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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. Experimental Set Up
NOTE: 12-week-old female Long-Evans rats were used in this procedure.
<ol>
	<li>Anesthetize the rat in an induction chamber using 4% isoflurane and 1 L per min oxygen (O2) flow rate. Confirm sufficient anesthesia by testing for the righting reflex and ensuring its absence before removing the animal from the induction chamber. Note that various anesthetic agents have differential effects on nerve excitability.</li>
	<li>Securely place the animal&#39;s snout in the nose cone attachment and deliver a maintenance dose of 2.5% isoflurane and 1 L per min O2 flow rate.</li>
	<li>Confirm adequate anesthesia by checking for both pedal withdrawal and corneal reflexes by pinching the animal&#39;s toes and gently touching its eye. 
	NOTE: Application of vet ointment on eyes, to prevent dryness while under anesthesia, is recommended but not essential as the procedure typically takes 30 min per animal.</li>
	<li>Maintain the body temperature of the rat at 37 &#176;C with the use of a feedback-controlled heating mat and a rectal thermometer probe. Set the heating mat and integrated body temperature sensor to 40 &#176;C to prevent any skin damage that occurs with higher temperatures. 
	NOTE: It is recommended to monitor and record physiological measures (heart rate, oxygen saturation, body temperature and respiratory rate) every 10s with the use of an animal physiological monitoring system. Optimal recording procedures should include local measurement of the limb temperature as the peripheral temperature can be colder than that of the core temperature (Figure 1).</li>
	<li>Wear antiseptic gloves and always use instruments that have been cleaned with 70% ethanol, to maintain a pathogen-free environment throughout the procedure.</li>
</ol>
2. Electrophysiological Set Up
<ol>
	<li>Use low impedance platinum electroencephalogram (EEG) needle electrodes for this procedure.</li>
	<li>Prepare the recording needle electrodes by inserting the recording needle electrode (Figure 1; indicated in purple) through the hypothenar muscle and the reference electrode through the dorsal aspect of the 4th digit (Figure 1; indicated in orange) to record compound muscle action potentials (CMAPs).</li>
	<li>Place the ground electrode through the skin on the superior aspect of the forearm between stimulating and recording electrodes (Figure 1; indicated in green). Take care when inserting the electrodes to avoid muscle tissue.</li>
	<li>Prepare the percutaneous stimulating needle electrodes by inserting the cathode (Figure 1; labeled in blue) approximately 4 mm distal to the cubital tunnel at the elbow. Insert the anode (Figure 1; labeled in red) approximately 1 cm proximally through the skin of the axillar region.</li>
</ol>
3. Axonal Excitability Procedures
<ol>
	<li>Perform the rodent motor nerve TRONDNF protocol using a semi-automated, computer controlled axonal excitability program (see the Table of Materials) linked to a constant current stimulator and an amplifier. Remove excess 50 Hz electrical noise using a 50/60 Hz noise eliminator.
	<ol>
		<li>Record the CMAP from the hypothenar muscle by simultaneously visualizing the CMAP morphology and applying a 1 ms square-wave pulse to the ulnar nerve with the cathode needle electrode.
		<ol>
			<li>To achieve optimal recordings, carefully adjust the angle and/or position of the cathode until an optimal biphasic response curve with constant amplitude is achieved (Figure 2A.). Once the optimal position has been determined, stabilize the cathode with a repositionable electrode holder. 
			NOTE: The software used automatically delivers the test stimuli described below and increases or decreases the current required to achieve the threshold.</li>
		</ol>
		</li>
		<li>Record a stimulus-response curve by incrementally increasing the stimulus intensity of a 1 ms impulse by 1 mA until a maximum response is achieved. 
		NOTE: The purple and green line in Figures 2B and 2C represents the incremental increase of the stimulus intensity and the automated system, respectively. The target amplitude for threshold tracking is automatically set to 40% of the maximal amplitude corresponding to the area of the steepest slope on the stimulus-response curve. The change in &#39;threshold&#39; (i.e. stimulus required to elicit 40% CMAP) induced by various test stimuli is the variable obtained throughout the remainder of the protocol.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>QTracS Program</td>
			<td>Digitimer Ltd.</td>
			<td></td>
			<td>Axonal excitability program</td>
		</tr>
		<tr>
			<td>AM-Systems 2200, Analog Stimulus Isolator, 2200V/50Hz</td>
			<td>SDR Scientific</td>
			<td>850005</td>
			<td>Stimulator</td>
		</tr>
		<tr>
			<td>High Performance AC Amplifier Model LP511</td>
			<td>Grass Technologies</td>
			<td></td>
			<td>Amplifier</td>
		</tr>
		<tr>
			<td>Humbug 50/60Hz Noise eliminator</td>
			<td>Quest Scientific Instruments</td>
			<td>726310</td>
			<td>Noise eliminator</td>
		</tr>
		<tr>
			<td>Low Impedance Platinum Monopolar Subdermal Needle Electrodes</td>
			<td>Grass Technologies</td>
			<td>F-E2-24</td>
			<td>Recording electrodes, 10 mm length, 30 gauge</td>
		</tr>
		<tr>
			<td>Low Impedance Platinum Electroencephalography Needle Electrodes</td>
			<td>Cephalon</td>
			<td>9013L0702</td>
			<td>Stimulating electrodes, 10 mm length, 30 gauge</td>
		</tr>
		<tr>
			<td>Multifunction I/O Device Model USB-6341</td>
			<td>National Instruments</td>
			<td></td>
			<td>Multifunction input/output device</td>
		</tr>
		<tr>
			<td>Iron Base Plate IP</td>
			<td>Narishige Scientific Instrument Laboratory</td>
			<td></td>
			<td>Used for holding stimulating needle electrode in place</td>
		</tr>
		<tr>
			<td>Rotating X-block X-4</td>
			<td>Narishige Scientific Instrument Laboratory</td>
			<td></td>
			<td>Used for holding stimulating needle electrode in place</td>
		</tr>
		<tr>
			<td>Magnetic Stand GJ-8</td>
			<td>Narishige Scientific Instrument Laboratory</td>
			<td></td>
			<td>Used for holding stimulating needle electrode in place</td>
		</tr>
		<tr>
			<td>Micromanipulator M-3333</td>
			<td>Narishige Scientific Instrument Laboratory</td>
			<td></td>
			<td>Used for holding stimulating needle electrode in place</td>
		</tr>
	</tbody>
</table>

