Exercise 6: Muscle Physiology II Twitch & Summation

Exercise 6: Muscle Physiology II Twitch & Summation Text Reading: Silverthorn, 5 th ed. 412 419, 425 427; 6 th ed. pg. 410 420 In this exercise, we will investigate the physiology of contraction in the gastrocnemius muscle of a frog. The Biopac system will produce the electrical stimulus to bring about muscle contraction and indirectly measure the force the muscle is able to generate under different conditions. There are several similarities between the excitability of neural tissue and the excitability of muscle tissue. Like a single neuron, a single muscle fiber will not respond to any stimulus impulse that is below threshold. Any single stimulus impulse above threshold will produce a contraction, or single twitch (see Fig. 1), of the same strength. There are no gradations in the strength of the muscle response for a single twitch. Muscles outside the laboratory, however, do not use single twitches to produce motion. A muscle twitch is usually divided into three phases: 1) the latent period; 2) the contraction period; 3) the relaxation period (see Figure 4). The latent period is the time from when the stimulus is delivered to the first indications of contraction in the muscle. The contraction period, or contraction time, is the time it takes the muscle to reach its peak contraction after the latent period. The relaxation period is the time the muscle takes to return to resting tension after reaching its peak contraction. Muscles rely on two physiological principles to bring about productive motion: recruitment and summation. Recruitment is increasing the number of Figure 1. A Single Twitch: An action potential on the muscle fiber is followed by contraction motor units responding to a single stimulus, which results in the increase in tension in whole muscle. Summation is the increase in tension that results when a muscle fiber is unable to relax between twitches. Individual twitches overlap and produce a continuous, smooth contraction of increasing strength. Recruitment requires increasing stimulus amplitude. Below threshold stimulus, there is no response from the muscle. As stimulus amplitude increases, only the muscle fibers with the March 29, 2013 page 65

lowest threshold initially respond and contract. As the stimulus amplitude continues to increase, an increasing number of motor units will be recruited so that the strength of the contraction, or tension, increases as well. Once all motor units in a muscle have been recruited, an increase in the stimulus amplitude will not result in any increase in tension. The contraction of a whole muscle can produce strong, weak or intermediate contractions depending upon how many motor units are stimulated. Figure 2. A single twitch (left) and two summated twitches in complete tetanus if there is no relaxation at all between stimulus impulses. Summation requires increasing stimulus frequency. At low frequency, the muscle fiber will relax before the next stimulus impulse occurs. As the stimulus frequency increases and the time between the stimuli decreases, the muscle fiber cannot fully relax before the next stimulus occurs (see Fig. 2). This loss of relaxation between stimuli is called tetanus. We say a muscle is in incomplete tetanus if the muscle fiber is able to partially relax between stimulus impulses (see Fig. 3). A muscle fiber is We can explain the phenomena of summation and tetanus by examining the molecular events that bring about muscle contraction. Remember that tension is generated in a muscle fiber when Ca +2 is released from the sarcoplasmic reticulum (SR), triggering formation of actin-myosin cross-bridges. In order for relaxation to occur, Ca +2 must be pumped back into the SR. Summation occurs because each subsequent stimulus releases Figure 3. Summation leading to incomplete tetanus, or unfused tetanus additional Ca +2 from the SR, which in turn increases the number of actin-myosin cross-bridges that form. As cytoplasmic Ca +2 increases, tension will increase until all possible actin-myosin cross-bridges have formed. At this point, the muscle fiber will reach maximal tension. This is illustrated in Figure 3. The black arrows along the X-axis indicate the occurrence of each stimulus, or action potential. With each stimulus, tension increases as Ca +2 accumulates in the muscle fiber until maximal tension is achieved. March 29, 2013 page 66

