Muscle Protocol

Muscle
In this experiment, you will explore how muscles work. You will also examine some of the properties of muscle fatigue. In this experiment, you will electrically stimulate the nerves in the forearm to demonstrate recruitment, summation, and tetanus.

Written by staff of ADInstruments.

Background
The skeleton provides support and articulation for the body. Bones act as support structures and joints function as pivot points. Skeletal, or striated, muscles are connected to the bones either directly or by tendons, strong bundles of collagen fibers. Two or more muscles usually work antagonistically. In this arrangement, a contraction of one muscle stretches, or elongates, the other. Skeletal muscle is composed of long, multinucleate cells called fibers. These fibers are innervated by motor nerves. An action potential in a motor axon produces an action potential in the muscle fibers it innervates. This muscle action potential allows for a brief increase in the intracellular concentration of calcium ions ([Ca2+]), and activates the contractile molecular machinery inside the fiber. The result is a brief contraction called a twitch.

A whole muscle is controlled by the firing of up to hundreds of motor axons. These motor nerves control movement in a variety of ways. One way the nervous system controls a muscle is by adjusting the number of motor axons firing, thus controlling the number of twitching muscle fibers. This process is called recruitment. A second way the nervous system controls a muscle contraction is to vary the frequency of action potentials in the motor axons. At stimulation frequencies of less than 5 Hz, intracellular [Ca2+] returns to normal levels between action potentials: the contraction consists of separate twitches. At stimulation frequencies between 5 and 15 Hz, [Ca2+] in the muscle has only partly recovered when the next action potential arrives. The muscle fiber produces a pulsing tension called a summation response with a force greater than that of a single twitch and that does not decay completely to zero between pulses. At even higher stimulation frequencies, the pulsing component becomes hard to discern and the muscle enters tetanus, a smooth contraction many times stronger than that in a single twitch.

In Exercise 1, you will observe muscular responses without recording them. In Exercises 2 and 3, you will use a force transducer to measure small forces generated by the adductor pollicis muscle. In the later exercises, the grip force exerted by the hand is recorded with a different transducer as you investigate the phenomenon of muscle fatigue.

Required Equipment
A computer system
Chart software, version 5.0 or later
PowerLab 4/20T
Finger pulse transducer
Bar stimulus electrode
Electrode cream
Hand dynamometer
Adhesive tape
Procedures
Warning
Some of these exercises involve application of electrical stimulation to muscle through electrodes placed on the skin. People who have cardiac pacemakers or who suffer from neurological or cardiac disorders should not volunteer for those exercises. If the volunteer feels major discomfort during the exercises, discontinue the exercise and consult your instructor.
A. Set up and calibration of equipment
1. Make sure the PowerLab is connected and your computer is turned on.
2. Connect the finger pulse transducer to the BNC socket on Input 1 of the PowerLab.
3. Place the finger pulse transducer diaphragm-side up on the top of the lab bench; tape the transducer in place along the Velcro strap (Figure 1).
4. Connect the bar stimulus electrode to the isolated stimulator output of the PowerLab. The leads are color-coded; plug the red lead into the red socket and the black lead into the black socket (Figure 1).
5. Place a small amount of electrode cream on the two silver contacts of the stimulating bar.
6. Turn on the PowerLab.
7. Locate Chart on your computer and launch the program. If the Experiments Gallery dialog box does not appear in front of the Chart window, choose the Experiments Gallery… command from the File menu.
8. In the Experiments Gallery dialog box, select Muscle from the left-hand list. Select the settings file “Nerve Effect Settings” in the right-hand list and click the Open button to apply those settings.
9. After a short time, the Chart window on the computer screen should be set up for the first exercise. Only one channel should be showing, Channel 1, and there should be a red cross through the Record/Monitor button next to the Start button at bottom right of the Chart window.
Exercise 1: The effects of nerve stimulation
Objectives
To explore the motor and sensory effects of electrical stimuli on a student volunteer, using the nerves of the forearm.
Procedure
In this first exercise, the PowerLab unit is used as a stimulator, but instead of recording, you will simply observe muscular responses by watching the hand of the volunteer. The finger pulse transducer is not used until Exercise 2. Chart is active only to control the Isolated Stimulator.

