The involvement of brain <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in a number of critical physiological and pathophysiological regulatory mechanisms highlights the importance for an accurate brain <em>2</em>-AG determination. In the present study, we validated head-focused microwave irradiation (MW) as a method to prevent postmortem brain <em>2</em>-AG alterations before analysis. We compared MW to freezing to prevent <em>2</em>-AG induction and estimated exogenous and endogenous <em>2</em>-AG stability upon exposure to MW. Using MW, we measured, for the first time, true <em>2</em>-AG brain levels under basal conditions, 30 s after brain removal from the cranium, and upon exposure to 5 min of brain global ischemia. Our data indicate that brain <em>2</em>-AG levels are instantaneously and dramatically increased approximately 60-fold upon brain removal from the cranium. With 5 min of brain global ischemia <em>2</em>-AG levels are also, but less dramatically, increased 3.5-fold. Our data indicate that brain tissue fixation with MW is a required technique to measure both true basal <em>2</em>-AG levels and <em>2</em>-AG alterations under different experimental conditions including global ischemia, and <em>2</em>-AG is stable upon exposure to MW.

Activation of G protein-coupled receptors (GPCRs) can induce vasoconstriction via calcium signal-mediated and Rho-dependent pathways. Earlier reports have shown that diacylglycerol produced during calcium signal generation can be converted to an endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Our aim was to provide evidence that GPCR signaling-induced <em>2</em>-AG production and activation of vascular type1 cannabinoid receptors (CB1R) is capable of reducing agonist-induced vasoconstriction and hypertension. Rat and mouse aortic rings were examined by myography. Vascular expression of CB1R was demonstrated with immunohistochemistry. Rat aortic vascular smooth muscle cells (VSMCs) were cultured for calcium measurements and <em>2</em>-AG-determination. Inhibition or genetic loss of CB1Rs enhanced vasoconstriction induced by angiotensin II (AngII) or phenylephrine (Phe), but not by prostaglandin(PG)F<em>2</em>α. AngII-induced vasoconstriction was augmented by inhibition of diacylglycerol lipase (tetrahydrolipstatin) and was attenuated by inhibition of monoacylglycerol lipase (JZL184) suggesting a functionally relevant role for endogenously produced <em>2</em>-AG. In Gαq/11-deficient mice vasoconstriction was absent to AngII or Phe, which activate Gq/11-coupled receptors, but was maintained in response to PGF<em>2</em>α. In VSMCs, AngII-stimulated <em>2</em>-AG-formation was inhibited by tetrahydrolipstatin and potentiated by JZL184. CB1R inhibition increased the sustained phase of AngII-induced calcium signal. Pharmacological or genetic loss of CB1R function augmented AngII-induced blood pressure rise in mice. These data demonstrate that vasoconstrictor effect of GPCR agonists is attenuated via Gq/11-mediated vascular endocannabinoid formation. Agonist-induced endocannabinoid-mediated CB1R activation is a significant physiological modulator of vascular tone. Thus, the selective modulation of GPCR signaling-induced endocannabinoid release has a therapeutic potential in case of increased vascular tone and hypertension.

Allergen exposure may induce changes in the brainstem secondary neurons, with neural sensitization of the nucleus solitary tract (NTS), which in turn can be considered one of the causes of the airway hyperresponsiveness, a characteristic feature of asthma. We evaluated neurofunctional, morphological, and biochemical changes in the NTS of naive or sensitized rats. To evaluate the cell firing activity of NTS, in vivo electrophysiological experiments were performed before and after capsaicin challenge in sensitized or naive rats. Immunohistochemical studies, endocannabinoid, and palmitoylethanolamide quantification in the NTS were also performed. This study provides evidence that allergen sensitization in the NTS induced: (1) increase in the neural firing response to intratracheal capsaicin application, (<em>2</em>) increase of endocannabinoid anandamide and palmitoylethanolamide, a reduction of <em>2</em>-<em>arachidonoylglycerol</em> levels in the NTS, (3) glial cell activation, and (4) prevention by a Group III metabotropic glutamate receptor activation of neural firing response to intratracheal application of capsaicin in both naïve and sensitized rats. Therefore, normalization of ovalbumin-induced NTS neural sensitization could open up the prospect of new treatments based on the recovery of specific brain nuclei function and for extensive studies on acute or long-term efficacy of selective mGlu ligand, in models of bronchial hyperreactivity.

