measuring compound muscle action potentials in mouse forelimb muscles in vivo

Measuring Compound Muscle Action Potentials in Mouse Forelimb Muscles In Vivo

To measure CMAP on the forelimbs, position the mouse on the heating pad in the supine position, and use adhesive tape to extend both forelimbs on the sides of the body. Next, place 5 millimeters of the stimulating electrode subcutaneously without puncturing the underlying muscles on both sides of the forelimb to align with the brachial plexus nerve.
Then, place the recording electrode subcutaneously above the biceps brachii muscle. Subsequently, place 3 millimeters of the reference electrode at a 30-degree angle on the walking pads, and insert the ground electrode subcutaneously on the side of the mouse.
Electrodes are in close proximity of each other in this setup. Prevent electrodes from touching each other as this distorts the recording.
Stimulate all axons using one pulse per second with 0.1 millisecond stimulus duration. To acquire data, start the stimulation by pushing the recurrent stimulus button in the controller unit and turn the intensity controller knob to increase the stimulus.
To reach supramaximal stimuli, apply increasing stimuli by turning the intensity controller knob until the amplitude of the CMAP response ceases to increase. From there, further, increase the stimulus by 20% to ensure that the CMAP amplitude has reached its maximal response. End the stimulation by pushing the recurrent stimulus button again.
Use the marker tool to indicate the following points in the recording; initiation of the response, maximum positive peak, and maximum negative peak. Initiation of the stimulus is determined automatically by the software.
Determine the latency as a delay from the initiation of the stimulus to the initiation of the response. Use the latency to evaluate demyelination in the axons. Measure the amplitude from the maximum negative to maximum positive peak, and use the magnitude of the amplitude to correlate the number of functional axons.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal Preparation and Anesthesia
<ol>
	<li>Induce anesthesia in the mouse with isoflurane/oxygen inhalation. Use 4% of isoflurane for the induction of the anesthesia and 2-3% for the maintenance at 2.5 L/min flow of oxygen. Adjust the isoflurane percentage for the maintenance of the anesthesia according to the condition of the mouse,&#160;i.e., small and weak mice require less anesthetics. Confirm adequate anesthesia&#160;e.g., by applying mild pressure to the hindlimb walking pad to check the absence of a pain withdrawal reflex.</li>
	<li>Control the mouse body temperature using a thermostatic heating plate at 37 &#176;C to prevent the decrease of body temperature during anesthesia.</li>
	<li>Fit the mouse with the nosecone for maintenance of the anesthesia. Ensure that the animal has sufficient delivery of oxygen by checking that the nosecone does not block airways and that the animal is breathing steadily.</li>
	<li>During the recording, monitor whether the mouse is sufficiently anesthetized by observing the breathing rate (approximately 1 Hz in anesthesia) and the absence of a withdrawal reflex on mild pressure. Increase the isoflurane concentration manually if the anesthesia is not deep enough.</li>
	<li>After the measurements, leave the mouse to recover on the heating plate or in the warmth of an infrared lamp until it has regained sufficient consciousness to maintain sternal recumbency, for approximately 2-5 min. Do not leave the mouse unattended and in the company of other mice until it has fully recovered from anesthesia.</li>
</ol>
2. Measurement of CMAP in Forelimbs
<ol>
	<li>Use the 27 G needle electrodes for forelimb CMAP measurements. See&#160;Figure 1&#160;for recommended places of electrode positioning.</li>
	<li>Place electrodes on the forelimbs as follows.
	<ol>
		<li>Position the mouse on the heating pad in the supine position and use adhesive tape to extend both forelimbs on the sides of the body (Figure 1B).</li>
		<li>Place the stimulating electrodes (1 = anode and 2 = cathode) subcutaneously on both sides of the forelimb to align with the brachial plexus nerve. Lift the skin to insert the needle perpendicularly through the skin and push approximately 5 mm of the needle under the skin without puncturing the underlying muscles.</li>
		<li>Place the recording electrode (3) subcutaneously on top of the biceps brachii muscle by lifting the skin. Place the reference electrode (4) on the walking pads in 3 mm depth at a 30-degree angle. Place the ground electrode (5) subcutaneously on the side of the mouse. 
		NOTE: Electrodes are in close proximity of each other in this setup. Prevent electrodes from touching each other as this distorts the recording.</li>
	</ol>
	</li>
</ol>
3. Data Acquisition
<ol>
	<li>Start the stimulation by pushing the&#160;recurrent stimulus&#160;button in the controller unit and turn the intensity controller knob to increase the stimulus. Stimulate all axons using 1 pulse/s with 0.1 ms stimulus duration. Select the correct frequency and duration from dropdown menus in the software.</li>
	<li>To reach supramaximal stimuli (5-20 mA; in demyelinating conditions up to 60 mA), apply increasing stimuli by turning the intensity controller knob until the amplitude of the CMAP response ceases to increase. From there, further increase the stimulus by 20% to ensure that the CMAP amplitude has reached its maximal response. End the stimulation by pushing the&#160;recurrent stimulus&#160;button again.</li>
	<li>Use the marker tool to indicate the following points in the recording: initiation of the stimulus, initiation of the response, maximum positive peak, and maximum negative peak (Figure 2).</li>
	<li>Determine the latency (in ms) as a delay from the initiation of the stimulus to the initiation of the response (Figure 2). Define the initiation of the response as the earliest point where the amplitude begins to increase. Use the latency to evaluate demyelination in the axons.</li>
	<li>Measure the amplitude (mV) from the maximum negative to the maximum positive peak (Figure 2). Use the magnitude of the amplitude to correlate the number of functional axons.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Resuable subdermal needle electrode, Pl/Ir</td>
			<td>Technomed</td>
			<td>TE/S61-434</td>
			<td>The Needle is 13 mm (0.51&#34;) in length, 0.4 mm (27G) in diameter</td>
		</tr>
		<tr>
			<td>Natus electrodiagnostic system</td>
			<td>Natus Neurology</td>
			<td>UltraPro S100</td>
			<td>EMG device</td>
		</tr>
		<tr>
			<td>Synergy</td>
			<td>Natus Neurology</td>
			<td>version 20.1.0.100</td>
			<td>EMG software for UltraPro S100</td>
		</tr>
		<tr>
			<td>Physitem Controller</td>
			<td>Rothacher-Medical GmbH</td>
			<td>TCAT-2LV</td>
			<td>Heating pad</td>
		</tr>
		<tr>
			<td>combi-vet Base Anesthesia System Digital Flowmeter with TEC 3 Vaporize</td>
			<td>Rothacher &#38; Partner</td>
			<td>CV 30-301-D</td>
			<td>Isoflurane Vaporizer and flowmeter</td>
		</tr>
		<tr>
			<td>Iso-Vet 1000 mg/g&#160;</td>
			<td>Piramal Healthcare UK Limited</td>
			<td>AP/DRUGS/220/96</td>
			<td>Isoflurane</td>
		</tr>
		<tr>
			<td>SOD1-G93A mice</td>
			<td>The Jackson Laboratory</td>
			<td>#002726</td>
			<td>ALS tg and non-tg control littermates, only females</td>
		</tr>
		<tr>
			<td>PrP-hFUS-WT3 mice</td>
			<td>The Jackson Laboratory</td>
			<td>#017916&#160;</td>
			<td>ALS tg and non-tg control littermates, all groups balanced for males and females</td>
		</tr>
		<tr>
			<td>C57BL/6Jax mice</td>
			<td>The Jackson Laboratory</td>
			<td>#000664</td>
			<td>Non-tg mice for axotomy, male and female</td>
		</tr>
		<tr>
			<td>C61-PMP22 mice</td>
			<td>Mouse line was generously donated&#160; by Prof. M. Sereda (The Max Planck Institute of Experimental Medicine, G&#246;ttingen, Germany).</td>
			<td></td>
			<td>CMT tg and non-tg control littermates, all groups balanced for males and female</td>
		</tr>
	</tbody>
</table>

