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).

This protocol was approved by and adheres to the animal care and ethics guidelines of the Ohio State University Wexner Medical Center.
1. Animal Preparation and Anesthesia&#160;
<ol>
	<li>Wear gloves while handling mice.</li>
	<li>Anesthetize mice with inhaled isoflurane and place in the prone position. Induce anesthesia using 3-5% isoflurane and 1 L&#160;per minute O2 flow rate. Following induction of anesthesia, maintain anesthesia at 2-3% and 1 L per minute O2 flow rate.
	<ol>
		<li>Adjust O2 flow and isoflurane percentage for adequate anesthesia according to animal&#8217;s disease state, age, and respiration rate. Smaller or weaker animals may require less isoflurane for adequate anesthesia (i.e.1.5- 2.5% isoflurane).</li>
		<li>Confirm adequate anesthesia by applying light hindlimb footpad pressure with an object such as forceps to demonstrate a lack of withdrawal response.&#160;</li>
	</ol>
	</li>
	<li>Maintain temperature at 37 &#176;C surface temperature with a thermostatic warming plate as variation in temperature can affect CMAP size and duration.</li>
	<li>Apply veterinarian petroleum-based ointment to eyes to prevent dryness. Monitor level of anesthesia observing respiration rate and assessing for withdrawal responses following pressure applied to the foot pad via forceps.</li>
	<li>Remove hair from the hindlimb to be studied using clippers. After removal of hair from the hindlimb(s) to be studied, lightly extend the hindlimbs at the knee, abduct at the hips and affix to the working surface using adhesive tape (as shown in Figure 1).</li>
	<li>Following the CMAP and MUNE recordings and discontinuation of anesthesia, do not leave animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return animal to the company of other animals until fully recovered.</li>
</ol>
2. Recording Setup and Equipment
<ol>
	<li>Place the electrodes for the CMAP and MUNE recordings as pictured in Figure 1.</li>
	<li>Use two fine ring electrodes for the recording electrodes.
	<ol>
		<li>Place the active (E1) ring electrode on the skin overlying the proximal portion of the gastrocnemius muscle of the hind limb, at the knee joint, and the reference (E2) ring electrode on the skin over mid-metatarsal portion of the foot.</li>
		<li>In order to reduce impedance, coat the skin underlying the ring electrodes with gel to sufficiently saturate residual hair and maximize electrode-skin contact. Avoid excessive application of electrode gel as this may cause an electrical bridge between electrodes and could prevent accurate recording.</li>
	</ol>
	</li>
	<li>For stimulation of the sciatic nerve at the proximal hind limb, use two insulated 28 G monopolar needles as the cathode and anode. Insert the cathode at the region of the proximal hind limb and insert the anode more proximally in the subcutaneous tissue overlying the sacrum.
	<ol>
		<li>Avoid inserting the stimulating electrodes overly close to the sciatic nerve or too deep that it would directly injury the sciatic nerve or other structure. Figure 1 illustrates electrode placement.</li>
	</ol>
	</li>
	<li>For the ground electrode, place a disposable surface electrode on the contralateral hind limb or tail.</li>
</ol>
3. Data Acquisition
<ol>
	<li>Sciatic CMAP
	<ol>
		<li>Obtain sciatic CMAP responses by stimulating the sciatic nerve with square-wave pulses of 0.1 ms duration and intensity ranging from 1-10 mA.</li>
		<li>Acquire CMAP responses with increasing stimulus intensity until the amplitude of the response no longer increases. Then, in order to ensure supramaximal stimulation, increase the stimulation to ~120% of the stimulus intensity utilized to obtain a maximal response and obtain an additional response. If there is no further increase in the CMAP size, record this response as the maximal CMAP.</li>
		<li>Record baseline-to-peak and peak-to-peak CMAP amplitudes in mV (Figure 2).</li>
	</ol>
	</li>
	<li>Average Single Motor Unit Potential (SMUP) Size and MUNE Calculation
	<ol>
		<li>Determine the average single motor unit potential (SMUP) size with an incremental stimulation technique1. To obtain incremental responses, deliver submaximal stimulation of 0.1 ms duration at a frequency of 1 Hz while increasing the intensity in 0.03 mA steps to obtain the minimal all-or-none responses. Obtain the initial response with stimulus intensity between 0.21 mA and 0.70 mA.
		<ol>
			<li>If the initial response does not occur with stimulus intensity between 0.21 mA and 0.70 mA, adjust the stimulating cathode position either closer or farther away from the position of the sciatic nerve in the proximal thigh to decrease or increase the required stimulus intensity, respectively.</li>
			<li>If the initial incremental response is obtained with a stimulus intensity between 0.21 mA and 0.70 mA and fulfills the criteria noted below (3.2.2), store and record additional increments with increasing stimulus intensities adjusting by steps of 0.03 mA to obtain a total of 9 additional increments that meet the established criteria.</li>
		</ol>
		</li>
		<li>During measurements of the incremental responses, ensure that each increment meets the following criteria.
		<ol>
			<li>Ensure that the initial negative peak of the incremental responses is aligned temporally within the negative peak of the maximal CMAP response shown as the shaded portion of the CMAP illustration in Figure 2.</li>
			<li>Ensure that each incremental response is stable and without fractionation, established by observing three duplicate responses. Distinguish visually incremental responses in real-time (superimposed on the previously recorded increments). 
			Note: Each increment should be visually distinct and larger compared with the preceding response (Figure 3). Analysis in real-time allows recognition of larger amplitude (incremental) responses compared to prior responses, and small changes attributable to background noise can be disregarded. A superimposed view of 10 increments is shown in Figure 4 (B and D) to further illustrate this point.</li>
