Neuropathic pain (NP) is a debilitating disease that is difficult to manage. Current treatments are only partially effective, in a small subset of patients. The development of effective analgesics for NP is stalled by our incomplete understanding of its underlying mechanisms. An important clue comes from our progress on this project over the past 20 years with studies of the neurobiology of neuropeptide Y (NPY) and its inhibitory actions at excitatory interneurons (IN) in the dorsal horn of the spinal cord that express the NPY Y1 receptor (Y1). Our long-term goal is the development of Y1 agonists that will engage and inhibit precise subpopulations of Y1-INs and thus reduce NP. To this end, we confirmed that Y1-INs co-express either cholecystokinin (Cck), gastrin-releasing peptide (Grp), or Neuropeptide FF (Npff) mRNA, and that chemogenetic inhibition or ablation of Y1-IN blocked multiple signs of NP in mice with peripheral nerve injury. Similarly, chemogenetic inhibition of protein kinase C type γ interneurons (PKCγ-INs) reversed touch-evoked allodynia. Our central hypothesis is that nerve injury recruits a pronociceptive dorsal horn microcircuit that includes excitatory presynaptic input from PKCγ-IN onto the subpopulations of Y1-INs that facilitate neuropathic pain. Specific Aim 1 will use patch clamp dorsal horn slice electrophysiology to record postsynaptic currents, and high-resolution confocal microscopy to measure appositions between pre and postsynaptic elements of synapse, at Y1eGFP neurons. We predict that nerve injury will produce a net ratio increase not only in presynaptic excitatory vs inhibitory drive, but also in the number of excitatory vs inhibitory synapses, leading to a potentiation of neuronal activation in the CCK, GRP and/or Npff subpopulations of Y1- INs. Specific Aim 2 tests the hypothesis that Y1-INs are necessary and sufficient for neuropathic pain. We predict that wireless in vivo optogenetic activation of spinal Npy1rcre (Y1cre) neurons will produce nocifensive and aversive behaviors in normal mice, while optogenetic inhibition will reverse mechanical and cold allodynia in nerve-injured mice. Specific Aim 3 tests the hypothesis that nerve injury recruits a pronociceptive PKCγ Y1-IN chronic pain circuit. We predict that chemogenetic or optogenetic activation of PKCγCreERT2 neurons will increase pERK+ immunoreactivity (a proxy for in vivo neuronal activity) and excitatory postsynaptic currents (EPSCs) in Y1eGFP reporter mice, while genetic inhibition of PKCγCreERT2 or pharmacological inhibition of PKCγ will prevent or reduce SNI potentiation of touch-evoked neuronal activation, spontaneous EPSCs and/or stimulus-evoked action potentials in Y1eGFP neurons. Specific Aim 4 will determine which subpopulations of excitatory Y1-INs are necessary for the antiallodynic actions of intrathecal Y1 agonists. We predict that conditional deletion of Y1 receptors in Lbx1cre spinal cord neurons as well as CckCre, GrpCre, and/or NpffCr...