AANT 318 Human Anatomy and Physiology Name:_____________________________Laboratory 7

Stimulus Response, Work, Summation, and Tetanus in Human Muscle

Background:

About 40% of the total body mass of a human is skeletal muscle. Skeletal muscle is intimately associated with the skeletal system and, combined, these muscles and bones are responsible for supportingand moving the body. While all skeletal muscle fibers have sarcomeresand the same banded appearance, different muscles can function in different ways. For example, some are relatively weak and fatigue resistant, while others are strong but fatigue quickly. These features may be explained in terms of biochemical properties.

The muscle fibers found in most mammalian skeletal muscles are either fast- or slow- twitch types. Each type contains a different type of myosin, with different rates of ATPase activity and cross-bridge binding. Within the group of fast-twitch fibers, there are fibers that use both glycolysis and oxidative phosphorylation. There are also fast-twitch fibers that just use glycolysis; this group tends to need less oxygen and is much stronger than the fibers that also use phosphorylation. However, these stronger glycolytic fibers breakdown glucose very inefficiently; a burst of contractile activityuses most of the available glucose, causes lactic acid to accumulate,and leads to fatigue more quickly.

Even though a motor neuron synapses with many muscle fibers, allthe muscle fibers connected to that motor neuron are the same type. So, the stimulation of one particular motor neuron will cause contractions of only one type of muscle fiber. Motor neurons that supply the weak, slow, oxidative fibers have the lowest threshold. Those that innervate fast, intermediate strength oxidative fibers have higher thresholds. And those that supply the fast, strong, glycolytic only fibers have the highest threshold. By increasing the amount of activity coming from the brain more motor neurons are progressively activated, and more of the stronger muscle fibers are

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brought into the response. All of this occurs through a single synapse on each fiber.

When a muscle tries to lift any weight, the muscle first shortens to put tension on the tendons which hold the muscle to the bones. Development of this tension before movement occurs takes time,known as the latency period, which is directly proportional to the weight attached to the muscle. Once the tension exceeds the weight ofthe object, any further muscle contraction produces a shortening of the muscle and a movement of the weight. The time that a muscle is inits active state (contracting) is finite; so, muscles have less time to shorten when they move heavier weights.

In this experiment, students will use a specialized displacementtransducer, the SMT-100 striated muscle transducer, to demonstrate the effect of increasing the stimulus strength on the strength of contraction of a muscle, the effect of increasing weight on twitch amplitude and work of a preloaded muscle, and the effect of increasing the frequency of stimulation on the contraction strength and muscle fatigue.

This experiment involves the application of stimulus pulses to the skin on the back of the hand to produce a twitch in the muscles that move the subject’s little finger. The stimulus pulses used in these experiments are generated by a SI-100 high voltage stimulus isolator. While the sensation of evoked muscle contractions may initially feel peculiar, electrical stimulation of the muscles in thehand is safe and painless. Standard safety precautions need to be observed. Persons with poor cardiac function, pacemakers, or any other condition that can be aggravated by electrical stimulation should not volunteer for this experiment.

Warning: Change the location of the stimulating electrodes if the subject finds the stimulus to be painful. Changing subjects might be necessary, if the subject is experiencing any pain.

Positioning of the Stimulating Electrodes

Before attaching the SMT-100 striated muscle transducer to the side of the subject’s hand, determine the positions for the stimulating electrode that cause the muscles of the little finger to create a strong twitch response.

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1 Make sure the SI-100 is turned off and the amplitude on the SI-100 is set to zero. Instruct the subject to remove all jewelry from the hand and wrist being used in the experiment.

2 Obtain a couple of disposable electrodes. Place the first electrodehalf way between the first knuckle of the middle finger and the wrist(see Figure below). This is the positive stimulating electrode. Placethe second electrode about one centimeter above the lateral edge of the hand, half way between the first knuckle of the little finger andthe wrist. This electrode is the negative stimulating electrode, and it is placed in an area that normally creates the largest twitch by the little finger.

