PROJECT SUMMARY Vulnerability to relapse remains a significant clinical hurdle in the treatment of addiction. Data from operant rodent models of addiction and relapse indicate that cocaine-conditioned cue exposure evokes a pronounced glutamate release in the nucleus accumbens core (NAcore). Cued glutamate release engages a cell type-specific transient synaptic potentiation in NAcore medium spiny neurons (MSNs), which does not occur during cued sucrose seeking. This transient synaptic potentiation consists of increased synaptic and structural plasticity in MSNs. Importantly, the magnitude of this plasticity positively correlates with relapse behavior. We posit that both the structural component (dendritic spine head expansion) and the synaptic component (increased insertion of glutamate receptors) of this plasticity are engaged by nitric oxide (NO); a gaseous transmitter produced by interneurons that express neuronal nitric oxide synthase (nNOS). Consistent with a role for these neurons in cued relapse, we have recently demonstrated that elevated NO release in the NAcore also occurs in parallel with glutamate release during cued cocaine seeking. The objectives of the proposed study are to reveal the inputs to the NAcore required for NO release during relapse, to determine which receptors on NAcore nNOS neurons regulate NO release and cued cocaine seeking, and to reveal how nNOS participates in the induction of the transient structural and synaptic plasticity in MSNs that drives cued cocaine seeking. In Aim 1, we will use chemogenetic inhibition of inputs to the NAcore while recording glutamate and NO release during cued cocaine seeking. We predict that inputs to the NAcore carrying discreet aspects of cue and contextual salience will differentially regulate NO release and cued relapse. In Aim 2, we will elucidate how glutamate and dopamine receptors, specifically expressed on NAcore nNOS interneurons, act in concert to regulate NO production and if they are required for cued cocaine seeking. To do this we will use transgenic mice that express Cre in nNOS neurons, Cre-dependent shRNA viral vectors, as well as cocaine self-administration and cued cocaine seeking trials. We predict that glutamate and dopamine receptor systems in nNOS neurons cooperatively regulate NO release and cued cocaine seeking. In Aim 3, we will determine if loss of nNOS in the NAcore will prevent cue- mediated synaptic and structural plasticity in MSNs. To do this we will use an shRNA vector to knockdown nNOS and concomitant viral labeling of D1 or D2 receptor expressing MSNs in the NAcore. This will be done using separate cohorts of D1-and D2-promoter driven Cre rats. In Aim 3, we will measure morphological and electrophysiological readouts synaptic plasticity induced by cues. We expect that loss of nNOS will prevent both forms of plasticity linked to relapse, predominantly in D1 MSNs. In conclusion, findings from these investigations will reveal the mechanistic aspects of ho...