This application addresses the question of """"""""what genes contribute to the variability in ethanol's psychomotor stimulant effect?"""""""" The goals of the application are to map the major QTLs for the activation response to a resolution of 10 cM and to confirm individual QTLs through the use of """"""""segregating congenic"""""""" strains. In addition, both the phenotypic extremes of the F2 intercross employed for QTL mapping and the congenic strains will be used to extend our understanding of the role that the central nucleus of the amygdala (CeA) has in the activation response.
The specific aims may be summarized: l. To conduct a genome wide search for QTLs associated with ethanol-induced psychomotor activation in C57BL/6J (B6) x DBA/2J (D2) F2 individuals. The focus on B6D2 genotypes is justified a) because the parental strains are highly polymorphic (Dietrich et al. 1992; 1996), b) because of marked differences in the activation response between the strains (e.g. Dudek and Tritto, 1994: Phillips et al. 1995) and c) because the heritability in the F2-intercross appears to be largely additive (Dudek and Tritto, 1994). The genotypic analysis will begin with pooled DNA samples from the phenotypic extremes of the first 900 individuals (males only) using polymorphic microsatellites spaced at 20 cM. QTL map location will then be estimated using the phenotypic extremes of the entire sample (N=1500); individual samples will be genotyped using a more dense genetic map (5-10 cM spacing) for the regions of interest. This analysis will reduce the 95% confidence interval for a QTL with a 5% effect size to approximately 10 cM. To confirm the location of the QTLs identified in this fashion, I propose. 2) To construct from B6D2 F2 intercross animals a series of """"""""segregating congenic strains in which each QTL is made homozygous in a single generation whereas the remainder of the genetic background is allowed to segregate"""""""" (Bennett et al 1997). Using flanking markers for the QTL of interest, homozygous male and female animals will be identified and mated to produce an F3 generation. To the extent possible, animals will be selected that are heterozygous for flanking markers at the residual QTLs; when this is not possible, heterozygous/homozygous matings will be performed and the heterozygous F3 animals identified for further mating. The congenic strains will also be tested for their activation response to the benzodiazepine, chlordiazepoxide, which shares some mechanisms of action with ethanol (Harris and Allan, 1989). In order to obtain additional confirmation of the QTLs detected in aims 1 and 2 and to investigate the mechanisms associated with the genetics of the activation response, I propose. 3. To conduct a 'short term' (5 generation) selective breeding study, beginning with B6D2 F2 intercross progeny. The segregation of the QTLs identified in aims 1 and 2 will be followed in the selected lines; by the fifth selected generation, all of the alleles with an effect size of 0.1 or greater will be fixed in the responsive and non-responsive lines (Falconer, 1989). In addition, the selected lines will be examined both for their response to chlordiazepoxide and for the relationship between the activation response and synaptic activity in the central nucleus of the amygdala (CeA).
