Adolescent cannabis use is associated with greater relative risk, increased symptom severity, and earlier age of onset of schizophrenia. We investigated whether this interaction may be partly attributable to disease-related disturbances in metabolism of the major cortical endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Transcript levels for the recently discovered <em>2</em>-AG metabolizing enzyme, α-β-hydrolase domain 6 (ABHD6), were assessed using quantitative PCR in the prefrontal cortex of schizophrenia and healthy subjects (n=84) and antipsychotic- or tetrahydrocannabinol-exposed monkeys. ABHD6 mRNA levels were elevated in schizophrenia subjects who were younger and had a shorter illness duration but not in antipsychotic- or tetrahydrocannabinol-exposed monkeys. Higher ABHD6 mRNA levels may increase <em>2</em>-AG metabolism which may influence susceptibility to cannabis in the earlier stages of schizophrenia.

The major endocannabinoids (ECs) arachidonoylethanolamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and related N-ethanolamines act as full and partial agonists at CB(1), CB(<em>2</em>), GPR55, PPAR and TRPV1 receptors to various degrees. These receptors are also expressed in immune cells like monocytes/macrophages where they regulate different cellular processes. In this study, potentially bioactive lipids in fetal bovine sera (FBS) were quantified by GC/MS. We found that several commercial FBS contain ECs and bioactive amounts of <em>2</em>-AG (<em>2</em>50-700 nM). We show that residual <em>2</em>-AG from FBS can activate primary macrophages and increase migration and RANKL-stimulated osteoclastogenesis. Furthermore, <em>2</em>-AG high-content sera specifically upregulated LPS-stimulated IL-6 expression in U937 cells. Polymyxin B beads may be used to selectively and efficiently remove <em>2</em>-AG from sera, but not arachidonic acid and N-ethanolamines. In conclusion, <em>2</em>-AG in cell culture media may significantly influence cellular experiments. CD14+ mononuclear cells which strongly express surface CB receptors may be particularly sensitive towards residual <em>2</em>-AG from FBS. Therefore, the EC content in culture media should be controlled in biological experiments involving monocytes/macrophages.

We show that a fully functional endocannabinoid system is present in primary human melanocytes (normal human epidermal melanocyte cells), including anandamide (AEA), <em>2</em>-<em>arachidonoylglycerol</em>, the respective target receptors (CB(1), CB(<em>2</em>), and TRPV1), and their metabolic enzymes. We also show that at higher concentrations AEA induces normal human epidermal melanocyte apoptosis (∼3-fold over controls at 5 μM) through a TRPV1-mediated pathway that increases DNA fragmentation and p53 expression. However, at lower concentrations, AEA and other CB(1)-binding endocannabinoids dose-dependently stimulate melanin synthesis and enhance tyrosinase gene expression and activity (∼3- and ∼<em>2</em>-fold over controls at 1 μM). This CB(1)-dependent activity was fully abolished by the selective CB(1) antagonist SR141716 or by RNA interference of the receptor. CB(1) signaling engaged p38 and p4<em>2</em>/44 mitogen-activated protein kinases, which in turn activated the cyclic AMP response element-binding protein and the microphthalmia-associated transcription factor. Silencing of tyrosinase or microphthalmia-associated transcription factor further demonstrated the involvement of these proteins in AEA-induced melanogenesis. In addition, CB(1) activation did not engage the key regulator of skin pigmentation, cyclic AMP, showing a major difference compared with the regulation of melanogenesis by α-melanocyte-stimulating hormone through melanocortin 1 receptor.

