This study was financially supported mainly by the Lundbeck Foundation.
Additionally, Knud og Edith Eriksens Mindefond, S&#248;ster og Verner Lipperts Fond, Fonden til L&#230;gevidenskabens Fremme, and Aage og Johanne Louis Hansens Fond supported this study.

All subjects must give their written consent prior to examination, and the recording protocol must be approved by the appropriate local ethical review board(s). All methods described here were approved by the Regional Scientific Ethical Committee and the Danish Data Protection Agency.
Note: The recordings are made with the &#8220;TRONDNF&#8221; recording protocol, which is a part of the software (see the Table of Materials). Other equipment used is a bipolar stimulator, a 50 Hz noise eliminator, an amplifier, and an analogue-to-digital (A/D) board (also recommended is an audio amplifier for feedback of electromyogram (EMG) activity, and a muting box to cut off the sound during electrical stimulation). Motor unit number estimation by the MScan method involves three stages: 1) preparation of the subject (as for nerve excitability studies), 2) recording the CMAP scan, and 3) analyzing the results with MScan software. The recording procedure described below is specific to the software and instruments we use (see the Table of Materials); these will need to be adapted for other software and hardware.
1. Preparation of the Subject
<ol>
	<li>Screen the subjects to ensure that they do not have any history of nervous system disorders (particularly neuropathy and carpal tunnel syndrome), other than the disease group that will be investigated.</li>
	<li>Instruct the subject in detail about the examinations and request written consent.
	<ol>
		<li>Inform the subject that when the recording starts, the power will gradually be increased to a maximum followed by a stepwise decrease, and the examinations will take about 5 - 6 min.</li>
		<li>Explain that the subject will experience a tickling feeling in the hand and fingers.</li>
		<li>Inform the subject that the power can be switched off immediately at any moment during the recording, if the subject feels too much discomfort.</li>
	</ol>
	</li>
	<li>Clean the subject&#8217;s hand and forearm with skin prep gel and alcohol.</li>
	<li>Place the active recording electrode over the abductor pollicis brevis muscle and the reference electrode on the metacarpo-phalangeal joint of the thumb (Figure 1).</li>
	<li>Place a ground electrode on the dorsum of the hand.</li>
	<li>Connect these electrodes to the pre-amplifier (Figure 1).</li>
	<li>Tape the fingers together to eliminate noise and artefacts due to voluntary movement (Figure 1).</li>
	<li>Maintain the skin temperature between 32&#160;&#176;C and 36 &#176;C with a warming lamp.</li>
</ol>
2. Recording the CMAP Scan
Note: All software actions described below are specific to the software and instruments we use (see the Table of Materials); these will need to be adapted for other software and hardware.
<ol>
	<li>Start the semi-automated computerized system.</li>
	<li>Select &#8216;MScan-R&#8217; recording protocol from the &#39;Select recording protocol&#39; form.</li>
	<li>Accept default settings for the pre-amplifier and the stimulator.</li>
	<li>On the &#39;Select recording parameters&#39; form, enter in the &#39;Output file&#39; box a 2- or 3-letter operator prefix, then click on the &#34;O.K.&#34; button.</li>
	<li>When the program displays the Raw EMG input, select either &#8216;MScan parameters&#8217; (scan step, interstimulus interval, and stimulus width) manually, or accept the default parameters: stimulus width of 0.2ms, scan step of 0.2%, and interstimulus interval of 0.5 s. Click on &#34;OK&#34; or press the &#8220;Escape&#8221; key to continue. 
	Note: The default parameters were accepted in this study. Filter settings were 3 Hz -3 kHz.</li>
	<li>Place a repositionable bipolar stimulating electrode on the median nerve at the wrist to find the site of lowest threshold. Click &#8220;OK&#8221; to start stimulation.</li>
	<li>The next display shows the stimulus (initially 15% max output) and the EMG response; adjust the electrode position to find site of lowest threshold (i.e. largest response). Adjust the position of the recording electrodes if necessary to ensure that the shape of the CMAP is diphasic with a single peak, if possible. If needed, adjust stimulus strength with the &#8216;Insert&#8217; and &#8216;Delete&#8217; keys. Then, click &#8220;OK&#8221; to continue.</li>
