Drugs of abuse cause changes in the mesocorticolimbic dopamine (DA) system, such as a long-term potentiation (LTP)-like phenomenon at glutamatergic synapses onto ventral tegmental area (VTA) DA neurons. Abolishing this LTP interferes with drug-seeking behavior. Endocannabinoids (ECs) can be released by DA neurons in response to repetitive activation, which can inhibit glutamate release. Therefore, we hypothesized that ECs may act as negative regulators of LTP. Here we tested the induction of LTP in DA neurons of the VTA in mice expressing enhanced green fluorescent protein under the control of the tyrosine hydroxylase promoter. Immunohistochemistry showed colocalization of CB1 receptors with vesicular glutamate transporter (VGLUT)1 in terminals near DA neuron dendrites, with less extensive colocalization with VGLUT<em>2</em>. In addition, a CB1 receptor agonist, as well as trains of stimulation leading to EC production, decreased glutamate release onto DA neurons. We found that blocking CB1 receptors or synthesis of the EC <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) was without effect on basal excitatory postsynaptic potential amplitude; however, it facilitated the induction of LTP. As previously reported, antagonizing γ-aminobutyric acid (GABA)(A) transmission also facilitated LTP induction. Combining GABA(A) and CB1 receptor antagonists did not lead to larger LTP. LTP induced in the presence of CB1 receptor blockade was prevented by an N-methyl-d-aspartate receptor antagonist. Our observations argue in favor of the hypothesis that <em>2</em>-AG acts as a negative regulator of LTP in the VTA. Understanding the factors that regulate long-term synaptic plasticity in this circuit is critical to aid our comprehension of drug addiction in humans.

The endocannabinoid system, including endogenous ligands ('endocannabinoids' ECs), their receptors, synthesizing and degrading enzymes, as well as transporter molecules, has been detected from the earliest stages of embryonic development and throughout pre- and postnatal development. ECs are bioactive lipids, which comprise amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are the best studied ECs, and act as agonists of cannabinoid receptors. Thus, AEA and <em>2</em>-AG mimic several pharmacological effects of the exogenous cannabinoid delta9-tetrahydrocannabinol (Delta(9)-THC), the psychoactive principle of cannabis sativa preparations like hashish and marijuana. Recently, however, several lines of evidence have suggested that the EC system may play an important role in early neuronal development as well as a widespread role in neurodegeneration disorders. Many of the effects of cannabinoids and ECs are mediated by two G protein-coupled receptors (GPCRs), CB1 and CB<em>2</em>, although additional receptors may be implicated. Both CB1 and CB<em>2</em> couple primarily to inhibitory G proteins and are subject to the same pharmacological influences as other GPCRs. This new system is briefly presented in this review, in order to put in a better perspective the role of the EC pathway from neurodevelopment to neurodegenerative disorders, like Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis. In addition, the potential exploitation of antagonists of CB1 receptors, or of inhibitors of EC metabolism, as next-generation therapeutics is discussed.

The use of marijuana, which today is the most used recreational drug, has been demonstrated to affect adversely reproduction. Marijuana smokers, both men and women, show impaired fertility, owing to defective signalling pathways, aberrant hormonal regulation, or wrong timing during embryo implantation. Anandamide (N-arachidonoylethanolamine, AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) mimic Delta(9)-tetrahydrocannabinol (THC), the psychoactive principle of Cannabis sativa, by binding to both the brain-type (CB(1)) and the spleen-type (CB(<em>2</em>)) cannabinoid receptors. These 'endocannabinoids' exert several actions either in the central nervous system or in peripheral tissues, and are metabolised by specific enzymes that synthesise or hydrolyse them. In this review, we shall describe the elements that constitute the endocannabinoid system (ECS), in order to put in a better perspective the role of this system in the control of human fertility, both in females and males. In addition, we shall discuss the interplay between ECS, sex hormones and cytokines, which generates an endocannabinoid-hormone-cytokine array critically involved in the control of human reproduction.

Pain perception can become altered in individuals with eating disorders and obesity for reasons that have not been fully elucidated. We show that leptin deficiency in ob/ob mice, or leptin insensitivity in the arcuate nucleus of the hypothalamus in mice with high-fat diet (HFD)-induced obesity, are accompanied by elevated orexin-A (OX-A) levels and orexin receptor-1 (OX1-R)-dependent elevation of the levels of the endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), in the ventrolateral periaqueductal gray (vlPAG). In ob/ob mice, these alterations result in the following: (i) increased excitability of OX1-R-expressing vlPAG output neurons and subsequent increased OFF and decreased ON cell activity in the rostral ventromedial medulla, as assessed by patch clamp and in vivo electrophysiology; and (ii) analgesia, in both healthy and neuropathic mice. In HFD mice, instead, analgesia is only unmasked following leptin receptor antagonism. We propose that OX-A/endocannabinoid cross talk in the descending antinociceptive pathway might partly underlie increased pain thresholds in conditions associated with impaired leptin signaling.

