Aberrant HDAC2-Mediated Histone Modifications and Synaptic Plasticity in the Amygdala Predisposes to Anxiety and Alcoholism

Background— Epigenetic mechanisms have been implicated in psychiatric disorders, including alcohol dependence. However, the epigenetic basis and role of specific histone deacetylase (HDAC) isoforms in the genetic predisposition to anxiety and alcoholism is unknown. Methods— We measured amygdaloid HDAC activity, levels of HDAC isoforms and histone H3 acetylation in selectively-bred alcohol-preferring (P) and -nonpreferring (NP) rats. We employed HDAC2 siRNA infusion into the central nucleus of amygdala (CeA) of P rats to determine the causal role of HDAC2 in anxiety-like and alcohol-drinking behaviors. Chromatin immunoprecipitation analysis was performed to examine the histone acetylation status of brain-derived neurotrophic factor (BDNF) and activity-regulated cytoskeleton associated protein (Arc) genes. Golgi-Cox staining was performed to measure dendritic spine density. Results— We found that P rats innately display higher nuclear HDAC activity and HDAC2, but not HDAC 1, 3, 4, 5, and 6 protein levels, and lower acetylation of H3-K9, but not H3-K14, in the CeA and medial nucleus of amygdala (MeA) compared with NP rats. Acute ethanol exposure decreased amygdaloid HDAC activity and HDAC2 protein levels, increased global and gene (BDNF and Arc)-specific histone acetylation and attenuated anxiety-like behaviors in P rats, but had no effects in NP rats. HDAC2 knockdown in the CeA attenuated anxiety-like behaviors and voluntary alcohol, but not sucrose, consumption in P rats and increased histone acetylation of BDNF and Arc with a resultant increase in protein levels that correlated with increased dendritic spine density. Conclusions— These novel data demonstrate the role of HDAC2-mediated epigenetic mechanisms in anxiety and alcoholism. As observed, higher expression of HDAC2 and lower acetylation of histone H3-K9, but not H3-K14 may be responsible for deficits in BDNF and Arc expression in the amygdaloid structures of P as compared with NP rats. Ethanol also inhibited HDAC activity, reduced HDAC2 protein levels and increased histone H3-K9 acetylation in the amygdaloid brain regions of P but not NP rats. These results are clearly suggestive of a specific role for CeA HDAC2-induced chromatin remodeling in mediating anxiety-like and alcohol drinking behaviors in a manner that could be related to downstream changes in BDNF-Arc signaling and dendritic spines. Our data also suggest that HDAC2-induced chromatin remodeling in the CeA and MeA, but not BLA may be involved in controlling baseline anxiety or anxiolytic effects of ethanol in P rats compared to NP rats. Recently, an optogenetic study indicated that manipulations of BLA projections in the CeA, but not the BLA somata, were able to control anxiety-like behaviors Also, abnormal neuropeptide Y (NPY) and GABA A α 2-regulated systems in CeA have been implicated in anxiety-like and alcohol-drinking behaviors of P rats (14, 48, 68). Future studies are needed to evaluate the epigenetic regulation of these and other genes in the amygdaloid circuitry of P rats contributing to anxiety and alcoholism. histone H3-K9: CeA, F 2, 17 = 70.0, p<0.001). HDAC2 and protein levels were lower in the CeA, but not MeA or BLA, of P rats infused with HDAC2 siRNA than those infused with vehicle or control siRNA. These results confirm that HDAC2 siRNA infusion causes a significant reduction in HDAC2 mRNA levels in the CeA. HDAC2 siRNA infusion into CeA also increased the levels of histone H3-K9 acetylation in the CeA, but not MeA or BLA. Values are represented as the mean ± SEM derived from 5–7 P rats per group. *Significantly different from control groups (p<0.001; one-way ANOVA followed by post hoc analysis by Tukey’s test).