1. Choose the Isolated Stimulator command from Chart’s Setup menu (Stimulator in Mac OS), and in the mini-window that appears, enter the following settings:
Frequency: 1 Hz
Pulse Duration: 200 µs
Amplitude: 10 mA
On button: highlighted

2. Turn the stimulator switch OFF on the front of the PowerLab (the switch should be pointing down). This disconnects the Isolated Stimulator terminals.
3. Click Start. Chart has been set up to monitor, not record. In this state, the Isolated Stimulator is active, but no recording is made (the data display area is grayed out to indicate that any signal appearing there is temporary). Observe the Isolated Stimulator Status light on the PowerLab. It should flash yellow, once per second.
Safety Note: The yellow light on the Isolated Stimulator indicates that the stimulator cannot pass the stimulus current requested, a condition known as “out of compliance”. This light flashes green when the stimulator switch is on, stimulus electrodes are correctly placed on the skin, and current is flowing properly.

Figure 2. Placement of the bar stimulus electrode on the left wrist.
4. Place the bar stimulus electrode over the volunteer’s ulnar nerve at the wrist (the approximate placing is shown in Figure 2). The bar stimulus electrode should be held in place along the axis of the arm, with the leads pointing towards the hand. The red dot on the back of the electrode indicates the anode (positive). The nerve impulse will be generated at the cathode end.
5. Set the stimulator switch on the PowerLab unit to the ON position (up). The stimulator status light should now flash green, indicating that the requested stimulus current is being passed through the subject’s skin.
6. Note the twitch contractions affecting the thumb and fingers. Examine the effect of small adjustments to the placing of the electrodes, and locate the position giving the largest twitches.
Note: If no twitch occurs, check that the electrodes are connected, and that the Isolated Stimulator is switched on. You may need to increase the stimulus amplitude to observe a twitch: do this in the Isolated Stimulator Panel. If the subject feels discomfort, you can at any time immediately stop stimulation by either turning the stimulator switch off on the PowerLab, or by removing the bar stimulus electrode from the subject’s wrist.

7. Explore the results of stimulating at other places in the forearm. Each time you move the electrode to another location, wipe away the residual electrode cream from the skin to prevent short-circuiting. (Stimulation will be ineffective if the current flows along a surface layer of electrode cream rather than through the arm.)
Note: You will probably find that effective stimulation will only occur when the two pads of the bar electrode are aligned along the arm’s length. If the stimulus status light changes in color from green to yellow, you will need to put more electrode cream on the pads.

Motor effects you may observe include:

Bending of the wrist (due to the flexor carpi radialis and flexor carpi ulnaris muscles)
Bending of the last segments of the fingers (due to the long finger flexor muscles)
Movement of all the fingers, combined with a pulling of the thumb towards the index finger (due to the intrinsic muscles of the hand innervated by the ulnar nerve)
Lifting of the thumb (due to the thenar muscles at the base of the thumb; innervated by the median nerve)
Note: Stimulation in most places gives rise to little discomfort. In some places, there is a substantial sensory effect: there may be a painful sensation in the forearm or hand, away from the site of stimulation (towards the fingers). At these places, a cutaneous sensory nerve is being stimulated.

