Alcoholism is a complex disorder caused by the influences of multiple genes, the environment, and interactions among them. The long-term goal of our studies is to provide targets for alcohol treatment by identifying genes that influence alcohol-drinking behavior. The objective of this proposal is to further study the two candidate genes, Neuropeptide Y (Npy) and Alpha-synuclein (Snca), underlying the Chromosome 4 quantitative trait locus (QTL) and previously identified to be differentially expressed in the inbred alcohol preferring (iP) and non-preferring (iNP) rats, to fine map the QTL to 2 cM, and to define other strong differentially expressed candidate genes in this fine mapped region. The proposal will employ inbred and congenic rat models for investigating the known and predicted genes in the QTL region. To study the effect of alcohol on the expression of Npy and Snca in discrete brain regions, mRNA and protein levels will be compared between the iP and iNP strains employing quantitative Real Time PCR (qRT-PCR) and Western blots, respectively. The functional significance of novel polymorphisms we identified in the Npy and Snca genes will be determined by transient transfection reporter assays. The proposal to narrow the Chr 4 QTL region to 2 cM will be accomplished by creating interval-specific congenic strains. Congenic strains, P.NP and NP.P, will be backcrossed to the respective inbred iP or iNP strain to identify animals recombinant within the map of markers placed at approximately 1 cM intervals throughout the QTL region. To identify other potential candidate genes in this region, microarray analysis will be employed as a means to identify those genes that are differentially expressed between the congenic and inbred lines. Those genes that are not only differentially expressed but are also in the narrowed 2 cM region will then be further confirmed using qRT-PCR. These experiments will combine an outstanding animal model of alcoholism and molecular techniques to provide a comprehensive approach that will identify with certainty candidate genes that influence alcohol drinking.