WDA is supported by grant funding from NIH-NICHD (5K12HD001097-17) and Cure SMA. SJK is supported by grant funding from NINDS (K08NS067282 and U01NS079163).

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

<ol>
	<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> Journal of neurology, neurosurgery, and psychiatry. 34 (2), 121-123 (1971).</li><li>Shefner, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+unit+number+estimation+in+human+neurological+diseases+and+animal+models.">Motor unit number estimation in human neurological diseases and animal models.</a> Clinical Neurophysiology. 112 (6), 955-964 (2001).</li><li>Shefner, J. M., Cudkowicz, M. E., Brown, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+incremental+with+multipoint+MUNE+methods+in+transgenic+ALS+mice.">Comparison of incremental with multipoint MUNE methods in transgenic ALS mice.</a> Muscle &amp; Nerve. 25 (1), 39-42 (2002).</li><li>Shefner, J. M., Cudkowicz, M., Brown, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+unit+number+estimation+predicts+disease+onset+and+survival+in+a+transgenic+mouse+model+of+amyotrophic+lateral+sclerosis.">Motor unit number estimation predicts disease onset and survival in a transgenic mouse model of amyotrophic lateral sclerosis.</a> Muscle Nerve. 34 (5), 603-607 (2006).</li><li>Arnold, W. D., 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=Electrophysiological+Biomarkers+in+Spinal+Muscular+Atrophy:+Preclinical+Proof+of+Concept.">Electrophysiological Biomarkers in Spinal Muscular Atrophy: Preclinical Proof of Concept.</a> Annals of clinical and translational neurology. 1 (1), 34-44 (2014).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+three+electrophysiological+methods+for+the+assessment+of+disease+status+in+a+mild+spinal+muscular+atrophy+mouse+model.">A comparison of three electrophysiological methods for the assessment of disease status in a mild spinal muscular atrophy mouse model.</a> PloS one. 9 (10), e111428 (2014).</li><li>Srivastava, A. K., 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=Mutant+HSPB1+overexpression+in+neurons+is+sufficient+to+cause+age-related+motor+neuronopathy+in+mice.">Mutant HSPB1 overexpression in neurons is sufficient to cause age-related motor neuronopathy in mice.</a> Neurobiology of disease. 47 (2), 163-173 (2012).</li><li>Yalvac, M., Arnold, E., D, W., Hussain, S. R., 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=VIP-expressing+dendritic+cells+protect+against+spontaneous+autoimmune+peripheral+polyneuropathy.">VIP-expressing dendritic cells protect against spontaneous autoimmune peripheral polyneuropathy.</a> Molecular therapy: the journal of the American Society of Gene Therapy. 22 (7), 1353-1363 (2014).</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 (6), 884-893 (2014).</li><li>Arnold, W. D., Kassar, D., Kissel, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+muscular+atrophy:+diagnosis+and+management+in+a+new+therapeutic+era.">Spinal muscular atrophy: diagnosis and management in a new therapeutic era.</a> Muscle Nerve. , (2014).</li><li>Swoboda, K. J., 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=Natural+history+of+denervation+in+SMA:+Relation+to+age,+SMN2+copy+number,+and+function).">Natural history of denervation in SMA: Relation to age, SMN2 copy number, and function).</a> Annals of Neurology. 57 (5), 704-712 (2005).</li><li>Finkel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+and+motor+function+scale+association+in+a+pre-symptomatic+infant+with+spinal+muscular+atrophy+type+I.">Electrophysiological and motor function scale association in a pre-symptomatic infant with spinal muscular atrophy type I.