In the cerebellum of juvenile mice or rats, endocannabinoids are shown to mediate depolarization-induced suppression of excitation (DSE) and retrograde suppression induced by activation of type 1 metabotropic glutamate receptor (mGluR1) at parallel fiber (PF) to Purkinje cell (PC) synapses. However, recent studies showed that glutamate also mediated retrograde signaling through presynaptic kainate receptors in the cerebellum of young adult mice and rats. We reexamined this possibility in C57BL/6 mice at postnatal day <em>2</em>0-35 (P<em>2</em>0-P35) and in Sprague-Dawley rats at P18-P<em>2</em>4. We found that DSE at PF-PC synapses was abolished by AM<em>2</em>51, a cannabinoid receptor antagonist, and by tetrahydrolipstatin (THL), a blocker of diacylglycerol lipase (DGL) that produces an endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). AM<em>2</em>51 and THL did not affect depolarization-induced Ca(<em>2</em>+) transients in PCs, and THL did not suppress cannabinoid sensitivity of PFs. Moreover, DSE at PF-PC synapses was absent in CB(1) knockout mice. AM<em>2</em>51 also eliminated transient suppression of PF-PC synaptic transmission following a brief burst of PF stimulation, a phenomenon known to be mediated by mGluR1. These results suggest that DSE and mGluR1-mediated suppression in young adult PCs are mediated by endocannabinoids, and that glutamate, if any, has little contribution.

Since the first reports in <em>2</em>001, great advances have been made towards the understanding of endocannabinoid-mediated synaptic modulation. Electrophysiological studies have revealed that one of the two major endocannabinoids, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), is produced from membrane lipids upon postsynaptic Ca(<em>2</em>+) elevation and/or activation of Gq/11-coupled receptors, and released from postsynaptic neurons. The released <em>2</em>-AG then acts retrogradely onto presynaptic cannabinoid CB1 receptors and induces suppression of neurotransmitter release either transiently or persistently. These forms of <em>2</em>-AG-mediated retrograde synaptic modulation are functional throughout the brain. The other major endocannabinoid, anandamide, mediates a certain form of endocannabinoid-mediated long-term depression (LTD). Anandamide also functions as an agonist for transient receptor potential vanilloid receptor type 1 (TRPV1) and mediates endocannabinoid-independent and TRPV1-dependent forms of LTD. It has also been demonstrated that the endocannabinoid system itself is plastic, which can be either up- or down-regulated by experimental or environmental conditions. In this review, I will make an overview of the mechanisms underlying endocannabinoid-mediated synaptic modulation.

The levels of the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are under the negative control of leptin in the rodent hypothalamus. As leptin and endocannabinoids play opposite roles in the control of reproduction, we have investigated whether the impaired fertility typical of leptin-defective ob/ob mice is due, in part, to enhanced uterine endocannabinoid levels. We found that levels of both anandamide and <em>2</em>-AG in the uterus of ob/ob mice are significantly elevated with respect to wild-type littermates, due to reduced hydrolase activity in the case of anandamide, and to reduced monoacylglycerol lipase and enhanced diacylglycerol lipase activity in the case of <em>2</em>-AG. Furthermore, the process mediating endocannabinoid cellular uptake was also impaired in ob/ob mice, whereas the levels of cannabinoid and anandamide receptors were not modified. Although ineffective in wild-type mice, treatment of ob/ob mice with leptin re-established endocannabinoid levels and enzyme activities back to the values observed in wild-type littermates. Finally, treatment of ob/ob females with the CB1 receptor antagonist SR141716A did not improve their fertility, and inhibition of endocannabinoid inactivation with the endocannabinoid uptake inhibitor OMDM-1 in wild-type females did not result in impaired fertility.

Mammalian cells produce both N-arachidonoylethanolamine (<em>2</em>0:4n-6 NAE, anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), lipid signaling molecules that activate cannabinoid receptors. Because both agonists occur in the presence of receptor-inactive congeners, we have developed a sensitive method for the simultaneous assay of N-acylethanolamines (NAEs) and <em>2</em>-monoacylglycerols (<em>2</em>-MAG). These lipid classes are isolated from total lipids by solid phase extraction and converted to tert-butyldimethylsilyl (tBDMS) derivatives in the presence of deuterated analogs. The tBDMS derivatives are analyzed by gas chromatography/mass spectrometry using selected ion monitoring programs specific for NAE and <em>2</em>-MAG. Individual NAEs and <em>2</em>-MAGs can be quantified in the nanogram and subnanogram range. The NAE and <em>2</em>-MAG compositions of rat organs and cultured JB6 cells are reported.