In the vascular system angiotensin II (Ang II) causes vasoconstriction via the activation of type 1 angiotensin receptors. Earlier reports have shown that in cellular expression systems diacylglycerol produced during type 1 angiotensin receptor signaling can be converted to <em>2</em>-<em>arachidonoylglycerol</em>, an important endocannabinoid. Because activation of CB(1) cannabinoid receptors (CB(1)R) induces vasodilation and reduces blood pressure, we have tested the hypothesis that Ang II-induced <em>2</em>-<em>arachidonoylglycerol</em> release can modulate its vasoconstrictor action in vascular tissue. Rat and mouse skeletal muscle arterioles and mouse saphenous arteries were isolated, pressurized, and subjected to microangiometry. Vascular expression of CB(1)R was demonstrated using Western blot and RT-PCR. In accordance with the functional relevance of these receptors WIN55<em>2</em>1<em>2</em>, a CB(1)R agonist, caused vasodilation, which was absent in CB(1)R knock-out mice. Inhibition of CB(1)Rs using O<em>2</em>050, a neutral antagonist, enhanced the vasoconstrictor effect of Ang II in wild type but not in CB(1)R knock-out mice. Inverse agonists of CB(1)R (SR141716 and AM<em>2</em>51) and inhibition of diacylglycerol lipase using tetrahydrolipstatin also augmented the Ang II-induced vasoconstriction, suggesting that endocannabinoid release modulates this process via CB(1)R activation. This effect was independent of nitric-oxide synthase activity and endothelial function. These data demonstrate that Ang II stimulates vascular endocannabinoid formation, which attenuates its vasoconstrictor effect, suggesting that endocannabinoid release from the vascular wall and CB(1)R activation reduces the vasoconstrictor and hypertensive effects of Ang II.

Stress and glucocorticoids stimulate the rapid mobilization of endocannabinoids in the basolateral amygdala (BLA). Cannabinoid receptors in the BLA contribute to anxiogenesis and fear-memory formation. We tested for rapid glucocorticoid-induced endocannabinoid regulation of synaptic inhibition in the rat BLA. Glucocorticoid application to amygdala slices elicited a rapid, nonreversible suppression of spontaneous, but not evoked, GABAergic synaptic currents in BLA principal neurons; the effect was also seen with a membrane-impermeant glucocorticoid, but not with intracellular glucocorticoid application, implicating a membrane-associated glucocorticoid receptor. The glucocorticoid suppression of GABA currents was not blocked by antagonists of nuclear corticosteroid receptors, or by inhibitors of gene transcription or protein synthesis, but was blocked by inhibiting postsynaptic G-protein activity, suggesting a postsynaptic nongenomic steroid signaling mechanism that stimulates the release of a retrograde messenger. The rapid glucocorticoid-induced suppression of inhibition was prevented by blocking CB1 receptors and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) synthesis, and it was mimicked and occluded by CB1 receptor agonists, indicating it was mediated by the retrograde release of the endocannabinoid <em>2</em>-AG. The rapid glucocorticoid effect in BLA neurons in vitro was occluded by prior in vivo acute stress-induced, or prior in vitro glucocorticoid-induced, release of endocannabinoid. Acute stress also caused an increase in anxiety-like behavior that was attenuated by blocking CB1 receptor activation and inhibiting <em>2</em>-AG synthesis in the BLA. Together, these findings suggest that acute stress causes a long-lasting suppression of synaptic inhibition in BLA neurons via a membrane glucocorticoid receptor-induced release of <em>2</em>-AG at GABA synapses, which contributes to stress-induced anxiogenesis.

We provide a cellular mechanism in the basolateral amygdala (BLA) for the rapid stress regulation of anxiogenesis in rats. We demonstrate a nongenomic glucocorticoid induction of long-lasting suppression of synaptic inhibition that is mediated by retrograde endocannabinoid release at GABA synapses. The rapid glucocorticoid-induced endocannabinoid suppression of synaptic inhibition is initiated by a membrane-associated glucocorticoid receptor in BLA principal neurons. We show that acute stress increases anxiety-like behavior via an endocannabinoid-dependent mechanism centered in the BLA. The stress-induced endocannabinoid modulation of synaptic transmission in the BLA contributes, therefore, to the stress regulation of anxiety, and may play a role in anxiety disorders of the amygdala.