</a> Neuromuscular Disorders. 23 (2), 112-115 (2013).</li><li>Kaufmann, P., 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=Prospective+cohort+study+of+spinal+muscular+atrophy+types+2+and+3.">Prospective cohort study of spinal muscular atrophy types 2 and 3.</a> Neurology. 79 (18), 1889-1897 (2012).</li><li>Arnold, W. D., Burghes, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+muscular+atrophy:+The+development+and+implementation+of+potential+treatments.">Spinal muscular atrophy: The development and implementation of potential treatments.</a> Annals of Neurology. 74 (3), 348-362 (2013).</li><li>Li, J., Sung, M., Rutkove, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiologic+biomarkers+for+assessing+disease+progression+and+the+effect+of+riluzole+in+SOD1+G93A+ALS+mice.">Electrophysiologic biomarkers for assessing disease progression and the effect of riluzole in SOD1 G93A ALS mice.</a> PloS one. 8 (6), e65976-65 (2013).</li><li>Ngo, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+Bayesian+motor+unit+number+estimation+and+histological+measurements+of+motor+neurons+in+wild-type+and+SOD1+(G93A)+mice.">The relationship between Bayesian motor unit number estimation and histological measurements of motor neurons in wild-type and SOD1 (G93A) mice.</a> Clin Neurophysiol. 123 (10), 2080-2091 (2012).</li><li>Shefner, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+MUNE+studies+in+animal+models+of+motor+neuron+disease.">Recent MUNE studies in animal models of motor neuron disease.</a> Supplements to Clinical neurophysiology. 60, 203-208 (2009).</li><li>Souayah, N., Potian, J. G., Garcia, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+unit+number+estimate+as+a+predictor+of+motor+dysfunction+in+an+animal+model+of+type+1+diabetes.">Motor unit number estimate as a predictor of motor dysfunction in an animal model of type 1 diabetes.</a> American journal of physiology Endocrinology and metabolism. 297 (3), E602-E608 (2009).</li><li>Zhou, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+comparison+in+monitoring+disease+progression+of+G93A+mouse+model+of+ALS.">A method comparison in monitoring disease progression of G93A mouse model of ALS.</a> Amyotrophic lateral sclerosis: official publication of the World Federation of Neurology Research Group on Motor Neuron Diseases. 8 (6), 366-3672 (2007).</li><li>Feng, X. H., Yuan, W., Peng, Y., Ss Liu, M., Cui, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+effects+of+dl-3-n-butylphthalide+in+a+transgenic+mouse+model+of+amyotrophic+lateral+sclerosis.">Therapeutic effects of dl-3-n-butylphthalide in a transgenic mouse model of amyotrophic lateral sclerosis.</a> Chinese medical journal. 125 (10), 1760-1766 (2012).</li><li>Mancuso, R., Santos-Nogueira, E., Osta, R., Navarro, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+analysis+of+a+murine+model+of+motoneuron+disease.">Electrophysiological analysis of a murine model of motoneuron disease.</a> Clinical neurophysiology : official journal of the International Federation of Clinical Neurophysiology. 122 (8), 1660-1670 (2011).</li><li>Lee, Y. i., Mikesh, M., Smith, I., Rimer, M., Thompson, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscles+in+a+mouse+model+of+spinal+muscular+atrophy+show+profound+defects+in+neuromuscular+development+even+in+the+absence+of+failure+in+neuromuscular+transmission+or+loss+of+motor+neurons.">Muscles in a mouse model of spinal muscular atrophy show profound defects in neuromuscular development even in the absence of failure in neuromuscular transmission or loss of motor neurons.</a> Developmental biology. 356 (2), 432-444 (2011).</li></ol>

