2/8 NADIA U01 Adolescent Alcohol Exposure: Neural Contributors to Sex- and Exposure Timing-Specific Anxiety

Research Component 2/8: Adolescent Alcohol Exposure: Neural Contributors to Sex- and Exposure Timing-Specific Anxiety

Principal Investigators: David F. Werner, Ph.D., Binghamton University, and Elena I. Varlinskaya, Ph.D., Binghamton University

Abstract

Our current work in the NIAAA consortium on the Neurobiology of Adolescent Drinking in Adulthood (NADIA) has revealed that long-lasting behavioral consequences of adolescent intermittent ethanol exposure (AIE) are sex- and exposure timing-dependent. When tested in adulthood, only male rats exposed to ethanol during early-mid adolescence (early AIE) demonstrate social alterations that include social anxiety and enhanced sensitivity to ethanol-induced social facilitation, with exposure to ethanol later in adolescence (late AIE) having no such consequences. Our most important translational finding is that a selective oxytocin receptor (OXTR) agonist reverses the male-specific social anxiety. At the cellular and molecular levels, sex-specific consequences of early AIE are evident as alterations in dendritic spine morphology and decreases in OXTR mRNA and protein expression in the hypothalamus. Our current proposal will address critical gaps arising from this work. Aim 1 will identify socially relevant regions differentially activated in adult males and females by social stimuli following early AIE and will test whether recruitment of neuronal ensembles in identified brain regions is required for male-specific social affective alterations. Aim 2 will further assess AIE-induced alterations of the OXT system contributing to male-specific social anxiety and will investigate neural mechanisms underlying the reversal effects of OXTR pharmacological activation. Aim 3 is designed to test whether AIE selectively disrupts epigenetic regulation of the OXT neuromodulatory peptide system in brain regions critical for normal social functioning.

Specific Aims

Our prior work has revealed that long-lasting behavioral consequences of adolescent intermittent ethanol exposure (AIE) are sex- and exposure timing-dependent. Specifically, when tested in adulthood, male (but not female) rats exposed to ethanol during early-mid adolescence (early AIE) demonstrate social affective alterations, with exposure to ethanol later in adolescence/emerging adulthood (late AIE) having no such consequences. Acute ethanol is able to diminish or even reverse social affective alterations evident in males following early AIE at doses that produce social inhibition in controls, suggesting enhanced sensitivity to socially facilitating and/or socially anxiolytic effects of ethanol that might make ethanol more appealing for socially anxious males. At the cellular level, sex-specific consequences of early AIE are evident as alterations in dendritic spine morphology in the lateral septum, with AIE decreasing spine density in males and increasing it in females. We have found that AIE decreases oxytocin receptor (OXTR) protein expression in the hypothalamus of males, but not females, suggesting that the OXT brain system is affected by AIE in a sex-specific manner. We have demonstrated that a selective OXTR agonist reverses male-specific social affective alterations as well as alterations in glutamate receptor expression. Since NADIA’s inception, this is our component’s most important translational finding, as OXT has been shown to improve social dysfunction in humans. Given our previous findings, we will further focus on neural contributors to the sex-specific consequences of early AIE with a particular emphasis on the contribution of the brain OXT system to this male-specific social anxiety. Our central hypothesis is that early AIE produces persistent, but reversible alterations of the OXT neuromodulatory peptide system in social brain regions of males, but not females, through epigenetic mechanisms, with pharmacological activation of the OXT system as well as modulation of epigenetic methylation effectively reversing social alterations associated with AIE. The following Aims will directly test this hypothesis.

AIM 1. Identify neural contributors to sex-specific social affective alterations associated with early AIE.

Our findings suggest that neural systems implicated in regulation of social interaction are more sensitive to repeated ethanol exposure during early adolescence in males than in females, with this sex difference in sensitivity to ethanol resulting in long-lasting sex-specific neural alterations in brain regions associated with social interaction and modulated by OXT. Therefore, we hypothesize that patterns of neural activation in the social brain regions will be altered by early AIE in males, but not in females. To test this hypothesis, transgenic cFos-LacZ rats exposed to ethanol during early-mid adolescence will be used to identify socially relevant regions differentially activated in adult males and females by social stimuli. Using inactivation procedures, we will further assess whether recruitment of neuronal ensembles in these identified brain regions is required for male-specific social affective alterations associated with early AIE. In this aim, we will also test whether an acute dose of ethanol that restores some social deficits in males following early AIE can also attenuate alterations in the pattern of neural activation.

AIM 2. Further assess AIE-induced alterations of the OXT system contributing to male-specific social anxiety and investigate neural mechanisms underlying the reversal effects of OXTR pharmacological activation. We will test whether pharmacological activation of the OXT system not only reverses social anxiety but also normalizes patterns of neural activation. Focusing on brain regions identified in Aim 1, fluorescence in situ hybridization (FISH) via RNAScope will be used to assess alterations in OXTR mRNA co-localization with c-fos in activated neurons and to explore whether the selective OXTR agonist can reverse these alterations. The ability of the OXTR agonist to reverse changes in dendritic spine morphology will also be examined. In collaboration with Component 5, we will also to test whether pharmacological activation of the OXT system can reverse AIE-associated increases in expression of neuroimmune markers previously reported by the NADIA in brain regions associated with social anxiety.

AIM 3. Determine whether epigenetic dysregulation of the OXT system is a major contributor to AIE-induced social anxiety. Our preliminary data indicated AIE-associated reductions in hypothalamic OXTR, suggesting impairment of the OXT modulatory role. We will test whether methylation-related suppression of OXTR is responsible for AIE-induced social alterations. RNA-Seq will be used to further assess sex-specific consequences of early AIE, and ATAC-Seq will be used to identify epigenetically regulated gene network pathways and to assess whether OXTR and other sex-specific genes are in “closed’ regions of the genome. Further enrichment studies will be performed to validate histone versus DNA methylation. We will also test whether DNMT inhibition will reverse AIE-associated social affective alterations. Finally, we will use viral-mediated CRISPR/dCas9 approaches to silence OXTR activity in order to mimic AIE-induced social deficits in non-exposed animals, as well as to reverse OXTR methylation following early AIE.