Project Summary / Abstract (30-line maximum) Spinal cord interneurons (INs) play indispensable roles in CNS circuit connectivity and function. Importantly, spinal IN populations are centrally involved in plasticity mechanisms responsible for mediating the limited degree of functional recovery that can occur spontaneously after spinal cord injury (SCI). Therefore, critical questions that need to be addressed are: Can therapeutic interventions further recruit these spinal cord INs into remodelled circuits to robustly restore lost function following SCI? If so, by which modes of circuit plasticity can this spinal cord IN recruitment occur? To address this highly important topic in this R01 Renewal application, we aim to examine whether therapeutically stimulating axon regeneration can promote respiratory circuit plasticity via the formation of pre-phrenic IN (PP-IN) relay circuits to drive recovery of diaphragm function after cervical SCI. A majority of SCI cases occur in the cervical spinal cord, resulting in persistent diaphragmatic respiratory dysfunction that is associated with mortality, dependence on mechanical ventilation, a host of morbidities such as respiratory infections, and greatly reduced quality of life. Diaphragm is directly controlled by phrenic motor neurons (PhMNs) located at cervical spinal cord levels C3, C4 and C5. PhMNs are monosynaptically activated by supraspinal brainstem neurons located in rostral Ventral Respiratory Group (rVRG). Cervical SCI results in the axotomy of descending rVRG fibers, denervation and silencing of spared PhMNs, and diaphragm paralysis. We have demonstrated that systemic administration of a blood brain barrier-permeable PTEN antagonist peptide (PAP) can regenerate rVRG axons after cervical SCI, resulting in significant restoration of diaphragm function. Excitingly, our findings suggest that PAP-induced rVRG axon regeneration is promoting a substantial portion this diaphragm recovery via formation of polysynaptic PP-IN relay circuitry. Specifically, we hypothesize that intersegmental PP-INs can be recruited to relay bulbospinal input from regenerating rVRG axons to PhMNs. In Aim 1 studies, we will determine whether selectively silencing C3-to-C5 projecting intersegmental PP- INs impacts recovery of diaphragm function stimulated by PAP-induced rVRG axon regeneration after C2 hemisection SCI. To do so, we will use an innovative dual-viral vector chemogenetic silencing approach, coupled with monosynaptic and polysynaptic retrograde and anterograde rVRG / PP-IN / PhMN circuit tracing. In Aim 2, we will determine whether therapeutically stimulating regeneration of C1-to-C5 projecting PP-IN axons across a SCI lesion promotes diaphragm recovery after C2 hemisection, again using a chemogenetic silencing strategy. In Aim 3, we will extend this work to the more clinically-associated cervical contusion SCI model to begin to assess the translational potential of targeting intersegmental PP-IN plasticity to pro...