Principles:

         A single stimulus to a muscle produces a simple twitch.  The twitch is significant for study because it is this type of single contraction from which all normal body contractions are built.  The force exerted by the twitch is determined by the number of motor units (a neuron and its muscle cells) responding at the same time.  Increasing the voltage used to stimulate the muscle will increase the number of motor units contracting at the same time.  This is referred to as multiple motor unit summation.

     Temporal summation is produced by stimulating the muscle rapidly enough so that one contraction is not finished before another begins.  Thus, the effect of the second contraction is added to at least a part of the first contraction, and so on.  If the muscle is stimulated rapidly enough, succeeding contractions may come so close together that no relaxation occurs between contractions.  When this occurs, the smooth state of contraction known as tetany has been achieved.  The frequency of stimulus necessary for tetanization varies depending on the particular muscle.

     In addition, the initial length of the muscle affects the strength of contraction.  The muscle is strongest at its "natural" length.  If the muscle is either stretched beyond its normal length or has been allowed to shorten passively, its strength of contraction is reduced.  The "natural" length is that length at which the maximum number of crossbridges are formed between filaments in muscle cells.

Procedures:

Calibration of the force transducer :  Force transducers are devices capable of transforming force into a proportional electrical signal.  Changing the attachment point changes the full scale range of the transducer from 50g to 1000g.  The SS12A has 5 different attachment points that determine the effective range of the force transducer.  These ranges are 50g, 100g, 200g, 500g, and 1000g.  We will begin in the 500g range.

Using the preset function for force recordings
    1.  Pull down the MP30 menu and select Setup Channels
    2.  Check the Analog option.
    3.  Activate the appropriate channel
    4.  Click on the Acquire, Plot and Values options
    5.  Click on the Setup button to generate an "Input channels" setup window.
    6.  Click on the Presets button and scroll down to select Force.

Calibration:
    1.  Find the optimal gain setting:
        a) Start with the software Presets (in this case a gain of 100)
        b) Load the transducer with the maximal weight expected
        c) collect data for a few seconds at these settings
        d) inspect the sample data to be sure the data is not "railed" or "clipped", i.e. that the plot does not bottom out or top out, respectively.  Be sure that small weights can be detected and that larger ones don't result in a "clipped" trace.
        e) adjust the gain as needed.

    2.  Actual Calibration:
        a)  Access the Channel Scaling dialog box  (MP30 menu, Setup Channels, Setup, Scaling).
        b)  Place the maximum expected weight on the pan.
        c)  Click on the Cal 1 button, then enter the mass and units in the appropriate boxes.
        d)  Remove all weight from the pan.
        e)  Click on the Cal 2 button, then enter the mass and units in the appropriate boxes.

Pithing and Muscle Preparation.  The method for pithing the frog and preparing the frog gastrocnemius muscle will be described by the instructor.  The object of the pithing operation is to destroy specified parts of the central nervous system.  In this case the frog is pithed as a means of immobilization and anaesthesia.  Generally, pithing the spinal cord only is adequate.

     To skin the frog leg, cut the skin all around the upper end of the leg.  Pull the skin down the leg, freeing it from any underlying muscles.  The removal of the skin is an operation resembling the removal of a stocking.  The skin turns wrong-side out as it is stripped off.

     The muscle preparation desired consists of the lower half of the femur bone and the attached gastrocnemius muscle.  Other muscles and the tibiofibula should be cut away (note: birds, reptiles and amphibians possess a single bone in the lower leg, the tibiofibula, whereas mammals have separate tibia and fibula bones).  Cut the tibiofibula loose by cutting through the knee joint.  Retain as much of the Achilles tendon as possible.  This structure should be separated from the other tissues around and beneath the heel.  Tie a thread securely around the tendon after cutting it free from beneath the heel.  The muscle may now be manipulated by holding the femur and/or the thread tied to the Achilles tendon.  Keep the preparation moist with Ringer's solution  at all times.

The instructor will assist in setting up the stand for mounting the muscle preparation.   A femur clamp is attached to the lower part of the stand and the stub of the femur bone clamped in place.  The thread from the Achilles tendon is tied to a hook on the variable range force transducer, using as short a length of the thread as possible.   Final adjustments can be made by moving the position of the clamps.

    Electric connections to the stimulator should be made as shown in the figure below.  Good contacts are essential.

    Minimal Stimulus and Recruitment.  Determine the minimal stimulus (voltage) required to produce a contraction.    Gradually increase the voltage and stimulate the muscle (single pulse mode) and observe the force of contraction.  Continue until the maximal stimulus (maximum force of contraction) is achieved.  You should be able to determine some voltage value which gives maximal contraction with the voltage no greater than necessary.  This voltage setting will be used throughout the rest of the exercise, except as instructed otherwise.

    Summation and Tetany.  Shock the muscle first at 1 time per second, allow the preparation to rest for 15 sec. and then repeat at 2 times per second.  Record a response for no more than 5 seconds.  Do not overstimulate you will fatique the muscle.  Repeat this procedure at 5 and 10 times per second.  You should observe summation, or an increased shortening of the muscle due to "overlap" of contractions.  Shock the muscle at 25 times per second and at 50 times per second.  Normally, tetany will occur at about 20-35 contractions per second.

     Fatigue and Contracture.  Set the frequency of stimulation control to one shock per second and record the muscle contractions.   Let this run for as long as any significant developments are occurring (5-10 minutes).  Interpret this tracing in terms of fatigue and contracture. Would the appearance of contracture be influenced by the amount of weight the muscle was lifting?  Explain.

Questions for Thought:

1.  If you had arranged your muscle preparation so that you stimulated the nerve leading to the muscle rather than the muscle directly, how would your results have been different?  Or would they have been?  Where would fatigue have occurred first?

2.  Define: Motor Unit, Summation, Tetany, Fatigue, and Maximal Stimulus.  What are the different types of summation?

3. Why does increasing the voltage and the frequency cause the muscle to contract more forcefully?