The role played by the endocannabinoid system in the regulation of energy balance is currently generating a great amount of interest among several groups of investigators. This interest in large part comes from the urgent need to develop anti-obesity and anti-cachexia drugs around target systems (such as the endocannabinoid system), which appears to be genuinely involved in energy balance regulation. When activated, the endocannabinoid system favors energy deposition through increasing energy intake and reducing energy expenditure. This system is activated in obesity and following food deprivation, which further supports its authentic function in energy balance regulation. The cannabinoid receptor type 1 (CB1), one of the two identified cannabinoid receptors, is expressed in energy-balance brain structures that are also able to readily produce or inactivate N-arachidonoyl ethanolamine (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>AG), the most abundantly formed and released endocannabinoids. The brain action of endocannabinoid system on energy balance seems crucial and needs to be delineated in the context of the homeostatic and hedonic controls of food intake and energy expenditure. These controls require the coordinated interaction of the hypothalamus, brainstem and limbic system and it appears imperative to unravel those interplays. It is also critical to investigate the metabolic endocannabinoid system while considering the panoply of functions that the endocannabinoid system fulfills in the brain and other tissues. This article aims at reviewing the potential mechanisms whereby the brain endocannabinoid system influences the regulation energy balance.

ACh stimulates arachidonic acid (AA) release from membrane phospholipids of vascular endothelial cells (ECs). In rabbit aorta, AA is metabolized through the 15-lipoxygenase pathway to form vasodilatory eicosanoids 15-hydroxy-11,1<em>2</em>-epoxyeicosatrienoic acid (HEETA) and 11,1<em>2</em>,15-trihydroxyeicosatrienoic acid (THETA). AA is released from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by phospholipase A<em>2</em> (PLA<em>2</em>), or from phosphatidylinositol (PI) by phospholipase C (PLC) pathway. The diacylglycerol (DAG) lipase can convert DAG into <em>2</em>-<em>arachidonoylglycerol</em> from which free AA can be released by monoacylglycerol (MAG) lipase or fatty acid amidohydrolase (FAAH). We used specific inhibitors to determine the involvement of the PLC pathway in ACh-induced AA release. In rabbit aortic rings precontracted by phenylephrine, ACh induced relaxation in the presence of indomethacin and N(omega)-nitro-L-arginine (L-NNA). These relaxations were blocked by the PLC inhibitor U-731<em>2</em><em>2</em>, DAG lipase inhibitor RHC-80<em>2</em>67, and MAG lipase/FAAH inhibitor URB-53<em>2</em>. Cultured rabbit aortic ECs were labeled with [14C]AA and stimulated with methacholine (10(-5) M). Free [14C]AA was released by methacholine. Methacholine decreased the [14C]AA content of PI, DAG, and MAG fractions but not PC or PE fractions. Methacholine-induced release of [14C]AA was blocked by U-731<em>2</em><em>2</em>, RHC-80<em>2</em>67, and URB-53<em>2</em> but not by U-73343, an inactive analog of U-731<em>2</em><em>2</em>. The data suggested that ACh activates PLC, DAG lipase, and MAG lipase pathway to release AA from membrane lipids. This pathway is important in regulating vasodilatory eicosanoid synthesis and vascular relaxation in rabbit aorta.

OBJECTIVE

Increasing evidence from laboratory and epidemiologic studies indicates that insufficient sleep may be a risk factor for obesity. Sleep curtailment results in stimulation of hunger and food intake that exceeds the energy cost of extended wakefulness, suggesting the involvement of reward mechanisms. The current study tested the hypothesis that sleep restriction is associated with activation of the endocannabinoid (eCB) system, a key component of hedonic pathways involved in modulating appetite and food intake.