Legend: “+” = red, “-“ = black.

3 Snap the ends of the lead wires onto the stimulating electrodes, sothat:• the red (+) lead is snapped on the electrode in the center of the back of the hand,• the black (-) lead is snapped on the electrode at the lateral edge of the hand,

Warning: Make sure the Amplitude knob on the front of the SI-100 is set to zero.

4 Flip the power switch on the back of the SI-100 to turn on the unit. The red lights on the front of the device should glow to indicate the SI-100 is properly powered and the high voltage source is ready to deliver a stimulus.5 Click on the Record button on the LabScribe Main window to activatethe IWX/214 and SI-100 stimulator. There should be no response from

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the subject’s finger since the current output is zero. Continue to record.Note: As the current output of the SI-100 is increased in the next couple of steps, ask the subject to indicate when he or she first feels any tingling under the negative stimulating electrode. Minor movement of the subject’s finger can usually be seen when the stimulus current is raised another milliamp or two.

6 Slowly rotate the Amplitude knob clockwise 2 turns, which is equal to a current output of 2 milliamperes (mA). Remind the subject to indicate the first occurrence of tingling. If no finger movement is seen, the stimulus current is below the threshold current of the mostsensitive muscle fibers controlling the finger’s movement. Threshold is the current level that is needed to create a muscle fiber contraction.

Note: The amplitude required to cause a finger twitch will differ between subjects. Some subjects require as low as 3 milliamperes of current to create a maximum response, while other subjects may require 7 or more milliamperes to create the strongest response.

7 If a finger twitch does not occur at 2 mA, rotate the Amplitude knob an additional half turn to increase the stimulus current by 0.5mA. Check for tingling and finger movement. Increase the current output in increments of 0.5mA until the subject’s finger twitches with the largest range of motion.

8 If the stimulus current has been raised to 5 mA and the subject feels tingling but no finger movement is observed, adjust the position of the negative stimulating electrode before making additional increases in the stimulus current.

• Lower the stimulus current to 2.5 mA before moving the negative stimulating electrode.• Move the negative stimulating electrode a centimeter closer to the positive stimulating electrode.• Stimulate the subject’s hand. Raise the stimulus current as high as5 mA. If the response from the subject’s hand is miniscule, or non-existent, with 5 mA of current, place the negative electrode at different points along an imaginary line on the back of the hand, from the base of the little finger to the lateral edge of the wrist.

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9 Find the lowest stimulus current that creates the largest twitch from the subject’s finger, click Stop to turn off the stimulator. Setthe current at this level for Exercise 1.

SMT-100 Setup

1 The SMT-100 striated muscle transducer has a sensor that is a flexible resistive element that generates a signal that is proportional to range of flexion of the sensor and the finger to which it is attached.2 The SMT-100 generates a signal only when flexed in one direction. Locate the label on the sensor that indicates the side of the sensor that will need to face outwardly from the finger.3 Align the SMT-100 along the side of the little finger so the outside of the sensor is oriented away from the finger (see previous Figure). Use a Velcro strap to attach the tip of the sensor to the end of the little finger. Use a longer Velcro strap to attach the base of the sensor along the lateral edge of the palm of the hand.

Exercise 1: Properties of a Finger Twitch

Aim: To become familiar with SI-100 and SMT-100 and to record a finger twitch with and without a weight attached to the finger.

Procedure1 Instruct the subject to relax and place the hand that is used for the experiment on the bench, with its palm up.2 Click the Stimulator Preferences icon on the LabScribe toolbar to open the stimulator control panel on the Main window (see figures below).

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3 Check the values for the stimulus parameters that are listed in thestimulator control panel on the Main window: the pulse amplitude (Amp) should be set to 5.0V, the pulse width (W) to 5 ms, the frequency (F) to 1 Hz; and, the number of pulses to 15. Click on the arrow buttons to change the value for a stimulus parameter. Click theApply button to finalize the change in any stimulus parameter.4 Use the Amplitude knob of the SI-100 to set the stimulus current tothe maximal level as determined previously.5 Click the Record button on the LabScribe Main window. Continue to record for 15 to 20 seconds. Click the Stop button.6 Leave all the stimulus parameters at the same settings.

