PROJECT SUMMARY/ABSTRACT – PROJECT 3 Project 3's objective is to address the role of microglia in modulating transcriptional adaptations in nucleus accumbens (NAc) and dorsal striatum (DS) neurons triggered by cocaine or opioid exposure. Microglia contribute to normal brain development and function by supporting neuronal survival and removing non- functional neurons and synapses. We found—with Project 2—that ablation of microglia increases behavioral responses to cocaine and to opioids, including drug self-administration (SA). These findings support the hypothesis that microglia may function homeostatically to oppose conditions of excessive dopaminergic transmission, such as seen with cocaine or opioid exposure. We have found as well that 10-15% of microglia in striatum, but not other brain regions, express the D1 receptor (D1R), raising the novel possibility of direct effects of drug (via increased dopaminergic transmission) on microglia, a possibility for which we now have robust evidence. As next steps, we propose to fully characterize the influence of microglia in controlling neural and behavioral responses to cocaine and opioids in mouse models. This will include delineating a role for all microglia in striatum as well as the selective role played by D1R+ microglia and by D1 receptor signaling within those microglia. We will next characterize the influence of microglia—again D1R+ and D1R- subpopulations— on the ability of cocaine or opioids to influence gene expression profiles in the major neuronal cell types of NAc and DS. This work includes characterizing a mechanism involving adenosine signaling that bidirectionally couples microglia to striatal neuron functioning. As well, we will characterize changes in gene expression induced by drug exposure in D1R+ and D1R- subpopulations of microglia themselves within striatum. These experiments are made possible by several novel lines of genetic mutant mice that enable the selective manipulation of microglial subpopulations within striatum combined with RNAseq of isolated neuronal and microglia cell types, including at the single cell level. These investigations of cocaine and opioids in mice set the stage for follow up studies of humans with substance use disorders. Overall, this Project is providing paradigm-shifting information about the role of microglia in the pathophysiology of drug addiction.