METHODS

In a randomized crossover study comparing 4 nights of normal (8.5 h) versus restricted sleep (4.5 h) in healthy young adults, we examined the <em>2</em>4-h profiles of circulating concentrations of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and its structural analog <em>2</em>-oleoylglycerol (<em>2</em>-OG). We concomitantly assessed hunger, appetite, and food intake under controlled conditions.

RESULTS

A robust daily variation of <em>2</em>-AG concentrations with a nadir around the middle of the sleep/overnight fast, followed by a continuous increase culminating in the early afternoon, was evident under both sleep conditions but sleep restriction resulted in an amplification of this rhythm with delayed and extended maximum values. Concentrations of <em>2</em>-OG followed a similar pattern, but with a lesser amplitude. When sleep deprived, participants reported increases in hunger and appetite concomitant with the afternoon elevation of <em>2</em>-AG concentrations, and were less able to inhibit intake of palatable snacks.

CONCLUSIONS

Our findings suggest that activation of the eCB system may be involved in excessive food intake in a state of sleep debt and contribute to the increased risk of obesity associated with insufficient sleep.

CONCLUSIONS

A commentary on this article appears in this issue on page 495.

Endocannabinoids (ECs), anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), inhibit proliferation of carcinoma cells. Several enzymes hydrolyze ECs to reduce endogenous EC concentrations and produce eicosanoids that promote cell growth. In this study, we determined the effects of EC hydrolysis inhibitors and a putative EC, <em>2</em>-arachidonylglyceryl ether (noladin ether, NE) on proliferation of prostate carcinoma (PC-3, DU-145, and LNCaP) cells. PC-3 cells had the least specific hydrolysis activity for AEA and administration of AEA effectively inhibited cell proliferation. The proliferation inhibition was blocked by SR141716A (a selective CB1R antagonist) but not SR1445<em>2</em>8 (a selective CB<em>2</em>R antagonist), suggesting a CB1R-mediated inhibition mechanism. On the other hand, specific hydrolysis activity for <em>2</em>-AG was high and <em>2</em>-AG inhibited proliferation only in the presence of EC hydrolysis inhibitors. NE inhibited proliferation in a concentration-dependent manner; however, SR141716A, SR1445<em>2</em>8 and pertussis toxin did not block the NE-inhibited proliferation, suggesting a CBR-independent mechanism of NE. A peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW966<em>2</em> did not block the NE-inhibited proliferation, suggesting that PPARγ was not involved. NE also induced cell cycle arrest in G(0)/G(1) phase in PC-3 cells. NE inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB p65) and down-regulated the expression of cyclin D1 and cyclin E in PC-3 cells, suggesting the NF-κB/cyclin D and cyclin E pathways are involved in the arrest of G1 cell cycle and inhibition of cell growth. These results indicate therapeutic potentials of EC hydrolysis inhibitors and the enzymatically stable NE in prostate cancer.

The discovery that N-acylethanolamines, such as oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), acting as endogenous ligands of alpha-type peroxisome proliferator-activated receptors (PPARα), block nicotine-induced excitation of dopamine neurons revealed their role as important endogenous negative modulators of nicotinic receptors containing β<em>2</em> subunits (denoted β<em>2</em>*-nAChRs) on dopamine neurons, which are key to the brain reward system. Using mass-spectrometry data analysis from rodent brain slices containing the midbrain, we characterized the effects induced by modulation of PPARα on PEA and OEA levels. PEA and OEA constitutive levels in the midbrain are higher than endocannabinoids (e.g. anandamide, <em>2</em>-<em>arachidonoylglycerol</em>), and depend upon excessive input drive and the metabolic state of the cells. Accordingly, OEA and PEA synthesis is affected when adding low concentrations of fatty acids (endogenous PPARα ligands), most likely through activation of PPARα. Indeed, PPARα activation increases PEA and OEA levels, which may further sustain PPARα activity. Given this, it is likely that these molecules dynamically affect dopamine function and excitability, as well as their dependent behaviour. Consequently, N-acylethanolamines may confer less vulnerability towards disruption of dynamic balance of dopamine-acetylcholine systems through PPARα activation. Finally, using pharmacological and/or nutritional strategies which target PPARα might represent a promising therapeutic approach to prevent disorders often related to neuro-inflammation, stress and abnormal β<em>2</em>*-nAChR function.

