In this report, we describe the localization of diacylglycerol lipase-α (DAGLα) in nuclei from adult cortical neurons, as assessed by double-immunofluorescence staining of rat brain cortical sections and purified intact nuclei and by western blot analysis of subnuclear fractions. Double-labeling assays using the anti-DAGLα antibody and NeuN combined with Hoechst staining showed that only nuclei of neuronal origin were DAGLα positive. At high resolution, DAGLα-signal displayed a punctate pattern in nuclear subdomains poor in Hoechst's chromatin and lamin B1 staining. In contrast, SC-35- and NeuN-signals (markers of the nuclear speckles) showed a high overlap with DAGLα within specific subdomains of the nuclear matrix. Among the members of the phospholipase C-β (PLCβ) family, PLCβ1, PLCβ<em>2</em>, and PLCβ4 exhibited the same distribution with respect to chromatin, lamin B1, SC-35, and NeuN as that described for DAGLα. Furthermore, by quantifying the basal levels of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) by liquid chromatography and mass spectrometry (LC-MS), and by characterizing the pharmacology of its accumulation, we describe the presence of a mechanism for <em>2</em>-AG production, and its PLCβ/DAGLα-dependent biosynthesis in isolated nuclei. These results extend our knowledge about subcellular distribution of neuronal DAGLα, providing biochemical grounds to hypothesize a role for <em>2</em>-AG locally produced within the neuronal nucleus.

The nucleus accumbens (NAc) plays a key role in drug-related behavior and natural reward learning. Synaptic plasticity in dopamine D1 and D<em>2</em> receptor medium spiny neurons (MSNs) of the NAc and the endogenous cannabinoid (eCB) system have been implicated in reward seeking. However, the precise molecular and physiological basis of reward-seeking behavior remains unknown. We found that the specific deletion of metabotropic glutamate receptor 5 (mGluR5) in D1-expressing MSNs (D1^miRmGluR5 mice) abolishes eCB-mediated long-term depression (LTD) and prevents the expression of drug (cocaine and ethanol), natural reward (saccharin), and brain-stimulation-seeking behavior. In vivo enhancement of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) eCB signaling within the NAc core restores both eCB-LTD and reward-seeking behavior in D1^miRmGluR5 mice. The data suggest a model where the eCB and glutamatergic systems of the NAc act in concert to mediate reward-seeking responses.

OBJECTIVE

Endocannabinoids and neuropeptide Y (NPY) promote energy storage via central and peripheral mechanisms. In the hypothalamus, the two systems were suggested to interact. To investigate such interplay also in non-hypothalamic tissues, we evaluated endocannabinoid levels in obese OE-NPY(DβH) mice, which overexpress NPY in the noradrenergic neurons in the sympathetic nervous system and the brain.

METHODS

The levels of the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) were measured in key regulatory tissues, that is, hypothalamus, pancreas, epididymal white adipose tissue (WAT), liver and soleus muscle, over the development of metabolic dysfunctions in OE-NPY(DβH) mice. The effects of a 5-week treatment with the CB1 receptor inverse agonist AM<em>2</em>51 on adiposity and glucose metabolism were studied.

RESULTS

<em>2</em>-AG levels were increased in the hypothalamus and epididymal WAT of pre-obese and obese OE-NPY(DβH) mice. Anandamide levels in adipose tissue and pancreas were increased at 4 months concomitantly with higher fat mass and impaired glucose tolerance. CB1 receptor blockage reduced body weight gain and glucose intolerance in OE-NPY(DβH) to the level of vehicle-treated wild-type mice.

CONCLUSIONS

Altered endocannabinoid tone may underlie some of the metabolic dysfunctions in OE-NPY(DβH) mice, which can be attenuated with CB1 inverse agonism suggesting interactions between endocannabinoids and NPY also in the periphery. CB1 receptors may offer a target for the pharmacological treatment of the metabolic syndrome with altered NPY levels.

Monoacylglycerol lipase (MAGL) has been recently proposed as the main enzymatic activity responsible for the in vivo hydrolysis of the most abundant endocannabinoid in the brain, the <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). The endocannabinoids, mainly anandamide (AEA) and <em>2</em>-AG, are a class of lipid messengers that modulate a broad number of physiological processes both in the central nervous system and in the periphery. To date, AEA has been by far the most studied endocannabinoid, although increasing evidence is pointing out the prominent, and sometimes underestimated, role of <em>2</em>-AG in the regulation of different functions. Therefore, it is of outmost importance to dissect the specific cellular pathways in which these two endocannabinoids are involved. Nonetheless, little is known about the structural require-ments of MAGL. Here we review the current knowledge on MAGL, with special focus on its structure and catalytic mechanism as the rational basis for the design of potent and selective compounds able to interact with it; the inhibitors that have been described to date, and the therapeutic applications that make MAGL an attractive therapeutic target.