BACKGROUND

Neuropathological, animal, and cell culture studies point to a role for the body's own endogenous cannabinoids (eCBs) system in Alzheimer's disease (AD) pathology and treatment. To date, no published studies have investigated the potential utility of circulating eCBs as diagnostic biomarkers for AD or the impact of central eCBs on cognition.

RESULTS

In comparison with healthy controls, there were no significant differences in measured eCB concentrations in plasma samples from patients with AD. Detectable eCBs in cerebrospinal fluid (CSF) had no relationship to cognitive performance in healthy controls at risk for AD. In pooled plasma samples, an inverse correlation was observed between plasma levels of the eCB <em>2</em>-AG (<em>2</em>-<em>arachidonoylglycerol</em>) and TNF-alpha (r = -0.41, p < 0.0<em>2</em>).

CONCLUSIONS

These results suggest that circulating endocannabinoids do not have utility as diagnostic biomarkers for AD and do not have a robust correlation with cognitive performance. Circulating levels of <em>2</em>-AG may downregulate TNF-alpha production.

BACKGROUND

Depression is known to be a risk factor for cardiovascular diseases but the underlying mechanisms remain unclear. Since recent preclinical evidence suggests that endogenous agonists of cannabinoid receptors (endocannabinoids) are involved in both cardiovascular function and depression, we asked whether endocannabinoids correlated with either in humans.

RESULTS

Resting blood pressure and serum content of endocannabinoids in ambulatory, medication-free, female volunteers with depression (n = <em>2</em>8) and their age- and ethnicity-matched controls (n = <em>2</em>7) were measured. In females with depression, both diastolic and mean arterial blood pressures were positively correlated with serum contents of the endocannabinoids, N-arachidonylethanolamine (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em>. There was no correlation between blood pressure and endocannabinoids in control subjects. Furthermore, depressed women had significantly higher systolic blood pressure than control subjects. A larger body mass index was also found in depressed women, however, it was not significantly correlated with serum endocannabinoid contents.

CONCLUSIONS

This preliminary study raises the possibility that endocannabinoids play a role in blood pressure regulation in depressives with higher blood pressure, and suggests an interrelationship among endocannabinoids, depression and cardiovascular risk factors in women.

Cannabinoid receptors are functionally operant at both glutamate and GABA synapses on hypothalamic magnocellular neuroendocrine cells; however, retrograde endocannabinoid actions are evoked at only glutamate synapses. We tested whether the functional targeting of evoked retrograde endocannabinoid actions to glutamate, and not GABA, synapses on magnocellular neurons is the result of the spatial restriction of extracellular endocannabinoids by astrocytes. Whole-cell GABA synaptic currents were recorded in magnocellular neurons in rat hypothalamic slices following manipulations to reduce glial buffering of extracellular signals. Depolarization- and glucocorticoid-evoked retrograde endocannabinoid suppression of synaptic GABA release was not detected under normal conditions, but occurred in both oxytocin and vasopressin neurons under conditions of attenuated glial coverage and depressed glial metabolic function, suggesting an emergent endocannabinoid modulation of GABA synapses with the loss of astrocyte function. Tonic endocannabinoid suppression of GABA release was insensitive to glial manipulation. Blocking cannabinoid transport mimicked, and increasing the extracellular viscosity reversed, the effect of suppressed glial buffering on the endocannabinoid modulation of GABA release. Evoked, but not tonic, endocannabinoid modulation of GABA synapses was mediated by <em>2</em>-<em>arachidonoylglycerol</em>. Therefore, depolarization- and glucocorticoid-evoked <em>2</em>-<em>arachidonoylglycerol</em> release from magnocellular neurons is spatially restricted to glutamate synapses by astrocytes, but spills over onto GABA synapses under conditions of reduced astrocyte buffering; tonic endocannabinoid modulation of GABA release, in contrast, is likely mediated by anandamide and is insensitive to astrocytic buffering. Astrocytes, therefore, provide dynamic control of stimulus-evoked <em>2</em>-<em>arachidonoylglycerol</em>, but not tonic anandamide, regulation of GABA synaptic inputs to magnocellular neuroendocrine cells under different physiological conditions.

