Elsevier

Experimental Neurology

Volume 249, November 2013, Pages 20-32
Experimental Neurology

Phrenic motoneuron discharge patterns following chronic cervical spinal cord injury

https://doi.org/10.1016/j.expneurol.2013.08.003Get rights and content

Highlights

  • First description of phrenic motoneuron burst patterns after chronic spinal injury

  • Most cells were either silent or active late in the inspiratory period.

  • Discharge patterns returned towards control values by 8 wks post-injury.

  • A novel pattern was noted in which some cells burst tonically during apnea.

Abstract

Cervical spinal cord injury (SCI) dramatically disrupts synaptic inputs and triggers biochemical, as well as morphological, plasticity in relation to the phrenic motor neuron (PhMN) pool. Accordingly, our primary purpose was to determine if chronic SCI induces fundamental changes in the recruitment profile and discharge patterns of PhMNs. Individual PhMN action potentials were recorded from the phrenic nerve ipsilateral to lateral cervical (C2) hemisection injury (C2Hx) in anesthetized adult male rats at 2, 4 or 8 wks post-injury and in uninjured controls. PhMNs were phenotypically classified as early (Early-I) or late inspiratory (Late-I), or silent according to discharge patterns. Following C2Hx, the distribution of PhMNs was dominated by Late-I and silent cells. Late-I burst parameters (e.g., spikes per breath, burst frequency and duration) were initially reduced but returned towards control values by 8 wks post-injury. In addition, a unique PhMN burst pattern emerged after C2Hx in which Early-I cells burst tonically during hypocapnic inspiratory apnea. We also quantified the impact of gradual reductions in end-tidal CO2 partial pressure (PETCO2) on bilateral phrenic nerve activity. Compared to control rats, as PETCO2 declined, the C2Hx animals had greater inspiratory frequencies (breaths  min 1) and more substantial decreases in ipsilateral phrenic burst amplitude. We conclude that the primary physiological impact of C2Hx on ipsilateral PhMN burst patterns is a persistent delay in burst onset, transient reductions in burst frequency, and the emergence of tonic burst patterns. The inspiratory frequency data suggest that plasticity in brainstem networks is likely to play an important role in phrenic motor output after cervical SCI.

Introduction

The injured spinal cord is, in principle, a “new spinal cord” in which neural networks and control mechanisms affecting virtually every functional domain are altered (Dimitrijevic, 1998, Edgerton et al., 2001). Accordingly, respiratory control mechanisms that have been firmly established in the spinal-intact condition are likely to be significantly altered after chronic spinal cord injury (SCI) (Sandhu et al., 2009). A basic understanding of how SCI and spontaneous neuroplastic processes impact respiratory motor control is important for developing and optimizing spinal rehabilitation and repair approaches.

Much of our current understanding of respiratory motor plasticity following SCI derives from experiments which have utilized a lateral cervical (C2) hemisection model (C2Hx) (Goshgarian, 2003, Goshgarian, 2009, Lane et al., 2008a, Sandhu et al., 2009, Zimmer et al., 2007). The C2Hx model has been used to carefully describe both the phrenic neurogram (i.e., compound action potentials) and diaphragm electromyogram (EMG) recovery profiles after incomplete cervical SCI (reviewed in Goshgarian, 2003, Goshgarian, 2009, Sandhu et al., 2009). However, it is unknown whether the gradual and functionally incomplete recovery process after chronic cervical SCI (Dougherty et al., 2012) coincides with fundamental changes in phrenic motoneuron (PhMN) recruitment profiles and discharge patterns (Lee and Fuller, 2011). In the spinal intact condition, respiratory-related PhMN bursting and recruitment are determined by both intrinsic motoneuron properties (i.e., resistance, rheobase (Berger, 1979, Dick et al., 1987, Webber and Pleschka, 1976)) and descending (i.e., bulbospinal) and possibly propriospinal synaptic inputs (Monteau et al., 1985). There is a debate as to whether PhMN recruitment patterns during breathing are driven exclusively by membrane properties (i.e., the “size principle” of motoneuron recruitment, (Henneman, 1957)), or if there is also some degree of selective synaptic input to cells recruited early vs. late in the inspiratory effort (reviewed in Lee and Fuller, 2011). In either case, since injuries to the cervical spinal cord are likely to alter both synaptic inputs (Goshgarian et al., 1989) and intrinsic motoneuron properties (Mantilla and Sieck, 2009, Sperry and Goshgarian, 1993), fundamental changes in how the central nervous system regulates PhMNs are likely after cervical SCI.

