Elsevier

Experimental Neurology

Volume 212, Issue 2, August 2008, Pages 348-357
Experimental Neurology

Glutamate receptor plasticity and activity-regulated cytoskeletal associated protein regulation in the phrenic motor nucleus may mediate spontaneous recovery of the hemidiaphragm following chronic cervical spinal cord injury

https://doi.org/10.1016/j.expneurol.2008.04.017Get rights and content

Abstract

High cervical spinal cord hemisection results in paralysis of the ipsilateral hemidiaphragm; however, functional recovery of the paralyzed hemidiaphragm can occur spontaneously. The mechanisms mediating this recovery are unknown. In chronic, experimental contusive spinal cord injury, an upregulation of the NMDA receptor 2A subunit and a downregulation of the AMPA receptor GluR2 subunit have been correlated with improved hind limb motor recovery. Therefore, we hypothesized that NR2A is upregulated, whereas GluR2 is down-regulated following chronic C2 hemisection to initiate synaptic strengthening in respiratory motor pathways. Since NMDA receptor activation can lead to the delivery of AMPA receptor subunits to the post-synaptic membrane, we also hypothesized that there would be an upregulation of the GluR1 AMPA receptor subunit and that activity-regulated cytoskeletal associated protein may mediate the post-synaptic membrane delivery. Female rats were hemisected at C2 and allowed to recover for different time points following hemisection. At these time points, protein levels of NR2A, GluR1, and GluR2 subunits were assessed via Western blot analysis. Western blot analysis revealed that there were increases in NR2A subunit at six and twelve weeks post C2 hemisection. At six, twelve, and sixteen weeks post hemisection, the GluR1 subunit was increased over controls, whereas the GluR2 subunit decreased sixteen weeks post hemisection. Immunocytochemical data qualitatively supported these findings. Results also indicated that activity-regulated cytoskeletal associated protein may be associated with the above changes. These findings suggest a role of NR2A, GluR1, and GluR2 in mediating chronic spontaneous functional recovery of the paralyzed hemidiaphragm following cervical spinal cord hemisection.

Introduction

C2 spinal cord hemisection (C2H) results in paralysis of the ipsilateral hemidiaphragm. This paralysis results from disruption of the pathways from medullary respiratory centers to the phrenic motor (PM) nucleus of the spinal cord (Goshgarian and Guth, 1977). Activation of a spared, latent respiratory pathway (i.e., the crossed phrenic pathway) (Moreno et al., 1992), restores activity to the once paralyzed hemidiaphragm (Fig. 1) (for review see Goshgarian, 2003). Chronically, expression of the crossed phrenic pathway occurs spontaneously without any intervention (Pitts, 1940, Nantwi et al., 1999).

The pathway driving PM neurons during inspiration is glutamatergic (McCrimmon et al., 1989, Liu et al., 1990, Chitravanshi and Sapru, 1996). Distinct glutamate receptor types and subunits are present on phrenic motor neurons (Robinson and Ellenber, 1997). Among those observed are the subunits of the N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors.

Distinguishing it from the AMPA and kainite glutamate receptors, the NMDA receptor has the capacity for calcium influx and a magnesium block, which is relieved through cell membrane depolarization (Moriyoshi et al., 1991). These features have led to the hypothesis that the NMDA receptor is a “coincidence detector” of simultaneous pre- and post-synaptic activity since NMDA receptor activation requires pre-synaptic glutamate release and post-synaptic membrane depolarization. This makes the NMDA receptor an ideal candidate for mediating long term potentiation (LTP) (Seeburg et al., 1995, Tang et al., 1999).

The NMDA receptor complex is formed by distinct subunits: the NR1, NR2A,B,C,D, and NR3 subunits. These subunits confer distinctive properties to the NMDA receptor (Monyer et al., 1992, Ishii et al., 1993, Monyer et al., 1994, Krupp et al., 1998). Following contusive spinal cord injury at T8, the mRNA levels of the NR2A subunit are upregulated caudal to the site of injury, and the upregulation has been correlated with improved hindlimb function, suggesting that the upregulation strengthens excitatory synaptic connections on hindlimb motor neurons (Grossman et al., 2000). Later studies have demonstrated that the NR2A subunit is a possible mediator of synaptic strengthening during LTP (Liu et al., 2004, Massey et al., 2004).

AMPA receptor complexes are composed of four genetically distinct subunits, GluR1, 2, 3, and 4, which confer distinct properties to the AMPA receptor. Specifically, the GluR2/GluR3 heteromer receptor complex is continuously delivered to the synapse, regardless of synaptic activity. However, the GluR1/GluR2 heteromer complex is delivered to the synapse on the basis of synaptic activity, particularly through NMDA receptor activation (Esteban, 2003). Furthermore, the GluR1 subunit is delivered to the post-synaptic membrane during LTP induction (Hayashi et al., 2000). The GluR2 subunit is downregulated following traumatic CNS injury, including T8 spinal cord injury (Grossman et al., 1999, Gorter et al., 1997, Alsbo et al., 2001).

Based on the above, we hypothesized that following C2H there may be an increase of the NR2A subunit and a downregulation of GluR2 at the level of the phrenic nucleus to initiate synaptic strengthening. Subsequently, an upregulation of GluR1 could result in enhanced glutamatergic drive to PM neurons bringing about spontaneous recovery of the hemidiaphragm in chronically injured animals. These hypotheses were tested in the present investigation.

Section snippets

Surgical procedures and C2 hemisection

Adult female Sprague Dawley rats (250–350 g) were anesthetized with a ketamine (70 mg/kg) and xylazine (7 mg/kg) solution administered i.p. Following administration of anesthesia, the animals were prepared for surgery by shaving and cleansing the dorsal neck area with betadine and 70% rubbing alcohol. Following the surgical prep, a dorsal midline incision approximately 4 cm on the neck was made. The paravertebral muscles were retracted and a laminectomy of the second cervical vertebra was

Functionally complete hemisection and recovery of diaphragmatic activity

One week following C2 hemisection, all hemisected animals included in this study displayed no hemidiaphragmatic activity on the injured side, revealed through bilateral EMG recordings, indicating a functionally complete hemisection (Fig. 2). The correlation between no EMG activity of the hemidiaphragm ipsilateral to an anatomically complete hemisection of the spinal cord has been observed previously in our lab (Moreno et al., 1992). Furthermore, as explained previously, waiting one full week

Discussion

The present data have demonstrated that following a C2 hemisection there are significant increases of the NR2A subunit at six and twelve weeks post injury at the ipsilateral C3–C6 level of the spinal cord compared to control animals. The NR2A subunit level decreases to amounts similar to control at sixteen weeks post C2 lesion. Through immunocytochemistry and retrograde WGA-HRP labeling, it was observed qualitatively that the upregulation of NR2A also occurred on PMNs. These changes were also

Conclusion

In summary, this report only begins to uncover the underlying mechanisms which may mediate spontaneous functional recovery following C2 hemisection. A potential role for the NR2A, GluR1 and GluR2 subunits, as well as the immediate early gene Arc protein, in mediating synaptic plasticity in the PM nucleus has been suggested. Understanding the roles of these receptors and their regulation following injury may prove to be beneficial in exploring new avenues of therapy following traumatic spinal

Acknowledgment

This work was supported by NIH Grant HD 31550 (HGG).

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    Support: NIH Grant HD31550.

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