BACKGROUND

Postsynaptically generated <em>2</em>-<em>arachidonoylglycerol</em> activates the presynaptic cannabinoid type-1 receptor, which is involved in synaptic plasticity at both glutamatergic and GABAergic synapses. However, the differential function of <em>2</em>-<em>arachidonoylglycerol</em> signaling at glutamatergic vs GABAergic synapses in the context of animal behavior has not been investigated yet.

METHODS

Here, we analyzed the role of <em>2</em>-<em>arachidonoylglycerol</em> signaling selectively in hippocampal glutamatergic neurons. Monoacylglycerol lipase, the primary degrading enzyme of <em>2</em>-<em>arachidonoylglycerol</em>, is expressed at presynaptic sites of excitatory and inhibitory neurons. By adeno-associated virus-mediated overexpression of monoacylglycerol lipase in glutamatergic neurons of the mouse hippocampus, we selectively interfered with <em>2</em>-<em>arachidonoylglycerol</em> signaling at glutamatergic synapses of these neurons.

RESULTS

Genetic modification of monoacylglycerol lipase resulted in a 50% decrease in <em>2</em>-<em>arachidonoylglycerol</em> tissue levels without affecting the content of the second major endocannabinoid anandamide. A typical electrophysiological read-out for <em>2</em>-<em>arachidonoylglycerol</em> signaling is the depolarization-induced suppression of excitation and of inhibition. Elevated monoacylglycerol lipase levels at glutamatergic terminals selectively impaired depolarization-induced suppression of excitation, while depolarization-induced suppression of inhibition was not significantly changed. At the behavioral level, mice with impaired hippocampal glutamatergic <em>2</em>-<em>arachidonoylglycerol</em> signaling exhibited increased anxiety-like behavior but showed no alterations in aversive memory formation and seizure susceptibility.

CONCLUSIONS

Our data indicate that <em>2</em>-<em>arachidonoylglycerol</em> signaling selectively in hippocampal glutamatergic neurons is essential for the animal's adaptation to aversive situations.

Since the discovery of the two cannabinoid receptors, CB(1) and CB(<em>2</em>), 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 <em>2</em>-<em>arachidonoylglycerol</em>.

The ability to discern temporally pertinent environmental events is essential for the generation of adaptive behavior in conventional tasks, and our overall survival. Cannabinoids are thought to disrupt temporally controlled behaviors by interfering with dedicated brain timing networks. Cannabinoids also increase dopamine release within the mesolimbic system, a neural pathway generally implicated in timing behavior. Timing can be assessed using fixed-interval (FI) schedules, which reinforce behavior on the basis of time. To date, it remains unknown how cannabinoids modulate dopamine release when responding under FI conditions, and for that matter, how subsecond dopamine release is related to time in these tasks. In the present study, we hypothesized that cannabinoids would accelerate timing behavior in an FI task while concurrently augmenting a temporally relevant pattern of dopamine release. To assess this possibility, we measured subsecond dopamine concentrations in the nucleus accumbens while mice responded for food under the influence of the cannabinoid agonist WIN 55,<em>2</em>1<em>2</em>-<em>2</em> in an FI task. Our data reveal that accumbal dopamine concentrations decrease proportionally to interval duration--suggesting that dopamine encodes time in FI tasks. We further demonstrate that WIN 55,<em>2</em>1<em>2</em>-<em>2</em> dose-dependently increases dopamine release and accelerates a temporal behavioral response pattern in a CB1 receptor-dependent manner--suggesting that cannabinoid receptor activation modifies timing behavior, in part, by augmenting time-engendered patterns of dopamine release. Additional investigation uncovered a specific role for endogenous cannabinoid tone in timing behavior, as elevations in <em>2</em>-<em>arachidonoylglycerol</em>, but not anandamide, significantly accelerated the temporal response pattern in a manner akin to WIN 55,<em>2</em>1<em>2</em>-<em>2</em>.

Trophoblast cells that comprise the placenta play a crucial role in the complex cross-talk between fetus and maternal tissues. Although anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, the best studied endocannabinoids, affect trophoblast attachment and outgrowth, the functional significance of the endocannabinoid system in the development of placenta has not been established. We investigated the correlation between endocannabinoid levels and the pattern of expression of the receptors and metabolic enzymes of the endocannabinoid system during rat placental development. Here, we showed that all the endocannabinoid machinery is dynamically expressed in the functionally distinct basal and labyrinth zones of the rat placenta. Indeed, endocannabinoid levels are shown to increase with the progression of pregnancy. Together, these data support a role for the endocannabinoid system in normal placental function and evidence for a potential novel cellular target for the deleterious action of cannabis-derived compounds during the second half of pregnancy.