Within the cervical spinal cord, neuroplastic changes occurring after C2Hx have been postulated to facilitate PhMN recruitment (Goshgarian, 2009, Tai et al., 1997a, Tai et al., 1997b) and lead to partial functional recovery. For example, increases in cervical spinal glutamatergic (Alilain and Goshgarian, 2008, Mantilla et al., 2012) and/or serotonergic receptor expression (Basura et al., 2001, Fuller et al., 2005, Mantilla et al., 2012) could both serve to increase PhMN excitability. A similar functional impact could result from reductions in PhMN soma dimensions (Mantilla and Sieck, 2009), changes in dendrodentritic appositions, or increased number of synaptic active zones (Goshgarian, 2003, Goshgarian et al., 1989, Sperry and Goshgarian, 1993). The primary purpose of the present work was to determine how and whether PhMN discharge patterns and recruitment profiles are altered in a model of chronic, incomplete cervical SCI. Our overall hypothesis was that PhMNs would show a time-dependent increase in inspiratory burst frequency that paralleled the gradual recovery in phrenic motor output which follows C2Hx.

Section snippets

Animals

Male Sprague–Dawley rats were purchased from Harlan Inc. (Indianapolis, IN, USA). Rats were randomly assigned to the following groups: control, uninjured (N = 18, 388 ± 9 g, age 121 ± 4 days) or C2Hx injury. Rats with C2Hx were studied at 2 wks (N = 8, 327 ± 6 g, 108 ± 1 days), 4 wks (N = 8, 367 ± 7 g, 118 ± 2 days) or 8 wks (N = 7, 380 ± 15 g, 145 ± 3 days) post-injury. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida.

Spinal cord injury

The C2Hx injury was induced at ~ 3 

Heart rate and blood pressure

Mean arterial blood pressure (MAP) was similar between control and C2Hx rats (Table 1). Furthermore, MAP declined in all groups when PETCO2 was reduced (P < 0.01, Table 1). Heart rate (HR) was similar between experimental groups, but some modest differences were noted (Table 1). While uninjured control rats showed an increase in HR as MAP decreased (P < 0.01), HR did not change with declining MAP in rats studied at 2 wks post-injury (Table 1). However, at 4 wks post-injury, the HR responses to

Discussion

The present results represent the first detailed analyses of PhMN firing patterns following chronic high cervical SCI. Using a C2Hx model, we observed that the distribution of PhMN bursting shifted from cells that predominantly initiated bursting early in the inspiratory effort (i.e., Early-I) to cells with a Late-I phenotype. Reductions in PhMN discharge duration and burst frequency were also prominent after C2Hx. In addition, a unique PhMN discharge pattern emerged after C2Hx in which tonic

Impact of C2Hx on PhMN discharge patterns

Respiratory-related PhMN burst patterns have been extensively studied in a variety of species in the spinal-intact condition (see reference Lee and Fuller, 2011 for a detailed review). Collectively, the literature indicates that inspiratory PhMN discharge patterns are not homogenous, but rather bursting can be separated into two groups based on the recruitment profile during inspiration. Thus, as in the current study (e.g., Fig. 1, Fig. 2), PhMN can be phenotypically classified as Early-I or

Mechanisms influencing PhMN activity following chronic C2Hx injury

The mechanisms underlying phrenic motor recovery following incomplete cervical SCI can be broadly considered either as “spinal” or “supraspinal” (Golder and Mitchell, 2005, Golder et al., 2001, Zimmer and Goshgarian, 2007). It must be emphasized, however, that spinal and supraspinal plasticity following SCI are not mutually exclusive. Indeed, the literature has documented neuroplastic changes in both the cervical spinal cord (Alilain and Goshgarian, 2008, Fuller et al., 2003, Mantilla et al.,

Tonic PhMN activity after C2Hx

The most unique aspect of PhMN activity after C2Hx was the appearance of tonic burst patterns. Tonic bursting occurred in some PhMNs throughout the respiratory cycle, and in other cells it was observed during periods of inspiratory apnea. Tonic PhMN bursting could reflect the loss of inhibitory synaptic inputs and/or the appearance of novel tonic excitatory inputs, or changes in intrinsic membrane properties. In the spinal intact condition, there is little evidence to support the prevalence of