Only a few studies have addressed the transport of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), a naturally occurring agonist for cannabinoid receptors. Based upon saturation kinetics, these early reports have proposed that <em>2</em>-AG enters the cell by a specific <em>2</em>-AG transporter, via the putative anandamide transporter, or by simple diffusion. In this review, the uptake of <em>2</em>-AG is discussed in light of the recent advances that have been made for anandamide transport, where the mechanism appears to be rate-limited diffusion through the membrane. Endocannabinoids may be a distinct class of agonists since they are hydrophobic and neutral, exhibiting similar biophysical properties to some anesthetics that freely diffuse through the membrane.

The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) enhances cell migration through the CB<em>2</em> cannabinoid receptor. In this study, using an immunoprecipitation and mass spectrometry-based proteomic approach, we first identified the 90-kDa heat shock protein (Hsp90), a chaperone protein with novel signaling functions, as a CB<em>2</em>-interacting protein. The CB<em>2</em>/Hsp90 interaction was confirmed in human embryonic kidney <em>2</em>93 cells expressing transfected CB<em>2</em> and in differentiated HL-60 cells expressing endogenous CB<em>2</em>, by coimmunoprecipitation and Western blot experiments, as well as by treatment with geldanamycin (GA), a specific Hsp90 inhibitor. Disruption of the CB<em>2</em>/Hsp90 interaction by treatment with GA or reducing Hsp90 levels with specific short interfering RNAs markedly inhibited <em>2</em>-AG-induced cell migration, demonstrating that Hsp90 is crucial for <em>2</em>-AG-induced cell migration. <em>2</em>-AG treatment resulted in a CB<em>2</em>-mediated stimulation of Rac1 activity, and treatment with GA blocked <em>2</em>AG-induced activation of Rac1. It is noteworthy that expression of the dominant-negative form of Rac1 reduced <em>2</em>-AG-induced cell migration. These data demonstrate that <em>2</em>-AG-induced activation of Rac1 is essential for <em>2</em>-AG-induced cell migration, and the CB<em>2</em>/Hsp90 interaction is needed for <em>2</em>-AG-induced activation of Rac1. Furthermore, <em>2</em>-AG-induced Rac1 activation was sensitive to pertussis toxin treatment, hence involving G(i) proteins. In addition, treatment with GA significantly inhibited the CB<em>2</em>/Galpha(i<em>2</em>) interaction. As a whole, our data indicate that Hsp90 may serve as scaffold to keep the CB<em>2</em> receptor and its signaling components, including Galpha(i<em>2</em>), in proximity, thus facilitating CB<em>2</em>-mediated signaling to cell migration through the G(i)-Rac1 pathway. By demonstrating that Hsp90 is essential for CB<em>2</em>-mediated signaling to cell migration, this study reveals a novel role of Hsp90 in the signaling events mediated by a G protein-coupled receptor.

Mounting evidence suggests that the endocannabinoid system regulates energy metabolism through direct effects on peripheral tissues as well as central effects that regulate appetite. Here we examined the effect of cannabinoid receptor 1 (CB1) signaling on insulin action in fat cells. We examined effects of the natural CB1 agonist, <em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG), and the synthetic CB1 antagonist, SR141716, on insulin action in cultured adipocytes. We used translocation of glucose transporter GLUT4 to plasma membrane (PM) as a measure of insulin action. <em>2</em>-AG activation of the CB1 receptor promoted insulin sensitivity whereas antagonism by SR141716 reduced insulin sensitivity. Neither drug affected GLUT4 translocation in the absence of insulin or with high doses of insulin. Consistent with these results we found that insulin-stimulated phosphorylation of the protein kinase Akt was increased by <em>2</em>-AG, attenuated by SR141716, and unaffected in the absence of insulin or by addition of high-dose insulin. These data provide a functional and molecular link between the CB1 receptor and insulin sensitivity, because insulin-stimulated phosphorylation of Akt is required for GLUT4 translocation to the PM. The sensitizing effects of <em>2</em>-AG were abrogated by SR141716 and Pertussis toxin, indicating that the effects are mediated by CB1 receptor. Importantly, neither <em>2</em>-AG nor SR141716 alone or in combination with maximal dose of insulin had effects on GLUT4 translocation and Akt phosphorylation. These data are consistent with a model in which the endocannabinoid system sets the sensitivity of the insulin response in adipocytes rather than directly regulating the redistribution of GLUT4 or Akt phosphorylation.