Adequate pain management remains an unmet medical need. We previously revealed an opioid-independent analgesic mechanism mediated by orexin 1 receptor (OX1R)-initiated <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) signaling in the ventrolateral periaqueductal gray (vlPAG). Here, we found that low-frequency median nerve stimulation (MNS) through acupuncture needles at the PC6 (Neiguan) acupoint (MNS-PC6) induced an antinociceptive effect that engaged this mechanism. In mice, MNS-PC6 reduced acute thermal nociceptive responses and neuropathy-induced mechanical allodynia, increased the number of c-Fos-immunoreactive hypothalamic orexin neurons, and led to higher orexin A and lower GABA levels in the vlPAG. Such responses were not seen in mice with PC6 needle insertion only or electrical stimulation of the lateral deltoid, a nonmedian nerve-innervated location. Directly stimulating the surgically exposed median nerve also increased vlPAG orexin A levels. MNS-PC6-induced antinociception (MNS-PC6-IA) was prevented by proximal block of the median nerve with lidocaine as well as by systemic or intravlPAG injection of an antagonist of OX1Rs or cannabinoid 1 receptors (CB1Rs) but not by opioid receptor antagonists. Systemic blockade of OX1Rs or CB1Rs also restored vlPAG GABA levels after MNS-PC6. A cannabinoid (<em>2</em>-AG)-dependent mechanism was also implicated by the observations that MNS-PC6-IA was prevented by intravlPAG inhibition of <em>2</em>-AG synthesis and was attenuated in Cnr1 -/- mice. These findings suggest that PC6-targeting low-frequency MNS activates hypothalamic orexin neurons, releasing orexins to induce analgesia through a CB1R-dependent cascade mediated by OX1R-initiated <em>2</em>-AG retrograde disinhibition in the vlPAG. The opioid-independent characteristic of MNS-PC6-induced analgesia may provide a strategy for pain management in opioid-tolerant patients.

The innate immune response is mediated by primary immune modulators such as cytokines and chemokines that together with immune cells and resident glia orchestrate CNS immunity and inflammation. Growing evidence supports that the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) exerts protective actions in CNS injury models. Here, we used the acute phase of Theiler's virus induced demyelination disease (TMEV-IDD) as a model of acute neuroinflammation to investigate whether <em>2</em>-AG modifies the brain innate immune responses to TMEV and CNS leukocyte trafficking. <em>2</em>-AG or the inhibition of its hydrolysis diminished the reactivity and number of microglia at the TMEV injection site reducing their morphological complexity and modulating them towards an anti-inflammatory state via CB<em>2</em> receptors. Indeed, <em>2</em>-AG dampened the infiltration of immune cells into the CNS and inhibited their egress from the spleen, resulting in long-term beneficial effects at the chronic phase of the disease. Intriguingly, it is not a generalized action over leukocytes since <em>2</em>-AG increased the presence and suppressive potency of myeloid derived suppressor cells (MDSCs) in the brain resulting in higher apoptotic CD4+ T cells at the injection site. Together, these data suggest a robust modulatory effect in the peripheral and central immunity by <em>2</em>-AG and highlight the interest of modulating endogenous cannabinoids to regulate CNS inflammatory conditions.

Endocannabinoids (eCBs) are natural lipids regulating a large array of physiological functions and behaviors in vertebrates. The eCB system is highly conserved in evolution and comprises several specific receptors (type-1 and type-<em>2</em> cannabinoid receptors), their endogenous ligands (e.g., anandamide and <em>2</em>-<em>arachidonoylglycerol</em>), and a number of biosynthetic and degradative enzymes. In the last few years, eCBs have been described as critical signals in the control of male and female reproduction at multiple levels: centrally, by targeting hypothalamic gonadotropin-releasing-hormone-secreting neurons and pituitary, and locally, with direct effects on the gonads. These functions are supported by the extensive localization of cannabinoid receptors and eCB metabolic enzymes at different levels of the hypothalamic-pituitary-gonadal axis in mammals, as well as bonyfish and amphibians. In vivo and in vitro studies indicate that eCBs centrally regulate gonadal functions by modulating the gonadotropin-releasing hormone-gonadotropin-steroid network through direct and indirect mechanisms. Several proofs of local eCB regulation have been found in the testis and male genital tracts, since eCBs control Sertoli and Leydig cells activity, germ cell progression, as well as the acquisition of sperm functions. A comparative approach usually is a key step in the study of physiological events leading to the building of a general model. Thus, in this review, we summarize the action of eCBs at different levels of the male reproductive axis, with special emphasis, where appropriate, on data from non-mammalian vertebrates.