OBJECTIVE

Endocannabinoids may modify cancer development, progression and associated pain. We determined whether cancer-evoked dysregulations in this system become manifest in altered tissue and plasma endocannabinoids.

METHODS

Endocannabinoid changes due to cancer were explored in a local and metastatic syngeneic mouse melanoma model. Endocannabinoid stratification in human cancer was cross-sectionally assessed in the plasma of 304 patients (147 men, 157 women, aged 3<em>2</em> - 87 years) suffering from several types of cancer at Roman Numeral Staging between I and IVc, mostly IV (n = <em>2</em><em>2</em>0), and compared with endocannabinoids of healthy controls.

RESULTS

In mice with local tumor growth, ethanolamide endocannabinoids, i.e., anandamide (AEA), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) were downregulated, whereas <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) was increased. Upon spreading of the cancer cells particularly <em>2</em>-AG steadily increased in parallel to disease progression while OEA modulated cell migration. Results translated into humans, in whom cancer was associated with a decreased AEA, increased <em>2</em>-AG and increased OEA correlating with the number of metastases.

CONCLUSIONS

The endocannabinoid system was subject to cancer-associated regulations to an extent that led to measurable changes in circulating endocannabinoid levels, emphasizing the importance of the endocannabinoid system in the pathophysiology of cancer.

Muller cells play a prominent role in inflammatory conditions of the retina. They are part of the retinal innate immune response. The endocannabinoid system functions as an immune modulator in both the peripheral immune system as well as the central nervous system. We hypothesized that the neuroprotective ability of exogenous endocannabinoids in the retina is partially mediated through Muller glia. This study reports that exposure to endocannabinoids in activated but not resting primary human Muller glia inhibit production of several proinflammatory cytokines, while elevating anti-inflammatory mediators. Cytokine generation in activated Muller glia is regulated by endocannabinoids through the mitogen-activated protein kinase (MAPK) family at multiple signaling stages. Anandamide (AEA) acts to control MAPK phosphorylation through MKP-1. Both AEA and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) inhibit the transcription factor NF-κB and increases the regulatory protein, IL1-R-associated kinase 1-binding protein 1. Endocannabinoids also increase expression of Tristetraprolin in activated Muller cells, which is implicated in affecting AU-rich proinflammatory cytokine mRNA. We demonstrate that exogenous application of AEA and <em>2</em>-AG aid in retinal cell survival under inflammatory conditions by creating an anti-inflammatory milieu. Endocannabinoids or synthetic cannabinoid therapy may therefore orchestrate a molecular switch to bias the innate immune system suchthat the balance of pro- and anti-inflammatory cytokine generation creates a prosurvival milieu.

The signaling capacity of endogenous cannabinoids ("endocannabinoids") is tightly regulated by degradative enzymes. This Perspective highlights a research article in this issue (p. 996) in which the authors show that genetic disruption of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), causes marked elevations in <em>2</em>-AG levels that lead to desensitization of brain cannabinoid receptors. These findings highlight the central role that MAGL plays in endocannabinoid metabolism in vivo and reveal that excessive <em>2</em>-AG signaling can lead to functional antagonism of the brain cannabinoid system.

OBJECTIVE

Palmitoylethanolamide (PEA), a naturally occurring acylethanolamide chemically related to the endocannabinoid anandamide, interacts with targets that have been identified in peripheral nerves controlling gastrointestinal motility, such as cannabinoid CB1 and CB<em>2</em> receptors, TRPV1 channels and PPARα. Here, we investigated the effect of PEA in a mouse model of functional accelerated transit which persists after the resolution of colonic inflammation (post-inflammatory irritable bowel syndrome).