	<li>&#39;Modified&#39; response appears, with &#39;window&#39;, in which response is measured, indicated by horizontal magenta line. Ensure that the short green line before the window (indicates the baseline) is a flat line between stimulus artifact and response. If necessary, set the window start by depressing right mouse button, drag the cursor to the right, and then release the button for window end. 
	Note: CMAP peak height is measured from baseline to the upward peak within window, and is indicated by vertical blue line. 
	Click &#34;OK&#34; to continue.</li>
	<li>Replace the repositionable electrode with a non-polarizable adhesive stimulating cathode electrode and place an anode 2 cm proximally along the median nerve; bottom trace now shows high gain EMG. Encourage the subject to find the most relaxed position for their hand to minimize spontaneous activity. Click &#34;OK&#34; to continue.</li>
	<li>Increase the stimulus intensity manually by pressing the &#8220;Insert&#8221; key until the stimulus current is above the level for the maximal amplitude of the CMAP; first, increase by steps of 3% in stimulus intensity, and then for fine adjustments use steps of 1%.</li>
	<li>Check the window and peak measurement before clicking on the &#8220;OK&#8221; button to start CMAP scan.</li>
	<li>Note that after the responses to 20 supramaximal stimuli (pre-scans) are recorded, the stimulus intensity is automatically decreased in small steps, from supramaximal stimulation until there is no longer a discernible motor response, then a further set of 20 CMAPs (post-scans) is recorded. 
	Note: The data are plotted as a &#8216;CMAP scan&#8217;, or a detailed stimulus response curve with the amplitude of the motor response on the y-axis and stimulus intensity on the x-axis. In healthy subjects, this creates an S-shaped curve, whereas in patients with reduced numbers of motor units, as in ALS, the curve develops a stepped appearance (Figure 2).</li>
	<li>Notice that in addition to the feedback achieved from the muting-box, it is possible to check in the middle panel on the left-hand side if the subject is relaxed. Look at the CMAP (modified) on the left top panel, and on the left bottom panel, the decrease in stimulus intensity (threshold). Follow the CMAP Scan on the right-hand side.</li>
	<li>Finish the recording by clicking on the &#8220;OK&#8221; button in the lower right corner unless a repeat scan is required and save the data. 
	Note: When completing the &#39;Legends and scaling&#39; form it is necessary to replace all the question marks.</li>
</ol>
3. MScan Analyses
Note: All software actions described below are specific to the software and instruments we use (see the Table of Materials); these will need to be adapted for other software and hardware.
<ol>
	<li>Fitting a model to the CMAP scan
	<ol>
		<li>To analyse the recording offline, start the analysis program and click on &#34;O.K.&#34; to select the the last recording for analysis.</li>
		<li>Click on the &#8220;select &#8216;Fit MScan QZD file&#8221; from the &#8220;MScanFit&#8220; menu. 
		Note: One can instead select &#8220;Fit MScan on MEM file&#8221;. It is alo possible to select &#8220;Fit MScan on dat (mA mV) file&#8221; in case the CMAP Scan was recorded with equipment different from that used here. See below how to create a DAT file.</li>
		<li>Observe that the program first generates a preliminary model, and then goes on to optimize the fit. No user intervention is required until the multi-coloured progress bar in the &#39;Optimization&#39; box is complete and the &#34;Stop&#34; button is grayed out.</li>
		<li>The preliminary model provides a first guess of a model, derived from the slope and variance over successive parts of the scan. Note that the original scan (in black) and a scan generated from the model (in magenta) may be plotted side by side. Follow the model and improvements in the model on the text display and alternative displays. 
		Note: Alternative displays may be selected at any time according to the options in the &#39;Data to plot&#39;, &#39;Plot amplitudes as&#39;, and &#39;Plot type&#39; boxes.</li>
