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In this experiment, you will investigate the physiological properties of skeletal muscle using the isolated
frog gastrocnemius. You will explore the single twitch, the graded response, the relationship between
muscle length and tension, muscle tetanus, and muscle fatigue. These experiments illustrate the
collective understanding of muscle physiology gained from over 400 years of research.
Background
The frog muscle preparation you will use in the laboratory is the earliest isolated tissue preparation. The
first experiments on muscle physiology appear to have been performed between 1661 and 1665 by Jan
Swammerdam, who demonstrated that an isolated frog muscle could be made to contract when the
sciatic nerve was irritated with a metal object. Later, Luigi Galvani (1737-1798) demonstrated that frog
muscle responded to electrical currents. This experiment focuses on the mechanical properties of
skeletal muscle. The invention of the kymograph (a rotating drum powered by a clockwork motor) in the
late 1840s, attributed to either Carlo Matteucci (1811-1868) or Carl Ludwig (1816-1895), revolutionized
experimental physiology for it enabled events such as muscle contractions to be recorded and analyzed
for the first time. Today, the computer has taken the place of the kymograph but physiology students of
the late 1800's would recognize these experiments. These demonstrate some of the important functional
characteristics of skeletal muscle.
The basic unit of a muscle is the muscle cell, or fiber. Whole muscles are made up of bundles of these
fibers. Unlike cardiac muscle cells, there are no gap junctions between adjacent cells. This means each
fiber behaves independently. A single muscle fiber has a very regular structure, defined by myofibrils
(Figure 1). 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 calcium ions (Ca2+) and ATP.
Skeletal muscle can be studied under isometric (constant length) or isotonic (constant load) conditions.
Here the force is measured isometrically. Action potentials in skeletal muscle, like those in nerve, last for
only a few milliseconds. In contrast the mechanical response of the muscle the muscle twitch last
significantly longer (Figure 4).
Figure 4. Temporal Relationship between Muscle Action Potential and Consequent Contraction
A second stimulus arriving before the muscle has relaxed again causes a second twitch on top of the first
so that greater peak tension is developed. This is called summation. With increasing frequency of
stimulation, there is less and less time for the muscle fiber to relax between stimuli, and eventually the
contractions fuse and a smooth powerful contraction tetanus is seen. Normally skeletal muscles are
activated by volleys of action potentials and operate in a state of fused contractions.
Required Equipment
LabChart 7 software
PowerLab Data Acquisition Unit
Bridge Pod
Force Transducer
Small weight between 550 grams
Ring Stand
Manipulator/Micropositioner and clamps
Strong thread
Petri dish
Pasteur pipette
One frog (Rana pipiens or Xenopus laevis)
Normal Frog Ringers solution
Cold (10 oC) Frog Ringers solution
Small millimeter ruler
Tape
Medium-sized beaker
Dissection tools:
o Glass fingerbowl
o Sharp scissors or scalpel
o Bone shears
o Blunt probe
o Dissection tray with wax or pad
o Dissection pins
Muscle Stimulation Equipment: You need one of the lists below (consult your instructor for more
information)
o LIST 1
Stimulator Cable (BNC to Alligator Clips)
Muscle Holder
o LIST 2
Animal Nerve Stimulating Electrode
Ring Stand clamp for the electrode
Femur clamp
Procedure
Equipment Setup and Calibration
1. Make sure the PowerLab is turned off and the USB cable is connected to the computer.
2. Connect the Force Transducer cable to the back of the Bridge Pod. Connect the Bridge Pod to Input
1 on the front panel of the PowerLab (Figure 7). Connect the Stimulating Electrodes to the output on
the front panel of the PowerLab. Follow the color scheme in Figure 7.
5. Ask your instructor whether you are using a Muscle Holder or femur clamp to secure the muscle.
Proceed to the proper setup after you calibrate the Force Transducer and dissect the frog.
6. Turn on the PowerLab.
Frog Dissection
1. Obtain a double-pithed frog from your instructor.
2. Use sharp scissors or a scalpel to cut the skin of the frog around its abdomen. Peel the skin down
and off the legs of the frog (Figure 11).
3. Remove the leg from the frog by severing at the hip joint. Carefully dissect the gastrocnemius
muscle away from the tibiofibula bone, but leave it attached to the knee and heel.
4. While the muscle is still attached, pass a 15 centimeter piece of strong thread under the Achilles
tendon at the heel of the frog. Tie this thread securely to the tendon (Figure 12). You will use this
thread to attach the muscle to the Force Transducer.
