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

We're interested in understanding normal and maladaptive responses of the neuromuscular system in the context of health, aging, and disease. Specifically, we are aiming to investigate how the respiratory motor system responds to motor neuron losses through compensatory mechanisms such as collateral sprouting. Currently, various laboring techniques, such as retrograde tracers, adenoviruses, and immunohistochemistry have been utilized to quantify the number of innervating motor neurons in rodent degenerative models.Labeling techniques, though valuable for motor neuron evaluations, have limitations in assessing the functionality of motor units and are not suitable for longitudinal assessments. The MUNE technique is a non-invasive approach in preclinical rodent studies that can be used longitudinally to quantify the phrenic motor units. Implementing the MUNE protocol has the potential to significantly aid and accelerate experimental research discoveries toward clinical testing, particularly in non-invasively and longitudinally assessing neuromuscular impairment of the phrenic nerve and diaphragm muscle.Our eclectic lab group focuses on investigating neuromuscular function in health and preclinical models of neurodegenerative disease. We are striving to utilize translational techniques to better understand whether underlying mechanisms of plasticity can be harnessed and adapted to preserve respiration to improve the quality of life in patients with neurodegenerative disease.

Quantifying Rat Diaphragm Motor Unit Connectivity Using Electrophysiology

To begin, prepare and position the anesthetized rat for electrophysiology. Apply monophasic cathartic square wave pulses to stimulate the phrenic nerve for recording CMAP responses. Increase the stimulus intensity progressively until the amplitude of the response ceases to increase.Then, raise the stimulus intensity to approximately 120%of the level required for a maximal response to ensure super maximal stimulation and record an additional response. Measure and document the peak-to-peak amplitudes of the CMAP in millivolts. Administer submaximal stimulation with a duration of 0.1 milliseconds at a frequency of 1 hertz.Gradually increase the intensity in 0.03 milliampere increments to elicit incremental responses until a minimal all or none response is achieved. Then, acquire the initial response with a stimulus intensity ranging between 2 and 10 milliamperes. After saving the first incremental response, acquire additional increments with progressively higher stimulus intensities in increments of 0.03 milliamperes.Ensure that the initial negative peak of the incremental responses aligns temporally with the negative peak of the maximal CMAP response. Given the diaphragm's involvement in respiration, check for stability and absence of fractionation in each incremental response to confirm consistency across three duplicate responses. Observe incremental responses in real time and overlay them on previous recordings for visual distinction.Then, confirm that the increment amplitude is at least 25 microvolts. After recording 10 incremental responses, verify that the amplitude of each increment does not exceed 1/3rd of the total amplitude of the final response. To estimate the average amplitude of the SMUP, average the values of 10 increments.Next, divide the maximum CMAP amplitude by the average SMUP amplitude to determine the MUNE. Seven days after intrapleural CTB-SAP injection, MUNE decreased to 60 functional motor units in adult rats compared to 74 in control rats. CMAP amplitude remained relatively stable, suggesting collateral sprouting as a possible compensatory mechanism.Diaphragm CMAP, SMUP and MUNE were measured over 28 days post-injection. CMAP showed no notable change. The average increase in SMUP for CTB-SAP rats was approximately 50 to 60%The changes were significant over time and attributed to CTB-SAP.MUNE was reduced by approximately 40%in CTB-SAP rats, indicating significant effects of time in CTB-SAP treatment.

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

Research

JoVE Journal

Neuroscience

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 ...

Electrode Placement to Record Neuromuscular Function in Rat Diaphragm Muscle

To begin, position the anesthetized and prepared rat for compound muscle action potential recording. Place the active needle electrode subcutaneously over the midclavicular line, inferior to the last rib border. Then, position the reference needle electrode subcutaneously at the angle between the xiphoid process and the last sternocostal cartilage.Next, use a pair of 28-gauge monopolar needle electrodes for phrenic nerve stimulation at the carotid sheath. Place the electrode subcutaneously on the lateral neck between the anterior and middle scalene muscles, with a separation of about one centimeter. Confirm that the placement of the stimulating needles is at the level below the fourth cervical vertebrae.Then, place a disposable surface electrode on the tail for the ground electrode.