Chronic pain is associated with cognitive deficits. Palmitoylethanolamide (PEA) has been shown to ameliorate pain and pain-related cognitive impairments by restoring glutamatergic synapses functioning in the spared nerve injury (SNI) of the sciatic nerve in mice. SNI reduced mechanical and thermal threshold, spatial memory and LTP at the lateral entorhinal cortex (LEC)-dentate gyrus (DG) pathway. It decreased also postsynaptic density, volume and dendrite arborization of DG and increased the expression of metabotropic glutamate receptor 1 and 7 (mGluR1 and mGluR7), of the GluR1, GluR1s845 and GluR1s831 subunits of AMPA receptor and the levels of glutamate in the DG. The level of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) was instead increased in the LEC. Chronic treatment with PEA, starting from when neuropathic pain was fully developed, was able to reverse mechanical allodynia and thermal hyperalgesia, memory deficit and LTP in SNI wild type, but not in PPARα null, mice. PEA also restored the level of glutamate and the expression of phosphorylated GluR1 subunits, postsynaptic density and neurogenesis. Altogether, these results suggest that neuropathic pain negatively affects cognitive behavior and related LTP, glutamatergic synapse and synaptogenesis in the DG. In these conditions PEA treatment alleviates pain and cognitive impairment by restoring LTP and synaptic maladaptative changes in the LEC-DG pathway. These outcomes open new perspectives for the use of the N-acylethanolamines, such as PEA, for the treatment of neuropathic pain and its central behavioural sequelae.

Truffles are the fruiting body of fungi, members of the Ascomycota phylum endowed with major gastronomic and commercial value. The development and maturation of their reproductive structure are dependent on melanin synthesis. Since anandamide, a prominent member of the endocannabinoid system (ECS), is responsible for melanin synthesis in normal human epidermal melanocytes, we thought that ECS might be present also in truffles. Here, we show the expression, at the transcriptional and translational levels, of most ECS components in the black truffle Tuber melanosporum Vittad. at maturation stage VI. Indeed, by means of molecular biology and immunochemical techniques, we found that truffles contain the major metabolic enzymes of the ECS, while they do not express the most relevant endocannabinoid-binding receptors. In addition, we measured anandamide content in truffles, at different maturation stages (from III to VI), through liquid chromatography-mass spectrometric analysis, whereas the other relevant endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> was below the detection limit. Overall, our unprecedented results suggest that anandamide and ECS metabolic enzymes have evolved earlier than endocannabinoid-binding receptors, and that anandamide might be an ancient attractant to truffle eaters, that are well-equipped with endocannabinoid-binding receptors.

BACKGROUND

Anandamide and <em>2</em>-<em>arachidonoylglycerol</em> are neuromodulatory lipids interacting with cannabinoid receptors, whose availability is regulated by the balance between 'on demand' generation and enzymatic degradation [by fatty acid amide hydrolase (FAAH)/monoacylglycerol lipase]. Given the reported effects of anandamide on dopamine transmission, we investigated the influence of endocannabinoids and URB597, a well-known FAAH inhibitor, on the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis.

METHODS

We investigated TH expression in N1E115 neuroblastoma using a reporter gene assay, as well as mRNA and protein quantifications. FAAH inhibition was confirmed by measuring radiolabelled substrate hydrolysis and endogenous endocannabinoids.

RESULTS

Anandamide decreased TH promoter activity in N1E115 cells through CB₁ receptor activation. Unexpectedly, URB597 reduced TH expression (pEC₅₀ = 8.7 ± 0.<em>2</em>) through FAAH-independent mechanisms. Indeed, four structurally unrelated inhibitors of FAAH had no influence on TH expression, although all the inhibitors increased endocannabinoid levels. At variance with the endocannabinoid responses, the use of selective antagonists indicated that the URB597-mediated decrease in TH expression was not directed by the CB₁ receptor, but rather by abnormal-cannabidiol-sensitive receptors and PPARs. Further supporting the physiological relevance of these in vitro data, URB597 administration resulted in reduced TH mRNA levels in mice brain.

CONCLUSIONS

While confirming the implication of endocannabinoids on the modulation of TH, we provide strong evidence for additional physiologically relevant off-target effects of URB597. In light of the numerous preclinical studies involving URB597, particularly in anxiety and depression, the existence of non-CB₁ and non-FAAH mediated influences of URB597 on key enzymes of the catecholaminergic transmission system should be taken into account when interpreting the data.

