Name: in vivo electrophysiological measurement of compound muscle action potential from the forelimbs in mouse models of motor neuron degeneration
Uploaded: 2018-06-15T14:30:00.000+00:00
Duration: 6 min 35 s
Description: Watch this Scientific Journal Video about In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration at JoVE.com

This research has been supported by KU Leuven (&#39;Opening the Future&#39; and C1), the Fund for Scientific Research Flanders (FWO-Vlaanderen), the Thierry Latran Foundation, the Association Belge contre les Maladies neuro-Musculaires (ABMM), the Muscular Dystrophy Association (MDA), the and ALS Association and the ALS Liga (Belgium). PVD holds a senior investigatorship of FWO-Vlaanderen. RP was supported by grants from the Central Remedial Clinic (CRC) Ireland and is currently supported the National University of Ireland (NUI) and the FWO.

All animals were housed under standard conditions according to the guidelines of the KU Leuven - University of Leuven and the associated European guidelines (European Union Directive 2010/63/EU for animal experiments). All animal experiments were approved by the local ethical committee of the KU Leuven.
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, i.e., small and weak mice require less anesthetics. Confirm adequate anesthesia 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 Hind- and Forelimbs
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57741/57741fig1.jpg" /> 
Figure 1. Positioning of the electrodes for CMAP measurements. 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. <a href="/files/ftp_upload/57741/57741fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Use the 27 G needle electrodes for hindlimb and forelimb CMAP measurements. See Figure 1 for recommended places of electrode positioning.</li>
	<li>Place electrodes on the hindlimb as follows.
	<ol>
		<li>Place the mouse on the heating pad in the prone position. Extend the hindlimb at the knee and attach the paw on the working surface using adhesive tape (Figure 1A).</li>
		<li>Place the stimulating electrodes subcutaneously on both sides of the sciatic notch with a distance of approximately 2 cm (1 = anode and 2 = cathode) between the electrodes. 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>Similarly, place the recording electrode (3) subcutaneously aligning the gastrocnemius muscle. Insert the reference electrode (4) subcutaneously next to the Achilles tendon in a 30-degree angle and leave 2-5 mm of the needle under the skin. Place the ground electrode (5) subcutaneously on the side of the mouse in a similar manner as the stimulating electrodes, but the position of this electrode is not critical for the measurement.</li>
	</ol>
	</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 recurrent stimulus 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 recurrent stimulus 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 maximum positive peak (Figure 2). Use the magnitude of the amplitude to correlate the number of functional axons.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57741/57741fig2.jpg" /> 
Figure 2. Representative image of CMAP response. A descriptive CMAP response indicating the points used for calculating the amplitude and latency (A). Latency is determined by the delay from the stimulation to the onset of the CMAP response. Peak-to-peak amplitude is measured from the maximum negative to the maximum positive peak of the biphasic wave. Representative recordings of a healthy non-transgenic animal (B) and a diseased animal with prolonged latency and reduced amplitude (C). <a href="/files/ftp_upload/57741/57741fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="6">
	<li>Since the exact placement of the electrodes can affect the outcome value of the recording,&#160;replace the electrodes and measure the same nerve for three times using supramaximal stimulus to ensure that the largest response is obtained. Use the average of the recordings.</li>
</ol>

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The authors have nothing to disclose.

