Project Summary Pathological substance use disorders are devastating psychiatric conditions that lead to patterns of increasing and out of control substance use. These disorders account for significant morbidity and mortality amongst patients and inflict untold costs on their families and loved ones as well as society at large. Of these disorders, pathological use of cocaine and other psychostimulants accounts for a significant proportion of morbidity and mortality. Despite tremendous advances in understanding of the neurobiology of stimulant use disorders, there are no FDA-approved pharmacotherapies for stimulant use disorders. In treating patients with stimulant use disorder, the largest hurdle to overcome is in preventing drug relapse. In recent years there has been a growing understanding that a number of systems outside of the brain can play a critical role in shaping brain and behavior. Among these is the resident population of bacteria in the intestinal tract – collectively referred to as the gut microbiome. Extensive research now demonstrates that changes in the gut microbiome play a critical role in both normal brain function, as well as in the development of pathological states. In our own lab we have previously published that acute depletion of the microbiome can alter the rewarding effects of cocaine and affect gene expression changes in the brain. More recently, we have begun investigating how depleting the microbiome with antibiotics affects the persistence of behavioral and brain gene expression changes in animal models of relapse. We find that animals that lack a complex microbiome have increased cocaine seeking behaviors after a prolonged period of abstinence. Genome-wide RNA-sequencing analyses demonstrate that these animals have robust changes in gene expression in important striatal gene networks that affect synaptic plasticity and behavior. Additional analyses demonstrate epigenetic changes in chromatin structure. Importantly, behavioral and molecular effects of microbiome depletion can be largely reversed by replenishment of specific bacterially derived small molecules. In this grant we will work in two comprehensive Aims to clarify the specificity and mechanisms of these effects and will work to further targeting the gut microbiome and its molecular byproducts. Aim 1 will define temporal specificity of microbiome depletion effects on drug seeking behaviors with targeted microbiome depletions and reconstitutions at different phases of behavior. Behavioral studies will be coupled with molecular analyses of synaptic plasticity related transcriptional and epigenetic effects. Additional analyses of microbiome composition will define bacterial populations associated with increased drug seeking. Aim 2 will further clarify the role of individual microbiome- derived small molecules in driving these behavioral and molecular changes. These studies will provide critical mechanistic insight into gut-brain signaling in a model of cocaine...