The proposed component takes an innovative approach to determine specific genes that modulate the severity of alcohol withdrawal identifying mRNAs regulated during chronic ethanol exposure and withdrawal in a model of withdrawal severity. The hypothesis to be tested is that altered regulation of brain gene expression that results from chronic ethanol exposure and withdrawal is a critical factor mediating neuroadaptation. Genes regulated by chronic ethanol exposure might promote withdrawal risk, or exert protection against withdrawal. We propose to directly identify putative ethanol target genes that are regulated in specific brain areas in chronically exposed and withdrawn mice, without bias about potential roles, using microarray hybridization analysis. Withdrawal Seizure-Prone or -Resistant selected lines of mice will be exposed to chronic ethanol vapor. At peak withdrawal, specific brain regions will be harvested from mice in the presence or absence of severe withdrawal. Gene expression profiling will then be accomplished with gene microarray hybridization analysis. Specifically, complex RNA probes will be prepared from either ethanol-withdrawn or control tissue from high withdrawal severity mice and sequentially hybridized initially to membranes containing >5000 cDNAs (60% EST and 40% characterized genes). Alcohol-withdrawn regulated genes will be identify by competed analysis of hybridization data (Specific Aim 1A). Candidate gene expression patterns and time course of regulation will be further characterized by: 1) in situ hybridization analysis for confirmation of regulation and for characterization of gene expression in distinct brain areas, and 2) quantification of changes in mRNA abundance using Northern analysis or RNase protection analysis, and 3) differential expression will be analyzed in additional animal models of withdrawal severity (Specific Aim 1B). Finally, we will also perform microarray hybridization analysis in lines of mice resistant to withdrawal (Specific Aim 2A) and similarly characterize the putatively regulated products (Specific Aim 2B). The strengths of this approach lies in the use of novel yet well-characterized lines of mice selected for high or low withdrawal severity and the application of innovative state-of-the-art gene discovery procedures to identify genes regulated by ethanol. We thus propose to use this integrated approach, as a complement to QTL mapping, to identify candidate proteins important in mediating the neuroadaptive changes that occur with chronic ethanol exposure and withdrawal. The ultimate goal of this research is related to a main theme of the Center: to understand mechanisms, influenced by differences in gene expression, that underlie deleterious responses to chronic ethanol exposure such as neuroadaptation, that can result in physical dependence and withdrawal.
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