Sensitive recording methods are essential for assessing the disease progression and especially the efficacy of a therapy in animal models of neuronal disorders. Determining the CMAPs is a minimally invasive electrophysiological technique, which is routinely used in clinics and in experimental setups to assess nerve conduction in neuromuscular and neuropathic disorders3,18. Here, we describe a novel application for CMAP recording in mice in order to measure the nerve conduction in the brachial plexus nerve of the forelimb. The presented method allows a more versatile and detailed longitudinal assessment of neuronal function in mouse models of neurodegeneration.
The needle electrodes are slightly more invasive than ring electrodes and especially in longitudinal studies care must be taken to minimize tissue damage. One possible drawback of the method is resulting injury from piercing a nerve or muscle. However, after careful subcutaneous placement of the electrodes, the injury and disruption of the muscles and nerves can be prevented. In contrast to the method using ring electrodes, the method presented here does not require shaving of the fur from large parts of the body. As a consequence, there is no discomfort or effect on thermoregulation for the animal.
The positioning of the electrodes is critical for the correct and consistent recording of the CMAP amplitudes and latencies. It is advisable to reposition the electrodes and to perform two to three measurements at each site to confirm that the maximal stimulation and responses are achieved. Correct recordings should produce biphasic curves as demonstrated in Figure 2. To standardize the method, non-transgenic mice without nerve injury are the best models to establish the proper and consistent electrode positioning for optimal stimulation.&#160;Reusable needle electrodes are suitable for repeated use if they are regularly sterilized, e.g. with glutaraldehyde for 20 minutes between animals, and inspected for sharpness.
In healthy adult mice, the CMAP amplitudes recorded with the presented method are typically 80-100 mV after stimulating the sciatic nerve and the brachial plexus. This is notably larger than the responses measured with ring electrodes, because there is a higher impedance caused by the skin for the ring electrodes that produces results of 20-40 mV8,19,20. In ALS mouse models, the CMAP amplitudes after stimulating the sciatic nerve or the brachial plexus in paralyzed limbs decrease to 10-30 mV. The magnitude of the CMAP amplitude is smaller in young animals since the CMAP amplitude increases during development21.
The method that we describe here is especially useful in mouse models of ALS, in which denervation, and subsequent motor deficits, occur earlier in the hindlimbs than in the forelimbs13. In addition to denervation, the method could detect reinnervation which is determined as prevented or retarded decline in the CMAP amplitude. The dramatic decrease in the CMAP amplitude in the muscles of the hindlimbs already at the age of symptom onset hinders the follow-up of further disease progression; as the CMAP amplitudes reach very low values at the early stage of the disease, they do not further decrease during the disease process. In contrast, axonal loss progresses at a slower pace in the brachial plexus nerve of the forelimbs and presents a more sensitive option for measuring disease progression over a longer duration of the disease. Moreover, the less degenerated forelimbs could provide a more potent site for assessing therapeutic approaches that aim at enhancing axonal function.
It is clear that the presented technique provides novel possibilities for the characterization of mouse models of neuromuscular disorders. CMAP recordings with needle electrodes from the sciatic nerve and the brachial plexus is a rapid and reproducible method to assess axonal loss and demyelination in hind- as well as in forelimbs. The sensitivity of the method enables detection of axonal deficits even before notable motor deficits can be recorded, and thus allows the early quantification of these defects. Moreover, the possibility of repeated testing reduces the number of required animals and provides a detailed overview of the progression of neuromuscular and neuropathic diseases at different sites in an individual animal.

Electrophysiology is used as a diagnostic tool in neuromuscular disorders such as motor neuron disorders, plexopathies, neuropathies, neuromuscular junction disorders, and myopathies. In amyotrophic lateral sclerosis (ALS), in which primarily the motor neurons are affected, the axonal damage and muscle paralysis1 are reflected in reduced CMAP amplitudes on nerve conduction studies (NCS). In Charcot-Marie-Tooth disease (CMT) both axonal degeneration and demyelination can be estimated in peripheral nerves using NCS2. This technique can be used for confirming the diagnosis as well as to evaluate the disease progression3,4. NCS enable the estimation of the axonal pathology, which is deduced from the magnitude of the action potential amplitude5, and the extent of demyelination - which results in reduced conduction velocity, prolonged distal latencies, or conduction block6.
CMAP measurement is a fast and sensitive method to evaluate nerve conduction both in humans and mice. Whereas in patients the NCS are performed routinely at various sites to record different nerves and muscles, in mice, the CMAP measurements are typically done only for the sciatic nerve to assess nerve functionality in the hindlimbs. However, in some animal studies it would be advantageous to record CMAP both in the fore- and hindlimbs, for example, to follow differential disease progression between fore- and hindlimbs in ALS mouse models.
Here, we introduce a method for recording CMAPs from the forelimbs of mice using needle electrodes. Additionally, we provide an approach to measure CMAPs&#160;from hindlimbs, likewise with needle electrodes. The measurement of CMAPs from hindlimbs with ring electrodes has been presented earlier7,8. The recording of CMAPs using needle electrodes is a rapid measuring method, it does not require shaving of the fur, and the procedure for measuring both hind- and forelimbs takes only 10 min per animal for an experienced researcher. Moreover, this minimally invasive approach is feasible for repeated measurements to allow longitudinal follow-up of multiple nerves in animals.

