{"id":95,"date":"2025-11-19T18:01:30","date_gmt":"2025-11-19T18:01:30","guid":{"rendered":"https:\/\/press.wpunj.edu\/animalphysiologylabmanual\/chapter\/__unknown__-3\/"},"modified":"2025-11-19T18:26:17","modified_gmt":"2025-11-19T18:26:17","slug":"musclecontraction","status":"publish","type":"chapter","link":"https:\/\/press.wpunj.edu\/animalphysiologylabmanual\/chapter\/musclecontraction\/","title":{"raw":"Lab 6: Frog Leg Muscle Contraction","rendered":"Lab 6: Frog Leg Muscle Contraction"},"content":{"raw":"
\r\n

In this lab activity, you will learn how to isolate the gastrocnemius muscle of the frog and its somatic motor nerve (sciatic nerve) and record the contractile responses resulting from an applied electrical stimulus to understand how neuronal impulses can elicit muscle contractions.<\/p>\r\n

Learning objectives for this lab activity:<\/strong><\/p>\r\n

1. Prepare a pithed frog for the study of muscle physiology.<\/p>\r\n

2. Describe how muscle can contract when an electrical stimulus is applied.<\/p>\r\n

3. Explain how motor nerves stimulate the contraction of skeletal muscles.<\/p>\r\n

4. To record the twitch Threshold, maximal twitch response, summation, tetanus and fatigue for frog skeletal muscle and its motor nerve.<\/p>\r\n

5. Examine the effects of the length of the sarcomere on the force of contraction.<\/p>\r\n

Background:<\/strong><\/p>\r\n

The ability to move is a basic property of life permitting an animal to respond to its environment. Depending upon the structural complexity of the organism, the mechanisms for motion can appear very different. For example, amoeboid movement, ciliary and flagellar motion, and muscular activity all appear very diverse. However, upon close inspection, the basic molecular mechanisms are very similar involving almost identical chemicals but in different spatial arrangements. For metazoan, contractile structures are organized into individual or groups of cells known as muscle fibers. Myocytes can efficiently transform chemical energy into a mechanical force that is capable of work.<\/strong> Muscle, perhaps more than any other physiological system, has been studied thoroughly. Of the three muscle types, smooth, cardiac and skeletal, the later is best understood. We know more about skeletal muscles at all levels of organization than any other system. In this laboratory, we will look at the contractile properties of the gastrocnemius muscle<\/strong> from the lower leg of the grass frog, Rana pipiens<\/em>. Muscle tissues are relatively easy to work with experimentally. They can be removed from an organism intact with little loss of function. Also, muscle tissue can be taken from an organism without removing other noncontractile tissue. Since muscular contractions are large in magnitude, they can be accurately measured with simple recording equipment.<\/strong><\/p>\r\n

For in vitro<\/em> contraction studies, there are two major preparations: 1) isotonic<\/strong> and 2) isometric<\/strong>. In the former, isotonic, the muscle can move exerting a constant force as the fiber changes length. In the latter, isometric, the length of the muscle is held constant as the force of contract varies. The physiology of contraction can be studied in muscles isolated from a pithed frog. The preparation can be stimulated directly by an electric shock or indirectly through activation of the appropriate motor nerve. In the gastrocnemius muscle experiments described here, we will examine properties of isotonic contractions.<\/strong><\/p>\r\n

Stimulation of Motor Nerve<\/strong><\/p>\r\n

In the vertebrates, skeletal muscle contractions are evoked in vivo by impulses in somatic motor neurons. The soma or cell body of motor or efferent neurons is located in the ventral gray horn of the spinal cord (Figure 1).<\/p>\r\n

