Physiological Science Lab: Frog Skeletal Muscle 2816w

Frog Skeletal Muscle 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.

Figure 1. Skeletal Muscle Structure A single motor neuron, and all the muscle fibers that it innervates, is known as a motor unit (Figure 2). Skeletal muscle is similar to nerve tissue in that the fiber responds to a stimulus in an all-or-none fashion. This response is called a twitch. One motor neuron supplies a number of muscle fibers to constitute a motor unit. Motor units vary greatly in size, from just a few muscle fibers innervated by a single neuron (small motor unit) up to thousands (large motor unit). The smaller the motor unit, the finer the control of movement in that muscle; thus, the muscles controlling the movements of the fingers and eyes have small motor units whereas those controlling the large limb muscles may have very large motor units. However, most muscle consists of a range of motor unit sizes.

Figure 2. Motor Unit Depending on the intensity and frequency of stimulation, greater numbers of fibers are activated. The strength of a muscle contraction, therefore, can be increased in two ways: by increasing the number of active motor units (termed recruitment) and by stimulating existing active motor units more frequently. The absolute force that a muscle can generate is dependent on the total number of muscle fibers. So muscles with large cross-sectional areas are able to generate larger forces than those with small crosssectional areas. Motor nerves release the neurotransmitter acetylcholine from their terminals, called motor end plates. The acetylcholine released into the junctional cleft binds to receptors on the muscle membrane that are directly coupled to cation-selective ion channels (Figure 3). Opening of these channels depolarizes the muscle fiber and leads to the release of intracellular calcium from the sarcoplasmic reticulum, a variant of smooth endoplasmic reticulum. The increased cytosolic calcium sets in motion the biochemical events that underlie contraction. The acetylcholine is rapidly hydrolyzed by acetylcholine esterase on the skeletal muscle membrane in this region and thus does not accumulate in the junctional cleft.

Figure 3. Neuromuscular Junction

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.

Figure 5. Effect of Frequency on Repeated Stimulation The strength of muscle contraction is also influenced by the degree of stretch of the muscle. When considering the force of the response to stimulation, it is necessary to separate out the ive and active forces. The ive forces reflect the contributions of elastic elements in the muscle, both extracellularly and within the fibers themselves. The active force is generated by the contractile machinery when the fibers are stimulated (Figure 6). Experimentally, what is measured at different degrees of stretch is the total force during nerve stimulation and contraction and ive force in the absence of nerve stimulation. The difference between the two is the active force at any muscle length.

Figure 6. Skeletal Muscle Length-Tension Relationship Skeletal muscle contraction requires metabolic energy. A depletion of energy stores results in fatigue. Some muscle fibers are more resistant to fatigue than others; these have a greater capacity for oxidative metabolism. Note that in the intact animal, fatigue occurs primarily because the motor drive from the brain is reduced, rather than as a result of an appreciable depletion of the muscle energy reserves.

Required Equipment • • • • • • • • • • • • • • • • •

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LabChart 7 software PowerLab Data Acquisition Unit Bridge Pod Force Transducer Small weight between 5–50 grams Ring Stand Manipulator/Micropositioner and clamps Strong thread Petri dish Pasteur pipette One frog (Rana pipiens or Xenopus laevis) Normal Frog Ringer’s solution Cold (10 oC) Frog Ringer’s 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 of the PowerLab (Figure 7). Connect the Stimulating Electrodes to the output on the front of the PowerLab. Follow the color scheme in Figure 7.

Figure 7. Equipment Setup for PowerLab 26T 3. Securely mount the Force Transducer and Manipulator/Micropositioner on the Ring Stand as shown in Figure 8.

Figure 8. Ring Stand, Manipulator/Micropositioner, and Force Transducer Setup 4. Tape a small millimeter ruler to the side of the Manipulator/Micropositioner to use as a reference when adjusting the height.

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.

Calibrating the Force Transducer Raw output from the Force Transducer is in millivolts (mV). It needs to be calibrated to give the more meaningful units of Newtons (N). The Force Transducer also has some residual offset voltage that needs to be corrected for. 1. Launch LabChart and open the settings file “Frog Muscle Settings” from the Experiments tab in the Welcome Center. It will be located in the folder for this experiment. 2. Select Bridge Pod from the Channel 1 Channel Function pop-up menu. Leave the Force Transducer undisturbed. Observe the signal (Figure 9) in the dialog. Zero this signal by turning the knob on the front of the Bridge Pod. Close the Bridge Pod pop-up menu.

Figure 9. Bridge Pod Dialog 3. Start recording. Record for five seconds, and then hang a known weight (between 5–50 grams, provided by your instructor) from the Force Transducer. Record for a further five seconds, and Stop. 4. Click-and-drag to select all the data. Select Units Conversion from the Channel 1 Channel Function pop-up menu (Figure 10).

Figure 10. Units Conversion Dialog 5. Select a small area when no weight was added, and click the arrow next to “Point 1.” 6. Select a small area when the weight was added, and click the arrow next to “Point 2.” 7. Enter the desired unit value in Newtons for each weight. Use the equation below:

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).

