratio schedule of reinforcement that, when unlocked, provided access to a running wheel for <em>2</em>-min periods. After baseline breakpoints were established, doses of <em>2</em>-AG (0.3-3 mg/kg) were administered before experimental sessions. Obese rats exhibited lower breakpoints for wheel activity, lower response rates, and fewer revolutions compared with lean rats. <em>2</em>-AG decreased breakpoints, response rates, and revolutions for obese rats, and revolutions only for lean rats. These data suggest that <em>2</em>-AG may reduce the reinforcing properties of activity, and that obese Zuckers may show a greater sensitivity to <em>2</em>-AG. The data also suggest that endocannabinoids may play a role in the reinforcing properties of exercise.

Growing evidence indicates that the endocannabinoid system is important for the acquisition and/or extinction of learning and memory. However, it is unclear which endocannabinoid(s) play(s) a crucial role in these cognitive functions, especially memory extinction. To elucidate the physiological role of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), a major endocannabinoid, in behavioral and cognitive functions, we conducted a comprehensive behavioral test battery in knockout (KO) mice deficient in monoacylglycerol lipase (MGL), the major hydrolyzing enzyme of <em>2</em>-AG. We found age-dependent increases in spontaneous physical activity (SPA) in MGL KO mice. Next, we tested the MGL KO mice using 5 hippocampus-dependent learning paradigms (i.e., Morris water maze (MWM), contextual fear conditioning, novel object recognition test, trace eyeblink conditioning, and water-finding test). In the MWM, MGL KO mice showed normal acquisition of reference memory, but exhibited significantly faster extinction of the learned behavior. Moreover, they showed faster memory acquisition on the reversal-learning task of the MWM. In contrast, in the contextual fear conditioning, MGL KO mice tended to show slower memory extinction. In the novel object recognition and water-finding tests, MGL KO mice exhibited enhanced memory acquisition. Trace eyeblink conditioning was not altered in MGL KO mice throughout the acquisition and extinction phases. These results indicate that <em>2</em>-AG signaling is important for hippocampus-dependent learning and memory, but its contribution is highly task-dependent.

Cannabinoids (CBs) are implicated in a number of physiological and pathological mechanisms in the central nervous system, but their exact role in post-ischemic brain injury is unclear. The toxic and neuroprotective effects of synthetic and endogenous CBs were evaluated in rat organotypic hippocampal slices exposed to <em>2</em>0 min oxygen-glucose deprivation (OGD) and in gerbils subjected to bilateral carotid occlusion for 5 min. When present in the incubation medium, the synthetic CB agonists WIN 55<em>2</em>1<em>2</em>-<em>2</em> and CP 55940 (1-30 μM) and the CB1 agonist ACEA exacerbated CA1 injury induced by OGD, whereas the CB1 receptor antagonists AM <em>2</em>51 and LY 3<em>2</em>0135 were neuroprotective with maximal activity at 1 μM. AM <em>2</em>51 (at 3 mg/kg, i.p.) also attenuated CA1 pyramidal cell death in gerbils in vivo. The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) reduced OGD injury in hippocampal slices at 0.1-1 μM, whereas anandamide (AEA) was neurotoxic at the same concentrations. The effects of WIN 55<em>2</em>1<em>2</em>-<em>2</em>, AEA and <em>2</em>-AG in slices were all dependent on the activation of CB1 but not CB<em>2</em> receptors, except for the toxic effects of AEA that were also dependent on vanilloid TRPV1 receptors. Our results suggest that exogenous administration of CB1 agonists and the production of endocannabinoids "on demand" may produce different, if not opposite, effects on the fate of neurons following cerebral ischemia.

Cannabis sativa, or marijuana, a popular recreational drug, alters sensory perception and exerts a range of potential medicinal benefits. The present study demonstrates that the endogenous cannabinoid receptor agonists <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide (AEA) activate a canonical cannabinoid receptor in Caenorhabditis elegans and also modulate monoaminergic signaling at multiple levels. <em>2</em>-AG or AEA inhibit nociception and feeding through a pathway requiring the cannabinoid-like receptor NPR-19. <em>2</em>-AG or AEA activate NPR-19 directly and cannabinoid-dependent inhibition can be rescued in npr-19-null animals by the expression of a human cannabinoid receptor, CB1, highlighting the orthology of the receptors. Cannabinoids also modulate nociception and locomotion through an NPR-19-independent pathway requiring an α<em>2</em>A-adrenergic-like octopamine (OA) receptor, OCTR-1, and a 5-HT1A-like serotonin (5-HT) receptor, SER-4, that involves a complex interaction among cannabinoid, octopaminergic, and serotonergic signaling. <em>2</em>-AG activates OCTR-1 directly. In contrast, <em>2</em>-AG does not activate SER-4 directly, but appears to enhance SER-4-dependent serotonergic signaling by increasing endogenous 5-HT. This study defines a conserved cannabinoid signaling system in C. elegans, demonstrates the cannabinoid-dependent activation of monoaminergic signaling, and highlights the advantages of studying cannabinoid signaling in a genetically tractable whole-animal model.SIGNIFICANCE STATEMENTCannabis sativa, or marijuana, causes euphoria and exerts a wide range of medicinal benefits. For years, cannabinoids have been studied at the cellular level using tissue explants with conflicting results. To better understand cannabinoid signaling, we have used the Caenorhabditis elegans model to examine the effects of cannabinoids on behavior. The present study demonstrates that mammalian cannabinoid receptor ligands activate a conserved cannabinoid signaling system in C. elegans and also modulate monoaminergic signaling, potentially affecting an array of disorders, including anxiety and depression. This study highlights the potential role of cannabinoids in modulating monoaminergic signaling and the advantages of studying cannabinoid signaling in a genetically tractable, whole-animal model.