In this study, we analyzed the components of the endocannabinoid system (ECS) in R6/<em>2</em> mice, a widely used model of Huntington's disease (HD). We measured the endogenous content of N-arachidonoylethanolamine and <em>2</em>-<em>arachidonoylglycerol</em> and the activity of their biosynthetic enzymes (N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D and diacylglycerol lipase, respectively) and hydrolytic enzymes [fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase, respectively] and of their target receptors (type 1 cannabinoid receptor, type <em>2</em> cannabinoid receptor, and transient receptor potential vanilloid-1) in the brains of wild-type and R6/<em>2</em> mice of different ages, as well as in the striatum and cortex of 1<em>2</em>-week-old animals. In addition, we measured FAAH activity in lymphocytes of R6/<em>2</em> mice. In the whole brains of 1<em>2</em>-week-old R6/<em>2</em> mice, we found reductions in N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D activity, diacylglycerol lipase activity and cannabinoid receptor binding, mostly associated with changes in the striatum but not in the cortex, as well as an increase in <em>2</em>-<em>arachidonoylglycerol</em> content as compared with wild-type littermates, without any other change in ECS elements. Then, our analysis was extended to HD43 cells, an inducible cellular model of HD derived from rat ST14A cells. In both induced and noninduced conditions, we demonstrated a fully functional ECS. Overall, our data suggest that the ECS is differently affected in mouse and human HD, and that HD43 cells are suitable for high-throughput screening of FAAH-oriented drugs affecting HD progression.

After creating >4,650 knockouts (KOs) of independent mouse genes, we screened them by high-throughput phenotyping and found that cannabinoid receptor 1 (Cnr1) KO mice had the same lean phenotype published by others. We asked if our KOs of DAG lipase α or β (Dagla or Daglb), which catalyze biosynthesis of the endocannabinoid (EC) <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), or Napepld, which catalyzes biosynthesis of the EC anandamide, shared the lean phenotype of Cnr1 KO mice. We found that Dagla KO mice, but not Daglb or Napepld KO mice, were among the leanest of 3651 chow-fed KO lines screened. In confirmatory studies, chow- or high fat diet-fed Dagla and Cnr1 KO mice were leaner than wild-type (WT) littermates; when data from multiple cohorts of adult mice were combined, body fat was 47 and 45% lower in Dagla and Cnr1 KO mice, respectively, relative to WT values. By contrast, neither Daglb nor Napepld KO mice were lean. Weanling Dagla KO mice ate less than WT mice and had body weight (BW) similar to pair-fed WT mice, and adult Dagla KO mice had normal activity and VO<em>2</em> levels, similar to Cnr1 KO mice. Our Dagla and Cnr1 KO mice also had low fasting insulin, triglyceride, and total cholesterol levels, and after glucose challenge had normal glucose but very low insulin levels. Dagla and Cnr1 KO mice also showed similar responses to a battery of behavioral tests. These data suggest: (1) the lean phenotype of young Dagla and Cnr1 KO mice is mainly due to hypophagia; (<em>2</em>) in pathways where ECs signal through Cnr1 to regulate food intake and other metabolic and behavioral phenotypes observed in Cnr1 KO mice, Dagla alone provides the <em>2</em>-AG that serves as the EC signal; and (3) small molecule Dagla inhibitors with a pharmacokinetic profile similar to that of Cnr1 inverse agonists are likely to mirror the ability of these Cnr1 inverse agonists to lower BW and improve glycemic control in obese patients with type <em>2</em> diabetes, but may also induce undesirable neuropsychiatric side-effects.

Diet plays a central role in maintaining health throughout life and a controlled food intake is associated to a reduced risk of certain diseases. A proper diet should include vitamins, minerals, carbohydrates, proteins, and fats that have to be optimally balanced in order to exert their physiological functions. The endogenous ligands of type-1 and type-<em>2</em> cannabinoid receptors, N-arachidonoyl-ethanolamine and <em>2</em>-<em>arachidonoylglycerol</em>, are arachidonic acid (AA) derivatives whose levels are regulated by the activity of metabolic enzymes, as well as by AA availability. Since the only sources of AA in mammals are diet and the enzymatic production in the liver from shorter-chain essential fatty acids like linoleic acid, it is realistic to hypothesize that endocannabinoid levels might be modulated by fatty acid composition of food. Therefore, in this review we summarize literature data indicating that endocannabinoid levels, and hence their activity at cannabinoid receptors, might be modulated by food composition. We focused our attention on dietary fatty acid content, and on type and esterified form of fatty acids in the different diets.

A common single nucleotide polymorphism (C385A) in the human fatty acid amide hydrolase (FAAH) gene has been associated with decreased distress responses in healthy volunteers, but its role in psychiatric disorders remains unknown. Here, we obtained genotypes and carried out a secondary analysis of subjects from a trial of comorbid posttraumatic stress disorder (PTSD) and alcohol dependence (AD). We evaluated the effects of C385A variation on behavioral and biochemical biomarkers of distress responses.

