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)&#160;&#160;</td>
			<td>QtracW</td>
			<td></td>
		</tr>
		<tr>
			<td>MScanFit</td>
			<td>Digitimer Ltd (copyright Institute of Neurology, University College, London)&#160;&#160;</td>
			<td>QtracW</td>
			<td></td>
		</tr>
		<tr>
			<td>DS5 bipolar stimulator</td>
			<td>Digitimer Ltd&#160;</td>
			<td>DS5</td>
			<td></td>
		</tr>
		<tr>
			<td>D440 amplifier&#160;</td>
			<td>Digitimer Ltd&#160;</td>
			<td>D440-2 (2 channel) or D440-4 (4 channel)</td>
			<td></td>
		</tr>
		<tr>
			<td>HumBug Noise Eliminator</td>
			<td>Digitimer Ltd&#160;</td>
			<td>Humbug</td>
			<td></td>
		</tr>
		<tr>
			<td>Analogue-to-digital (A/D) board&#160;&#160;</td>
			<td>National Instruments</td>
			<td>NI-6221&#160;</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

Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

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 ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed. 
This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.

A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic Brain Injury (mTBI). The mechanisms responsible for its complex pathological changes remain largely elusive, despite a recent consensus to define the neuropathological criteria. Here, we describe a novel method to develop a model of CTE in Drosophila melanogaster&#160;(Drosophila&#160;)&#160;in an attempt to identify the key genes and pathways that lead to the characteristic hyperphosphorylated tau accumulation and neuronal death in the brain. Adjustable-strength impacts to inflict mild closed injury are delivered directly to the fly head, subjecting the head to rapid acceleration and deceleration. Our method eliminates the potential problems inherent with other Drosophila mTBI models (e.g.,animal death might be induced by damage to other parts of the body or to internal organs). The less labor- and cost-intensive animal care, short life span, and extensive genetic tools make the fruit fly ideal to study CTE pathogenesis and make it possible to perform large-scale, genome-wide forward genetic and pharmacological screens. We anticipate that the ongoing characterization of the model will generate important mechanistic insights on disease prevention and therapeutic approaches.

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Here, we describe a new approach to inflict closed-head traumatic brain injury in Drosophila melanogaster. Our method has the advantage of directly delivering repetitive impacts with adjustable strength to the head alone. Further exploration of the invertebrate system will help to illuminate the pathogenesis of chronic traumatic encephalopathy.

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the substantia nigra, is essential. To estimate the total dopaminergic neuron number, unbiased stereology using the optical fractionator method is currently considered the gold standard. Because the theory behind the optical fractionator method is complex and because stereology is difficult to achieve without specialized equipment, several commercially available complete stereology systems that include the necessary software do exist, purely for cell counting reasons. Since purchasing a specialized stereology setup is not always feasible, for many reasons, this report describes a method for the stereological estimation of dopaminergic neuronal cell counts using standard microscopy equipment, including a light microscope, a motorized object table (x, y, z plane) with imaging software, and a computer for analysis. A step-by-step explanation is given on how to perform stereological quantification using the optical fractionator method, and pre-programmed files for the calculation of estimated cell counts are provided. To assess the accuracy of this method, a comparison to data obtained from a commercially available stereology apparatus was performed. Comparable cell numbers were found using this protocol and the stereology device, thus demonstrating the precision of this protocol for unbiased stereology.

This work presents a step-by-step protocol for the unbiased stereological estimation of dopaminergic neuronal cell numbers in the mouse substantia nigra using standard microscopy equipment (i.e., a light microscope, a motorized object table (x, y, z plane), and public domain software for digital image analysis.

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...