		<li>Perform optimization to improve the fit to the recorded CMAP by making serial adjustments to minimize the difference between the simulated and recorded CMAP scans16. 
		Note: This runs several optimizations in succession, starting with the preliminary model, as previously described in detail15. If the optimization increases the number of units, then the next attempt at fitting the scan starts by generating a model with more units, whereas if the first optimization reduced the number of units, the second attempt starts with a model with fewer units. If all works well, the optimization procedure zeroes in on the best number of units to fit the scan.</li>
		<li>Notice that when running the optimization procedure, the optimization panel also shows a multi-colored progress bar.</li>
		<li>Use different plotting options at the &#8220;plot type&#8221; box, i.e., contour plots, error v N units, model units, cumulative amplitude to follow the optimization process. 
		Note: The changes in the model during the optimization procedure are continually updated according to the latest model and the improvements in the model may be followed by different plotting options and on the text display.
		<ol>
			<li>Contour maps
			<ol>
				<li>Utilize contour maps to assess the accuracy of the model by blurring the points and generating an error score based on the difference in the x-y distributions15. Select &#8220;Diff&#8221; and &#8220;Contour map&#8221; displays to see the differences between the recorded and model CMAP scans as a contour map. It is these differences that the optimization process tries to minimize. 
				Note: &#39;Contour maps&#39; enables the probability density of obtaining a given response with a given stimulus. The error score depends on the difference between the original and modeled contour map, and this can be visualized by selecting the &#8216;Diff&#8217; option in the top panel and the &#8216;Contour plot&#8217; in the Plot type panel. The red lines indicate that there was a greater density of points in the original scan, and the green lines indicate more points in the modeled scan. The difference plot at the top indicates the stimulus intensities at which the greatest errors are found.</li>
			</ol>
			</li>
			<li>Error v N units
			<ol>
				<li>Follow the optimization procedure by selecting the log-log &#8216;Error v N units&#8217; plot. Each stage of the optimization procedure is plotted in a different color, corresponding to the bars in the &#8216;Optimization&#8217; panel.</li>
			</ol>
			</li>
			<li>Model units
			<ol>
				<li>Use this to see how the mean CMAP amplitude is made up by the recruitment of the different axons in the model; the top part shows the peak amplitudes and threshold distributions of the individual motor units.</li>
			</ol>
			</li>
			<li>Cumulative amplitude
			<ol>
				<li>Note that the units are plotted in order of increasing size, rather than threshold. The black curve plots the cumulative unit number, while the red curve shows cumulative amplitude.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>When the optimization process is complete, view the results of the analysis on the &#39;MScanFit text display&#39;. The available MScan parameters, i.e., number of units, median amplitude, largest unit size are shown on the text display.</li>
	<li>Click on the &#8220;OK&#8221; button in the &#39;Save fit to MEM file&#39; box to save the model in the MEM file for further analyses.</li>
</ol>
4. CMAP Scan MUNE Using .DAT Files
Note: An alternative and free version of the software enables analysis of CMAP Scans recorded by other equipment.
<ol>
	<li>Generate a.DAT file containing the CMAP scan to be analyzed. 
	Note: This must be a text file in a standard 2-column format, with the stimulus intensity in mA in one column and CMAP amplitude in mV in another column.</li>
	<li>Generate a suitable.DAT file by copying two columns from a spreadsheet file into a text editing software, and then designating the extension as.DAT rather than.TXT.
	<ol>
		<li>Perform all the other steps of the MScan analysis as previously described, after starting the stand-alone program and selecting the.DAT file.</li>
	</ol>
	</li>
	<li>Find the freeware program, manual, and specimen data on the University College London FTP site (Host: 144.82.46.62, User name: QtracW, Password: Hg32wK5e).</li>
</ol>