Data Analysis

1 Scroll through the recording of the finger twitches (see figure on next page). Notice each finger twitch has two phases, a quick contraction phase followed by a slow relaxation phase. Since the duration of the stimulus pulses are short and their frequency is low,the muscles of the finger have the opportunity to relax between twitches as indicated by the return of the twitch amplitude to the same baseline between twitches.

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2 Use the Display Time icons to adjust the Display Time of the Main window to show at least three complete twitches on the Main window. Three adjacent twitches can also be selected by:• Placing the cursors on either side of a group of three complete twitches; and• Clicking the Zoom between Cursors button on the LabScribe toolbar to expand the three twitches to the width of the Main window.

3 Click on the Analysis window icon in the LabScribe toolbar.4 Look at the Function Table that is above the uppermost channel displayed in the Analysis window. The mathematical functions V2-V1 and T2-T1 should appear in this table. Values for V2-V1 and T2-T1 on each channel are seen in the table across the top margin of each channel.5 Once the cursors are placed in the correct positions for determining the amplitude and times of each finger twitch, record thevalues of the parameters from the Function Table in the table on the next page.6 On the Finger Twitch channel, use the mouse to click on and drag the cursors to specific points on the recording to measure the following parameters:

• Twitch Amplitude. To measure it, place one cursor on the baseline at the point just before the twitch starts to develop, and the secondcursor on the peak of the twitch. On the Finger Twitch channel, the

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value for V2-V1 is the amplitude of that twitch (figure on next page).

• Contraction Time, which is the time it takes the amplitude of the twitch to rise to its peak. To measure it, keep the cursors in the same positions used to measure the twitch amplitude. On the Finger Twitch channel, the value for T2-T1 is the contraction time of that twitch.

• Relaxation Time, which is the time it takes the amplitude of the twitch to return to baseline. To measure it, place one cursor on the peak of the twitch, and the second cursor at the point where the amplitude returns to the baseline. On the Finger Twitch channel, the value for T2- T1 is the relaxation time of that twitch.

7 Enter the values for the three parameters in the table below:

Twitch Amplitude (mV) Contraction time (s)

Relaxation time (s)

Twitch 1Twitch 2Twitch 3

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Question:

How does the contraction time of a typical finger twitch compare to the relaxation time?

Contraction time is shorter than relaxation time.

Exercise 2: The Effect of Weight on Finger Twitches

Aim: To determine the amount of work performed by the finger with different weights, or loads, attached to the finger. In this exercise, we will use a 10-gram paperclip attached to a thread, and 5screws, each weighting 10 grams, that will be successively added to the load system.

Procedure

1 Check the stimulus parameters set in the Stimulator control panel on the LabScribe Main window. These settings should be the same as the ones used in Exercise 1.2 Use the Amplitude knob of the SI-100 to set the stimulus current tothe maximal level as used in Exercise 1.3 Attach the thread with the paperclip to the last joint of the finger being used in the experiment. The thread should go across the ring, middle, and index fingers of the same hand, so that weight can hang over the edge of the table. 4 Have the subject move his or her hand to the edge of the table, so the palm is up and the thread with the weight is hanging along the side of the table (see Figure 1). Another possible setup is one in which the hand is turned so that the small finger faces upwardly, andthe load system hanging freely.5 Type the weight of the object that is on the thread in the Mark boxthat is to the right of the Mark button. Click the Record button on the LabScribe Main window. Press the Enter key on the keyboard to mark the recording. Continue to record for 15 seconds. Click the Stopbutton.

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6 Add another 10 gram weight (a screw) to end of the thread and repeat Step 5.7 Continue to add weight to the thread in 10 gram increments. Repeat Step 5 for the 5 screws. The amplitude of the twitch should continuously decrease with increasing weight.8 Remove the weights and thread from the subject’s finger.