			<li>After visually confirming each increment, ensure that the measured amplitude difference (confirmed response-amplitude of the prior response=amplitude difference) is at least 25 &#181;V.</li>
			<li>If the increment is less than 25 &#181;V, discard and re-measure the response. After recording 10 incremental responses, assess the increments to ensure that the amplitude of each individual incremental response is not greater than 1/3 of the sum of all ten increments (i.e. the total amplitude of the final response). If this condition is not met, re-measure the incremental responses.&#160;</li>
		</ol>
		</li>
		<li>Average the 10 incremental values to give an estimation of the average single motor unit potential (SMUP) amplitude (Figure 3). Note: Figure 3 details the basis of the average SMUP calculation, but the average SMUP amplitude can be simply calculated by dividing the entire amplitude of the final incremental response by the total number of increments (i.e., 10). 
		Example individual SMUP calculations (illustrated in Figure 3): 
		SMUP 1=peak-to-peak amplitude of increment 1 
		SMUP 1=0.050 mV 
		SMUP 2= (peak-to-peak amplitude of increment 2) &#8211; (peak-to-peak amplitude of increment 1) 
		SMUP 2=0.150 mV-0.050 mV=0.100 mV
		<ol>
			<li>Calculate each subsequent increment (up to a total of 10), and make an average of the ten increments.</li>
		</ol>
		</li>
		<li>Calculate MUNE by dividing the maximum CMAP amplitude (peak-to-peak) by the average SMUP amplitude (peak-to-peak). (MUNE=CMAP/average SMUP). In some electrophysiological systems, the SMUP increments are measured in &#956;V whereas CMAP is typically provided in mV. When necessary, convert CMAP and SMUP results to similar units prior to MUNE calculation.</li>
	</ol>
	</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+incremental+with+multipoint+MUNE+methods+in+transgenic+ALS+mice.">Comparison of incremental with multipoint MUNE methods in transgenic ALS mice.</a> Muscle &amp; Nerve. 25 (1), 39-42 (2002).</li><li>Shefner, J. M., Cudkowicz, M., Brown, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+unit+number+estimation+predicts+disease+onset+and+survival+in+a+transgenic+mouse+model+of+amyotrophic+lateral+sclerosis.">Motor unit number estimation predicts disease onset and survival in a transgenic mouse model of amyotrophic lateral sclerosis.</a> Muscle Nerve. 34 (5), 603-607 (2006).</li><li>Arnold, W. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+muscular+atrophy:+The+development+and+implementation+of+potential+treatments.">Spinal muscular atrophy: The development and implementation of potential treatments.</a> Annals of Neurology. 74 (3), 348-362 (2013).</li><li>Li, J., Sung, M., Rutkove, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiologic+biomarkers+for+assessing+disease+progression+and+the+effect+of+riluzole+in+SOD1+G93A+ALS+mice.">Electrophysiologic biomarkers for assessing disease progression and the effect of riluzole in SOD1 G93A ALS mice.</a> PloS one. 8 (6), e65976-65 (2013).</li><li>Ngo, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+Bayesian+motor+unit+number+estimation+and+histological+measurements+of+motor+neurons+in+wild-type+and+SOD1+(G93A)+mice.">The relationship between Bayesian motor unit number estimation and histological measurements of motor neurons in wild-type and SOD1 (G93A) mice.</a> Clin Neurophysiol. 123 (10), 2080-2091 (2012).</li><li>Shefner, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+MUNE+studies+in+animal+models+of+motor+neuron+disease.">Recent MUNE studies in animal models of motor neuron disease.</a> Supplements to Clinical neurophysiology. 60, 203-208 (2009).</li><li>Souayah, N., Potian, J. G., Garcia, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+unit+number+estimate+as+a+predictor+of+motor+dysfunction+in+an+animal+model+of+type+1+diabetes.">Motor unit number estimate as a predictor of motor dysfunction in an animal model of type 1 diabetes.</a> American journal of physiology Endocrinology and metabolism. 297 (3), E602-E608 (2009).</li><li>Zhou, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+comparison+in+monitoring+disease+progression+of+G93A+mouse+model+of+ALS.">A method comparison in monitoring disease progression of G93A mouse model of ALS.</a> Amyotrophic lateral sclerosis: official publication of the World Federation of Neurology Research Group on Motor Neuron Diseases. 8 (6), 366-3672 (2007).</li><li>Feng, X. H., Yuan, W., Peng, Y., Ss Liu, M., Cui, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+effects+of+dl-3-n-butylphthalide+in+a+transgenic+mouse+model+of+amyotrophic+lateral+sclerosis.">Therapeutic effects of dl-3-n-butylphthalide in a transgenic mouse model of amyotrophic lateral sclerosis.</a> Chinese medical journal. 125 (10), 1760-1766 (2012).</li><li>Mancuso, R., Santos-Nogueira, E., Osta, R., Navarro, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+analysis+of+a+murine+model+of+motoneuron+disease.">Electrophysiological analysis of a murine model of motoneuron disease.</a> Clinical neurophysiology : official journal of the International Federation of Clinical Neurophysiology. 122 (8), 1660-1670 (2011).</li><li>Lee, Y. i., Mikesh, M., Smith, I., Rimer, M., Thompson, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscles+in+a+mouse+model+of+spinal+muscular+atrophy+show+profound+defects+in+neuromuscular+development+even+in+the+absence+of+failure+in+neuromuscular+transmission+or+loss+of+motor+neurons.">Muscles in a mouse model of spinal muscular atrophy show profound defects in neuromuscular development even in the absence of failure in neuromuscular transmission or loss of motor neurons.</a> Developmental biology. 356 (2), 432-444 (2011).</li></ol>

