Size-dependent alterations in phrenic motor neuron perineuronal nets following cervical spinal hemisection

Phrenic motor neurons (PhMNs) innervate the diaphragm muscle (DIAm), which is essential for ventilation and airway defense maneuvers. "Inspiratory drive" for PhMNs arises primarily from ipsilateral, descending glutamatergic inputs originating from the medulla. Unilateral cervical hemisection (C2SH) disrupts this drive, reducing DIAm activation on the injured side. Neuroplastic changes, increasing synaptic signaling via spared pathways, can enhance the spontaneous recovery from C2SH. However, synaptic neuroplasticity is highly regulated by the perineuronal net, an extracellular matrix structure. Here, we evaluated perineuronal nets on retrogradely labeled PhMNs in control (sham) and C2SH rats. After confirming reduced DIAm EMG activity at 3 days following C2SH, rats were euthanized and perfused with paraformaldehyde, and the cervical spinal cord was extracted and then processed to perform free-floating immunolabeling 7 days postinjury. Horizontal sections of the cervical spinal cord were cut and probed with antibodies against aggrecan and choline acetyltransferase (ChAT). Three-color confocal images (2 µm z-steps) of PhMNs (alexa-647nm-conjugated CTB), aggrecan (488 nm), and ChAT (594 nm) were obtained in control (386 PhMNs) and C2SH (292 PhMNs) rats. Aggrecan intensity was quantified around the soma of PhMNs, with ChAT immunolabeling used to identify the somal boundary. Overall, less aggrecan was present on the injured ipsilateral side of C2SH rats. In control rats, the aggrecan intensity was comparable across PhMNs of varying size. In C2SH rats, aggrecan intensity was dependent on PhMN size, consistent with increased reconfiguring of the ipsilateral PhMN neural network. In addition, C2SH rats showed contralateral increase in perineuronal nets (PNNs) consistent with increased extracellular matrix components near the region of the denervated target. These findings have important implications for neuroplastic mechanisms following cervical spinal cord injury.NEW & NOTEWORTHY Neuroplastic changes in phrenic motor neurons that underlie recovery from spinal cord injury may be mediated by perineuronal nets. We show that changes in perineuronal nets around phrenic motor neurons occur within 7 days after cervical spinal cord injury, and that the increase in perineuronal nets around larger phrenic motor neurons was more pronounced. These changes are consistent with substantial reorganization of the neural network onto intact phrenic motor neurons following cervical spinal cord injury.