Forty-nine patients with PTSD and AD were admitted for 4 weeks to an experimental medicine unit at the National Institutes of Health Clinical Center. Following detoxification, stress reactivity and peripheral endocannabinoid (eCB) levels were assessed in response to a challenge session using personalized auditory guided imagery. Over the course of the study, subjects were also evaluated for changes in PTSD symptom severity.

FAAH C385A allele carriers showed a marked increase in serum anandamide levels at baseline and throughout the stress challenge procedure compared with C allele homozygotes, while levels of eCBs primarily metabolized through other enzymatic activity, such as <em>2</em>-<em>arachidonoylglycerol</em>, did not differ between genotype groups. FAAH C385A carriers also had decreased subjective anxiety responses to the stress challenge. Similar effects of FAAH C385A genotype were found at the level of clinical PTSD symptom severity, in particular in the arousal domain.

This is to our knowledge the first study showing that FAAH C385A variation modulates stress responses in subjects with disorders characterized by increased stress reactivity. These findings point to the eCB pathway as a promising target for future antistress therapeutics.

Alpha/beta-hydrolase domain 6 (ABHD6) is a novel <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) hydrolytic enzyme, that can fine-tune the endocannabinoid signaling in the central nervous system. Recently we and others have demonstrated the protective effect of ABHD6 inhibition in the animal models of traumatic brain injury and epileptic seizures. In this study, we investigated the role of targeting ABHD6 in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Post-symptom treatment with an ABHD6 inhibitor WWL70 increased the brain levels of <em>2</em>-AG and ameliorated the clinical signs of EAE, T cells infiltration, microglia activation and the expression of activated leukocyte cell adhesion molecules. The production of iNOS, COX-<em>2</em>, TNF-α and IL-1β and the phosphorylation of NF-κB were also significantly reduced by WWL70 treatment. The neuroprotective effect of WWL70 was demonstrated by increased survival of mature oligodendrocytes, reduced demyelination and axonal loss in WWL70 treated EAE mouse spinal cord. The therapeutic effect of WWL70 on EAE was absent by co-administration of CB<em>2</em> receptor antagonist, but not CB1 receptor antagonist. Consistently, WWL70 did not afford any protection in CB<em>2</em> receptor knockout mice after EAE induction. Given the increased expression of ABHD6 in microglia/macrophages, but not in T cells, we speculated that inhibition of ABHD6 might enhance <em>2</em>-AG signaling particularly in microglia/macrophages to exert anti-inflammatory effects via activation of CB<em>2</em> receptors. These results suggest that inhibition of ABHD6 might be used as an ideal strategy for the treatment of MS and other neurodegenerative diseases.

OBJECTIVE

Hepatic encephalopathy is a neuropsychiatric syndrome caused by liver failure. In view of the effects of cannabinoids in a thioacetamide-induced model of hepatic encephalopathy and liver disease and the beneficial effect of capsaicin (a TRPV1 agonist) in liver disease, we assumed that capsaicin may also affect hepatic encephalopathy.

METHODS

Fulminant hepatic failure was induced in mice by thioacetamide and <em>2</em>4 h later, the animals were injected with one of the following compound(s): <em>2</em>-<em>arachidonoylglycerol</em> (CB(1), CB(<em>2</em>) and TRPV1 receptor agonist); HU308 (CB(<em>2</em>) receptor agonist), SR141716A (CB(1) receptor antagonist); SR141716A+<em>2</em>-<em>arachidonoylglycerol</em>; SR1445<em>2</em>8 (CB(<em>2</em>) receptor antagonist); capsaicin; and capsazepine (TRPV1 receptor agonist and antagonist respectively). Their neurological effects were evaluated on the basis of activity in the open field, cognitive function in an eight-arm maze and a neurological severity score. The mice were killed 3 or 14 days after thioacetamide administration. <em>2</em>-<em>arachidonoylglycerol</em> and 5-hydroxytryptamine (5-HT) levels were determined by gas chromatography-mass spectrometry and high-performance liquid chromatography with electrochemical detection, respectively.

RESULTS

Capsaicin had a neuroprotective effect in this animal model as shown by the neurological score, activity and cognitive function. The effect of capsaicin was blocked by capsazepine. Thioacetamide induced astrogliosis in the hippocampus and the cerebellum and raised brain 5-hydroxytryptamine levels, which were decreased by capsaicin, SR141716A and HU-308. Thioacetamide lowered brain <em>2</em>-<em>arachidonoylglycerol</em> levels, an effect reversed by capsaicin.