BACKGROUND

Under some conditions, stress, rather than directly triggering cocaine seeking, potentiates reinstatement to other stimuli, including a subthreshold cocaine dose. The mechanisms responsible for stress-potentiated reinstatement are not well defined. Endocannabinoid signaling is increased by stress and regulates synaptic transmission in brain regions implicated in motivated behavior.

OBJECTIVE

The objective of this study was to test the hypothesis that cannabinoid type 1 receptor (CB1R) signaling is required for stress-potentiated reinstatement of cocaine seeking in rats.

METHODS

Following i.v. cocaine self-administration (<em>2</em> h access/day) and extinction in male rats, footshock stress alone does not reinstate cocaine seeking but reinstatement is observed when footshock is followed by an injection of an otherwise subthreshold dose of cocaine (<em>2</em>.5 mg/kg, i.p.). CB1R involvement was tested by systemic administration of the CB1R antagonist AM<em>2</em>51 (0, 1, or 3 mg/kg, i.p.) prior to testing for stress-potentiated reinstatement.

RESULTS

Stress-potentiated reinstatement was blocked by both 1 and 3 mg/kg AM<em>2</em>51. By contrast, AM<em>2</em>51 only attenuated food-reinforced lever pressing at the higher dose (i.e., 3 mg/kg) and did not affect locomotor activity at either dose tested. Neither high-dose cocaine-primed reinstatement (10 mg/kg, i.p.) nor footshock stress-triggered reinstatement following long-access cocaine self-administration (6 h access/day) was affected by AM<em>2</em>51 pretreatment. Footshock stress increased concentrations of both endocannabinoids, N-arachidonylethanolamine and <em>2</em>-<em>arachidonoylglycerol</em>, in regions of the prefrontal cortex.

CONCLUSIONS

These findings demonstrate that footshock stress increases prefrontal cortical endocannabinoids and stress-potentiated reinstatement is CB1R-dependent, suggesting that CB1R is a potential therapeutic target for relapse prevention, particularly in individuals whose cocaine use is stress-related.

We demonstrated that the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) activated dose-dependently washed human platelets and increased intracellular calcium levels. Moreover <em>2</em>-AG activated protein kinase C measured as p47pleckstrin phosphorylation. These parameters were prevented by the tromboxane A<em>2</em> receptor antagonist SQ<em>2</em>9548, by phospholipase C pathway (U731<em>2</em><em>2</em>) and protein kinase C (GF109<em>2</em>03X) inhibitors. No effect on <em>2</em>-AG-induced platelet activation and calcium elevation in the presence of inhibitors of fatty acid amide hydrolase or monoacylglycerol lipase was observed. In addition we have shown that <em>2</em>-AG dose-dependently increased NO and cGMP levels. These effects were abolished by U731<em>2</em><em>2</em>, GF109<em>2</em>03X, EGTA and the intracellular calcium chelator BAPTA/AM. Moreover, <em>2</em>-AG enhanced eNOS activity through the phosphorylation of its positive regulatory residue ser1177 and by dephosphorylation of the negative one thr495. The eNOS ser1177 phosphorylation was inhibited by U731<em>2</em><em>2</em> and GF109<em>2</em>03X but it was unaffected by the PI3K/AKT pathway inhibitors LY<em>2</em>9400<em>2</em> and MK<em>2</em><em>2</em>06. The dephosphorylation of thr495 was reversed by low concentrations of calyculin A. Taken together these data suggest that <em>2</em>-AG behaves as a true platelet agonist stimulating PKC activation and calcium elevation. Likely <em>2</em>-AG can modulate platelet activation by increasing NO levels through eNOS activation.

Chronic pain is often a symptom of knee osteoarthritis (OA) for which current analgesics are either inadequate or are associated with serious side effects. The endocannabinoid system may offer alternative targets for pain relief. We evaluated the effects of a potent and selective monoacylglycerol (MAG) lipase inhibitor (MJN110) on OA pain behaviour, spinal mechanisms of action and joint histopathology in the rat.

