Dysfunctional breathing is a significant cause of morbidity and mortality after cervical SCI (cSCI). The ability to restore breathing in the chronic phase after cervical SCI (cSCI) is an overwhelming yet important goal. The diaphragm, the major inspiratory muscle, is innervated by phrenic motoneurons (PMNs) located in the cervical spinal cord (C3-C5). Cervical interneurons synapse onto PMNs and discharge in synchrony with phrenic inspir- atory output and modulate breathing. Premotor neurons within the rostral ventral respiratory group of neurons (rVRG) in the brainstem provide the main inspiratory drive to the spinal respiratory circuitry. Cervical spinal cord injury (SCI) disrupts the communication between rVRG and the spinal respiratory circuitry resulting in significant respiratory compromise. We recently demonstrated that pharmacogenetic stimulation of cervical excitatory in- terneurons (eINs) immediately after cSCI rescues breathing in mice; however, it is not known if this strategy will be effective in improving breathing in the chronic phase of cSCI. At the chronic stage, the respiratory network is known to undergo significant modifications. Moreover, our previous work in non-traumatic compressive injury to the cervical spinal cord demonstrated that cervical eINs play an integral role in promoting plasticity and main- taining ventilation despite a significant loss of PMNs. Given that cervical eINs integrate into the respiratory net- work, and they are necessary for the spontaneous respiratory recovery, they emerge as critical therapeutic tar- gets for respiratory recovery in the chronic phase after traumatic cSCI. We hypothesize that providing selec- tive excitatory input to surviving PMNs at the late stage of cSCI will enhance PMNs output and respiratory recovery. The first objective of this proposal aims to gain more significant insights into the status of the respira- tory neural network after chronic cSCI. The second objective of this proposal will examine a novel treatment strategy involving selective stimulation of cervical eINs to promote respiratory recovery in the chronic phase after cSCI. In aim 1, we will assess the survival of PMNs following C2 hemisection injury (C2Hx) using the monosynaptic retrograde tracer Cholera Toxin Subunit B to specifically track the PMNs. In the second part of Aim 1, we will simultaneously map the input-output connectivity of the cervical respiratory elements, the PMNs and the prephrenic cervical eINs, in the naïve-uninjured state and after cSCI. In Aim 2, we will selectively stimulate the cervical eINs in the region of the phrenic nuclei after chronic cSCI using the DREADD technology. This technique has innovative applications due to its ability to activate or silence neuronal populations non-invasively. The effect of stimulating cervical eINs on promoting respiratory recovery after chronic cSCI will be assessed using electro- myography and whole-body plethysmography. The expected outcomes of the pro...