Endogenous and exogenous cannabinoids bind to central cannabinoid receptors to control a multitude of behavioral functions, including aggression. The first main objective of this review is to dissect components of the endocannabinoid system, including cannabinoid 1 and cannabinoid <em>2</em> receptors; the endogenous cannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em>; and the indirect cannabinoid modulators fatty acid amide hydrolase and monoacylglycerol lipase; that have shown abnormalities in basic research studies investigating mechanisms of aggression. While most human research has concluded that the active ingredient of marijuana, Δ9-tetrahydrocannabinol, tends to dampen rather than provoke aggression in acute doses, recent evidence supports a relationship between the ingestion of synthetic cannabinoids and emergence of violent or aggressive behavior. Thus, another objective is to evaluate the emerging clinical data. This paper also discusses the relationship between prenatal and perinatal exposure to cannabis as well as use of cannabis in adolescence on aggressive outcomes. A final objective of the paper is to discuss endocannabinoid abnormalities in psychotic and affective disorders, as well as clinically aggressive populations, such as borderline personality disorder and antisocial personality disorder. With regard to the former condition, decreased anandamide metabolites have been reported in the cerebrospinal fluid, while some preliminary evidence suggests that fatty acid amide hydrolase genetic polymorphisms are linked to antisocial personality disorder and impulsive-antisocial psychopathic traits. To summarize, this paper will draw upon basic and clinical research to explain how the endocannabinoid system may contribute to the genesis of aggressive behavior.

Monoacylglycerol lipase (MAGL) is the enzyme hydrolyzing the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) to free arachidonic acid and glycerol. Therefore, MAGL is implicated in many physiological processes involving the regulation of the endocannabinoid system and eicosanoid network. MAGL inhibition represents a potential therapeutic target for many diseases, including cancer. Nowadays, most MAGL inhibitors inhibit this enzyme by an irreversible mechanism of action, potentially leading to unwanted side effects from chronic treatment. Herein, we report the discovery of long-chain salicylketoxime derivatives as potent and reversible MAGL inhibitors. The compounds herein described are characterized by a good target selectivity for MAGL and by antiproliferative activities against a series of cancer cell lines. Finally, modeling studies suggest a reasonable hypothetical binding mode for this class of compounds.

BACKGROUND

Monoacylglycerol lipase is a serine hydrolase that plays a major role in the degradation of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>. Because of this key role, selective inactivation of MAGL represents an interesting approach to obtain desirable effects in several diseases. Furthermore, MAGL is upregulated in cancer cells and primary tumors and its inhibition in aggressive breast, ovarian, and melanoma cancer cells impairs cell migration, invasiveness, and tumorigenicity. Areas covered: This review covers patent literature on MAGL inhibitors and their applications published from <em>2</em>013 to <em>2</em>017. Expert opinion: MAGL inhibition has gained considerable importance in many therapeutic fields and one compound has been subjected to Phase I studies. Even if a reasonable number of patents have been recently reported, novel MAGL inhibitors are still required, especially novel chemical classes displaying a reversible mechanism of action.

Currently, the involvement of the endocannabinoid system in cancer development and possible options for a cancer-regressive effect of cannabinoids are controversially discussed. In recent decades, a number of preclinical studies have shown that cannabinoids have an anticarcinogenic potential. Therefore, especially against the background of several legal simplifications with regard to the clinical application of cannabinoid-based drugs, an extended basic knowledge about the complex network of the individual components of the endocannabinoid system is required. The canonical endocannabinoid system consists of the endocannabinoids <i>N</i>-arachidonoylethanolamine (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> as well as the G_i/o protein-coupled transmembrane cannabinoid receptors CB₁ and CB_<em>2</em>. As a result of extensive studies on the broader effect of these factors, other fatty acid derivatives, transmembrane and intracellular receptors, enzymes and lipid transporters have been identified that contribute to the effect of endocannabinoids when defined in the broad sense as "extended endocannabinoid system." Among these additional components, 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 such as GPR55, members of the transient receptor family, and peroxisome proliferator-activated receptors were identified as targets for possible strategies to combat cancer progression. Other endocannabinoid-related fatty acids such as <em>2</em>-arachidonoyl glyceryl ether, <i>O</i>-arachidonoylethanolamine, <i>N</i>-arachidonoyldopamine and oleic acid amide showed an effect via cannabinoid receptors, while other compounds such as endocannabinoid-like substances exert a permissive action on endocannabinoid effects and act via alternative intracellular target structures. This review gives an overview of the modulation of the extended endocannabinoid system using the example of anticancer cannabinoid effects, which have been described in detail in preclinical studies.