<ol>
	<li>Sherrington, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REMARKS+ON+THE+FOREGOING+LETTER.">REMARKS ON THE FOREGOING LETTER.</a> Can. Med. Assoc. J. 20, 66-67 (1929).</li><li>Gooch, C. L., 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+estimation:+a+technology+and+literature+review.">Motor unit number estimation: a technology and literature review.</a> Muscle Nerve. 50, 884-893 (2014).</li><li>McComas, A. J., Fawcett, P. R., Campbell, M. J., Sica, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+estimation+of+the+number+of+motor+units+within+a+human+muscle.">Electrophysiological estimation of the number of motor units within a human muscle.</a> J Neurol Neurosur Ps. 34, 121-131 (1971).</li><li>Oge, A. E., 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+estimation+in+transected+peripheral+nerves.">Motor unit number estimation in transected peripheral nerves.</a> Neurol Res. 32, 1072-1076 (2010).</li><li>Doherty, T. J., Brown, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+estimated+numbers+and+relative+sizes+of+thenar+motor+units+as+selected+by+multiple+point+stimulation+in+young+and+older+adults.">The estimated numbers and relative sizes of thenar motor units as selected by multiple point stimulation in young and older adults.</a> Muscle Nerve. 16, 355-366 (1993).</li><li>Bromberg, M. B. <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+estimation:+reproducibility+of+the+spike-triggered+averaging+technique+in+normal+and+ALS+subjects.">Motor unit estimation: reproducibility of the spike-triggered averaging technique in normal and ALS subjects.</a> Muscle Nerve. 16, 466-471 (1993).</li><li>Lomen-Hoerth, C., Slawnych, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+motor+unit+number+estimation:+from+theory+to+practice.">Statistical motor unit number estimation: from theory to practice.</a> Muscle Nerve. 28, 263-272 (2003).</li><li>Unlusoy Acar, Z., 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=Decline+of+compound+muscle+action+potentials+and+statistical+MUNEs+during+Wallerian+degeneration.">Decline of compound muscle action potentials and statistical MUNEs during Wallerian degeneration.</a> Neurophysiol. Clin. 44, 257-265 (2014).</li><li>Henderson, R. D., Ridall, P. G., Hutchinson, N. M., Pettitt, A. N., McCombe, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bayesian+statistical+MUNE+method.">Bayesian statistical MUNE method.</a> Muscle Nerve. 36, 206-213 (2007).</li><li>Ridall, P. G., Pettitt, A. N., Henderson, R. D., McCombe, P. A. <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--a+Bayesian+approach.">Motor unit number estimation--a Bayesian approach.</a> Biometrics. 62, 1235-1250 (2006).</li><li>Nandedkar, S. D., Nandedkar, D. S., Barkhaus, P. E., Stalberg, E. V. <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+index+(MUNIX).">Motor unit number index (MUNIX).</a> IEEE Trans. Biomed. Eng. 51, 2209-2211 (2004).</li><li>Neuwirth, 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+Index+(MUNIX):+a+novel+neurophysiological+marker+for+neuromuscular+disorders;+test-retest+reliability+in+healthy+volunteers.">Motor Unit Number Index (MUNIX): a novel neurophysiological marker for neuromuscular disorders; test-retest reliability in healthy volunteers.</a> Clin. Neurophysiol. 122, 1867-1872 (2011).</li><li>Jacobsen, A. B., 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=Reproducibility,+and+sensitivity+to+motor+unit+loss+in+amyotrophic+lateral+sclerosis,+of+a+novel+MUNE+method:+MScanFit+MUNE.">Reproducibility, and sensitivity to motor unit loss in amyotrophic lateral sclerosis, of a novel MUNE method: MScanFit MUNE.</a> Clin neurophysiol. 128, 1380-1388 (2017).</li><li>Maathuis, E. M., Drenthen, J., Visser, G. H., Blok, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+of+the+CMAP+scan.">Reproducibility of the CMAP scan.</a> J. Electromyogr. Kinesiol. 21, 433-437 (2011).</li><li>Sleutjes, B. T. H. M., 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=CMAP+scan+discontinuities:+automated+detection+and+relation+to+motor+unit+loss.">CMAP scan discontinuities: automated detection and relation to motor unit loss.</a> Clin Neurophysiol. 125, 388-395 (2014).</li><li>Bostock, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+motor+unit+numbers+from+a+CMAP+scan.">Estimating motor unit numbers from a CMAP scan.</a> Muscle Nerve. 53, 889-896 (2016).</li><li>DeLong, E. R., DeLong, D. M., Clarke-Pearson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+the+areas+under+two+or+more+correlated+receiver+operating+characteristic+curves:+a+nonparametric+approach.">Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach.</a> Biometrics. 44, 837-845 (1988).</li><li>Hanley, J. A., McNeil, B. J. <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+of+comparing+the+areas+under+receiver+operating+characteristic+curves+derived+from+the+same+cases.">A method of comparing the areas under receiver operating characteristic curves derived from the same cases.</a> Radiology. 148, 839-843 (1983).</li><li>Farschtschi, S., 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=Muscle+action+potential+scans+and+ultrasound+imaging+in+neurofibromatosis+type+2+:+CMAP+Scans+and+Nerve+Imaging+in+NF2.">Muscle action potential scans and ultrasound imaging in neurofibromatosis type 2 : CMAP Scans and Nerve Imaging in NF2.</a> Muscle Nerve. 55, 350-358 (2017).</li></ol>