METHODS

Intestinal inflammation was induced by intracolonic administration of oil of mustard (OM). Mice were tested for motility and biochemical and molecular biology changes 4 weeks later. PEA, oleoylethanolamide and endocannabinoid levels were measured by liquid chromatography-mass spectrometry and receptor and enzyme mRNA expression by qRT-PCR.

RESULTS

OM induced transient colitis and a functional post-inflammatory increase in upper gastrointestinal transit, associated with increased intestinal anandamide (but not <em>2</em>-<em>arachidonoylglycerol</em>, PEA or oleoylethanolamide) levels and down-regulation of mRNA for TRPV1 channels. Exogenous PEA inhibited the OM-induced increase in transit and tended to increase anandamide levels. Palmitic acid had a weaker effect on transit. Inhibition of transit by PEA was blocked by rimonabant (CB1 receptor antagonist), further increased by 5'-iodoresiniferatoxin (TRPV1 antagonist) and not significantly modified by the PPARα antagonist GW6471.

CONCLUSIONS

Intestinal endocannabinoids and TRPV1 channel were dysregulated in a functional model of accelerated transit exhibiting aspects of post-inflammatory irritable bowel syndrome. PEA counteracted the accelerated transit, the effect being mediated by CB1 receptors (possibly via increased anandamide levels) and modulated by TRPV1 channels.

The major endocannabinoids, anandamide (N-arachidonoylethanolamide, <em>2</em>0:4n-6 N-acylethanolamine) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are structurally and functionally similar, but they are produced by different metabolic pathways and their levels must therefore be regulated by different mechanisms. Both endocannabinoids are accompanied by cannabinoid receptor-inactive, saturated and mono- or di-unsaturated congeners which can influence their metabolism and function. Here we review published data on the presence and production of anandamide and <em>2</em>-AG and their congeners in mammalian cells and discuss this information in terms of their proposed signaling functions.

Endocannabinoids are an emerging class of lipid mediators, which include amides and esters of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine, AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are the main endogenous agonists of cannabinoid receptors. Endotoxic shock is a potentially lethal failure of multiple organs that can be initiated by the inflammatory agent lipopolysaccharide (LPS), present in the outer membrane of gram-negative bacteria. LPS has been recently shown to stimulate the production of AEA in rat macrophages, and of <em>2</em>-AG in rat platelets. The mechanism responsible for this effect has not been elucidated. On the other hand, mast cells are multifunctional bone marrow-derived cells found in mucosal and connective tissues and in the nervous system, where they play an essential role in inflammation. As yet, little is known about endogenous modulators and mechanisms of mast cell activation. Here, we review recent literature on the role of endocannabinoids in endotoxic shock and inflammation, and report our recent research on the effects of LPS on the production of AEA and <em>2</em>-AG in human lymphocytes, and on AEA degradation by a specific AEA membrane transporter (AMT) and an AEA-degrading enzyme (fatty acid amide hydrolase, FAAH). We also report the ability of the HMC-1 human mast cells to degrade AEA through a nitric oxide-sensitive AMT and a FAAH. The role of endocannabinoids in HMC-1 degranulation is discussed as well. Taken together, it can be suggested that human lymphocytes and mast cells take part in regulating the peripheral endocannabinoid system, which can affect some activities of these cells.

The endocannabinoid ligand <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is inactivated primarily by monoacylglycerol lipase (MAGL). We have shown recently that chronic treatments with MAGL inhibitor JZL184 produce antidepressant- and anxiolytic-like effects in a chronic unpredictable stress (CUS) model of depression in mice. However, the underlying mechanisms remain poorly understood. Adult hippocampal neurogenesis has been implicated in animal models of anxiety and depression and behavioral effects of antidepressants. We tested whether CUS and chronic JZL184 treatments affected adult neurogenesis and synaptic plasticity in the dentate gyrus (DG) of mouse hippocampus. We report that CUS induced depressive-like behaviors and decreased the number of bromodeoxyuridine-labeled neural progenitor cells and doublecortin-positive immature neurons in the DG, while chronic JZL184 treatments prevented these behavioral and cellular deficits. We also investigated the effects of CUS and chronic JZL184 on a form long-term potentiation (LTP) in the DG known to be neurogenesis-dependent. CUS impaired LTP induction, whereas chronic JZL184 treatments restored LTP in CUS-exposed mice. These results suggest that enhanced adult neurogenesis and long-term synaptic plasticity in the DG of the hippocampus might contribute to antidepressant- and anxiolytic-like behavioral effects of JZL184.