Animal studies point to an implication of the endocannabinoid system on executive functions. In humans, several studies have suggested an association between acute or chronic use of exogenous cannabinoids (Δ9-tetrahydrocannabinol) and executive impairments. However, to date, no published reports establish the relationship between endocannabinoids, as biomarkers of the cannabinoid neurotransmission system, and executive functioning in humans. The aim of the present study was to explore the association between circulating levels of plasma endocannabinoids N-arachidonoylethanolamine (AEA) and <em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) and executive functions (decision making, response inhibition and cognitive flexibility) in healthy subjects. One hundred and fifty seven subjects were included and assessed with the Wisconsin Card Sorting Test; Stroop Color and Word Test; and Iowa Gambling Task. All participants were female, aged between 18 and 60 years and spoke Spanish as their first language. Results showed a negative correlation between <em>2</em>-AG and cognitive flexibility performance (r = -.37; p<.05). A positive correlation was found between AEA concentrations and both cognitive flexibility (r = .59; p<.05) and decision making performance (r = .<em>2</em>3; P<.05). There was no significant correlation between either <em>2</em>-AG (r = -.17) or AEA (r = -.08) concentrations and inhibition response. These results show, in humans, a relevant modulation of the endocannabinoid system on prefrontal-dependent cognitive functioning. The present study might have significant implications for the underlying executive alterations described in some psychiatric disorders currently associated with endocannabinoids deregulation (namely drug abuse/dependence, depression, obesity and eating disorders). Understanding the neurobiology of their dysexecutive profile might certainly contribute to the development of new treatments and pharmacological approaches.

Core deficits in social functioning are associated with various neuropsychiatric and neurodevelopmental disorders, yet biomarker identification and the development of effective pharmacological interventions has been limited. Recent data suggest the intriguing possibility that endogenous cannabinoids, a class of lipid neuromodulators generally implicated in the regulation of neurotransmitter release, may contribute to species-typical social functioning. Systematic study of the endogenous cannabinoid signaling could, therefore, yield novel approaches to understand the neurobiological underpinnings of atypical social functioning. This article provides a critical review of the major components of the endogenous cannabinoid system (for example, primary receptors and effectors-Δ9-tetrahydrocannabinol, cannabidiol, anandamide and <em>2</em>-<em>arachidonoylglycerol</em>) and the contributions of cannabinoid signaling to social functioning. Data are evaluated in the context of Research Domain Criteria constructs (for example, anxiety, chronic stress, reward learning, motivation, declarative and working memory, affiliation and attachment, and social communication) to enable interrogation of endogenous cannabinoid signaling in social functioning across diagnostic categories. The empirical evidence reviewed strongly supports the role for dysregulated cannabinoid signaling in the pathophysiology of social functioning deficits observed in brain disorders, such as autism spectrum disorder, schizophrenia, major depressive disorder, posttraumatic stress disorder and bipolar disorder. Moreover, these findings indicate that the endogenous cannabinoid system holds exceptional promise as a biological marker of, and potential treatment target for, neuropsychiatric and neurodevelopmental disorders characterized by impairments in social functioning.

<AbstractText>Despite the growing knowledge on the functional relationship between an altered endocannabinoid (eCB) system and development of anorexia nervosa (AN), to date no studies have investigated the central eCB tone in the activity-based anorexia (ABA) model that reproduces key aspects of human AN.</AbstractText><AbstractText>We measured levels of two major eCBs, anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), those of two eCB-related lipids, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and the cannabinoid type-1 receptor (CB1R) density in the brain of female ABA rats, focusing on areas involved in homeostatic and rewarding-related regulation of feeding behavior (i.e., prefrontal cortex, nucleus accumbens, caudato putamen, amygdala, hippocampus and hypothalamus). Analysis was carried out also at the end of recovery from the ABA condition.</AbstractText><AbstractText>At the end of the ABA induction phase, <em>2</em>-AG was significantly decreased in ABA rats in different brain areas but not in the caudato putamen. No changes were detected in AEA levels in any region, whereas the levels of OEA and PEA were decreased exclusively in the hippocampus and hypothalamus. Furthermore, CB1R density was decreased in the dentate gyrus of hippocampus and in the lateral hypothalamus. After recovery, both <em>2</em>-AG levels and CB1R density were partially normalized in some areas. In contrast, AEA levels became markedly reduced in all the analyzed areas.</AbstractText><AbstractText>These data demonstrate an altered brain eCB tone in ABA rats, further supporting the involvement of an impaired eCB system in AN pathophysiology that may contribute to the maintenance of some symptomatic aspects of the disease.</AbstractText>

