Date

6-2015

Document Type

Thesis

Degree Name

Ph.D.

Department

Dept. of Behavioral Neuroscience

Institution

Oregon Health & Science University

Abstract

Alcoholism is a debilitating disease, influenced by both biological (genetic) and environment factors. Unfortunately, the genetic determinants of risk remain largely unknown, hindering effective prevention and treatment of dependent individuals. Recent quantitative trait mapping studies have identified the Mpdz gene, which encodes the multiple Post-synaptic density-95, Discs Large, Zona Occludens protein 1 MUPP1, in predisposition to ethanol withdrawal (EWD) in mice. The aim of my dissertation was to assess the caudolateral substantia nigra pars reticulata (clSNr) as a region through which Mpdz affects EWD, and assess the potential role of the Mpdz gene in predisposition to binge-like ethanol drinking. Further, the aim of my dissertation was to assess the effect of MUPP1 on γ-aminobutyric acid type B receptor (GABABR) mediated responses, as an important step towards identifying how this gene may influence EWD and potentially binge-like ethanol drinking. Although no animal model exactly duplicates clinically defined alcoholism, models for specific traits, such as risk for excessive intake and withdrawal, have proven beneficial for identifying potential genetic determinants of liability in humans. The handling induced convulsion (HIC) scale is a sensitive and quantitative measure of central nervous system (CNS) hyperexcitability, that can be used to assess EWD severity, and the drinking in the dark (DID) procedure is a validated model of binge-like ethanol drinking in mice. I implemented these procedures to assess two alcohol phenotypes that may be associated with the negative and positive reinforcement, respectively. Further, I used Mpdz targeted RNA interference (RNAi), as well as Mpdz heterozygote knockout (Mpdz+/-) and Mpdz transgenic (MpdzTg) mice to begin to elucidate the role of Mpdz/MUPP1 in influencing risk for EWD and binge-like ethanol drinking. In Chapter 1, I introduce research identifying the Mpdz gene as a quantitative trait gene (QTG) in predisposition to EWD, with reduced expression predicted to be associated with an enhancement in EWD severity. Further, I introduce evidence supporting the clSNr as a region that may mediate the effects of Mpdz on EWD. Finally, I introduce evidence supporting an effect of MUPP1 on GABABR function, and evidence for a role of both of these proteins in EWD and ethanol drinking phenotypes. In Chapter 2, I describe how I used the method of RNAi to assess the role of Mpdz expression in the clSNr on predisposition to EWD. I found that reduced expression of Mpdz in the clSNr was associated with a significant enhancement in EWD severity, with no changes in baseline HICs or pentylenetetrazol-enhanced HICs. Consistent with previous work, these data provide further confirmation of Mpdz as a QTG for EWD, and demonstrated that its expression in the clSNr is involved. In Chapter 3, I describe how I used behavioral, pharmacological, and molecular techniques to assess whether clSNr GABABRs mediate EWD severity, and whether expression of Mpdz/MUPP1 was associated with an enhancement in GABABR-mediated responses both behaviorally and neurophysiologically. I demonstrated that activation of clSNr GABABRs enhanced HICs and inhibition of these receptors during EWD significantly reduced EWD severity. This represents the first evidence that GABABRs within the clSNr are involved in EWD, suggesting that GABABRs in this region may play a role in mediating CNS hyperexcitability. Further, using novel Mpdz+/- and MpdzTg genetic models, I demonstrated an association between Mpdz/MUPP1 expression and GABABR-mediated HICs, with reduced expression associated with greater HICs and vice versa. Further, I demonstrated a greater enhancement in SNr GABABR response to baclofen through G-protein inwardly rectifying potassium (GIRK) channels in Mpdz+/- compared to wild-type (WT) littermates. Using radioligand saturation binding and gene expression analyses, no differences in GABABR density, binding affinity, or expression in whole brain or the clSNr of Mpdz+/- and WT littermates were detected. In Chapter 4, I describe how I used three variations of the DID procedure to assess the potential effect of Mpdz on predisposition to binge-like ethanol intake. No association between reduced Mpdz/MUPP1 expression and binge-like ethanol intake or ethanol preference was detected. Further, reduced Mpdz/MUPP1 expression was not associated with a difference in the effectiveness of baclofen to reduce binge-like ethanol drinking. In Chapter 5, I discuss the interpretation of my findings, the relationship to the broader literature, and future directions of this research. Together, these experiments provided additional confirmation for a role of Mpdz in predisposition to EWD and demonstrated the clSNr to be involved. My data also demonstrated an association between reduced Mpdz/MUPP1 expression and heightened GABABR-mediated responses, but were unable to detect an association between Mpdz/MUPP1 expression and binge-like ethanol drinking. The results of this research may inform future studies of genetic susceptibility to alcohol use disorders in human clinical populations and response to pharmacotherapeutic treatments for dependent individuals.

Identifier

doi:10.6083/M40P0XS6

School

School of Medicine

Included in

Neurosciences Commons

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