Phrenic motoneuron expression of serotonergic and glutamatergic receptors following upper cervical spinal cord injury

Abstract

Following cervical spinal cord injury at C2 (SH hemisection model) there is progressive recovery of phrenic activity. Neuroplasticity in the postsynaptic expression of neurotransmitter receptors may contribute to functional recovery. Phrenic motoneurons express multiple serotonergic (5-HTR) and glutamatergic (GluR) receptors, but the timing and possible role of these different neurotransmitter receptor subtypes in the neuroplasticity following SH are not clear. The current study was designed to test the hypothesis that there is an increased expression of serotonergic and glutamatergic neurotransmitter receptors within phrenic motoneurons after SH. In adult male rats, phrenic motoneurons were labeled retrogradely by intrapleural injection of Alexa 488-conjugated cholera toxin B. In thin (10 μm) frozen sections of the spinal cord, fluorescently-labeled phrenic motoneurons were visualized for laser capture microdissection (LCM). Using quantitative real-time RT-PCR in LCM samples, the time course of changes in 5-HTR and GluR mRNA expression was determined in phrenic motoneurons up to 21 days post-SH. Expression of 5-HTR subtypes 1b, 2a and 2c and GluR subtypes AMPA, NMDA, mGluR1 and mGluR5 was evident in phrenic motoneurons from control and SH rats. Phrenic motoneuron expression of 5-HTR2a increased ~8-fold (relative to control) at 14 days post-SH, whereas NMDA expression increased ~16-fold by 21-days post-SH. There were no other significant changes in receptor expression at any time post-SH. This is the first study to systematically document changes in motoneuron expression of multiple neurotransmitter receptors involved in regulation of motoneuron excitability. By providing information on the neuroplasticity of receptors expressed in a motoneuron pool that is inactivated by a higher-level spinal cord injury, appropriate pharmacological targets can be identified to alter motoneuron excitability.

Localized Delivery of Brain-Derived Neurotrophic Factor-Expressing Mesenchymal Stem Cells Enhances Functional Recovery following Cervical Spinal Cord Injury

“…8,9,11,12,14,21 Briefly, the cervical spinal cord was exposed with a bilateral dorsal laminectomy at C2, and the right anterolateral cord was transected at C2 (i.e., involving the lateral and ventral funiculi, and preserving the dorsal funiculus). All animals were observed daily after surgery, and intramuscular buprenorphine and oral acetaminophen were administered for the first 3 days.…”

“…12,48,49 At SH 14D, rats were anesthetized, euthanized by exsanguination after administration of heparin, and transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). The cervical spinal cord was excised and post-fixed in 4% paraformaldehyde overnight, and then transferred to 24% sucrose in PBS for 24-72 h. Longitudinal 50 lm thick sections were cut with a Reichert-Jung Frigocut cryostat (Reichert Microscope Services, Depew, NY).…”

“…Retrogradely labeled phrenic motoneurons in rats injected with CTB had a staining pattern identical to previous studies. 12,46,48,49 186 GRANSEE ET AL.…”

“…Retrograde labeling with CTB allowed for visualization of phrenic motoneurons, as in previous studies, 2,12,[48][49][50] in order to localize transplanted MSCs relative to the phrenic motor pool. As expected, expression of GFP by transplanted BDNF-MSCs was localized near the injection sites (i.e., both rostral and caudal to C2), and primarily in the white matter (Fig.…”

“…5,6 SH involves a unilateral transection of anterolateral funiculi at C2, which removes premotor drive to phrenic motoneurons, paralyzing the ipsilateral diaphragm muscle. [7][8][9][10][11][12][13][14] Following SH, there is gradual recovery of rhythmic diaphragm activity ipsilateral to injury, [15][16][17][18][19][20][21] suggesting neuroplasticity and strengthening of spared contralateral synaptic inputs to phrenic motoneurons. Importantly, recovery of ipsilateral diaphragm activity after SH can be enhanced by intrathecal delivery of BDNF at the level of the phrenic motor nucleus.…”