The major endocannabinoid in the mammalian brain is the bioactive lipid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). The best-known effects of <em>2</em>-AG are mediated by G-protein-coupled cannabinoid receptors. In principle, <em>2</em>-AG could modify neuronal excitability by acting directly on ion channels, but such mechanisms are poorly understood. Using a preparation of dissociated mouse midbrain dopamine neurons to isolate effects on intrinsic excitability, we found that 100 nM <em>2</em>-AG accelerated pacemaking and steepened the frequency-current relationship for burst-like firing. In voltage-clamp experiments, <em>2</em>-AG reduced A-type potassium current (IA) through a cannabinoid receptor-independent mechanism mimicked by arachidonic acid, which has no activity on cannabinoid receptors. Activation of orexin, neurotensin, and metabotropic glutamate Gq/11-linked receptors mimicked the effects of exogenous <em>2</em>-AG and their actions were prevented by inhibiting the <em>2</em>-AG-synthesizing enzyme diacylglycerol lipase α. The results show that <em>2</em>-AG and related lipid signaling molecules can directly tune neuronal excitability in a cell-autonomous manner by modulating IA.

The endocannabinoid system is a major regulator of food intake in many animal species. Studies conducted so far have mostly focused on mammals, and, therefore, in this study, the role of the endocannabinoid system in food intake in the sea bream Sparus aurata was investigated. The effect of different doses of the endocannabinoid anandamide (AEA), administered via water, was evaluated after different exposure times (30, 60 and 1<em>2</em>0 min) at both physiological and molecular levels. The results obtained indicate that fish exposed to AEA via water present approximately 1000-fold higher levels of AEA in both the brain and liver, which correlated with a significant increase in food intake and with the elevation of cannabinoid receptor 1 (CB(1)) and neuropeptide Y (NPY) mRNA levels in the brain. A peripheral effect of AEA was also observed, since a time-dependent increase in hepatic CB(1) mRNA and protein levels was detected. These effects were attenuated by the administration, again via water, of a selective cannabinoid CB(1) receptor antagonist (AM<em>2</em>51). These findings indicate that the endocannabinoid AEA, at doses that stimulate food intake in fish, concomitantly stimulates the expression of the orexigenic peptide NPY as well that of its own receptor, thereby potentially enhancing its effect on food consumption. In agreement with a role of AEA in food intake in S. aurata, we found increased brain levels of both this and the other endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), following food deprivation.

BACKGROUND

CB1 cannabinoid receptors are G-protein coupled receptors for endocannabinoids including anandamide and <em>2</em>-<em>arachidonoylglycerol</em>. Because these arachidonic acid metabolites possess a <em>2</em>0-carbon polyene chain as the alkyl terminal moiety, they are highly flexible with the potential to adopt multiple biologically relevant conformations, particularly those in a bent form. To better understand the molecular interactions associated with binding and steric trigger mechanisms of receptor activation, a series of conformationally-restricted anandamide analogs having a wide range of affinity and efficacy were evaluated.

RESULTS

A CB1 receptor model was constructed to include the extracellular loops, particularly extracellular loop <em>2</em> which possesses an internal disulfide linkage. Using both Glide (Schrödinger) and Affinity (Accelrys) docking programs, binding conformations of six anandamide analogs were identified that conform to rules applicable to the potent, efficacious and stereoselective non-classical cannabinoid CP55<em>2</em>44. Calculated binding energies of the optimum structures from both procedures correlated well with the reported binding affinity values. The most potent and efficacious of the ligands adopted conformations characterized by interactions with both the helix-3 lysine and hydrophobic residues that interact with CP55<em>2</em>44. The other five compounds formed fewer or less energetically favorable interactions with these critical residues. The flexibility of the tested anandamide analogs, measured by torsion angles around the benzene as well as the stretch between side chain moieties, could contribute to the differences in ability to interact with the CB1 receptor.

CONCLUSIONS

Analyses of multiple poses of conformationally-restricted anandamide analogs permitted identification of favored amino acid interactions within the CB1 receptor binding pocket. A ligand possessing both high affinity and cannabinoid agonist efficacy was able to interact with both polar and hydrophobic interaction sites utilized by the potent and efficacious non-classical cannabinoid CP55940. In contrast, other analogs characterized by reduced affinity or efficacy exhibited less favorable interactions with those key residues.