Conflict of interest: HB receives royalties from UCL for sales of his Qtrac software used in this study. The other authors have no potential conflicts of interest. All authors have approved the final article.

Critical steps within the protocol: MScan is a highly automated procedure, but as with all EMG methods, care should be taken to obtain consistent results. In the preparation stage, it is important to achieve relaxation, since spontaneous activity or movement artefacts during the CMAP scan introduce spurious variance in the CMAP and confound the generation of the preliminary model.
Modifications and troubleshooting: We found that taping of the fingers, and use ...

Motor system movement is dependent on the motor unit, which refers to an individual motor nerve fiber together with the muscle fibers it activates, and motor unit number is the number of anterior horn cells or axons innervating a single muscle1. During the denervation and reinnervation processes, healthy axons take over the role of axons that are lost by collateral sprouting. Therefore, compound muscle action potential (CMAP) amplitude does not give the necessary information about the degree of motor unit loss. CMAP amplitude may only start to fall when more than 50% of motor units are lost. Similarly, the magnitude of abnormal spontaneous activity or motor unit potential (MUP) changes does not correlate with the degree of denervation.
Overall, there is no electrophysiological technique that allows for simple, direct measurements of motor unit number. Instead, an estimate of motor unit number (MUNE) is used to assess lower motor neuron loss2. Several MUNE methods have been developed since the implementation of the first method, incremental stimulation MUNE, which was introduced in 1971 by McComas3. Most methods have been based on measuring several surface-recorded motor unit potentials (sMUP) and dividing the maximal CMAP by the average sMUP amplitude. Such methods include incremental stimulation4, multiple point stimulation (MPS)5, and spike-triggered averaging6. Other MUNE methods have used statistical techniques based on the probabilistic nature of the firing of a motor unit in response to a stimulus7,8,9,10. This variability means that firing different combinations of motor units leads to variability in the size of the CMAP responses. Motor unit number index (Munix) is a more recently introduced method, which uses the surface interference patterns recorded during voluntary contractions to estimate the average size of sMUP11,12.
These MUNE methods all suffer from one or more limitations, such as the presence of subjectivity, dependence on the absolute CMAP amplitude, bias in the selection of units, the long time needed to sample enough units, or the long time required to analyze the results. A new MUNE method has recently been developed, &#39;CMAP scan MUNE` (MScan), to overcome these limitations13. This method avoids the problems inherent in unit selection by taking account of the contribution of all units to the CMAP, as measured in a detailed stimulus-response curve, or CMAP scan14,15. It also avoids the extended analysis time of a similar, model-fitting method9,10, by using new algorithms16. In a recent study, the reproducibility of MScan in estimating the number of motor units was better than two more traditional methods, MPS MUNE and Munix13. Additionally, MScan could show motor unit loss in earlier stages of Amyotrophic Lateral Sclerosis (ALS) than MPS MUNE and Munix. MScan was faster than MPS MUNE and as fast as Munix13.
This paper describes the methodology of MScan in detail. It also summarizes the previously reported intra- and inter-rater reproducibility of MScan in patients with ALS and healthy control subjects13, which may enable the reader to judge whether the method would be appropriate for a planned study.