8. Try stimulating the ulnar nerve at the level of the elbow. The nerve passes behind a bony prominence (the medial epicondyle) on the humerus. At this location, the nerve is exposed to minor mechanical injury and is known to children as the “funny bone”. Stimulation at this site gives large and obvious motor effects.
9. Click the Stop button to stop Chart monitoring, and turn the stimulator switch OFF on the PowerLab. It doesn’t matter if you close the Chart window or not.
Exercise 2: Twitch response and recruitment
Objectives
In this part of the laboratory, you will measure the muscular twitch response to nerve stimulation, and to show recruitment in the twitch response as the stimulus strength increases.
Procedure
1. Open the settings file Stimuli Settings from the Experiments Gallery. After a short time, the Chart window on the computer screen should be set up, with two channels appearing. Channel 1 (Force) shows the raw signal from the finger pulse transducer, and Channel 2 (Stimulus) shows the occurrence of stimuli.
2. The volunteer should place their hand as shown in Figure 3, with the fingers under the edge of the table, and the edge of the thumb resting lightly on the pulse transducer. If the table edge is too thick for the subject’s hand, a plank or shelf may have to be used.
3. Choose Input Amplifier… from the “Force” Channel Function pop-up menu. The Input Amplifier dialog box should show a stable baseline reading in its display. You should see a deflection of the trace when you press lightly on the pulse transducer.
4. Wipe the electrode cream from the subject’s wrist. Apply a small amount of electrode cream to the pads of the bar stimulus electrode, and place the electrode at the site for stimulation of the ulnar nerve at the wrist (Figure 3), and hold it firmly in place. (The subject can hold the electrode in place with their other hand if this is easier.) Ensure that the edge of the subject’s thumb is resting lightly on the transducer.
5. Turn the stimulator switch located on the front of the PowerLab, to ON (the up position).
6. The Isolated Stimulator mini-window should be floating in front of the Chart window. If not, choose the Isolated Stimulator command from the Setup menu (Win), or the Stimulator Panel command (Mac). The stimulus amplitude should be set to 0 mA. If necessary, move the mini-window to a convenient position that does not obscure your view of the Chart window.
7. Click Start. Chart will record for a fixed duration of 0.5 seconds, then stop automatically.
8. Increase the amplitude to 1.0 mA and click Start. Continue to increase the amplitude in 1 mA steps, and click Start until a response is recorded. For most subjects, this threshold stimulus is in the range 3–8 mA. When you first see a response, add a comment to the recording to note the stimulus amplitude used.
9. Reduce the amplitude by 1 mA, and then increase it in 0.5 mA steps, adding a comment each time to note the amplitude used. Continue this until the response no longer increases. For most subjects, this maximal stimulus is in the range 6–15 mA.
10. Close the Isolated Stimulator Panel. Turn the stimulator switch OFF on the PowerLab.
11. If you are saving your files, choose Save from the File menu to save the recording. Your data should resemble Figure 4. (If you find the block boundaries distracting for such short blocks, they can be hidden. Choose the Display Settings… command from the Setup menu and alter the settings in the dialog box that appears.)

Figure 4. Typical results from Exercise 2, showing the effect of increasing stimulus strength.
Exercise 3: Summation and tetanus
Objectives
In this exercise, you will demonstrate the effects of changing the interval between paired stimulus pulses and observe a short tetanic contraction.
Procedure
1. Close the open document, and choose “Experiments Gallery…” from the File menu. Open the settings file “Summation Settings”. After a short time, the Chart window on the computer screen should be set up, with two channels appearing. Channel 1 should be named ‘Force’ and Channel 2 should be named ‘Stimulus’, as they were in the last exercise.
2. Windows users: From the Setup menu, choose Isolated Stimulator. This will open the Isolated Stimulator window (Mac OS users choose the Stimulator command to open the Stimulator window). Ensure that the settings are the same as in Figure 5.
3. Windows users: Proceed to step 4. Mac users: Close the Isolated Stimulator window, choose the Stimulator Panel command from the Setup menu, open the Isolated Stimulator Panel and then proceed to step 5.
4. Ensure that the volunteer’s hand and stimulus electrodes are placed as shown in Figure 2, and turn the stimulator switch ON.
5. In the Isolated Stimulator Panel, set the pulse current to about 5 mA greater than the maximal stimulus value you determined in Exercise 2.
6. Windows users: Click Start and then immediately click the “Stimulate” button in the Isolated Stimulator window. Chart will record for a fixed duration of 3 seconds, and then stop automatically. Mac OS users: The stimulus will automatically occur 0.5 seconds after the recording begins and Chart will record for 3 seconds. When the recording has stopped, add a comment called “1 Hz” in the new block of data to note the stimulus frequency used.
7. Increase the stimulus frequency to 2 Hz in the Stimulator Panel, and then click Start. Note the stimulus frequency (2 Hz) in a comment, as in the previous step.
8. Repeat the stimulation for the frequencies 5, 10 and 20 Hz, noting the values in comments as above.
9. Open the Isolated Stimulator window once more, change the number of pulses from 2 to 3, then close the dialog box. Be careful with this setting: a prolonged tetanus with a large number of pulses may be painful for the subject.
10. Click Start. The volunteer should receive a burst of three stimuli at 20 Hz. Add the comment “Tetanic stimuli (3)” to the new block of data. If it is not causing too much discomfort, you could try again with four pulses.
11. Set the stimulus pulse current to 0 mA in the Isolated Stimulator Panel, then close it.
12. From the front of the PowerLab, turn the stimulator switch OFF. Disconnect the finger pulse transducer and stimulus electrodes from the PowerLab.
13. If you are saving your files, choose Save from the File menu to save the recording. Your data should resemble Figure 6.