\"\"<\/p>\r\n

Normally, electrical activity in the somatomotor fibers originates in the spinal cord and travels over a single axon to a myocyte or to a group of myocytes known as a motor unit. The motor neurons<\/strong> are activated either by other neurons in the brain or spinal cord, or by local sensory neurons<\/strong>. Impulses in efferent neurons may also be stimulated by damaging fibers peripherally. This damage produces an injury current, which stimulates action potentials. This is why a muscle twitch<\/strong> occurs when a nerve is pinched. Once the axon of motor neurons leaves the spinal cord, it joins a nerve<\/strong> along with hundreds of other motor and sensory neurons. Each efferent somatic neuron transmits impulses from the spinal cord directly to a specific group of myocytes evoking contraction. The form of the contraction expressed by skeletal myocytes, or motor unit depends upon the pattern of motor neuron stimulation. For example, the gastrocnemius is innervated by hundreds of motor neurons in the sciatic nerve<\/strong>. Each motor neuron has a threshold voltage<\/strong> for activation. If a neuron is stimulated with a single, small supra-threshold voltage<\/strong>, a single all-or-none contraction<\/strong> occurs in all myocytes of a motor unit. This is a twitch<\/strong>. When more neurons are activated by increasing stimulus strength, additional motor units contract increasing muscular strength, and the twitch is larger. When all efferent neurons are activated for a muscle, a maximum-sized twitch occurs indicating all motor units have been recruited<\/strong>. Supra-maximal voltages<\/strong> can recruit no additional motor units and do not increase muscular strength. This relationship between neuron stimulation, motor unit activation, and muscle contraction is known as excitation-contraction coupling<\/strong>. Because increasing stimulus amplitude recruits more motor units, this pattern or kind of response is known as spatial<\/strong> or motor-unit summation<\/strong>.<\/p>\r\n

In addition to recruitment of motor units by increasing stimulus amplitude (i.e. voltage,) variation in contraction can be achieved by temporal summation<\/strong>. Action potentials in the motor nerve fibers elicit the release of a chemical neurotransmitter called acetylcholine<\/strong> (ACh) from the axon endings. This transmitter combines with nicotinic receptors<\/strong> to produce action potentials on the membrane of the skeletal myocyte. An electrical impulse in the muscle cell, in turn, causes Ca++ to be released from the sarcoplasmic reticulum<\/strong> (SR). At the molecular level, for contraction, the release of Ca++ into the cytosol permits actin and myosin to interact in the presence of ATP. Actin-myosin interactions and contractions are relaxed when Ca++ is re-absorbed into the SR by a Ca++ pump.<\/p>\r\n

The rate<\/strong> at which myocytes are stimulated influences how long Ca++ lingers in the cytoplasm. If a myocyte receives a single, sub-maximum stimulus, Ca++ is released and quickly re-absorbed. If the myocyte receives a second impulse before relaxation is fully affected, the amount of cytoplasmic Ca++ in the second contraction is greater than the first. Consequently, the strength (i.e. force in grams) of the 2nd contraction is greater (Figure 2).<\/p>\r\n

\"\"<\/p>\r\n

If no other impulse appears, all Ca++ is returned to the SR. If a train or volley of sub-maximal impulses is used to stimulate a myocyte, the strength of the contraction is proportional to the frequency of stimulation. At the maximum rate, the myocyte remains in a persistent, sustained contraction known as tetanus<\/strong>. If the rate of stimulation (pulses\/second) is increased above the tetanizing frequency<\/strong>, no greater increase in strength occurs. At frequencies of stimulation between initial temporal response and tetany, contractions express partial relaxations. From its appearance, this response is called treppe<\/strong> or stair-step<\/strong>. In temporal summation, by varying the stimulating frequency we manipulate cytoplasmic Ca++ and molecular interactions within motor units to control the strength of contraction.<\/p>\r\n

For the frog gastrocnemis, there are two modes or ways to simulate the muscle with electrodes. The way most resembling a physiological state is to apply an exogenous electrical signal indirectly via the sciatic nerve. Alternatively, the electrodes can be inserted directly into the muscle which applies the stimulus directly to the myocytes.\u00a0 If you are able to activate muscular contraction via the sciatic nerve, DO NOT use excessively large supra-maximal stimuli. These large voltages will destroy the neurons.<\/strong><\/p>\r\n

In striated myocytes, contractile proteins are assembled in linear, parallel arrays stabilized by z-discs<\/strong>, titan<\/strong> and nebulin<\/strong> in each sarcomere<\/strong>. Sarcomeres are arranged in a series form myofibrils, hundreds of which fill the sarcoplasm<\/strong> of the muscle cell. When Ca++ is abundant in the sarcoplasm, actin and myosin bind forming the rigor complex<\/strong>. In the presence of ATP, the two molecules cycle continuously between the bound and unbound states. As a consequence, filamentous actin and myosin macromolecules slide past each other in opposite directions producing the force of contraction. When Ca++ is removed, in lieu <\/em>of the ATP concentration, the actinomyosin<\/strong> complex is blocked and the rigor complex relaxes. The force generated during contraction of a sarcomere is related to its shape. If a myocyte is extirpated from tendons and elastic components of the fascia or aponeuroses<\/strong> contract, thick and thin filaments overlap maximally in each sarcomere. When the myocyte is stimulated, no force is generated. On the other hand, if the fiber is stretched to its limits just before tearing, actin and myosin filaments do not overlap. Again no force can be generated by the fiber with stimulation. At intermediate lengths, the thick and thin filaments overlap to various degrees depending upon degree of stretch. Thus, muscle strength (in grams) is related to length (in mm) in a very precise way. A plot of gastrocnemius muscle force versus length<\/strong> will illustrate an optimum length of the muscle for generating a force. This is usually a dome-shaped curve with maximum force generated at an intermediate length. In situ<\/em>, skeletal muscles are usually stretched to intermediate length by ligaments anchoring them to bone so they can generate maximum force if it is desired.<\/p>\r\n