<table><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&quot;) 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 &amp;amp; Partner</td><td>CV 30-301-D</td><td>Isoflurane Vaporizer and flowmeter</td></tr><tr><td>Iso-Vet 1000 mg/g&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp; by Prof. M. Sereda (The Max Planck Institute of Experimental Medicine, G&amp;ouml;ttingen, Germany).</td><td></td><td>CMT tg and non-tg control littermates, all groups balanced for males and females</td></tr></tbody></table>

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.

Electrophysiological measurements of CMAPs using needle electrodes is a minimally invasive and very sensitive method to follow neuromuscular function over time. The technique described here allows the assessment of forelimb nerve conduction in mice, and thus, provides insights into the functionality of the nerve.
The CMAP amplitudes and latencies were measured from hind- and forelimbs during disease course in two mouse models of ALS, SOD1-G93A9 and PrP-hFUS-WT310 (Figure 3), and in a mouse model of CMT, C61-PMP2211,12 (Figure 4). ALS mouse models were created by overexpression of ALS-related human genes, namely either mutated SOD1 or wild type FUS. In both models, mice develop ALS resembling progressive motor neuron degeneration leading to paralysis. In non-transgenic littermate controls, the CMAP amplitude of both hind- and forelimbs did not change over time (Figure 3A). On the other hand, the CMAP amplitude of the sciatic nerve from the hindlimb was dramatically decreased in the SOD1-G93A mice, even before symptom onset around the age of 60 days (whereas the first motor symptoms are usually observed at the age of three months)13. The amplitude was 90 mV at that age in non-transgenic (non-tg) littermates, whereas in SOD1-G93A mice it was only 30 mV. There was only minimal further decline in the amplitude as the disease progressed to late symptomatic stage at the age of 150 days. The decline in CMAP amplitude, and hence the degeneration of axons, was delayed in the brachial plexus nerve of the forelimbs in comparison to the sciatic nerve from the hindlimbs. In the forelimbs, the disease progression was also more noticeable as the CMAP amplitude decreased from 70 mV to 30 mV when measured before and after the manifestation of the motor deficits in these mice.
In the PrP-hFUS-WT3 mouse model of ALS, the onset of motor deficits starts approximately at the age of 28 days10, which coincides with the initiation of the decline in the CMAP amplitude. This is a more accelerated disease model as the mice reach end-stage approximately at the age of 65 days. The decline in the CMAP amplitude occurred more rapidly in the sciatic nerve of the hindlimb in comparison to the brachial plexus nerve in the forelimb, which indicates an earlier axonal degeneration in the hindlimbs (Figure 3D). This observation supports the clinical observation in both of these mouse models as the hindlimbs are paralyzed notably earlier than the forelimbs that remain functional up to the late stages of the disease process.
In general, the latency from stimulus to initiation of the action potential was shorter in the forelimbs compared to hindlimbs (Figure 3B, E). This is simply due to the shorter distance between the stimulating and the recording electrodes. The latency provides an indication of the myelination level of the axons. Our observation is that CMAP latencies are prolonged during the disease progression in mouse models of ALS, although ALS is not a demyelinating disease. This is most likely due to the loss of larger, faster conducting motor axons.
The C61-PMP22 mice overexpressing 3-4 copies of the human PMP22 and the heterozygote mice recapitulate a very mild CMT1A disease phenotype with mild demyelination and reduced CMAPs, but with no visible phenotype11,12. In 1.5-2 years of age C61-PMP22 mice, the CMAP amplitudes are reduced and latencies prolonged both in the hindlimbs and forelimbs (Figure 4). Representative recordings displaying diminished amplitude and delayed response in comparison to a recording from a healthy subject are presented in Figure 2C, B, respectively. The CMAP latencies in the forelimbs are not affected as much as in the hind limbs. This is consistent with CMT1A patients, as more often patients have severely reduced or undetectable CMAPs in the lower limbs due to the pathophysiological nature of CMT as a length-dependent disorder14. Additionally, the degree of disease severity is correlated with CMAP amplitude, rather than latency or conduction velocity, as amplitudes correlate with the degree of axonal integrity14,15. Nevertheless, results indicate that this method is sensitive enough to the detect demyelinating process such as those observed in CMT1A.
