Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

The overall goal of this procedure is to isolate the levator auris longus muscle and its nerve in order to record synaptic currents at the neural muscular junction. This method can help answer key questions in synaptic physiology such as the end plate current size, quantal content, probability of release and relationships between neurotransmission and muscle excitability. The main advantage of this technique is the detailed electrophysiological recordings from a single synapse can easily be combined with live cell optical experiments.

Though this method can provide insight into synaptic function and feedback communication between nerve and muscle, it can also be applied to other systems ranging from drosophila to mammals. Generally, individuals new to this method will struggle because the mammalian nerve muscle prep is fragile, particularly the nerve. Carefully acquiring and interpreting the subsequent voltage clamp data can also be challenging.

Once mastered, this technique can be done in one hour if it is performed properly. Demonstrating the procedure will be Steve Burke from my laboratory. To begin this procedure, under a stereo-dissecting microscope, make a small incision in the skin on the back of the mouse at the level of the scapulae.

Use a pair of microdissection scissors. Cut the skin over the head and the back. Next, pull up the skin along the incision near the scapulae.

Cut the muscles that are inferior to the LAL by starting at the right scapula. Then, gently lift the cut tissue immediately to the right of the midline. Make a cut toward the left ear with the blades of the scissors pressed against the cranium to remove several layers of muscle that are inferior to the left LAL.

Avoid cutting the nerve that innervates the LAL which wraps around the ear canal and enter the muscles on the medial side of the ear. Continue cutting through the ear canal keeping as much of the nerve attached as possible. Then, cut the fatty tissue behind the ear canal along the ventrolateral portion of the ear.

Cut along the left scapula similar to the procedures done on the right side to remove the muscle completely from the mouse. Next, cut off the pinna of the ear along the base leaving the cartilaginous part of the ear attached to the LAL. Flip the muscle so that the inferior side is facing up.

To isolate LAL muscle, place the LAL and the surrounding tissue into a Petri dish with a silicon elastomer bottom and pin the dissected tissue to the bottom. Wash the tissue frequently in a physiological saline solution. Then, place an insect pin through the ear canal to hold the prep in place.

Using smaller pins, pin the remaining tissue on the opposite side of the midline of the LAL. Use a pair of forceps. Gently pull the skin up on the lateral portion of the ear to stretch the muscle out and place a small pin through the skin.

Repeat this step until the tissue is well-secured to the dish. Remove the muscles that are covering the LAL and those bound to the LAL via connective tissue and are especially tight near the midline. Subsequently, pull up the overlying muscle layer and cut the connective tissue with the blades oriented toward the muscle layer being pulled.

Cut toward the midline until about 3/4 of the way to the midline and keep removing muscle layers until only the LAL remains. During the cleaning process, ensure that the nerves are not damaged. Following that, remove some of the remaining connective tissue that is covering the LAL to aid in impalement of the electrodes.

Only remove tissue that can be done easily without risk of damaging the LAL in the process. To identify the nerve that innervates the LAL, using a nerve stimulator, touch the nerves with the nerve stimulator. When the muscle contracts, the correct nerve has been identified.

Then, carefully grab the tissue near the nerve and use spring scissors to separate the nerve from the tissue surrounding the ear. To minimize damage, keep most of the nerve embedded in some surrounding tissue which will be used later to secure the nerve to the recording dish. At this point, the investigator can take an hour break as long as the LAL is bathed in 20 milliliters or more of a physiological saline solution.

Next, unpin and transfer the muscle to a stage insert under the dissection microscope for electrophysiology experiments. Pin the muscle at the ends and along its edge. Position the nerve perpendicular to the muscle fibers and pin it to the bottom of the dish through some excess tissue that was left intact at the end of the nerve.

Keep the tissue bathed in a physiological saline solution at all times. For electrophysiology experiment, secure the perfusion chamber with the LAL to the microscope stage. Place the reference electrode into a cup filled with three-molar potassium chloride which is connected to the recording chamber via an agar bridge.

At this point, position the nerve-stimulating electrode on the nerve. Expose the LAL prep to 4-Di-2-Asp for 10 minutes to achieve adequate florescence for neuromuscular junction visualization. After 10 minutes, exchange the 4-Di-2-Asp solution with the normal calcium solution.

In the meantime, fill the glass capillaries with the appropriate solutions for the voltage-sensing and current-passing electrodes. Gently tap the capillary to remove any air bubbles and secure the filled capillary into the electrode holder on the headstage. Using an upright microscope with standard bright field and florescence illumination, look for a bright band of fluorescent green neuromuscular junctions running perpendicular to the muscle fibers along the prep with a low-magnification water-emersion objective.

Then, switch to a higher magnification water-emersion objective to identify a neuromuscular junction on the top layer of muscle in order to examine with electrophysiology. Using primarily bright field, position the electrode above the muscle membrane within 100 microns of the identified neuromuscular junction. This figure shows an example of the current pulses and the voltage responses from one LAL fiber under current clamp from a 12-week-old wild-type R62 mouse.

The records were obtained in normal physiological saline solution and the presence of mEPPs indicates that these records were taken from the motor end plate. Here is a representative recording of an EPC and two mEPCs obtained under voltage clamp condition. This figure shows the superimposed EPCs and mEPCs from a representative fiber.

Once mastered, this technique can be done in one hour if it is performed properly. Following this procedure, other methods like live-cell imaging with membrane dyes such as FM143 or histology can be performed in order to answer questions related to vesicle release uptake or morphological changes. After its development, this technique paved the way for researchers in the field of physiology to explore synaptic transmission and nerve-cell communication in organisms ranging from drosophila to mammals.

After watching this video, you should have a good understanding of how to isolate the innervated levator auris longus muscle in order to record synaptic currents at the neuromuscular junction. While attempting this procedure, it's important to remember to take great care in removing the muscle tissue adjoined to the LAL and isolating the nerve.