Intra-articular injection of monosodium iodoacetate (MIA) models OA pain and mimics clinical joint pathology. Effects of MJN110 on MIA-induced weight-bearing asymmetry and lowered paw withdrawal thresholds (PWTs), changes in spinal gene expression and brain levels of relevant lipids were determined.

Acute MJN110 (5 mg·kg-1 ) significantly reversed MIA-induced weight-bearing asymmetry (MIA/vehicle: 68 ± 6 g; MIA/MJN110: 35 ± 4 g) and lowered ipsilateral PWTs (MIA/vehicle: 7 ± 0.8 g; MIA/MJN110: 11 ± 0.6 g), via both CB1 and CB<em>2</em> receptors. Repeated treatment with MJN110 (5 mg·kg-1 ) resulted in anti-nociceptive tolerance. A lower dose of MJN110 (1 mg·kg-1 ) acutely inhibited pain behaviour, which was maintained for 1 week of repeated administration but had no effect on joint histology. MJN110 significantly inhibited expression of membrane-associated PGE synthase-1 in the ipsilateral dorsal horn of the spinal cord of MIA rats, compared with vehicle-treated MIA rats. Both doses of MJN110 significantly elevated brain levels of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>.

Our data support further assessment of the therapeutic potential of MAG lipase inhibitors for the treatment of OA pain.

OBJECTIVE

Marijuana smoking is widespread in many countries, and the use of smoked synthetic cannabinoids is increasing. Smoking a marijuana joint leads to bronchodilation in both healthy subjects and asthmatics. The effects of Δ(9) -tetrahydrocannabinol and synthetic cannabinoids on human bronchus reactivity have not previously been investigated. Here, we sought to assess the effects of natural and synthetic cannabinoids on cholinergic bronchial contraction.

METHODS

Human bronchi isolated from 88 patients were suspended in an organ bath and contracted by electrical field stimulation (EFS) in the presence of the phytocannabinoid Δ(9) -tetrahydrocannabinol, the endogenous <em>2</em>-<em>arachidonoylglycerol</em>, the synthetic dual CB1 and CB<em>2</em> receptor agonists WIN55,<em>2</em>1<em>2</em>-<em>2</em> and CP55,940, the synthetic, CB<em>2</em> -receptor-selective agonist JWH-133 or the selective GPR55 agonist O-160<em>2</em>. The receptors involved in the response were characterized by using selective CB1 and CB<em>2</em> receptor antagonists (SR141716 and SR1445<em>2</em>8 respectively).

RESULTS

Δ(9) -tetrahydrocannabinol, WIN55,<em>2</em>1<em>2</em>-<em>2</em> and CP55,940 induced concentration-dependent inhibition of cholinergic contractions, with maximum inhibitions of 39, 76 and 77% respectively. JWH-133 only had an effect at high concentrations. <em>2</em>-Arachidonoylglycerol and O-160<em>2</em> were devoid of any effect. Only CB1 receptors were involved in the response because the effects of cannabinoids were antagonized by SR141716, but not by SR1445<em>2</em>8. The cannabinoids did not alter basal tone or contractions induced by exogenous Ach.

CONCLUSIONS

Activation of prejunctional CB1 receptors mediates the inhibition of EFS-evoked cholinergic contraction in human bronchus. This mechanism may explain the acute bronchodilation produced by marijuana smoking.

The entourage effect was a proposed explanation for biological observations that endocannabinoid ligand activities can be modified by other lipids released from cells at the same time. An increasing volume of anecdotal reports and interest in the plant have provoked research into the activity of minor chemical constituents of the plant-including volatile terpenoids such as myrcene, α- and β- pinene, β-caryophyllene, and limonene. However, to date, no clear interaction has been identified. The current study was designed to determine whether terpenes in the cannabis plant have detectable receptor-mediated activity, or modify the activity of Δ⁹-tetrahydrocannabinol, cannabidiol, or the endocannabinoid <em>2</em>-arachidonylglycerol at the cannabinoid receptors. In addition, we have utilized a standard radioligand binding paradigm with ability to detect orthosteric and allosteric interactions of test compounds. With the possible exception of a weak interaction of β-caryophyllene with CB<em>2</em>, no data were produced to support the hypothesis that any of the five terpenes tested (either alone or in mixtures) have direct interactions with CB1 or CB<em>2</em>, as the binding of radioligand ([³H]-CP55,940), Δ⁹-tetrahydrocannabinol, and cannabidiol were unaltered by the presence of terpenes. Similarly, terpene functional effects were also not detected, either alone or in combination with Δ⁹-tetrahydrocannabinol, cannabidiol, or <em>2</em>-<em>arachidonoylglycerol</em>. This study adds to the evidence that the putative entourage effect cannot be explained by direct effects at CB1 or CB<em>2</em>.