Variation in amplitude and latency was lowest in non-transgenic groups (coefficient of variation 2-15% and 1-13%, respectively). In all transgenic cases, there was more variation in the measurements (coefficient of variation for amplitude 8-51% and for latency 1-21%), which most likely is caused by the differences in disease progression among the animals. In all cases, the variation was similar in hind- and forelimbs. The variation in the use of needle and surface electrodes has been reported to be similar16.
The required stimulus intensities did not vary greatly between non-transgenic and ALS models (Figure 3C, F). Likewise, the required stimulus to reach supramaximal stimulus in these cases was similar for fore- and hindlimbs and varied between 5-12 mA. In CMT, the requirement for increased stimulus intensities has been recognized17 and the same phenotype was seen in C61-PMP22 mice (Figure 4C). The phenomenon has been explained by increased electrical impedance from hypertrophic endoneurial changes17.
To confirm that the CMAP amplitude recorded from the forelimbs was due to nerve stimulation and not muscle stimulation, we performed unilateral partial axotomy on the brachial plexus nerve in 5 months old non-transgenic C57BL/6Jax mice (male and female) (Figure 5). Axotomy reduced the CMAP amplitude from 90 mV to 20 mV, indicating that most of the axons were disconnected in the operation. There was no change in the amplitude in the contralateral forelimb or in the hindlimbs. This result strongly indicates that the response detected in the biceps brachii was due to nerve stimulation and did not result from muscle stimulation.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57741/57741fig3.jpg" /> 
Figure 3. CMAP amplitude, latency, and required stimulus over the disease course in the hind- and forelimbs in ALS mouse models. SOD1-G93A (A-C) and PrP-hFUS-WT3 (D-F) transgenic (tg) mice and non-transgenic (non-tg) littermates were measured at the onset of the motor symptoms, at the symptomatic stage, and in the late-symptomatic phase of the disease process, at ages 57, 91, and 147 days (d) or at 29, 38, and 53 days for SOD1-G93A and PrP-hFUS-WT3 mice, respectively. Black: Non-transgenic hindlimb, black dashed: non-transgenic forelimb, grey: transgenic hindlimb, grey dashed: transgenic forelimb. Results are presented as mean &#177; SD. Amplitudes (A, D) were stable over time in the non-transgenic animals both in hind- and forelimbs. In transgenic animals, amplitudes decreased during the disease process. Latencies (B, E) were less affected by the disease and major differences were observed between hind- and forelimbs, regardless of the genotype. Variation in the required stimulus (C, F) was minimal in all groups. For SOD1-G93A N = 4 in all groups except for tg 147 d, N = 3. For PrP-hFUS-WT3 mice in age groups 29, 38, and 53, N is for non-tg 4, 5, and 4, and for tg 7, 5, and 3, respectively. Symbols denote the difference between groups as follows: *: non-tg hindlimb vs. tg hindlimb, #: non-tg forelimb vs. tg forelimb, &#164;: non-tg hindlimb vs. non-tg forelimb, grey *: tg hindlimb vs. tg forelimb. Two-way ANOVA with Tukey&#39;s multiple comparisons test, *: p &#60; 0.05, **: p &#60; 0.01, ***: p &#60; 0.001, ****: p &#60; 0.0001.#: p &#60; 0.05, ##: p &#60; 0.01, ###: p &#60; 0.001, ####: p &#60; 0.0001.&#164;: p &#60; 0.05, &#164;&#164;: p &#60; 0.01, &#164;&#164;&#164;: p &#60; 0.001, &#164;&#164;&#164;&#164;: p &#60; 0.0001. <a href="/files/ftp_upload/57741/57741fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57741/57741fig4.jpg" /> 
Figure 4. CMAP amplitude, latency, and required stimulus in the hind- and forelimbs in CMT1A mice. C61-PMP22 transgenic (tg) mice and non-transgenic (non-tg) littermates were measured at 1.5-2 years of age. Amplitude (A) was decreased both in hind- and forelimbs in transgenic mice. Latency (B) was prolonged in all limbs in CMT mice and even subtle change in forelimbs was detected with this measurement. Requirement for stimulus intensity (C) was increased in C61-PMP22 mice, which resembles the detected phenotype in CMT1A patients. Results are presented as mean &#177; SD, for non-tg N = 4 and tg N = 3. Two-way ANOVA with Sidak&#39;s multiple comparisons test,**: p &#60; 0.01, ****: p &#60; 0.0001. <a href="/files/ftp_upload/57741/57741fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57741/57741fig5.jpg" /> 
Figure 5. Forelimb action potentials are caused by nerve stimulation. To exclude the possibility that the observed CMAP response was caused by muscle stimulation, (partial) axotomy was performed on the brachial plexus nerve. CMAP amplitude (A) and latency (B) were recorded before (pre) and 4 days after (post) the axotomy of the brachial plexus in adult non-transgenic mice. Axotomy diminished the CMAP amplitude indicating that the response was due to nerve stimulation. Black: hindlimb, grey: contralateral forelimb, grey dashed: ipsilateral forelimb. Results are presented as mean &#177; SD, N = 2. <a href="/files/ftp_upload/57741/57741fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration at JoVE.com

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.