Fatigue<\/strong> is a physiology state commonly observed in biological preparations. It is a decline or decrease in the ability to respond when repeatedly presented the same stimulus. In muscle preparations, fatigue can occur for several reasons. First, impulse frequency in motor neurons can decrease. Recall that the action potentials in neurons and myocytes are all-or-none signals, reoccurring with the same amplitude (or height). Since the frequency of impulses regulates amount of neurotransmitter released, a decline in neuronal impulse frequency results in fewer impulses and less neurotransmitter to the myocyte. Second, the neuron and myocyte communicate via a synapse at the neuromuscular junction<\/strong> (or endplate).<\/p>\r\n

If the pre-synaptic membrane does not replace vesicle containing neurotransmitter, the ability to produce a post-synaptic impulse in the myocyte will decrease. And third, the myocyte can become less responsive with prolonged stimulation. This occurs by either of two ways, decrease in Ca++ availability or decrease in ATP (or other energetic molecules). Since it is abundant is external solutions and stored in the sarcoplasmic reticulum, Ca++ depletion occurs infrequently and is not usually related to fatigue. However, a sudden influx Ca++ without an impulse produces a spontaneous twitch known as a \u201cspasm<\/strong>\u201d or \u201ccramp.<\/strong>\u201d Most often fatigue results from a decline in the availability of biological energy. When a myocyte is stimulated rapidly to tetany, ATP is consumed rapidly sustaining the process. Since the myocyte manufactures ATP at a finite rate, energy supply cannot match demand and consumption. Consequently, when stimulation produces tetany, fatigue results from a decline in available ATP. If tetany is interrupted for a short period (few seconds), energy production will restore maximum muscular performance. You can observe this in your gastrocnemis preparation.<\/p>\r\n

In the following experiment, the frog sciatic and gastrocnemius will be exposed to study basic physiological properties of muscle contraction. Initially, you will determine Threshold and examine the components of muscular contraction: latent, contraction <\/strong>and relaxation phases<\/strong> or periods. Also, you will study motor unit summation<\/strong> and temporal summation<\/strong> by independently changing stimulus amplitude and frequency while observing the effect on muscle responses. Within the vertebrate body, fine motor control is a product of the simultaneous integration<\/strong> of temporal and spatial summation (Figure 3).<\/p>\r\n

\"\"<\/p>\r\n\r\n

Experimental setup:<\/strong><\/h5>\r\n

\"\"<\/p>\r\n

Experiment AM-10: Summation, Tetanus, and Fatigue in an Intact Nerve-Muscle Prep<\/strong>\r\nBy: RJ Cooper, Ph.D., Margaret A. Weck, D.A., and Dayton J. Ford, Ph.D.: at the St. Louis College of <\/em>Pharmacy. Adapted from: Hoff, H.E. and L.A. Geddes, Experimental Physiology (1965)<\/em><\/p>\r\n

Equipment Required<\/strong><\/p>\r\n

PC or Mac Computer\r\nIWX\/214, USB cable, IWX\/214 power supply\r\nFT-104 Force transducer\r\nC-STIM-BNC-N2 Needle-type stimulating electrodes\r\nC-BNC-SE Sleeve-type stimulating electrodes\r\n<\/span>Female BNC to Dual Banana Adapter\r\nRingstand and clamps\r\nMuscle tension adjuster\r\nThread\r\nFrog board\r\nDissection tray\r\nGlass dissection hooks\r\n1cc tuberculin syringe with needle\r\nAmphibian Ringer\u2019s solution (see Appendix)\r\n1% Tubocurarine solution in Ringer\u2019s<\/p>\r\n

For details of the setup please see the \u201cFrogNerveMuscle_Setup214\u201d PDF document.<\/strong><\/p>\r\n\r\n