Several linear fatty acid dopamides (N-acyldopamines) have been identified recently in the brain. Among them, N-arachidonoyldopamine (NADA) is an endogenous lipid mediator sharing endocannabinoid and endovanilloid biological activities. We have reported previously that NADA exerts some of its biological activities through inhibition of the NF-kappaB pathway and, because this transcription factor plays a key role in HIV-1-long terminal repeat (LTR) trans activation, we have evaluated the anti-HIV-1 activity of NADA. In this study, we show that NADA inhibits vesicular stomatitis virus-pseudotyped HIV-1 infection in the human leukemia T cell line Jurkat, in primary T cells, and in the human astrocytic cell line U373-MG. Other endocannabinoids such as anandamide, <em>2</em>-<em>arachidonoylglycerol</em>, and noladin ether did not show inhibitory activity in the HIV-1 replication assays. The anti-HIV-1 activity of NADA was independent of known cannabinoid and vanilloid receptor activation. In addition, NADA did not affect reverse transcription and integration steps of the viral cycle, and its inhibitory effect was additive with that of the reverse transcriptase inhibitor azidothymidine. NADA inhibited both TNF-alpha and HIV-1 trans activator protein-induced HIV-1-LTR activation. We also show that NADA counteracts the TNF-alpha-mediated trans activation capacity of the p65 NF-kappaB subunit without affecting its physical association to the HIV-1-LTR promoter. Moreover, NADA inhibited the p65 transcriptional activity by specifically targeting the phosphorylation of this NF-kappaB subunit at Ser(536). These findings provide new mechanistic insights into the biological activities of NADA, and highlight the potential of lipid mediators for the management of AIDS.

The endocannabinoid system is dysregulated in schizophrenia. Mice with heterozygous deletion of neuregulin 1 (Nrg1 HET mice) provide a well-characterised animal model of schizophrenia, and display enhanced sensitivity to stress and cannabinoids during adolescence. However, no study has yet determined whether these mice have altered brain endocannabinoid concentrations. Nrg1 application to hippocampal slices decreased <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) signalling and disrupted long-term depression, a form of synaptic plasticity critical to spatial learning. Therefore we specifically aimed to examine whether Nrg1 HET mice exhibit increased <em>2</em>-AG concentrations and disruption of spatial learning. As chronic stress influences brain endocannabinoids, we also sought to examine whether Nrg1 deficiency moderates adolescent stress-induced alterations in brain endocannabinoids. Adolescent Nrg1 HET and wild-type (WT) mice were submitted to chronic restraint stress and brain endocannabinoid concentrations were analysed. A separate cohort of WT and Nrg1 HET mice was also assessed for spatial learning performance in the Morris Water Maze. Partial genetic deletion of Nrg1 increased anandamide concentrations in the amygdala and decreased <em>2</em>-AG concentrations in the hypothalamus. Further, Nrg1 HET mice exhibited increased <em>2</em>-AG concentrations in the hippocampus and impaired spatial learning performance. Chronic adolescent stress increased anandamide concentrations in the amygdala, however, Nrg1 disruption did not influence this stress-induced change. These results demonstrate for the first time in vivo interplay between Nrg1 and endocannabinoids in the brain. Our results demonstrate that aberrant Nrg1 and endocannabinoid signalling may cooperate in the hippocampus to impair cognition, and that Nrg1 deficiency alters endocannabinoid signalling in brain stress circuitry.

