Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

The overall goal of this procedure is to administer muscarinic antagonists to LAL muscles of adult mice and examine morphological changes of njs in response to inhibition of muscarinic acetylcholine receptors. First muscarinic antagonists are subcutaneously administered at the desired frequency and duration. Following euthanasia, the treated LAL muscles are carefully dissected out and any connective tissue is cleaned off.

The muscle tissue is then triple immuno labeled to visualize the motor nerve schwan cells and nicotinic acetylcholine receptors using standard immunohistochemical methods. Finally, confocal microscopy is used to examine morphological changes of njs in muscles treated with muscarinic antagonists. Ultimately, results can be obtained that show that the morphological organization of the tripartite NMJ is severely perturbed through inhibition of muscarinic acetylcholine receptors.

The main advantage of this technique over existing methods like administration of pharmaceutical compounds to skeletal muscles of the rodent hy limb, such as the gastric anemia and tib anterior is that the LAL muscle is more accessible and relatively thin. This permits repeated local application of drugs and visualization of almost all njs within the muscle On a bench top that has been cleaned with ethanol, dissolve the appropriate dose of muscarinic acetylcholine receptor antagonist in sterile physiological saline in a 1.5 milliliter reaction tube. Draw up 50 microliters of solution into a one cc insulin syringe using a separate syringe for each mouse.

Also prepare syringes containing physiological saline only for each mouse in the control group, keeping all syringes on ice until ready to inject Next anesthetize mice with a ketamine xylazine cocktail. Confirm anesthesia using the toe pinch technique after proper sedation, shave off the hair covering the rostral region of the head to the coddle region of the neck and wipe remaining hair away with 70%ethanol. Place the mouse under a stereo microscope and insert the syringe needle parallel to the LAL muscle so that the tip covers the codal aspect.

Pull up lightly on the needle of the syringe to ensure that the needle is inserted into overlying connective tissue and does not directly damage the LAL muscle itself. Very slowly begin to inject solution over the right LAL muscle while withdrawing the syringe over the course of approximately one minute. If injected properly, the solution should form a dome in the overlying skin that remains for at least one hour.

Repeat the procedure every 12 hours for one to seven days prior to muscle dissection euthanize, and then pin down the mouse in a dissection dish dorsal side up. Make the first incision through the skin only using small spring scissors to cut along the left LAL from the region just proximal to the ear to the region around the left shoulder blade. Carefully remove the skin in this region, but avoid cutting too close to the right ear as this is one of the points of attachment For the right LAL muscle.

Locate the centrally positioned adipose tissue strip of lipid on the superficial surface of the head where the right and left LAL muscles meet using small spring scissors. Cut one centimeter to the right of the fat tissue and towards the left ear from the proximal edge of the left LAL muscle until reaching the shoulder. Once the incision is made, peel back the right LAL muscle with small forceps to expose the ventral side of the muscle.

Trim the connective tissue and fascia while carefully pulling up on the right LAL muscle with forceps. Cut around the coddle end of the right LAL at the base of the ear and to move towards the rostral end of the right ear. Keeping the right LAL turned over.

It is better to include more tissue at this step than risk damaging the LAL itself. If the muscle begins to dry out. Pipette one XPBS over the muscle.

Place the dissected right LAL in a 30 millimeter culture cigar dish containing one XPBS dorsal side up. Pin down four corners of the muscle with small insect pins using small forceps and spring scissors. Clean connective tissue from the dorsal surface of the right LAL muscle.

Turn the muscle over and re-pin in order to clean off the ventral surface. Two to three muscles may be placed in the same 30 millimeter dish for triple immuno staining after overnight. Primary antibody incubation.

Wash the muscles in one XPB S3 times for 10 minutes and then add the cocktail of secondary antibodies in blocking solution for one hour. At room temperature, keep the incubating muscles protected from light to prevent photobleaching after an hour incubation, wash the muscles in one XPB S3 times for 10 minutes each. Keeping the muscles covered in fresh one XPBS after the last wash under the stereo microscope, clean off any remaining connective tissue and then cut the lateral borders of the LAL.

Mount the muscle tissue on the slide dorsal side up. Then add two or three drops of mounting media. Apply glass cover slips and use clear nail polish to secure them in place.

Protect the slides from light and store them at minus 20 degrees Celsius until ready for analysis here. High magnification confocal views of a triple immunostain mouse NMJ show, its tripartite organization. The fine terminal branches that elaborate from a single axon stained with NFS V two are tightly covered by terminal schwan cell bodies and their processes as revealed by S 100 staining post synaptically.

A muscle fiber, a cluster of nicotinic acetylcholine receptors is precisely opposed to the branches of nerve terminals and terminal schwan cells. The LAL muscle of a vehicle treated mouse displays njs, whose tripartite organization is unperturbed and have the typical highly differentiated arbors of the nerve terminal as shown by triple immuno staining. In contrast, the structural and functional integrity of this tripartite organization is severely perturbed by the daily application of four damp.

A subtype specific muscarinic acetylcholine receptor inhibitor. Here four damp evokes the selective elimination of nerve terminals from numerous njs throughout the muscle surface. In addition, terminal schwan cells are abnormally quiescent as evidenced by bright S 100 labeling without process extension.

After watching this video, you should have a good understanding of how to administer muscarinic antagonists to LAL muscles and triple immunostain, the muscles to examine morphological changes of njs in response to inhibition of these receptors.