The authors have nothing to disclose.

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 &#956;V 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.

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&#160;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.

<table><tbody><tr><td>Pro trimmer Pet Grooming Kit</td><td>Oster</td><td>078577-010-003</td><td>clippers for hair removal</td></tr><tr><td>Synergy T2 EMG system</td><td>Natus Neurology</td><td>Model no longer available</td><td>portable electrodiagnostic system</td></tr><tr><td>monopolar needles 28 G</td><td>Teca</td><td>017K121</td><td>cathode and anode stimulating electrodes</td></tr><tr><td>Alpine Biomed Digital Ring Electrode with twisted wires and 1.5 mm TP connectors.</td><td>Alpine Biomed</td><td>9013S0312</td><td>recording electrodes</td></tr><tr><td>Helping Hands alligator clip with iron base</td><td>Radio Shack</td><td>64-079</td><td>Maintaining recording electrode placement&amp;nbsp;</td></tr><tr><td>Spectra 360 Electrode Gel&amp;nbsp;</td><td>Parker Laboratories</td><td>9013G5012</td><td>applied to reduce skin impedance</td></tr><tr><td>monoject curved tip irrigating syringe</td><td>Covidien</td><td>81412012</td><td>utilized for application of electrode gel</td></tr><tr><td>EMG needle cable</td><td>Teca</td><td>902-RLC-TP&amp;nbsp;</td><td>to connect monopolar electrodes to electrodiagnostic stimulator</td></tr><tr><td>Disposable 2&quot; x 2&quot; Electrode or similar trimmed as needed</td><td>Carefusion</td><td>019-415000&amp;nbsp;</td><td>ground electrode</td></tr><tr><td>Small Heating Plate with built-in RTD sensor, 15 x10 cm</td><td>World Precision Instruments</td><td>61830</td><td>warming plate used with animal temperature controller to transmit heat to animal</td></tr><tr><td>Silicone pad for use with ATC2000</td><td>World Precision Instruments</td><td>503573</td><td>conductive removable pad to cover warming plate for easy cleaning</td></tr><tr><td>Animal temperature controller</td><td>World Precision Instruments</td><td>ATC2000</td><td>low noise animal heating system for maintaining animal temperature</td></tr><tr><td>Veterinarian petroleum-based ophthalmic ointment&amp;nbsp;</td><td>Puralube</td><td>26870</td><td>applied during anesthesia to avoid corneal injury</td></tr></tbody></table>

