Project Summary/Abstract Recent evidence demonstrates that inhibitory interneurons in the spinal dorsal horn (DH) can be classified into multiple subpopulations, based on their expression of a variety of genes encoding neuropeptides, neurotransmitter receptors and enzymes, which play distinct roles in somatosensation. However, none of the genes identified to date are selectively expressed within the DH, and instead often exhibit a widespread pattern of distribution across the peripheral and central nervous systems. As a result, there is currently a lack of straightforward genetic strategies to selectively potentiate the activity of inhibitory DH interneurons as a means to suppress pain and itch without influencing other neuronal populations in the sensory ganglia, spinal cord or brain. This constitutes an important gap in knowledge that must be addressed in order to identify new candidate approaches to restrict the output of the spinal nociceptive network without unwanted side effects on sensorimotor processing, cognitive function, emotional regulation or reward-seeking behavior. The long-term goal is to advance our understanding of the cellular and molecular mechanisms governing nociceptive processing in the central nervous system (CNS). The objective of this application is to identify and characterize novel targets that strongly regulate spinal nociceptive signaling but are absent throughout the brain. The central hypothesis is that guanylate cyclase D (GC-D) suppresses pain and itch sensitivity via mechanisms that are selectively localized to a subset of dynorphin-expressing GABAergic interneurons in the spinal superficial dorsal horn (SDH) which directly inhibit ascending projection neurons. The rationale of the proposed research is that these studies will identify novel molecular strategies to manipulate pain and itch signaling in the CNS with unparalleled spatial specificity. Guided by strong preliminary data, the central hypothesis will be tested and the overall objective of this application achieved by pursuing the following specific aims: (1) Identify the role of guanylate cyclase D (GC-D) in the regulation of pain and itch; and (2) Elucidate the functional connectivity of Gucy2d-expressing DH neurons and their role in somatosensation. These aims will be accomplished by using a multidisciplinary experimental approach that includes the generation of novel Gucy2dCreERT2 knock-in mice combined with in vitro electrophysiological, optogenetic, chemogenetic, behavioral and immunohistochemical techniques. The proposed work is innovative because it will be the first to identify a role for GC-D in somatosensation, and it will also reveal Gucy2d (encoding GC-D) as a novel marker of a highly specific subset of inhibitory DH interneurons that are dedicated to the suppression of pain and itch. The outcome of these investigations will be the identification of unique molecular mechanisms that regulate nociceptive processing only at the level of the sp...