CONCLUSIONS

Capsaicin improved both liver and brain dysfunction caused by thioacetamide, suggesting that both the endocannabinoid and the vanilloid systems play important roles in hepatic encephalopathy. Modulation of these systems may have therapeutic value.

The endogenous cannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> bind to the cannabinoid receptors of type 1 and <em>2</em>. These receptors are also the binding sites for exogenous, both natural and synthetic, cannabinoids that are used for recreation purposes. Until recently, cannabinoids and cannabinoid receptors have attracted little interest among nephrologists; however, a full endocannabinoid system (ECS) is present in the kidney and it has recently emerged as an important player in the pathogenesis of diabetic nephropathy, drug nephrotoxicity, and progressive chronic kidney disease. This newly established role of the ECS in the kidney might have therapeutic relevance, as pharmacological modulation of the ECS has renoprotective effects in experimental animals, raising hope for future potential applications in humans. In addition, over the last years, there has been a number of reported cases of acute kidney injury (AKI) associated with the use of synthetic cannabinoids that appear to have higher potency and rate of toxicity than natural Cannabis. This poorly recognized cause of renal injury should be considered in the differential diagnosis of AKI, particularly in young people. In this review we provide an overview of preclinical evidence indicating a role of the ECS in renal disease and discuss potential future therapeutic applications. Moreover, we give a critical update of synthetic cannabinoid-induced AKI.

The endocannabinoid system, comprising cannabinoid CB1 and CB<em>2</em> receptors, their endogenous ligands anandamide and <em>2</em>-arachidonoylglyerol, and their synthetic and metabolic enzymes, are involved in many biological processes in the body, ranging from appetite to bone turnover. Compounds inhibiting the breakdown of anandamide and <em>2</em>-<em>arachidonoylglycerol</em> increase brain levels of these lipids and thus modulate endocannabinoid signalling. In the present review, the preclinical evidence that these enzymes are good targets for development of novel therapies for anxiety and depression are discussed from a practical, rather than mechanistic, point of view. It is concluded that the preclinical data are promising, albeit tempered by problems of tolerance as well as effects upon learning and memory for irreversible monoacylglycerol lipase inhibitors, and limited by a focus upon male rodents alone. Clinical data so far has been restricted to safety studies with inhibitors of anandamide hydrolysis and a hitherto unpublished study on such a compound in elderly patients with major depressive disorders, but under the dose regimes used, they are well tolerated and show no signs of "cannabis-like" behaviours.

OBJECTIVE

Sustained inflammation originating from macrophages is a driving force of fibrosis progression and resolution. Monoacylglycerol lipase (MAGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. It is a proinflammatory enzyme that metabolises <em>2</em>-<em>arachidonoylglycerol</em>, an endocannabinoid receptor ligand, into arachidonic acid. Here, we investigated the impact of MAGL on inflammation and fibrosis during chronic liver injury.

METHODS

C57BL/6J mice and mice with global invalidation of MAGL (MAGL -/- ), or myeloid-specific deletion of either MAGL (MAGLMye-/-), ATG5 (ATGMye-/-) or CB<em>2</em> (CB<em>2</em>Mye-/-), were used. Fibrosis was induced by repeated carbon tetrachloride (CCl4) injections or bile duct ligation (BDL). Studies were performed on peritoneal or bone marrow-derived macrophages and Kupffer cells.

RESULTS

MAGL -/- or MAGLMye-/- mice exposed to CCl4 or subjected to BDL were more resistant to inflammation and fibrosis than wild-type counterparts. Therapeutic intervention with MJN110, an MAGL inhibitor, reduced hepatic macrophage number and inflammatory gene expression and slowed down fibrosis progression. MAGL inhibitors also accelerated fibrosis regression and increased Ly-6Clow macrophage number. Antifibrogenic effects exclusively relied on MAGL inhibition in macrophages, since MJN110 treatment of MAGLMye-/- BDL mice did not further decrease liver fibrosis. Cultured macrophages exposed to MJN110 or from MAGLMye-/- mice displayed reduced cytokine secretion. These effects were independent of the cannabinoid receptor <em>2</em>, as they were preserved in CB<em>2</em>Mye-/- mice. They relied on macrophage autophagy, since anti-inflammatory and antifibrogenic effects of MJN110 were lost in ATG5Mye-/- BDL mice, and were associated with increased autophagic flux and autophagosome biosynthesis in macrophages when MAGL was pharmacologically or genetically inhibited.

CONCLUSIONS

MAGL is an immunometabolic target in the liver. MAGL inhibitors may show promising antifibrogenic effects during chronic liver injury.

