Stimulation of cannabinoid receptor agonist 2-arachidonylglycerol by chronic ethanol and its modulation by specific neuromodulators in cerebellar granule neurons

AbstractIn an earlier study, we reported that chronic ethanol (EtOH) stimulates the formation of anandamide in human SK-N-SH cells. In the present study, we investigated the effect of chronic EtOH on the formation of yet another cannabinoid receptor (CB1) agonist, 2-arachidonylglycerol (2-AG), in cerebellar granule neurons (CGNs). The formation of 2-[3H]AG without any stimulation was more pronounced in the older cultures than in younger cultures. Exposure of CGNs to EtOH led to a significant increase in the level of 2-[3H]AG (P<0.05). Incubation with the anandamidehydrolase inhibitor phenylmethylsulfonyl fluoride and EtOH did result in an additive increase in 2-[3H]AG, but did not with E-6-(bromomethylene)tetrahydro-3-(1-naphthelenyl)-2H-pyran-2-one. The formation of 2-[3H]AG was enhanced by ionomycin in both the control and EtOH-exposed CGNs, and the ionomycin-stimulated 2-[3H]AG synthesis was inhibited by the intracellular chelating agent 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. Further, glutamate increased the formation of 2-[3H]AG only in control CGNs. MK-801 inhibited the EtOH-induced 2-[3H]AG synthesis, suggesting the participation of intracellular Ca2+ in EtOH-induced 2-[3H]AG synthesis. The dopamine receptor (D2) agonist did not modify the 2-AG synthesis in either the control or EtOH-exposed CGNs. However, the D2 receptor antagonist inhibited the EtOH-induced formation of 2-[3H]AG. The EtOH-induced 2-[3H]AG formation was inhibited by SR141716A and pertussis toxin, suggesting the CB1 receptor- and Gi/o-protein-mediated regulation of 2-AG. The observed increase in 2-AG level in CGNs is possibly a mechanism for neuronal adaptation to the continuous presence of EtOH. These findings indicate that some of the pharmacological actions of EtOH may involve alterations in the endocannabinoid signaling system

Dysfunction in Fatty Acid Amide Hydrolase Is Associated with Depressive-Like Behavior in Wistar Kyoto Rats

BACKGROUND: While the etiology of depression is not clearly understood at the present time, this mental disorder is thought be a complex and multifactorial trait with important genetic and environmental contributing factors. METHODOLOGY/PRINCIPAL FINDINGS: The role of the endocannabinoid (eCB) system in depressive behavior was examined in Wistar Kyoto (WKY) rat strain, a genetic model of depression. Our findings revealed selective abnormalities in the eCB system in the brains of WKY rats compared to Wistar (WIS) rats. Immunoblot analysis indicated significantly higher levels of fatty acid amide hydrolase (FAAH) in frontal cortex and hippocampus of WKY rats with no alteration in the level of N-arachidonyl phosphatidyl ethanolamine specific phospholipase-D (NAPE-PLD). Significantly higher levels of CB1 receptor-mediated G-protein coupling and lower levels of anandamide (AEA) were found in frontal cortex and hippocampus of WKY rats. While the levels of brain derived neurotropic factor (BDNF) were significantly lower in frontal cortex and hippocampus of WKY rats compared to WIS rats, pharmacological inhibition of FAAH elevated BDNF levels in WKY rats. Inhibition of FAAH enzyme also significantly increased sucrose consumption and decreased immobility in the forced swim test in WKY rats. CONCLUSIONS/SIGNIFICANCE: These findings suggest a critical role for the eCB system and BDNF in the genetic predisposition to depressive-like behavior in WKY rats and point to the potential therapeutic utility of eCB enhancing agents in depressive disorder