The endocannabinoid system remains an attractive molecular target for pharmacological intervention due to its roles in the central nervous system in learning, thinking, emotional function, regulation of food intake or pain sensation, as well as in the peripheral nervous system, where it modulates the action of cardiovascular, immune, metabolic or reproductive function. α/β hydrolase domain containing 6 (ABHD6)--an enzyme forming part of the endocannabinoid system--is a newly discovered post-genomic protein acting as a <em>2</em>-AG (<em>2</em>-<em>arachidonoylglycerol</em>) serine hydrolase. We have recently reported a series of 1,<em>2</em>,5-thiadiazole carbamates as potent and selective ABHD6 inhibitors. Here, we present comparative molecular field analysis (CoMFA) and molecular dynamics studies of these compounds. First, we performed a homology modeling study of ABHD6 based on the assumption that the catalytic triad of ABHD6 comprises Ser148-His306-Asp <em>2</em>78 and the oxyanion hole is formed by Met149 and Phe80. A total of 4<em>2</em> compounds was docked to the homology model using the Glide module from the Schrödinger suite of software and the selected docking poses were used for CoMFA alignment. A model with the following statistics was obtained: R(<em>2</em>) = 0.98, Q(<em>2</em>) = 0.55. In order to study the molecular interactions of the inhibitors with ABHD6 in detail, molecular dynamics was performed with the Desmond program. It was found that, during the simulations, the hydrogen bond between the inhibitor carbonyl group and the main chain of Phe80 is weakened, whereas a new hydrogen bond with the side chain of Ser148 is formed, facilitating the possible formation of a covalent bond. Graphical Abstract Left-right: Docking pose of 1 in the binding pocket of α/β hydrolase domain containing 6 (ABHD6) selected for molecular alignment; CoMFA steric and electrostatic contour fields; changes in potential energy of the complex during simulations for the complex of 6 and ABHD6.

Systemic Sclerosis (SSc) is a chronic autoimmune disease whose origin and pathogenesis are not yet well known. Recent studies are allowing a better definition of the disease. However, few studies have been performed based on metabolomics. In this way, this study aims to find altered metabolites in SSc patients in order to improve their diagnosis, prognosis and treatment. For that, 59 SSc patients and <em>2</em>8 healthy volunteers participated in this study. Urine and plasma samples were analysed by a fingerprinting metabolomic approach based on HPLC-ESI-QTOF-MS. We observed larger differences in urine than plasma metabolites. The main deregulated metabolic families in urine were acylcarnitines, acylglycines and metabolites derived from amino acids, specifically from proline, histidine and glutamine. These results indicate perturbations in fatty acid beta oxidation and amino acid pathways in scleroderma patients. On the other hand, the main plasma biomarker candidate was <em>2</em>-<em>arachidonoylglycerol</em>, which is involved in the endocannabinoid system with potential implications in the induction and propagation of systemic sclerosis and autoimmunity.

The elucidation of Δ9-tetrahydrocannabinol as the active principal of Cannabis sativa in 1963 initiated a fruitful half-century of scientific discovery, culminating in the identification of the endocannabinoid signaling system, a previously unknown neuromodulatory system. A primary function of the endocannabinoid signaling system is to maintain or recover homeostasis following psychological and physiological threats. We provide a brief introduction to the endocannabinoid signaling system and its role in synaptic plasticity. The majority of the article is devoted to a summary of current knowledge regarding the role of endocannabinoid signaling as both a regulator of endocrine responses to stress and as an effector of glucocorticoid and corticotrophin-releasing hormone signaling in the brain. We summarize data demonstrating that cannabinoid receptor 1 (CB1R) signaling can both inhibit and potentiate the activation of the hypothalamic-pituitary-adrenal axis by stress. We present a hypothesis that the inhibitory arm has high endocannabinoid tone and also serves to enhance recovery to baseline following stress, while the potentiating arm is not tonically active but can be activated by exogenous agonists. We discuss recent findings that corticotropin-releasing hormone in the amygdala enables hypothalamic-pituitary-adrenal axis activation via an increase in the catabolism of the endocannabinoid N-arachidonylethanolamine. We review data supporting the hypotheses that CB1R activation is required for many glucocorticoid effects, particularly feedback inhibition of hypothalamic-pituitary-adrenal axis activation, and that glucocorticoids mobilize the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>. These features of endocannabinoid signaling make it a tantalizing therapeutic target for treatment of stress-related disorders but to date, this promise is largely unrealized. © <em>2</em>017 American Physiological Society. Compr Physiol 7:1-15, <em>2</em>017.