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

<img alt="Figure 1" src="/files/ftp_upload/22729/22729_Figure1.jpg" /> 
Figure 1. Positioning of the electrodes for CMAP measurements.&#160;The position of the electrodes is presented for hind- (A) and forelimbs (B). The electrodes are numbered as follows: 1: anode and 2: cathode stimulating electrodes, 3: active recording electrode, 4: reference electrode, and 5: grounding electrode.&#160;
<p class="jove_content" fo:keep-togethe...

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

Secure an anesthetized mouse in a supine position.&#160;
Subcutaneously insert the stimulating electrodes, aligning them with the nerve innervating the forelimb muscle.
Subcutaneously insert the recording electrode on top of the upper arm muscle, the reference electrode into the walking pads, and the ground electrode on the side.
Apply electrical pulses to stimulate the forelimb neurons, inducing a positive ion influx and action potential generation.
The action potential induces excitatory neurotransmitter release at the neuromuscular junction, leading to muscle fiber activation and contraction.
Record the combined response from all muscle fibers &#8212; the Compound Muscle Action Potential, or CMAP.
Apply maximal stimuli to ensure stimulation of all muscle fibers.
End the stimulation and determine the latency, the time delay between the initiation of the stimulus and the response.
Increased latency indicates decreased conduction speed due to neuronal demyelination.
Measure the CMAP amplitude; a decreased magnitude indicates the loss of functional neurons.

32 vues. Mesure des potentiels d’action musculaire composés dans les muscles des membres antérieurs de souris in vivo

Vidéo: Mesure des potentiels d’action musculaire composés dans les muscles des membres antérieurs de souris in vivo - Expérience

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

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

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

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

Nombre électrophysiologique Motor Unit Estimation (MUNE) Mesure composé Muscle Potentiel d&#39;action (CMAP) dans les muscles de souris membres postérieurs

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

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Sciences du comportement

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

In Vivo Mesures électrophysiologiques du nerf cubital Rat avec test d’excitabilité axonale

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

Neurosciences

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

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

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

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

CMAP Scan MUNE (MScan) - une méthode d’Estimation (MUNE) Numéro de bloc moteur roman

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

Measuring Compound Muscle Action Potentials in Mouse Forelimb Muscles In Vivo

Transcription

Measuring Compound Muscle Action Potentials in Mouse Forelimb Muscles In Vivo