Procedure:<\/strong><\/h5>\r\n

Start the Software<\/p>\r\n

1. Click on LabScribe\r\n2. Click Settings _ Animal Muscle _ FrogNerveMuscle<\/p>\r\n\r\n

Exercise 1: Twitch Threshold Determined by Direct Stimulation<\/strong><\/h3>\r\n

Aim:<\/strong> To determine the threshold stimulus of the muscle when it is stimulated directly with single pulses of constant duration.<\/p>\r\n

Approximate Time: 15 minutes<\/p>\r\n

\u2022 Plug in the Pin stimulator into the SI port, Leave the sleeve stimulator on the nerve but not plugged in to the IWORX box<\/strong>\r\n\u2022 DO NOT PIN THE FOOT, Pin skin around the knee<\/strong><\/p>\r\n

1. Click the Stimulator Preferences icon on the LabScribe toolbar (Figure AM-10-L1) to open the stimulator control panel (Figure AM-10-L2) on the Main window.<\/p>\r\n

\"\"<\/p>\r\n

Figure AM-10-L1: The <\/em>LabScribe<\/em> toolbar.<\/em><\/p>\r\n

2. Check the values for the stimulus parameters that are listed in the stimulator control panel on the Main window:<\/p>\r\n

\u2022 the pulse amplitude (Amp) should be set to 0.000 V;\r\n\u2022 the number of pulses (#pulses) to 1;\r\n\u2022 the frequency (F(Hz)) to 1;\r\n\u2022 and, the pulse width (W) to 5 ms.\r\n\u2022 Click APPLY in the upper left of the control panel<\/p>\r\n

3. The value for a stimulus parameter can be changed by either of two methods: click on the arrow buttons to the right of the window that displays the value of the parameter to increase or decrease the value; or, type the value of the parameter in the window next to the label of the parameter. Click the Apply button to finalize the change in any stimulus parameter.<\/p>\r\n

\"\"<\/p>\r\n

Figure AM-10-L2: The stimulator control panel<\/em><\/p>\r\n

4. Type 0.000V in the Mark box to the right of the Mark button. Click Record to stimulate the nerve with 0.000V and press the mark button to attach the comment to the recording.<\/p>\r\n

5. Apply a light load to the muscle by raising the force transducer with the tension adjuster until the trace moves a few mV above the baseline. If you are using a FT-302 force transducer, the initial baseline can be adjusted to 0 mV by rotating the offset knob on top of the FT-302 transducer.<\/p>\r\n

6. Once a light load is applied to the muscle, Click on the Stop.<\/p>\r\n

7. Change the stimulus amplitude (Amp) to 0.1V using one of the techniques described in Step 2. Click the Apply button on the Stimulator control panel to effect the change in amplitude.<\/p>\r\n

8. Type 0.1V in the Mark box to the right of the Mark button. Click Record to stimulate the nerve with 0.1V and press the mark button to attach the comment to the recording. Stop the recording as soon as the muscle twitch is finished, or after a couple of seconds if no twitch is detected.<\/p>\r\n

Note: Be sure to have at least one member of your lab group <\/em>LOOKING at the specimen at all times<\/em>.<\/em> It is possible for contractions to occur and not be recorded. Click <\/em>AutoScale<\/em> to make sure the<\/em> recording is displayed properly. Check the tautness of the thread going from the tendon to the<\/em> transducer, the transducer itself, and check the connection from the transducer to the <\/em>iWorx<\/em> recorder.<\/em><\/strong><\/p>\r\n

9. Determine the threshold voltage for direct muscle stimulation by increasing the stimulus voltage in 0.1V increments. DON\u2019T SKIP A VALUE and DO NOT EXCEED 1V.<\/span> Remember to click the Apply button on the Stimulator control panel each time it is changed. Record and mark the muscle response as described in Steps 3 and 7.<\/p>\r\n

10. Once the threshold voltage has been determined, test the muscle\u2019s response to direct stimulation at additional stimulus voltages (Figure AM-10-L3). Increase the stimulus amplitude in 1.0V increments, up to 5 V. Record the muscle response and mark the recording as described in Steps 3 and 6.<\/p>\r\n

11. Select Save As in the File menu, type a name for the file. Click on the Save button to save the data file.<\/p>\r\n

\"\"<\/p>\r\n

Figure AM-10-L3: Muscle twitches resulting from direct stimulation of the muscles.<\/em><\/p>\r\n\r\n