Figure 6. Typical results from paired stimuli, viewed with 2:1 horizontal compression (the stimulus delay is about a second here).
Exercise 4: Grip force measurement
Objective
In this part of the laboratory you will calibrate the hand dynamometer with respect to a volunteer’s maximal grip strength.
Procedure
1. Remove any transducers and electrodes from the PowerLab, and then connect the plug of the grip force transducer to the Pod Input 1 (Figure 7).
2. Close the open Chart data file. If a warning dialog box appears, click the No button. From the Experiments Gallery, open the settings file “Grip Settings”. After a short time, the Chart View on the computer screen should be set up, with one channel appearing. Channel 1 should be named “Grip”.
3. The volunteer should loosely grip the hand dynamometer in the fist, as shown in Figure 7.

Figure 7. Connections for measuring grip strength and muscle fatigue.
4. Click Start to begin recording. The volunteer should squeeze the dynamometer as hard as possible for a second or two, and then relax their grip. After recording for a few seconds, have the volunteer repeat the maximum grip and then relax. Click the Stop button.
5. Drag over the largest response to select a range of data that includes both the relaxed and maximum-force signals (Figure 8). Choose the Units Conversion… item from the Channel Function pop-up menu for Channel 1.

Figure 8. Selection of trace for calibrating to relative grip strength.
6. In the Units Conversion dialog box (Figure 9) a roughly correct conversion has already been set. Now you need to calibrate correctly for the strength of the volunteer. Select part of the trace where the force was zero, and click the top Point 1 arrow button. Then select part of the trace at the peak, and click the bottom Point 2 arrow button. Click the OK button to return to the Chart View.

Figure 9. The Units Conversion dialog box, with relaxed signal selected.
Exercise 5: Muscle fatigue
Objectives
In this part of the experiment, you will observe the decline in maximal force during a sustained contraction, and to examine some properties of muscular fatigue.
Procedure
The grip force transducer should already be calibrated for the volunteer, as described in Exercise 4.

1. Adjust the scale for Channel 1 (Grip) to show –20 to 120%.
2. Allow the volunteer to view the computer screen. Click the Start button, and ask the volunteer to maintain 20% maximal grip strength while watching the recorded trace (the Range/Amplitude display for Channel 1 shows the percentage force applied).
3. After 20 seconds, tell the volunteer to relax.
4. Click Stop.
5. Wait for 30 seconds to allow recovery of muscle function.
6. Repeat steps 2–4 above for contractions of 40%, 60%, 80% and 100% of maximal grip strength.
7. Allow the volunteer to rest for two minutes.
8. Turn the volunteer away so that they cannot see the computer screen.
9. Click Start, and ask the volunteer to produce a sustained maximal contraction. After 8 to 10 seconds, or when the force has obviously declined, instruct them to try harder. After another 8 to 10 seconds, repeat the encouragement. After a few seconds, ask the volunteer to relax, and click Stop.
Note: Nearly all subjects can produce temporary increases in muscle force during a fatiguing contraction, when sufficiently motivated by verbal encouragement.
10. Click Start and ask the volunteer to produce a sustained maximal contraction. Every 8 to 10 seconds, allow the volunteer to relax very briefly (half a second), and then return to maximal contraction. Click the Stop button to terminate recording after 30 to 40 seconds.
Note: Even brief periods of relaxation allow substantial recovery from fatigue, but the recovery is only temporary (Figure 10).

