Injection of WGA-Alexa 488 into the ipsilateral hemidiaphragm of acutely and chronically C2 hemisected rats reveals activity-dependent synaptic plasticity in the respiratory motor pathways

•Transsynaptic transport of WGA-Alexa 488 goes across physiologically active synapses.•Plasticity following spinal cord injury occurs caudally and supraspinally.•Hyperactivity is selective to the circuitry of the respiratory system.•Tracer may identify alternate circuitry mediating diaphragmatic recovery after SCI.

WGA-Alexa 488 is a fluorescent neuronal tracer that demonstrates transsynaptic transport in the central nervous system. The transsynaptic transport occurs over physiologically active synaptic connections rather than less active or silent connections. Immediately following C2 spinal cord hemisection (C2Hx), when WGA-Alexa 488 is injected into the ipsilateral hemidiaphragm, the tracer diffuses across the midline of the diaphragm and retrogradely labels the phrenic nuclei (PN) bilaterally in the spinal cord. Subsequently, the tracer is transsynaptically transported bilaterally to the rostral Ventral Respiratory Groups (rVRGs) in the medulla over physiologically active connections. No other neurons are labeled in the acute C2Hx model at the level of the phrenic nuclei or in the medulla. However, with a recovery period of at least 7 weeks (chronic C2Hx), the pattern of WGA-Alexa 488 labeling is notably changed. In addition to the bilateral PN and rVRG labeling, the chronic C2Hx model reveals fluorescence in the ipsilateral ventral and dorsal spinocerebellar tracts, and the ipsilateral reticulospinal tract. Furthermore, interneurons are labeled bilaterally in laminae VII and VIII of the spinal cord as well as neurons in the motor nuclei bilaterally of the intercostal and forelimb muscles. Moreover, in the chronic C2Hx model, there is bilateral labeling of additional medullary centers including raphe, hypoglossal, spinal trigeminal, parvicellular reticular, gigantocellular reticular, and intermediate reticular nuclei. The selective WGA-Alexa 488 labeling of additional locations in the chronic C2Hx model is presumably due to a hyperactive state of the synaptic pathways and nuclei previously shown to connect with the respiratory centers in a non-injured model. The present study suggests that hyperactivity not only occurs in neuronal centers and pathways caudal to spinal cord injury, but in supraspinal centers as well. The significance of such injury-induced plasticity is that hyperactivity may be a mechanism to re-establish lost function by compensatory routes which were initially physiologically inactive.