Exercise 2: Twitch Threshold Determined by Nerve Stimulation<\/strong><\/h3>\r\n

Aim<\/strong>: In this exercise, the muscle will be stimulated by action potentials generated in the nerve attached to the muscle. The nerve will be stimulated by single pulses of constant duration.<\/p>\r\n

Approximate Time: 30 minutes<\/p>\r\n

1. On the iWorx box, disconnect the BNC connector of the pin electrodes used for direct<\/p>\r\n

stimulation of the muscle. Leave the pin electrodes in place in the muscle.<\/p>\r\n

2. On the iWorx box, connect the BNC connector of the sleeve electrodes to the BNC-banana adapter on the stimulator output of the IWX\/214 or to the BNC output of the stimulator on the front of the IXTA.<\/p>\r\n

3. Reset the pulse amplitude (Amp) to 0.000V. Remember to click the Apply button on the stimulator control panel to finalize the change.<\/p>\r\n

4. Repeat Exercise 1 while stimulating the gastrocnemius muscle through the sciatic nerve with single stimulus pulses of increasing amplitude (Figure AM-10-L4). Label the recording at each voltage attempted.<\/p>\r\n

5. Determine the threshold voltage for nerve stimulation by increasing the stimulus voltage in 0.1V increments. Above threshold, test the muscle\u2019s response to nerve stimulation in 1.0V increments.<\/p>\r\n

6. Click the Save button to save the file.<\/p>\r\n

\"\"<\/p>\r\n

Figure AM-10-L4: Muscle twitches resulting from nerve stimulation.<\/em><\/p>\r\n\r\n

Exercise 3: Effect of Stimulus Frequency on Twitch Amplitude<\/strong><\/h3>\r\n

Aim<\/strong>: In this exercise, the muscle will be stimulated by action potentials generated in the nerve attached to the muscle. The nerve will be stimulated for short periods at different stimulus frequencies.<\/p>\r\n

Approximate Time: 30 minutes<\/p>\r\n

1. On the iWorx box, leave the BNC connector of the sleeve electrodes attached to the BNC banana adapter on the stimulator output of the IWX\/214 or on the stimulator output on the front of the IXTA.<\/p>\r\n

2. Set the stimulus frequency (F(Hz)) to 2 Hz, the number of pulses (#pulses) to zero (0), and the pulse amplitude (Amp) to the lowest value that still gave strong contractions in the previous exercise. This voltage will usually be in the range of 0.400V to 1.000 V, but your frog may be different. Keep the pulse width (W) at 5 ms. Remember to click the Apply button on the Stimulator control panel with each change.<\/p>\r\n

Warning: Since the number of <\/em>pulses (#<\/em>pulses) is set to zero (0), the stimulus pulses will be delivered<\/em> continuously at the frequency selected. It is important to stop recording once you see the effect of<\/em> stimulating at a certain frequency.<\/em><\/strong><\/p>\r\n

3. Type 2 Hz in the Mark box to the right of the Mark button. Click Record to stimulate the nerve and click the mark button to mark the recording. Stop the recording after 5 seconds<\/p>\r\n

4. Change the stimulus frequency to 4 Hz. Repeat Step 3. Continue the recording for stimulus frequencies of 5, 10, 25, 50, and 100 Hz (Figure AM-10-L5).<\/p>\r\n

5. Click the Save button to save the file.<\/p>\r\n

6. When you analyze the data for this exercise, note the frequency at which wave summation begins, the frequencies at which incomplete and complete tetanus occur, and the shape of the contraction waves and any indication of fatigue.<\/p>\r\n

\"\"<\/p>\r\n

Figure AM-10-L5: Muscle response from nerve stimulation with increased stimulus frequencies.<\/em><\/p>\r\n\r\n

Exercise 4: Fatigue with Repeated Stimuli<\/strong><\/h3>\r\n

Approximate Time: 45 minutes<\/p>\r\n

1. On the iWorx box, disconnect the sleeve electrodes from the stimulator output, and connect the pin electrodes to the stimulator. Leave the sleeve electrodes in place on the nerve.<\/p>\r\n

2. Set the Display Time to 20 sec, the stimulus frequency to 4 Hz, the number of pulses (#pulses) to zero (0), and pulse amplitude (Amp) to an adequate value between 0.4V and 1 V. Remember to click the Apply button.<\/p>\r\n

3. Type 4Hz-Direct in the Mark box to the right of the Mark button. Click Record to stimulate the nerve with 4 Hz and press the mark button to attach the comment to the recording. Stop the recording after 5 seconds<\/p>\r\n

