Susceptibility to physiological dependence on ethanol (EtOH), which is manifested as a withdrawal syndrome, is influenced by multiple genes and environmental factors. We have identified three chromosomal regions that contain genes that influence risk for physiological dependence on EtOH in mice. These chromosomal regions are referred to as quantitative trait loci (QTL). This application focuses on the Chr 4 QTL (LOD>8, p<10[-9]). We have fine mapped this QTL to a 1.5 Mb (0.7 cM) interval that contains 3 known genes and at least 2 novel genes. This proposal will use novel genetic animal models for rigorous, unbiased evaluations of the known and predicted genes in this QTL interval as potential candidate genes. In addition, we will evaluate the influence of the QTL (gene) on neuronal activation related to physiological dependence on EtOH and associated withdrawal. Using a novel congenic strain that isolates the QTL on a uniform (inbred) genetic background, we propose the following. (1) Evaluate the predicted genes in the QTL interval for expression to identify novel expressed genes. Expression will be assessed in the brain, peripheral tissues, and in embryonic tissue. (2) Evaluate the expressed genes to identify promising candidates that show genotype-dependent (i.e., congenic vs. background strain mice) differences in expression. Expressed genes will also be assessed for differential regulatory and coding sequence (structural differences) between these strains. Whenever feasible, their protein products will be assessed immunohistochemically to determine the anatomical distribution of their expression. Genotype-dependent differences in protein abundance will be assessed using Western blot analysis. (3) Evaluate neuronal activation to identify the circuit(s) that show genotype dependent activation in EtOH withdrawn mice. (4) Develop an innovative, transgenic model to rigorously test the hypothesis that a promising candidate gene (Mpdz) underlies the QTL. An innovative feature of this proposal is to combine a robust behavioral model of physiological dependence on EtOH with sophisticated molecular techniques to identify candidate genes of high quality as well as the circuit(s) involved in their influence on EtOH withdrawal, and novel transgenic models that can establish with certainty that a promising candidate gene in fact underlies the QTL.