Abstract/Project Summary (30 lines of text) Life expectancy of ventilator-dependent cervical spinal cord injury (cSCI) patients is significantly reduced by respiratory complications. Despite recent advances in pulmonary medicine, including phrenic nerve stimulation and diaphragm pacing, there are no long-term solutions for cSCI-induced ventilator dependency. This project addresses this profound therapeutic gap by testing and developing translational principles to amplify diaphragm contraction strength via selective neuromodulation of phrenic sympathetic innervation to reduce or eliminate ventilator dependency. While sympathetic fibers are widely associated with vasomotor control, recent studies provide compelling evidence that sympathetic innervations of skeletal muscles provide essential trophic support for maintaining healthy neuromuscular junctions (NMJs) and facilitates neurotransmission. In humans and in animals, the phrenic nerve is a conduit for sympathetic fibers innervating the diaphragm. Phrenic sympathetics (PS) originate from extra-spinal post-ganglionic neurons and are undamaged with direct injuries to the spinal cord. Notably, the diaphragm compared to all muscles studied, has among the richest supply of sympathetic axonal terminals colocalizing with NMJs. This study will develop principles to translation for PS neuromodulation, a biologically-based but untested therapeutic strategy, for enhancing or restoring diaphragm function following cervical spinal cord injury. In Aim 1, the effects of chronic selective PS stimulation on long-term diaphragm function and NMJ health will be studied in an optogenetic C4-5 hemocontusion cSCI mouse model with photoexcitable PS fibers. Chronic in-vivo photostimulation of diaphragm PS fibers with blue-light will be delivered by an implanted miniature optoelectronic device. Terminal experiments will be performed to assess diaphragm contractile properties and phrenic-diaphragm histology to evaluate the extent to which chronic PS recruitment can mitigate observed cSCI-induced physiological and histological changes. Aim 2 will develop a recombinant adeno-associated virus (rAAV) gene-therapy approach using luminopsin technology to impart selective opto- chemogenetic control over PS neuronal activity. Luminopsins are fusion proteins with a light-sensing channelrhodopsin ionotropic channel and a light-emitting luciferase imparting two modes of neuronal control; 1) photoactivation of opsin and 2) molecular-activation of luciferase generating opsin-activating bioluminescence light. The luminopsin approach permits photoactivation control over neuronal activity but obviates the need for internal light source. Survival surgeries will be performed to deliver the engineered rAAV to the diaphragm via a transabdominal approach. Terminal experiments, 2 weeks following transduction, will compare the effects of photo- and molecular luminopsin activation on diaphragm contraction force and recruited PS activity. Histolog...