4. After recording at 4Hz with the pin electrodes, switch to the sleeve electrodes.<\/p>\r\n

5. Type 4Hz-Nerve in the Mark box to the right of the Mark button. Click on the Record button to stimulate the nerve with 4 Hz and press the mark button to attach the comment to the recording. Stop the recording after 5 seconds.<\/p>\r\n

6. Change the stimulus frequency to 50 Hz. Remember to click the Apply button on the Stimulator control panel.<\/p>\r\n

7. Type 50Hz-Nerve in the Mark box to the right of the Mark button. Click Record to stimulate the nerve with 50Hz and press the mark button to attach the comment to the recording. Continue to stimulate through the nerve until the force of the muscle contraction drops to an amplitude that is only 25% of the maximum force at the beginning of this tetanic contraction.<\/p>\r\n

8. Continue to record after the contraction force of the muscle has dropped below 25% of maximum. Quickly disconnect the BNC connector of the sleeve electrodes from stimulator, and reconnect the BNC connector of the pin electrodes to the stimulator.<\/p>\r\n

9. Stimulate the muscle directly for 3 seconds, with the same voltage, duration, and frequency that was used to fatigue the muscle.<\/p>\r\n

Note: If no contraction is seen while using these stimulus parameters, increase the stimulus amplitude<\/em> until a contraction is measured.<\/em><\/strong><\/p>\r\n

10. Continue to record after the direct stimulation of the muscle. Quickly disconnect the BNC connector of the pin electrodes from the stimulator, and reconnect the BNC connector of the sleeve electrodes to the stimulator.<\/p>\r\n

11. Apply the same stimulus used in Step 9 to the nerve for a period of 3 seconds.<\/p>\r\n

12. After you stop the recording, label the recording with marks and notations to indicate the site of stimulation (muscle or nerve) and the stimulus voltage that generated each response.<\/p>\r\n

13. Click the Save button to save the file.<\/p>\r\n

14. When you analyze the data for this exercise:<\/p>\r\n

\u2022 Note the shape of the contraction waves and any indication of fatigue.\r\n\u2022 Note whether the muscle contraction was stronger when the stimulus was applied directly to the muscle or when the stimulus was applied to the nerve. What does this indicate about the location of the fatigue observed during nerve stimulation?<\/p>\r\n

\"\"<\/p>\r\n

Figure AM-10-L6: Decrease in muscle force (fatigue) with continuous, high frequency stimulation<\/em> through the nerve innervating the muscle. The period of fatigue occurring during nerve stimulation is<\/em> followed by a large response by the muscle when it is stimulated directly.<\/em><\/p>\r\n

Note: As you complete each exercise, make sure to record your data and answer the questions in the lab report document.<\/strong><\/span><\/p>\r\n

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<\/p>\r\n\r\n<\/div>","rendered":"

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In this lab activity, you will learn how to isolate the gastrocnemius muscle of the frog and its somatic motor nerve (sciatic nerve) and record the contractile responses resulting from an applied electrical stimulus to understand how neuronal impulses can elicit muscle contractions.<\/p>\n

Learning objectives for this lab activity:<\/strong><\/p>\n

1. Prepare a pithed frog for the study of muscle physiology.<\/p>\n

2. Describe how muscle can contract when an electrical stimulus is applied.<\/p>\n

3. Explain how motor nerves stimulate the contraction of skeletal muscles.<\/p>\n

4. To record the twitch Threshold, maximal twitch response, summation, tetanus and fatigue for frog skeletal muscle and its motor nerve.<\/p>\n

5. Examine the effects of the length of the sarcomere on the force of contraction.<\/p>\n

Background:<\/strong><\/p>\n

The ability to move is a basic property of life permitting an animal to respond to its environment. Depending upon the structural complexity of the organism, the mechanisms for motion can appear very different. For example, amoeboid movement, ciliary and flagellar motion, and muscular activity all appear very diverse. However, upon close inspection, the basic molecular mechanisms are very similar involving almost identical chemicals but in different spatial arrangements. For metazoan, contractile structures are organized into individual or groups of cells known as muscle fibers. Myocytes can efficiently transform chemical energy into a mechanical force that is capable of work.<\/strong> Muscle, perhaps more than any other physiological system, has been studied thoroughly. Of the three muscle types, smooth, cardiac and skeletal, the later is best understood. We know more about skeletal muscles at all levels of organization than any other system. In this laboratory, we will look at the contractile properties of the gastrocnemius muscle<\/strong> from the lower leg of the grass frog, Rana pipiens<\/em>. Muscle tissues are relatively easy to work with experimentally. They can be removed from an organism intact with little loss of function. Also, muscle tissue can be taken from an organism without removing other noncontractile tissue. Since muscular contractions are large in magnitude, they can be accurately measured with simple recording equipment.<\/strong><\/p>\n