Inflammatory bowel disease (IBD) is a lifelong disease of the gastrointestinal tract whose annual incidence and prevalence is on the rise. Current immunosuppressive therapies available for treatment of IBD offer limited benefits and lose effectiveness, exposing a significant need for the development of novel therapies. In the clinical setting, cannabis has been shown to provide patients with IBD symptomatic relief, although the underlying mechanisms of their anti-inflammatory effects remain unclear.

This review reflects our current understanding of how targeting the endocannabinoid system, including cannabinoid receptors 1 and <em>2</em>, endogenous cannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, atypical cannabinoids, and degrading enzymes including fatty acid amide hydrolase and monoacylglycerol lipase, impacts murine colitis. In addition, the impact of cannabinoids on the human immune system is summarized.

Cannabinoid receptors 1 and <em>2</em>, endogenous cannabinoids, and atypical cannabinoids are upregulated in inflammation, and their presence and stimulation attenuate murine colitis, whereas cannabinoid receptor antagonism and cannabinoid receptor deficient models reverse these anti-inflammatory effects. In addition, inhibition of endocannabinoid degradation through monoacylglycerol lipase and fatty acid amide hydrolase blockade can also attenuate colitis development, and is closely linked to cannabinoid receptor expression.

Although manipulation of the endocannabinoid system in murine colitis has proven to be largely beneficial in attenuating inflammation, there is a paucity of human study data. Further research is essential to clearly elucidate the specific mechanisms driving this anti-inflammatory effect for the development of therapeutics to target inflammatory disease such as IBD.

Glucagon-like peptide-1 (GLP-1), a metabolic signal molecule, regulates reproduction, although, the involved molecular mechanisms have not been elucidated, yet. Therefore, responsiveness of gonadotropin-releasing hormone (GnRH) neurons to the GLP-1 analog Exendin-4 and elucidation of molecular pathways acting downstream to the GLP-1 receptor (GLP-1R) have been challenged. Loose patch-clamp recordings revealed that Exendin-4 (100 nM-5 μM) elevated firing rate in hypothalamic GnRH-GFP neurons of male mice via activation of GLP-1R. Whole-cell patch-clamp measurements demonstrated increased excitatory GABAergic miniature postsynaptic currents (mPSCs) frequency after Exendin-4 administration, which was eliminated by the GLP-1R antagonist Exendin-3(9-39) (1 μM). Intracellular application of the G-protein inhibitor GDP-β-S (<em>2</em> mM) impeded action of Exendin-4 on mPSCs, suggesting direct excitatory action of GLP-1 on GnRH neurons. Blockade of nitric-oxide (NO) synthesis by Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME; 100 μM) or N(5)-[Imino(propylamino)methyl]-L-ornithine hydrochloride (NPLA; 1 μM) or intracellular scavenging of NO by <em>2</em>-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO; 1 mM) partially attenuated the excitatory effect of Exendin-4. Similar partial inhibition was achieved by hindering endocannabinoid pathway using cannabinoid receptor type-1 (CB1) inverse-agonist 1-(<em>2</em>,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl) pyrazole-3-carboxamide (AM<em>2</em>51; 1 μM). Simultaneous blockade of NO and endocannabinoid signaling mechanisms eliminated action of Exendin-4 suggesting involvement of both retrograde machineries. Intracellular application of the transient receptor potential vanilloid 1 (TRPV1)-antagonist <em>2</em>E-N-(<em>2</em>, 3-Dihydro-1,4-benzodioxin-6-yl)-3-[4-(1, 1-dimethylethyl)phenyl]-<em>2</em>-Propenamide (AMG9810; 10 μM) or the fatty acid amide hydrolase (FAAH)-inhibitor PF3845 (5 μM) impeded the GLP-1-triggered endocannabinoid pathway indicating an anandamide-TRPV1-sensitive control of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) production. Furthermore, GLP-1 immunoreactive (IR) axons innervated GnRH neurons in the hypothalamus suggesting that GLP-1 of both peripheral and neuronal sources can modulate GnRH neurons. RT-qPCR study confirmed the expression of GLP-1R and neuronal NO synthase (nNOS) mRNAs in GnRH-GFP neurons. Immuno-electron microscopic analysis revealed the presence of nNOS protein in GnRH neurons. These results indicate that GLP-1 exerts direct facilitatory actions via GLP-1R on GnRH neurons and modulates NO and <em>2</em>-AG retrograde signaling mechanisms that control the presynaptic excitatory GABAergic inputs to GnRH neurons.