in vivo measurement of the compound muscle action potential in a rat

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

<img alt="Figure 1" src="/files/ftp_upload/22741/22741_Figure1.jpg" /> 
Figure 1: A schematic diagram of the needle electrode placements in the rat forelimb. The cathode (blue) is inserted approximately 4 mm distal to the cubital tunnel at the elbow, and the anode (red) is inserted approximately 1 cm proximally through the skin of the axillar region. The ground needle electrode (green) is inserted through the skin on the superior aspect of the forearm between stimulating and recording electrodes. The recording (purple) and reference (orange) needle electrodes are inserted through the hypothenar muscle and through the dorsal aspect of the 4th digit. The temperature probe (grey) is placed on the superior aspect of the thenar muscle.
<img alt="Figure 2" src="/files/ftp_upload/22741/22741_Figure2.jpg" /> 
Figure 2: Raw data from an axonal excitability test. Panel A demonstrates a biphasic CMAP response curve after an incremental increase of stimulation to the ulnar nerve. Panels B and C represent the stimulus intensity (mA) and amplitude of CMAP (mV), respectively. The green components of B and C depict the automated incremental decrease in stimulus intensity and the associated sigmoid-shaped decrease in CMAP required for threshold tracking.

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

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

Place an anesthetized long-Evans rat on a heating mat.
Insert a reference electrode into the fourth finger and a recording electrode into the hypothenar muscles.
These are a group of muscle fibers in the palm that are innervated by the ulnar nerve.
Next, position a ground electrode on the upper forearm, a stimulating cathode near the elbow, and an anode near the armpit.
Apply a constant electrical stimulus through the cathode to the ulnar nerve.
This stimulation induces an action potential across the axon, leading to neurotransmitter release at the neuromuscular junction.
The neurotransmitter release causes the muscle fiber to depolarize and contract, generating electrical activity.
Record the combined electrical activity from all muscle fibers, called the compound muscle action potential, or CMAP.
Adjust the cathode&#39;s position and stabilize the cathode with an electrode holder to acquire optimum recording.
Gradually increase the electrical stimulation until a maximum response is obtained from all muscle fibers.

in-vivo-measurement-of-the-compound-muscle-action-potential-in-a-rat

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

130 Views. Source: Wild, B. M., et al. In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing. J. Vis. Exp. (2018) This video demonstrates the procedure for recording compound muscle action potential (CMAP) in rats by applying electrical stimuli to the ulnar nerve, adjusting electrodes for optimal signal, and increasing stimulation to assess nerve functionality. This method enables the in vivo investigation of the peripheral nerve functions in neurological disorde...

Video: In Vivo Measurement of the Compound Muscle Action Potential in a Rat - Concept

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

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

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

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

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

JoVE Journal

Behavior

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

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

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

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

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

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

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

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

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

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

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

Transcript

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

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

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

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