<em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is an endogenous ligand of the cannabinoid CB1 receptor. This endocannabinoid and its hydrolyzing enzyme, monoacylglycerol lipase (MAGL), are present in encephalic regions related to psychiatric disorders, including the midbrain dorsolateral periaqueductal grey (dlPAG). The dlPAG is implicated in panic disorder and its stimulation results in defensive responses proposed as a model of panic attacks. The present work verified if facilitation of <em>2</em>-AG signalling in the dlPAG counteracts panic-like responses induced by local chemical stimulation. Intra-dlPAG injection of <em>2</em>-AG prevented panic-like response induced by the excitatory amino acid N-methyl-d-aspartate (NMDA). This effect was mimicked by the <em>2</em>-AG hydrolysis inhibitor (MAGL preferring inhibitor) URB60<em>2</em>. The anti-aversive effect of URB60<em>2</em> was reversed by the CB1 receptor antagonist, AM<em>2</em>51. Additionally, a combination of sub-effective doses of <em>2</em>-AG and URB60<em>2</em> also prevented NMDA-induced panic-like response. Finally, immunofluorescence assay showed a significant increase in c-Fos positive cells in the dlPAG after local administration of NMDA. This response was also prevented by URB60<em>2</em>. These data support the hypothesis that <em>2</em>-AG participates in anti-aversive mechanisms in the dlPAG and reinforce the proposal that facilitation of endocannabinoid signalling could be a putative target for developing additional treatments against panic and other anxiety-related disorders.

The effect of <em>2</em>-<em>arachidonoylglycerol</em>, a cannabimimetic eicosanoid, was studied on mucosa-free longitudinal muscle strips isolated from the guinea-pig distal colon. In the presence of indomethacin (3 microM) and N(G)-nitro-L-arginine (100 microM), <em>2</em>-<em>arachidonoylglycerol</em> (10 nM-10 microM) produced concentration-dependent and tetrodotoxin (1 microM)-sensitive contractions of the longitudinal muscle strips. The contractions were markedly attenuated in the presence of atropine (0.<em>2</em> microM), and partially by hexamethonium (100 microM) pretreatment. The response to <em>2</em>-<em>arachidonoylglycerol</em> was mimicked with N-arachidonoylethanolamine (anandamide, 0.1-30 microM), another cannabimimetic eicosanoid, but the cannabinoid CB(1)/CB(<em>2</em>) receptor agonist, R-[<em>2</em>,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,<em>2</em>,3,-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,<em>2</em>1<em>2</em>-<em>2</em>) (0.1-10 microM), and the vanilloid receptor agonist, (all Z)-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide (AM 404) (10-30 microM), were without effect. The cannabinoid CB(1) receptor antagonist, N-piperidino-5-(4-chlorophenyl)-l-(<em>2</em>,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A) (1 microM), the cannabinoid CB(<em>2</em>) receptor antagonist, [N-[1S]-endo-1,3,3-trimethyl bicyclo [<em>2</em>.<em>2</em>.1] heptan-<em>2</em>-yl]-5-(4-chloro-3-methylphenyl)-l-(4-methylbenzyl)-pyrazole-3-carboxamide (SR1445<em>2</em>8) (1 microM), and the vanilloid receptor antagonist, capsazepine (10 microM), did not shift the concentration-response curve for <em>2</em>-<em>arachidonoylglycerol</em> to the right. The contractile action of <em>2</em>-<em>arachidonoylglycerol</em> was also partially attenuated in the presence of nordihydroguaiaretic acid (10 microM), a lipoxygenase inhibitor. These results indicate that <em>2</em>-<em>arachidonoylglycerol</em> produces contraction of longitudinal muscle of the guinea-pig distal colon via mainly stimulation of myenteric cholinergic neurones, and that neither cannabinoid CB(1)/CB(<em>2</em>) receptors nor vanilloid receptors contributed to the response. The present results suggest the possibility that lipoxygenase metabolites may also contribute, at least in part, to the contractile action of <em>2</em>-<em>arachidonoylglycerol</em>.