Today s Objectives 1. Analyze the phases of a single muscle twitch. 2. Investigate the effect of stimulus strength on whole muscle response. 3. Investigate the effect of stimulus frequency on whole muscle response. 4. Correlate the molecular events within a muscle cell with your observations. Setup I. Equipment The equipment for today s experiment is listed below. Instructions for set up will follow. Do not turn on any equipment until you have finished reading the entire set up instructions. 1. Computer 2. BIOPAC MP35 Acquisition unit (big blue box). This will be referred to as "the MP35 unit". 3. Finger Twitch Transducer (SS61L). This will be referred to as "the transducer" (see Figure 4). Figure 4. Finger Twitch Transducer (SS61L) 4. BSLSTM Stimulator (small blue box). This will be referred to as "the stimulator" (see Figure 5). The stimulator has two connector cables. One cable is labeled BSLSTMB reference output and the other cable is labeled BSLSTMB trigger. March 29, 2013 Figure 5. BSLSTM Stimulator page 67

5. Human-safe Stimulating Electrode (HSTM01). This will be referred to as "the electrode" (see Figure 6). You should be able to explain the purpose of each of these pieces of equipment. Figure 6. Stimulating Electrode (HSTM01) II. Equipment Set Up: 1. Turn the computer on. 2. Plug the finger twitch transducer into Channel 1 of the big blue box. 3. Set up the stimulator. NOTE: DO NOT PLACE THE STIMULATOR ON TOP OF THE MP35 UNIT. Figures 7 & 8 are to reference cable placement only. a. Plug the BSLSTM Reference Output into Channel 2 (see Fig. 7) b. Plug the BSLSTM Trigger into Analog Out port on the back of the MP35 unit (see Fig. 8) c. Plug the electrode into the Stimulus Output port on the front of the simulator by positioning the small metal knob on the front of the box in the metal groove of the connector and rotating the metal connector, not the rubber part, clockwise. d. Settings on the front of the stimulator: Turn the Level knob counterclockwise until it stops (this is the 0 volts setting) Check that Range is set at 100V (the key hole should be pointing to the left). If it is not, use the key attached to the cable and set the Range. Check that the Reference switch to set to Fixed (the toggle switch should be pointing in the down position). March 29, 2013 page 68

Figure 7. Front of the stimulator and MP35 unit Figure 8. Back of the stimulator and MP35 unit 4. After the computer has finished booting up, turn on both the MP35 unit and the stimulator. 5. When the busy light on the MP35 unit goes off, launch the appropriate Biopac application. Mac users will launch the BSL PRO 3.7 application, NOT the BSL Student Lessons like last week. The icon for launching can be found on the right side of the dock. PC users will launch the usual BSL 3.7.7 application by double-clicking on the BSL 3.7.7 icon on the desktop (NOT the BSL PRO icon). 6. The file you need to open is called "h06a.gtl". For Mac users, you may need to transfer the h06a.gtl file from the thumb drive provided by the instructor. Once it is downloaded to the desktop, double click the h06.gtl icon. For PC users, click the "PRO" tab located to the right of the "BSL Lessons" tab and below "Choose a file to open:". Double click on "H06 Finger Twitch (SS61L).gtl". The stimulator window will open (see Fig. 9). March 29, 2013 page 69

Figure 9. Stimulator Window (ho6a.gtl) III. Getting the subject ready: Attach the transducer to the palmer surface of the nondominant hand (see Fig. 10) with the "UP" label facing out. Make sure the "UP" label is positioned so that the transducer bends towards the label. Secure the transducer to the finger by positioning the Velcro between the middle and distal phalanges. The transducer can be placed on either the pointer or middle finger. The finger chosen will determine where the electrode should be positioned on the forearm. Figure 10. Palmar placement of the transducer March 29, 2013 page 70

