Download Role of Epigenetic Regulation in Rapid Tolerance to Anxiolytic

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).