For in vitro<\/em> contraction studies, there are two major preparations: 1) isotonic<\/strong> and 2) isometric<\/strong>. In the former, isotonic, the muscle can move exerting a constant force as the fiber changes length. In the latter, isometric, the length of the muscle is held constant as the force of contract varies. The physiology of contraction can be studied in muscles isolated from a pithed frog. The preparation can be stimulated directly by an electric shock or indirectly through activation of the appropriate motor nerve. In the gastrocnemius muscle experiments described here, we will examine properties of isotonic contractions.<\/strong><\/p>\n

Stimulation of Motor Nerve<\/strong><\/p>\n

In the vertebrates, skeletal muscle contractions are evoked in vivo by impulses in somatic motor neurons. The soma or cell body of motor or efferent neurons is located in the ventral gray horn of the spinal cord (Figure 1).<\/p>\n

\"\"<\/p>\n

Normally, electrical activity in the somatomotor fibers originates in the spinal cord and travels over a single axon to a myocyte or to a group of myocytes known as a motor unit. The motor neurons<\/strong> are activated either by other neurons in the brain or spinal cord, or by local sensory neurons<\/strong>. Impulses in efferent neurons may also be stimulated by damaging fibers peripherally. This damage produces an injury current, which stimulates action potentials. This is why a muscle twitch<\/strong> occurs when a nerve is pinched. Once the axon of motor neurons leaves the spinal cord, it joins a nerve<\/strong> along with hundreds of other motor and sensory neurons. Each efferent somatic neuron transmits impulses from the spinal cord directly to a specific group of myocytes evoking contraction. The form of the contraction expressed by skeletal myocytes, or motor unit depends upon the pattern of motor neuron stimulation. For example, the gastrocnemius is innervated by hundreds of motor neurons in the sciatic nerve<\/strong>. Each motor neuron has a threshold voltage<\/strong> for activation. If a neuron is stimulated with a single, small supra-threshold voltage<\/strong>, a single all-or-none contraction<\/strong> occurs in all myocytes of a motor unit. This is a twitch<\/strong>. When more neurons are activated by increasing stimulus strength, additional motor units contract increasing muscular strength, and the twitch is larger. When all efferent neurons are activated for a muscle, a maximum-sized twitch occurs indicating all motor units have been recruited<\/strong>. Supra-maximal voltages<\/strong> can recruit no additional motor units and do not increase muscular strength. This relationship between neuron stimulation, motor unit activation, and muscle contraction is known as excitation-contraction coupling<\/strong>. Because increasing stimulus amplitude recruits more motor units, this pattern or kind of response is known as spatial<\/strong> or motor-unit summation<\/strong>.<\/p>\n

In addition to recruitment of motor units by increasing stimulus amplitude (i.e. voltage,) variation in contraction can be achieved by temporal summation<\/strong>. Action potentials in the motor nerve fibers elicit the release of a chemical neurotransmitter called acetylcholine<\/strong> (ACh) from the axon endings. This transmitter combines with nicotinic receptors<\/strong> to produce action potentials on the membrane of the skeletal myocyte. An electrical impulse in the muscle cell, in turn, causes Ca++ to be released from the sarcoplasmic reticulum<\/strong> (SR). At the molecular level, for contraction, the release of Ca++ into the cytosol permits actin and myosin to interact in the presence of ATP. Actin-myosin interactions and contractions are relaxed when Ca++ is re-absorbed into the SR by a Ca++ pump.<\/p>\n

The rate<\/strong> at which myocytes are stimulated influences how long Ca++ lingers in the cytoplasm. If a myocyte receives a single, sub-maximum stimulus, Ca++ is released and quickly re-absorbed. If the myocyte receives a second impulse before relaxation is fully affected, the amount of cytoplasmic Ca++ in the second contraction is greater than the first. Consequently, the strength (i.e. force in grams) of the 2nd contraction is greater (Figure 2).<\/p>\n

