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☆
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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Cervical spinal hemisection alters phrenic motor neuron glutamatergic mRNA receptor expression
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2018, Experimental NeurologyCitation Excerpt :It is unclear whether the mechanisms underlying the sCPP differ from the CPP, but recovery has been attributed to progressive actiavtion of crossed bulbospinal pathways, axonal sprouting, rerouting of bulbospinal projections (Darlot et al., 2012; Vinit et al., 2005; Vinit et al., 2011) and forming new polysynaptic connections with phrenic motoneurons via cervical spinal interneurons (Darlot et al., 2012; Fuller et al., 2009; Lane et al., 2009; Lane et al., 2008b; Sandhu et al., 2009; Zholudeva et al., 2017). However, diaphragm recovery via sCPP has been shown to be dependent on plasticity of glutamatergic (Alilain and Goshgarian, 2007, 2008; Gransee et al., 2017; Mantilla et al., 2012, 2017), serotonergic (Basura et al., 2001; Fuller et al., 2005; Lee and Gonzalez-Rothi, 2017; Mantilla et al., 2012, 2017) and adenosinergic (Golder et al., 2008; Minic et al., 2017; Nantwi, 2009; Nantwi and Goshgarian, 2002) inputs to phrenic motoneurons. Glutamate is the primary excitatory neurotransmitter in the CNS and mediates respiratory synaptic inputs to phrenic motoneurons from the medulla (Alheid and McCrimmon, 2008; Chitravanshi and Sapru, 1996; Liu et al., 1990; McCrimmon et al., 1989).
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Support: NIH Grant HD31550.