Project Summary/Abstract Pain is a debilitating condition that severely impacts quality of life. Despite a growing need for new treatments, an incomplete understanding of the neural circuitry underlying pain has limited the development of novel pain therapeutics. Spinal projection neurons in the superficial dorsal horn channel pain signals from the spinal cord into the brain. These neurons may represent a viable target for future pain management strategies. While virtually all the spinal output neurons mediating nociception target the contralateral parabrachial nucleus, subsets of these neurons also project to other supraspinal structures, such as the ipsilateral parabrachial nucleus (spino- PBN neurons) or the contralateral periaqueductal gray (spino-PAG neurons). Given the distinct collateralization patterns of these neurons, I hypothesize that the spino-PBN and spino-PAG neurons have distinct nociceptive functions. I will test this hypothesis using anatomical (Aim 1), functional (Aim 2), and behavioral (Aim 3) approaches. Aim 1 will characterize the projection patterns of the spino-PAG and spino-PBN neurons through retrograde viral tracing, optical clearing, and ribbon scanning confocal microscopy. Aim 2 will determine the functional response properties of the spino-PAG and spino-PBN neurons using two-photon calcium imaging and natural stimuli applied to an ex vivo preparation. Aim 3 will uncover the role of the spino-PAG and spino- PBN neurons in mediating nocifensive behavior using chemogenetics and behavioral assays of nociception. The experiments described in this proposal have the potential to advance our basic understanding of the spinal output circuitry mediating nociception and help determine the efficacy of spinal projection neurons as new targets for clinical intervention. To complete this proposal, I will receive rigorous intellectual and technical training from a team of expert scientist and clinician mentors. This training will enable me to become an independent investigator, allowing me to accomplish my long-term goal of becoming a physician-scientist specializing in pain.