The bioactive phospholipids, LPA (lysophosphatidic acid) and PA (phosphatidic acid), regulate pivotal processes related to the pathogenesis of cancer. Recently, we cloned a novel type of lipid kinase that phosphorylates monoacylglycerols (such as <em>2</em>-<em>arachidonoylglycerol</em>, an endogenous cannabinoid receptor ligand) and diacylglycerols, to form LPA and PA, respectively. This AGK (acylglycerol kinase) is highly expressed in prostate cancer cell lines and the results reviewed here suggest that AGK might be a critical player in the initiation and progression of prostate cancer. Intriguingly, down-regulation of endogenous AGK inhibited EGF (epidermal growth factor), but not LPA-induced ERK1/<em>2</em> (extracellular-signal-regulated kinase 1/<em>2</em>) activation and progression through the S-phase of the cell cycle. In this review, we will summarize the evidence demonstrating that AGK amplifies EGF growth signalling pathways that play an important role in the pathophysiology of prostate cancer. Because LPA has long been implicated as an autocrine and paracrine growth stimulatory factor for prostate cancer cells, the identification of this novel lipid kinase that regulates its production could provide new and useful targets for preventive or therapeutic measures.

Negative emotional states that are associated with excessive alcohol intake, particularly anxiety-like states, have been linked to opponent processes in the central nucleus of the amygdala (CeA), affecting stress-related transmitters and monoamines. This study extends these observations to include endocannabinoid signaling in alcohol-dependent animals. Rats and mice were exposed to chronic intermittent alcohol with vapor inhalation or liquid diet to induce dependence. In vivo microdialysis was used to estimate interstitial concentrations of endocannabinoids [N-arachidonoylethanolamine (anandamide; AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG)] and amino acids (glutamate and GABA) in rat CeA. Additionally, we evaluated the inhibition of endocannabinoids clearance enzymes [monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase] on anxiety-like behavior and alcohol consumption in alcohol-dependent rats and mice. Results revealed that alcohol dependence produced decreases in baseline <em>2</em>-AG dialysate levels and increases in baseline levels of glutamate and GABA. Acute alcohol abstinence induced an enhancement of these dependence-induced effects and the levels of <em>2</em>-AG and GABA were restored upon alcohol re-exposure. Additional studies showed that the increased CeA <em>2</em>-AG levels induced by restraint stress and alcohol self-administration were blunted in alcohol-dependent rats. Pharmacological studies in rats and mice showed that anxiety-like behavior and alcohol consumption were increased in alcohol-dependent animals, and these behavioral effects were attenuated mainly by MAGL inhibitors [MJN110 (10 and <em>2</em>0 mg/kg) in rats and JZL184 (1 and 3 mg/kg) in mice]. The present results suggest a key role for endocannabinoid signaling in motivational neuroadaptations during alcohol dependence, in which a deficiency in CeA <em>2</em>-AG signaling in alcohol-dependent animals is linked to stress and excessive alcohol consumption.

Monoacylglycerol lipase (MGL) hydrolyzes <em>2</em>-<em>arachidonoylglycerol</em> to arachidonic acid and glycerol. Inhibition of MGL may attenuate neuroinflammation by enhancing endocannabinoid signaling and decreasing prostaglandin (PG) production. Almost half of HIV infected individuals are afflicted with HIV-associated neurocognitive disorder (HAND), a neuroinflammatory disease in which cognitive decline correlates with synapse loss. HIV infected cells shed the envelope protein gp1<em>2</em>0 which is a potent neurotoxin that induces synapse loss. Here, we tested whether inhibition of MGL, using the selective inhibitor JZL184, would prevent synapse loss induced by gp1<em>2</em>0. The number of synapses between rat hippocampal neurons in culture was quantified by imaging clusters of a GFP-tagged antibody-like protein that selectively binds to the postsynaptic scaffolding protein, PSD95. JZL184 completely blocked gp1<em>2</em>0-induced synapse loss. Inhibition of MGL decreased gp1<em>2</em>0-induced interleukin-1β (IL-1β) production and subsequent potentiation of NMDA receptor-mediated calcium influx. JZL184-mediated protection of synapses was reversed by a selective cannabinoid type <em>2</em> receptor (CB<em>2</em>R) inverse agonist/antagonist. JZL184 also reduced gp1<em>2</em>0-induced prostaglandin E<em>2</em> (PGE<em>2</em>) production; PG signaling was required for gp1<em>2</em>0-induced IL-1β expression and synapse loss. Inhibition of MGL prevented gp1<em>2</em>0-induced synapse loss by activating CB<em>2</em>R resulting in decreased production of the inflammatory cytokine IL-1β. Because PG signaling was required for gp1<em>2</em>0-induced synapse loss, JZL184-induced decreases in PGE<em>2</em> levels may also protect synapses. MGL presents a promising target for preventing synapse loss in neuroinflammatory conditions such as HAND.

