The overall goal of this procedure is to study neuromuscular junction functionality by means of an ex vivo experimental approach. This is accomplished by stimulating the muscle nerve preparation in two ways, directly on the muscle membrane and through the nerve. Since membrane stimulation bypasses neuro transmission signaling, any differences between the two contractile responses can be considered as an indirect measurement of neuromuscular junction functionality.

Here we present this procedure in soleus sciatic nerve preparation. The muscle is dissected together with its nerve and placed into a perfused tissue bath. It is fixed to a force and length controller and a platinum pair of electrodes are placed parallel to the muscle.

A glass suction electrode is then moved close to the cut end of the nerve. A comprehensive testing protocol is then applied to thoroughly evaluate both neuromuscular junction and muscle functionality. The functional connection between muscle and nerve is conclusion for both part and two surviving faction.

In the first region of the level of each of the two tissue communicate that these neuromuscular junction, which normally displays a pressure like morphology. Nevertheless in several pathological conditions the functionality interplayed between muscle and nerve is severely compromised and neuromuscular junction lose the complex morphologic organization. The overall goal of our procedure is to study neuromuscular junction functionality by using and ex vivo experimental approach.

This is accomplished by stimulating the muscle nerve preparation in two ways. One by directly stimulating the muscle membrane and the other by stimulating the nerve and analyze the muscle properties. Turn on the circulation water bath and adjust the temperature to 30 degrees celsius.

Fill the bath with the Krebs-Ringer solution. Allow O point four bar of gas mixture to flow through the oxitube and into the bath. Turn on the actuator transducer and the two pulse stimulators.

Set the current values to 300 million pair for the membrane stimulation and to five million pair for the nerve stimulation. After sacrificing the mouse by cervical dislocation, remove the skin from the legs. Now cut the achilles tendon and while tightly clamping the tendon, pull the gastrocnemius muscle and the soleus together upwards.

Once the proximal tendon of the soleus is exposed cut the entire calf above it and quickly place the sample in the preparatory tissue bath located under the stereo microscope. Using a pair of forceps, tightly clamp the proximal tendon of the soleus and gently pull it to expose the sciatic innervation. Once the innervation is exposed remove the surrounding tissues to reveal about five millimeters of the nerve.

Then use a fine pair of scissors to carefully cut the nerve. Complete the muscle, nerve excision by cutting the achilles tendon to separate the soleus from the gastrocnemius. Now the muscle, nerve preparation is ready to be mounted onto the testing apparatus.

Create a slip knot at the end of the nylon thread and tighten it around the achilles tendon. Clamp the proximal tendon within the fixed clamp and tie the nylon wire around the lever arm of the force transducer. Allow the muscle to equilibrate in the solution.

To determine the initial optimal length, stimulate the muscle with a series of single pulses while gently changing the creep load value. The optimal length is obtained when the twitch force is maximum. Place the suction electrodes near the muscle and pull the nerve in.

Then, while gently altering the pulse current value, stimulate the muscle with a series of single pulses. The twitch force generated by the muscle when stimulated through the nerve should be equal to the values measured when stimulating it on the membrane. Once the optimal current value is determined, push the nerve out of the electrode and deliver a few current pulses.

If the amount of previously selected current is excessive, the current pulses delivered through the suction electrode elicit the muscle contraction by conducting current through the bath. Using a home-made software we devised an automated testing protocol for the study of soleus neuromuscular junction functionality. The protocol lasts about 65 minutes and is made up of four different parts.

In the first part, the muscle is stimulated with four single pulses. Two delivered directly and two through the nerve. Time to peak, half relaxation time, maximum value of force derivative and twitch force are then measured from the twitch responses.

In the second part, the muscle is stimulated with a series of pulse trains ranging from 20 hertz to 80 hertz, which is the tetanic frequency. To compute the force, frequency curves for both nerve and direct stimulations. In the third and fourth parts of the protocol the muscle is subjected to two fatigue paradigms to measure neuro transmission failure and intra tetanic fatigue.

During these fatigue paradigms, the muscle is continuously stimulated with one pulse train delivered on the membrane followed by 14 pulse trains delivered through the nerve. The entire sequence is repeated 20 times. The first paradigm is delivered at a firing frequency of 35 hertz.

The second at the tetanic frequency of 80 hertz. Neuro transmission failure is believed to play an important role in the development of fatigue as it is related to the external block of action potential propagation, decreased transmitter release and decreased and plate excitability of junction fatigue ability. Another aspect of neuromuscular junction fatigue ability is clearly expressed by intratetanic fatigue which is an estimate of the muscle's ability to maintain force during a single tetanic contraction and reflects high frequency fatigue.

At the end of the protocol, net muscle length and weight are measured using an analog caliper and a precision scale to compute the muscle cross sectional area. Studies on the SOD1 transgenic mouse model of amyotrophic lateral sclerosis have highlighted the potential of this methodology. In fact, the transgenic soleus muscles yield a reduced contractile response for both the force derivative and tetanic force when directly stimulated and an even greater reduction when stimulated through the nerve.

As regards to tetanic force for example, these experiments have shown that muscle contractility accounts for 25%of the damage while a further 45%is related to defects in neuro transmission. Another interesting point is the absence of any difference in control muscles when stimulated directly or indirectly. This finding proves that the methodology does not induce any technical artifacts since the neuromuscular junction is expected to be fully functional in control animals.

As regards intratetanic fatigue, the results have shown significantly lower values in transgenic soleus muscles than in their control counterparts. Interestingly, transgenic soleus muscle is significantly damaged by repetitive stimulation, which means that neuromuscular junction functionality can be evaluated for a maximum stimulation time of eight minutes. After eight minutes, the transgenic muscle returns to an almost null value of force when stimulated.

After watching the video you should have got understanding how to measure neuromuscular junction functionality and soleus muscle of mouse. Given that this technique based on indirect measurement of neuromuscular junction functionality, it does not allow to be made where reported defects are related to morphologic or biochemical changes. On the other hand, this approach represents an essential way to assess whether these aggressions affect at user functionality of the neuro transmission signal.

Finally, the protocol proposal can be easily adopted to measure neuromuscular junction functionality of the diaphragm, another muscle often involved in pathological diseases.