BACKGROUND

The incidence of melanoma is considerably increasing worldwide. Frequent failing of classical treatments led to development of novel therapeutic strategies aiming at managing advanced forms of this skin cancer. Additionally, the implication of the endocannabinoid system in malignancy is actively investigated.

METHODS

We investigated the cytotoxicity of endocannabinoids and their hydrolysis inhibitors on the murine B16 melanoma cell line using a MTT test. Enzyme and receptor expression was measured by RT-PCR and enzymatic degradation of endocannabinoids using radiolabeled substrates. Cell death was assessed by Annexin-V/Propidium iodine staining. Tumors were induced in C57BL/6 mice by s.c. flank injection of B16 melanoma cells. Mice were injected i.p. for six days with vehicle or treatment, and tumor size was measured each day and weighted at the end of the treatment. Haematoxylin-Eosin staining and TUNEL assay were performed to quantify necrosis and apoptosis in the tumor and endocannabinoid levels were quantified by HPLC-MS. Tube formation assay and CD31 immunostaining were used to evaluate the antiangiogenic effects of the treatments.

RESULTS

The N-arachidonoylethanolamine (anandamide, AEA), <em>2</em>-<em>arachidonoylglycerol</em> and N- palmitoylethanolamine (PEA) reduced viability of B16 cells. The association of PEA with the fatty acid amide hydrolase (FAAH) inhibitor URB597 considerably reduced cell viability consequently to an inhibition of PEA hydrolysis and an increase of PEA levels. The increase of cell death observed with this combination of molecules was confirmed in vivo where only co-treatment with both PEA and URB597 led to decreased melanoma progression. The antiproliferative action of the treatment was associated with an elevation of PEA levels and larger necrotic regions in the tumor.

CONCLUSIONS

This study suggests the interest of targeting the endocannabinoid system in the management of skin cancer and underlines the advantage of associating endocannabinoids with enzymatic hydrolysis inhibitors. This may contribute to the improvement of long-term palliation or cure of melanoma.

Systemic activation of cannabinoid receptors often induces biphasic effects on emotional memory and anxiety depending on the levels of emotional arousal associated to the experimental context. The basolateral nucleus of the amygdala (BLA) represents a crucial structure for the ability of endocannabinoid (eCB) signaling to modulate emotional behaviour, and receives dense projections from brainstem arousal system nuclei. We examined whether changes in emotional arousal state would influence the ability of acute eCB manipulations within the BLA to modulate anxiety. Rats were tested in an elevated plus maze (EPM) under low or high arousal conditions. The low emotional arousal group was extensively handled and habituated to the experimental room and tested under red light condition, the high emotional arousal group was not handled or habituated and tested under high light condition. We examined amygdalar eCB anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) levels immediately after the EPM and the effects of intra-BLA administration of the AEA hydrolysis inhibitor URB597 or the <em>2</em>-AG hydrolysis inhibitor KML<em>2</em>9 on anxiety behaviour. The modulation of anxiety-like behaviour by eCBs in the BLA was strictly dependent on the environmental-associated emotional arousal. Pharmacologically-induced elevations of AEA or <em>2</em>-AG in the BLA decreased anxiety under conditions of low emotional arousal. Conversely, when the level of emotional arousal increased, local eCB manipulation was ineffective in the modulation of the emotional arousal-induced anxiety response. These findings suggest that, depending on the emotional arousal state, eCB system is differentially activated to regulate the anxiety response in the amygdala and help to understand the state-dependency of many interventions on anxiety.