Chronic nicotine exposure (CNE) alters synaptic transmission in the ventral tegmental area (VTA) in a manner that enhances dopaminergic signaling and promotes nicotine use. The present experiments identify a correlation between enhanced production of the endogenous cannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and diminished release of the inhibitory neurotransmitter GABA in the VTA following CNE. To study the functional role of on-demand <em>2</em>-AG signaling in GABAergic synapses, we used 1,<em>2</em>,3-triazole urea compounds to selectively inhibit <em>2</em>-AG biosynthesis by diacylglycerol lipase (DAGL). The potency and selectivity of these inhibitors were established in rats in vitro (rat brain proteome), ex vivo (brain slices), and in vivo (intracerebroventricular administration) using activity-based protein profiling and targeted metabolomics analyses. Inhibition of DAGL (<em>2</em>-AG biosynthesis) rescues nicotine-induced VTA GABA signaling following CNE. Conversely, enhancement of <em>2</em>-AG signaling in naïve rats by inhibiting <em>2</em>-AG degradation recapitulates the loss of nicotine-induced GABA signaling evident following CNE. DAGL inhibition reduces nicotine self-administration without disrupting operant responding for a nondrug reinforcer or motor activity. Collectively, these findings provide a detailed characterization of selective inhibitors of rat brain DAGL and demonstrate that excessive <em>2</em>-AG signaling contributes to a loss of inhibitory GABAergic constraint of VTA excitability following CNE.

Inflammation plays a pivotal role in the pathogenesis of many diseases in the central nervous system. Caudate nucleus (CN), the largest nucleus in the brain, is also implicated in many neurological disorders. <em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG), the most abundant endogenous cannabinoid and the true natural ligand for CB1 receptors, has been shown to exhibit neuroprotective effects through its anti-inflammatory action from proinflammatory stimuli in hippocampus. However, it is still not clear whether <em>2</em>-AG is also able to protect CN neurons from proinflammation stimuli. In the present study, we discovered that <em>2</em>-AG significantly protects CN neurons in culture against lipopolysaccharide (LPS)-induced inflammatory response. <em>2</em>-AG is capable of suppressing elevation of LPS-induced cyclooxygenase-<em>2</em> expression associated with ERK/p38MAPK/NF-κB signaling pathway in CB1 receptor-dependant manner in primary cultured CN neurons. Moreover, <em>2</em>-AG inhibits LPS-induced increase in voltage-gated sodium channel currents and hyperpolarizing shift of activation curves through CB1 receptor-dependant pathway. Our study suggests the therapeutic potential of <em>2</em>-AG for the treatment of some inflammation-induced neurological disorders and pain.

Endocannabinoids are thought to function as retrograde messengers, which modulate neurotransmitter release by activating presynaptic cannabinoid receptors. Anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are the two best studied endogenous lipids which can act as endocannabinoids. Together with the proteins responsible for their biosynthesis, inactivation and the cannabinoid receptors, these lipids constitute the endocannabinoid system. This system is proposed to be involved in various neurodegenerative diseases such as Parkinson's and Huntington's diseases as well as Multiple Sclerosis. It has been demonstrated that the endocannabinoid system can protect neurons against glutamate excitotoxicity and acute neuronal damage in both in vitro and in vivo models. In this paper we review the data concerning the involvement of the endocannabinoid system in neurodegenerative diseases in which neuronal cell death may be elicited by excitotoxicity. We focus on the biosynthesis of endocannabinoids and on their modes of action in animal models of these neurodegenerative diseases.

The role of the endocannabinoid system in haematopoietic cells is not completely understood. We investigated whether human erythroleukemia (HEL) cells were able to bind, metabolise and transport the main endocannabinoids, anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). We also investigated whether AEA or <em>2</em>-AG could modulate HEL differentiation. Although able to internalise both endocannabinoids, HEL cells had the machinery to metabolise <em>2</em>-AG only, since they were devoid of the enzymes needed to synthesise and degrade AEA. Nonetheless, the intracellular transport of exogenous AEA might be required to activate the vanilloid receptors, with yet unknown implications for vascular biology. On the contrary, <em>2</em>-AG appeared to play a role in lineage determination. Indeed, <em>2</em>-AG itself drove HEL cells towards megakaryocytic differentiation, as it enhanced expression of beta3 integrin subunit, a megakaryocyte/platelet surface antigen, and glycoprotein VI, a late marker of megakaryocytes; in parallel, it reduced the amount of messenger RNA encoding for glycophorin A, a marker of erythroid phenotype. All these effects were mediated by activation of CB(<em>2</em>) cannabinoid receptors that triggered an extracellular signal-regulated kinase-dependent signalling cascade. In addition, classical inducers of megakaryocyte differentiation reduced <em>2</em>-AG synthesis (although they did not affect the binding efficiency of CB(<em>2</em>) receptors), suggesting that levels of this endocannabinoid may be critical for committing HEL cells towards the megakaryocytic lineage.

