In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration
June 15th, 2018
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.
With this method we can estimate the degeneration of the axons as well as the demyelination in the peripheral nerves of mouse models of neurodegeneration. The main advantage of this technique is that this minimally invasive method is feasible for repeated measurements only one did not follow up of the multiple nerves in the same animals. The sensitivity of this method enables early detection of axon loss in the modulation even before notable more defects can be recorded thus allowing early detection of these defects.
To begin this procedure, anesthetize the mouse with isoflurane oxygen inhalation. Confirm adequate anesthesia by applying mild pressure to the hind limb walking pad to check the absence of a pain withdrawal reflex. Then place a nose cone on the animal's face to maintain anesthesia.
Ensure that the nose cone does not block the airway and the animal is breathing steadily. Next, maintain the animal's body temperature at 37 degrees Celsius using a thermostatic heating plate. To measure CMAP on the hind limb, position the mouse in the prone position.
Extend the hind limb and attach the paw on the work surface using adhesive tape. Then place five millimeters of the 27 gauge stimulating electrodes subcutaneously on both sides of the sciatic nodge without puncturing the underlying muscles with two centimeters between the electrodes. Similarly place the recording electrode subcutaneously above and parallel to the gastrocnemius muscle.
Afterward place the reference electrode subcutaneously next to the Achilles tendon in a 30 degree angle and leave two to five millimeters of the needle under the skin. Place the ground electrode subcutaneously on the side of the mouse in a similar manner as the stimulating electrodes. To measure CMAP on the forelimbs, position the mouse on the heading pad in the supine position and use adhesive tape to extend both forelimbs on the sides of the body.
Next, place five millimeters of the stimulating electrode subcutaneously without puncturing the underlying muscles on both sides of the forelimb to align with brachial plexus nerve. Then place the recording electrode subcutaneously above the biceps brachii muscle. Subsequently, place three 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 milliseconds 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 super maximal 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 fro 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. Since the exact placement of the electrodes can affect the outcome value of the recording, replace the electrodes and measure the same nerve for three times using super maximal stimulus to ensure that the largest response is obtained. Use the average of the recordings.
After the measurements remove all the electrodes and leave the mouse to recover on the heating plate or under an infrared lamp for approximately two to five minutes until it has regained sufficient consciousness. Do not leave the mouse unattended and in the company of other mice until it has fully recovered from anesthesia. The C61 PMP22 mice over expressing three to four copies of the human PMP22 and heterozygote mice recapitulate a very mild CMT1A disease phenotype with mild demyelination and reduced CMAPs but with no visible phenotype.
In one and a half to two years of age of C61 PMP22 transgenic mice the CMAP amplitudes are reduced and latencies are prolonged both in the hind limbs and forelimbs. Amplitude was decreased both in hind and forelimbs in transgenic mice, and latency was prolonged in all limbs in CMT transgenic mice, and even subtle change in forelimbs was detected with this measurement. Requirement for stimulus intensity was increased in C61 PMP22 mice, which resembles the detected phenotype in CMT1A patients.
Once mastered CMAP recording can be done for both hind and forelimbs in 15 minutes when performed properly. The presented technique provides novel possibilities of the characterization of mouse models of neurodegenerative disorders such as amyotrophic lat sclerosis and Charcot-Marie-Tooth disease. Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders.
Similar recordings are used in clinical settings emphasizing the translational potential of this method.
Overview
This study presents a technique to measure compound muscle action potential (CMAP) in vivo in mouse forelimb muscles innervated by the brachial plexus, expanding the assessment of neurodegeneration in mouse models. The method enables the detection of axon degeneration and demyelination in peripheral nerves using a minimally invasive approach.
Key Study Components
Area of Science
- Neuroscience
- Electrophysiology
- Neurodegeneration
Background
- Assessment of nerve conduction is crucial in studying neurodegenerative diseases.
- Traditional methods predominantly focus on hindlimb measurements.
- There is a need for improved techniques that provide insights into forelimb nerve function.