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 ...

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Neuroscience

19.1K Views.  KU Leuven - University of Leuven. 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.

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

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

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

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

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

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

Behavior

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4 , including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice5,6. However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9, or reproducting human disease10,11, we expect this technique to be an essential tool for the study of spinal motor system.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

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

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

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

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

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

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.

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

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Medicine

Fabrication and Surgical Implantation of Electromyographic Electrodes in Mice

Recording Forelimb Muscle Activity in Head&#45;Fixed Mice with Chronically Implanted EMG Electrodes

Powerful genetic and molecular tools available in mouse systems neuroscience research have enabled researchers to interrogate motor system function with unprecedented precision in head-fixed mice performing a variety of tasks. The small size of the mouse makes the measurement of motor output difficult, as the traditional method of electromyographic (EMG) recording of muscle activity was designed for larger animals like cats and primates. Pending commercially available EMG electrodes for mice, the current gold-standard method for recording muscle activity in mice is to make electrode sets in-house. This article describes a refinement of established procedures for hand fabrication of an electrode set, implantation of electrodes in the same surgery as headplate implantation, fixation of a connector on the headplate, and post-operative recovery care. Following recovery, millisecond-resolution EMG recordings can be obtained during head-fixed behavior for several weeks without noticeable changes in signal quality. These recordings enable precise measurement of forelimb muscle activity alongside in vivo neural recording and/or perturbation to probe mechanisms of motor control in mice.

Author Spotlight: Investigating Mouse Motor Cortex Interactions from Muscle Activity to Neural Dynamics

This protocol describes the hand fabrication and surgical implantation of electromyographic (EMG) electrodes in the forelimb muscles of mice to record muscle activity during head-fixed behavior experiments.

Powerful genetic and molecular tools available in mouse systems neuroscience research have enabled researchers to interrogate motor system function with ...

In vivo Measurement of Knee Extensor Muscle Function in Mice

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the ...

Biology

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

Reliable assessment of skeletal muscle strength is arguably the most important outcome measure in neuromuscular and musculoskeletal disease and injury studies, particularly when evaluating regenerative therapies&#39; efficacy. Additionally, a critical aspect of translating many regenerative therapies is the demonstration of scalability and effectiveness in a large animal model. Various physiological preparations have been established to evaluate intrinsic muscle function properties in basic science studies, primarily in small animal models. The practices may be categorized as: in vitro (isolated fibers, fiber bundles, or whole muscle), in situ (muscle with intact vascularization and innervation but distal tendon attached to a force transducer), and in vivo (structures of the muscle or muscle unit remain intact). There are strengths and weaknesses to each of these preparations; however, a clear advantage of in vivo strength testing is the ability to perform repeated measurements in the same animal. Herein, the materials and methods to reliably assess isometric torque produced by the hindlimb dorsiflexor muscles in vivo in response to standard peroneal electrical stimulation in anesthetized pigs are presented.

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

The present protocol describes concise experimental details on the evaluation and interpretation of in vivo torque data obtained via electrical stimulation of the common peroneal nerve in anesthetized pigs.

Reliable assessment of skeletal muscle strength is arguably the most important outcome measure in neuromuscular and musculoskeletal disease and injury ...

Measuring Compound Muscle Action Potentials in Mouse Forelimb Muscles In Vivo

Source: Pollari, E. et al., <a href="https://appjove.unicartagenaproxy.elogim.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.

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

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

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Chapters in this video

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

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measuring Neuromuscular Junction Functionality

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

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Recording Forelimb Muscle Activity in Head-Fixed Mice with Chronically Implanted EMG Electrodes

In vivo Measurement of Knee Extensor Muscle Function in Mice

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

Measuring Compound Muscle Action Potentials in Mouse Forelimb Muscles In Vivo