Figure 10. Fatiguing contraction, with brief periods of relaxation.
11. Allow the volunteer to use their other hand if gripping the transducer has become painful. Turn the volunteer so that they can see the computer screen. Click the Start button, and ask the volunteer to produce a 40% contraction while watching the trace. After 10 seconds, press the Enter key to enter a comment (to mark the time).
12. Have the volunteer close their eyes, and attempt to maintain exactly the same contraction force for the next 30 seconds.
13. After the elapsed time, the volunteer should open their eyes, and adjust the contraction force back to 40%.
14. Click Stop and examine the trace.
Note: Almost all subjects will show a declining force (pseudo-fatigue) while their eyes are shut, that is very similar to fatigue. This is, however, not true fatigue, because the full 40% force can be exerted easily, as can be seen when the subject’s eyes are opened again.
Analysis
Exercise 2: Twitch response and recruitment
1. Use the Waveform Cursor to measure the amplitude of each peak: place it over the peak and read off the force in the Range/Amplitude display above the channel title. This is easiest if you use the View buttons to expand the horizontal display to 1:1.
2. Refer to the comments in the Chart View to determine the current applied to produce each response.
3. Write down your data in Table 1 of your Data Notebook. Graph the relationship between stimulus current and response size. Note the stimulus intensity at which the maximal response first appears.

Exercise 3: Summation and tetanus
1. Calculate the stimulus interval in seconds for each stimulation frequency. Enter your data in Table 2 of the Data Notebook.
2. Using the Marker and Waveform Cursor, measure the amplitude of the first two responses at each stimulus interval. Enter your results in Table 2 of the Data Notebook.
3. Examine the tetanic response. Calculate the stimulation interval in seconds and enter your value in Table 3 of the Data Notebook.
4. Drag across the tetanic response to select it, and examine the selected data in the Zoom window. Determine the maximum force amplitude using the Marker and Waveform Cursor and enter your results in Table 3 of the Data Notebook.
5. If you repeated the tetanus experiment with four pulses, repeat your analysis and add it to Table 3. Otherwise, leave the second line blank in Table 3.
Data Notebook
Table 1. The effects of varying stimulus strength on twitch force.
Stimulus
Response
Stimulus
Response
Stimulus
Response
0.0 mA

7.0 mA

14.0 mA

0.5 mA

7.5 mA

14.5 mA

1.0 mA

8.0 mA

15.0 mA

1.5 mA

8.5 mA

15.5 mA

2.0 mA

9.0 mA

16.0 mA

2.5 mA

9.5 mA

16.5 mA

3.0 mA

10.0 mA

17.0 mA

3.5 mA

10.5 mA

17.5 mA

4.0 mA

11.0 mA

18.0 mA

4.5 mA

11.5 mA

18.5 mA

5.0 mA

12.0 mA

19.0 mA

5.5 mA

12.5 mA

19.5 mA

6.0 mA

13.0 mA

20.0 mA

6.5 mA

13.5 mA

Table 2. Results for summation experiment in Exercise 3.
Stimulus frequency (Hz)
Stimulus interval (sec)
Amplitude of first response (mV)
Amplitude of second response (mV)
1

2

5

10

20

Table 3. Results from the tetanus experiment in Exercise 3.
Stimulus frequency (Hz)
Stimulus interval (sec)
Number of pulses
Amplitude of response (mV)
20

3

20

4

Study Questions
Exercise 2
1. Did you get a measurable twitch with a stimulus of 0 mA? What does this tell you about the number of muscle fibers contracting at this stimulus current?

2. What was the smallest current required to produce a contraction (the threshold current)? What proportion of the fibers in the muscle do you think were contracting to produce this small response?

3. What was the smallest current required to produce the maximum (largest) contraction? What proportion of the fibers in the muscle do you think were contracting to produce this maximal response?

4. What do you conclude happened to the number of fibers contracting as the current was raised from threshold to that required to produce a maximal contraction?

5. Why does varying the stimulus strength affect the twitch force?
Exercise 3
6. What are the two ways by which the nervous system can control the force generated by a muscle?

7. Electromyography, with needle electrodes inserted through the skin into a muscle, has been used to study the frequency of muscle fiber activation during voluntary contraction in humans. During weak contractions, the firing frequency is low, so that each fiber produces distinct twitches. The force produced by the whole muscle, however, is relatively smooth. How do you think this occurs?

Exercise 5
Fatigue is not well understood. Some factors that have been proposed to explain the fall in force during fatigue include: changes in the “sense of effort”, loss of “central drive”, failure of neuromuscular propagation, reduction in calcium release in excitation–contraction coupling, metabolic changes in the muscle, and reduction in muscle blood flow owing to compression of blood vessels.

8. Do your experiments help to decide which factors are important?

9. What explanations can you think of for pseudo-fatigue?

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