The endogenous cannabinoid (EC) system has been recently implicated in several neuropsychiatric disorders. This study analyzed post-mortem brain regions of Cloninger type 1 (n=9) and <em>2</em> (n=8) alcoholics and non-alcoholic controls (n=10) for ECs by quantitative liquid chromatography with triple quadrupole mass spectrometric detection. A significant difference was found in anandamide (AEA) levels in nucleus accumbens (NAcc) between the three groups (p=0.047). AEA levels were significantly lower when compared to controls in both perigenual anterior cingulate (p=0.017) and frontal cortices (p=0.018) of type 1 alcoholics. Similar trends were observed for dihomo-gamma-linolenoyl ethanolamide and docosahexaenoyl ethanolamide, but not for <em>2</em>-<em>arachidonoylglycerol</em>, palmitoyl ethanolamide, or oleoyl ethanolamide. Although preliminary, and from diagnostic groups with a relatively small number of subjects and substantially different mean ages for each group, these results suggest that the EC system may be hyperactive in type <em>2</em> alcoholics and hypoactive in type 1 alcoholics.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG (1)) is an endogenous ligand for the cannabinoid receptors (CB1 and CB<em>2</em>). There is growing evidence that <em>2</em>-<em>arachidonoylglycerol</em> plays important physiological and pathophysiological roles in various mammalian tissues and cells, though the details remain to be clarified. In this study, we synthesized several remarkable analogs of <em>2</em>-<em>arachidonoylglycerol</em>, closely related in chemical structure to <em>2</em>-<em>arachidonoylglycerol</em>: an analog containing an isomer of arachidonic acid with migrated olefins (<em>2</em>-AGA118 (3)), an analog containing a one-carbon shortened fatty acyl moiety (<em>2</em>-AGA113 (4)), an analog containing an one-carbon elongated fatty acyl moiety (<em>2</em>-AGA114 (5)), a hydroxy group-containing analog (<em>2</em>-AGA105 (6)), a ketone group-containing analog (<em>2</em>-AGA109 (7)), and a methylene-linked analog (<em>2</em>-AGA104 (8)). We evaluated their biological activities as cannabinoid receptor agonists using NG108-15 cells which express the CB1 receptor and HL-60 cells which express the CB<em>2</em> receptor. Notably, these structural analogs of <em>2</em>-<em>arachidonoylglycerol</em> exhibited only weak agonistic activities toward either the CB1 receptor or the CB<em>2</em> receptor, which is in good contrast to <em>2</em>-<em>arachidonoylglycerol</em> which acted as a full agonist at these cannabinoid receptors. These results clearly indicate that the structure of <em>2</em>-<em>arachidonoylglycerol</em> is strictly recognized by the cannabinoid receptors (CB1 and CB<em>2</em>) and provide further evidence that the cannabinoid receptors are primarily the intrinsic receptors for <em>2</em>-<em>arachidonoylglycerol</em>.

Whether adolescent exposure to chronic delta-9-tetrahydrocannabinol (THC) facilitates progression to opioid consumption is still controversial. In a maternal deprivation model (3 h daily from postnatal day 1-14), we previously reported that adolescent exposure to chronic THC blocks morphine dependence in maternally deprived (D) rats. Owing to the existence of a functional cross-interaction between the opioid and cannabinoid systems in reward, we evaluated if the vulnerability to opiate reward in D rats, may involve an alteration of the endocannabinoid system. Anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), were quantified in the striatum and mesencephalon of adolescent and adult D and non-deprived (animal facility rearing, AFR) rats by isotope dilution liquid chromatography-mass spectrometry. Oral morphine self-administration behavior was analyzed for 14 weeks, <em>2</em>4 days after chronic injection of the cannabinoid CB1 receptor antagonist/inverse agonist, SR141716A (3 mg/kg) for <em>2</em> weeks during adolescence (PND 35-48). Adolescent D rats exhibited higher basal levels of anandamide than adolescent AFR rats in the nucleus accumbens (38%), the caudate-putamen nucleus (6<em>2</em>%) and the mesencephalon (3<em>2</em>0%), whereas adult D rats showed an increase of anandamide and <em>2</em>-AG levels in the nucleus accumbens (50% and <em>2</em>4%, respectively) and of <em>2</em>-AG in the caudate-putamen nucleus (48%), compared to adult AFR rats. Chronic administration of SR141716A to adolescent D rats blocked the escalation behavior in the morphine consumption test. Our data suggest that altered brain endocannabinoid levels may contribute to the escalation behavior in the morphine consumption test in a maternal deprivation model.

Social play behavior is a highly rewarding, developmentally important form of social interaction in young mammals. However, its neurobiological underpinnings remain incompletely understood. Previous work has suggested that opioid and endocannabinoid neurotransmission interact in the modulation of social play. Therefore, we combined behavioral, pharmacological, electrophysiological, and genetic approaches to elucidate the role of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in social play, and how cannabinoid and opioid neurotransmission interact to control social behavior in adolescent rodents. Systemic administration of the <em>2</em>-AG hydrolysis inhibitor JZL184 or the opioid receptor agonist morphine increased social play behavior in adolescent rats. These effects were blocked by systemic pretreatment with either CB1 cannabinoid receptor (CB1R) or mu-opioid receptor (MOR) antagonists. The social play-enhancing effects of systemic morphine or JZL184 treatment were also prevented by direct infusion of the CB1R antagonist SR141716 and the MOR antagonist naloxone into the nucleus accumbens core (NAcC). Searching for synaptic correlates of these effects in adolescent NAcC excitatory synapses, we observed that CB1R antagonism blocked the effect of the MOR agonist DAMGO and, conversely, that naloxone reduced the effect of a cannabinoid agonist. These results were recapitulated in mice, and completely abolished in CB1R and MOR knockout mice, suggesting that the functional interaction between CB1R and MOR in the NAcC in the modulation of social behavior is widespread in rodents. The data shed new light on the mechanism by which endocannabinoid lipids and opioid peptides interact to orchestrate rodent socioemotional behaviors.