electrophysiological motor unit number estimation (mune) measuring compound muscle action potential (cmap) in mouse hindlimb muscles

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.

The techniques of CMAP and MUNE described in this report allow recording of neuromuscular function of the sciatic innervated hind limb muscles utilizing minimally invasive electrode placement (Figure 1). Supramaximal CMAP size, which represents the total output from a muscle group, can be described using the parameters of amplitude and area (Figure 2), however, in the current methods, we use amplitude to quantify the CMAP and the SMUP sizes. Since the CMAP response measures summated depolarization of muscle fibers within a muscle, pathology anywhere from the motor neuron to the muscle fiber can result in reduction in CMAP size. Therefore, CMAP gives an excellent measure of the total functional status. As expected, CMAP size will increase during development5. Due to compensatory changes that can occur following denervation (i.e. collateral sprouting), CMAP size may be maintained despite processes of motor neuron or motor axonal loss. Therefore, the technique of MUNE is required to determine the motor neuron or axon input to the muscle or group of muscles being tested. Recording of individual increments (Figure 3) allows estimation of the average output of single motor units (SMUP size) to give more detailed information about functional status of motor units.
CMAP and MUNE can be utilized to measure neuromuscular function in various mouse models of neuromuscular disease. In Figure 4, findings in an adult control mouse and an adult mouse 11 weeks following sciatic nerve crush are contrasted. Following sciatic nerve crush, MUNE is severely reduced at 50 estimated functional motor units compared with normal findings of 278 functional motor units in the control mouse. In contrast, the CMAP amplitude in the crushed animal (39.6 mV baseline-to-peak, 74.9 mV peak-to-peak) shows only mild reduction compared to control (49.0 mV baseline-to-peak, 84.2 mV peak-to-peak) due to collateral sprouting.
<img alt="Animal physiology study with electrodes connected to rat legs for neuromuscular research experiment." src="/files/ftp_upload/52899/52899fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Electrode Placement. The black (E1) “active” electrode (A) and red (E2) “reference” recording electrode (B) are placed over the gastrocnemius at the proximal portion of the gastrocnemius at the knee. The stimulating cathode (black) (C) and anode (red) (D) are inserted subcutaneously proximal to the recording electrodes to generate distal responses. A disposable disk electrode (D) is placed on the hind limb, tail or sacrum as a ground to minimize artifact. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52899/52899fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Transient absorption spectroscopy; graph of spectral data with analyzed absorption peak and baseline." src="/files/ftp_upload/52899/52899fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Compound Muscle Action Potential. Pictured is an illustration of a representative CMAP response. (A) The baseline-to-peak amplitude is measured from the isoelectric baseline to the initial negative peak (negative voltage is depicted above the baseline). (B) The peak-to-peak amplitude is measured from negative peak voltage to positive peak voltage. The grey-shaded area denotes the negative peak area. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52899/52899fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Voltage increment graph, superimposed waveforms, showing potential differences, diagram." src="/files/ftp_upload/52899/52899fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Incremental responses. Two representative incremental responses are shown superimposed and in isolation. For MUNE calculation amplitudes of each increment are measured peak-to-peak. Increment #1 is the initial all-or-none response recorded and represents a single motor unit potential (SMUP). Each subsequent increment (#2-10) represents a quantal increase superimposed on the prior response. Therefore to obtain the SMUP amplitudes for increments 2-10, the amplitude of the prior response is subtracted from the amplitude of the increment obtained. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52899/52899fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Electrophysiology graph: sciatic nerve signals; comparison of normal and reinnervated conditions." src="/files/ftp_upload/52899/52899fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Example Sciatic CMAP and MUNE. (A) Sciatic compound muscle action potential (CMAP) in an adult (6 months of age) control mouse with baseline-to-peak amplitude of 49.0 mV and peak-to-peak amplitude of 84.2 mV. Screen sensitivity=10 mV per division and screen duration 10 ms. (B) Ten corresponding incremental responses (in the control mouse) with total amplitude of 3.028 mV are divided by 10 to determine average SMUP size (0.3028 mV). Screen sensitivity=0.5 mV and sweep speed of 1 ms per division. Calculated MUNE=278 (MUNE=CMAP/average SMUP(84.2 mV/ 0.3028 mV)) (C) Sciatic CMAP 11 weeks following sciatic nerve crush in an adult mouse (6 months of age) showing mildly reduced baseline-to-peak amplitude (39.6 mV) and peak-to-peak amplitude (74.9 mV). Screen sensitivity=10 mV per division and sweep speed of 1 ms per division. (D) Ten corresponding incremental responses (in the mouse with nerve crush) with total peak–to–peak amplitude of 14.923 mV divided by 10 to obtain an average SMUP size of 1.4923 mV. Screen sensitivity=2 mV per division and a sweep speed of 1 ms per division. Calculated MUNE=50 (MUNE=CMAP/average SMUP (74.9 mV/1.4923 mV)). (**Note the different sensitivity for the incremental responses of the sciatic crush mouse). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52899/52899fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Electrophysiological Motor Unit Number Estimation MUNE Measuring Compound Muscle Action Potential CMAP in Mouse Hindlimb Muscles at JoVE.com

