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Research Component 4: GABAergic Corticolimbic Circuit Mechanisms of Ethanol Dependence 

Primary Investigator: Dr. Leslie Morrow

Co-Investigator: Dr. Melissa Herman

Chronic ethanol exposure induces dependence-like behavior and produces receptor subtype-specific changes in GABA-mediated inhibition and sensitivity to GABAA receptor modulators. The pathological consequences of these changes include heightened CNS excitability, dysphoria, anxiety, and tremor. We previously demonstrated that ethanol exposure promotes internalization of synaptic GABAA a1g2 receptors and extrasynaptic GABAA a4d receptors, while increasing surface expression of synaptic a4g2 receptors. These changes are accompanied by adaptations in synaptic and tonic inhibition. Further, preliminary data show that chronic ethanol effects on synaptic a1 receptors are prevented by histone deacetylase (HDAC) inhibitors, including trichostatin A (TSA), which also prevented dependence-associated anxiety-like behavior. It is unknown, however, if these adaptations in GABAA receptor expression and function lead to altered circuit function in the brain. Current models of alcohol addiction implicate prefrontal cortex (PFC) and amygdala dysregulation and subsequent loss of inhibitory control in the development of dependence. The overall goal of this component is to test the hypothesis that chronic ethanol exposure alters local inhibition and produces GABAA receptor adaptations in medial prefrontal cortex (mPFC) and central amygdala (CeA) as well as mPFC à CeA circuitry using electrophysiological and molecular methods. We will further examine if HDAC inhibition prevents ethanol-induced changes in GABAA receptor circuit function as a potential new avenue for therapeutic intervention.

Specific Aim 1. Test the hypothesis that chronic ethanol exposure alters prelimbic mPFC inhibitory microcircuits in a TSA-dependent manner in ethanol-dependent male and female rats. We will investigate phasic and tonic inhibition in GABAergic microcircuits in mPFC Layer V/VI prelimbic principal neurons and interneurons. Phasic inhibition will be measured by inhibitory postsynaptic currents (IPSCs) and tonic inhibition will be measured by the change in holding current with the GABAA receptor antagonist gabazine. GABAA receptor gene/protein expression will be measured by qPCR and Western blot analysis following microdissection. Interneurons will be differentiated using single cell qPCR to identify parvalbumin, somatostatin, or calretinin markers. Effects of the HDAC inhibitor TSA on GABAA receptor expression and microcircuit function will be assessed. We predict that ethanol will alter GABAA receptor expression and microcircuit function in the mPFC, which will be sensitive to the HDAC inhibitor TSA.

Specific Aim 2. Test the hypothesis that chronic ethanol exposure alters CeA microcircuit function via changes in local GABAA receptor expression that can be prevented by HDAC inhibition in male and female rats. Previous data from Dr. Herman demonstrated that ethanol dependence produces cell type-specific reductions in phasic and tonic GABAA receptor inhibition in the CeA. Preliminary data suggest that these effects are associated with concomitant changes in synaptic and extrasynaptic a1 and δ GABAA receptor subunit expression. We propose to: 1) examine ethanol-induced changes in local phasic and tonic inhibition and the expression of synaptic and extrasynaptic GABAA receptors in CeA neurons and 2) determine if HDAC inhibition prevents the receptor and circuit adaptations observed following chronic ethanol exposure. We predict that chronic ethanol exposure will produce parallel changes in GABAA receptor expression and function that will be prevented by TSA.

Specific Aim 3. Test the hypothesis that chronic ethanol exposure alters the activity and functional connectivity of the mPFC à CeA projection in male and female rats in a TSA-dependent manner. We will examine the effects of chronic ethanol exposure in the absence or presence of TSA on the inhibitory control and intrinsic excitability of retrobead-labeled prelimbic mPFC Layer V/VI neurons that project to CeA. Inhibitory control will be assessed by baseline tonic and phasic inhibition while changes in spiking characteristics will be used to measure intrinsic excitability. In collaboration with the Scientific Resource Core, we will further probe changes in mPFC-amygdala functional connectivity following ethanol exposure ± TSA by resting state functional connectivity magnetic resonance imaging (rs-fcMRI). This analysis will extend our cellular findings into an intact, in vivo system as well as contribute to the ARC-wide assessment of functional connectivity across species and models of ethanol pathogenesis. We predict that chronic ethanol exposure will enhance functional connectivity between prelimbic mPFC and CeA, due to a loss of inhibition in both mPFC and amygdala, predicted by our previous studies.

These studies will delineate the molecular and circuit mechanisms relating to ethanol dependence in local mPFC and amygdalar microcircuits as well as the mPFC projection to amygdalar subregions. These studies may lead to microcircuit and molecular targets for reversal of ethanol dependence pathology.