Located between the median eminence, the portal vessels, and the pars distalis (PD) of the hypophysis, the hypophysial pars tuberalis (PT) is an important center for transmission of photoperiodic information to neuroendocrine circuits involved in the control of reproduction, metabolism and behavior. Despite enormous and long lasting efforts, output pathways and messenger molecules from the PT have been unraveled only recently. Most interestingly, the PT sends its signals in two directions: via a "retrograde" pathway to the hypothalamus and via an "anterograde" pathway to the PD. TSH has been identified as a messenger of the "retrograde" pathway. As discovered in Japanese quail, TSH triggers molecular cascades mediating thyroid hormone conversion in the mediobasal hypothalamus (MBH) to activate the gonadal axis. These molecular mechanisms are conserved in photoperiodic mammals, and even in non-photoperiodic laboratory mice. The search for molecules of the "anterograde" pathway was for a long time focused on PT-specific neuropeptides, the so-called "tuberalins". The discovery of a PT-intrinsic endocannabinoid system in hamsters which is regulated by the photoperiod provides strong experimental evidence that the PT also synthesizes lipidergic messengers. To date, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) appears as the most important lipidergic messenger from the PT. The primary target of <em>2</em>-AG, the cannabinoid receptor 1 (CB1) is expressed in the hamster PD. A PT-intrinsic endocannabinoid system also exists in man and CB1 receptors are demonstrated in ACTH-producing cells and folliculo-stellate cells of the human PD. These data lend support to the hypothesis that endocannabinoids function as messengers of the anterograde pathway.

Phytocannabinoids, such as THC and endocannabinoids, are well known to promote feeding behavior and to control energy metabolism through cannabinoid type 1 receptors (CB1R). However, the underlying mechanisms are not fully understood. Generally, cannabinoid-conducted retrograde dis-inhibition of hunger-promoting neurons has been suggested to promote food intake, but so far it has not been demonstrated due to technical limitations.

We applied immunohistochemical labeling of CB1R for light microscopy and electron microscopy combined with three-dimensional reconstruction from serial sections in CB1R-expressing and CB1R-null mice, which served as a negative control. Hunger-promoting neurons expressing Agouti-related protein and neuropeptide Y (AgRP/NPY) in the hypothalamic arcuate nucleus were identified in NPY-GFP and NPY-hrGFP mice.

Using three-dimensional reconstruction from serial sections we demonstrated numerous discontinuous segments of anti-CB1R labeling in the synaptic boutons and axonal shafts in the arcuate nucleus. We observed CB1R in the symmetric, presumed GABAergic, synaptic boutons innervating AgRP/NPY neurons. We also detected CB1R-containing axons producing symmetric and asymmetric synapses onto AgRP/NPY-negative neurons. Furthermore, we identified CB1R in close apposition to the endocannabinoid (<em>2</em>-<em>arachidonoylglycerol</em>)-synthesizing enzyme diacylglycerol lipase-alpha at AgRP/NPY neurons.

Our immunohistochemical and ultrastructural study demonstrates the morphological substrate for cannabinoid-conducted feeding behavior via retrograde dis-inhibition of hunger-promoting AgRP/NPY neurons.

Endocannabinoid system has attracted the researchers to investigate into its ever fascinating roles governing many of the physiological functions in the human body. The prime endogenous cannabinoids are arachidonoylethanolamide also called anandamide and <em>2</em>-<em>arachidonoylglycerol</em>. Recent pharmacological advances point out that this system of molecules are in initial stages and by updating our current knowledge, we could innovatively design molecules for suitable interventions that could be potentially beneficial to the mankind.

