Date of Award

Summer 7-26-2023

Embargo Period

8-8-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Neuroscience

College

College of Graduate Studies

First Advisor

L. Judson Chandler

Second Advisor

Michael D. Scofield

Third Advisor

John J. Woodward

Fourth Advisor

Jim S. Otis

Fifth Advisor

Mario A. Penzo

Abstract

Dependence is a hallmark of alcohol use disorder characterized by excessive alcohol intake and negative withdrawal symptoms. The central nucleus of the amygdala (CeA) is a key brain structure necessary for synaptic and behavioral consequences of ethanol dependence. Accumulating evidence suggests that astrocytes regulate synaptic transmission and behavior, however, a lack a basic understanding of the role of astrocytes in ethanol dependence. Therefore, the following experiments examined the effects of chronic intermittent ethanol (CIE) exposure on astrocyte-neuron interactions in the CeA. Astrocytes, the synaptic marker, synaptojanin1 (SJ1), and astrocytic GABA transporter, GAT3, were immunolabeled and analyzed using super-resolution confocal microscopy and Imaris software. Co-registration of SJ1 with the astrocyte surface was quantified as an index of synaptic proximity. Co-registration with GAT3 was quantified to determine astrocytic expression of GAT3. Triple co-registration of astrocyte-SJ1-GAT3 was quantified to determine the proportion of GAT3-containing astrocyte processes proximal to the synapse. CIE-exposed rats exhibited increased synaptic proximity, GAT3 expression, and increased synaptic proximity of GAT3+ astrocyte processes, indicating that, in the CeA, CIE exposure promotes protrusion of GAT3-containing astrocyte processes toward synapses. Increased synaptic proximity and GAT3 expression was also observed 7 days into withdrawal, suggesting CIE induces persistent alterations in structural astrocytic plasticity. Further, voluntary ethanol intake during withdrawal was positively associated with higher synaptic proximity and GAT3 expression in CIE-exposed rats. Complementary to the morphological data, in vitro slice electrophysiology demonstrated that CIE-exposed rats exhibit indices of increased GABA release, increased tonic current, and increased GAT3-mediated current, supporting the theory that chronic ethanol exposure induces GABA spillover in the CeA. The role of astrocytic GAT3 in dependence-escalated ethanol intake and somatic withdrawal was assessed using viral-mediated GAT3 overexpression and knockdown approaches. GAT3 knockdown resulted in a persistent elevation only in post-dependent ethanol intake, an effect primarily observed in females. In contrast, GAT3 overexpression had no effect in any dependent measures assessed. Together, these findings indicate that CIE exposure remodels astrocyte-neuron interactions in the CeA and implicate CeA astrocytes as a key substrate dysregulated by ethanol dependence.

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Copyright is held by the author. All rights reserved.

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