IV. Calibration of Transducer (SS61L) 1. From the pull down menus at the top of the computer screen, select MP35 > Setup Channels, then click the Channel 1 wrench icon and choose Scaling at the bottom of the window. A calibration window will open (see Fig. 11). 2. The Input Value is the electrical signal produced by the twitch transducer. The Scale Value is in units of centimeters (cm). For the zero point in your calibration, the Scale Value is set to 0. Lay the subject's hand palm up with the entire forearm resting on the table and the hand relaxed. The transducer should be relatively straight, although the relaxed palm may cause it to curve a bit. Click Cal1. 3. Set the Cal2 Scale value to 5 cm by highlighting 8.89 in the second Scale Value text box and typing in "5". Have the subject bend his or her finger until the twitch transducer is at a 90º angle (perpendicular to the palm) and click Cal2. NOTE: if you get an error message, check that the transducer is correctly attached to the hand. The bar must bend towards the "UP" label. The input value should be a positive number. 4. Click OK to close the calibration window and the Input Channel Parameters window. Close the Set Up Channels window. NOTE: the transducer must be recalibrated each time you open a new h06a.gtl file. Figure 11. Scaling Window for calibrating the SS61L twitch transducer V. Electrode (HSTM01) Preparation 1. Controlling the stimulator: The ON/OFF switch for the stimulator is found at the top of computer screen in the red box labeled "Output Settings" (see Fig. 9). This red box is the Stimulator Window. To the right of the ON/OFF switch is the frequency setting. Confirm that frequency of the stimulator is set to 1.00 Hz. Recording of the stimulus and response is controlled by the START button located at the bottom right corner of the screen. The stimulator and the recorder are independent of each other. The voltage level is controlled by turning the knob on the front of the stimulator. Adjust this setting to 15 V to begin with. When you turn on the stimulator by clicking the ON switch, a stimulus will be produced at a frequency of 1 stimulus per second and a red light will flash March 29, 2013 page 71

on the front of the stimulator in time with the stimulus pulse. The stimulus will be delivered to the electrode when the subject depresses the red button. 2. The subject will hold the electrode in their dominant hand and control the stimulus by depressing the red button. With the subject's forearm resting on the table and palm up, smear some electrode gel in the center of the forearm to lubricate the electrodes. A glob of gel is better than a smooth thin layer. Gently press the electrodes against the skin with the two stimulating electrodes positioned lengthwise, parallel with the tendons of the wrist and forearm. 3. Click the ON switch and slowly turn up the voltage of the stimulator. The subject must depress and hold down the red button on the electrode to deliver the stimulus to the forearm. The subject can stop the stimulus at any time by releasing the red button. When the subject can feel the stimulus, slowly move the electrode around the forearm, maintaining the two points of the electrode in a lengthwise orientation, until the finger wearing the transducer begins to twitch. A typical stimulus needed to produce a twitch is between 25 V and 30 V. Adjust the stimulus level to a voltage setting that produces a twitch without being uncomfortable for the subject. Try not to move the electrode, however, in order to maintain the correct position for the finger twitch. Recording The Single Twitch 1. Remember that the stimulator and the recorder are controlled independently of each other. The recorder is turned on when you click the word START, found at the bottom right corner of the screen. The stimulus will be recorded in the bottom half of the window (blue trace) and the finger twitch response will be recorded in the upper half of the window (red trace). The finger twitch response is measured in centimeters of displacement or bending of the finger, which indirectly reflects the force of the twitch. The actual tension generated in the muscle, usually measured in gram or kilogram units, cannot be measured with this type of transducer. 2. When you are ready, click START and record for about 10 seconds so that you have a good sampling to analyze. Click the word STOP to stop the recording. Don't forget to have your subject stop the stimulation once you have stopped recording! 3. Your data can be analyzed immediately after recording when using the Biopac PRO software. Select three individual twitches to analyze, using the zoom tool. Choose Autoscale Waveforms in the Display menu to fit the peaks in the recording window. 4. At the top of the screen and below the red Stimulator Window, change the first channel box from SC to either Ch. 1. To the right of the channel box, set the measurement function box to Delta T to measure time on the X-axis. 5. Use the I-beam tool to measure the time of each segment of the twitch (see Fig. 1 to review the twitch segments). Record your data in Table 1. Measure each period for three separate twitches and average the values. March 29, 2013 page 72

The latent period, which is the time between the stimulus, recorded in the bottom window, and the beginning of the twitch response, recorded in the upper window. The contraction time, which is the time between the start of the twitch and the peak. The relaxation time, which is the time between the peak of the twitch and complete relaxation. It will be difficult to tell where to end the relaxation period. Just eyeball it the best you can. Note that the units of time may change from msec to sec when the values increase above 100 msec. Latent Period Contraction Time Relaxation Time Time of Total Contraction Table 1. Data for Muscle Twitch #1 #2 #3 Average Questions: 1. What were the two pieces of equipment used by the subject and what did they do? 2. How does direct electrical stimulation produce contractions of the muscle? 3. How closely did the Delta T values (time periods) you measured compare to the time values of a twitch as described in the textbook? 4. Describe the molecular events in the muscle fiber that produce a twitch in response to the stimulus voltage. Specifically, what molecular events occur during the latent period? Which events occur in the contraction period? Which events occur in the relaxation period? Be sure to include the role of calcium in your description. March 29, 2013 page 73

