The injured spinal cord is now recognized to have a robust capacity for neuroplasticity, and it is desirable to therapeutically harness that in ways tht will enhance respiratory outcomes after cervical spinal cord injury (SCI). Fundamental to rehabilitation and repair approaches is a basic understanding of the spinal respiratory circuit and the control of spinal respiratory neurons after chronic SCI. In principle, the injured spinal cord s essentially a new spinal cord in which neural networks and control mechanisms affecting virtually every functional domain are significantly altered. Our group recently characterized the spinal respiratory circuit anatomically after cervical SCI, but functional-anatomical correlates remain to be determined. We propose a series of experiments which will neurophysiologically define the spinal respiratory circuit after cervical SCI, examine the influence of a key neuromodulator - serotonin (5-HT) - on the circuit, and determine the impact of a promising spinal cord transplantation approach on spinal respiratory neurons and recovery of ventilation after cervical SCI. The overall hypothesis guiding this proposal is that the regulation of phrenic motoneuron (PMN) activity following chronic cervical SCI is influenced by spinal pre-phrenic interneurons and that spinal 5-HT is a critically important modulator of the spinal respiratory circuitry following chronic SCI. A rat model of high cervical SCI (lateral C2 hemisection) will be used to address three specific aims. Aim 1 will test the hypothesis that following chronic cervical SCI, PMNs retain a robust capacity for plasticity, and their bursting patterns are partly regulated by cervical interneurons. PMN and cervical interneuron activity will be measured using a multi- electrode array; anatomical and immunohistochemical methods will be used to evaluate the spinal respiratory circuit. Aim 2 will use neurophysiological, pharmacological, immunochemical, and molecular techniques to test the hypothesis that spinal 5-HT receptor activation is an integral part of phrenic motor recovery after chronic cervical SCI. Lastly, Aim 3 will test the hypothesis that transplantation of serotonergic cells can enhance or restore serotonergic modulation of spinal respiratory neurons thereby improving respiratory recovery after SCI. One week following C2 hemisection injury, adult rats will receive an intraspinal transplant of serotonergic cells derived from fetal rat raph neurons. The impact of the grafts on the phrenic motor system will be assessed using behavioral, neurophysiological, pharmacological, immunohistochemical, and molecular techniques. This proposal brings together a unique and synergistic combination of expertise in respiratory neurophysiology, multi-unit recording approaches, neural transplantation, and cervical SCI modeling. Innovative aspects include: 1) the first descriptive and mechanistic studies of PMN burst patterns after chronic cervical SCI; 2) the first neurophysiological studies of respiratory-related cervical interneurons after cervical SC; 3) the use of multi- array electrodes to describe the spinal respiratory circuitry, and 4) the firs use of transplant strategies to enhance serotonergic innervation of the spinal respiratory circuit. PUBLIC HEALTH RELEVANCE: The impact of cervical spinal cord injury on the neural control of spinal respiratory motoneurons - including the cells which control the diaphragm - is unclear. We propose a series of experiments to determine how chronic cervical spinal cord injury alters the neural regulation of respiratory motoneurons and interneurons in the spinal cord. The work will then transition to translationally relevant studies aimed at enhancing respiratory recovery by transplanting serotonergic cells into the injured spinal cord.