Drug addiction is a massive public health concern that inflicts extensive burdens on our economy and society. The harmful consequences of drug abuse extend far beyond the addicts and gravely impact their families. A growing body of evidence suggests that the children of fathers who consumed drugs around the time of conception show altered brain function and behavioral abnormalities. We have established a highly translational paradigm of paternal opioid drug taking, using morphine self-administration in rats to study this phenomenon. Our results demonstrate that the male progeny of fathers (sires) that took morphine chronically are more susceptible to develop addiction-like traits and self-administer morphine. This multigenerational animal model offers a rare window into a pool of subjects that are more vulnerable to develop addiction, a population that has been historically difficult to identify. Here, we can reliably and systematically produce animals that show increased drug taking behavior, which offers a unique opportunity to delve into the mechanisms underlying addiction susceptibility. This multifaceted project will combine behavioral and molecular biological approaches to identify functionally relevant mechanisms that confer a higher propensity to develop addiction. The proposed studies will address two major questions: (1) which germline epigenetic reprogramming events are critical for shaping development toward addiction vulnerability into adulthood? (2) what are the functionally relevant neuro-epigenetic processes that increase addiction-like behavior in our multigenerational model of drug taking? This proposal will delineate biomarkers of addiction by identifying changes in sperm miRNA expression and DNA methylation caused by chronic paternal morphine exposure. We hypothesize that the opioid- derived sperm molecular signature is predictive of addiction susceptibility in the resulting progeny. We will directly test this possibility by assessing the functional relevance of specific sperm methyl marks and individual sperm miRNAs in shaping neurodevelopment toward higher drug taking and elevated drug reinforcing efficacy in adult animals. We will also probe covalent modifications of nuclear histone proteins that package DNA in the brains of adult morphine-sired progeny that show increased drug taking. Gene-targeted epigenetic editing will be used to characterize histone marks that regulate drug taking behavior in neural reward circuits. This research will establish a strategy to delineate functional mechanisms associated with addiction susceptibility and develop a platform to study how environmental insults can shape and affect the likelihood of individuals to develop psychiatric diseases.