Alcohol use disorder (AUD) is a major health concern in the VA population as well as in the general population. An integral part of the mission of the VA is to provide care for VA patients suffering from alcohol and/or drug abuse problems. Cessation of chronic ethanol consumption leads to development of negative withdrawal symptoms that promote increased alcohol drinking and maintain alcohol addictive behaviors. Amygdaloid brain structures are known to play an important role in anxiety behaviors as well as in alcohol addiction. The molecular mechanisms, and particularly the role of non-coding microRNAs (miRNAs; 21-23 nt), in specific neural circuits of the brain that are involved in the development of anxiety-like behaviors during ethanol withdrawal after chronic ethanol exposure are not well understood. Using microarray analysis, we recently reported that acute ethanol exposure produces differential miRNA expression in the amygdala. This data revealed that expression of miRNA-494 (miR-494) was decreased by acute ethanol exposure and its target gene mRNA levels were increased and involved in the regulation of anxiolytic effects of ethanol. This proposal will extend these studies and establish the role of miR-494 in the regulation of chromatin and synaptic remodeling in the amygdala, leading to negative affective state of alcoholism. In addition, this proposal will identify changes in the expression of other miRNAs during alcohol dependence and then propose experiments to investigate how these changes in miRNAs coordinate shifts in the expression of target gene pathways that regulate anxiety-like behaviors during withdrawal after chronic ethanol exposure. We hypothesize that ethanol withdrawal after chronic exposure will produce differential expression of several miRNAs, and one of these may be miR-494. The upregulation of miR-494 will induce changes in target genes that regulate synaptic plasticity and dendritic spines in the amygdala which may underlie the development of anxiety-like behaviors during ethanol withdrawal. The specific aims of the proposal are: 1) To perform miRNA expression profiling using a microarray approach in the amygdala of rats during ethanol withdrawal after chronic ethanol exposure. The emerging miRNAs will be validated and changes in the expression of their putative target genes will also be examined. 2) To examine if a) miR-494 antagomir infusion in the CeA or acute ethanol challenge will attenuate anxiety-like behaviors, deficits in the expression (mRNA and protein) of direct targets (Cbp, p300, Cited2) and indirect targets (Bdnf, Arc, and synaptophysin) as well as deficits in synapses and dendritic spines in the amygdala of rats during ethanol withdrawal after chronic ethanol exposure and b) synthetic mimics of miR-494 infused into CeA of control rats will provoke anxiety-like behaviors and reduce the expression of target genes in the amygdala. 3) To examine if the effects of CeA infusion of a Cited2 siRNA in control rats will a) produce anxiety-like behaviors, thus mimicking withdrawal- associated anxiety and b) prevent the anxiolytic effects produced by acute ethanol in control rats. Behavioral changes will be due to decreased expression of Cited2 and associated reductions in synapses and dendritic spines in the CeA of rats. The proposed studies will provide new information on the regulation of miRNA expression and their target genes in the amygdala that may be involved in the abnormal synaptic plasticity observed during alcohol dependence, and will also identify novel miRNA targets for the development of potential drugs for the treatment of AUD.

Public Health Relevance: Alcohol use and anxiety disorders are major health concerns in the VA population. Veterans suffering from anxiety disorders alone or in combination with post traumatic stress disorder are more prone to developing alcoholism and drug addiction. This proposal will enhance our understanding of the molecular mechanisms of alcohol dependence and will identify molecular and epigenetic targets that can be used to develop future therapeutic agents for treating alcohol use disorders (AUD) with and without anxiety disorders.