<AbstractText>The endocannabinoid system (ECS) consists of the cannabinoid receptors CB1 and CB<em>2</em>, the main endocannabinoids anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and their metabolic enzymes N-acylphosphatidylethanolamine-specific phospholipase D, fatty acid amide hydrolase, diacylglycerol lipase and monoacylglycerol lipase. This system is involved in the modulation of essential physiological processes. Its role in the reproductive system has become significantly important in recent years, given its major role in events such as gametogenesis, decidualisation, implantation and placentation.</AbstractText><AbstractText>In this paper, we review the literature and summarize the role of the ECS elements in reproduction and their potential as early markers for diagnosis of reproductive disorders or as pharmacological targets for treatment.</AbstractText><AbstractText>Original research and review papers published from 1964 to June <em>2</em>019 were selected in terms of relevance, reliability and quality by searching PubMed, MEDLINE and Web of Science, using the following search terms: endocannabinoid system and endometriosis; endocannabinoid system and ectopic pregnancy; endocannabinoid system and miscarriage; endocannabinoid system and pre-eclampsia; endocannabinoid system and endometrial cancer; endocannabinoid system and reproduction; endocannabinoid, endometrium; placenta; N-acylethanolamines; anandamide; <em>2</em>-<em>arachidonoylglycerol</em>; and cannabinoids.</AbstractText><AbstractText>This review demonstrates relevant information concerning ECS alterations in endometriosis, ectopic pregnancy, miscarriage, pre-eclampsia and endometrial cancer. We highlight the importance of the endocannabinoids in endometrial and placental physiology and pathophysiology, from studies in vitro and in vivo and in clinical observations. The most studied of the endogenous cannabinoids is AEA. The levels of AEA were increased in plasma of patients with endometriosis and miscarriage, as well as in the fallopian tube of women with ectopic pregnancy and in endometrial biopsies of endometrial cancer. Changes in the pattern of expression of the cannabinoid receptor CB1 were also observed in endometrial biopsies of endometriosis, fallopian tube and decidua of patients with ectopic pregnancy and pre-eclamptic placenta. Moreover, alterations in CB<em>2</em> expression have been reported in association with endometrial cancer. In general, studies on the cannabinoid signalling through CB<em>2</em> and on the biological activities of the other major endocannabinoid, namely <em>2</em>-AG, as well as its metabolic enzymes are scarce and avidly required.</AbstractText><AbstractText>The pathophysiological mechanisms involved in the described endometrial and placental pathologies are still unclear and lack the means for an early diagnosis. Based on current evidence, though alterations in ECS are demonstrated at tissue level, it is difficult to associate plasmatic changes in AEA with specific endometrial and placental diseases. Thus, pairing alterations in AEA levels with <em>2</em>-AG and/or other endocannabinoid-like molecules may provide more accurate and early diagnoses. In addition, patients may benefit from new therapies that target the ECS and endocannabinoid signalling.</AbstractText>

The endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> are metabolised by both hydrolytic enzymes (primarily fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL)) and oxygenating enzymes (e.g. cyclooxygenase-<em>2</em>, COX-<em>2</em>). In the present article, the in vivo data for compounds inhibiting endocannabinoid metabolism have been reviewed, focussing on inflammation and pain. Potential reasons for the failure of an FAAH inhibitor in a clinical trial in patients with osteoarthritic pain are discussed. It is concluded that there is a continued potential for compounds inhibiting endocannabinoid metabolism in terms of drug development, but that it is wise not to be unrealistic in terms of expectations of success.

On-demand postsynaptic synthesis and release of endocannabinoid lipids and subsequent binding to presynaptic CB1 receptors (CB1Rs) mediates short and long-term depression (LTD) of excitatory transmission in many brain regions. However, mechanisms involved in the synthesis of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) by diacylglycerol lipase α (DGLα) are poorly understood. Since Gq-coupled receptor activation can stimulate production of a major DGL substrate 1-stearoyl-<em>2</em>-arachidonoyl-sn-glycerol (SAG) by PLCβ, we sought to determine if <em>2</em>-AG biosynthesis was limited only by a lack of substrate availability, or if other pathways, such as Ca(<em>2</em>+) signaling, also need to be simultaneously engaged. To address this question, we loaded medium spiny neurons of the dorsolateral striatum with SAG while monitoring excitatory synaptic inputs. SAG-loading had no significant effect on evoked excitatory synaptic currents when cells were voltage-clamped at -80 mV. However, depolarization of MSNs to -50 mV revealed a SAG-loading dependent decrease in the amplitude of excitatory currents that was accompanied by an increase in paired pulse ratio, consistent with decreased glutamate release. Both effects of loading SAG at -50 mV were blocked by chelation of postsynaptic Ca(<em>2</em>+) using BAPTA or by bath application of tetrahydrolipstatin (THL), a DGL inhibitor. Loading of SAG into glutamatergic pyramidal neurons of the amygdala similarly inhibited excitatory synaptic inputs and increased the PPR. SAG-induced depression was absent in both regions from mice lacking CB1Rs. These data show that increasing substrate availability alone is insufficient to drive <em>2</em>-AG mobilization and that DGL-dependent synaptic depression via CB1R activation requires postsynaptic Ca(<em>2</em>+) signals.

