Chronic itch is a severe clinical problem that afflicts a large number of humans and it is very difficult to treat. Understanding the chronic itch circuitry and molecular mechanisms is critical to developing new therapies for this intractable disease. Mechanical itch sensitization (alloknesis) is one common symptom in many of chronic itch patients. Our previous work has identified neuropeptide Y-positive (NPY+) spinal inhibitory interneurons that gates mechanical itch. Our findings raise two fundamental questions: 1) What are the specific excitatory neurons in the dorsal spinal cord that transmit mechanical itch? 2) Does dysregulation of this pathway lead to chronic itch? Recently we have identified that spinal excitatory interneurons expressing Urocortin 3::Cre (Ucn3+) are mechanical itch-transmission neurons, which do not transmit touch, pain and chemical itch. Retrograde rabies virus tracing showed that NPY+ neurons monosynaptically connect to Ucn3+ neurons in the dorsal spinal cord. The goal of this project is to elucidate the spinal circuits that transmit and gate mechanical itch, and to study how the circuits are altered in chronic itch conditions. Aim 1: Delineate the functional organization of the spinal microcircuit that processes mechanical itch. We will examine the functional connections from NPY+ neurons onto Ucn3+ neurons. We will map the sensory inputs from dorsal root ganglion (DRG) neurons onto Ucn3+ and NPY+ neurons. Aim 2: Determine the mechanisms of mechanical itch sensitization in chronic itch conditions. We will test the mechanical itch sensitization and spontaneous itch behaviors in various chronic itch conditions after ablating spinal Ucn3+ interneurons. We will characterize the electrical properties of Ucn3+ and NPY+ neurons and synaptic transmission in the spinal mechanical itch circuits in chronic itch conditions. We will investigate whether disinhibition of spinal mechanical itch circuits is a common mechanism of mechanical itch sensitization in various chronic itch conditions. Finally, we will determine the disinhibition mechanisms in chronic itch.