SPECIAL ISSUE ARTICLE ROLE OF THE ENDOCANNABINOID SYSTEM IN THE DEVELOPMENT OF TOLERANCE TO ALCOHOL

Abstract — The present review evaluates the evidence that the endocannabinoid system plays in the development of tolerance to alcohol. The identification of a G-protein-coupled receptor, namely, the cannabinoid receptor (CB 1 receptor), which was activated by ∆ 9-tetrahydrocannabinol ( ∆ 9-THC), the major psychoactive component of marijuana, led to the discovery of endogenous cannabinoid agonists. Until now, four fatty acid derivatives identified to be arachidonylethanolamide (AEA), 2-arachidonylglycerol (2-AG), 2-arachidonylglycerol ether (noladin ether) and virodhamine have been isolated from both nervous and peripheral tissues. Both AEA and 2-AG have been shown to mimic the pharmacological and behavioural effects of ∆ 9-THC. The role of the endocannabinoid system in the development of tolerance to alcohol was not known until recently. Recent studies from our laboratory have implicated for the first time a role for the endocannabinoid system in development of tolerance to alcohol. Chronic alcohol treatment has been shown to down-regulate CB 1 receptors and its signal transduction. The observed downregulation of CB 1 receptor function results from the persistent stimulation of the receptors by AEA and 2-AG, the synthesis of which has been shown to be increased by chronic alcohol treatment. The enhanced formation of endocannabinoids may subsequently influence the release of neurotransmitters. It was found that the DBA/2 mice, known to avoid alcohol intake, have significantly reduced CB 1 receptor function in the brain, consistent with other studies in which the CB 1 receptor antagonist SR 141716A has been shown to block voluntary alcohol intake in rodents. Similarly, activation of the CB 1 receptor system promoted alcohol craving, suggesting a role for the C

Manipulation of fatty acid amide hydrolase functional activity alters sensitivity and dependence to ethanol

The aim of this study was to examine the role of fatty acid amide hydrolase (FAAH) on ethanol sensitivity, preference, and dependence. The deletion of FAAH gene or the inhibition of FAAH by carbamoyl-biphenyl-3-yl-cyclohexylcarbamate (URB597) (0.1 mg/kg) markedly increased the preference for ethanol. The study further reveals that URB597 specifically acts through FAAH and that cannabinoid-1 (CB1) receptor is critical for N-arachidonoyl ethanolamide (AEA) mediated ethanol-reinforced behavior as revealed by lack of URB597 effect in both FAAH and CB1−/− mice compared with vehicle-treated −/− mice. The FAAH −/− mice displayed a lower sensitivity to hypothermic and sedative effects to acute ethanol challenge. The FAAH −/− mice also exhibited a reduction in the severity of handling-induced convulsions following withdrawal from chronic ethanol exposure. The CB1 receptor and proenkephalin gene expressions, and CB1 receptor and μ-opioid (MO) receptor-mediated G-protein activation were found to be significantly lower in the caudate-putamen, nucleus accumbens core and shell of FAAH −/− than +/+ mice. Interestingly, the MO receptor-stimulated G-protein signaling was greater in the striatum of FAAH −/− than +/+ mice following voluntary ethanol consumption. These findings suggest that an elevation in the AEA content and its action on the limbic CB1 receptor and MO receptor might contribute to ethanol-reinforced behavior. Treatment with drugs that decrease AEA tone might prove useful in reducing excessive ethanol consumption.This study was supported by grants from NIH (AA13003 and AA22008).Peer reviewe

Effect of FAAH inhibition on depressive-like behavior in WKY rats.

<p>Treatment with URB597 (0.3 mg/kg, i.p. for 7 days) elicited a significant decrease in total time spent in immobility (50%, p<0.01; A) and a marked increase in sucrose intake (48%, p<0.05; B) without any effect on the spontaneous locomotor activity in the open field (C) in WKY rats compared to vehicle treated WKY rats.</p

Effect of FAAH inhibition on BDNF, AEA and CB1 function in the brain of WKY rats.