The authors have nothing to disclose.

MUNE and CMAP are clinically relevant measures frequently utilized in research studies and in monitoring patients with neuromuscular disorders such as ALS and spinal muscular atrophy (SMA)9, 10. For instance, in SMA, CMAP and MUNE correlate well with age, severity and clinical measures of function10-14. Both measures are minimally invasive and allow assessment of function longitudinally in the same individual. Importantly, these measures cannot measure activation or recruitment of the motor unit by cortical motor neurons, but they provide a clinically-relevant assessment of the integrity of the motor neuron and its functional counterpart, the motor unit.
Animal models of neuromuscular disease are critical to an understanding of pathogenic mechanisms of human disease and to the preclinical development of potentially effective therapeutic agents. The ability to translate outcome measures and biomarkers that can be utilized across species can facilitate and hasten the translation of promising preclinical findings to human clinical trials. Several groups have previously utilized both electrophysiological and force (mechanical) measurements to estimate motor unit function in mouse models2-4, 15-22. Due to the relative complexity of the measures, we have refined these techniques in a visual format to allow more widespread use and implementation in mice. The format of video demonstration and instruction, allows key steps of the procedure to be highlighted and potential pitfalls to be addressed. The application of these techniques to preclinical testing of potential therapies in motor neuron diseases may improve the translation of putative therapies from mice to human disease.
There are several critical steps in the process of acquiring the CMAP and MUNE responses. Proper and consistent recording electrode placement and sufficient electrode contact with the hind limb are critical for reproducible measurement of amplitude and to decrease background noise. Therefore, close contact between hind limb skin and electrodes should be consistently confirmed. We have found that surface electrodes offer more consistent CMAP and MUNE recordings than needle electrodes. Due to very thin subcutaneous tissues, small movements of needle recording surface may lead to wide variation in CMAP amplitudes. Additionally, the more invasive nature of needle electrodes is not optimal for neonatal mice or longitudinal studies due to potential muscle disruption and injury. One potential drawback of non-selective, surface electrode recordings relates to the possibility of diminished phenotype resolution if a particular muscle is more or less involved compared with another, and this has been reported in an ALS mouse model21.
Acquiring the average SMUP size is technically more challenging compared to the CMAP. Due to the smaller response size (in the range of &#956;V rather than mV) background noise can be more problematic. Background noise can be reduced by adjusting the ground electrode, cathode, anode, and checking other electrical equipment near the experimental setup. A Faraday cage, typically used for intracellular electrophysiology applications, is not required. Visual determination of the individual SMUP responses is the most difficult skill to acquire and takes practice for consistent results with adequate repeatability. It is important to ensure that the SMUPs that are being recorded initiate within the duration of the maximal CMAP response. We have defined criteria for acceptance of individual incremental responses to make this process more straightforward to perform and to increase intra- and inter-rater reliability.
One potential drawback of the incremental MUNE technique includes the possibility of overestimating the number of functional motor units due to alternation of motor units. We have used a technique similar to Shefner et al. in that each response should be reproducibly seen a total of 3 times to reduce the impact of this phenomenon3.
In our experience, clinical electrodiagnostic systems are optimized for the studies described herein due to improved examiner-electrodiagnostic system interface ergonomics allowing ease of control. The two-channel system utilized in our lab is equipped with two non-switched amplifier channels using an amplifier with 24 bit Analog to Digital Converter and a sampling rate of 48 kHz per channel. Hardware gain can be adjusted from 10nV to 100 mV/division. The low frequency filter has a range from 0.2 Hz-5 kHz, and the high frequency filter settings range from 30 Hz-10 kHz. A constant-current stimulator is used (intensity: 0-100 mA; duration: 0.02-1 ms). Most clinical systems have similar appropriate features and can be adjusted to adequately record CMAP and MUNE responses. Additionally, standard electrophysiological rigs can be assembled to adequately record CMAP and MUNE, but the interface may need to be adjusted for ease of stimulation adjustment and rapid identification of CMAP and SMUP responses.
We have previously utilized the techniques of CMAP and MUNE described here to allow rapid and reproducible assessment of the sciatic innervated muscle of the hind limb in mice during the early postnatal period to adulthood5. These techniques allow assessment in mouse models when behavioral testing for motor function is not feasible or is less reliable. Application of this technique to neonatal mice facilitates the study of motor unit development and has the potential to expand our understanding of motor neuron innervation and pruning. For instance, we have shown that the number of functional motor units recorded with MUNE will increase during pruning from polyneuronal to mononeuronal innervation during the first two weeks of life in neonatal mice5. The ability to test mice over long periods of time with this technique lends itself to the study of motor unit response to peripheral nerve injury, hereditary neuromuscular disorders and aging.

