Project Summary Severe pain caused by osteoarthritis (OA) affects over 15 million individuals in the United States alone. This degenerative joint disorder not only poses a significant healthcare burden for the country’s aging society, but also fuels the national opioid crisis, as no effective treatments exist to treat OA-induced joint pain. Disease- modifying therapeutic development for OA typically involves mechanistic and preclinical studies in small animal models (mouse, rat), followed by studies in large animal models (horse, pig) to confirm effectivity and safety. Unfortunately, pain-modifying therapeutic development for OA has thus far been hampered by a limited knowledge of the types of neurons that innervate the joint, the connections they form, and their functional or circuit dynamics in the presence of joint disease. The RE-JOIN consortium strives to fill these knowledge gaps by adapting and optimizing cutting-edge, multidisciplinary technologies to label, image, and profile joint- innervating neurons and by applying them in small animal models and patients with OA. Here, we propose a set of experiments that will be complimentary to RE-JOIN’s ongoing efforts. In this proposal, we aim to leverage neuronal tracing and profiling technologies developed under parent grant (UC2-AR082200), applying them to the equine carpal joint to identify and characterize joint-innervating neurons. We aim to identify the dorsal root ganglia (DRG) that harbor carpal joint-innervating neurons (Viral neural tracing; Aim 1), develop high-quality, complete transcriptomes of these DRGs that identify all tissue-specific isoforms (long-read RNA sequencing; Aim 2), and perform multi-omic analysis of these DRGs to develop a spatial single cell reference map that identifies the tissue’s individual cell types and their molecular fingerprints (spatial transcriptomics and single nucleus RNA sequencing; Aim 3). The equine osteochondral chip model of post-traumatic OA has been an excellent bridging translational model from small rodent to humans. The successful completion of research proposed here will yield high-quality resources that will be shared with the scientific community, enabling future studies to identify pain-mediating molecular and cellular changes that occur in the equine carpal joint innervation during the development of joint disease. These resources will also enable translational researchers to expand preclinical studies in equine models of OA to also include studies with chronic joint pain-modifying therapeutic candidates, thereby increasing the likelihood of finding relevant new therapeutic targets to effectively treat joint pain and/or solve the national opioid public health crisis.