We present refined protocols that allow in vivo monitoring of motor unit function in the mouse. Techniques to measure compound muscle action potential (CMAP) and motor unit number estimation (MUNE) in the mouse hind limb muscles innervated by the sciatic nerve are described.
Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the functional status of a motor unit pool in vivo. These measures can provide insight into the normal development and degeneration of the neuromuscular system. These measures have clear translational potential because they are routinely applied in diagnostic and clinical human studies. We present electrophysiological techniques similar to those employed in humans to allow recordings of mouse sciatic nerve function. The CMAP response represents the electrophysiological output from a muscle or group of muscles following supramaximal stimulation of a peripheral nerve. MUNE is an electrophysiological technique that is based on modifications of the CMAP response. MUNE is a calculated value that represents the estimated number of motor neurons or axons (motor control input) supplying the muscle or group of muscles being tested. We present methods for recording CMAP responses from the proximal leg muscles using surface recording electrodes following the stimulation of the sciatic nerve in mice. An incremental MUNE technique is described using submaximal stimuli to determine the average single motor unit potential (SMUP) size. MUNE is calculated by dividing the CMAP amplitude (peak-to-peak) by the SMUP amplitude (peak-to-peak). These electrophysiological techniques allow repeated measures in both neonatal and adult mice in such a manner that facilitates rapid analysis and data collection while reducing the number of animals required for experimental testing. Furthermore, these measures are similar to those recorded in human studies allowing more direct comparisons.
Motor unit number estimation (MUNE) was originally described by McComas et al. over three decades ago1. The original technique was a modification of the compound muscle action potential (CMAP) recording technique that employed a gradual increase of stimulation to obtain submaximal increments. These increments were summed and averaged to determine an estimated size of a single motor unit potential (SMUP). This size was divided into the CMAP response to estimate the number of motor units innervating the muscle being tested. Following the original description, numerous variations using both electrophysiological responses and incremental force (mechanical) measurements have been used in both human studies and animal models2. The MUNE technique was modified by Shefner and colleagues to investigate mouse models of Amyotrophic Lateral Sclerosis (ALS)3, 4.
In the current description, we detail simplified modifications of the MUNE techniques that are rapid to perform. Importantly, CMAP and MUNE allow reliable measures in both neonatal and adult mice5-8. Experienced individuals can perform these measures in 10-20 min per animal, and repeated measures are feasible which permits the acquisition of longitudinal data5. In the current studies, we employ a clinical electrodiagnostic system. In our experience, clinical electrodiagnostic systems are optimized for rapid and efficient capture of electrophysiological data in vivo, nevertheless standard electrophysiological rigs can easily be adapted for this application.
MUNE and CMAP are clinically relevant measures frequently utilized in research studies and in monitoring patients with neuromuscular disorders such as ALS and spinal muscular atrophy (SMA)9, 10. For instance, in SMA, CMAP and MUNE correlate well with age, severity and clinical measures of function10-14. Both measures are minimally invasive and allow assessment of function longitudinally in the same individual. Importantly, these measures cannot measure activation or recruitment of the motor unit by cortical motor neurons, but they provide a clinically-relevant assessment of the integrity of the motor neuron and its functional counterpart, the motor unit.
Animal models of neuromuscular disease are critical to an understanding of pathogenic mechanisms of human disease and to the preclinical development of potentially effective therapeutic agents. The ability to translate outcome measures and biomarkers that can be utilized across species can facilitate and hasten the translation of promising preclinical findings to human clinical trials. Several groups have previously utilized both electrophysiological and force (mechanical) measurements to estimate motor unit function in mouse models2-4, 15-22. Due to the relative complexity of the measures, we have refined these techniques in a visual format to allow more widespread use and implementation in mice. The format of video demonstration and instruction, allows key steps of the procedure to be highlighted and potential pitfalls to be addressed. The application of these techniques to preclinical testing of potential therapies in motor neuron diseases may improve the translation of putative therapies from mice to human disease.
There are several critical steps in the process of acquiring the CMAP and MUNE responses. Proper and consistent recording electrode placement and sufficient electrode contact with the hind limb are critical for reproducible measurement of amplitude and to decrease background noise. Therefore, close contact between hind limb skin and electrodes should be consistently confirmed. We have found that surface electrodes offer more consistent CMAP and MUNE recordings than needle electrodes. Due to very thin subcutaneous tissues, small movements of needle recording surface may lead to wide variation in CMAP amplitudes. Additionally, the more invasive nature of needle electrodes is not optimal for neonatal mice or longitudinal studies due to potential muscle disruption and injury. One potential drawback of non-selective, surface electrode recordings relates to the possibility of diminished phenotype resolution if a particular muscle is more or less involved compared with another, and this has been reported in an ALS mouse model21.