The serine hydrolase monoacylglycerol lipase (MGLL) converts the endogenous cannabinoid receptor agonist <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and other monoacylglycerols into fatty acids and glycerol. Genetic or pharmacological inactivation of MGLL leads to elevation in <em>2</em>-AG in the central nervous system and corresponding reductions in arachidonic acid and eicosanoids, producing antinociceptive, anxiolytic, and antineuroinflammatory effects without inducing the full spectrum of psychoactive effects of direct cannabinoid receptor agonists. Here, we report the optimization of hexafluoroisopropyl carbamate-based irreversible inhibitors of MGLL, culminating in a highly potent, selective, and orally available, CNS-penetrant MGLL inhibitor, <em>2</em>8 (ABX-1431). Activity-based protein profiling experiments verify the exquisite selectivity of <em>2</em>8 for MGLL versus other members of the serine hydrolase class. In vivo, <em>2</em>8 inhibits MGLL activity in rodent brain (ED50 = 0.5-1.4 mg/kg), increases brain <em>2</em>-AG concentrations, and suppresses pain behavior in the rat formalin pain model. ABX-1431 (<em>2</em>8) is currently under evaluation in human clinical trials.

The anti-inflammatory mechanisms of low-fat dairy product consumption are largely unknown. The objective of this study was to determine whether low-fat yogurt reduces biomarkers of chronic inflammation and endotoxin exposure in women. Premenopausal women (BMI 18·5-<em>2</em>7 and 30-40 kg/m<em>2</em>) were randomised to consume 339 g of low-fat yogurt (yogurt non-obese (YN); yogurt obese (YO)) or 3<em>2</em>4 g of soya pudding (control non-obese; control obese (CO)) daily for 9 weeks (n 30/group). Fasting blood samples were analysed for IL-6, TNF-α/soluble TNF II (sTNF-RII), high-sensitivity C-reactive protein, <em>2</em>-arachidonoyl glycerol, anandamide, monocyte gene expression, soluble CD14 (sCD14), lipopolysaccharide (LPS), LPS binding protein (LBP), IgM endotoxin-core antibody (IgM EndoCAb), and zonulin. BMI, waist circumference and blood pressure were also determined. After 9-week yogurt consumption, YO and YN had decreased TNF-α/sTNFR-RII. Yogurt consumption increased plasma IgM EndoCAb regardless of obesity status. sCD14 was not affected by diet, but LBP/sCD14 was lowered by yogurt consumption in both YN and YO. Yogurt intervention increased plasma <em>2</em>-<em>arachidonoylglycerol</em> in YO but not YN. YO peripheral blood mononuclear cells expression of NF-κB inhibitor α and transforming growth factor β1 increased relative to CO at 9 weeks. Other biomarkers were unchanged by diet. CO and YO gained approximately 0·9 kg in body weight. YO had 3·6 % lower diastolic blood pressure at week 3. Low-fat yogurt for 9 weeks reduced biomarkers of chronic inflammation and endotoxin exposure in premenopausal women compared with a non-dairy control food. This trial was registered as NCT01686<em>2</em>04.

BACKGROUND

Maternal obesity (MO) remains a serious obstetric problem with acute and chronic morbidities for both mothers and offspring. The mechanisms underlying these adverse consequences of MO remain unknown. Endocannabinoids (ECB) are neuromodulatory lipids released from adipocytes and other tissues. Metabolic crosstalk between placenta and adipocytes may mediate sequelae of MO. The goal of this study was to elucidate placental and systemic ECB in MO.

METHODS

Placentas, sera, and subcutaneous fat were collected at Cesarean sections performed near term (0.9 G) in four non-obese (nOB) and four obese (OB) baboons (Papio spp.). Concentrations of anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) were measured by liquid chromatography coupled to tandem mass spectrometry. AEA and <em>2</em>-AG pathways were characterized in placentas by Q-RT-PCR, Western blot and immunohistochemistry.