Motor unit number estimation (MUNE) was originally described by McComas et al. over three decades ago1. The original technique was a modification of the compound muscle action potential (CMAP) recording technique that employed a gradual increase of stimulation to obtain submaximal increments. These increments were summed and averaged to determine an estimated size of a single motor unit potential (SMUP). This size was divided into the CMAP response to estimate the number of motor units innervating the muscle being tested. Following the original description, numerous variations using both electrophysiological responses and incremental force (mechanical) measurements have been used in both human studies and animal models2. The MUNE technique was modified by Shefner and colleagues to investigate mouse models of Amyotrophic Lateral Sclerosis (ALS)3, 4.
In the current description, we detail simplified modifications of the MUNE techniques that are rapid to perform. Importantly, CMAP and MUNE allow reliable measures in both neonatal and adult mice5-8. Experienced individuals can perform these measures in 10-20 min&#160;per animal, and repeated measures are feasible which permits the acquisition of longitudinal data5. In the current studies, we employ a clinical electrodiagnostic system. In our experience, clinical electrodiagnostic systems are optimized for rapid and efficient capture of electrophysiological data in vivo, nevertheless standard electrophysiological rigs can easily be adapted for this application.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pro trimmer Pet Grooming Kit</td>
			<td>Oster</td>
			<td>078577-010-003</td>
			<td>clippers for hair removal</td>
		</tr>
		<tr>
			<td>Synergy T2 EMG system</td>
			<td>Natus Neurology</td>
			<td>Model no longer available</td>
			<td>portable electrodiagnostic system</td>
		</tr>
		<tr>
			<td>monopolar needles 28 gauge</td>
			<td>Teca</td>
			<td>017K121</td>
			<td>cathode and anode stimulating electrodes</td>
		</tr>
		<tr>
			<td>Alpine Biomed Digital Ring Electrode with twisted wires and 1.5 mm TP connectors.</td>
			<td>Alpine Biomed</td>
			<td>9013S0312</td>
			<td>recording electrodes</td>
		</tr>
		<tr>
			<td>Helping Hands alligator clip with iron base</td>
			<td>Radio Shack</td>
			<td>64-079</td>
			<td>Maintaining recording electrode placement&#160;</td>
		</tr>
		<tr>
			<td>Spectra 360 Electrode Gel&#160;</td>
			<td>Parker Laboratories</td>
			<td>9013G5012</td>
			<td>applied to reduce skin impedance</td>
		</tr>
		<tr>
			<td>monoject curved tip irrigating syringe</td>
			<td>Covidien</td>
			<td>81412012</td>
			<td>utilized for application of electrode gel</td>
		</tr>
		<tr>
			<td>EMG needle cable</td>
			<td>Teca</td>
			<td>902-RLC-TP&#160;</td>
			<td>to connect monopolar electrodes to electrodiagnostic stimulator</td>
		</tr>
		<tr>
			<td>Disposable 2&#34; x 2&#34; Electrode or similar trimmed as needed</td>
			<td>Carefusion</td>
			<td>019-415000&#160;</td>
			<td>ground electrode</td>
		</tr>
		<tr>
			<td>Small Heating Plate with built-in RTD sensor, 15x10cm</td>
			<td>World Precision Instruments</td>
			<td>61830</td>
			<td>warming plate used with animal temperature controller to transmit heat to animal</td>
		</tr>
		<tr>
			<td>Silicone pad for use with ATC2000</td>
			<td>World Precision Instruments</td>
			<td>503573</td>
			<td>conductive removable pad to cover warming plate for easy cleaning</td>
		</tr>
		<tr>
			<td>Animal temperature controller</td>
			<td>World Precision Instruments</td>
			<td>ATC2000</td>
			<td>low noise animal heating system for maintaining animal temperature</td>
		</tr>
		<tr>
			<td>Veterinarian petroleum-based ophthalmic ointment&#160;</td>
			<td>Puralube</td>
			<td>26870</td>
			<td>applied during anesthesia to avoid corneal injury</td>
		</tr>
	</tbody>
</table>

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

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the functional status of a motor unit pool in vivo. These measures can provide insight into the normal development and degeneration of the neuromuscular system. These measures have clear translational potential because they are routinely applied in diagnostic and clinical human studies. We present electrophysiological techniques similar to those employed in humans to allow recordings of mouse sciatic nerve function. The CMAP response represents the electrophysiological output from a muscle or group of muscles following supramaximal stimulation of a peripheral nerve. MUNE is an electrophysiological technique that is based on modifications of the CMAP response. MUNE is a calculated value that represents the estimated number of motor neurons or axons (motor control input) supplying the muscle or group of muscles being tested. We present methods for recording CMAP responses from the proximal leg muscles using surface recording electrodes following the stimulation of the sciatic nerve in mice. An incremental MUNE technique is described using submaximal stimuli to determine the average single motor unit potential (SMUP) size. MUNE is calculated by dividing the CMAP amplitude (peak-to-peak) by the SMUP amplitude (peak-to-peak). These electrophysiological techniques allow repeated measures in both neonatal and adult mice in such a manner that facilitates rapid analysis and data collection while reducing the number of animals required for experimental testing. Furthermore, these measures are similar to those recorded in human studies allowing more direct comparisons.