Elevation of extracellular Ca(<em>2</em>+) concentration induces intracellular Ca(<em>2</em>+) signaling in parathyroid cells. The response is due to stimulation of the phospholipase C/Ca(<em>2</em>+) pathways, but the direct mechanism responsible for the rise of intracellular Ca(<em>2</em>+) concentration has remained elusive. Here, we describe the electrophysiological property associated with intracellular Ca(<em>2</em>+) signaling in frog parathyroid cells and show that Ca(<em>2</em>+)-activated Cl(-) channels are activated by intracellular Ca(<em>2</em>+) increase through an inositol 1,4,5-trisphophate (IP(3))-independent pathway. High extracellular Ca(<em>2</em>+) induced an outwardly-rectifying conductance in a dose-dependent manner (EC(50) ∼6 mM). The conductance was composed of an instantaneous time-independent component and a slowly activating time-dependent component and displayed a deactivating inward tail current. Extracellular Ca(<em>2</em>+)-induced and Ca(<em>2</em>+) dialysis-induced currents reversed at the equilibrium potential of Cl(-) and were inhibited by niflumic acid (a specific blocker of Ca(<em>2</em>+)-activated Cl(-) channel). Gramicidin-perforated whole-cell recording displayed the shift of the reversal potential in extracellular Ca(<em>2</em>+)-induced current, suggesting the change of intracellular Cl(-) concentration in a few minutes. Extracellular Ca(<em>2</em>+)-induced currents displayed a moderate dependency on guanosine triphosphate (GTP). All blockers for phospholipase C, diacylglycerol (DAG) lipase, monoacylglycerol (MAG) lipase and lipoxygenase inhibited extracellular Ca(<em>2</em>+)-induced current. IP(3) dialysis failed to induce conductance increase, but <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), arachidonic acid and 1<em>2</em>S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (1<em>2</em>(S)-HPETE) dialysis increased the conductance identical to extracellular Ca(<em>2</em>+)-induced conductance. These results indicate that high extracellular Ca(<em>2</em>+) raises intracellular Ca(<em>2</em>+) concentration through the DAG lipase/lipoxygenase pathway, resulting in the activation of Cl(-) conductance.

Monoacylglycerol lipase (MAGL) is a major serine hydrolase that hydrolyzes <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) to arachidonic acid (AA) and glycerol in the brain. Because <em>2</em>-AG and AA are endogenous biologically active ligands in the brain, inhibition of MAGL is an attractive therapeutic target for CNS disorders, particularly neurodegenerative diseases. In this study, we report the structure-based drug design of novel piperazinyl pyrrolidin-<em>2</em>-ones starting from our hit compounds <em>2</em>a and <em>2</em>b. By enhancing the interaction of the piperazinyl pyrrolidin-<em>2</em>-one core and its substituents with the MAGL enzyme via design modifications, we identified a potent and reversible MAGL inhibitor, compound ( R)-3t. Oral administration of compound ( R)-3t to mice decreased AA levels and elevated <em>2</em>-AG levels in the brain.

Adipose tissue (AT) plays a major role in metabolic adaptations in postpartum (PP) dairy cows. The endocannabinoid (eCB) system is a key regulator of metabolism and energy homeostasis; however, information about this system in ruminants is scarce. Therefore, this work aimed to assess the eCB system in subcutaneous AT, and to determine its relation to the metabolic profile in peripartum cows. Biopsies of AT were performed at 14 d prepartum, and 4 and 30 d PP from 18 multiparous peripartum cows. Cows were categorized retrospectively according to those with high body weight (BW) loss (HWL, 8.5 ± 1.7% BW loss) or low body weight loss (LWL, <em>2</em>.9 ± <em>2</em>.5% BW loss) during the first month PP. The HWL had higher plasma non-esterified fatty acids and a lower insulin/glucagon ratio PP than did LWL. Two-fold elevated AT levels of the main eCBs, N-arachidonoylethanolamine (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), were found 4 d PP compared with prepartum in HWL, but not in LWL cows. AT levels of the eCB-like molecules oleoylethanolamide, palmitoylethanolamide, and of arachidonic acid were elevated PP compared with prepartum in all cows. The abundance of monoglyceride lipase (MGLL), the <em>2</em>-AG degrading enzyme, was lower in HWL vs. LWL AT PP. The relative gene expression of the cannabinoid receptors CNR1 and CNR<em>2</em> in AT tended to be higher in HWL vs. LWL PP. Proteomic analysis of AT showed an enrichment of the inflammatory pathways' acute phase signaling and complement system in HWL vs. LWL cows PP. In summary, eCB levels in AT were elevated at the onset of lactation as part of the metabolic adaptations in PP dairy cows. Furthermore, activating the eCB system in AT is most likely associated with a metabolic response of greater BW loss, lipolysis, and AT inflammation in PP dairy cows.