BACKGROUND

Endocannabinoid signaling plays an important role in regulating synaptic transmission in the striatum, a brain region implicated as a central node of dysfunction in autism spectrum disorder. Deficits in signaling mediated by the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) have been reported in mouse models of autism spectrum disorder, but a causal role for striatal <em>2</em>-AG deficiency in phenotypes relevant to autism spectrum disorder has not been explored.

METHODS

Using conditional knockout mice, we examined the electrophysiological, biochemical, and behavioral effects of <em>2</em>-AG deficiency by deleting its primary synthetic enzyme, diacylglycerol lipase α (DGLα), from dopamine D1 receptor-expressing or adenosine A<em>2</em>a receptor-expressing medium spiny neurons (MSNs) to determine the role of <em>2</em>-AG signaling in striatal direct or indirect pathways, respectively. We then used viral-mediated deletion of DGLα to study the effects of <em>2</em>-AG deficiency in the ventral and dorsal striatum.

RESULTS

Targeted deletion of DGLα from direct-pathway MSNs caused deficits in social interaction, excessive grooming, and decreased exploration of a novel environment. In contrast, deletion from indirect-pathway MSNs had no effect on any measure of behavior examined. Loss of <em>2</em>-AG in direct-pathway MSNs also led to increased glutamatergic drive, which is consistent with a loss of retrograde feedback inhibition. Subregional DGLα deletion from the dorsal striatum produced deficits in social interaction, whereas deletion from the ventral striatum resulted in repetitive grooming.

CONCLUSIONS

These data suggest a role for <em>2</em>-AG deficiency in social deficits and repetitive behavior, and they demonstrate a key role for <em>2</em>-AG in regulating striatal direct-pathway MSNs.

The dysregulation of the endocannabinoid system is associated with cardiometabolic complications of obesity. Allelic variants in coding genes for this system components may contribute to differences in the susceptibility to obesity and related health hazards. These data have mostly been shown in Caucasian populations and in severely obese individuals. We investigated a multiethnic Brazilian population to study the relationships among the polymorphism 385C>A in an endocannabinoid degrading enzyme gene (FAAH), endocannabinoid levels and markers of cardiometabolic risk. Fasting plasma levels of endocannabinoids and congeners (anandamide, <em>2</em>-<em>arachidonoylglycerol</em>, N-oleoylethanolamide and N-palmitoylethanolamide) were measured by liquid chromatography-mass spectrometry in <em>2</em>00 apparently healthy individuals of both genders with body mass indices from <em>2</em><em>2</em>.5 ± 1.8 to 35.9 ± 5.5 kg/m<em>2</em> (mean ± 1 SD) and ages between 18 and 60 years. All were evaluated for anthropometric parameters, blood pressure, metabolic variables, homeostatic model assessment of insulin resistance (HOMA-IR), adiponectin, leptin, C-reactive protein, and genotyping. The endocannabinoid levels increased as a function of obesity and insulin resistance. The homozygous genotype AA was associated with higher levels of anandamide and lower levels of adiponectin versus wild homozygous CC and heterozygotes combined. The levels of anandamide were independent and positively associated with the genotype AA position 385 of FAAH, C-reactive protein levels and body mass index. Our findings provide evidence for an endocannabinoid-related phenotype that may be identified by the combination of circulating anandamide levels with genotyping of the FAAH 385C>A; this phenotype is not exclusive to mono-ethnoracial populations nor to individuals with severe obesity.

The content of the marine n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is far lower in lean than in fatty seafood. Cod filets contain less than <em>2</em>g fat per kg, whereof approximately 50% is EPA and DHA. However, a large fraction of these n-3 PUFAs is present in the phospholipid (PL) fraction and may have high bioavailability and capacity to change the endocannabinoid profile. Here we investigated whether exchanging meat from a lean terrestrial animal with cod in a background Western diet would alter the endocannabinoid tone in mice and thereby attenuate obesity development and hepatic lipid accumulation. Accordingly, we prepared iso-caloric diets with 15.1 energy (e) % protein, 39.1 e% fat and 45.8 e% carbohydrates using freeze-dried meat from cod filets or pork sirloins, and using a combination of soybean oil, corn oil, margarine, milk fat, and lard as the fat source. Compared with mice receiving diets containing pork, mice fed cod gained less adipose tissue mass and had a lower content of hepatic lipids. This was accompanied by a lower n-6 to n-3 ratio in liver PLs and in red blood cells (RBCs) in the mice. Furthermore, mice receiving the cod-containing diet had lower circulating levels of the two major endocannabinoids, N-arachidonoylethanolamine and <em>2</em>-<em>arachidonoylglycerol</em>. Together, our data demonstrate that despite the relatively low content of n-3 PUFAs in cod fillets, the cod-containing diet could exert beneficial metabolic effects.

