7. Project Summary/Abstract Opioid receptors (ORs), consisting of ?-, ?-, and ?-ORs, are neuropeptide receptors that are broadly involved in regulating analgesia, mood, reward, and motor coordination. Opioid signaling is correspondingly implicated in a variety of behavioral disorders, including depression, drug addiction, stress, and dyskinesia. Understanding how OR circuit activity influences behavior is therefore critical to developing better treatments for disorders such as depression and addiction. Since these neural circuits are deeply embedded together in the brain at high density, very high resolution tools are needed for modulating and studying individual circuits in detail. Current high resolution optical methods for dissecting neuronal circuits are limited to altering membrane ion gradients. However, neuropeptide receptors, including ORs, are G-protein coupled receptors (GPCRs) that trigger multiple biochemical signaling cascades inside the cell when activated. These signaling cascades affect diverse processes in the neuron from membrane potential and firing rates to DNA transcription and growth. Thus, a critical barrier to understanding OR signaling is the lack of high-resolution tools that can fully replicate both membrane electrophysiological and biochemical consequences of OR activation. The Avena sativa Light, Oxygen, Voltage sensing domain 2 (LOV2) is an optogenetic platform that has been successfully utilized to photoregulate interactions between peptides and their partners by fusing peptides to LOV2 in a way that couples the peptide binding reaction to a light-dependent conformational change in LOV2. This LOV2-peptide caging strategy will be used in this proposal to overcome the current barriers to opioid signaling research through the following specific aims: 1) develop a screening platform for the rapid design of LOV2-based optogenetic opioid peptide modulators, and 2) develop a LOV2-opioid peptide (LOV-OP) fusion protein capable of light-dependent activation of ORs in cell culture and brain slice models. OR activation by LOV-OP proteins will be evaluated in Aim 1 using a high-throughput GPCR cAMP activation assay. Aim 2 will optimize a LOV-OP protein to show light-dependent OR activation via mutagenesis, creating a high resolution tool for studying OR activation. Electrophysiological measurements of light-dependent depression of neuron firing by ORs activated by LOV-OP will validate LOV-OP function in primary neuron culture and brain slice. The broad, long-term objectives of this proposal are to enable high spatiotemporal resolution studies of opioid peptide activity in the brain that lead to breakthroughs in the understanding of how specific opioid peptidergic circuits contribute to cognition and behavioral disorders. The high-throughput screening methods developed in the proposal can also be rapidly extended to studying circuits of other neuropeptides in the brain.