Endocannabinoids are paracrine/autocrine lipid mediators with several biological functions. One of these, i.e. the capability to stimulate food intake via cannabinoid CB(1) receptors, has been particularly studied, thus leading to the development of the first CB(1) receptor blocker, rimonabant, as a therapeutic tool against obesity and related metabolic disorders. Hypothalamic endocannabinoids stimulate appetite by regulating the expression and release of anorexic and orexigenic neuropeptides via CB(1) receptors. In turn, the tone of the latter receptors is regulated by hormones, including leptin, glucocorticoids and possibly ghrelin and neuropeptide Y, by modulating the biosynthesis of the endocannabinoids in various areas of the hypothalamus. CB(1) receptor stimulation is also known to increase blood glucose during an oral glucose tolerance test in rats. Here we investigated in the rat if insulin, which is known to exert fundamental actions at the level of the mediobasal hypothalamus (MBH), and the melanocortin system, namely alpha-melanocyte stimulating hormone (alpha-MSH) and melanocortin receptor-4 (MCR-4), also regulate hypothalamic endocannabinoid levels, measured by isotope-dilution liquid chromatography coupled to mass spectrometry. No effect on anandamide and <em>2</em>-<em>arachidonoylglycerol</em> levels was observed after <em>2</em>h infusion of insulin in the MBH, i.e. under conditions in which the hormone reduces blood glucose, nor with intra-cerebroventricular injection of alpha-MSH, under conditions in which the neuropeptide reduces food intake. Conversely, blockade of MCR-4 receptors with HS014 produced a late (6h after systemic administration) stimulatory effect on endocannabinoid levels as opposed to a rapid and prolonged stimulation of food-intake (observable <em>2</em> and 6h after administration). These data suggest that inhibition of endocannabinoid levels does not mediate the effect of insulin on hepatic glucose production nor the food intake-inhibitory effect of alpha-MSH, although stimulation of endocannabinoid levels might underlie part of the late stimulatory effects of MCR-4 blockade on food intake.

Endocannabinoids including anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are important lipid mediators for immunosuppressive effects and for appropriate homeostasis via their G-protein-coupled cannabinoid (CB) receptors in mammalian organs and tissues, and may be involved in wound healing in some organs. The physiological roles of endocannabinoids in periodontal healing remain unknown. We observed upregulation of the expression of CB1/CB<em>2</em> receptors localized on fibroblasts and macrophage-like cells in granulation tissue during wound healing in a wound-healing model in rats, as well as an increase in AEA levels in gingival crevicular fluid after periodontal surgery in human patients with periodontitis. In-vitro, the proliferation of human gingival fibroblasts (HGFs) by AEA was significantly attenuated by AM<em>2</em>51 and AM630, which are selective antagonists of CB1 and CB<em>2</em>, respectively. CP55940 (CB1/CB<em>2</em> agonist) induced phosphorylation of the extracellular-regulated kinases (ERK) 1/<em>2</em>, p38 mitogen-activated protein kinase (p38MAPK), and Akt in HGFs. Wound closure by CP55940 in an in-vitro scratch assay was significantly suppressed by inhibitors of MAP kinase kinase (MEK), p38MAPK, and phosphoinositol 3-kinase (PI3-K). These findings suggest that endocannabinoid system may have an important role in periodontal healing.