OBJECTIVE

Cannabinoids, such as Δ9-THC, act through an endogenous signaling system in the vertebrate eye that reduces IOP via CB1 receptors. Endogenous cannabinoid (eCB) ligand, <em>2</em>-arachidonoyl glycerol (<em>2</em>-AG), likewise activates CB1 and is metabolized by monoacylglycerol lipase (MAGL). We investigated ocular <em>2</em>-AG and its regulation by MAGL and the therapeutic potential of harnessing eCBs to lower IOP.

METHODS

We tested the effect of topical application of <em>2</em>-AG and MAGL blockers in normotensive mice and examined changes in eCB-related lipid species in the eyes and spinal cord of MAGL knockout (MAGL-/-) mice using high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). We also examined the protein distribution of MAGL in the mouse anterior chamber.

RESULTS

<em>2</em>-Arachidonoyl glycerol reliably lowered IOP in a CB1- and concentration-dependent manner. Monoacylglycerol lipase is expressed prominently in nonpigmented ciliary epithelium. The MAGL blocker KML<em>2</em>9, but not JZL184, lowered IOP. The ability of CB1 to lower IOP is not desensitized in MAGL-/- mice. Ocular monoacylglycerols, including <em>2</em>-AG, are elevated in MAGL-/- mice but, in contrast to the spinal cord, arachidonic acid and prostaglandins are not changed.

CONCLUSIONS

Our data confirm a central role for MAGL in metabolism of ocular <em>2</em>-AG and related lipid species, and that endogenous <em>2</em>-AG can be harnessed to reduce IOP. The MAGL blocker KML<em>2</em>9 has promise as a therapeutic agent, while JZL184 may have difficulty crossing the cornea. These data, combined with the relative specificity of MAGL for ocular monoacylglycerols and the lack of desensitization in MAGL-/- mice, suggest that the development of an optimized MAGL blocker offers therapeutic potential for treatment of elevated IOP.

Monoacylglycerol lipase (MAGL) is a 33 kDa member of the serine hydrolase superfamily that preferentially degrades <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) to arachidonic acid in the endocannabinoid system. Inhibition of MAGL is not only of interest for probing the cannabinoid pathway but also as a therapeutic and diagnostic target for neuroinflammation. Limited attempts have been made to image MAGL in vivo and a suitable PET ligand for this target has yet to be identified and is urgently sought to guide small molecule drug development in this pathway. Herein we synthesized and evaluated the physiochemical properties of an array of eleven sulfonamido-based carbamates and ureas with a series of terminal aryl moieties, linkers and leaving groups. The most potent compounds were a novel MAGL inhibitor, N-((1-(1H-1,<em>2</em>,4-triazole-1-carbonyl)piperidin-4-yl) methyl)-4-chlorobenzenesulfonamide (TZPU; IC50 = 35.9 nM), and the known inhibitor 1,1,1,3,3,3-hexafluoropropan-<em>2</em>-yl 4-(((4-chlorophenyl)sulfonamido) methyl)piperidine-1-carboxylate (SAR1<em>2</em>7303; IC50 = 39.3 nM), which were also shown to be selective for MAGL over fatty acid amide hydrolase (FAAH), and cannabinoid receptors (CB1 & CB<em>2</em>). Both of these compounds were radiolabeled with carbon-11 via [(11)C]COCl<em>2</em>, followed by comprehensive ex vivo biodistribution and in vivo PET imaging studies in normal rats to determine their brain permeability, specificity, clearance and metabolism. Whereas TZPU did not show adequate specificity to warrant further evaluation, [(11)C]SAR1<em>2</em>7303 was advanced for preliminary PET neuroimaging studies in nonhuman primate. The tracer showed good brain permeability (ca. 1 SUV) and heterogeneous regional brain distribution which is consistent with the distribution of MAGL.