<p>Basal BDNF levels were found to be significantly lower in frontal cortex (27%) and hippocampus (26%) of WKY compared to WIS rats (p<0.05; A). Subchronic treatment with URB597 (0.3 mg/kg, i.p. for 7 days) significantly elevated BDNF levels in frontal cortex (64%) and hippocampus (45%) of WKY rats compared to vehicle treated WKY rats (p<0.05; B). Inhibition of FAAH was accompanied by significant increase in AEA levels in frontal cortex (31%, p<0.01; C) and hippocampus (42%, p<0.001; C), and a subsequent decrease in CB1 receptor-mediated G-protein activation in frontal cortex of WKY rats (21%, p<0.05; D). Hippo; Hippocampus.</p

Basal differences in CB1 receptor in the brain of WKY rats.

<p>The CB1 receptor agonist-stimulated [³⁵S]GTPγS binding was significantly higher in frontal cortex (24%, p<0.05) and hippocampus (44%, p<0.01) of WKY rats compared to WIS rats (A). Data is presented as percentage of stimulation over basal binding. Western blot analysis revealed significantly higher levels of CB1 receptors in hippocampus (45%, p<0.05), while they were found to be slightly higher in frontal cortex of WKY rats (18%, B). Hippo; Hippocampus.</p

Basal differences in AEA and FAAH levels in the brain of WKY rats.

<p>The level of eCB, AEA was found to be significantly lower in hippocampus of WKY rats compared to WIS rats (31%, p<0.01; A). Conversely, the level of FAAH enzyme was significantly higher in frontal cortex (40%, p<0.05) and hippocampus (40%, p<0.05; B) of WKY rats. A representative immunoblot for hippocampus is provided in the upper panel (B). The qPCR analysis also indicated higher levels of mFAAH in hippocampus of WKY rats (24%, p<0.05; C). The qPCR data on FAAH, normalized to β-Actin (internal standard) is presented as the fold change relative to the control value of 1.0. The FAAH activity was slightly higher in frontal cortex (15%, p<0.05) and hippocampus (17%, p<0.05) of WKY rats compared to WIS rats (D). Hippo; Hippocampus.</p

Basal levels of NAPE-PLD in the brain of WKY rats.

<p>There were no significant differences in the levels of NAPE-PLD enzyme in frontal cortex and hippocampus of WKY rats compared to WIS rats (A). A representative immunoblot for hippocampus is provided in the upper panel (B). Hippo; Hippocampus.</p

Innate difference in the endocannabinoid signaling and its modulation by alcohol consumption in alcohol-preferring sP rats

The present study was undertaken to examine whether genetically predetermined differences in components of the endocannabinoid system were present in the brain of Sardinian alcohol-preferring (sP) and Sardinian alcohol-non-preferring (sNP) rats, a pair of rat lines selectively bred for opposite alcohol preference. The effects of acquisition and maintenance of alcohol drinking, alcohol withdrawal, and alcohol re-exposure on the endocannabinoid system was also assessed in the striatum of sP rats. The findings revealed significantly higher density of the CB1 receptors and levels of CB1 receptor mRNA, CB1 receptor-mediated G-protein coupling, and endocannabinoids in the cerebral cortex, hippocampus and striatum of alcohol-naive sP rats than sNP rats. A significantly lower expression of mFAAH enzyme was evident in the hippocampus of alcohol-naive sP rats. Alcohol drinking (during both acquisition and maintenance phases) in sP rats resulted in a significant reduction in striatal CB1 receptor-mediated G-protein coupling whereas alcohol withdrawal attenuated this effect. Alcohol consumption was also associated with markedly increased levels of endocannabinoids in the striatum. Co-administration of the CB1 receptor antagonist, rimonabant (SR141716A) reduced alcohol intake, and reversed alcohol-induced changes in CB1 receptor-mediated G-protein activation. These findings provided a new insight into a potential genetic basis of excessive alcohol consumption, suggesting innate differences in the endocannabinoid system might be associated with higher alcohol preference in sP rats. The data also indicate a modulation of CB1 receptor-mediated signaling following alcohol consumption, and further strengthen the potential of the endocannabinoid system as a target for the treatment of alcohol related behaviors.This study was supported by grants from NIH (AA015525) to BLH and RETICS (Red de Trastornos Adictivos; RD06/0001/1004, Spain) to JM. MSG was supported by a fellowship from the Spanish Ministry of Health.Peer reviewe