Project Summary/Abstract The opioid epidemic is a persistent public health crisis in the United States and abroad, historically dominated by the misuse of prescription opioids and heroin; however, those illicit substances have been overtaken by a surge in the synthetic opioid fentanyl. Fentanyl’s unmatched potency and addictiveness makes it more difficult to treat with pharmaceuticals that act on opiate receptors directly; as a result, opioid overdose deaths in the U.S. skyrocketed to more than 80,000 in 2021. Novel therapeutic approaches to manage this crisis are urgently needed, making understanding basic opiate signaling mechanisms in the brain critically important. A potential target to mitigate opioid use disorder (OUD) are the cannabinoid receptors (CBRs), which are co-expressed alongside opiate receptors in the brain’s reward centers. Recent studies indicate that CBRs can modulate opioid-reward behaviors; however, our understanding is challenged by multiple CBR subtypes that have dynamic and overlapping expression in the brain. Antibodies for CBRs can be nonspecific and provide only a snapshot of receptor localization, and conventional approaches to manipulate CBRs have poor spatial and temporal resolution: Genetic knock-outs are prone to compensation, while microinjection or wash-on of hydrophobic cannabinoid ligands suffer from poor kinetics and don’t distinguish between CBRs on overlapping circuits. To delineate the role(s) of CBRs in opioid reward and as a potential therapeutic target for OUD, there is a critical need for new tools to probe their localization and manipulate their signaling with high resolution. The long-term goal of my lab is to build chemical tools for deconvoluting the molecular components of substance use disorder, and I have pioneered several chemical biology technologies to study CBRs with enhanced spatiotemporal precision. In collaboration with Erick Carreira’s lab, we developed fluorescent probes that label CB1 and CB2 cannabinoid receptors with a fluorophore. These overcome the poor selectivity of antibodies and can be applied to live-cell imaging experiments. We also developed chemical photoswitches allowing acute reversible optical manipulation of CB1 and CB2, which permits control of downstream signaling by a precise optical stimulus. The objective of this proposal is to expand our cannabinoid chemical biology toolkit towards the atypical cannabinoid receptor GPR55, and then apply these tools in ex vivo and in vivo models of opioid administration. We will explore the interactions between cannabinoid and opiate receptors in the ventral tegmental area (VTA), a critical hub in the brain’s reward system. This proposal has a strong scientific premise built on our published studies, preliminary data, and a careful review of the literature. It is innovative because it applies our state-of-the-art tools to illuminate basic signaling mechanisms relevant to opioid reward and could generate novel therapeutic avenues f...