Name: Video: Quantifying Rat Diaphragm Motor Unit Connectivity Using Electrophysiology
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quantifying rat diaphragm motor unit connectivity using electrophysiology

Quantifying Phrenic Motor Unit Function in Rodent Models of Neurodegeneration

Phrenic motor neurons (MNs), extending from C3 to C6 myotome levels, form the final link from the central nervous system (CNS) to the diaphragm muscle1. Phrenic motor units (MUs) are comprised of a single spinal MN and its innervated diaphragm muscle fibers forming the smallest functional unit of the respiratory neuromuscular system. The ventilatory function requires adequate contraction of the diaphragm muscle achieved through coordinated activation of the phrenic MU pool2,3. Many neurological diseases, including amyotrophic lateral sclerosis (ALS), result in severe ventilatory impairment, ultimately contributing to the cause of death4.
Several electrophysiological approaches can be employed to evaluate and monitor the integrity of the motor unit (MU) pool in vivo. Compound muscle action potential (CMAP) reflects the summated depolarization of all muscle fibers in a specific muscle or muscle group after peripheral nerve stimulation and is sensitive to a range of neuromuscular conditions, including ALS5,6 and spinal muscular atrophy (SMA)7,8,9. A limitation of CMAP assessment is that collateral sprouting can lead to maintained CMAP amplitude and area even in the presence of MU loss10. To overcome this limitation, modifications have been made to the CMAP technique to evaluate both motor unit number and size11. Additionally, an in vivo study investigating the functional assessment of diaphragm CMAP by an electrophysiological system suggested that it may also be feasible to utilize the described diaphragm CMAP recording technique for motor unit number estimation12.
The incremental motor unit number estimation (MUNE) technique was initially introduced in the early 1970s by McComas et al. for the extensor digitorum brevis muscle in humans13. The incremental MUNE approach was a modification of the traditional CMAP recording technique during which a gradually increasing stimulation was delivered to record quantal, all-or-none submaximal increments as indices of single motor unit responses. The summed and averaged increments were used to calculate an estimate for the size of a single motor unit potential (SMUP). This calculated size was then divided into the CMAP amplitude to estimate the number of MUs innervating the muscle under examination11. MUNE demonstrates high sensitivity in detecting and monitoring motor unit loss, allowing for the identification of motor unit dysfunction before observable changes in measures such as CMAP amplitude or area14,15. In ALS patients, MUNE has proven to be exceptionally sensitive, serving as a prominent biomarker for disease onset, progression, and prognosis16,17.
Numerous adaptations of MUNE have been developed and widely used to assess MU function in conditions such as neurodegeneration, neural injury, and the natural aging process18,19,20,21. Since the initial description, various adaptations utilizing both electrophysiological responses and incremental force (mechanical) measurements have been employed in both human studies and animal models22. MUNE provides a non-invasive functional assessment of motor neuron connectivity with the muscle. Longitudinally applying MUNE enables the understanding of disease or induced phenotype progression and the evaluation of protective or regenerative effects of therapeutic interventions, both in clinical and preclinical settings. Regardless of the effectiveness of MUNE measures reproducibility and the clinical relevance of the technique for MU pools throughout most of the human body, efforts have largely focused on limb muscles in rodent muscles10,23,24,25.
Therefore, the objectives of this study were to describe an approach to obtaining compound muscle action potential (CMAP), SMUP, and phrenic motor unit number (MUNE) as in vivo assessments that can be used longitudinally in preclinical rodent studies to quantify the MUNE, motor unit size (represented as SMUP), and CMAP. Furthermore, we present representative data that highlights the loss of diaphragm MU number following intrapleural administration of a phrenic MN degenerative agent, cholera toxin B fragment conjugated to saporin (CTB-SAP).

Author Spotlight: Studying Neuromuscular Responses and Motor Neuron Plasticity in Neurodegenerative Diseases

Assessing Rat Diaphragm Motor Unit Connectivity Outcome Measures as Quantitative Biomarkers of Phrenic Motor Neuron Degeneration and Compensation

Quantifying Rat Diaphragm Motor Unit Connectivity Using Electrophysiology

quantifying-rat-diaphragm-motor-unit-connectivity-using

Research

JoVE Journal

Neuroscience

35 Views. Quantifying Rat Diaphragm Motor Unit Connectivity Using Electrophysiology

Video: Quantifying Rat Diaphragm Motor Unit Connectivity Using Electrophysiology

Loss of ventilatory muscle function is a consequence of motor neuron injury and neurodegeneration (e.g., cervical spinal cord injury and amyotrophic lateral sclerosis, respectively). Phrenic motor neurons are the final link between the central nervous system and muscle, and their respective motor units (groups of muscle fibers innervated by a single motor neuron) represent the smallest functional unit of the neuromuscular ventilatory system. Compound muscle action potential (CMAP), single motor unit potential (SMUP), and motor unit number estimation (MUNE) are established electrophysiological approaches that enable the longitudinal assessment of motor unit integrity in animal models over time but have mostly been applied to limb muscles. Therefore, the objectives of this study are to describe an approach in preclinical rodent studies that can be used longitudinally to quantify the phrenic MUNE, motor unit size (represented as SMUP), and CMAP, and then to demonstrate the utility of these approaches in a motor neuron loss model. Sensitive, objective, and translationally relevant biomarkers for neuronal injury, degeneration, and regeneration in motor neuron injury and diseases can significantly aid and accelerate experimental research discoveries to clinical testing.

In this study, we present an in vivo method for estimating motor unit number and size to quantify rat diaphragm motor unit connectivity. A step-by-step approach to these techniques is described.

Loss of ventilatory muscle function is a consequence of motor neuron injury and neurodegeneration (e.g., cervical spinal cord injury and amyotrophic ...

Methods Article

Quantifying Rat Diaphragm Motor Unit Connectivity Using Electrophysiology