The techniques of CMAP and MUNE described in this report allow recording of neuromuscular function of the sciatic innervated hind limb muscles utilizing minimally invasive electrode placement (Figure 1). Supramaximal CMAP size, which represents the total output from a muscle group, can be described using the parameters of amplitude and area (Figure 2), however, in the current methods, we use amplitude to quantify the CMAP and the SMUP sizes. Since the CMAP response measures summated depolarization of muscle fibers within a muscle, pathology anywhere from the motor neuron to the muscle fiber can result in reduction in CMAP size. Therefore, CMAP gives an excellent measure of the total functional status. As expected, CMAP size will increase during development5. Due to compensatory changes that can occur following denervation (i.e. collateral sprouting), CMAP size may be maintained despite processes of motor neuron or motor axonal loss. Therefore, the technique of MUNE is required to determine the motor neuron or axon input to the muscle or group of muscles being tested. Recording of individual increments (Figure 3) allows estimation of the average output of single motor units (SMUP size) to give more detailed information about functional status of motor units.
CMAP and MUNE can be utilized to measure neuromuscular function in various mouse models of neuromuscular disease. In Figure 4, findings in an adult control mouse and an adult mouse 11 weeks following sciatic nerve crush are contrasted. Following sciatic nerve crush, MUNE is severely reduced at 50 estimated functional motor units compared with normal findings of 278 functional motor units in the control mouse. In contrast, the CMAP amplitude in the crushed animal (39.6 mV baseline-to-peak, 74.9 mV peak-to-peak) shows only mild reduction compared to control (49.0 mV baseline-to-peak, 84.2 mV peak-to-peak) due to collateral sprouting.
<img alt="Figure 1" src="/files/ftp_upload/52899/52899fig1.jpg" /> 
Figure 1.&#160;Electrode Placement.&#160;The black (E1) &#8220;active&#8221; electrode (A) and red (E2) &#8220;reference&#8221; recording electrode (B) are placed over the gastrocnemius at the proximal portion of the gastrocnemius at the knee. The stimulating cathode (black) (C) and anode (red) (D) are inserted subcutaneously proximal to the recording electrodes to generate distal responses. A disposable disk electrode (D) is placed on the hind limb, tail or sacrum as a ground to minimize artifact. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52899/52899fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52899/52899fig2.jpg" /> 
Figure 2.&#160;Compound Muscle Action Potential.&#160;Pictured is an illustration of a representative CMAP response. (A)&#160;The baseline-to-peak amplitude is measured from the isoelectric baseline to the initial negative peak (negative voltage is depicted above the baseline). (B) The peak-to-peak amplitude is measured from negative peak voltage to positive peak voltage. The grey-shaded area denotes the negative peak area. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52899/52899fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52899/52899fig3.jpg" /> 
Figure 3.&#160;Incremental responses.&#160;Two representative incremental responses are shown superimposed and in isolation. For MUNE calculation amplitudes of each increment are measured peak-to-peak. Increment #1 is the initial all-or-none response recorded and represents a single motor unit potential (SMUP). Each subsequent increment (#2-10) represents a quantal increase superimposed on the prior response. Therefore to obtain the SMUP amplitudes for increments 2-10, the amplitude of the prior response is subtracted from the amplitude of the increment obtained. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52899/52899fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52899/52899fig4.jpg" /> 
Figure 4.&#160;Example Sciatic CMAP and MUNE.&#160;(A)&#160;Sciatic compound muscle action potential (CMAP) in an adult (6 months of age) control mouse with baseline-to-peak amplitude of 49.0 mV and peak-to-peak amplitude of 84.2 mV. Screen sensitivity=10 mV per division and screen duration 10 ms. (B) Ten corresponding incremental responses (in the control mouse) with total amplitude of 3.028 mV are divided by 10 to determine average SMUP size (0.3028 mV). Screen sensitivity=0.5 mV and sweep speed of 1 ms per division. Calculated MUNE=278 (MUNE=CMAP/average SMUP(84.2 mV/ 0.3028 mV)) (C) Sciatic CMAP 11 weeks following sciatic nerve crush in an adult mouse (6 months of age) showing mildly reduced baseline-to-peak amplitude (39.6 mV) and peak-to-peak amplitude (74.9 mV). Screen sensitivity=10 mV per division and sweep speed of 1 ms per division. (D) Ten corresponding incremental responses (in the mouse with nerve crush) with total peak&#8211;to&#8211;peak amplitude of 14.923 mV divided by 10 to obtain an average SMUP size of 1.4923 mV. Screen sensitivity=2 mV per division and a sweep speed of 1 ms per division. Calculated MUNE=50 (MUNE=CMAP/average SMUP (74.9 mV/1.4923 mV)). (**Note the different sensitivity for the incremental responses of the sciatic crush mouse). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52899/52899fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

We present refined protocols that allow in vivo monitoring of motor unit function in the mouse. Techniques to measure compound muscle action potential (CMAP) and motor unit number estimation (MUNE) in the mouse hind limb muscles innervated by the sciatic nerve are described.