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 <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-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 CNR<em>2</em> 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 CB<em>2</em>A) were also differentially distributed, mainly in germ cells. The cannabinoid-metabolising enzymes were abundantly present; the α/β-hydrolase domain-containing protein <em>2</em> (ABHD<em>2</em>) 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.

Exposure to a traumatic event may result in the development of post-traumatic stress disorder (PTSD). Endocannabinoids are crucial modulators of the stress response, interfere with excessive retrieval and facilitate the extinction of traumatic memories. Exposure therapy, combined with pharmacotherapy, represents a promising tool for PTSD treatment. We investigated whether pharmacological manipulations of the endocannabinoid system during extinction learning ameliorates the behavioral changes induced by trauma exposure. Rats were exposed to inescapable footshocks paired with social isolation, a risk factor for PTSD. One week after trauma, rats were subjected to three spaced extinction sessions, mimicking human exposure therapy. The anandamide hydrolysis inhibitor URB597, the <em>2</em>-<em>arachidonoylglycerol</em> hydrolysis inhibitor JZL184 or the cannabinoid agonist WIN55,<em>2</em>1<em>2</em>-<em>2</em> were administered before or after the extinction sessions. Rats were tested for extinction retention 16 or 36 days after trauma and <em>2</em>4-h later for social interaction. Extinction training alone reduced fear of the trauma-associated context but did not restore normal social interaction. Traumatized animals not exposed to extinction sessions exhibited reductions in hippocampal anandamide content with respect to home-cage controls. Noteworthy, all drugs exerted beneficial effects, but URB597 (0.1 mg/kg) induced the best improvements by enhancing extinction consolidation and restoring normal social behavior in traumatized rats through indirect activation of CB1 receptors. The ameliorating effects remained stable long after treatment and trauma exposure. Our findings suggest that drugs potentiating endocannabinoid neurotransmission may represent promising tools when combined to exposure-based psychotherapies in the treatment of PTSD.

The aim of the current study was to investigate the interaction of the nitric oxide and cannabinoidergic systems on feeding behaviour in neonatal chicken. A total of 6 experiments were designed to evaluate the interaction between cannabinoidergic and nitrergic systems on food intake in 3-h food-deprived (FD3) neonatal chickens. In Experiment 1, chickens received intracerebroventricular (ICV) injections of saline, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) (a CB1 receptor agonist, <em>2</em> µg), l-arginine (nitric oxide precursor, <em>2</em>00 nmol) and co-administration of <em>2</em>-AG + l-arginine. In Experiment <em>2</em>, ICV injection of saline, <em>2</em>-AG (<em>2</em> µg), l-NAME (a nitric oxide synthesis inhibitor, 100 nmol) and their combination (<em>2</em>-AG + l-NAME) were applied to the birds. In Experiment 3, injections were saline, CB65 (a CB<em>2</em> receptor agonist, 1.<em>2</em>5 µg), l-arginine (<em>2</em>00 nmol) and CB65 + l-arginine. In Experiment 4, birds received ICV injection of saline, CB65 (1.<em>2</em>5 µg), l-NAME (100 nmol) and CB65 + l-NAME. In Experiment 5, chickens were ICV injected with saline, l-arginine (800 nmol), SR141716A (a selective CB1 receptor antagonist, 6.<em>2</em>5 µg) and l-arginine + SR141716A. In Experiment 6, birds were injected with saline, l-arginine (800 nmol), AM630 (a selective CB<em>2</em> receptor antagonist, 5 µg) and l-arginine + AM630. Cumulative food intake was recorded until <em>2</em>-h post injection. ICV injection of CB1 and CB<em>2</em> receptor agonists increased food intake. Co-injection of <em>2</em>-AG + l-NAME increased the hyperphagic effects of CB1 receptors. CB<em>2</em> receptor-induced food intake was not affected by co-administration of CB65 + l-NAME. l-Arginine decreased food intake and this effect was amplified by co-injection of l-arginine + SR141716A. However; CB<em>2</em> receptor antagonists had no effect on l-arginine-induced hypophagia. The results suggest that there is an interaction between endogenous nitric oxide and the cannabinoidergic system on feeding behaviour which is mediated via CB1 receptors in the neonatal chicken.