Phytocannabinoids, such as Δ9-tetrahydrocannabinol (THC), bind and activate cannabinoid (CB) receptors, thereby "piggy-backing" on the same pathway's endogenous endocannabinoids (ECs). The recent discovery that liver fatty acid binding protein-1 (FABP1) is the major cytosolic "chaperone" protein with high affinity for both Δ9-THC and ECs suggests that Δ9-THC may alter hepatic EC levels. Therefore, the impact of Δ9-THC or EC treatment on the levels of endogenous ECs, such as N-arachidonoylethanolamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), was examined in cultured primary mouse hepatocytes from WT and Fabp1 gene-ablated (LKO) mice. Δ9-THC alone or <em>2</em>-AG alone significantly increased AEA and especially <em>2</em>-AG levels in WT hepatocytes. LKO alone markedly increased AEA and <em>2</em>-AG levels. However, LKO blocked/diminished the ability of Δ9-THC to further increase both AEA and <em>2</em>-AG. In contrast, LKO potentiated the ability of exogenous <em>2</em>-AG to increase the hepatocyte level of AEA and <em>2</em>-AG. These and other data suggest that Δ9-THC increases hepatocyte EC levels, at least in part, by upregulating endogenous AEA and <em>2</em>-AG levels. This may arise from Δ9-THC competing with AEA and <em>2</em>-AG binding to FABP1, thereby decreasing targeting of bound AEA and <em>2</em>-AG to the degradative enzymes, fatty acid amide hydrolase and monoacylglyceride lipase, to decrease hydrolysis within hepatocytes.

OBJECTIVE

Endogenous cannabinoids (endocannabinoids) in the periaqueductal grey (PAG) play a vital role in mediating stress-induced analgesia. This analgesic effect of endocannabinoids is enhanced by pharmacological inhibition of their degradative enzymes. However, the specific effects of endocannabinoids and the inhibitors of their degradation are largely unknown within this pain-modulating region.

METHODS

In vitro electrophysiological recordings were conducted from PAG neurons in rat midbrain slices. The effects of the major endocannabinoids and their degradation inhibitors on inhibitory GABAergic synaptic transmission were examined.

RESULTS

Exogenous application of the endocannabinoid, anandamide (AEA), but not <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), produced a reduction in inhibitory GABAergic transmission in PAG neurons. This AEA-induced suppression of inhibition was enhanced by the fatty acid amide hydrolase (FAAH) inhibitor, URB597, whereas a <em>2</em>-AG-induced suppression of inhibition was unmasked by the monoacylglycerol lipase (MGL) inhibitor, JZL184. In addition, application of the CB1 receptor antagonist, AM<em>2</em>51, facilitated the basal GABAergic transmission in the presence of URB597 and JZL184, which was further enhanced by the dual FAAH/MGL inhibitor, JZL195.

CONCLUSIONS

Our results indicate that AEA and <em>2</em>-AG act via disinhibition within the PAG, a cellular action consistent with analgesia. These actions of AEA and <em>2</em>-AG are tightly regulated by their respective degradative enzymes, FAAH and MGL. Furthermore, individual or combined inhibition of FAAH and/or MGL enhanced tonic disinhibition within the PAG. Therefore, the current findings support the therapeutic potential of FAAH and MGL inhibitors as a novel pharmacotherapy for pain.

Inflammation is an important part of the innate immune response and is involved in the healing of many disease processes; however, chronic inflammation is a harmful component of many diseases. The regulatory mechanisms of inflammation are incompletely understood. One possible regulatory mechanism is the endocannabinoid system. Endocannabinoids such as <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide (AEA) are generally anti-inflammatory via engagement of the cannabinoid receptor <em>2</em> (CB<em>2</em>) on innate cells; therefore, preventing the degradation of endocannabinoids by specific serine hydrolases such as fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL), and carboxylesterases (CES) might decrease inflammation. We hypothesized that the activities of these catabolic enzymes would decrease with a subsequent increase in <em>2</em>-AG and AEA in a model of inflammation. Mice were injected with lipopolysaccharide (LPS) for 6 or <em>2</em>4h, and inflammation was confirmed by an increase in interleukin-6 (il6) and il17 gene expression. Activity-based protein profiling (ABPP) of serine hydrolases showed no significant difference in various serine hydrolase activities in brain or liver, whereas a modest decrease in Ces activity in spleen after LPS administration was noted. <em>2</em>-AG hydrolase activity in the spleen was also decreased at 6h post LPS, which was corroborated by LPS treatment of splenocytes ex vivo. ABPP-MudPIT proteomic analysis suggested that the decreased <em>2</em>-AG hydrolysis in spleen was due to a reduction in Ces<em>2</em>g activity. These studies suggest that the endocannabinoid system could be activated via suppression of a <em>2</em>-AG catabolic enzyme in response to inflammatory stimuli as one mechanism to limit inflammation.

