PROJECT SUMMARY/ABSTRACT – PROJECT 2 Project 2 provides unique characterization of microRNAs in drug addiction, a focus pioneered by my lab. In previous work, we identified two closely related microRNAs, miR-132 and miR-212, which are encoded by the same gene and induced in nucleus accumbens (NAc) and dorsal striatal (DS) neurons by cocaine self- administration (SA). While miR-212 signaling in striatal neurons potently regulates motivational responses to cocaine, the actions of miR-132 remain unknown. In extensive preliminary work, we discovered a highly novel mechanism of action for miR-132 in striatal neurons, where its expression is controlled by microglia through release of the cytokine, TNFα, thus connecting this Project with Project 3. Brain-wide depletion of microglia dramatically increases cocaine SA and disrupts extinction of cocaine-seeking behavior. As well, such microglia depletion disinhibits neuroplasticity-related transcriptional programs in NAc neurons, including programs regulated by miR-132 and by drug exposure in mice and humans. TNFα downregulates AMPA receptor (AMPAR) signaling in striatal neurons via a mechanism that requires miR-132, and local application of TNFα into NAc abolishes cocaine-seeking, while disruption of miR-132 signaling in NAc neurons enhances AMPAR signaling and increases cocaine-seeking. We will now test our hypothesis that microglia—acting in part via TNFα—control miR-132 signaling in NAc neurons to oppose the physiological and behavioral actions of cocaine. We are using a single-cell spatial transcriptomics platform to define subregions of NAc and DS that respond to cocaine in a microglia-dependent manner. We will perform initial studies with opioids as well, where we expect shared actions of the two drugs. We will then delete miR-132 selectively from NAc or DS neuronal subtypes and determine miR-132’s role in controlling the neurophysiology of the neurons and in mediating the protective effect of TNFα. We will next use HITS-CLIP to identify mRNAs that are regulated by miR-132 in these neurons in a drug- and microglia-dependent manner. This will identify prioritized mRNAs that we will link back to changes in cell and synaptic physiology and SA behavior. This proposed scheme is innovative because microRNA involvement in microglia protection against cocaine action has not been explored.