The overall goal of this procedure is to study the electrophysiological and contractile properties of a single mouse motor unit by performing intracellular recordings of motor neurons in vivo. This is accomplished by first exposing the spinal cord and the muscle of interest. Then a force transducer is attached to the tendon of the muscle.
Next emoto neuron is stimulated with an intracellular electrode thereby causing the contraction of the recorded motor unit. Ultimately, the electrophysiological properties of the motor neuron can be characterized at the same time as the contractile properties of its motor unit. Visual demonstration of this technique is critical because the microsurgery steps are very difficult to learn due to the small size of the animal.
To begin the procedure, anesthetize a mouse by injecting pentobarbital sodium or a mixture of ketamine and xylazine intraperitoneal. Make sure the mouse has no toe pinch reflex. Next, make a cut over the trachea using a pair of blunt scissors and pull the skin away on both sides.
Then tear the salivary gland apart with a pair of blunt forceps. To expose two thin muscles covering the trachea, separate the two muscles to reveal the trachea. Using a pair of Dumont seven forceps.
Slide two threads of four aut silk suture under the trachea. Then make a transversal cut in the trachea in between two cartilaginous rings. But be careful not to completely section the trachea.
Now, insert the tracheal tube down the trachea. Secure both sides of the insertion opening by tying the sutures over it. After that, connect the tracheal tube to a mouse ventilator and a capnograph.
Then connect the ventilator to the pure oxygen through a compliance bag. Adjust the parameters of the ventilator so that the mouse is not fighting against the artificial ventilation and the end tidal carbon dioxide. Partial pressure is stable between four and 5%In this step, expose the jugular vein on one side of the neck using blunt dissection techniques.
The jugular vein splits into two major trunks, the anterior and posterior facial veins. Carefully separate the vein from its surrounding connective tissue. Then slide two threads of six aut silk suture under the vein, and have them separated as far as possible along the length of the vein.
Next, place a small vessel clip on the proximal side of the vein and tie off the distal side of the vein. Using the very fine iris scissors, make a small transverse incision in the vein. Be very careful not to section the vein completely.
After that, insert a prefilled one French catheter in the opening up to the vessel clip holding the vein and the catheter together Carefully remove the vessel clip. Then push the catheter a few more millimeters into the vein. Secure the catheter by tying it with the sutures on both sides of the insertion.
Next, connect one of the catheters to a syringe or a syringe pump to inject supplemental doses of anesthetics such as pentobarbital, sodium or ketamine xylazine. Repeat the procedure for another vein and connect the other catheter to a syringe pump for a slow intravenous infusion of a 4%glucose solution containing 1%sodium bicarbonate and 14%plasmon. At the end, close the neck skin with needle with suture.
After that, return the mouse to prone position. Now make an incision from the top of the thigh to the achilles tendon. Separate the skin from the underlying muscles and be careful not to damage the blood vessels.
Next, identify the anterior edge of the biceps femoris, which appears as a white line running down the thigh. Then carefully open along the line from the knee all the way to the hip bone. Separate the muscles to expose the sciatic nerve under the biceps femoris.
Carefully dissect the biceps femoris and remove it. To expose the sciatic nerve and the tricep Siri muscles, identify the tibial nerve in between the common peroneal and sural nerves. Among the different branches of the tibial nerve, identify the branches innervating the tricep sury from the branches that go deeper.
Using eight OTT silk thread, tie all the branches of the tibial nerve together while keeping the branches innervating the tricep sury intact. Cut the tibial nerve as far as possible from the knot. Identify vertebrae T 13 and L one, whereas T 13 is the last vertebra to have ribs attached.
Then immobilize the vertebral column with the Cunningham spinal cord vertebral clamps on each side of T 13 and L one. And be careful not to compress the spinal cord, but apply a little bit of tension on the longitudinal axis. Make sure that the spinal column is well secured by pressing it down gently with forceps.
Using the fine R jurors, remove the spinal processes and then the laminate over T 13 and L one to expose the spinal cord. Then place a custom made plastic bath to surround the spinal cord. After that, secure the bath in place using a four aught silk thread.
Lastly, ensure that the bath is waterproof by sealing it with quick cast sealant. Once the quick cast has dried, remove the cotton covering the spinal cord and fill the bath with mineral oil. Use another vertebral clamp to clamp a spinous process of the sacral region.
In order to support the back region of the animal, use a third clamp to immobilize the right hind limb and ankle flexed at a 90 degree angle at the knee with the limb bent at 90 degrees at the knee and at the ankle. Remove the gauze covering the hind limb area. Dissect the achilles tendon from the surrounding tissue.
Cut the tricep from the surrounding tissue as much as possible using a threaded needle. Thread a six aught silk suture through the achilles tendon and make a triple knot around the tendon. Then place the force transducer close to the knot.
After that, attach the tendon to the force transducer using the silk thread, cut the distal part of the tendon. Next, insert two stainless steel wires under the fascia of the tricep sury muscles. These wires are connected to an extracellular AC amplifier.
For EMG recording, place the tricep sury nerve on the cathode of a hook electrode with the anode touching a nearby muscle. Then stimulate the tricep sury nerve using a 50 microsecond square pulse of increasing intensity at a low frequency until the maximum twitch amplitude is observed. Then stimulate the tricep sury nerve using a 50 microsecond square pulse of increasing intensity at a low frequency until the maximum twitch amplitude is observed.
These figures show how to identify a moto neuron from the tricep sury group. After penetration at low stimulation intensity, only a monos synaptic EPSP can be observed at higher intensity. The EPSP might be large enough to trigger an orthotropic spike at even higher stimulation intensity, and all or none.
Antidromic spike appears with a shorter latency than the monos synaptic EPSP. If enough current is injected through the micro electrode to trigger a spike. An EMG activity can be recorded on the muscle after a short delay followed by a muscle twitch.
After the identification of the motor neuron, its electrophysiological properties can be characterized. For example, this figure illustrates how the input resistance can be measured using hyperpolarizing and depolarizing pulses of current and plotting the variations of the membrane potential versus the amount of injected current. The contractile properties of the motor unit can also be studied using various protocols of stimulation.
For example, the force frequency relationship can be obtained by injecting short pulses of current at various frequencies in the motor neuron. The steady State force can then be plotted against the frequency of the current pulses to reveal a sigmoidal force frequency curve. Once mastered the surgery, part of this technique can be done in three hours if perfect.Properly.
