A long-standing challenge in basic and translational neuroscience and in clinical practice is to understand the vast inter-individual differences in vulnerability to substance abuse and addiction. To address this challenge we propose to study the genetic and functional basis of novelty-seeking behavior in two lines of rats that offer a uniquely powerful model for understanding the neural mechanisms of drug seeking, addiction and relapse. We developed these lines by selecting for high and low propensity to explore a mildly stressful novel environment, respectively. After 37 generations, the bred High Responders (bHRs) and bred Low Responders (bLRs) show contrasting spectra of behaviors, which are heritable in both lines. Compared to bLRs and outbred rats, bHRs exhibit higher novelty seeking and impulsive behaviors, lower anxiety, greater propensity to sensitize to psychostimulants, and lower thresholds for drug- and cue-induced relapse, reminiscent of human “externalizing disorders”. The bLRs are more prone to anxious and depressive behaviors, more responsive to psychosocial stress, which triggers drug-seeking behavior. Thus, the two lines exemplify two extremes of emotional reactivity that map onto human temperamental differences and underlie two paths to drug abuse—novelty seeking and reactivity to psychosocial stress. Our working hypothesis is that functional DNA variants in a limited number of genes, initially derived from outbred Sprague Dawley (SD) founders, account for the current molecular and behavioral divergence of the two lines. Our goal is to identify these causal genes through (1) mapping of quantitative trait loci (QTL) in both an F2 cohort already collected and in SD animals that represent the founders, and (2) further integration of functional genomic data. We propose to apply several sequencing-based technologies and analytical tools under the following specific aims (SAs): SA1: Conduct genome sequencing and quantitative trait loci (QTL) analyses in a bHR-bLR intercross population (n~636, males and females) using a low-cost Genotype by Sequencing method. SA2: Identify eQTLs, allele-specific expression, and transcripts/pathways associated with the traits by using RNAseq analysis of key neural structures– the nucleus accumbens and hippocampus. As some genes may exert their primary influence during development, we will analyze both young F2 rats (age: 28 days) and adults. SA3: Perform genomewide association study of 1,000 outbred SD rats, followed by integration of all strands of data to identify putatively causal variants and provide initial validation using qPCR, in situ hybridization, and further behavioral tests. We expect to find functional alleles at multiple genes that existed in the SDs and have evolved further apart in the two lines. Many of these genes may be directly relevant to the corresponding human phenotypes or, at a minimum, provide clues to important pathways that could explain or predict the differential vulnerability to addiction and relapse in humans. Our ultimate goal is to gain a deeper mechanistic understanding of addiction, and translate this knowledge to more precise and effective treatment for patients.