PAK1 inhibitors are known to markedly improve social and cognitive function in several animal models of brain disorders, including autism, but the underlying mechanisms remain elusive. We show here that disruption of PAK1 in mice suppresses inhibitory neurotransmission through an increase in tonic, but not phasic, secretion of endocannabinoids (eCB). Consistently, we found elevated levels of anandamide (AEA), but not <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) following PAK1 disruption. This increased tonic AEA signaling is mediated by reduced cyclooxygenase-<em>2</em> (COX-<em>2</em>), and COX-<em>2</em> inhibitors recapitulate the effect of PAK1 deletion on GABAergic transmission in a CB1 receptor-dependent manner. These results establish a novel signaling process whereby PAK1 upregulates COX-<em>2</em>, reduces AEA and restricts tonic eCB-mediated processes. Because PAK1 and eCB are both critically involved in many other organ systems in addition to the brain, our findings may provide a unified mechanism by which PAK1 regulates these systems and their dysfunctions including cancers, inflammations and allergies.

Following binding to cannabinoid receptors, endocannabinoids regulate a variety of central nervous system processes including appetite and mood. Recent evidence suggests that the systemic release of these lipid metabolites can be altered by acute exercise and that their levels also vary across the <em>2</em>4-h sleep-wake cycle. The present study utilized a within-subject design (involving 16 normal-weight men) to determine whether daytime circulating endocannabinoid concentrations differ following three nights of partial sleep deprivation (4.<em>2</em>5-h sleep opportunity, <em>2</em>:45-7a.m. each night) vs. normal sleep (8.5-h sleep opportunity, 10:30p.m.-7a.m. each night), before and after an acute bout of ergometer cycling in the morning. In addition, subjective hunger and stress were measured. Pre-exercise plasma concentrations of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>AG) were 80% higher 1.5h after awakening (vs. normal sleep, p<0.05) when participants were sleep-deprived. This coincided with increased hunger ratings (+<em>2</em>5% vs. normal sleep, p<0.05). Moreover, plasma <em>2</em>AG was elevated 15min post-exercise (+44%, p<0.05). Sleep duration did not however modulate this exercise-induced rise. Finally, subjective stress was generally lower on the day after three nights of short sleep vs. normal sleep, especially after exercise (p<0.05). Given that activation of the endocannabinoid system has been previously shown to acutely increase appetite and mood, our results could suggest that behavioral effects of acute sleep loss, such as increased hunger and transiently improved psychological state, may partially result from activation of this signaling pathway. In contrast, more pronounced exercise-induced elevations of endocannabinoids appear to be less affected by short sleep duration.

Diacylglycerol lipases (DGLs) mainly catalyze "on-demand" biosynthesis of bioactive monoacylglycerols (MAGs) with different long fatty acyl chains, including <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), <em>2</em>-linoleoylglycerol (<em>2</em>-LG), <em>2</em>-oleoylglycerol (<em>2</em>-OG) and <em>2</em>-palmitoylglycerol (<em>2</em>-PG). Enzymatic characterization of DGLs, their expression and distribution, and functional features has been elucidated from microorganisms to mammals in some extent. In mammals, biosynthesis, degradation and metabolism of these bioactive lipids intertwine and form a complicated biochemical pathway to affect the mammal neuromodulation of central nervous system and also other physiological processes in most peripheral organs and non-nervous tissue cells, and yet we still do not know if the neuromodulatory role of mammal DGL and MAGs is similar to invertebrates. Tracing the evolutionary history of DGLs from microorganisms to vertebrates will be an essential method to infer DGL and MAG research in organisms. In this review, we give an exhaustive explanation of the ancestral origin, divergence and evolutionary pattern through systemic searching of DGL orthologs in different species. Finally, we also summarize our recent work on the structural and functional studies of DGL in order to explore usage of DGLs in industry and the development of inhibitors for clinical intervention.

