Frog Gastrocnemius muscle/sciatic nerve preparation
Double pith a frog. Remove the skin from the lower part of the animal as before. Then tie a ligature around the lower part of the Gastrocnemius muscle, and cut the muscle away from the foot leaving as much of the Achilles tendon as possible. Clear the thigh muscles away from the sciatic nerve and put a thread around the nerve for later use. Do not tie the thread tight just yet.
1. Hardware
Set up the transducer and Biopac data collection system/Mac as instructed. Familiarize yourself with the software and hardware. Follow the directions in the Student Lab Pro manual, page E13. Further information may be obtained from Chapter 3 (Page B5). Calibrate the transducer with a 100g weight. It is probably easiest to use the Autosave mode in the Aquisitions setup. This will save your trials to the hard drive without having to name each one.
2. Stimulation of the sciatic nerve
Do not cut the muscle away from the animal just yet. Stimulate the nerve with stimulating electrodes looking for the lowest current that will produce a perceptible twitch in the muscle. Use the oscilloscope to determine the voltage levels as before. This is the threshold voltage for this nerve/muscle preparation.
Q. How does this threshold compare with the AP threshold from the Sciatic nerve lab?
Gastrocnemius muscle clamped for the examination of contractions.
Prepare the muscle for the transducer system by cutting the femur around the hip and securing the bone in a femur clamp. Fix the other end (with the thread) to the spring of the transducer and adjust the position until slight tension is placed upon the muscle.
3. Effects of stimulus strength applied directly to the muscle.
Set the stimulator to 1msec pulses. Touch the two electrodes to the muscle about midway between the knee and the Achilles tendon. Do not allow the the electrodes to touch one another. Use one leaf on the transducer. Stimulate with a single pulse. Slowly turn up up the stimulus strength until the first perceptible twitch occurs. This is the threshold voltage. Gradually increase the stimulus strength and record the twitches obtained. Continue until no further increase in twitch height occurs. You may have to adjust the scale of the deflection to get it to register on scale at the highest level. Do not exceed this level by very much or you risk damaging the muscle. Do not forget to make notes as you go along. You will want to go back and analyse this data later so it is important to know which file contains which treatment.
Q. How does direct stimulation of the muscle produce contractions?
Q. Low intensity shocks produced no contractions. Why not?
Q How did the threshold for direct stimulation compare to the threshold for sciatic nerve /contraction stimulation?
4. Effects of stimulus frequency.
Stimulate the muscle with a supra-threshold voltage long enough to obtain ten twitches at the following frequencies; 0.5/sec, 1/sec, 2sec, 3/sec, 4/sec and so on until tetanus is reached. Allow about 15 seconds between trials to give the muscle a rest.
5. Effects of load (or stretch) on muscle contractility.
Swing the remaining force leaves into position so that the transducer allows for isometric contraction measurement. The muscle contracts isometrically and this transducer measures the tension produced. Use the tension adjuster (on the clamp post) to increase the tension on the muscle until a supra-threshold stimulus produces a small pen deflection. Record this as 0mm of stretch. Using the tension adjuster, increase the stretch on the muscle by 1mm increments (use a slow paper speed), and stimulate the muscle. Allow at least 10 seconds on relaxation time between the stimulation and the next stretching. KEEP THE PREPARATION MOIST WITH RINGER'S DURING THE ENTIRE EXPERIMENT. Continue until no response of the muscle is noted.
You may have to keep changing the position of the trace to keep it visible on the computer screen.
Plot the relationship of total tension (load) against length. Also plot the passive and active tensions against length. Passive- the distance between the the baseline and the pre-contraction (after stretching) level. Active- the distance between the pre-contraction level and the post-contraction level.
Q. At what length does the muscle have the greatest active tension? At what stretched length is the active tension reduced to zero? What would you predict is the resting length of the frog gastrocnemius muscle?
Save your data to floppy discs so that you can analyse it later.