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

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

electrophysiological-motor-unit-number-estimation-mune-measuring

Nanyang Technological University

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Behavior

Unit 1

Muscle Tissue

SCIENTISTS IN ACTION

21.0K Views.  The Ohio State University Wexner Medical Center. 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.

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

Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism

Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental disorder characterized by decreased reciprocal social interaction, abnormal communication, and repetitive behaviors with restricted interest. As diagnosis is based on clinical criteria, any potentially relevant rodent models of this heterogeneous disorder should ideally recapitulate these diverse behavioral traits. The BTBR T+ Itpr3tf/J (BTBR) mouse is an established animal model of ASD, displaying repetitive behaviors such as increased grooming, as well as cognitive inflexibility. With respect to social interaction and interest, the juvenile play test has been employed in multiple rodent models of ASD. Here, we show that when BTBR mice are tested in a juvenile social interaction enclosure containing sawdust bedding, they display a repetitive synchronous digging motion. This repetitive motor behavior, referred to as &#34;inchworming,&#34; was named because of the stereotypic nature of the movements exhibited by the mice while moving horizontally across the floor. Inchworming mice must use their fore- and hind-limbs in synchrony to displace the bedding, performing a minimum of one inward and one outward motion. Although both BTBR and C56BL/6J (B6) mice exhibit this behavior, BTBR mice demonstrate a significantly higher duration and frequency of inchworming and a decreased latency to initiate inchworming when placed in a bedded enclosure. We conclude that this newly described behavior provides a measure of a repetitive motor stereotypy that can be easily measured in animal models of ASD.

Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism

Inchworming is a highly repetitive synchronous digging motion displayed by BTBR T+ Itpr3tf/J (BTBR) mice when placed in a testing cage with sufficient sawdust bedding. The procedure is a modification of the juvenile social interaction protocol and is used here to assess repetitive motor stereotypies relevant to Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental disorder characterized by decreased reciprocal social interaction, abnormal ...

Corticospinal Excitability Modulation During Action Observation

This study used the transcranial magnetic stimulation/motor evoked potential (TMS/MEP) technique to pinpoint when the automatic tendency to mirror someone else's action becomes anticipatory simulation of a complementary act. TMS was delivered to the left primary motor cortex corresponding to the hand to induce the highest level of MEP activity from the abductor digiti minimi (ADM; the muscle serving little finger abduction) as well as the first dorsal interosseus (FDI; the muscle serving index finger flexion/extension) muscles. A neuronavigation system was used to maintain the position of the TMS coil, and electromyographic (EMG) activity was recorded from the right ADM and FDI muscles. Producing original data with regard to motor resonance, the combined TMS/MEP technique has taken research on the perception-action coupling mechanism a step further. Specifically, it has answered the questions of how and when observing another person's actions produces motor facilitation in an onlooker's corresponding muscles and in what way corticospinal excitability is modulated in social contexts.

Single-pulse transcranial magnetic stimulation over the primary motor cortex, neuronavigation, and registration of electromyographic activity of hand muscles were used in this study to explore corticospinal excitability while participants were observing action sequences.

This study used the transcranial magnetic stimulation/motor evoked potential (TMS/MEP) technique to pinpoint when the automatic tendency to mirror someone ...

Methods to Explore the Influence of Top-down Visual Processes on Motor Behavior

Kinesthetic awareness is important to successfully navigate the environment. When we interact with our daily surroundings, some aspects of movement are deliberately planned, while others spontaneously occur below conscious awareness. The deliberate component of this dichotomy has been studied extensively in several contexts, while the spontaneous component remains largely under-explored. Moreover, how perceptual processes modulate these movement classes is still unclear. In particular, a currently debated issue is whether the visuomotor system is governed by the spatial percept produced by a visual illusion or whether it is not affected by the illusion and is governed instead by the veridical percept. Bistable percepts such as 3D depth inversion illusions (DIIs) provide an excellent context to study such interactions and balance, particularly when used in combination with reach-to-grasp movements. In this study, a methodology is developed that uses a DII to clarify the role of top-down processes on motor action, particularly exploring how reaches toward a target on a DII are affected in both deliberate and spontaneous movement domains.