Monoacylglycerol lipase (MGL) is the last enzymatic step in triglyceride degradation, hydrolyzing monoglycerides into glycerol and fatty acids (FA) and converting <em>2</em>-<em>arachidonoylglycerol</em> into arachidonic acid (AA), thus providing ligands for nuclear receptors (NRs) as key regulators of hepatic bile acid (BA)/lipid metabolism and inflammation. We aimed to explore the role of MGL in the development of cholestatic liver and bile duct injury in mouse models of sclerosing cholangitis (SC), a disease so far lacking effective pharmacological therapy. To this aim we analyzed the effects of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding to induce SC in wild type (WT) and knockout (MGL^-/- ) mice and tested pharmacological inhibition with JZL184 in the Mdr<em>2</em>^-/- mouse model of SC. Cholestatic liver injury and fibrosis were assessed by serum biochemistry, liver histology, gene expression and western blot characterization of BA and FA synthesis/transport. Moreover, intestinal FAs and fecal microbiome were analyzed. Transfection and silencing were performed in Caco<em>2</em> cells. MGL^-/- mice were protected from DDC-induced biliary fibrosis and inflammation with reduced serum liver enzymes, increased FA/BA metabolism and β-oxidation. Notably, pharmacological (JZL184) inhibition of MGL ameliorated cholestatic injury in DDC-fed WT mice and protected Mdr<em>2</em>^-/- from spontaneous liver injury, with improved liver enzymes, inflammation and biliary fibrosis. In vitro experiments confirmed that silencing of MGL decreases prostaglandin E<em>2</em> accumulation in the intestine upregulating peroxisome proliferator activated receptor (PPAR) -α and -γ activity, thus reducing inflammation. Conclusions: Collectively, our study unravels MGL as a novel metabolic target, demonstrating that MGL inhibition may be considered as potential therapy for SC.

<AbstractText>This study aimed to investigate and compare the magnitude of exercise-induced hypoalgesia (EIH) with low intensity blood flow restriction (BFR) resistance exercise (RE) at varying pressures to other intensities of resistance exercise and examine endogenous mechanisms of pain reduction.</AbstractText><AbstractText>Twelve individuals performed four experimental trials involving unilateral leg press exercise in a randomised crossover design: low load RE at 30% of one repetition maximum (1RM), high load RE (70% 1RM) and BFR-RE (30% 1RM) at a low and high pressure. BFR pressure was prescribed relative to limb occlusion pressure at 40% and 80% for the low- and high-pressure trials. Pressure pain thresholds (PPT) were assessed before, 5-min and <em>2</em>4-h following exercise in exercising and non-exercising muscles. Venous blood samples were collected at the same timepoints to determine plasma concentrations of beta-endorphin and <em>2</em>-<em>arachidonoylglycerol</em>.</AbstractText><AbstractText>High pressure BFR-RE increased PPTs in the exercising limb to a greater extent than all other trials. Comparable systemic EIH effects were observed with HLRE and both BFR-RE trials. PPTs in the exercising limb remained elevated above baseline at <em>2</em>4-h post-exercise following both BFR-RE trials. Post-exercise plasma beta-endorphin concentration was elevated during the BFR-RE trials. No changes to <em>2</em>-<em>arachidonoylglycerol</em> concentration were observed.</AbstractText><AbstractText>High pressure BFR-RE causes a greater EIH response in the exercising limb that persists for up to <em>2</em>4-h following exercise. The reduction in pain sensitivity with BFR-RE is partly driven by endogenous opioid production of beta-endorphin. BFR-RE should be introduced as a possible pain-modulation tool in individuals with acute and chronic pain.</AbstractText>

BACKGROUND

There is compelling evidence in humans that peripheral endocannabinoid signaling is disrupted in obesity. However, little is known about the corresponding central signaling. Here, we have investigated the relationship between gender, leptin, body mass index (BMI) and levels of the endocannabinoids anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in the serum and cerebrospinal fluid (CSF) of primarily overweight to obese patients with osteoarthritis.

RESULTS

Patients (<em>2</em>0 females, 15 males, age range 44-78 years, BMI range <em>2</em>4-4<em>2</em>) undergoing total knee arthroplasty for end-stage osteoarthritis were recruited for the study. Endocannabinoids were quantified by liquid chromatography - mass spectrometry. AEA and <em>2</em>-AG levels in the serum and CSF did not correlate with either age or BMI. However, <em>2</em>-AG levels in the CSF, but not serum, correlated negatively with CSF leptin levels (Spearman's ρ -0.48, P=0.0076, n=30). No such correlations were observed for AEA and leptin.

CONCLUSIONS

In the patient sample investigated, there is a negative association between <em>2</em>-AG and leptin levels in the CSF. This is consistent with pre-clinical studies in animals, demonstrating that leptin controls the levels of hypothalamic endocannabinoids that regulate feeding behavior.