Electrophysiological Motor Unit Number Estimation MUNE Measuring Compound Muscle Action Potential CMAP in Mouse Hindlimb Muscles

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.

Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the ...

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21.1K Views.  The Ohio State University Wexner Medical Center.  We presenteren verfijnde protocollen waarmee in vivo opvolging van motoreenheid functie in de muis. Technieken om verbinding spier actiepotentiaal (CMAP) en de motor-eenheid nummer schatting (MUNE) in de muis achterste ledematen spieren geïnnerveerd door de nervus vagus te meten beschreven. 

Video: Electrophysiological Motor Unit Number Estimation MUNE Measuring Compound Muscle Action Potential CMAP in Mouse Hindlimb Muscles

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4 , including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice5,6. However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9, or reproducting human disease10,11, we expect this technique to be an essential tool for the study of spinal motor system.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

Neuroscience

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

CMAP Scan MUNE (MScan) - A Novel Motor Unit Number Estimation (MUNE) Method

Like other methods for motor unit number estimation (MUNE), compound muscle action potential (CMAP) scan MUNE (MScan) is a non-invasive electrophysiologic method to estimate the number of functioning motor units in a muscle. MUNE is an important tool for the assessment of neuropathies and neuronopathies. Unlike most MUNE methods in use, MScan assesses all the motor units in a muscle, by fitting a model to a detailed stimulus-response curve, or CMAP scan. It thereby avoids the bias inherent in all MUNE methods based on extrapolating from a small sample of units. Like &#39;Bayesian MUNE,&#39;&#160;MScan analysis works by fitting a model, made up of motor units with different amplitudes, thresholds, and threshold variabilities, but the fitting method is quite different, and completed within five minutes, rather than several hours. The MScan off-line analysis works in two stages: first, a preliminary model is generated based on the slope and variance of the points in the scan, and second, this model is then refined by adjusting all the parameters to improve the fit between the original scan and scans generated by the model.
This new method has been tested for reproducibility and recording time on 22 amyotrophic lateral sclerosis (ALS) patients and 20 healthy controls, with each test repeated twice by two blinded physicians. MScan showed excellent intra- and inter-rater reproducibility with ICC values of &#62;0.98 and a coefficient of variation averaging 12.3 &#177; 1.6%. There was no difference in the intra-rater reproducibility between the two observers. Average recording time was 6.27 &#177; 0.27 min.
This protocol describes how to record a CMAP scan and how to use the MScan software to derive an estimate of the number and sizes of the functioning motor units. MScan is a fast, convenient, and reproducible method, which may be helpful in diagnoses and monitoring disease progression in neuromuscular disorders.