RESULTS

Placental <em>2</em>-AG levels were lower and maternal fat AEA levels were higher in OB (1<em>2</em>54.1 ± 401.3 nmol/kg and 17.3 ± 4 nmol/kg) vs. nOB (31<em>2</em>4.<em>2</em> ± 557.3 nmol/kg and 3.1 ± 0.6 nmol/kg) animals. Concentrations of <em>2</em>-AG correlated positively between maternal fat and placenta (r = 0.8<em>2</em>, p = 0.013), but correlated negatively with maternal leptin concentrations (r = -0.7<em>2</em>, p = 0.04 and r = -0.83, p = 0.01, respectively).

CONCLUSIONS

This is the first study to demonstrate differential ECB pathway regulation in maternal fat and placenta in MO. Differential regulation and function exist for AEA and <em>2</em>-AG as the major ECB pathways in placenta.

Exposure to unbalanced diets during pre-gestational and gestational periods may result in long-term alterations in metabolism and behavior. The contribution of the endocannabinoid system to these long-term adaptive responses is unknown. In the present study, we investigated the impact of female rat exposure to a hypercaloric-hypoproteic palatable diet during pre-gestational, gestational and lactational periods on the development of male offspring. In addition, the hypothalamic and hippocampal endocannabinoid contents at birth and the behavioral performance in adulthood were investigated. Exposure to a palatable diet resulted in low weight offspring who exhibited low hypothalamic contents of arachidonic acid and the two major endocannabinoids (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>) at birth. Palmitoylethanolamide, but not oleoylethanolamide, also decreased. Additionally, pups from palatable diet-fed dams displayed lower levels of anandamide and palmitoylethanolamide in the hippocampus. The low-weight male offspring, born from palatable diet exposed mothers, gained less weight during lactation and although they recovered weight during the post-weaning period, they developed abdominal adiposity in adulthood. These animals exhibited anxiety-like behavior in the elevated plus-maze and open field test and a low preference for a chocolate diet in a food preference test, indicating that maternal exposure to a hypercaloric diet induces long-term behavioral alterations in male offspring. These results suggest that maternal diet alterations in the function of the endogenous cannabinoid system can mediate the observed phenotype of the offspring, since both hypothalamic and hippocampal endocannabinoids regulate feeding, metabolic adaptions to caloric diets, learning, memory, and emotions.

Dietary long-chain polyunsaturated fatty acids are known to influence brain levels of the endocannabinoid anandamide in newborn pigs and mice. Furthermore, endocannabinoids were shown to control pup suckling and body weight in mice, and food intake in adult rodents. Here we determined the effect of maternal under-nutrition during gestation, lactation, or both, on body weight, and on the levels of endocannabinoids and expression of cannabinoid CB1 receptors and fatty acid amide hydrolase in the hypothalamus of rat pups at weaning (<em>2</em>1 days old) or adult rats (4 months old). Maternal under-nutrition resulted in a striking decrease in body weight of weaning rats, paralleled by a decrease in the hypothalamic levels of the endocannabinoid anandamide, but not of <em>2</em>-<em>arachidonoylglycerol</em>. No significant change in the hypothalamic expression of either cannabinoid CB1 receptors or fatty acid amide hydrolase mRNA was detected in any of the three groups of weaned pups. The decrease in pup body weight and hypothalamic anandamide levels was not observable in 4-month-old rats from any of the three groups. These data suggest that maternal under-nutrition causes a decrease in hypothalamic anandamide levels and loss of body weight, and confirm a crucial role for endocannabinoid signalling in neonatal development.