Parathion (PS) and chlorpyrifos (CPF) are organophosphorus insecticides (OPs) that elicit acute toxicity by inhibiting acetylcholinesterase (AChE). Endocannabinoids (eCBs, N-arachidonoylethanolamine, AEA; <em>2</em>-<em>arachidonoylglycerol</em>, <em>2</em>AG) can modulate neurotransmission by inhibiting neurotransmitter release. We proposed that differential inhibition of eCB-degrading enzymes (fatty acid amide hydrolase, FAAH, and monoacylglycerol lipase, MAGL) by PS and CPF leads to differences in extracellular eCB levels and toxicity. Microdialysis cannulae were implanted into hippocampus of adult male rats followed by treatment with vehicle (peanut oil, <em>2</em> ml/kg, sc), PS (<em>2</em>7 mg/kg) or CPF (<em>2</em>80 mg/kg) 6-7 days later. Signs of toxicity, AChE, FAAH and MAGL inhibition, and extracellular levels of AEA and <em>2</em>AG were measured <em>2</em> and 4 days later. Signs were noted in PS-treated rats but not in controls or CPF-treated rats. Cholinesterase inhibition was extensive in hippocampus with PS (89-90%) and CPF (78-83%) exposure. FAAH activity was also markedly reduced (88-91%) by both OPs at both time-points. MAGL was inhibited by both OPs but to a lesser degree (35-50%). Increases in extracellular AEA levels were noted after either PS (about <em>2</em>-fold) or CPF (about 3-fold) while lesser treatment-related <em>2</em>-AG changes were noted. The cannabinoid CB1 receptor antagonist/inverse agonist AM<em>2</em>51 (3mg/kg, ip) had no influence on functional signs after CPF but markedly decreased toxicity in PS-treated rats. The results suggest that extracellular eCBs levels can be markedly elevated by both PS and CPF. CB1-mediated signaling appears to play a role in the acute toxicity of PS but the role of eCBs in CPF toxicity remains unclear.

<em>2</em>-O-Arachidonoyl-1-O-stearoyl-sn-glycerol is the most abundant molecular species of the 1,<em>2</em>-diacyl-sn-glycerol signaling lipids in neural tissue. The facile preparation of <em>2</em>-O-[1'-(14)C]arachidonoyl-1-O-stearoyl-sn-glycerol from <em>2</em>-O-[1'-(14)C]arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine at a hexane and phosphate buffer interface with phospholipase C was demonstrated on a <em>2</em>0 µCi scale in 83% radiochemical yield. The specific activity of the product <em>2</em>-O-[1'-(14)C]arachidonoyl-1-O-stearoyl-sn-glycerol was 57.0 mCi/mmol and the radiochemical purity was determined to be >99% by TLC. The hydrolysis of this lipid biosynthetic intermediate with lipoprotein lipase was shown to produce <em>2</em>-O-[1'-(14)C]<em>arachidonoylglycerol</em> (<em>2</em>-AG). The (14)C-radiolabeled monoacylglycerol <em>2</em>-AG is an endogenous cannabinoid receptor-signaling molecule (endocannabinoid).

