Impact of glutamatergic and serotonergic neurotransmission on diaphragm muscle activity after cervical spinal hemisection

Abstract

Incomplete cervical spinal cord hemisection at C2 (SH) disrupts descending excitatory drive to phrenic motoneurons, paralyzing the ipsilateral diaphragm muscle. Spontaneous recovery over time is associated with increased phrenic motoneuron expression of glutamatergic N-methyl-d-aspartate (NMDA) and serotonergic 5-HT2A receptors. We hypothesized that NMDA and 5-HT2A receptor-mediated neurotransmission play a role in ipsilateral diaphragm muscle activity post-SH. Adult male Sprague-Dawley rats were implanted with bilateral diaphragm EMG electrodes for chronic EMG recordings up to 28 days post-SH (SH 28D). The extent of recovery was calculated by peak root-mean-square (RMS) EMG amplitude. In all animals, absence of ipsilateral activity was verified at 3 days post-SH. Diaphragm EMG activity was also recorded during exposure to hypoxia-hypercapnia (10% O₂-5% CO₂). In SH animals displaying recovery of ipsilateral diaphragm EMG activity at SH 28D, cervical spinal cord segments containing the phrenic motor nucleus (C3-C5) were surgically exposed and either the NMDA receptor antagonist d-2-amino-5-phosphonovalerate (d-AP5; 100 mM, 30 μl) or 5-HT2A receptor antagonist ketanserin (40 mM, 30 μl) was instilled intrathecally. Following d-AP5, diaphragm EMG amplitude was reduced ipsilaterally, during both eupnea (42% of pre-d-AP5 value; P = 0.007) and hypoxia-hypercapnia (31% of pre-d-AP5 value; P = 0.015), with no effect on contralateral EMG activity or in uninjured controls. Treatment with ketanserin did not change ipsilateral or contralateral RMS EMG amplitude in SH animals displaying recovery at SH 28D. Our results suggest that spinal glutamatergic NMDA receptor-mediated neurotransmission plays an important role in ipsilateral diaphragm muscle activity after cervical spinal cord injury.NEW & NOTEWORTHY Spontaneous recovery following C2 spinal hemisection (SH) is associated with increased phrenic motoneuron expression of glutamatergic and serotonergic receptors. In this study, we show that pharmacological inhibition of glutamatergic N-methyl-d-aspartate (NMDA) receptors blunts ipsilateral diaphragm activity post-SH. In contrast, pharmacological inhibition of serotonergic 5-HT2A receptors does not change diaphragm EMG activity post-SH. Our results suggest that NMDA receptor-mediated glutamatergic neurotransmission plays an important role in enhancing rhythmic respiratory-related diaphragm activity after spinal cord injury.

Keywords: d-AP5; ketanserin; neurotransmitter; respiration; spinal cord injury.

Fig. 1.

Diaphragm EMG activity ipsilateral to injury and contralateral to injury, after acute treatment with N-methyl-d-aspartate (NMDA) receptor antagonist d-2-amino-5-phosphonovalerate (d-AP5). Representative raw diaphragm EMG recordings (Raw) and root mean square (RMS) EMG tracings during eupnea (A) and hypoxia-hypercapnia (B) at pre-SH, SH 3D, SH 28D, and after d-AP5 treatment. Diaphragm EMG tracings from 2 animals (1 ipsilateral, 1 contralateral) represent chronic recordings obtained for the same animal on each side. Note the lack of ipsilateral diaphragm EMG activity at SH 3D. The NMDA receptor antagonist d-AP5 was given only to animals displaying recovery during eupnea at SH 28D (n = 7). C: at 30 min after d-AP5 treatment, ipsilateral RMS EMG amplitude during eupnea decreased in all animals (each symbol represents a different animal), on average by ~60% (*P = 0.007 compared with SH 28D). D: similarly, ipsilateral RMS EMG amplitude during hypoxia-hypercapnia decreased in all animals, on average by ~70% (P = 0.015 compared with SH 28D hypoxia-hypercapnia).

Fig. 2.

Ipsilateral and contralateral diaphragm EMG activity after acute treatment with 5-HT2A receptor antagonist ketanserin. Representative raw diaphragm EMG recordings (Raw) and root mean square (RMS) EMG tracings during eupnea (A) and hypoxia-hypercapnia (B) at pre-SH, SH 3D, SH 28D, and after ketanserin treatment. The 5-HT2A receptor antagonist ketanserin was given only to animals displaying recovery during eupnea at SH 28D (n = 6). C: at 30 min after ketanserin treatment, ipsilateral RMS EMG amplitude during eupnea changed inconsistently across animals (each symbol represents a different animal). The average change was not significant (P = 0.575 compared with SH 28D). D: similarly, ipsilateral RMS EMG amplitude during hypoxia-hypercapnia on average was not affected by ketanserin treatment (P = 0.531 compared with SH 28D hypoxia-hypercapnia).