Measuring Compound Muscle Action Potentials in Mouse Forelimb Muscles In Vivo

Secure an anesthetized mouse in a supine position. 

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 — 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.

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.