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LAB 12: MUSCLE PHYSIOLOGY
The basic unit of a muscle is the muscle cell, or fiber. Whole muscles are made up of bundles of these fibers. A single muscle fiber has a very regular structure, defined by myofibrils. Each myofibril consists of an arrangement of the contractile proteins actin and myosin, which are able to slide past each other in the presence of Ca2+ and ATP.
Skeletal muscle is similar to nerve tissue in that it responds to a stimulus in an all-or-none fashion. This response is called a twitch. Depending on the intensity and frequency of stimulation, greater numbers of fibers are activated. By increasing the number of active muscle fibers, the muscle is able to increase the force it generates. Muscles with large cross-sectional areas are able to generate larger forces than those with small cross-sectional areas.
The muscle fibers and their associated motor nerves are called a motor unit. Greater numbers of motor nerves associated with a muscle give finer control over that muscle. Motor nerves release the neurotransmitter acetylcholine from their synaptic bulbs onto the muscle. This junction between a nerve and a muscle is called the motor end plate.
The release of acetylcholine at the motor end plate depolarizes the muscle tissue and in turn leads to the release of intracellular calcium from the sarcoplasmic reticulum, a variant of smooth endoplasmic reticulum. This release of intracellular calcium sets in motion the biochemical events that allow actin and myosin to move past each other and hydrolyze ATP for energy.
In this experiment, you will examine the basic principles of skeletal muscle physiology, including the all-or-none response, the effect of stimulus intensity and frequency on contraction force and the phenomenon of muscle fatigue. These experiments illustrate the collective understanding of muscle physiology gained from over 400 years of research.
Pre-Lab Questions:
1. Where is the gastrocnemius muscle and sciatic nerve in the frog and in you?
2. How does an action potential move in a bundle of neurons like the sciatic nerve?
3. What is the function of the PowerLab hardware and software
4. What type of signal does the Powerlab hardware receive?
5. What is tetanus?
Procedures
A. Frog Dissection
1. Kill a frog as described or demonstrated by your instructor. 2. Using a scalpel or sharp scissors, cut the skin of the frog around its abdomen. 3. Peel the skin down and off the legs of the frog. 4. Keep the tissue moist at all times with Ringer’s solution. 5. Use the glass probe to expose the sciatic nerve from between the thigh muscles down to the knee. Be careful not to handle the sciatic nerve with metal instruments. 6. Slip a 10” piece of strong thread under the Achilles tendon at the heel of the frog. Tie this thread securely to the tendon. 7. Carefully dissect the gastrocnemius muscle away from the tibio-fibula bone, but leave it attached to the knee. 8. Remove the foot, thigh muscles, and the other muscles from the tibio-fibula, leaving the sciatic nerve and gastrocnemius attached at the knee. 9. Using bone shears or strong scissors, cut the tibio-fibula bone 8-10 mm below the knee. 10. Cut the femur close to the hip joint. 11. Keep the muscle in a Petri dish of cold Frog Ringer’s until you are ready to mount it. B. Mounting the muscle for recording
1. Securely mount the MLT500 force transducer in the positioner mounting bracket. Plug the MLT500 cable into the back of the ML301 Bridge Pod. 2. Connect the Bridge Pod to the Pod Port on Input 1 of the PowerLab. 3. Mount the bipolar stimulator handle to the ring stand using a clamp. 4. Clamp the femur of the frog to the ring stand using a femur clamp or strong metal clamp. 5. Attach the thread through the hole in the metal tab of the force transducer. Make sure that there is some slack in the thread. 6. Adjust the angle of the positioner so that the gastrocnemius is perpendicular to the table (Figure 2). 7. Raise the positioner so that the muscle is vertical but not under tension. The thread should not be loose, but have a slight amount of slack in it. Make sure that there is room to increase the height of the force transducer by at least 10 mm. 8. Rinse the muscle with Ringer’s solution to keep it moist. 9. Position the bipolar stimulator so that both stimulus leads are touching the middle of the muscle. 10. Attach the BNC +/- leads from the stimulator handle to the Output (+/-) connectors on the front of the PowerLab.
Figure 2. Set-up of the mounted gastrocnemius muscle.
C. Setting up the software
1. Make sure the computer is running and turn on the PowerLab.
2. Launch Chart 5 from your computer.
3. You will only be using one channel so turn off and minimize the window for the inactive channel.
4. Open the stimulator and the output for a duration of 1ms, delay of 100ms, pulse mode. You will have to determine the output level (amplitude) starting at low levels.
Exercise 1: The Graded Response
In this experiment, you will give the muscle a series of stimuli of increasing amplitude to examine the effects of stimulus amplitude on contraction force.
1. Make sure that the muscle and nerve are moist and that both leads from the stimulator are touching the nerve without shorting themselves out or stretching the nerve too much.
Exercise 2: Summation, the effect of pulse frequency on contraction force
In this experiment, you will stimulate the muscle with twin pulses at different pulse intervals and observe their effect on muscle contractions.
1. Make sure that the muscle is moist and the stimulator leads are positioned correctly.
2. Set up the stimulator to deliver twin pulses at decreasing intervals between the pulses, something like 400ms, 200 ms, 100ms, 50 ms and 20 ms apart.
Exercise 4: Tetanus
In this part of the experiment, you will examine the muscle’s response to a continuous stimulus at different frequencies.
1. Make sure the muscle is moist and the stimulator leads are positioned correctly.
2. Set the stimulator to continuous pulse for one second and select different pulse intervals, something like pulses at intervals of 400 ms, 200 ms, 100 ms, 50 ms and 20 ms. Continue until you can demonstrate tetanus and then fatigue.
Study Questions
1. In light of the “all or none” law of muscle contraction, how can you explain the graded response?
2. What effect does stretching the muscle have on contraction strength? Is this effect linear? What preload force resulted in the highest contraction force?
3. What effect does varying the stimulation frequency have on contraction force? Which stimulus interval caused the greatest contraction force?
4. Define tetanus. At which stimulus interval did you observe tetanus?
5. At what time point did your muscle begin to fatigue? Calculate the % decrease in contraction force by comparing the force at the end of the experiment with the maximal contraction force.
6. In your own words, explain a possible mechanism for why the muscle was unable to maintain a prolonged contraction in this experiment. .
Materials
5+ computers loaded with Chart 5 software 5+ Powerlab modules MLT500/A Force Transducer MLT301 Bridge Pod Bipolar stimulator electrodes MLA40 Ring Stand with micropositioner Ring stand clamps Femur clamp Dissecting tools (scalpel, scissors, forceps) Frog Ringer’s solution Petri dish Glass rod for blunt dissection Strong nylon thread Double-pithed frog or toad Small millimeter ruler Pins for anchoring knee to pan
References:
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