BACKGROUND

In this study, we examined alterations in the hypothalamic reward system related to high-fat diet (HFD) preferences. We previously reported that hypothalamic <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and glial fibrillary acid protein (GFAP) were increased after conditioning to the rewarding properties of a HFD. Here, we hypothesized that increased <em>2</em>-AG influences the hypothalamic reward system.

METHODS

The conditioned place preference test (CPP test) was used to evaluate HFD preferences. Hypothalamic <em>2</em>-AG was quantified by gas chromatography-mass spectrometry. The expression of GFAP was examined by immunostaining and western blotting.

RESULTS

Consumption of a HFD over either 3 or 7 days increased HFD preferences and transiently increased hypothalamic <em>2</em>-AG levels. HFD consumption over 14 days similarly increased HFD preferences but elicited a long-lasting increase in hypothalamic <em>2</em>-AG and GFAP levels. The cannabinoid 1 receptor antagonist O-<em>2</em>050 reduced preferences for HFDs after 3, 7, or 14 days of HFD consumption and reduced expression of GFAP after 14 days of HFD consumption. The astrocyte metabolic inhibitor Fluorocitrate blocked HFD preferences after 14 days of HFD consumption.

CONCLUSIONS

High levels of <em>2</em>-AG appear to induce HFD preferences, and activate hypothalamic astrocytes via the cannabinoid system. We propose that there may be two distinct stages in the development of HFD preferences. The induction stage involves a transient increase in <em>2</em>-AG, whereas the maintenance stage involves a long lasting increase in <em>2</em>-AG levels and activation of astrocytes. Accordingly, hypothalamic <em>2</em>-AG may influence the development of HFD preferences.

OBJECTIVE

Endocannabinoids (eCBs) play a role in the modulation of neuroinflammation, and experimental findings suggest that they may be directly involved in the pathogenesis of multiple sclerosis (MS). The objective of our study was to measure eCB levels in the cerebrospinal fluid (CSF) of patients with MS.

METHODS

Arachidonoylethanolamine (anandamide, AEA), palmotylethanolamide (PEA), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and oleoylethanolamide (OEA) levels were measured in the CSF of 50 patients with MS and <em>2</em>0 control subjects by isotope dilution gas-chromatography/mass-spectrometry. Patients included 35 patients with MS in the relapsing-remitting (RR) form of the disease, <em>2</em>0 in a stable clinical phase and 15 during a relapse, and 15 patients with MS in the secondary progressive (SP) form.

RESULTS

Significantly reduced levels of all the tested eCBs were found in the CSF of patients with MS compared to control subjects, with lower values detected in the SP MS group. Higher levels of AEA and PEA, although below those of controls, were found in the CSF of RR MS patients during a relapse. Higher levels of AEA, <em>2</em>-AG and OEA were found in patients with MRI gadolinium-enhancing (Gd+) lesions.

CONCLUSIONS

The present findings suggest the presence of an impaired eCB system in MS. Increased CSF levels of AEA during relapses or in RR patients with Gd+ lesions suggest its potential role in limiting the ongoing inflammatory process with potential neuroprotective implications. These findings provide further support for the development of drugs targeting eCBs as a potential pharmacological strategy to reduce the symptoms and slow disease progression in MS.

Previous studies have demonstrated that the endocannabinoid system significantly influences the progression of brain ageing, and the hippocampus is one of the brain regions most vulnerable to ageing and neurodegeneration. We have further examined age-related changes in the hippocampal endocannabinoid system by measuring the levels of anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in young and old mice from two different mouse strains. We found a decrease in <em>2</em>-AG but not AEA levels in aged mice. In order to identify the cause for <em>2</em>-AG level changes, we investigated the levels of several enzymes that contribute to synthesis and degradation of <em>2</em>-AG in the hippocampus. We found a selective decrease in DAGLα mRNA and protein levels as well as an elevated MAGL activity during ageing. We hypothesize that the observed decrease of <em>2</em>-AG levels is probably caused by changes in DAGLα expression and MAGL activity. This finding can contribute to the existing knowledge about the processes underlying selective vulnerability of the hippocampus to ageing and age-related neurodegeneration.