Data Analysis

1 Scroll to the beginning of the recording for this exercise. Locate the section of data in which finger twitches appeared when the stimulus threshold was reached. Click AutoScale to maximize the size of the twitch on the window.

2 Use the Display Time icons to adjust the Display Time of the Main window to show a segment with four or five twitches on the Main window. The twitches can also be selected by:• Placing the cursors on either side of the selected twitches; and• Clicking the Zoom between Cursors button on the LabScribe toolbar to expand the segment of twitches to the width of the Main window.

3 Click on the Analysis window. Use the same techniques used in Exercise 1 to measure and record the amplitudes (V2-V1) of three twitches recorded at each weight (use Table below). For each weight, determine the average amplitude of three finger twitches. Use the average twitch amplitude at each weight to calculate the work performed on that weight. For this exercise, use the following equation:

Work = Weight (g) x Average Amplitude of Twitch (mV) with that Weight

Load weight (g) Amplitude (mV) Work (g. mV)1 (paperclip alone)111 Average2 (1 screw)222 Average3 (2 screws)

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333 Average4 (3 screws)444 Average5 (4 screws)555 Average6 (5 screws)666 Average

Questions:

1 How do the amplitudes of the finger twitches with different weightscompare to each other? Plot the work performed at each weight (g.mV) as a function of the weight attached to the finger (g).

The amplitude of the finger twitches should increase with heavier weight because more fibers are recruited, thus more EMG activity is generated.

2 Why did the amount of work decrease when heavier weights were used?

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This was not observed because the weight was not heavy enough. But ifthe amount of work decreases this should be due to eccentric contraction, or a contraction that takes place when the muscle actually lengthens. If the sarcomeres are too long, then not enough cross-bridges can be formed between them, and thus not enough force can be developed.

3 Is there any correlation between the weight moved and the contraction or relaxation times of the twitches? Do not record values, just eyeball the data, and see whether the left and right slopes of each spike become steeper (occurring in a shorter amount oftime) or less steep (taking longer).

If the weight gets progressively heavier, it is expected that it takes longer to develop enough tension for muscle contraction to occur. So it should take longer to produce a contraction. As explained in the introduction, the latency period corresponding to the development of tension is directly proportional to the weight attached to the muscle I am not sure how muscle relaxation should be affected if it is affected at all.

Exercise 3: Stimulus-Response

Aim: To examine the effect of stimulus strength on the amplitude of finger movement.

Procedure1 Remind the subject to relax and place the hand that is used for theexperiment on the bench, with its palm up.2 Check the stimulus parameters set in the stimulator control panel on the LabScribe Main window. The duration of the stimulus pulses should be 5 milliseconds (ms), their frequency should be 1 Hz, and the number of pulses should be 0. Setting the number of pulses to zero makes the SI-100 stimulus isolator fire continuously. Click the Apply button on the Stimulator control panel.3 Make sure the Amplitude knob on the front of the SI-100 is set to zero. Type Zero in the Mark box that is to the right of the Mark button. Click the Record button on the LabScribe Main window. Press the Enter key on the keyboard to mark the recording.

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4 While recording, type 1 mA in the Mark box that is to the right of the Mark button. Increase the current output of the SI-100 by 1 mA byrotating the Amplitude knob one turn. Press the Enter key on the keyboard to mark the recording. Record at this stimulus current for afew (5) seconds.5 Repeat Step 4, in increments of 1 mA, until the stimulus current reaches the maximal level (10 mA). Click the Stop button.

Data Analysis1 Scroll to the beginning of the data for this exercise. Click AutoScale to maximize the size of the twitches on the window. 2 Use the Display Time icons to adjust the display time of the Main window to show a segment with the majority of the twitches.4 For each stimulus strength determine the average amplitude of 2 twitches. Use the table below. 5 Plot the average amplitude of the finger twitch at each stimulus current as a function of stimulus current.