Alcohol dependence is associated with anxiety during withdrawal. The endocannabinoid (ECB) system participates in the neuroendocrine and behavioral response to stress and changes in corticolimbic ECB signaling may contribute to alcohol withdrawal-induced anxiety. Moreover, symptoms of alcohol withdrawal differ between sexes and sexual dimorphism in withdrawal-induced ECB recruitment may be a contributing factor. Herein, we exposed intact male and female rats and ovariectomized (OVX) female rats with or without estradiol (E<em>2</em>) replacement to 6 weeks of chronic intermittent alcohol vapor and measured anxiety-like behavior, ECB content, and ECB-related mRNA in the basolateral amygdala (BLA) and ventromedial prefrontal cortex (vmPFC). Acute alcohol withdrawal increased anxiety-like behavior, produced widespread disturbances in ECB-related mRNA, and reduced anandamide (AEA) content in the BLA and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) content in the vmPFC of male, but not female rats. Similar to males, alcohol-exposed OVX females showed reductions in Napepld mRNA in the BLA, decreased AEA content in the BLA and vmPFC, and reductions in all ECB-related genes measured in the vmPFC. Importantly, E<em>2</em> replacement prevented withdrawal-induced alterations in ECB content (but not mRNA) in OVX females, and although alcohol-exposed OVX females failed to exhibit more anxiety compared to their respective control, chronic alcohol exposure abolished the anxiolytic properties of E<em>2</em> in OVX rats. These data indicate that ovarian sex hormones (but not E<em>2</em> alone) protect against withdrawal-induced alterations in corticolimbic ECB signaling but do not impart resilience to withdrawal-induced anxiety. Thus, the mechanisms implicated in the manifestation of alcohol withdrawal-induced anxiety are most likely sex-specific. This article is part of the Special Issue entitled "A New Dawn in Cannabinoid Neurobiology".

BACKGROUND

α/β-Hydrolase domain containing (ABHD)1<em>2</em> is a recently discovered serine hydrolase that acts in vivo as a lysophospholipase for lysophosphatidylserine. Dysfunctional ABHD1<em>2</em> has been linked to the rare neurodegenerative disorder called PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, cataract). In vitro, ABHD1<em>2</em> has been implicated in the metabolism of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Further studies on ABHD1<em>2</em> function are hampered as no selective inhibitor have been identified to date. In contrast to the situation with the other endocannabinoid hydrolases, ABHD1<em>2</em> has remained a challenging target for inhibitor development as no crystal structures are available to facilitate drug design.

RESULTS

Here we report the unexpected discovery that certain triterpene-based structures inhibit human ABHD1<em>2</em> hydrolase activity in a reversible manner, the best compounds showing submicromolar potency. Based on structure activity relationship (SAR) data collected for 68 natural and synthetic triterpenoid structures, a pharmacophore model has been constructed. A pentacyclic triterpene backbone with carboxyl group at position 17, small hydrophobic substituent at the position 4, hydrogen bond donor or acceptor at position 3 accompanied with four axial methyl substituents was found crucial for ABHD1<em>2</em> inhibitor activity. Although the triterpenoids typically may have multiple protein targets, we witnessed unprecedented selectivity for ABHD1<em>2</em> among the metabolic serine hydrolases, as activity-based protein profiling of mouse brain membrane proteome indicated that the representative ABHD1<em>2</em> inhibitors did not inhibit other serine hydrolases, nor did they target cannabinoid receptors.

CONCLUSIONS

We have identified reversibly-acting triterpene-based inhibitors that show remarkable selectivity for ABHD1<em>2</em> over other metabolic serine hydrolases. Based on SAR data, we have constructed the first pharmacophore model of ABHD1<em>2</em> inhibitors. This model should pave the way for further discovery of novel lead structures for ABHD1<em>2</em> selective inhibitors.

In mammalian brain, monoacylglycerol lipase (MAGL) is the primary enzyme responsible for terminating signaling function of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Previous in vivo studies with mice indicate that both genetic and chronic pharmacological inactivation of MAGL result in 8-30-fold increase of <em>2</em>-AG concentration in the brain, causing desensitization and downregulation of cannabinoid CB1 receptor (CB1R) activity, leading to functional and behavioral tolerance. However, direct evidence for reduced CB1R activity in the brain is lacking. In this study, we used functional autoradiography to assess basal and agonist-stimulated CB1R-dependent Gi/o protein activity in multiple brain regions of MAGL-KO mice in comparison to their wild-type (WT) littermates. In addition, the role of endogenous cannabinoids in basal CB1R signaling was assessed after comprehensive pharmacological blockade of <em>2</em>-AG hydrolysis by determining the contents of endocannabinoids (eCBs) in WT and MAGL-KO brain tissues by LC/MS/MS technology. To show whether lack of MAGL cause compensatory alterations in the serine hydrolase activity, we compared serine hydrolase pattern of WT and MAGL-KO using activity-based protein profiling. Consistent with studies using chronic pharmacological MAGL inactivation in vivo, we observed a statistically significant decrease of CB1R-Gi/o signaling in most of the studied brain regions. In MAGL-KO brain sections, elevated <em>2</em>-AG levels were mirrored to heightened basal CB1R-dependent Gi/o-activity, as well as, dampened agonist-evoked responses in several brain regions. The non-selective serine hydrolase inhibitor methylarachidonoylfluorophosphonate (MAFP) was able to significantly elevate <em>2</em>-AG levels in brain sections of MAGL-KO mice, indicating that additional serine hydrolases possess <em>2</em>-AG hydrolytic activity in MAGL-KO brain sections.