It is unclear how top-down signals from the ventral visual stream affect movement. We developed a paradigm to test motor behavior towards a target on a 3D depth inversion illusion. Significant differences are reported in both deliberate, goal-directed movements and automatic actions under illusory and veridical viewing conditions.

Kinesthetic awareness is important to successfully navigate the environment. When we interact with our daily surroundings, some aspects of movement are ...

Irrelevant Stimuli and Action Control: Analyzing the Influence of Ignored Stimuli via the Distractor-Response Binding Paradigm

Selection tasks in which simple stimuli (e.g. letters) are presented and a target stimulus has to be selected against one or more distractor stimuli are frequently used in the research on human action control. One important question in these settings is how distractor stimuli, competing with the target stimulus for a response, influence actions. The distractor-response binding paradigm can be used to investigate this influence. It is particular useful to separately analyze response retrieval and distractor inhibition effects. Computer-based experiments are used to collect the data (reaction times and error rates). In a number of sequentially presented pairs of stimulus arrays (prime-probe design), participants respond to targets while ignoring distractor stimuli. Importantly, the factors response relation in the arrays of each pair (repetition vs. change) and distractor relation (repetition vs. change) are varied orthogonally. The repetition of the same distractor then has a different effect depending on response relation (repetition vs. change) between arrays. This result pattern can be explained by response retrieval due to distractor repetition. In addition, distractor inhibition effects are indicated by a general advantage due to distractor repetition. The described paradigm has proven useful to determine relevant parameters for response retrieval effects on human action.

The distractor-response binding paradigm is described. It can be used to shed light on the influence irrelevant stimuli, competing with targets for a response, can have on human action. Both response retrieval effects and distractor inhibition effects can be analyzed within the paradigm.

Selection tasks in which simple stimuli (e.g. letters) are presented and a target stimulus has to be selected against one or more distractor stimuli are ...

Topographical Estimation of Visual Population Receptive Fields by fMRI

Visual cortex is retinotopically organized so that neighboring populations of cells map to neighboring parts of the visual field. Functional magnetic resonance imaging allows us to estimate voxel-based population receptive fields (pRF), i.e., the part of the visual field that activates the cells within each voxel. Prior, direct, pRF estimation methods1 suffer from certain limitations: 1) the pRF model is chosen a-priori and may not fully capture the actual pRF shape, and 2) pRF centers are prone to mislocalization near the border of the stimulus space. Here a new topographical pRF estimation method2 is proposed that largely circumvents these limitations. A linear model is used to predict the Blood Oxygen Level-Dependent (BOLD) signal by convolving the linear response of the pRF to the visual stimulus with the canonical hemodynamic response function. PRF topography is represented as a weight vector whose components represent the strength of the aggregate response of voxel neurons to stimuli presented at different visual field locations. The resulting linear equations can be solved for the pRF weight vector using ridge regression3, yielding the pRF topography. A pRF model that is matched to the estimated topography can then be chosen post-hoc, thereby improving the estimates of pRF parameters such as pRF-center location, pRF orientation, size, etc. Having the pRF topography available also allows the visual verification of pRF parameter estimates allowing the extraction of various pRF properties without having to make a-priori assumptions about the pRF structure. This approach promises to be particularly useful for investigating the pRF organization of patients with disorders of the visual system.

It is important to obtain unbiased estimates of visual population receptive fields (pRFs) by functional magnetic resonance imaging. We use mild regularization constraints to estimate pRF topography without a-priori assumptions about pRF shape, allowing us to choose specific pRF models post-hoc. This is particularly advantageous in subjects with visual-pathway lesions.