The presence of fatty acid derived oxylipins, endocannabinoids and related compounds in human milk may be of importance to the infant. Presently, clinically relevant protocols for storing and handling human milk that minimize error and variability in oxylipin and endocannabinoid concentrations are lacking. In this study, we compared the individual and combined effects of the following storage conditions on the stability of these fatty acid metabolites in human milk: state (fresh or frozen), storage temperature (4 °C, -<em>2</em>0 °C or -80 °C), and duration (1 day, 1 week or 3 months). Thirteen endocannabinoids and related compounds, as well as 37 oxylipins were analyzed simultaneously by liquid chromatography coupled to tandem mass spectrometry. Twelve endocannabinoids and related compounds (<em>2</em>-111 nM) and 31 oxylipins (1.<em>2</em> pM-1<em>2</em>4<em>2</em> nM) were detected, with highest levels being found for <em>2</em>-<em>arachidonoylglycerol</em> and 17(R)hydroxydocosahexaenoic acid, respectively. The concentrations of most endocannabinoid-related compounds and oxylipins were dependent on storage condition, and especially storage at 4 °C introduced significant variability. Our findings suggest that human milk samples should be analyzed immediately after, or within one day of collection (if stored at 4 °C). Storage at -80 °C is required for long-term preservation, and storage at -<em>2</em>0 °C is acceptable for no more than one week. These findings provide a protocol for investigating the oxylipin and endocannabinoid metabolome in human milk, useful for future milk-related clinical studies.

The endocannabinoid system is widely expressed in the limbic system, prefrontal cortical areas, and brain structures regulating neuroendocrine stress responses, which explains the key role of this system in the control of emotions. In this review, we update recent advances on the function of the endocannabinoid system in determining the value of fear-evoking stimuli and promoting appropriate behavioral responses for stress resilience. We also review the alterations in the activity of the endocannabinoid system during fear, stress, and anxiety, and the pathophysiological role of each component of this system in the control of these protective emotional responses that also trigger pathological emotional disorders. In spite of all the evidence, we have not yet taken advantage of the therapeutic implications of this important role of the endocannabinoid system, and possible future strategies to improve the treatment of these emotional disorders are discussed. .

El sistema endocannabinoide tiene una amplia expresión en el sistema límbico, las áreas corticales prefrontales y las estructuras cerebrales que regulan las respuestas al estrés neuroendocrino, lo que explica el papel clave de este sistema en el control de las emociones. En esta revisión, se actualizan los avances recientes sobre la función del sistema endocannabinoide para determinar el valor de los estímulos que provocan miedo y promueven respuestas conductuales apropiadas para la resiliencia frente al estrés. También se revisan las alteraciones en la actividad del sistema endocannabinoide durante el miedo, el estrés y la ansiedad, y el papel fisiopatológico de cada componente de este sistema en el control de estas respuestas emocionales protectoras que también desencadenan trastornos emocionales patológicos. A pesar de todas las evidencias, todavía no se han aprovechado las traducciones terapéuticas de este importante papel del sistema endocannabinoide. También se discuten las posibles estrategias futuras para mejorar el tratamiento de estos trastornos emocionales.

Abondamment retrouvé dans le système limbique, le cortex préfrontal et les structures cérébrales régulant les réponses neuroendocriniennes au stress, le système endocannabinoïde joue un rôle clé dans le contrôle des émotions. Sa fonction dans la détermination de l’intensité des stimuli déclenchant la peur et dans l’amélioration des réponses comportementales adaptées à la résistance au stress est étudiée ici à l’aune des données récentes actualisées. Nous reconsidérons également les modifications de l'activité du système endocannabinoïde face à la peur, au stress et à l'anxiété, ainsi que le rôle physiopathologique de chaque composante de ce système dans le contrôle de ces réponses émotionnelles protectrices, elles-mêmes génératrices de troubles émotionnels pathologiques. Malgré toutes les données disponibles, le rôle crucial du système endocannabinoïde sur le plan thérapeutique n’a pas encore été exploité. Nous analysons les futures stratégies possibles pour améliorer le traitement de ces troubles émotionnels.

<strong class="sub-title"> Keywords: </strong> <em>2</em>-<em>arachidonoylglycerol</em>; CB1 receptor; CB<em>2</em> receptor; HPA axis; THC; amygdala; anandamide; emotional response; limbic system.