Purpose of Study
- To develop a method for measuring CMAP in mouse forelimb muscles.
- To evaluate axonal health and demyelination in neurodegeneration models.
- To facilitate repeated measurements in the same animal.
Methods Used
- The study employs in vivo electrophysiological recordings.
- Mice are anesthetized using isoflurane and positioned for maximal recording efficacy.
- Electrodes are strategically placed to record CMAP from both hindlimbs and forelimbs.
- Data acquisition involves stimulating the nerves and measuring resultant electrical responses.
- Multiple measurements ensure reliability and accuracy of the data collected.
Main Results
- The method demonstrated sensitivity in detecting early signs of axonal loss and demyelination.
- Measurements revealed reduced CMAP amplitudes and prolonged latencies in transgenic mice.
- This technique allows for early identification of neuropathological changes associated with conditions like CMT1A.
Conclusions
- This study establishes a novel framework for assessing mouse models of neurodegenerative disorders.
- The CMAP recording technique has significant translational potential for understanding human neuromuscular disorders.
- Insights from this methodology could enhance our grasp of neuronal mechanisms and disease progression.
Frequently Asked Questions
Executive Industry Relevance
This method enables longitudinal assessment of peripheral nerve function in mouse models, supporting early detection of axonal degeneration and demyelination. By providing translatable electrophysiological readouts, it enhances predictive confidence in preclinical target validation for neuromuscular disorders. The technique facilitates go/no-go decisions by quantifying functional axon integrity and myelination status across disease progression.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by quantifying axon loss and demyelination in vivo.
- Operational Value: Supports repeated measurements in the same animal, reducing variability and improving statistical power in target validation studies.
- Predictive Value: Detects early neuropathological changes before phenotypic onset, enabling mechanistic de-risking of targets in ALS and CMT models.
Screening & Assay Development
- Scientific Value: Generates quantitative CMAP amplitude and latency data for assessing compound effects on nerve conduction.
- Operational Value: Enables standardized, reproducible electrophysiological assays across forelimb and hindlimb nerves.
- Assay Readiness: Provides scalable, minimally invasive readouts suitable for high-frequency longitudinal screening in neurodegeneration models.
Translational & Preclinical Research
- Translational Continuity: Mirrors clinical nerve conduction studies, strengthening preclinical-to-clinical extrapolation in neuromuscular disease models.
- Disease Relevance: Applicable to both axonal and demyelinating neuropathies, supporting biomarker-aligned target evaluation.
- Risk-Adjusted Advancement: Enables longitudinal tracking of nerve function to inform dose selection and therapeutic window definition.
Pipeline & Workflow Integration
The method integrates into discovery workflows by providing electrophysiological phenotyping that supports target validation and lead optimization in neurodegeneration programs.
- Discovery Biology: Supports hypothesis testing on axonal integrity and myelination in peripheral nerves of disease models.
- Screening: Delivers quantitative, reproducible nerve conduction metrics for compound screening campaigns.
- Analytics: Provides CMAP amplitude and latency readouts that enable functional comparison across genotypes and treatment groups.
- Translational Research: Aligns with clinical electrophysiology, enhancing predictive validity of preclinical findings.
- Enterprise Reuse: Establishes a reusable electrophysiological platform for multi-target assessment in neuromuscular portfolios.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity by directly measuring axon count and myelination status.
- Operational Value: Enables standardization across labs through defined electrode placement and stimulation protocols.
- Strategic Value: Improves capital efficiency by identifying ineffective targets early via functional nerve readouts.
- Portfolio Impact: Supports risk-adjusted prioritization through longitudinal, quantitative neuropathy progression data.
Implementation Considerations
- Requires expertise in in vivo electrophysiology and rodent surgical techniques.
- Dependent on stimulator and recording equipment capable of sub-millisecond pulse delivery.
- Necessitates standardized electrode placement to minimize inter-session variability.
- Must account for anesthesia depth and temperature control to ensure consistent nerve excitability.
- Limited by signal-to-noise ratio in severely atrophied muscles, requiring validation in advanced disease stages.