CMAP Scan MUNE MScan - A Novel Motor Unit Number Estimation MUNE Method

This protocol describes a new method to estimate the number of functioning motor units in a muscle, by fitting a model to a detailed stimulus-response curve of the compound muscle action potential. It is quick and easy to perform and analyze and has excellent reproducibility.

Like other methods for motor unit number estimation (MUNE), compound muscle action potential (CMAP) scan MUNE (MScan) is a non-invasive electrophysiologic ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Medicine

Functional and Physiological Methods of Evaluating Median Nerve Regeneration in the Rat

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different methods of simulating postoperative physiotherapy are presented. Multiple standardized strategies are used to assess motor and sensory recovery using an MN model of peripheral nerve lesion and repair, thus permitting easy comparison of the results. Several options are included for providing a postoperative physiotherapy-like environment to rats that have undergone MN injuries. Finally, the paper provides a method to evaluate the recovery of the MN using several noninvasive tests (i.e., grasping test, pin prick test, ladder rung walking test, rope climbing test, and walking track analysis), and physiological measurements (infrared thermography, electroneuromyography, flexion strength evaluation, and flexor carpi radialis muscle weight determination). Hence, this model seems particularly appropriate to replicate a clinical scenario, facilitating extrapolation of results to the human species.
Although the sciatic nerve is the most studied nerve in peripheral nerve research, analysis of the rat MN presents various advantages. For example, there is a reduced incidence of joint contractures and automutilation of the affected limb in MN lesion studies. Furthermore, the MN is not covered by muscle masses, making its dissection easier than that of the sciatic nerve. In addition, MN recovery is observed sooner, because the MN is shorter than the sciatic nerve. Also, the MN has a parallel path to the ulnar nerve in the arm. Hence, the ulnar nerve can be easily used as the nerve graft for repairing MN injuries. Finally, the MN in rats is located in the forelimb, akin to the human upper limb; in humans, the upper limb is the site of most peripheral nerve lesions.

Presented is a protocol to produce different types of median nerve (MN) lesions and repair in the rat. Additionally, the protocol shows how to evaluate the functional recovery of the nerve using several noninvasive behavioral tests and physiological measurements.

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different ...

Functional Isolation of Single Motor Units of Rat Medial Gastrocnemius Muscle

This work outlines functional isolation of motor units (MUs), a standard electrophysiological method for determining characteristics of motor units in hindlimb muscles (such as the medial gastrocnemius, soleus, or plantaris muscle) in experimental rats. A crucial element of the method is the application of electrical stimuli delivered to a motor axon isolated from the ventral root. The stimuli may be delivered at constant or variable inter-pulse intervals. This method is suitable for experiments on animals at varying stages of maturity (young, adult or old). Moreover, this protocol can be used in experiments studying variability and plasticity of motor units evoked by a large spectrum of interventions. The results of these experiments may both augment basic knowledge in muscle physiology and be translated into practical applications. This procedure focuses on the surgical preparation for the recording and stimulation of MUs, with an emphasis on the necessary steps to achieve preparation stability and reproducibility of results.

This method allows the recording of the force of twitch and tetanic contractions and action potentials in three types of motor units in the rat medial gastrocnemius muscle. The functional isolation of a single motor unit is induced by electrical stimulation of the axon.

This work outlines functional isolation of motor units (MUs), a standard electrophysiological method for determining characteristics of motor units in ...

Quantifying Phrenic Motor Unit Function in Rodent Models of Neurodegeneration

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

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.

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

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 ...

Measuring Compound Muscle Action Potentials in Mouse Forelimb Muscles In Vivo

Source: Pollari, E. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57741">In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration.</a>&#160;J. Vis. Exp. (2018).
This video demonstrates an in vivo technique for measuring compound muscle action potential (CMAP) in mice. It outlines the steps for setting up electrodes, stimulating forelimb neurons that innervate muscle fibers, and recording muscle responses. The technique assesses neuronal demyelination and functional neuron loss.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

In Vivo Measurement of the Compound Muscle Action Potential in a Rat

Source: Wild, B. M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56102">In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing.</a> J. Vis. Exp. (2018)
This video demonstrates the procedure for recording compound muscle action potential (CMAP) in rats by applying electrical stimuli to the ulnar nerve, adjusting electrodes for optimal signal, and increasing stimulation to assess nerve functionality. This method enables the in vivo investigation of the peripheral nerve functions in neurological disorders.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

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