Mast cells (MCs) contribute to the control of local inflammatory reactions and become hyporesponsive after prolonged TLR4 activation by bacterial LPS. The molecular mechanisms involved in endotoxin tolerance (ET) induction in MCs are not fully understood. In this study, we demonstrate that the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and its receptor, cannabinoid receptor <em>2</em> (CB<em>2</em>), play a role in the establishment of ET in bone marrow-derived MCs from C57BL/6J mice. We found that CB<em>2</em> antagonism prevented the development of ET and that bone marrow-derived MCs produce <em>2</em>-AG in a TLR4-dependent fashion. Exogenous <em>2</em>-AG induced ET similarly to LPS, blocking the phosphorylation of IKK and the p65 subunit of NF-κB and inducing the synthesis of molecular markers of ET. LPS caused CB<em>2</em> receptor trafficking in Rab11-, Rab7-, and Lamp<em>2</em>-positive vesicles, indicating recycling and degradation of the receptor. <em>2</em>-AG also prevented LPS-induced TNF secretion in vivo, in a MC-dependent model of endotoxemia, demonstrating that TLR4 engagement leads to <em>2</em>-AG secretion, which contributes to the negative control of MCs activation. Our study uncovers a functional role for the endocannabinoid system in the inhibition of MC-dependent innate immune responses in vivo.

<AbstractText>Palmitoylethanolamide (PEA) is a pleiotropic endogenous lipid mediator currently used as a "dietary food for special medical purposes" against neuropathic pain and neuro-inflammatory conditions. Several mechanisms underlie PEA actions, among which the "entourage" effect, consisting of PEA potentiation of endocannabinoid signaling at either cannabinoid receptors or transient receptor potential vanilloid type-1 (TRPV1) channels. Here, we report novel molecular mechanisms through which PEA controls mast cell degranulation and substance P (SP)-induced histamine release in rat basophilic leukemia (RBL-<em>2</em>H3) cells, a mast cell model.</AbstractText><AbstractText>RBL-<em>2</em>H3 cells stimulated with SP were treated with PEA in the presence and absence of a cannabinoid type-<em>2</em> (CB<em>2</em>) receptor antagonist (AM630), or a diacylglycerol lipase (DAGL) enzyme inhibitor (OMDM188) to inhibit the biosynthesis of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). The release of histamine was measured by ELISA and β-hexosaminidase release and toluidine blue staining were used as indices of degranulation. <em>2</em>-AG levels were measured by LC-MS. The mRNA expression of proposed PEA targets (Cnr1, Cnr<em>2</em>, Trpv1, Ppara and Gpr55), and of PEA and endocannabinoid biosynthetic (Napepld, Dagla and Daglb) and catabolic (Faah, Naaa and Mgl) enzymes were also measured. The effects of PEA on the activity of DAGL-α or -β enzymes were assessed in COS-7 cells overexpressing the human recombinant enzyme or in RBL-<em>2</em>H3 cells, respectively.</AbstractText><AbstractText>SP increased the number of degranulated RBL-<em>2</em>H3 cells and triggered the release of histamine. PEA counteracted these effects in a manner antagonized by AM630. PEA concomitantly increased the levels of <em>2</em>-AG in SP-stimulated RBL-<em>2</em>H3 cells, and this effect was reversed by OMDM188. PEA significantly stimulated DAGL-α and -β activity and, consequently, <em>2</em>-AG biosynthesis in cell-free systems. Co-treatment with PEA and <em>2</em>-AG at per se ineffective concentrations downmodulated SP-induced release of histamine and degranulation, and this effect was reversed by OMDM188.</AbstractText><AbstractText>Activation of CB<em>2</em> underlies the inhibitory effects on SP-induced RBL-<em>2</em>H3 cell degranulation by PEA alone. We demonstrate for the first time that the effects in RBL-<em>2</em>H3 cells of PEA are due to the stimulation of <em>2</em>-AG biosynthesis by DAGLs.</AbstractText>

BACKGROUND

The ability to effectively cope with stress is a critical determinant of disease susceptibility. The lateral habenula (LHb) and the endocannabinoid (ECB) system have independently been shown to be involved in the selection of stress coping strategies, yet the role of ECB signaling in the LHb remains unknown.

METHODS

Using a battery of complementary techniques in rats, we examined the localization of type-1 cannabinoid receptors (CB1Rs) and assessed the behavioral and neuroendocrine effects of intra-LHb CB1R manipulations. We further tested the extent to which the ECB system in the LHb is impacted following chronic unpredictable stress or social defeat stress, and whether manipulation of LHb CB1Rs can bias coping strategies in rats with a history of chronic stress.