Stimulus Strength and Recruitment In this section, we will demonstrate the response to increasing stimulus intensity (progressively higher voltage). Remember that motor units within a whole muscle have variable thresholds. The slow twitch motor units have the lowest threshold and will be the first to respond as the voltage of the stimulus is increased. Intermediate motor units are recruited next. The fast twitch motor units have the highest thresholds and are the last to be recruited. As more motor units are recruited with increased stimulus voltage, the tension produced by each twitch increases. 1. Click the ON button in the Stimulator Window if the stimulator is not already on. Adjust the voltage setting and check that the position of the electrode is generating a finger response before you begin to record. 2. Determine Threshold: Restore your recording window using Autoscale Horizontal and Autoscale Waveforms. Click the START button to begin recording (bottom right corner). Watch the subject's finger for the twitch. Slowly reduce the voltage until the twitch is no longer observed, then increase the voltage one volt at a time until the twitch occurs again. Record the observed voltage that corresponds to the reoccurrence of the twitch in Table 2. The is the observed Threshold Stimulus. Observed Threshold Measured values: Twitch #1 Twitch #2 Twitch #3 Average Table 2. Recruitment Data Stimulus (volts) Threshold Response (cm) Voltage (volts) Maximum Response (cm) 3. Demonstrate Recruitment: Increase the stimulus in approximately 5 V increments, allowing five or six twitches to occur before increasing the stimulus voltage again. Observe response of the subject's finger as the voltage increases. The voltage can be increased until the finger does not appear to bend any further (full recruitment) or as March 29, 2013 page 74

high as the subject chooses, up to a maximum voltage of 100V. Most people can tolerate anywhere from 65 V to 90 V. Remember, the subject can stop the stimulus at any time by releasing the red button on the electrode. Once the finger appears to be fully recruited or the subject chooses to stop, click STOP to end the recording and the OFF button to turn off the stimulator. Reduce the stimulus voltage to 20 V. Your data should look something like Figure 12. Figure 12. Recruitment data ANALYSIS 1. Below the red Stimulator Window, set the channel boxes and function boxes for analysis: The first measurement function box has already been set to Delta T. Change the second channel box from SC to Channel 2 (blue) and set the measurement function box P-P (point to point measures the difference between the lowest point and the highest point in a highlighted region). This measures the stimulus in volts. Change the third channel box to Channel 1 (red) and the measurement function box P-P. This measures the magnitude of the twitch response in centimeters. 2. Measure Threshold: Expand your display by choosing first Autoscale Horizontal, then Autoscale Waveforms from the Display menu. March 29, 2013 page 75

Use the zoom tool to select the area where threshold appears to occur. Choose Autoscale Waveforms to adjust your image, if necessary. Use the I-beam to highlight the stimulus that corresponds with the smallest measureable response. Record in Table 2 the values that appear in the measurement function boxes for the threshold voltage (Ch. 2 blue) and the threshold response (Ch. 1 red) for first three adjacent twitches that occur at threshold. Two significant digits are sufficient. 3. Recruitment: Once again expand your display by choosing first Autoscale Horizontal, then Autoscale Waveforms. Use the zoom tool to select an area where maximum recruitment appears to occur. Measure and record the stimulus and response for three maximal twitches. NOTE: It may be difficult to quantitate (measure) the increase in force produced during recruitment, but you will see a qualitative increase in strength of the twitches as the stimulus is increased when you look at the recording as a whole. Expand your trace using Autoscale Horizontal, Autoscale Waveforms and print a copy of your data window. 4. If printing is not possible from your computer, take a screen shot of the stimulator window and transfer a copy of it to a USB thumb drive. Your instructor can print from the instructor's computer. For Macs, hold down shift-command-4, then click and drag the crosshairs that appear to draw a box around the part of the screen you would like to include in your screen shot. The screen shot will be automatically saved to the desktop, but will be erased when the computer is shut down. For PCs, use the screenshot key at the top of the number pad. This puts the screenshot in the clip board. Paste the image into the Paint application found under Programs, then name and save the image to the desktop. Questions: 5. Explain why the muscle doesn t respond to low stimulus voltages. 6. How does the twitch response vary with increasing voltage? Include the concepts of both threshold and recruitment in your discussion. March 29, 2013 page 76

