Differential effects of single versus repeated alcohol withdrawal on the expression of endocannabinoid system-related genes in the rat amygdala

Background: Endogenous cannabinoids such as anandamide and 2‐arachidonoylglycerol (2‐AG) exert important regulatory influences on neuronal signaling, participate in short‐ and long‐term forms of neuroplasticity, and modulate stress responses and affective behavior in part through the modulation of neurotransmission in the amygdala. Alcohol consumption alters brain endocannabinoid levels, and alcohol dependence is associated with dysregulated amygdalar function, stress responsivity, and affective control. Methods: The consequence of long‐term alcohol consumption on the expression of genes related to endocannabinoid signaling was investigated using quantitative RT‐PCR analyses of amygdala tissue. Two groups of ethanol (EtOH)‐exposed rats were generated by maintenance on an EtOH liquid diet (10%): the first group received continuous access to EtOH for 15 days, whereas the second group was given intermittent access to the EtOH diet (5 d/wk for 3 weeks). Control subjects were maintained on an isocaloric EtOH‐free liquid diet. To provide an initial profile of acute withdrawal, amygdala tissue was harvested following either 6 or 24 hours of EtOH withdrawal. Results: Acute EtOH withdrawal was associated with significant changes in mRNA expression for various components of the endogenous cannabinoid system in the amygdala. Specifically, reductions in mRNA expression for the primary clearance routes for anandamide and 2‐AG (fatty acid amide hydrolase FAAH] and monoacylglycerol lipase MAGL], respectively) were evident, as were reductions in mRNA expression for CB₁, CB₂, and GPR55 receptors. Although similar alterations in FAAH mRNA were evident following either continuous or intermittent EtOH exposure, alterations in MAGL and cannabinoid receptor‐related mRNA (e.g., CB₁, CB₂, GPR55) were more pronounced following intermittent exposure. In general, greater withdrawal‐associated deficits in mRNA expression were evident following 24 versus 6 hours of withdrawal. No significant changes in mRNA expression for enzymes involved in 2‐AG biosynthesis (e.g., diacylglicerol lipase‐α/β) were found in any condition. Conclusions: These findings suggest that EtOH dependence and withdrawal are associated with dysregulated endocannabinoid signaling in the amygdala. These alterations may contribute to withdrawal‐related dysregulation of amygdalar neurotransmission.