Helminths have coevolved with their hosts, resulting in the development of specialized host immune mechanisms and parasite-specific regulatory products. Identification of new pathways that regulate helminth infection could provide a better understanding of host-helminth interaction and may identify new therapeutic targets for helminth infection. Here we identify the endocannabinoid system as a new mechanism that influences host immunity to helminths. Endocannabinoids are lipid-derived signaling molecules that control important physiologic processes, such as feeding behavior and metabolism. Following murine infection with Nippostrongylus brasiliensis, an intestinal nematode with a life cycle similar to that of hookworms, we observed increased levels of endocannabinoids (<em>2</em>-<em>arachidonoylglycerol</em> [<em>2</em>-AG] or anandamide [AEA]) and the endocannabinoid-like molecule oleoylethanolamine (OEA) in infected lung and intestine. To investigate endocannabinoid function in helminth infection, we employed pharmacological inhibitors of cannabinoid subtype receptors 1 and <em>2</em> (CB1R and CB<em>2</em>R). Compared to findings for vehicle-treated mice, inhibition of CB1R but not CB<em>2</em>R resulted in increased N. brasiliensis worm burden and egg output, associated with significantly decreased expression of the T helper type <em>2</em> cytokine interleukin 5 (IL-5) in intestinal tissue and splenocyte cultures. Strikingly, bioinformatic analysis of genomic and transcriptome sequencing (RNA-seq) data sets identified putative genes encoding endocannabinoid biosynthetic and degradative enzymes in many parasitic nematodes. To test the novel hypothesis that helminth parasites produce their own endocannabinoids, we measured endocannabinoid levels in N. brasiliensis by mass spectrometry and quantitative PCR and found that N. brasiliensis parasites produced endocannabinoids, especially at the infectious larval stage. To our knowledge, this is the first report of helminth- and host-derived endocannabinoids that promote host immune responses and reduce parasite burden.

Endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) regulates several important physiological processes in the brain. <em>2</em>-AG is commonly quantified by gas chromatography mass spectrometry after an initial purification step. The most precise and rapid purification utilizes C(18) solid-phase extraction, but quantification problems can arise with acyl migration from <em>2</em>-AG to 1-<em>arachidonoylglycerol</em>. We found that extraction with methanol promoted this migration, but acetone and diethyl ether (Et(<em>2</em>)O) did not. Acetone and Et(<em>2</em>)O were used to develop a purification method for the direct determination of <em>2</em>-AG.

Endocannabinoids serve as retrograde messengers that are released by postsynaptic cells to regulate neurotransmitter release from presynaptic boutons. A new study in this issue of Neuron by Tanimura et al. establishes that most endocannabinoid signaling in the brain is a consequence of the calcium-dependent or receptor-driven generation of the retrograde signal <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) by diacylglycerol lipase alpha (DGLalpha).

In rat thalamic paraventricular nucleus of thalamus (PVT) neurons, activation of thyrotropin-releasing hormone (TRH) receptors enhances excitability via concurrent decrease in G protein-coupled inwardly-rectifying potassium (GIRK)-like and activation of transient receptor potential cation (TRPC)4/5-like cationic conductances. An exploration of intracellular signaling pathways revealed the TRH-induced current to be insensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitors, but reduced by D609, an inhibitor of phosphatidylcholine-specific PLC (PC-PLC). A corresponding change in the I-V relationship implied suppression of the cationic component of the TRH-induced current. Diacylglycerol (DAG) is a product of the hydrolysis of PC. Studies focused on the isolated cationic component of the TRH-induced response revealed a reduction by RHC80<em>2</em>67, an inhibitor of DAG lipase, the enzyme involved in the hydrolysis of DAG to the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Further investigation revealed enhancement of the cationic component in the presence of either JZL184 or WWL70, inhibitors of enzymes involved in the hydrolysis of <em>2</em>-AG. A decrease in the TRH-induced response was noted in the presence of rimonabant or SR1445<em>2</em>8, membrane permeable CB1 and CB<em>2</em> receptor antagonists, respectively. A decrease in the TRH-induced current by intracellular, but not by bath application of the membrane impermeable peptide hemopressin, selective for CB1 receptors, suggests a postsynaptic intracellular localization of these receptors. The TRH-induced current was increased in the presence of arachidonyl-<em>2</em>'-chloroethylamide (ACEA) or JWH133, CB1 and CB<em>2</em> receptor agonists, respectively. The PI3-kinase inhibitor LY<em>2</em>9400<em>2</em>, known to inhibit TRPC translocation, decreased the response to TRH. In addition, a TRH-induced enhancement of the low-threshold spike was prevented by both rimonabant, and SR1445<em>2</em>8. TRH had no influence on excitatory or inhibitory miniature postsynaptic currents, suggesting presynaptic CB receptors are not involved in this situation. Collectively, the data imply that activation of TRH receptors in these midline thalamic neurons engages novel signaling pathways that include postsynaptic intracellular CB1 and CB<em>2</em> receptors in the activation of TRPC4/5-like channels.