RESULTS

Electron microscopy studies revealed CB1R expression on presynaptic axon terminals, postsynaptic membranes, mitochondria, and glial processes in the rat LHb. In vivo microdialysis experiments indicated that acute stress increased the amount of <em>2</em>-<em>arachidonoylglycerol</em> in the LHb, while intra-LHb CB1R blockade increased basal corticosterone, augmented proactive coping strategies, and reduced anxiety-like behavior. Basal LHb <em>2</em>-<em>arachidonoylglycerol</em> content was similarly elevated in rats that were subjected to chronic unpredictable stress or social defeat stress and positively correlated with adrenal weight. Finally, intra-LHb CB1R blockade increased proactive behaviors in response to a novel conspecific, increasing approach behaviors irrespective of stress history and decreasing the latency to be attacked during an agonistic encounter.

CONCLUSIONS

Alterations in LHb ECB signaling may be relevant for development of stress-related pathologies in which LHb dysfunction and stress-coping impairments are hallmark symptoms.

Prenatal alcohol exposure often results in fetal alcohol syndrome and fetal alcohol spectrum disorders. Mechanisms of fetal brain damage by alcohol remain unclear. We used baboons (Papio spp.) to study alcohol-driven changes in the fetal cerebral artery endocannabinoid system. Pregnant baboons were subjected to binge alcohol exposure via gastric infusion three times during a period equivalent to the second trimester of human pregnancy. A control group was infused with orange-flavored drink that was isocaloric to the alcohol-containing solution. Cesarean sections were performed at a time equivalent to the end of the second trimester of human pregnancy. Fetal cerebral arteries were harvested and subjected to in vitro pressurization followed by pharmacological profiling. During each alcohol-infusion episode, maternal blood alcohol concentrations (BAC) reached 80 mg/dL, that is, equivalent to the BAC considered legal intoxication in humans. Circulating anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) remained unchanged. Ultrasound studies on pregnant mothers revealed that fetal alcohol exposure decreased peak systolic blood velocity in middle cerebral arteries when compared to pre-alcohol levels. Moreover, ethanol-induced dilation was observed in fetal cerebral arteries pressurized in vitro. This dilation was abolished by the mixture of AM<em>2</em>51 and AM630, which block cannabinoid receptors 1 and <em>2</em>, respectively. In the presence of AM<em>2</em>51, the cannabinoid receptor agonist AEA evoked a higher, concentration-dependent dilation of cerebral arteries from alcohol-exposed fetuses. The difference in AEA-induced cerebral artery dilation vanished in the presence of AM630. CB1 and CB<em>2</em> receptor mRNA and protein levels were similar in cerebral arteries from alcohol-exposed and control-exposed fetuses. In summary, alcohol exposure dilates fetal cerebral arteries via endocannabinoid receptors and results in an increased function of CB<em>2</em>.

Monoacylglycerol lipase (MAGL) has been characterized as the main enzyme responsible for the inactivation of the most abundant brain endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Besides this role, MAGL has progressively acquired a growing importance as an integrative metabolic hub that controls not only the in vivo levels of <em>2</em>-AG but also of other monoacylglycerides and, indirectly, the levels of free fatty acids derived from their hydrolysis as well as other lipids with pro-inflammatory or pro-tumorigenic effects, coming from the further metabolism of fatty acids. All these functions have only started to be elucidated in the last years due to the progress made in the knowledge of the structure of MAGL and in the development of genetic and chemical tools. In this review we report the advances made in the field with a special focus on the last decade and how MAGL has become a promising therapeutic target for the treatment of several diseases that currently lack appropriate therapies.

Cocaine is an addictive drug that acts in brain reward areas. Recent evidence suggests that cocaine stimulates synthesis of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in midbrain, increasing dopamine neuron activity via disinhibition. Although a mechanism for cocaine-stimulated <em>2</em>-AG synthesis is known, our understanding of <em>2</em>-AG release is limited. In NG108 cells and mouse midbrain tissue we find that <em>2</em>-AG is localized in non-synaptic extracellular vesicles (EVs) that are secreted in the presence of cocaine via interaction with the chaperone protein sigma-1 receptor (Sig-1R). The release of EVs occurs when cocaine causes dissociation of the Sig-1R from ADP-ribosylation factor (ARF6), a G-protein regulating EV trafficking, leading to activation of myosin light chain kinase (MLCK). Blockade of Sig-1R function, or inhibition of ARF6 or MLCK also prevented cocaine-induced EV release and cocaine-stimulated <em>2</em>-AG-modulation of inhibitory synapses in DA neurons. Our results implicate the Sig-1R-ARF6 complex in control of EV release and demonstrate that cocaine-mediated <em>2</em>-AG release can occur via EVs.