PROJECT ABSTRACT With the ongoing opioid crisis, there is a tremendous need for an in-depth understanding of opioid actions and the underlying mechanisms at the cellular and circuit levels. The striatum integrates excitatory inputs from the interconnected cortex and thalamus to form a triangular circuit that mediates critical brain functions, including motor control, affective pain, decision-making, and reward. Opioids impose strong modulation of this circuit, but their specific actions, such as “where” and “how” they act, are not fully understood. The overarching goal of our proposal is to comprehensively elucidate how individual elements in the thalamo-cortico-striatal triangular circuit are modulated by distinct opioid receptor agonists and how these modulations alter the function of the circuit. The thalamo-cortico-striatal circuit is organized based on specific subregions within the cortex, thalamus, and striatum. During the previous funding period, we established the first comprehensive thalamo-cortico-striatal circuit wiring diagram, which allowed us to identify and delineate subregion- specific connectivity. In our preliminary studies, we have identified the exact convergent sites of the anterior cingulate cortex (ACC) and the mediodorsal (MD) thalamus, both of which play critical roles in affective pain and reward, in the dorsomedial striatum (DMS). This MD-ACC-DMS circuit presumably drives pain and reward-associated executive functions. Different subtypes of opioid receptors are expressed in all three of these brain regions, making this circuit a likely substrate for opioids. However, the precise actions of agonists in the context of specific opioid receptor types, cell types, and brain subregions are poorly characterized in this circuit. In the current proposal, we will use cutting-edge tools to dissect subregion-specific, cell type-specific, opioid receptor type-specific, and synapse-specific modulation of the synapses in the MD-ACC-DMS circuit. Specifically, we will take advantage of our unique research strengths, including the novel connectomic information we acquired during the previous funding period, our novel imaging capability for directly visualizing subcellular cAMP/PKA signaling downstream of opioid receptors in living tissue, and our establishment of novel brain slice preparations for monitoring opioid modulation of multi-synaptic information propagation. Using these approaches, we will identify the action sites (Aim 1), the underlying intracellular signaling mechanisms (Aim 2), and the functional impacts (Aim 3) of distinct activated opioid receptors. Our proposed experiments will result in an in-depth, mechanistic understanding of the actions of opioid receptors in the MD-ACC-DMS circuit that may facilitate the development of strategies to more effectively address the role of opioids in analgesia and addiction.