Intrathecal oxytocin (OT) is in clinical trials as an opioid alternative for chronic pain treatment. Our work in mice suggests that coupling intrathecal oxytocin with manual therapies (i.e. massage) optimizes the analgesic properties of oxytocin. This project provides the framework to support this combination therapy by concentrating on the spinal cord circuit mechanisms by which oxytocin alleviates pain. Our preliminary studies suggest that oxytocin-specific spinal cord circuits are embedded within a previously uncharacterized dorsal horn nociceptive/affective touch circuit. We will carry out three complementary sets of experiments to test the overall hypothesis that oxytocin alleviates pain by balancing excitation, inhibition, nociception, and affective touch to sculpt the activity of spinal projections systems that carry both negative valences (associated with noxious stimuli), and signals associated with positive valence (like the pleasurable properties of touch). Pharmacological and behavioral studies in rodents suggest that spinal cord oxytocin receptors (OTRs) mediate intrathecal oxytocin-induced analgesia. In Aim 1 we map the distribution of OTR+ interneurons within the dorsal horn of female and male mice, rats, and humans. In Aim 2, we map the specific input/output profiles of OTR+INs. Here we test the hypothesis that inhibitory and excitatory OTR+INs integrate peripheral nociceptive/affective touch information with OT to differentially regulate the activity of molecularly defined Lamina I projection neurons. In Aim 3 we assay the contribution of OT spinal cord circuits to both sensory-evoked reflexes and affective- motivational pain. For Aim 3 we implement our recently developed computational approaches to scale sensory- reflexive and affective-motivational pain. Results from our human tissue studies will inform how our interpretations of our rodent studies may be applied to human therapies. Based on our unique expertise in touch- specific spinal cord circuits, access to a large repertoire of spinal cord-specific tools, and behavior analytics that match the granularity of our circuit dissection techniques, we are uniquely poised to provide the theoretical framework for this combination therapy. In addition to informing context and condition for OT delivery, this work may also be used in the clinic to adjust OT dosage and delivery method. This project is impactful for several other reasons: 1) using computer vision/machine learning we will uncover the specific aspects of the pain experience that are alleviated by spinal cord OT, and assess efficacy against other analgesics; 2) our computational approaches to objectively scale rodent pain can be easily shared and implemented across research groups, serving as a blueprint to standardize rodent pain assessment (see Resource Sharing); 3) our general approach and model can serve as a basic blueprint for testing how other neuromodulators are functionally integrated into spinal cord circuits of...