Figure 11. How to Remove the Skin Note: Keep the tissue moist at all times with normal Ringer’s solution.

3. Remove the leg from the frog by severing at the hip t. 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, 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.

Figure 12. Thread Positioned under the Achilles Tendon 5. Sever the Achilles tendon below the attached thread. 6. Using bone shears or strong scissors, cut the tibiofibula bone below the knee, and cut the femur bone above the knee. If you are using the Muscle Holder, cut the femur close to the knee t, leaving little femur remaining. If you are using the femur clamp, cut the femur close to the hip t, leaving as much femur bone as possible for clamping (Figure 13).

Figure 13. Locations to Cut the Leg Bones Note: Keep the muscle in a Petri dish of cold Frog Ringer’s solution until you are ready to mount it in the Muscle Holder or femur clamp.

Equipment Setup if Using a Muscle Holder 1. Attach the Muscle Holder to the Ring Stand. 2. Fix the muscle in the Muscle Holder by positioning the knee t just below the constriction in the Perspex molding, as shown in Figure 14. Connect the Alligator Clips to the two inner connectors at the top of the Muscle Holder.

Figure 14. Muscle Holder 3. Secure the thread attached to the Achilles tendon of the muscle through the hole in the metal tab of the Force Transducer. Make sure there is some slack in the thread. 4. Raise the Manipulator/Micropositioner using the adjustment knob so that the muscle is vertical but not under tension. The thread should not be loose but should have some slack in it. Make sure there is room to increase the height of the Force Transducer by at least 10 mm (Figure 15).

Figure 15. Equipment Setup with Muscle Holder

5. Rotate the base of the Muscle Holder and insert an appropriately-sized beaker to collect waste solution. Rinse the muscle with regular Ringer’s solution to keep it moist. 6. Make sure the silver wires on the Muscle Holder are still in with the muscle. Check that everything is connected correctly.

Equipment Setup if Using a Femur Clamp 1. Attach the femur clamp to the Ring Stand. 2. Fix the muscle in the femur clamp with the knee t side of the muscle in the clamp. 3. Secure the thread attached to the Achilles tendon of the muscle through the hole in the metal tab of the Force Transducer. Make sure there is some slack in the thread. 4. Raise the Manipulator/Micropositioner using the adjustment knob so that the muscle is vertical but not under tension. The thread should not be loose but should have some slack in it. Make sure there is room to increase the height of the Force Transducer by at least 10 mm (Figure 16). 5. Connect the stimulating electrode to a Ring Stand clamp to keep it in place. Touch the electrode tip to the belly of the muscle. (It is referred to as a bipolar stimulator in Figure 16 because there are two electrodes on the tip.)

Figure 16. Equipment Setup with Femur Clamp 6. Insert an appropriately-sized beaker to collect waste solution under the femur clamp. Rinse the muscle with regular Ringer’s solution to keep it moist. 7. Make sure the stimulating electrode is still in with the muscle. Check that everything is connected correctly. Turn on the PowerLab.

Exercise 1: Twitch Recruitment In this exercise, you will examine the effects of stimulus amplitude (strength) on contractile force as the muscle is given a series of stimuli of increasing amplitude. Note: It is essential you watch what is happening to the muscle. You will be asked to describe your observations in the Data Notebook. 1. LabChart should be open. If not, open the settings file “Frog Muscle Settings.” 2. Make sure the muscle is moist and is in with the electrodes. 3. Zero the Bridge Pod. Use the same procedure as before. You do not need to calibrate the data. Note: For this exercise, you will be running a macro to apply a series of increasing stimuli. A macro is a recorded set of commands and operations that can be executed with a single command. 4. Go to the Macro menu and select Recruitment to start the macro. Alternatively, you can press F2. LabChart will start recording, increase the stimulus on its own, and stop recording. (Do not click Start before playing the macro.) 5. When the macro is finished, save your data, but do not close the file. 6. Scroll through your data and Autoscale, if necessary. Start at the end of the data and move toward the beginning. Determine the minimum voltage required to elicit the maximum contraction; this is the maximum excitation voltage. 7. Multiply this voltage by 1.5. This new value is the supramaximal stimulus voltage. Record these values in Table 2 of the Data Notebook. The supramaximal stimulus voltage will be used in each of the following exercises. 8. Wait at least 30 seconds before moving on to the next exercise. Keep the muscle moist with Ringer’s solution.

Exercise 2: Effect of Stretch on Contractile Force In this exercise, you will examine the effect of stretch (also called preload) on muscle contractile force, by raising the Manipulator/Micropositioner. 1. Make sure the muscle is moist and the electrodes are still positioned correctly. 2. Zero the Bridge Pod as before. You do not need to calibrate the data. 3. Note the position of the Manipulator/Micropositioner using the ruler. This is the reference point. Record this value in Table 3 of the Data Notebook. 4. Click on the data at the end of the last data block, and go to the Commands menu and select Add comment. Type “exercise 2” and click Add. 5. Go to the Setup menu and select Stimulator . In the Pulse Height text box, enter the value (in volts) for the supramaximal stimulus voltage calculated in Exercise 1. Do not adjust any other settings. You can close the or keep it open.