The endogenous cannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) plays an important role in a variety of physiologic processes, but its rapid breakdown by monoacylglycerol lipase (MAGL) results in short-lived actions. Initial MAGL inhibitors were limited by poor selectivity and low potency. In this study, we tested JZL184 [4-nitrophenyl 4-[bis(<em>2</em>H-1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate] and MJN110 [<em>2</em>,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate], MAGL inhibitors that possess increased selectivity and potency, in mouse behavioral assays of neuropathic pain [chronic constriction injury (CCI) of the sciatic nerve], interoceptive cannabimimetic effects (drug-discrimination paradigm), and locomotor activity in an open field test. MJN110 (1.<em>2</em>5 and <em>2</em>.5 mg/kg) and JZL184 (16 and 40 mg/kg) significantly elevated <em>2</em>-AG and decreased arachidonic acid but did not affect anandamide in whole brains. Both MAGL inhibitors significantly reduced CCI-induced mechanical allodynia with the following potencies [ED50 (95% confidence limit [CL]) values in mg/kg: MJN110 (0.43 [0.30-0.63])>> JZL184 (17.8 [11.6-<em>2</em>7.4])] and also substituted for the potent cannabinoid receptor agonist CP55,940 [<em>2</em>-[(1R,<em>2</em>R,5R)-5-hydroxy-<em>2</em>-(3-hydroxypropyl)cyclohexyl]-5-(<em>2</em>-methyloctan-<em>2</em>-yl)phenol] in the drug-discrimination paradigm [ED50 (95% CL) values in mg/kg: MJN110 (0.84 [0.69-1.0<em>2</em>])>> JZL184 (<em>2</em>4.9 [14.6-4<em>2</em>.5])]; however, these compounds elicited differential effects on locomotor behavior. Similar to cannabinoid 1 (CB1) receptor agonists, JZL184 produced hypomotility, whereas MJN110 increased locomotor behavior and did not produce catalepsy or hypothermia. Although both drugs substituted for CP55,940 in the drug discrimination assay, MJN110 was more potent in reversing allodynia in the CCI model than in producing CP55,940-like effects. Overall, these results suggest that MAGL inhibition may alleviate neuropathic pain, while displaying limited cannabimimetic effects compared with direct CB1 receptor agonists.

Endocannabinoids are implicated in the control of glucose utilization and energy homeostasis by orchestrating pancreatic hormone release. Moreover, in some cell niches, endocannabinoids regulate cell proliferation, fate determination, and migration. Nevertheless, endocannabinoid contributions to the development of the endocrine pancreas remain unknown. Here, we show that α cells produce the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in mouse fetuses and human pancreatic islets, which primes the recruitment of β cells by CB1 cannabinoid receptor (CB1R) engagement. Using subtractive pharmacology, we extend these findings to anandamide, a promiscuous endocannabinoid/endovanilloid ligand, which impacts both the determination of islet size by cell proliferation and α/β cell sorting by differential activation of transient receptor potential cation channel subfamily V member 1 (TRPV1) and CB1Rs. Accordingly, genetic disruption of TRPV1 channels increases islet size whereas CB1R knockout augments cellular heterogeneity and favors insulin over glucagon release. Dietary enrichment in ω-3 fatty acids during pregnancy and lactation in mice, which permanently reduces endocannabinoid levels in the offspring, phenocopies CB1R(-/-) islet microstructure and improves coordinated hormone secretion. Overall, our data mechanistically link endocannabinoids to cell proliferation and sorting during pancreatic islet formation, as well as to life-long programming of hormonal determinants of glucose homeostasis.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is an endogenous ligand for cannabinoid receptors. There are two types of cannabinoid receptors, CB1 and CB<em>2</em>. We investigated the chemotactic activity of <em>2</em>-AG using mouse lymphocytes because cells in the immune system are known to express CB<em>2</em> . Spleen cell migration toward <em>2</em>-AG was observed, which was completely inhibited by SR1445<em>2</em>8, a CB<em>2</em>-specific antagonist. <em>2</em>-AG has been reported to induce a preferential B cell chemotaxis. We examined whether there is any difference in responsiveness during the activation of B cells. When spleen cells from immunized mice were tested, naive B cells but not germinal center B cells (GL7-positive) were increased in the fraction attracted by <em>2</em>-AG. Furthermore, when Peyer's patch lymphocytes were tested after oral administration of cholera toxin, the number of IgA* B cells was increased in the fraction attracted by <em>2</em>-AG. These results suggested that <em>2</em>-AG preferentially attracts unstimulated naive B cells rather than activated and/or class-switched B cells. This property may influence the structure of B cell compartments in secondary lymphoid tissues.

The visceral insular cortex (VIC) has previously been shown to play a critical role during acute nausea-induced conditioned gaping in rats. Specifically, localized administration of the conventional anti-emetic, ondansetron or the synthetic cannabinoid, HU<em>2</em>10, interferes with the establishment of conditioned gaping, likely by reducing the effects of an illness-inducing treatment. However the precise role of the VIC in endocannabinoid-suppression of nausea remains unknown; thus we investigated the potential of localized intra-VIC endocannabinoid administration to interfere with acute nausea-induced conditioned gaping behavior in male Sprague-Dawley rats. Animals received an intraoral infusion of saccharin (0.1%) followed by intra-VIC exogenous N-arachidonoylethanolamide (AEA; 0.4, 4 μg) or <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG; 0.5, 1 μg), and were subsequently injected with nausea-inducing LiCl (0.15M) 15 min later. Bilateral intra-VIC infusions of <em>2</em>-AG (1 μg, but not 0.5 μg) dose-dependently suppressed conditioned gaping, whereas exogenous AEA was without effect. Interestingly, <em>2</em>-AG reduced conditioned gaping despite additional pretreatment with the selective cannabinoid receptor type 1 (CB1) antagonist, AM-<em>2</em>51; however, concomitant pretreatment with the cyclooxygenase inhibitor, indomethacin (0.5 μg), blocked the suppressive effects of intra-VIC <em>2</em>-AG. These findings suggest that the modulatory role of the endocannabinoid system during nausea is driven largely by the endocannabinoid, <em>2</em>-AG, and that its anti-nausea effects may be partly independent of CB1-receptor signaling through metabolic products of the endocannabinoid system.

