The rat is the most commonly used model organism for behavioral studies of addiction. We propose to establish an innovative hybrid rat diversity panel (HRDP) in this work. The HRDP is unique in that it integrates: 1) a high level of genetic diversity similar to that of admixed human populations; 2) a way to control drug exposures and to systematically study gene-by-environment and gene-by-drug interactions; and 3) a way to integrate "addictome" data across scale: from genetics, genomics, and other molecular data together with key addiction related risks. This work will be a step toward developing experimental resources for precision medicine. The HRDP consists of 91 highly diverse genomes of rats that are all open access and can be used by any investigators to study facet of addiction and in different environments or under different treatments. We will use the HRDP to identify sequence variants that control motivational effects of nicotine with a menthol cue. Approximately 25% of smokers prefer mentholated cigarettes. Clinical studies have shown that menthol facilitates initiation, enhances dependence and makes quitting more difficult. Given the large sample size needed in human studies to identify key sequence variants associated with drug addiction, we argue that animal models provide an efficient means to define and test genetic and molecular mechanisms that contribute to the addiction-enhancing effects of menthol. We developed a rat model of nicotine i.v. self-administration (IVSA) with an oral menthol cue. We found that 1) menthol facilitates the acquisition of nicotine IVSA, 2) rats that receive the menthol cue for nicotine show a strong extinction burst, a model for drug craving, and 3) these rats also demonstrate a strong cue-induced reinstatement, a model of relapse. We also showed that the cooling sensation of menthol functions as a conditioned cue for nicotine reward, and that oral menthol treatment increases brain nicotine accumulation. Critically, in the context of this U01 mechanism, we estimate that heritability of these traits are greater than 0.6. We have three aims: In Aim 1 we conduct whole genome sequencing of the HRDP. We will define all sequence variants that underlie heritable variation using innovative linked-read libraries and de novo assemblies. In Aim 2 we phenotype nicotine IVSA with a menthol cue in adolescent HRDP animals of both sexes with deep replication. We will phenotype nicotine IVSA with a visual cue as a control. Effects of oral menthol on brain nicotine level will be measured. In Aim 3 we use systems genetics methods to map and integrated behavioral phenotypes. Both forward (QTL) and reverse (PheWAS) genetic methods will be used. We will use new linear mixed models to map and test candidate genes with key cofactors (i.e., different cues). Finally, we evaluate the translational relevance of candidate genes and biomarkers by comparison to GWAS cohorts and longitudinal reports of addiction in humans. This U01 w...