Investigating cytoskeletal dynamics in astrocyte structure and cocaine seeking behavior

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ABSTRACT Cocaine abuse presents a significant public health concern across the United States, as the number of cocaine-related deaths in the United States has almost tripled since 2013. Despite an urgent need for intervention, an FDA-approved treatment for Cocaine Use Disorder is lacking. Hence, there is a considerable need for investigations into the mechanisms that drive relapse vulnerability. Recent advances indicate that cocaine-induced structural adaptations in astrocytes may contribute to relapse vulnerability. Astrocytes are the most abundant glial cell in the brain and regulate varied critical functions, including synaptic transmission and plasticity. Research in the Reissner lab has revealed that astrocytes in the nucleus accumbens are significantly structurally impaired following cocaine self-administration and extinction. In addition, preliminary data from our lab indicate that rat long-access (LgA, 6h/day) self-administration followed by prolonged abstinence (45d) leads to a significant ~40% decrease in astrocyte volume, surface area, and synaptic colocalization. However, the mechanisms driving these observations are unknown. I hypothesize that downregulation of astrocyte cytoskeletal dynamics is a major contributor to these effects. Accordingly, this proposal will examine how astrocyte cytoskeletal dynamics are altered following chronic cocaine self-administration and prolonged abstinence, and how manipulation of cytoskeletal proteins can influence cocaine seeking. Of note, phosphorylation of ezrin, an actin-cytoskeleton linker protein that is abundantly and preferentially expressed in astrocytes, is downregulated by acute cocaine exposure. Thus, the overarching hypothesis of this proposal is that cocaine and abstinence-induced astrocyte structural deficits result from alterations in astrocyte cytoskeletal dynamics, which further contribute to cocaine-seeking behaviors that are exacerbated following long-term abstinence. To test this hypothesis, in Aim 1 I will measure the effects of cocaine self-administration and abstinence on cytoskeletal proteins, including ezrin, and I will use an astrocyte-specific AAV to manipulate ezrin expression, to examine its role astrocyte structure and further in cocaine-seeking behaviors. In Aim 2, I will use a RiboTag AAV, to isolate astrocyte-specific mRNAs in conjunction with RNAseq as an unbiased approach to measure cocaine and abstinence-induced changes in the astrocyte transcriptome that may contribute to cocaine-seeking behavior. I will specifically analyze results to measure relative expression profiles of cytoskeletal proteins among these astrocyte mRNAs. These results will provide a greater understanding of how dysfunctions in astrocyte structure actively contribute to cocaine abuse and relapse.