BACKGROUND

Levels of cannabinoid 1 receptor (CB1R) messenger RNA (mRNA) and protein, which are expressed most heavily in the cholecystokinin class of γ-aminobutyric acid (GABA) neurons, are lower in the dorsolateral prefrontal cortex in schizophrenia, and the magnitude of these differences is strongly correlated with that for glutamic acid decarboxylase 67 (GAD(67)) mRNA, a synthesizing enzyme for GABA. However, whether this correlation reflects a cause-effect relationship is unknown.

METHODS

Using quantitative in situ hybridization, we measured CB1R, GAD(67), and diacylglycerol lipase alpha (the synthesizing enzyme for the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>) mRNA levels in the medial prefrontal cortex of genetically engineered GAD(67) heterozygous (GAD(67)(+/-)), CB1R heterozygous (CB1R(+/-)), CB1R knockout (CB1R(-/-)), and matched wild-type mice.

RESULTS

In GAD(67)(+/-) mice, GAD(67) and CB1R mRNA levels were significantly reduced by 37% and 16%, respectively, relative to wild-type mice and were significantly correlated across animals (r = .61; p = .01). In contrast, GAD(67) mRNA levels were unaltered in CB1R(+/-) andCB1R(-/-) mice. Expression of diacylglycerol lipase alpha mRNA, which is not altered in schizophrenia, was also not altered in any of the genetically engineered mice.

CONCLUSIONS

The findings that reduced GAD(67) mRNA expression can induce lower CB1R mRNA expression support the hypothesis that lower cortical levels of CB1Rs in schizophrenia may partially compensate for deficient GAD(67)-mediated GABA synthesis by reducing endogenous cannabinoid suppression of GABA release.

Several studies have implicated endocannabinoids in various forms of shock. However, the role of endocannabinoids in hepatic ischemia-reperfusion injury remains unclear. The purpose of this study was to evaluate the changes of two endocannabinoidsin hepatic ischemia-reperfusion injury: anandamide (ANA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Male Sprague-Dawley rats were divided into <em>2</em> groups: the short (15 min) ischemic group and the long (60 min)ischemic group in the segmental (70%) hepatic tissue. Blood levels of aspartate transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH), ANA, and <em>2</em>-AG were examined. Serum lev-els of AST, ALT, and LDH were significantly higher in the long-ischemia group than in the short-ischemia group. Plasma levels of <em>2</em>-AG showed similar augmentation prior to and after reperfusion in both the short- and long-ischemia groups, although plasma <em>2</em>-AG lev-els tended to be higher in the long-ischemia group than in the short-ischemia group. Plasma levels of ANA were augmented in the early phase of reperfusion in the short-ischemia group and did not differ significantly from the normal level with time after reperfusion in the long-ischemia group. These results suggest that the endocannabinoid <em>2</em>-AG increases in hepatic ischemia-reperfusion injury of rats, rather than ANA.

Borderline personality (BPD) and complex posttraumatic stress disorders (PTSD) are both powerfully associated with the experience of interpersonal violence during childhood and adolescence. The disorders frequently co-occur and often result in pervasive problems in, e.g., emotion regulation and altered pain perception, where the endocannabinoid system is deeply involved. We hypothesize an endocannabinoid role in both disorders. We investigated serum levels of the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> and related fatty acid ethanolamides (FAEs) in BPD, PTSD, and controls. Significant alterations were found for both endocannabinoids in BPD and for the FAE oleoylethanolamide in PTSD suggesting a respective link to both disorders.