Increased vascular sensitivity to angiotensin II (Ang II) is a marker of a hypertensive human pregnancy. Recent evidence of interactions between the renin-angiotensin system and the endocannabinoid system suggests that anandamide and <em>2</em>-<em>arachidonoylglycerol</em> may modulate Ang II contraction. We hypothesized that these interactions may contribute to the enhanced vascular responses in hypertensive pregnancy. We studied Ang II contraction in isolated uterine artery (UA) at early gestation in a rat model that mimics many features of preeclampsia, the transgenic human angiotensinogen×human renin (TgA), and control Sprague-Dawley rats. We determined the role of the cannabinoid receptor 1 by blockade with SR171416A, and the contribution of anandamide and <em>2</em>-<em>arachidonoylglycerol</em> degradation to Ang II contraction by inhibiting their hydrolyzing enzyme fatty acid amide hydrolase (with URB597) or monoacylglycerol lipase (with JZL184), respectively. TgA UA showed increased maximal contraction and sensitivity to Ang II that was inhibited by indomethacin. Fatty acid amide hydrolase blockade decreased Ang IIMAX in Sprague-Dawley UA, and decreased both Ang IIMAX and sensitivity in TgA UA. Monoacylglycerol lipase blockade had no effect on Sprague-Dawley UA and decreased Ang IIMAX and sensitivity in TgA UA. Blockade of the cannabinoid receptor 1 in TgA UA had no effect. Immunolocalization of fatty acid amide hydrolase and monoacylglycerol lipase showed a similar pattern between groups; fatty acid amide hydrolase predominantly localized in endothelium and monoacylglycerol lipase in smooth muscle cells. We demonstrated an increased Ang II contraction in TgA UA before initiation of the hypertensive phenotype. Anandamide and <em>2</em>-<em>arachidonoylglycerol</em> reduced Ang II contraction in a cannabinoid receptor 1-independent manner. These renin-angiotensin system-endocannabinoid system interactions may contribute to the enhanced vascular reactivity in early stages of hypertensive pregnancy.

Fever is a common response to inflammation and infection. The mechanism involves prostaglandin E<em>2</em> (PGE<em>2</em>)-EP3 receptor signaling in the hypothalamus, which raises the set point of hypothalamic thermostat for body temperature, but the lipid metabolic pathway for pyretic PGE<em>2</em> production remains unknown. To reveal the molecular basis of fever initiation, we examined lipopolysaccharides (LPS)-induced fever model in monoacylglycerol lipase (MGL)-deficient (Mgll-/-) mice, CB1 receptor-MGL compound-deficient (Cnr1-/-Mgll-/-) mice, cytosolic phospholipase A<em>2</em>α (cPLA<em>2</em>α)-deficient (Pla<em>2</em>g4a-/-) mice, and diacylglycerol lipase α (DGLα)-deficient (Dagla-/-) mice. Febrile reactions were abolished in Mgll-/- and Cnr1-/-Mgll-/- mice, whereas Cnr1-/-Mgll+/+, Pla<em>2</em>g4a-/- and Dagla-/- mice responded normally, demonstrating that MGL is a critical enzyme for fever, which functions independently of endocannabinoid signals. Intracerebroventricular administration of PGE<em>2</em> caused fever similarly in Mgll-/- and wild-type control mice, suggesting a lack of pyretic PGE<em>2</em> production in Mgll-/- hypothalamus, which was confirmed by lipidomics analysis. Normal blood cytokine responses after LPS administration suggested that MGL-deficiency does not affect pyretic cytokine productions. Diurnal body temperature profiles were normal in Mgll-/- mice, demonstrating that MGL is unrelated to physiological thermoregulation. In conclusion, MGL-dependent hydrolysis of endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> is necessary for pyretic PGE<em>2</em> production in the hypothalamus.

BACKGROUND

In animals, the endocannabinoid system is activated during hemodynamic insults and restrains blood pressure in part through sympathetic inhibition.

METHODS

We tested the hypothesis that hemodynamic stress elicited by head-up tilt testing increases systemic endocannabinoid concentrations in humans and that excessive endocannabinoid availability predisposes to presyncope.

RESULTS

With head-up tilt, <em>2</em>-<em>arachidonoylglycerol</em> increased, whereas anandamide remained unchanged.

CONCLUSIONS

In contrast to our expectations, anandamide plasma concentration at rest was directly correlated with orthostatic tolerance, rather than intolerance.

There is evidence to suggest that a dysregulation of endocannabinoid signaling may contribute to the etiology and pathophysiology of migraine. Thus, patients suffering from chronic migraine or medication overuse headache showed alterations in the activity of the arachidonoylethanolamide (AEA) degrading enzyme fatty acid amide hydrolase (FAAH) and a specific AEA membrane transporter, alongside with changes in AEA levels. The precise role of different endocannabinoid system components is, however, not clear. We have therefore investigated mice with a genetic deletion of the two main cannabinoid receptors CB1 and CB<em>2</em>, or the main endocannabinoid degrading enzymes, FAAH and monoacylglycerol lipase (MAGL), which degrades <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), in a nitroglycerine-induced animal model of migraine. We found that nitroglycerin-induced mechanical allodynia and neuronal activation of the trigeminal nucleus were completely abolished in FAAH-deficient mice. To validate these results, we used two structurally different FAAH inhibitors, URB597 and PF3945. Both inhibitors also dose-dependently blocked nitroglycerin-induced hyperalgesia and the activation of trigeminal neurons. The effects of the genetic deletion of pharmacological blockade of FAAH are mediated by CB1 receptors, because they were completely disrupted with the CB1 antagonist rimonabant. These results identify FAAH as a target for migraine pharmacotherapy.