Platelets play a central role in atherosclerosis and atherothrombosis, and circulating endocannabinoids might modulate platelet function. Previous studies concerning effects of anandamide (N-arachidonylethanolamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) on platelets, mainly performed on isolated cells, provided conflicting results. We therefore investigated the action of three main endocannabinoids [anandamide, <em>2</em>-AG and virodhamine (arachidonoylethanolamine)] on human platelets in blood and platelet-rich plasma (PRP). <em>2</em>-AG and virodhamine induced platelet aggregation in blood, and shape change, aggregation and adenosine triphosphate (ATP) secretion in PRP. The EC50 of <em>2</em>-AG and virodhamine for platelet aggregation in blood was 97 and 160 µM, respectively. Lower concentrations of <em>2</em>-AG (<em>2</em>0 µM) and virodhamine (50 µM) synergistically induced aggregation with other platelet stimuli. Platelet activation induced by <em>2</em>-AG and virodhamine resembled arachidonic acid (AA)-induced aggregation: shape change, the first platelet response, ATP secretion and aggregation induced by <em>2</em>-AG and virodhamine were all blocked by acetylsalicylic acid (ASA) or the specific thromboxane A<em>2</em> (TXA<em>2</em>) antagonist daltroban. In addition, platelet activation induced by <em>2</em>-AG and virodhamine in blood and PRP were inhibited by JZL184, a selective inhibitor of monoacylglycerol lipase (MAGL). In contrast to <em>2</em>-AG and virodhamine, anandamide, a substrate of fatty acid amidohydrolase, was inactive. Synthetic cannabinoid receptor subtype 1 (CB1) and <em>2</em> (CB<em>2</em>) agonists lacked stimulatory as well as inhibitory platelet activity. We conclude that <em>2</em>-AG and virodhamine stimulate platelets in blood and PRP by a MAGL-triggered mechanism leading to free AA and its metabolism by platelet cyclooxygenase-1/thromboxane synthase to TXA<em>2</em>. CB1, CB<em>2</em> or non-CB1/CB<em>2</em> receptors are not involved. Our results imply that ASA and MAGL inhibitors will protect platelets from activation by high endocannabinoid levels, and that pharmacological CB1- and CB<em>2</em>-receptor ligands will not affect platelets and platelet-dependent progression and complications of cardiovascular diseases.

The endocannabinoid system is implicated in numerous physiopathological processes while more and more pieces of evidence wave the link between this complex machinery and cancer related phenomenon. In these lines, we confirmed the effects of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), the main endocannabinoid, on neuroblastoma cells proliferation in vitro, and proved that some N-phenylmaleimide compounds that were previously shown as MAGL inhibitors can also inhibit type <em>2</em> topoisomerase. We also shed light on their antiproliferative effects on a neuroblastoma cell line. In order to establish a link between MAGL inhibition, topoisomerase inhibition and the effects on N1E-115 cells, we tested combinations of maleimides or known endocannabinoid metabolism inhibitors and <em>2</em>-AG, the major MAGL substrate, on N1E-115 cells. However, none of the inhibitors tested, except the carbamate CAY10499, managed to increase <em>2</em>-AG's effects. Even the MAGL reference inhibitor JZL184 failed to induce a stronger inhibition of proliferation.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide are two major endocannabinoids produced, released and eliminated by metabolic pathways. Anticonvulsive effect of <em>2</em>-AG and CB1 receptor is well-established. Herein, we designed to investigate the anticonvulsive influence of key components of the <em>2</em>-AG and anandamide metabolism. Tonic-clonic seizures were induced by an injection of Pentylenetetrazol (80 mg/kg, i.p.) in adult male Wistar rats. Delay and duration for the seizure stages were considered for analysis. Monoacylglycerol lipase blocker (JJKK048; 1 mg/kg) or alpha/beta hydroxylase domain 6 blocker (WWL70; 5 mg/kg) were administrated alone or with <em>2</em>-AG to evaluate the anticonvulsive potential of these enzymes. To determine the CB1 receptor involvement, its blocker (MJ15; 3 mg/kg) was administrated associated with JJKK048 or WWL70. To assess anandamide anticonvulsive effect, anandamide membrane transporter blocker (LY<em>2</em>1813<em>2</em>40; <em>2</em>.5 mg/kg) was used alone or associated with MJ15. Also, fatty acid amide hydrolase blocker (URB597; 1 mg/kg; to prevent intracellular anandamide hydrolysis) were used alone or with AMG<em>2</em>16<em>2</em>9 (transient receptor potential vanilloid; TRPV1 antagonist; 3 mg/kg). All compounds were dissolved in DMSO and injected i.p., before the Pentylenetetrazol. Both JJKK048 and WWL70 revealed anticonvulsive effect. Anticonvulsive effect of JJKK048 but not WWL70 was CB1 receptor dependent. LY<em>2</em>183<em>2</em>40 showed CB1 receptor dependent anticonvulsive effect. However, URB597 revealed a TRPV1 dependent proconvulsive effect. It seems extracellular accumulation of <em>2</em>-AG or anandamide has anticonvulsive effect through the CB1 receptor, while intracellular anandamide accumulation is proconvulsive through TRPV1.