Mammalian diacylglycerol kinases are a family of enzymes that catalyze the phosphorylation of diacylglycerol to produce phosphatidic acid. The extent of interaction of these enzymes with monoacylglycerols is the focus of the present study. Because of the structural relationship between mono- and diacylglycerols, one might expect the monoacylglycerols to be either substrates or inhibitors of diacylglycerol kinases. This would have some consequence to lipid metabolism. One of the lipid metabolites that would be affected is <em>2</em>-arachidonoyl glycerol, which is an endogenous ligand for the CB1 cannabinoid receptor. We determined if the monoglycerides <em>2</em>-arachidonoyl glycerol or <em>2</em>-oleoyl glycerol affected diacylglycerol kinase activity. We found that <em>2</em>-arachidonoyl glycerol is a very poor substrate for either the epsilon or the zeta isoforms of diacylglycerol kinases. Moreover, <em>2</em>-arachidonoyl glycerol is an inhibitor for both of these diacylglycerol kinase isoforms. <em>2</em>-oleoyl glycerol is also a poor substrate for these two isoforms of diacylglycerol kinases. As an inhibitor, <em>2</em>-oleoyl glycerol inhibits diacylglycerol kinase ε less than does <em>2</em>-arachidonoyl glycerol, while for diacylglycerol kinase ζ, these two monoglycerides have similar inhibitory potency. These results have implications for the known role of diacylglycerol kinase ε in neuronal function and in epilepsy since the action of this enzyme will remove 1-stearoyl-<em>2</em>-<em>arachidonoylglycerol</em>, the precursor of the endocannabinoid <em>2</em>-arachidonoyl glycerol.

A simple and efficient synthesis of <em>2</em>-arachidonoyl glycerol, an endogenous agonist for cannabinoid receptors was achieved using Novozym 435, immobilized lipase from Candida Antarctica.

Follicle-stimulating hormone (FSH) is a glycoprotein that transmits its signals via a G protein-coupled receptor. As yet, not many targets of FSH have been identified, able to justify the critical role of this hormone on reproductive events. On the other hand, among the biological activities of the endocannabinoid anandamide (AEA), growing interest has been attracted by the regulation of mammalian fertility. Recently, we have shown that treatment of mouse primary Sertoli cells with FSH enhances the activity of the AEA hydrolase (fatty acid amide hydrolase, FAAH), whereas it does not affect the enzymes that synthesize AEA, nor the level of the AEA-binding type-<em>2</em> cannabinoid and type-1 vanilloid receptors. In addition, diacylglycerol lipase and monoacylglycerol lipase, which, respectively, synthesize and degrade the other major endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>, were not regulated by FSH. Interestingly, FAAH stimulation by FSH occurred through protein kinase A and aromatase-dependent pathways that were able to modulate FAAH activity (via phosphorylation of accessory proteins) and faah gene expression (via an estrogen response element on the promoter region). Taken together, these data identify FAAH as the only target of FSH among the elements of the endocannabinoid system, with a critical impact on Sertoli cell proliferation, and thus spermatogenesis and male reproduction.

A number of studies have examined the ability of the endogenous cannabinoid anandamide to elicit Δ(9)-tetrahydrocannabinol (THC)-like subjective effects, as modeled through the THC discrimination paradigm. In the present study, we compared transgenic mice lacking fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide catabolism, to wildtype counterparts in a THC discrimination procedure. THC (5.6 mg/kg) served as a discriminative stimulus in both genotypes, with similar THC dose-response curves between groups. Anandamide fully substituted for THC in FAAH knockout, but not wildtype, mice. Conversely, the metabolically stable anandamide analog O-181<em>2</em> fully substituted in both groups, but was more potent in knockouts. The CB1 receptor antagonist rimonabant dose-dependently attenuated THC generalization in both groups and anandamide substitution in FAAH knockouts. Pharmacological inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme for the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), with JZL184 resulted in full substitution for THC in FAAH knockout mice and nearly full substitution in wildtypes. Quantification of brain endocannabinoid levels revealed expected elevations in anandamide in FAAH knockout mice compared to wildtypes and equipotent dose-dependent elevations in <em>2</em>-AG following JZL184 administration. Dual inhibition of FAAH and MAGL with JZL195 resulted in roughly equipotent increases in THC-appropriate responding in both groups. While the notable similarity in THC's discriminative stimulus effects across genotype suggests that the increased baseline brain anandamide levels (as seen in FAAH knockout mice) do not alter THC's subjective effects, FAAH knockout mice are more sensitive to the THC-like effects of pharmacologically induced increases in anandamide and MAGL inhibition (e.g., JZL184).