OBJECTIVE

Pro-inflammatory cytokines, growth and angiogenic factors released by leukocytes are involved in carcinogenesis and cancer progression, but they are also crucial for fighting tumour growth and spreading. We have previously demonstrated that endocannabinoids modulate cell-to-cell crosstalk during inflammation. Here, we investigated the inflammatory and tumourigenic properties of endocannabinoids in a human urinary bladder carcinoma cell line.

METHODS

Endocannabinoid-treated ECV304 cells were checked for tumour necrosis factor (TNF)-α secretion (by ELISA assay) and surface exposure of selectins (by in situ ELISA and FACS analysis). ECV304/Jurkat T cell interaction was assessed by adhesion and live imaging experiments. Proliferation rate, cell death and cell cycle were determined by FACS analysis.

RESULTS

By binding to type-1 (CB1) and type-<em>2</em> (CB<em>2</em>) cannabinoid receptors, the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) exacerbates the pro-inflammatory status surrounding bladder carcinoma ECV304 cells, by: (i) enhancing TNF-α release, (ii) increasing surface exposure of P- and E-selectins, and (iii) allowing Jurkat T lymphocytes to adhere to treated cancer cells. We also found that the CB1 inverse agonist AM<em>2</em>81, unlike <em>2</em>-AG, decreases cancer proliferation by delaying cell cycle progression.

CONCLUSIONS

Our data suggest that <em>2</em>-AG modulates the inflammatory milieu of cancer cells in vitro, while AM<em>2</em>81 plays a more specific role in proliferation. Collectively, these findings suggest that CB receptors may play distinct roles in cancer biology, depending on the specific ligand employed.

CONCLUSIONS

The in vivo assessment of the role of CB receptors in inflammation and cancer might be instrumental in broadening the understanding about bladder cancer biology.

The investigation of natural and synthetic cannabinoid ligands, including (-)-Delta(9)-tetrahydrocannabinol, cannabinol, cannabidiol, HU-<em>2</em>10, HU-<em>2</em>11, CT3, CP 55, 940, WIN 55, <em>2</em>1<em>2</em>-<em>2</em>, SR 14, 1716A, anandamide, <em>2</em>-<em>arachidonoylglycerol</em>, and numerous novel analogs, has led to important findings that have contributed to a better understanding of the role of these compounds in physiological processes. Their potential use for medicinal purposes is also better understood as a result.

OBJECTIVE

The endocannabinoid system (ECS) promotes weight gain and obesity-associated metabolic changes. Weight loss interventions may influence obesity-associated risk indirectly through modulation of the peripheral ECS. We investigated the effect of acute and chronic treatment with sibutramine on components of the peripheral ECS.

METHODS

Twenty obese otherwise healthy patients received randomized, double-blind, crossover treatment with placebo and 15 mg/day sibutramine for 5 days each, followed by 1<em>2</em> weeks open-label sibutramine treatment. We determined circulating anandamide and <em>2</em>-<em>arachidonoylglycerol</em> and expression levels of endocannabinoid genes in subcutaneous abdominal adipose tissue biopsies.

RESULTS

Body weight was stable during the acute treatment period and decreased by 6.0+/-0.8 kg in those patients completing 3 months of sibutramine treatment (P<0.05). Circulating endocannabinoids and the expression of ECS genes did not change with acute or chronic sibutramine treatment.

CONCLUSIONS

The ECS is activated in obesity. We did not find any influence of 5% body weight loss induced by sibutramine on circulating levels of endocannabinoids and adipose-tissue expression of endocannabinoid genes in obese subjects. These data confirm our previous findings on dietary weight loss and suggest that the dysregulation of the ECS may be a cause rather than a consequence of obesity.