DDHD<em>2</em> has been reported to exhibit phospholipase A1, triacylglycerol (TG) lipase and diacylglycerol (DG) lipase activities. However, the detailed enzymatic properties of DDHD<em>2</em> have not yet been elucidated. In the current study, the substrate specificity of DDHD<em>2</em> towards DG, TG and phosphatidic acid (PA) has been examined using highly purified recombinant rat DDHD<em>2</em> (rDDHD<em>2</em>) with a liquid chromatography mass spectrometer. The k cat/Km value for DG (18:0/<em>2</em>0:4) was much higher than those for TG (18:1/18:1/18:1), and PA (18:0/<em>2</em>0:4) in the presence of sodium deoxycholate. The enzyme activity of rDDHD<em>2</em> towards DG (18:0/<em>2</em>0:4) was highest among all of the substrates tested. In addition, rDDHD<em>2</em> was highly specific to DG substrates with a polyunsaturated fatty acid at their sn-<em>2</em> position. The levels of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in CHO cells were quantified by gas chromatography-tandem mass spectrometry, showing that CHO cells expressing recombinant rDDHD<em>2</em> contained higher levels of <em>2</em>-AG when cells were treated with a monoacylglycerol lipase inhibitor, URB60<em>2</em>. These results therefore support the idea that DDHD<em>2</em> functions as a DG lipase in vivo and produces <em>2</em>-AG.

Exposure to inadequate nutritional conditions in critical windows of development has been associated to disturbances on metabolism and behavior in the offspring later in life. The role of the endocannabinoid system, a known regulator of energy expenditure and adaptive behaviors, in the modulation of these processes is unknown. In the present study, we investigated the impact of exposing rat dams to diet restriction (<em>2</em>0% less calories than standard diet) during pre-gestational and gestational periods on: (a) neonatal outcomes; (b) endocannabinoid content in hypothalamus, hippocampus and olfactory bulb at birth; (c) metabolism-related parameters; and (d) behavior in adult male offspring. We found that calorie-restricted dams tended to have a reduced litter size, although the offspring showed normal weight at birth. Pups from calorie-restricted dams also exhibited a strong decrease in the levels of anandamide (AEA), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), arachidonic acid (AA) and palmitoylethanolamide (PEA) in the hypothalamus at birth. Additionally, pups from diet-restricted dams displayed reduced levels of AEA in the hippocampus without significant differences in the olfactory bulb. Moreover, offspring exhibited increased weight gain, body weight and adiposity in adulthood as well as increased anxiety-related responses. We propose that endocannabinoid signaling is altered by a maternal caloric restriction implemented during the preconceptional and pregnancy periods, which might lead to modifications of the hypothalamic and hippocampal circuits, potentially contributing to the long-term effects found in the adult offspring.

The endocannabinoid system is currently under intense investigation due to the therapeutic potential of cannabinoid-based drugs as treatment options for a broad variety of diseases including cancer. Besides the canonical endocannabinoid system that includes the cannabinoid receptors CB1 and CB<em>2</em> and the endocannabinoids N-arachidonoylethanolamine (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em>, recent investigations suggest that other fatty acid derivatives, receptors, enzymes, and lipid transporters likewise orchestrate this system as components of the endocannabinoid system when defined as an extended signaling network. As such, fatty acids acting at cannabinoid receptors (e.g. <em>2</em>-arachidonoyl glyceryl ether [noladin ether], N-arachidonoyldopamine) as well as endocannabinoid-like substances that do not elicit cannabinoid receptor activation (e.g. N-palmitoylethanolamine, N-oleoylethanolamine) have raised interest as anticancerogenic substances. Furthermore, the endocannabinoid-degrading enzymes fatty acid amide hydrolase and monoacylglycerol lipase, lipid transport proteins of the fatty acid binding protein family, additional cannabinoid-activated G protein-coupled receptors, members of the transient receptor potential family as well as peroxisome proliferator-activated receptors have been considered as targets of antitumoral cannabinoid activity. Therefore, this review focused on the antitumorigenic effects induced upon modulation of this extended endocannabinoid network.