Conclusion

A unilateral cervical SCI which removes descending synaptic inputs to the ipsilateral phrenic motor pool (i.e., C2Hx) results in alterations in the recruitment patterns and burst profiles of PhMNs. The delay in PhMN burst onset and reductions in burst frequency are likely to reflect the persistent reduction in bulbospinal excitatory synaptic inputs to the phrenic pool, although spinal neuroplasticity, including formation of de novo intraspinal circuits, may play a role. We hypothesize that the

Acknowledgments

Support for this work was provided by grants from the National Institutes of Health (NIH): 1R01NS080180-01A1 (DDF). KZL was supported by the Paralyzed Veterans of America Research Foundation (#2691), the National Science Council (NSC) NSC100-2320-B-110-003-MY2, the National Health Research Institutes (NHRI-EX102-10223NC) and the NSYSU-KMU Joint research Project (2013-I006).

References (70)

  • M.A. Lane

    Spinal respiratory motoneurons and interneurons

    Respir. Physiol. Neurobiol.

    (2011)
  • M.A. Lane et al.

    Respiratory neuroplasticity and cervical spinal cord injury: translational perspectives

    Trends Neurosci.

    (2008)
  • M.A. Lane et al.

    Spinal circuitry and respiratory recovery following spinal cord injury

    Respir. Physiol. Neurobiol.

    (2009)
  • M.A. Lane et al.

    Respiratory function following bilateral mid-cervical contusion injury in the adult rat

    Exp. Neurol.

    (2012)
  • K.Z. Lee et al.

    Neural control of phrenic motoneuron discharge

    Respir. Physiol. Neurobiol.

    (2011)
  • C.B. Mantilla et al.

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

    Exp. Neurol.

    (2012)
  • C.B. Mantilla et al.

    Neuromuscular adaptations to respiratory muscle inactivity

    Respir. Physiol. Neurobiol.

    (2009)
  • C.B. Mantilla et al.

    Phrenic motor unit recruitment during ventilatory and non-ventilatory behaviors

    Respir. Physiol. Neurobiol.

    (2011)
  • R. Monteau et al.

    Central determination of recruitment order: intracellular study of phrenic motoneurons

    Neurosci. Lett.

    (1985)
  • K.L. Rowley et al.

    Respiratory muscle plasticity

    Respir. Physiol. Neurobiol.

    (2005)
  • M.S. Sandhu et al.

    Respiratory recovery following high cervical hemisection

    Respir. Physiol. Neurobiol.

    (2009)
  • G.C. Sieck et al.

    Role of neurotrophins in recovery of phrenic motor function following spinal cord injury

    Respir. Physiol. Neurobiol.

    (2009)
  • M.A. Sperry et al.

    Ultrastructural changes in the rat phrenic nucleus developing within 2 h after cervical spinal cord hemisection

    Exp. Neurol.

    (1993)
  • W.M. St John et al.

    Comparison of phrenic motoneuron activity in eupnea and apneusis

    Respir. Physiol.

    (1985)
  • N. Sukiasyan et al.

    Distribution of calcium channel Ca(V)1.3 immunoreactivity in the rat spinal cord and brain stem

    Neuroscience

    (2009)
  • M.B. Zimmer et al.

    Spinal cord injury in neonates alters respiratory motor output via supraspinal mechanisms

    Exp. Neurol.

    (2007)
  • W.J. Alilain et al.

    Light-induced rescue of breathing after spinal cord injury

    J. Neurosci.

    (2008)
  • F.M. Bareyre et al.

    The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats

    Nat. Neurosci.

    (2004)
  • M.C. Bellingham

    Synaptic inhibition of cat phrenic motoneurons by internal intercostal nerve stimulation

    J. Neurophysiol.

    (1999)
  • A.J. Berger

    Phrenic motoneurons in the cat: subpopulations and nature of respiratory drive potentials

    J. Neurophysiol.

    (1979)
  • T. Costa et al.

    Antagonists with negative intrinsic activity at delta opioid receptors coupled to GTP-binding proteins

    Proc. Natl. Acad. Sci. U. S. A.

    (1989)
  • G. Courtine et al.

    Transformation of nonfunctional spinal circuits into functional states after the loss of brain input

    Nat. Neurosci.

    (2009)
  • G. Courtine et al.

    Recovery of supraspinal control of stepping via indirect propriospinal relay connections after spinal cord injury

    Nat. Med.

    (2008)
  • T.E. Dick et al.

    Correlation of recruitment order with axonal conduction velocity for supraspinally driven diaphragmatic motor units

    J. Neurophysiol.

    (1987)
  • M.R. Dimitrijevic

    Motor control in human spinal cord injury

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