Visual cortex is retinotopically organized so that neighboring populations of cells map to neighboring parts of the visual field. Functional magnetic ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Using a Split-belt Treadmill to Evaluate Generalization of Human Locomotor Adaptation

Understanding the mechanisms underlying locomotor learning helps researchers and clinicians optimize gait retraining as part of motor rehabilitation. However, studying human locomotor learning can be challenging. During infancy and childhood, the neuromuscular system is quite immature, and it is unlikely that locomotor learning during early stages of development is governed by the same mechanisms as in adulthood. By the time humans reach maturity, they are so proficient at walking that it is difficult to come up with a sufficiently novel task to study de novo locomotor learning. The split-belt treadmill, which has two belts that can drive each leg at a different speed, enables the study of both short- (i.e., immediate) and long-term (i.e., over minutes-days; a form of motor learning) gait modifications in response to a novel change in the walking environment. Individuals can easily be screened for previous exposure to the split-belt treadmill, thus ensuring that all experimental participants have no (or equivalent) prior experience. This paper describes a typical split-belt treadmill adaptation protocol that incorporates testing methods to quantify locomotor learning and generalization of this learning to other walking contexts. A discussion of important considerations for designing split-belt treadmill experiments follows, including factors like treadmill belt speeds, rest breaks, and distractors. Additionally, potential but understudied confounding variables (e.g., arm movements, prior experience) are considered in the discussion.

We describe a protocol for investigating human locomotor adaptation using the split-belt treadmill, which has two belts that can drive each leg at a different speed. We specifically focus on a paradigm designed to test the generalization of adapted locomotor patterns to different walking contexts (e.g., gait speeds, walking environments).

Understanding the mechanisms underlying locomotor learning helps researchers and clinicians optimize gait retraining as part of motor rehabilitation. ...

The "Motor" in Implicit Motor Sequence Learning: A Foot-stepping Serial Reaction Time Task

This protocol describes a modified serial reaction time (SRT) task used to study implicit motor sequence learning. Unlike the classic SRT task that involves finger-pressing movements while sitting, the modified SRT task requires participants to step with both feet while maintaining a standing posture. This stepping task necessitates whole body actions that impose postural challenges. The foot-stepping task complements the classic SRT task in several ways. The foot-stepping SRT task is a better proxy for the daily activities that require ongoing postural control, and thus may help us better understand sequence learning in real-life situations. In addition, response time serves as an indicator of sequence learning in the classic SRT task, but it is unclear whether response time, reaction time (RT) representing mental process, or movement time (MT) reflecting the movement itself, is a key player in motor sequence learning. The foot-stepping SRT task allows researchers to disentangle response time into RT and MT, which may clarify how motor planning and movement execution are involved in sequence learning. Lastly, postural control and cognition are interactively related, but little is known about how postural control interacts with learning motor sequences. With a motion capture system, the movement of the whole body (e.g., the center of mass (COM)) can be recorded. Such measures allow us to reveal the dynamic processes underlying discrete responses measured by RT and MT, and may aid in elucidating the relationship between postural control and the explicit and implicit processes involved in sequence learning. Details of the experimental set-up, procedure, and data processing are described. The representative data are adopted from one of our previous studies. Results are related to response time, RT, and MT, as well as the relationship between the anticipatory postural response and the explicit processes involved in implicit motor sequence learning.

We introduce the foot-stepping serial reaction time (SRT) task. This modified SRT task, complementing the classic SRT task that involves only finger-pressing movement, better approximates daily sequenced activities and allows researchers to study the dynamic processes underlying discrete response measures and disentangle the explicit process operating in implicit sequence learning.

This protocol describes a modified serial reaction time (SRT) task used to study implicit motor sequence learning. Unlike the classic SRT task that ...

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal&#39;s free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals.

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...

Measuring Active and Passive Tameness Separately in Mice

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two behavioral components: a reluctance to avoid humans (passive tameness) and a motivation to approach humans (active tameness). To quantify these components in mice, we previously developed behavioral tests for active tameness, passive tameness, and the willingness to stay on a human hand, each designed to be completed within 3 min. The data obtained were used for selective breeding, with a large number of mice analyzed per generation. The active tameness test measures the movement of the mouse toward a human hand and the contact it engages in. The passive tameness test measures the duration of time that a mouse tolerates human touch. In the stay-on-hand test, a mouse is placed on a human hand and touched slowly using the thumb of that hand; the duration of time that the animal remains on the hand is measured. Here, we describe the test set-up and apparatus, explain the procedures, and discuss the appropriate data analysis. Finally, we explain how to interpret the results.

Tameness in animals includes a reduction of avoidance responses to humans (passive tameness) and an increase in active approaches to humans (active tameness). Here, we describe detailed protocols for three behavioral tests (active tameness, passive tameness, and stay-on-hand tests) to separately measure the active and passive tameness of mice.

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two ...