ABSTRACT In 2017 the U.S. Department of Health and Human Services declared the ongoing opioid epidemic a public health crisis after more than 47,000 Americans died from opioid overdoses during that year. A critical part of the solution is to understand the fundamental reaction and adaptation of brain circuits to stimulation by opioids. For example, the desensitization of opioid receptors is a critical problem in pain management because it requires increasing doses of analgesic compounds, which could contribute to developing a drug addiction. Recently, it has been shown that the activation of mu and kappa opioid receptors in neurons cause the production of reactive oxygen species (ROS) through a pathway involving NADPH oxidase and c-Jun N-terminal kinase. Therefore, this distinct response, downstream from the receptor, could be utilized to detect specific opioid receptor activation and modulation. Current studies of opioid receptors rely either on in vitro experiments in cell cultures or analysis of ex-vivo brain tissue to monitor them under drug exposure. We currently lack sensitive fluorescent sensors, which would allow us to utilize state-of-the-art fiber photometry to directly monitor mu-opioid receptor (MOR) activity in real-time and in vivo. Current limitations of contemporary sensors are slow response times, low specificity, low signal output, toxicity, or dependency on ex vivo tissue preparation. Our goal is to develop a genetically encoded sensor protein that detects ROS levels at endogenous levels with response time and signal amplitudes that will enable in vivo monitoring of neuronal systems upon MOR activation. We have recently developed a novel fluorescent ROS sensor by fusing a green fluorescent protein to a bacterial hydrogen peroxide binding protein. Signal kinetics, ROS sensitivity, and signal amplitudes are significantly enhanced compared to other available tools. We hypothesize that we can further increase the fidelity of this tool by additional structure-guided protein design at the hydrogen-peroxide binding site and the interface between the green fluorescent reporter and the sensing domain. Our objective is to express this novel tool in vivo in MOR positive neurons and to link ROS signals to MOR activity pharmacologically. We hypothesize that ROS signals in MOR neurons will increase under drug exposure. Second, we hypothesize that we will observe a decrease of ROS transients under repeated drug exposure reflecting the desensitization of MORs. At the end of this project, we will have a novel and highly specific sensor for monitoring opioid receptor activity and adaptivity. Our proposal is significant because, for the first time, we will be able to monitor the adaptation of this clinically relevant signaling pathway to opioid exposure in vivo. Our approach is innovative because we combine structure-guided protein engineering and in vivo monitoring of opioid-triggered signals to dissect a difficult-to- access neuronal signaling ...