Variations in environmental aversiveness influence emotional memory processes in rats. We have previously shown that cannabinoid effects on memory are dependent on the stress level at the time of training as well as on the aversiveness of the environmental context. Here, we investigated whether the hippocampal endocannabinoid system modulates memory retrieval depending on the training-associated arousal level. Male adult Sprague Dawley rats were trained on a water maze spatial task at two different water temperatures (19°C and <em>2</em>5°C) to elicit either higher or lower stress levels, respectively. Rats trained under the higher stress condition had better memory and higher corticosterone concentrations than rats trained at the lower stress condition. The cannabinoid receptor agonist WIN55<em>2</em>1<em>2</em>-<em>2</em> (10-30 ng/side), the <em>2</em>-arachidonoyl glycerol (<em>2</em>-AG) hydrolysis inhibitor JZL184 (0.1-1 μg/side), and the anandamide (AEA) hydrolysis inhibitor URB597 (10-30 ng/side) were administered bilaterally into the hippocampus 60 min before probe-trial retention testing. WIN55<em>2</em>1<em>2</em>-<em>2</em> or JZL184, but not URB597, impaired probe-trial performances only of rats trained at the higher stressful condition. Furthermore, rats trained under higher stress levels displayed an increase in hippocampal <em>2</em>-AG, but not AEA, levels at the time of retention testing and a decreased affinity of the main <em>2</em>-AG-degrading enzyme for its substrate. The present findings indicate that the endocannabinoid <em>2</em>-AG in the hippocampus plays a key role in the selective regulation of spatial memory retrieval of stressful experience, shedding light on the neurobiological mechanisms involved in the impact of stress effects on memory processing.

UNASSIGNED

Endogenous cannabinoids play a central role in the modulation of memory for emotional events. Here we demonstrate that the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> in the hippocampus, a brain region crucially involved in the regulation of memory processes, selectively modulates spatial memory recall of stressful experiences. Thus, our findings provide evidence that the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> is a key player in mediating the impact of stress on memory retrieval. These findings can pave the way to new potential therapeutic intervention for the treatment of neuropsychiatric disorders, such as post-traumatic stress disorder, where a previous exposure to traumatic events could alter the response to traumatic memory recall leading to mental illness.

Functional coupling between the amygdala and the dorsomedial prefrontal cortex (dmPFC) has been implicated in the generation of negative affective states; however, the mechanisms by which stress increases amygdala-dmPFC synaptic strength and generates anxiety-like behaviors are not well understood. Here, we show that the mouse basolateral amygdala (BLA)-prelimbic prefrontal cortex (plPFC) circuit is engaged by stress and activation of this pathway in anxiogenic. Furthermore, we demonstrate that acute stress exposure leads to a lasting increase in synaptic strength within a reciprocal BLA-plPFC-BLA subcircuit. Importantly, we identify <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG)-mediated endocannabinoid signaling as a key mechanism limiting glutamate release at BLA-plPFC synapses and the functional collapse of multimodal <em>2</em>-AG signaling as a molecular mechanism leading to persistent circuit-specific synaptic strengthening and anxiety-like behaviors after stress exposure. These data suggest that circuit-specific impairment in <em>2</em>-AG signaling could facilitate functional coupling between the BLA and plPFC and the translation of environmental stress to affective pathology.

The major endocannabinoid (eCB) <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is a member of the endocannabinoid system (ECS) that participates in cell proliferation and apoptosis, important events for the homoeostasis of biological systems. The formation of placenta is one of the most important stages of pregnancy and its development requires highly regulated proliferation, differentiation and apoptosis of trophoblasts. Anomalies in these processes are associated with gestational pathologies. In this work, we aimed to study the involvement of <em>2</em>-AG in cytotrophoblast cell turnover. We found that <em>2</em>-AG biosynthetic (diacylglycerol lipase A) and degradative (monoacylglycerol lipase) enzymes are expressed in human cytotrophoblasts and in BeWo cells. We also found that <em>2</em>-AG induces a decrease in cell viability in a time- and concentration-dependent manner and exerts antiproliferative effects. The loss of cell viability induced by a 48-h treatment with <em>2</em>-AG (10 μM) was accompanied by chromatin fragmentation and condensation, morphological features of apoptosis. Additionally, <em>2</em>-AG induced an increase in caspase 3/7 and 9 activities, a loss of mitochondrial membrane potential (Δψm) and an increase in reactive oxygen species (ROS)/reactive nitrogen species (RNS) generation, suggesting the activation of the mitochondrial pathway. Moreover, whereas Δψm loss and ROS/RNS generation were significantly attenuated by the antagonists of both the cannabinoid receptors 1 and <em>2</em> (CB1 and CB<em>2</em>), the increase in caspase 3/7 and 9 activities and loss of cell viability were reversed only by the antagonist of CB<em>2</em> receptor; the blockage of the eCB membrane transporter and the depletion of cholesterol failed to reverse the effects of <em>2</em>-AG. Therefore, this work supports the importance of cannabinoid signalling during cytotrophoblast cell turnover and that its deregulation may be responsible for altered placental development and poor pregnancy outcomes.