Acquiring the average SMUP size is technically more challenging compared to the CMAP. Due to the smaller response size (in the range of μV rather than mV) background noise can be more problematic. Background noise can be reduced by adjusting the ground electrode, cathode, anode, and checking other electrical equipment near the experimental setup. A Faraday cage, typically used for intracellular electrophysiology applications, is not required. Visual determination of the individual SMUP responses is the most difficult skill to acquire and takes practice for consistent results with adequate repeatability. It is important to ensure that the SMUPs that are being recorded initiate within the duration of the maximal CMAP response. We have defined criteria for acceptance of individual incremental responses to make this process more straightforward to perform and to increase intra- and inter-rater reliability.
One potential drawback of the incremental MUNE technique includes the possibility of overestimating the number of functional motor units due to alternation of motor units. We have used a technique similar to Shefner et al. in that each response should be reproducibly seen a total of 3 times to reduce the impact of this phenomenon3.
In our experience, clinical electrodiagnostic systems are optimized for the studies described herein due to improved examiner-electrodiagnostic system interface ergonomics allowing ease of control. The two-channel system utilized in our lab is equipped with two non-switched amplifier channels using an amplifier with 24 bit Analog to Digital Converter and a sampling rate of 48 kHz per channel. Hardware gain can be adjusted from 10nV to 100 mV/division. The low frequency filter has a range from 0.2 Hz-5 kHz, and the high frequency filter settings range from 30 Hz-10 kHz. A constant-current stimulator is used (intensity: 0-100 mA; duration: 0.02-1 ms). Most clinical systems have similar appropriate features and can be adjusted to adequately record CMAP and MUNE responses. Additionally, standard electrophysiological rigs can be assembled to adequately record CMAP and MUNE, but the interface may need to be adjusted for ease of stimulation adjustment and rapid identification of CMAP and SMUP responses.
We have previously utilized the techniques of CMAP and MUNE described here to allow rapid and reproducible assessment of the sciatic innervated muscle of the hind limb in mice during the early postnatal period to adulthood5. These techniques allow assessment in mouse models when behavioral testing for motor function is not feasible or is less reliable. Application of this technique to neonatal mice facilitates the study of motor unit development and has the potential to expand our understanding of motor neuron innervation and pruning. For instance, we have shown that the number of functional motor units recorded with MUNE will increase during pruning from polyneuronal to mononeuronal innervation during the first two weeks of life in neonatal mice5. The ability to test mice over long periods of time with this technique lends itself to the study of motor unit response to peripheral nerve injury, hereditary neuromuscular disorders and aging.
The authors have nothing to disclose.
WDA is supported by grant funding from NIH-NICHD (5K12HD001097-17) and Cure SMA. SJK is supported by grant funding from NINDS (K08NS067282 and U01NS079163).
Pro trimmer Pet Grooming Kit | Oster | 078577-010-003 | clippers for hair removal |
Synergy T2 EMG system | Natus Neurology | Model no longer available | portable electrodiagnostic system |
monopolar needles 28 gauge | Teca | 017K121 | cathode and anode stimulating electrodes |
Alpine Biomed Digital Ring Electrode with twisted wires and 1.5 mm TP connectors. | Alpine Biomed | 9013S0312 | recording electrodes |
Helping Hands alligator clip with iron base | Radio Shack | 64-079 | Maintaining recording electrode placement |
Spectra 360 Electrode Gel | Parker Laboratories | 9013G5012 | applied to reduce skin impedance |
monoject curved tip irrigating syringe | Covidien | 81412012 | utilized for application of electrode gel |
EMG needle cable | Teca | 902-RLC-TP | to connect monopolar electrodes to electrodiagnostic stimulator |
Disposable 2" x 2" Electrode or similar trimmed as needed | Carefusion | 019-415000 | ground electrode |
Small Heating Plate with built-in RTD sensor, 15x10cm | World Precision Instruments | 61830 | warming plate used with animal temperature controller to transmit heat to animal |
Silicone pad for use with ATC2000 | World Precision Instruments | 503573 | conductive removable pad to cover warming plate for easy cleaning |
Animal temperature controller | World Precision Instruments | ATC2000 | low noise animal heating system for maintaining animal temperature |
Veterinarian petroleum-based ophthalmic ointment | Puralube | 26870 | applied during anesthesia to avoid corneal injury |