Stimulus Current and Twitch AmplitudeTwitch

1 2 3 4 5 6 7 8 9 10

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2

Avg.

Twitch Amplitude(mV)

1 2 3 4 5 6 7 8 9 10Stimulus Current (mA)

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Questions

1 At which stimulus current did the finger first show a twitch? Why didn’t a finger twitch occur at a lower stimulus current?

Around 3-4 mV. Because threshold was not reached.

2 Above a certain stimulus current, the amplitude of the finger twitch no longer increases. Why does this happen?

Because at this point all of the muscle fibers have been recruited.

Exercise 4: Summation and Tetanus

Aim: To monitor the contraction and relaxation of the finger twitch in relation to the stimulus frequency.

Procedure1 Remind the subject to relax and place the hand that is used for theexperiment on the bench, with its palm up.

2 Check the stimulus parameters set in the stimulator control panel on the LabScribe Main window and the current output on the amplitude knob of the SI-100. The duration of the stimulus pulses should be 5 milliseconds, their frequency should be 1 Hz, the number of pulses should be set to 15.

3 During the course of this exercise, the stimulus frequency and the number of stimulus pulses will be changed to demonstrate summation and tetanus. After any stimulus parameter is changed, click the Applybutton on the Stimulator control panel to effect the change.

4 Use the Amplitude knob of the SI-100 to set the stimulus current tothe maximal level as used in Exercise 1.

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5 Type 1 Hz in the Mark box to the right of the Mark button. Click the Record button on the LabScribe Main window. Press the Enter key on the keyboard. Click Stop when the stimulus pulse stop firing. There should be fifteen twitches on the recording.

6 Change the stimulus frequency to 2 Hz. Go to the Stimulator controlpanel on the LabScribe Main window, and change the value in the stimulus frequency box, labeled F(Hz), from 1 to 2. Click the up arrow in the box to increase the frequency. The frequency can also bechanged by typing the new value into box. Click the Apply button to put any frequency change into effect.

7 Repeat Step 5 with the stimulus frequency set to 2. Type 2 in the Mark box that is to the right of the Mark button.

8 Repeat Step 5 for stimulus frequencies of 3, 4, 5, 10, 15, and 20 Hz. Remember to click the Apply button to effect changes.

9 When performing the experiment at the highest frequency keep recording after complete tetanus is reached. Remove the cable from the stimulator to stop the SI-100 from firing. Continue to record as the tension in the muscles of the fingers begins to relax.

10 Use the Display Time icons to adjust the Display Time of the Mainwindow to show the twitches of interest. You should observe somethingsimilar to the diagrams below.

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Left: summation and tetanus; right: summation.

11 Summation occurs when the time between twitches is not long enoughto allow the muscle to completely relax to its baseline level of tension. Another twitch or contraction, following in quick successionto the first, will add on the first twitch at the current level of tension for the first twitch. The third twitch will add on to the current tension of the second twitch, and so on. 12 If a level of relatively constant tension occurs during contractions at high frequency, the phenomenon is called tetanus. If small relaxations are still detectable along the level of constant tension, a state of incomplete tetanus exists. If there are no detectable relaxations along the region of constant tension, a state of complete tetanus exists(20 Hz).

Questions

1 At which frequency did summation first appear? At which frequenciesdoes incomplete tetanus occur? At which frequencies does complete tetanus first occur?

Around 10 Hz. Around 11-13 Hz. We did not observe a complete tetanus.

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2 After the stimulator is turned off, the relaxation time after tetanus is much longer than the relaxation time after a single twitch. Why?Because fatigue was achieved. The mechanism behind fatigue is not fully understood. Scientists think that it is due to the decreased ability of the sarcoplasmic reticulum to release Ca++ leading to failure of the excitation-contraction coupling and due to a depletionof muscle glycogen, or to the accumulation of K+ in the extracellularspace. If the latter is true then action potentials cannot be initiated due to the very low membrane potential.

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