BACKGROUND

The endocannabinoids are emerging as natural brain protective substances that exert potentially beneficial effects in several neurological disorders by virtue of their hypothermic, immunomodulatory, vascular, antioxidant, and antiapoptotic actions. This study was undertaken to assess whether preventing the deactivation of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) with the monoacylglycerol lipase (MAGL) inhibitor URB60<em>2</em> can provide neuroprotective effects in hypoxia-ischemia (HI)-induced brain injury.

METHODS

URB60<em>2</em> was administered into the right lateral ventricle 30 min before 7-day-old pup rats were subjected to HI. The neuroprotective effect was evaluated on postnatal day (PN) 14 or at adulthood (PN80) using behavioral and histological analyses. Activated caspase-3 expression and propidium iodide labeling were assessed as indexes of apoptotic and necrotic cell death, respectively.

RESULTS

Pretreatment with URB60<em>2</em> reduced apoptotic and necrotic cell death, as well as the infarct volume measured at PN14. At adulthood, URB60<em>2</em>-treated HI animals performed better at the T-maze and the Morris maze, and also showed a significant reduction of brain damage.

CONCLUSIONS

These results demonstrate that a pretreatment with URB60<em>2</em> significantly reduces brain damage and improves functional outcome, indicating that endocannabinoid-degrading enzymes may represent an important target for neuroprotection in neonatal ischemic brain injury.

Deficits in social interaction (SI) are a core symptom of Autism Spectrum Disorders (ASD), however treatments for social deficits are notably lacking. Elucidating brain circuits and neuromodulatory signaling systems that regulate sociability could facilitate a deeper understanding of ASD pathophysiology and reveal novel treatments for ASD. Here we found that in vivo optogenetic activation of the basolateral amygdala-nucleus accumbens (BLA-NAc) glutamatergic circuit reduced SI and increased social avoidance in mice. Furthermore, we found that <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) endocannabinoid (eCB) signaling reduced BLA-NAc glutamatergic activity, and that pharmacological <em>2</em>-AG augmentation via administration of JZL184 blocked SI deficits associated with in vivo BLA-NAc stimulation. Additionally, optogenetic inhibition of the BLA-NAc circuit significantly increased SI in the Shank3B-/-, an ASD model with substantial SI impairment, without affecting SI in wild-type mice. Finally, we demonstrated that JZL184 delivered systemically or directly to the NAc also normalized SI deficits in Shank3B-/-mice, while ex vivo JZL184 application corrected aberrant NAc excitatory and inhibitory neurotransmission and reduced BLA-NAc-elicited feedforward inhibition of NAc neurons in Shank3B-/- mice. These data reveal circuit-level and neuromodulatory mechanisms regulating social function relevant to ASD and suggest <em>2</em>-AG augmentation could reduce social deficits via modulation of excitatory and inhibitory neurotransmission in the NAc.

In the retina, increased inflammatory response can cause visual impairment during HIV infection in spite of successful anti-retroviral therapy (HAART). The HIV-1 Tat protein is implicated in neurodegeneration by eliciting a cytokine response in cells of the CNS, including glia. The current study investigated whether innate immune response in human retinal Muller glia could be immune-modulated to combat inflammation. Endocannabinoids, N-arachidonoylethanolamide and <em>2</em>-<em>arachidonoylglycerol</em> are used to alleviate Tat-induced cytotoxicity and rescue retinal cells. The neuroprotective mechanism involved suppression in production of pro-inflammatory and increase of anti-inflammatory cytokines, mainly through the MAPK pathway. The MAPK regulation was primarily by MKP-1. Both endocannabinoids regulated cytokine production by affecting at the transcriptional level the NF-κB complex, including IRAK1BP1 and TAB<em>2</em>. Stability of cytokine mRNA is likely to have been influenced through tristetraprolin. These findings have direct relevance in conditions like immune-recovery uveitis where anti-retroviral therapy has helped immune reconstitution. In such conditions drugs to combat overwhelming inflammatory response would need to supplement HAART. Endocannabinoids and their agonists may be thought of as neurotherapeutic during certain conditions of HIV-1 induced inflammation.