Design of genetically encoded sensors for detecting endogenous opioid peptides Opioids that target the mu-opioid receptors (MOR) remain the most effective pain medication but with severe side effects, such as addiction, constipation and respiratory suppression. The side effects result from a lack of specificity for the MOR in the pain modulation circuit by currently available opioids. Since endogenous opioid peptides do not lead to such adverse effects, understanding their role in different neuronal circuits could advance our knowledge of how opioid peptides act differently, and possibly facilitate the design of novel pain medications with reduced side effects. To study how endogenous opioid peptides exert their effects on different neural circuits, we need to detect when and where the endogenous opioid peptides are released in the brain at a high spatiotemporal resolution and at the circuit level. Microdialysis, the best available method for detecting opioid peptides in the mouse brain, can detect opioid peptides with a spatial resolution of ~ 400 µm and a temporal resolution of ~20 minutes. However, neuron somas are ~ 20 µm and neuromodulating peptides are usually released and function on the order of seconds to minutes. There is a need of methods to detect the endogenous opioid peptide release with higher spatiotemporal resolution. Therefore, we propose to design two classes of opioid sensors: 1) A transcriptional reporter that will enable the detection of the endogenous opioid peptides at a cellular resolution across a large volume of the brain tissue for studying how endogenous opioid peptides exert their effects at the circuit level; 2) Real time fluorescent sensors that will enable the detection of the endogenous opioid peptides with subcellular spatial resolution and a temporal resolution on the order of seconds. These two sensors will complement each other to address the long unanswered questions regarding the endogenous opioid peptide regulation and signaling. For example, what kind of pain and reward stimuli will stimulate the opioid peptide release? Where exactly are the opioid peptides released at a cellular or sub-cellular resolution in response to different pain and reward stimuli? How soon after pain or reward stimuli are endogenous opioid peptides released? Completion of this proposal will contribute to our long-term goal of designing tools to advance our understanding of the endogenous opioid signaling for designing pain medications with mini- mum side effects.