An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

The overall goal of this procedure is to study muscle spindle NT activity in vitro in an adult mouse. This is accomplished by first dissecting the extensor digitorum longest muscle and deep perineal nerve and placing them into a perfused tissue bath. The second step is to affix the muscle to a force and length controller and place a glass suction electrode onto the cut end of the nerve to record muscle afferent activity.

Then the recorded ENT is identified as a muscle spindle afferent by looking for a pause in afferent firing during twitch contraction. The final step is to record the responses of the identified muscle spindle afferent to ramp and hold stretches. Ultimately, this in vitro preparation can be used to study the effect of a perturbation, such as a disease model on muscle spindle afferent response properties.

The main advantage to using the in vitro preparation to study muscle spindle afferent is that it gives you precise control over the muscle environment and can eliminate the potential confounds of anesthesia and muscle perfusion status. Demonstrating the Dissection will be Remi manway an undergraduate student from the laboratory After anesthetizing and decapitating an adult mouse. Remove the internal organs and the skin as described in the text protocol.

Next, remove the legs by cutting above the hips and placing the skin to legs into a dish with chilled low calcium high magnesium saline solution. After arranging the legs dorsal side up in the dish, place one needle in both ends of one foot and one needle on the anterior and posterior thigh. Then pin out the other foot and thighs in a similar manner.

When finished, the knee and ankle joints are at a 90 degree angle. Next, under a dissecting scope, lift up the top layer of muscle on the thighs. Use gastro Viejo spring scissors to make a midline cut.

To expose the sciatic nerve directly below. The sciatic nerve is located just below the top muscle layer and runs above the femur from its exit near the hips until it branches into the common perineal and tibial nerve just before the knee joint. Then remove the muscle above the sciatic nerve to expose the point at which the deep perineal nerve branch dives into the flexor hallis longus or FHL muscle.

Using number 55 forceps, dissect the connective tissue around the perineal branch to free it from the gastro and sous muscles, which are on the medial side of the tibia. Cut the tendons of the gastro and soleus muscles and carefully remove them from under the nerve. Next, remove the superficial muscle on the lateral side of the tibia to expose three distinct tendon bundles at the ankle joint from medial to lateral the FHL, the extensor digitorum longus or EDL and the tibias anterior or ta.

Note that the EDL is hidden underneath the ta. Now cut the TA tendon at the ankle joint. Lift the TA up and away from the EDL and cut the muscle near the knee to remove it.

Then cut the FHL tendon at the ankle and lift it back to reach the area where the nerve enters the FHL. Cut just below that point and remove approximately two thirds of the FHL muscle using large spring scissors. Cut the EDL tendons at both the ankle and knee joints.

Next, cut the sciatic nerve as close to the hip joint as possible, and gently strip away all nerve branches except for the deep perineal branch. Then using sharp scissors, cut through the tibia bone at the knee and cut midway through the thigh so as to remove the EDL, the remaining FHL and the nerve from the surrounding tissue. Finally, cut away the remaining tibia bone so that just the EDL part of the FHL and the nerve remain.

The nerve is quite tiny and easy to pull from the muscle. Leaving part of the FHL muscle gives you something to manipulate and helps to protect the EDL and the nerve. The tissue bath used for the experiment is a commercially available bath 25 milliliter capacity.

Two stimulating electrodes are fixed to the bottom of the bath. There is a mounted tissue post and amount for a force in length controller. Begin profusing the bath with oxygenated synthetic interstitial fluid at a flow rate of 15 to 30 milliliters per minute.

Place a small piece ofs guard on the bottom of the dish and insert an insect pin through the remaining FHL tissue to stabilize the muscle. Then transfer the muscle nerve preparation into the tissue bath to facilitate attaching the muscle. In a later step, bend a small piece of wire into a JS shaped hook and use epoxy to fix it to the lever arm of the force and length controller.

Now tie a six oh silk suture around each tendon affix one suture to the tissue post and the other suture to the JS shaped wire of the lever arm. Remove the insect pin and sal guard after tying the tendons. Next, select the glass suction micro electrode with an inner tip diameter of 10 to a hundred micrometers, and fill it with enough synthetic interstitial fluid to touch the inner silver wire.

Then suction the cut end of the nerve into the electrode. Next, connect the suction electrode to the positive port of a differential amplifier. Wrap the electrode with a chloride silver wire that connects to the negative port of the head stage.

Ground the profusion bath by running a second chloride silver wire from the bath to the head stage's ground port. Also ground the profusion tubing to the Faraday cage at multiple points to mitigate electrical noise introduced via the perfusion pumps. Begin stimulation of the muscle by mounting electrodes on either side of the muscle in the tissue bath to induce a twitch contraction.

Increase the stimulating voltage until a peak contractile force is observed, and then increase the voltage by an additional 15%to reach supra maximal voltage. Continue the twitch contractions at the SRA maximal voltage with a ten second rest in between, but vary the length of the muscle until a peak contractual force is reached. To find the optimal length or L knot of the muscle to collect data at a temperature other than room temperature.

Place a temperature probe into the tissue bath near the muscle. Slowly increase the bath temperature by pumping heated water through the tissue bath base plate. Wrap clay microwavable heating pads around the synthetic interstitial fluid reservoir to help maintain a steady temperature.

At this point, wait an hour to allow the tissue to reach the bath temperature or for normal synaptic transmission to recover. Following the dissection in the low calcium solution. To identify an afferent as a muscle spindle, afferent deliver a 0.5 millisecond super maximal voltage stimulus once every second to induce repeated twitch contractions.

If the neuronal activity pauses during the twitch contraction, this indicates that the recording is likely from a spindle afferent. Next, use data acquisition software to apply length changes at different speeds and to different lengths. A custom script is available for download from the JO website To automate this task, apply ramp and hold stretches for four seconds from L Knot to stretch the muscle to an additional 2.5%5%or 7.5%of L knot.

Use stretch speeds of 2040 or 60%L knot per second. At the end of the experiment, determine the muscle health at 24 degrees Celsius using maximal isometric tetin contractions as described in the text protocol, neuronal activity and muscle tension from 30 twitch contractions are superimposed in this image. The afferent activity pauses during a contraction induced tension increase, which is a characteristic response of muscle spindle ens shown here is the raw neural activity of two ens during a ramp and hold stretch apply to the EDL.

The spike histogram feature of lab chart is used to identify the spikes as belonging to two separate ens. The afferent indicated in blue had a baseline firing rate and increase its response during the stretch. The RIN activity in orange had no activity at rest, but did respond to the stretch.

The instantaneous firing frequency of the blue coated unit exhibiting activity at resting length is shown here. The instantaneous firing frequency of the smaller orange coated unit that only fires during the stretch is shown here. Note that both units exhibit the spike frequency adaptation during stretch that is characteristic of muscle spindle ens.

After watching this video, you should have a good understanding of how to record spindle ENS activity using an in vitro preparation. This procedure can be used to study the response of muscle spindle ENS to a perturbation like injury or disease. It can also be easily modified to study additional subtypes of muscle sensory ens.