5. Rotate the base of the Muscle Holder and insert an appropriately-sized beaker to collect waste
solution. Rinse the muscle with regular Ringers solution to keep it moist.
6. Make sure the silver wires on the Muscle Holder are still in contact with the muscle. Check that
everything is connected correctly.
Note: For this exercise, you will be running another macro to set up the Stimulator and record the data.
Do not start recording before playing the macro.
6. Go to the Macro menu and select Muscle Tension to start the macro. Alternatively, you can press
F3. LabChart will start recording and will prompt you with messages. Follow the on-screen
instructions to record data.
7. After each recording, there will be a prompt to raise the Manipulator/Micropositioner by one
millimeter. Before doing so, wait 30 seconds to allow the muscle to recover. Turn the adjustment
knob at the top to raise the instrument. Do not try to reposition the entire unit on the Ring Stand.
During the exercise, the Manipulator/Micropositioner will increase a total of 10 millimeters.
8. At the end of the macro, immediately return the Manipulator/Micropositioner to its original position
and release the tension on the muscle by turning the adjustment knob.
9. Save your data. Do not close the file.
10. Wait at least two minutes before moving on to the next exercise to give the muscle time to recover.
Make sure you keep it moist with Ringers solution.
1. Make sure the muscle is moist and the electrodes are still positioned correctly.
2. Zero the Bridge Pod as before, but do not calibrate the data.
3. Click on the data at the end of the last data block, and go to the Commands menu and select Add
comment. Type exercise 4 and click Add.
Note: For this exercise, you will be running a different macro to set up the Stimulator and record the
data. Do not click Start before playing the macro.
4. Go to the Macro menu and select Tetanus to start the macro. Alternatively, you can press F5.
LabChart will start recording and will prompt you with messages.
5. Follow the on-screen instructions. The PowerLab will stimulate the muscle for one second with
repetitive pulses at intervals of 400 ms, 200 ms, 100 ms, 50 ms, and then 20 ms. Each recording will
appear in a separate block.
6. When the macro has finished, add a comment to each block of data with the stimulus interval as
indicated above. Click on the data at the beginning of a data block, and go to the Commands menu
and select Add comment. Type the stimulus interval and select the comment to be inserted at the
selection.
7. Save your data. Do not close the file.
8. Wait at least 30 seconds before moving on to the next exercise. Make sure you keep the muscle
moist with Ringers solution.
Analysis
Exercise 1: Twitch Recruitment
1. Examine the data in the Chart View. Autoscale, if necessary. Up to 20 contractions should be seen
in the Force channel, each in a separate data block.
2. Place the Marker on the baseline of the waveform in the Force channel.
3. Place the Waveform Cursor at the top of the last contraction peak.
4. Record the peak height for each of the peaks in Table 1 of the Data Notebook, starting at the last
point and working backwards to the beginning of the trace. Do not move the Marker, only the
Waveform Cursor. Fill out Table 1 starting at the bottom.
Note: The PowerLab will have stimulated the muscle 20 times, but not all of the stimuli may have elicited
a twitch. If there are fewer than 20 contractions, enter a zero in Table 1 for those stimulus intensities
without a twitch.
t
t
t
t
t
t
t
=
=
=
=
=
=
=
1s
5s
10 s
15 s
20 s
25 s
30 s
Data Notebook
Exercise 1 Observations
a) Describe the contractile force of the muscle when the stimulus strength was increased.
Supramaximal Stimulus
Voltage (V)
Raw Twitch
Force (N)
Net Twitch
Force (N)
Block 1
(Reference Point
___ mm)
Block 2
(___ mm)
Block 3
(___ mm)
Block 4
(___ mm)
Block 5
(___ mm)
Block 6
(___ mm)
Block 7
(___ mm)
Block 8
(___ mm)
Block 9
(___ mm)
Block 10
(___ mm)
Force of First
Peak (N)
Force of Second
Peak (N)
Contractile
Force (N)
200
100
50
20
Maximum Force
(N)
Contractile Force
(N)
5
10
15
20
25
30
Study Questions
1.
In light of the all or none law of muscle contraction, how can you explain twitch
recruitment (also called the graded response)?
2.
What effect does stretching the muscle have on contractile strength? Is this effect
linear? What preload force resulted in the highest contractile force?
3.
What effect does varying the stimulation frequency have on contractile force? Which
stimulus interval caused the greatest contractile force?
4.
5.
At what time point did your muscle begin to fatigue? Calculate the percent decrease in
contractile force by comparing the force at the end of the experiment with the
maximum contractile force.
6.
In your own words, explain a possible mechanism for why the muscle was unable to
maintain a prolonged contraction in Exercise 5.