This article outlines a method for injecting fluorescent tracers into the motor end plates of rat muscles to study neuromuscular junctions. The technique facilitates the retrograde transport of tracers to motor neuron cell bodies, highlighting potential neurotherapeutic applications.
Take an anesthetized rat and apply eye lubricant.
Shave the targeted limb, sterilize the skin, and position the rat to ensure optimal access to the targeted muscle.
Lift and incise the skin to expose the muscle.
Refer to photographs of motor end plates or MEPs, the regions where motor neuron axon terminals synapse with muscle fibers to form neuromuscular junctions.
Mentally map the MEP region onto the exposed muscle.
Take a glass micropipette containing a fluorescent tracer.
Inject the tracer intramuscularly at multiple points along the MEP region.
Remove any excess tracer, close the incision, and administer a pain suppressant.
The injection enhances tracer availability at neuromuscular junctions, facilitating its endocytosis at axon terminals.
The tracers enclosed within vesicles are then transported retrogradely to motor neuron cell bodies in the spinal cord.
The presence of tracer-labeled motor neurons in the spinal cord suggests the neurotherapeutic potential of MEP-targeted delivery.
To inject the live muscle at the motor end plates, first use a micropipette puller to pull graded glass micropipettes equipped with plungers. Next, using a dissecting microscope, break the tips of the micropipettes with a pair of forceps such that the internal diameter of each lumen is approximately 0.5 millimeters. Then, fill the pulled pipettes with fluoro-gold.
Now, apply eye lubricant to an anesthetized animal and shave the limb to be injected. Use gauze to wipe the shaved area with three alternating scrubs of chlorhexidine and 70% alcohol. Then, place the animal on a clean under pad to allow access to the targeted muscle. When the animal is in position, use a pair of forceps with tooth grips to lift the skin over the targeted muscle away from the underlying musculature, and use surgical scissors to make an incision in the skin.
Take care that the incision is large enough to completely expose the muscle of interest, with a minimal disruption to the fascia. Then, using the motor end plate photographs to mentally transpose the location and shape of the motor endplate region of interest onto the muscle, and administered 3 to 4 injections of 1 to 2 microliters of fluoro-gold along the full length of the motor endplate region.
After the last injection, gently wipe the muscle to remove any seepage. Then use blunt forceps to bring the two ends of the incised skin close together. Close the wound with surgical clips, and infiltrate the entire span of the wound with the appropriate local anesthetic.
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