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UNIVERSITY OF ILLINOIS AT CHICAGO Department of Psychiatry Fifth Annual Research Forum – Extravaganza 2014 POSTER TITLE ROLE OF EPIGENETIC REGULATION IN RAPID TOLERANCE TO ANXIOLYTIC EFFECTS OF ETHANOL EXPOSURE DISEASE/KEY WORDS: Alcohol Tolerance, Anxiety, Amygdala, Histone Acetylation, DNA methylation, Synaptic plasticity, BDNF AUTHORS: Harish R. Krishnan, Huaibo Zhang, Amul J. Sakharkar, and Subhash C. Pandey MENTEE CATEGORY: Research Associate BACKGROUND: Epigenetic regulation of neuronal pathways has been shown to regulate the transcriptome and modify behavioral outcomes in response to abused drugs such as ethanol. We have shown that rats display an anxiolytic response after acute ethanol exposure, whereas a subsequent second exposure produces rapid tolerance to this effect. Here we investigated whether epigenetic regulation at the level of DNA methylation and histone acetylation of the synaptic plasticity associated genes, activity-regulated cytoskeleton-associated protein (Arc) and brain-derived neurotrophic factor (BDNF) play a role in mediating these responses. We measured the effects of one (1g/kg intraperitoneal) and two doses of ethanol (1g/kg; 24h apart) on anxiety-like behaviors in adult male Sprague-Dawley rats. Following ethanol administration, the rats were sacrificed and the amygdala collected for the following biochemical measures. DNA Methyltransferase (DNMT) activity measurement, evaluation of 5-methylcytosine levels at the gene promoters of Arc and BDNF using the MethylMiner™ protocol followed by quantitative real-time PCR. Evaluation of AcH3 using Chromatin immunoprecipitation (ChIP) assays at the same promoters. BDNF and Arc mRNA and protein measurement using in situ PCR and Gold Immunolabeling. We also performed central amygdaloid infusion of BDNF in rats that received two doses of ethanol. It was found that a single ethanol exposure produced anxiolytic behavioral effects whereas two doses of ethanol normalized the anxiety-like behavior back to control levels indicating tolerance to the effect. One dose of ethanol decreased DNMT activity, which correlated with decreased 5-Methylcytosine (5mc) levels within specific promoter regions of Arc and BDNF (Exons I & IV). There were also increased acetylated-H3 (AcH3 K9-14) levels in these same regions. Additionally Arc and BDNF mRNA and protein levels were found to be increased, which substantiate the above epigenetic findings. Interestingly, after two doses of ethanol (tolerance group), we observed that amygdaloid DNMT activity and corresponding 5mc levels at the promoters of Arc and BDNF (Exons I & IV) remained decreased in the tolerance group. On the other hand AcH3 levels at the promoters of Arc and BDNF (Exons I & IV) normalized to control levels and this also resulted in the normalization of mRNA and protein levels of these METHODS: RESULTS: RESEARCH MENTOR: Subhash C. Pandey UNIVERSITY OF ILLINOIS AT CHICAGO Department of Psychiatry genes. However, central amygdaloid infusion of BDNF was able to circumvent the tolerant phenotype and cause anxiolytic effects even after two doses of ethanol, thus mimicking rats that received one dose. CONCLUSIONS: Acute ethanol leads to the following epigenetic changes at the promoters of Arc and BDNF decreased DNA methylation and increased histone H3 acetylation, resulting in increased mRNA and protein levels of Arc and BDNF, thereby regulating synaptic plasticity leading to the anxiolytic response. Interestingly in the tolerance group the mRNA and protein levels of Arc and BDNF are normalized, and we show that histone acetylation, rather than DNA methylation, could be the predominant factor responsible for this effect. This leads to the development of rapid tolerance to anxiolytic effects of ethanol. However we were able to circumvent this by increasing BDNF through Central Amygdaloid exogenous infusion, which abolished the tolerance phenotype leading to anxiolytic effects. This study has revealed the importance of epigenetic signaling pathways in the amygdala that presumably play a role in regulating tolerance to anxiolytic-like effects following acute ethanol exposure (Supported by NIH-NIAAA grants and VA Merit and Career Scientist Grants to SCP).