Project Summary Opioid abuse remains a costly epidemic in the US, prompting research into new and effective interventions to curb addiction-like behavior. Amongst the therapeutic characteristics of opioids, their associated withdrawal effects are substantial and recognized to contribute to relapse and prolonged intake. While the acute somatic symptoms of opioid withdrawal are relatively well characterized, their prolonged affective counterparts are less understood. This proposal will investigate opioid-induced motivational changes following prolonged abstinence from morphine by focusing on mesolimbic dopamine (DA), or the `reward system'. Regulating a broad array of affective behaviors, the mesolimbic dopamine system has been demonstrated as both necessary and sufficient for reward-related behavior towards opioids. Despite this, limitations of previous studies concerning heterogeneity of mesolimbic circuitry and a shortage of assessments of opioid-induced changes to motivated behavior have stifled clear demonstrations of motivational dysfunction following opioid dependence. Here, we aim to expand upon our lab's novel pilot data demonstrating both increased inhibitory tone and decreased excitability selective to a lateral-specific mesolimbic sub-circuit of DA neurons following 14 days of abstinence from a dependence-inducing regimen of morphine injections. Our pilot data also show that this plasticity aligns temporally with a reduction in motivation for natural rewards but an increase in motivation for morphine and an overall escalation in drug intake compared previously non-dependent animals. Aim1 will build this data and test morphine dependence induces a persistent shift in motivation that drives escalation of intake, in part by establishing a novel choice model with both rewards simultaneously present. Aim2 will investigate the necessity and sufficiency of the lateral mesolimbic subcircuit in this bidirectional shift in motivated behavior using excitatory and inhibitory chemogenetic approaches in DAT-IRES-Cre transgenic mice. Finally, Aim 3 will seek to identify the locus of increased inhibition to latVTA-latShell DA sub-circuit under identical conditions using ex vivo electrophysiology to assess both pre- and postsynaptic changes in GABAergic signaling arising from the opioid sensitive, rostral tegmental nucleus, and if this increased inhibition aligns with reductions in basal DA transmission using the DA sensor, dLight. The outcomes of these studies will further our understanding of neural circuits that regulate motivational states and inform us of adaptations in these circuits produced by opioid dependence that may confer enduring susceptibility. the hypothesis that