Endocannabinoids are the endogenous ligands of the G protein-coupled cannabinoid receptors. The principal brain endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), is enzymatically produced by postsynaptic neurons and then activates presynaptic CB1 receptors in a retrograde manner. The primary pathway for <em>2</em>-AG generation is believed to be conversion from the diacylglycerols (DAGs) by two sn-1-specific lipases, DAGLα and DAGLβ. Previous studies with DAGL-deficient mice indicated that DAGLα is the major enzyme needed for retrograde synaptic <em>2</em>-AG signalling. The current study investigated whether the CB1 receptor-mediated Gi/o protein activity is altered in brain cryosections of DAGL-deficient mice when compared to wild-type mice and whether the sn-1-specific DAGLs are able to generate <em>2</em>-AG in brain cryosections. Functional autoradiography indicated that brain regional CB1 receptor-Gi/o-activity largely remained unaltered in DAGLα-knockout and DAGLβ-knockout mice when compared to wild-type littermates. Following comprehensive pharmacological blockade of <em>2</em>-AG hydrolysis, brain sections generated sufficient amounts of <em>2</em>-AG to activate CB1 receptors throughout the regions endowed with these receptors. As demonstrated by LC/MS/MS, this pool of <em>2</em>-AG was generated via tetrahydrolipstatin-sensitive enzymatic pathways distinct from DAGLα or DAGLβ. We conclude that in addition to the sn-1-specific DAGLs, additional <em>2</em>-AG generating enzymatic pathways are active in brain sections.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is the most abundant endogenous cannabinoid in the brain and an agonist at two cannabinoid receptors (CB1 and CB<em>2</em>). The synthesis, degradation and signaling of <em>2</em>-AG have been investigated in detail but its relationship to other endogenous monoacylglycerols has not been fully explored. Three congeners that have been isolated from the CNS are <em>2</em>-linoleoylglycerol (<em>2</em>-LG), <em>2</em>-oleoylglycerol (<em>2</em>-OG), and <em>2</em>-palmitoylglycerol (<em>2</em>-PG). These lipids do not orthosterically bind to cannabinoid receptors but are reported to potentiate the activity of <em>2</em>-AG, possibly through inhibition of <em>2</em>-AG degradation. This phenomenon has been dubbed the 'entourage effect' and has been proposed to regulate synaptic activity of <em>2</em>-AG. To clarify the activity of these congeners of <em>2</em>-AG we tested them in neuronal and cell-based signaling assays. The signaling profile for these compounds is inconsistent with an entourage effect. None of the compounds inhibited neurotransmission via CB1 in autaptic neurons. Interestingly, each failed to potentiate <em>2</em>-AG-mediated depolarization-induced suppression of excitation (DSE), behaving instead as antagonists. Examining other signaling pathways we found that <em>2</em>-OG interferes with agonist-induced CB1 internalization while <em>2</em>-PG modestly internalizes CB1 receptors. However in tests of pERK, cAMP and arrestin recruitment, none of the acylglycerols altered CB1 signaling. Our results suggest 1) that these compounds do not serve as entourage compounds under the conditions examined, and <em>2</em>) that they may instead serve as functional antagonists. Our results suggest that the relationship between <em>2</em>-AG and its congeners is more nuanced than previously appreciated.

Endocannabinoids - primarily anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) - are lipophilic molecules that bind to cannabinoid receptors (CB1 and CB<em>2</em>). They affect neuroendocrine activity inhibiting gonadotropin releasing hormone (GnRH) secretion and testosterone production in rodents, through a molecular mechanism supposed to be hypothalamus dependent. In order to investigate such a role, we choose the seasonal breeder, the anuran amphibian Rana esculenta, an experimental model in which components of the endocannabinoid system have been characterized. In February, at the onset of a new spermatogenetic wave, we carried out in vitro incubations of frog testis with AEA, at 10(-9)M dose. Such a treatment had no effect on the expression of cytochrome P450 17alpha hydroxylase/17,<em>2</em>0 lyase (cyp17) nor 3-β-hydroxysteroid dehydrogenase/Δ-5-4 isomerase (3β-HSD), key enzymes of steroidogenesis. To understand whether or not the functionality of the hypothalamus-pituitary axis could be essential to support the role of endocannabinoids in steroidogenesis, frogs were injected with AEA, at 10(-8)M dose. Differently from in vitro experiment, the in vivo administration of AEA reduced the expression of cyp17 and 3β-HSD. Whereas the effect on 3β-HSD was counteracted by SR141716A (Rimonabant) - a selective antagonist of CB1, thus indicating a CB1 dependent modulation - the effect on cyp17 was not, suggesting a possible involvement of receptors other than CB1, probably the type-1 vanilloid receptor (TRPV1), since AEA works as an endocannabinoid and an endovanilloid as well. In conclusion our results indicate that endocannabinoids, via CB1, inhibit the expression of 3β-HSD in frog testis travelling along the hypothalamus-pituitary axis.