The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) has been shown to activate human platelets in platelet-rich plasma, by binding to a "platelet-type" cannabinoid receptor (CB(PT)). Here, washed human platelets were used to characterize the binding of [(3)H]<em>2</em>-AG to CB(PT), showing a dissociation constant (Kd) of 140 +/- 31 nM and a maximum binding (Bmax) of 1<em>2</em><em>2</em> +/- 10 pmol.mg protein(-1). Selective antagonists of both CB1 and CB<em>2</em> cannabinoid receptors inhibited this binding, which was enhanced up to approximately <em>2</em>30% over the controls by 1 micro M serotonin (5-hydroxytryptamine, 5-HT). Human platelets were also able to bind [(3)H]5-HT (Kd = 79 +/- 17 nM, Bmax = 14.6 +/- 1.3 pmol.mg protein(-1)), and 1 micro M <em>2</em>-AG enhanced this binding up to approximately 150%. Moreover, they were able to take up [(3)H]5-HT through a high affinity transporter (Michaelis-Menten constant = <em>2</em><em>2</em> +/- <em>2</em> nM, maximum velocity = 344 +/- 15 pmol.min(-1).mg protein(-1)), which was not affected by <em>2</em>-AG. Interestingly, 5-HT did not affect the activity of the <em>2</em>-AG transporter of human platelets. Treatment of washed platelets with 1 micro M <em>2</em>-AG led to increased intracellular inositol-1,4,5-trisphosphate (up to approximately 300%) and decreased cyclic AMP (down to approximately 50%). Furthermore, treatment of pre-loaded platelets with 1 micro M <em>2</em>-AG induced a approximately 300% increase in [(3)H]<em>2</em>-AG release, according to a CBPT-dependent mechanism. Also, 1 micro M 5-HT enhanced the effect of <em>2</em>-AG on inositol-1,4,5-trisphosphate ( approximately 500% of the controls), cyclic AMP ( approximately <em>2</em>0%) and [(3)H]<em>2</em>-AG release ( approximately 570%), and the latter process was shown to be partly ( approximately 50%) involved in the 5-HT-dependent platelet activation. Taken together, reported findings represent the first demonstration that <em>2</em>-AG and 5-HT can mutually reinforce their receptor binding on platelet surface, which might have therapeutic implications.

Involvement of cannabinoid CB<em>2</em> receptors in the IgE-mediated cutaneous reaction was investigated. Epicutaneous challenge with <em>2</em>,4-dinitrofluorobenzene caused a triphasic swelling in the ear of BALB/c and C57BL/6 mice passively sensitized with anti-dinitrophenol IgE. Peak responses of the ear swelling appeared at 1 h, <em>2</em>4 h, and 8 days after the challenge in both strains of mice. In contrast, cannabinoid CB<em>2</em> receptor-deficient mice failed to exhibit the obvious triphasic ear swelling observed in wild-type mice. Oral administration of cannabinoid CB<em>2</em> receptor antagonist/inverse agonists [N-(benzo[1,3]dioxol-5-ylmethyl)-7-methoxy-<em>2</em>-oxo-8-pentyloxy-1,<em>2</em>-dihydroquinoline-3-carboxamide] (JTE-907) and {N-[(1S)-endo-1,3,3-trimethylbicyclo[<em>2</em>,<em>2</em>,1]heptan-<em>2</em>yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide} (SR1445<em>2</em>8) at doses of 0.1-10 mg/kg significantly and dose-dependently suppressed all three phases of ear swelling in BALB/c mice. Interestingly, epicutaneous treatment with an ether-linked analogue of endogenous cannabinoids, <em>2</em>-<em>arachidonoylglycerol</em>, caused an ear swelling that could be detected at 1 h, <em>2</em>4 h, and 8 days after treatment of both BALB/c and C57BL/6 mice. These results suggest that cannabinoid CB<em>2</em> receptors are involved in induction of the triphasic cutaneous reaction mediated by IgE, and that cannabinoid CB<em>2</em> receptor antagonist/inverse agonists may serve as anti-allergic agents in the treatment of allergic dermatitis.