We previously reported the involvement of brain diacylglycerol lipase and cyclooxygenase in intracerebroventricularly (i.c.v.) administered bombesin-induced secretion of noradrenaline and adrenaline from the adrenal medulla in rats. Diacylglycerol can be hydrolyzed by diacylglycerol lipase into <em>2</em>-<em>arachidonoylglycerol</em>, which may be further hydrolyzed by monoacylglycerol lipase into arachidonic acid, a substrate of cyclooxygenase. <em>2</em>-<em>Arachidonoylglycerol</em> is a major endocannabinoid, which can inhibit synaptic transmission by presynaptic cannabinoid CB(1) receptors. Released <em>2</em>-<em>arachidonoylglycerol</em> is rapidly inactivated by uptake into cells and enzymatic hydrolysis. In the present study, we examined the involvement of brain <em>2</em>-<em>arachidonoylglycerol</em> and its regulatory role in the bombesin-induced central activation of adrenomedullary outflow using anesthetized rats. The elevation of plasma noradrenaline and adrenaline induced by a sub-maximal dose of bombesin (1 nmol/animal, i.c.v.) was reduced by MAFP (monoacylglycerol lipase inhibitor) (0.<em>2</em>8 and 0.7 μmol/animal, i.c.v.), JZL184 (selective monoacylglycerol lipase inhibitor) (0.7 and 1.4 μmol/animal, i.c.v.), ACEA (CB(1) receptor agonist) (0.7 and 1.4 μmol/animal, i.c.v.) and AM 404 (endocannabinoid uptake-inhibitor) (80 and <em>2</em>50 nmol/animal, i.c.v.), while AM <em>2</em>51 (CB(1) receptor antagonist) (90 and 180 nmol/animal, i.c.v.) potentiated the response induced by a small dose of bombesin (0.1 nmol/animal, i.c.v.). These results suggest a possibility that <em>2</em>-<em>arachidonoylglycerol</em> is endogenously generated in the brain during bombesin-induced activation of central adrenomedullary outflow, thereby inhibiting the peptide-induced response by activation of brain CB(1) receptors in rats.

Spinal cord injury (SCI) induces a cascade of processes that may further expand the damage (secondary injury) or, alternatively, may be part of a safeguard response. Here we show that after a moderate-severe contusive SCI in rats there is a significant and very early increase in the spinal cord content of the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and arachidonoyl ethanolamide (anandamide, AEA). Since <em>2</em>-AG and AEA act through CB1 and CB<em>2</em> cannabinoid receptors, we administered at <em>2</em>0 minutes after lesion a single injection of their respective antagonists AM<em>2</em>81 and AM630 alone or in combination to block the effects of this early endocannabinoid accumulation. We observed that AM<em>2</em>81, AM630 or AM<em>2</em>81 plus AM630 administration impairs the spontaneous motor recovery of rats according to the Basso-Beattie-Bresnahan (BBB) locomotor scale. However, blockade of CB1, CB<em>2</em> or both receptors produced different effects at the histopathological level. Thus, AM630 administration results at 90 days after lesion in increased MHC-II expression by spinal cord microglia/monocytes and reduced number of serotoninergic fibres in lumbar spinal cord (below the lesion). AM<em>2</em>81 exerted the same effects but also increased oedema volume estimated by MRI. Co-administration of AM<em>2</em>81 and AM630 produced the effects observed with the administration of either AM<em>2</em>81 or AM630 and also reduced white matter and myelin preservation and enhanced microgliosis in the epicentre. Overall, our results suggest that the endocannabinoids acting through CB1 and CB<em>2</em> receptors are part of an early neuroprotective response triggered after SCI that is involved in the spontaneous recovery after an incomplete lesion.