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 Ringer’s solution.

Exercise 3: Muscle Summation In this exercise, you will stimulate the muscle with twin pulses at different pulse intervals and will observe their effect on muscle contractions. 1. Make sure the muscle is moist and the electrodes are still positioned correctly. 2. Zero the Bridge Pod as before. 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 3” and click Add. Note: For this exercise, you will be running a 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 Summation to start the macro. Alternatively, you can press F4. LabChart will start recording and will prompt you with messages. 5. Follow the on-screen instructions. The PowerLab will stimulate the muscle with twin pulses 400 ms, 200 ms, 100 ms, 50 ms, and 20 ms apart. 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, select “Insert at selection” and click Add. 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 Ringer’s solution.

Exercise 4: Muscle Tetanus In this exercise, you will examine the muscle’s response to repetitive stimulation at five different frequencies, each for a 1 second duration.

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 Ringer’s solution.

Exercise 5: Muscle Fatigue In this exercise, you will examine muscle fatigue, evoked by prolonged stimulation of the muscle at a high frequency (50 Hz for 30 seconds). 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 5” and click Add. Note: For this exercise, you will be running a final macro to stimulate the muscle repetitively. Do not click Start before playing the macro. 4. Go to the Macro menu and select Fatigue to start the macro. Alternatively, you can press F6. LabChart will start recording and will prompt you with messages. 5. Follow the on-screen instructions. The PowerLab will stimulate the muscle with a stimulus interval of 20 ms for 30 seconds. The recording duration of the macro is 45 seconds. 6. Save your data. Do not close the file.

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.

Exercise 2: Effect of Stretch on Contractile Force 1. Examine the data in the Chart View, and Autoscale, if necessary. 2. If the Marker is still in the Chart View, click in the lower left corner to return it to its Marker box. 3. Using the Waveform Cursor, measure the baseline value. This is the preload force. 4. Determine the raw twitch force with the Waveform Cursor by placing the cursor on the maximum value in the data block. Record this value in Table 3 of the Data Notebook. 5. Repeat steps 3 and 4 for the other data blocks in this exercise. 6. Calculate the net twitch force by subtracting the preload (baseline) value from the raw twitch force value. 7. Record these values in Table 3 of the Data Notebook.

Exercise 3: Muscle Summation 1. Examine the data in the Chart View, and Autoscale, if necessary. There will be five blocks of recorded data. 2. For each data block, determine the maximum contractile force of the first peak and second peak using the Marker and Waveform Cursor. Position the Marker on the baseline immediately before the first contraction. Use the Waveform Cursor to measure the maximum heights of the two contractions. 3. Record these values in Table 4 of the Data Notebook. Note: When the peaks merge, put a single measurement in the second response column of Table 4.

Exercise 4: Muscle Tetanus 1. Examine the data in the Chart View, and Autoscale, if necessary. There should be five blocks of data. 2. Determine the maximum contractile force using the Marker and Waveform Cursor, as done for Exercise 3. Follow the steps above. 3. Record these values in Table 5 of the Data Notebook.

Exercise 5: Muscle Fatigue 1. Examine the data in the Chart View, and Autoscale, if necessary. 2. Place the Marker on the waveform baseline in the Force channel immediately prior to stimulation. 3. Use the Waveform Cursor to determine the maximum contractile force. Record this value in Table 6 of the Data Notebook. 4. Record the time of maximum stimulation in Table 6 of the Data Notebook. 5. Determine the contractile force at the following times: • • • • • • •

t t t t t t t

= = = = = = =

1s 5s 10 s 15 s 20 s 25 s 30 s

6. Record each of these values in Table 6 of the Data Notebook.

Data Notebook Exercise 1 Observations a) Describe the contractile force of the muscle when the stimulus strength was increased.

Table 1. Effect of Stimulus Intensity on Contractile Force Stimulus Amplitude (V) 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.80 0.85 0.90 0.95 1.00

Contractile Force (N)

Table 2. Supramaximal Stimulus Voltage Minimum Voltage Required For Maximal Contraction (V)

Supramaximal Stimulus Voltage (V)

Table 3. Effect of Preload on Contractile Force Reference position of micropostitioner (mm) = Preload (N)

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)

Table 4. Effect of Stimulus Frequency on Contractile Force Stimulus Interval (ms) 400 200 100 50 20

Force of First Peak (N)

Force of Second Peak (N)

Table 5. Muscle Tetanus Stimulus Interval (ms) 400

Contractile Force (N)

200 100 50 20

Table 6. Muscle Fatigue Time at Maximum Force (s, post-stimulation)

Maximum Force (N)

Time Since Stimulation (s) 1

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.

Define tetanus. At which stimulus interval did you observe tetanus?

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.