<table><tbody><tr><td>QtracW software</td><td>Digitimer Ltd (copyright Institute of Neurology, University College, London)</td><td>QtracW</td><td></td></tr><tr><td>MScanFit</td><td>Digitimer Ltd (copyright Institute of Neurology, University College, London)</td><td>QtracW</td><td></td></tr><tr><td>DS5 bipolar stimulator</td><td>Digitimer Ltd</td><td>DS5</td><td></td></tr><tr><td>D440 amplifier</td><td>Digitimer Ltd</td><td>D440-2 (2 channel) or D440-4 (4 channel)</td><td></td></tr><tr><td>HumBug Noise Eliminator</td><td>Digitimer Ltd</td><td>Humbug</td><td></td></tr><tr><td>Analogue-to-digital (A/D) board</td><td>National Instruments</td><td>NI-6221</td><td></td></tr></tbody></table>

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.

The following results were obtained in a recent study, in which the MScan method was compared with two established techniques: multiple point stimulation MUNE (MPS) and motor unit number index (Munix)13. The results show that with the technique described in this protocol, consistent results with excellent reproducibility can be achieved. The method can differentiate ALS patients from healthy controls in an earlier stage of the disease than CMAP and it is quick and ...

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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.

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 ...

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12.2K Views.  Aarhus University Hospital. 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.

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

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.

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.

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

Behavior

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 ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

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, ...

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 Modulation and Robotic Mapping of Motor Cortex in the Developing Brain

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries unique challenges in children. Similarly, transcranial direct current stimulation (tDCS) can improve motor learning in adults but has only recently been applied to children. The use of tDCS and emerging techniques like high&#8211;definition tDCS (HD-tDCS) require special methodological considerations in the developing brain. Robotic TMS motor mapping may confer unique advantages for mapping, particularly in the developing brain. Here, we aim to provide a practical, standardized approach for two integrated methods capable of simultaneously exploring motor cortex modulation and motor maps in children. First, we describe a protocol for robotic TMS motor mapping. Individualized, MRI-navigated 12x12 grids centered on the motor cortex guide a robot to administer single-pulse TMS. Mean motor evoked potential (MEP) amplitudes per grid point are used to generate 3D motor maps of individual hand muscles with outcomes including map area, volume, and center of gravity. Tools to measure safety and tolerability of both methods are also included. Second, we describe the application of both tDCS and HD-tDCS to modulate the motor cortex and motor learning. An experimental training paradigm and sample results are described. These methods will advance the application of non-invasive brain stimulation in children.

We demonstrate protocols for the modulation (tDCS, HD-tDCS) and mapping (robotic TMS) of the motor cortex in children.

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries ...

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 ...

Medicine

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 ...

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

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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

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

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

Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain

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

Functional Isolation of Single Motor Units of Rat Medial Gastrocnemius Muscle

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