Stimulus Frequency and Summation In this section, we will demonstrate how a muscle responds to increases in stimulus frequency (the number of stimuli per second). The stimulus voltage will remain constant throughout this experiment. As frequency increases, summation and tetanus will begin to occur. Summation can be measured as the increase in the muscle response along the Y-axis. Tetanus will be observed as the loss of relaxation between twitches. 1. Open a new window using the h06a.gtl template file. Calibrate the finger twitch transducer as you did previously. 2. Set the voltage on the stimulator to the lowest voltage needed to produce a small finger twitch, about 20 V to 25 V. Frequency should be set to 1 Hz (1 stimulus per second) in the Stimulator Window. 3. Click the ON button in the Stimulator Window. Have the subject depress the red button on the electrode and adjust the electrode so that a twitch is produced. Click Start to begin recording. 4. Let the recorder run for about 10 seconds before beginning to increase the frequency. Increase the stimulus frequency in 1 Hz increments by clicking on the right pointing arrow underneath the frequency setting window. Typing a number into the frequency setting window WILL NOT change the frequency. Observe how the subject's finger responds to the increasing frequency. Continue to increase the frequency at approximately 5 to 10 second intervals. 5. When the recording plateaus, stop stimulating the finger by releasing the red button and allow the finger to relax before you stop recording. The subject should take care not to move their hand. Maximum summation usually occurs somewhere around 11 Hz. 6. Click Autoscale Horizontal to display the entire recording in the window. Print a copy of your data. It should resemble Figure 13. On your printout, use an arrow to indicate where summation begins. Indicate where unfused tetanus becomes fused. Remember, the terms fused and unfused are interchangeable with the terms complete and incomplete. March 29, 2013 page 77

Figure 13. Summation Data So What Happened? Remember what causes contraction in a muscle fiber. An action potential causes calcium ions to be released from the sarcoplasmic reticulum into the sarcoplasm. This allows myosin to form cross bridges with actin filaments of the sarcomere and generate the movement associated with the contraction. The more cross bridges formed, the stronger the contraction. Relaxation is brought about by the removal of the Ca ++ from the sarcoplasm. If a second stimulus is delivered to a fiber before all of the Ca ++ are removed (the fiber has not relaxed), more Ca ++ are released and the fiber starts a second contraction without completely relaxing from the first. If there is no relaxation at all between stimuli, but rather a smooth sustained contraction, it is called Fused Tetanus, Complete Tetanus or just plain Tetanus. If there is some relaxation between the stimuli, the contraction is referred to as Unfused Tetanus" or Imcomplete Tetanus". If a muscle completely relaxes in between successive stimuli, there is no tetanus at all. As a muscle responds to successive stimuli, the contractions increase in strength. This property is called Summation of Contractions, or simply "summation." Summation makes sense when you remember that the increased frequency of stimuli causes increased amounts of Ca 2+ to be deposited in the sarcomeres. The increased Ca 2+ means there will be increased numbers of myosin-actin cross bridges, and thus, an increase in the strength of contraction. Sample data can be seen in Figure 6. The data has been squished into one window so that the results for each increase in frequency can be compared side by side. Note the difference in the amplitude of the single twitches on the left versus the amplitude of the contractions showing March 29, 2013 page 78

fused tetanus on the right. The I-bar added to the figure indicates summation, or the increase in force that results from fused tetanus. Questions: 7. What do the terms unfused and fused tetanus refer to? What do the terms incomplete and complete tetanus refer to? 8. Define the terms tetanus and summation. 9. Explain the cellular mechanism that produces tetanus and summation. March 29, 2013 page 79