Endocannabinoids are bioactive lipids that have the potential to signal through cannabinoid receptors to modulate the functional activities of a variety of immune cells. Their activation of these seven-transmembranal, G protein-coupled receptors sets in motion a series of signal transductional events that converge at the transcriptional level to regulate cell migration and the production of cytokines and chemokines. There is a large body of data that supports a functional relevance for <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) as acting through the cannabinoid receptor type <em>2</em> (CB<em>2</em>R) to inhibit migratory activities for a diverse array of immune cell types. However, unequivocal data that supports a functional linkage of anandamide (AEA) to a cannabinoid receptor in immune modulation remains to be obtained. Endocannabinoids, as typical bioactive lipids, have a short half-life and appear to act in an autocrine and paracrine fashion. Their immediate effective action on immune function may be at localized sites in the periphery and within the central nervous system. It is speculated that endocannabinoids play an important role in maintaining the overall "fine-tuning" of the immune homeostatic balance within the host.

OBJECTIVE

A decreased sensitivity to 6-n-propylthiouracil (PROP) has been shown to be associated with increased energy intake and therefore an increased body mass index, although other studies have not confirmed this association, suggesting the involvement of other factors. We investigated whether the endocannabinoid system, which also modulates hunger/satiety and energy balance, plays a role in modulating eating behavior influenced by a sensitivity to PROP.

METHODS

The plasma profile of the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), anandamide (AEA), and congeners of AEA, palmitoylethanolamide and oleylethanolamide (OEA), was determined in normal-weight PROP supertasters (STs) and PROP non-tasters (NTs). A cognitive eating behavior disorder was assessed by the Three-Factor Eating Questionnaire, which estimates dietary restraint, disinhibition, and perceived hunger.

RESULTS

The disinhibition score of NTs was higher than those of STs (P = 0.0<em>2</em>). Moreover, in NTs, OEA was inversely correlated to the perceived hunger score (r = -0.7, P = 0.00<em>2</em>), and AEA was positively correlated to the restraint score (r = 0.5, P = 0.04) and negatively to the perceived hunger score, although the latter correlation was at the limit of statistical significance (r = -0.47, P = 0.05). In addition, we found lower concentrations of AEA and <em>2</em>-AG in the plasma of NT compared with ST subjects (AEA, P = 0.034; <em>2</em>-AG, P = 0.003).

CONCLUSIONS

Our data suggest that a higher disinhibition behavior in NTs may be compensated in part, in normal-weight subjects, by the decrease of peripheral endocannabinoids to downregulate the hunger-energy intake circuitry.

Preclinical investigations have demonstrated that drugs of abuse alter the levels of lipid-based signalling molecules, including endocannabinoids (eCBs) and N-acylethanolamines (NAEs), in the rodent brain. In addition, several drugs targeting eCBs and/or NAEs are implicated in reward and/or seeking behaviours related to the stimulation of dopamine systems in the brain. In our study, the brain levels of eCBs (anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG)) and NAEs (oleoylethanolamide (OEA) and palmitoylethanolamide (PEA)) were analyzed via an LC-MS/MS method in selected brain structures of rats during cocaine self-administration and after extinction training according to the "yoked" control procedure. Repeated (14days) cocaine (0.5mg/kg/infusion) self-administration and yoked drug delivery resulted in a significant decrease (ca. 5<em>2</em>%) in AEA levels in the cerebellum, whereas levels of <em>2</em>-AG increased in the frontal cortex, the hippocampus and the cerebellum and decreased in the hippocampus and the dorsal striatum. In addition, we detected increases (>150%) in the levels of OEA and PEA in the limbic areas in both cocaine treated groups, as well as an increase in the tissue levels of OEA in the dorsal striatum in only the yoked cocaine group and increases in the tissue levels of PEA in the dorsal striatum (both cocaine groups) and the nucleus accumbens (yoked cocaine group only). Compared to the yoked saline control group, extinction training (10days) resulted in a potent reduction in AEA levels in the frontal cortex, the hippocampus and the nucleus accumbens and in <em>2</em>-AG levels in the hippocampus, the dorsal striatum and the cerebellum. The decreases in the limbic and subcortical areas were more apparent for rats that self-administered cocaine. Following extinction, there was a region-specific change in the levels of NAEs in rats previously injected with cocaine; a potent increase (ca. 100%) in the levels of OEA and PEA was detected in the prefrontal cortex and the hippocampus, whilst a drop was noted in the striatal areas versus yoked saline yoked animals. Our findings support the previous pharmacological evidence that the eCB system and NAEs are involved in reinforcement and extinction of positively reinforced behaviours and that these lipid-derived molecules may represent promising targets for the development of new treatments for drug addiction.