\"\"<\/p>\n

If no other impulse appears, all Ca++ is returned to the SR. If a train or volley of sub-maximal impulses is used to stimulate a myocyte, the strength of the contraction is proportional to the frequency of stimulation. At the maximum rate, the myocyte remains in a persistent, sustained contraction known as tetanus<\/strong>. If the rate of stimulation (pulses\/second) is increased above the tetanizing frequency<\/strong>, no greater increase in strength occurs. At frequencies of stimulation between initial temporal response and tetany, contractions express partial relaxations. From its appearance, this response is called treppe<\/strong> or stair-step<\/strong>. In temporal summation, by varying the stimulating frequency we manipulate cytoplasmic Ca++ and molecular interactions within motor units to control the strength of contraction.<\/p>\n

For the frog gastrocnemis, there are two modes or ways to simulate the muscle with electrodes. The way most resembling a physiological state is to apply an exogenous electrical signal indirectly via the sciatic nerve. Alternatively, the electrodes can be inserted directly into the muscle which applies the stimulus directly to the myocytes.\u00a0 If you are able to activate muscular contraction via the sciatic nerve, DO NOT use excessively large supra-maximal stimuli. These large voltages will destroy the neurons.<\/strong><\/p>\n

In striated myocytes, contractile proteins are assembled in linear, parallel arrays stabilized by z-discs<\/strong>, titan<\/strong> and nebulin<\/strong> in each sarcomere<\/strong>. Sarcomeres are arranged in a series form myofibrils, hundreds of which fill the sarcoplasm<\/strong> of the muscle cell. When Ca++ is abundant in the sarcoplasm, actin and myosin bind forming the rigor complex<\/strong>. In the presence of ATP, the two molecules cycle continuously between the bound and unbound states. As a consequence, filamentous actin and myosin macromolecules slide past each other in opposite directions producing the force of contraction. When Ca++ is removed, in lieu <\/em>of the ATP concentration, the actinomyosin<\/strong> complex is blocked and the rigor complex relaxes. The force generated during contraction of a sarcomere is related to its shape. If a myocyte is extirpated from tendons and elastic components of the fascia or aponeuroses<\/strong> contract, thick and thin filaments overlap maximally in each sarcomere. When the myocyte is stimulated, no force is generated. On the other hand, if the fiber is stretched to its limits just before tearing, actin and myosin filaments do not overlap. Again no force can be generated by the fiber with stimulation. At intermediate lengths, the thick and thin filaments overlap to various degrees depending upon degree of stretch. Thus, muscle strength (in grams) is related to length (in mm) in a very precise way. A plot of gastrocnemius muscle force versus length<\/strong> will illustrate an optimum length of the muscle for generating a force. This is usually a dome-shaped curve with maximum force generated at an intermediate length. In situ<\/em>, skeletal muscles are usually stretched to intermediate length by ligaments anchoring them to bone so they can generate maximum force if it is desired.<\/p>\n

Fatigue<\/strong> is a physiology state commonly observed in biological preparations. It is a decline or decrease in the ability to respond when repeatedly presented the same stimulus. In muscle preparations, fatigue can occur for several reasons. First, impulse frequency in motor neurons can decrease. Recall that the action potentials in neurons and myocytes are all-or-none signals, reoccurring with the same amplitude (or height). Since the frequency of impulses regulates amount of neurotransmitter released, a decline in neuronal impulse frequency results in fewer impulses and less neurotransmitter to the myocyte. Second, the neuron and myocyte communicate via a synapse at the neuromuscular junction<\/strong> (or endplate).<\/p>\n

If the pre-synaptic membrane does not replace vesicle containing neurotransmitter, the ability to produce a post-synaptic impulse in the myocyte will decrease. And third, the myocyte can become less responsive with prolonged stimulation. This occurs by either of two ways, decrease in Ca++ availability or decrease in ATP (or other energetic molecules). Since it is abundant is external solutions and stored in the sarcoplasmic reticulum, Ca++ depletion occurs infrequently and is not usually related to fatigue. However, a sudden influx Ca++ without an impulse produces a spontaneous twitch known as a \u201cspasm<\/strong>\u201d or \u201ccramp.<\/strong>\u201d Most often fatigue results from a decline in the availability of biological energy. When a myocyte is stimulated rapidly to tetany, ATP is consumed rapidly sustaining the process. Since the myocyte manufactures ATP at a finite rate, energy supply cannot match demand and consumption. Consequently, when stimulation produces tetany, fatigue results from a decline in available ATP. If tetany is interrupted for a short period (few seconds), energy production will restore maximum muscular performance. You can observe this in your gastrocnemis preparation.<\/p>\n

In the following experiment, the frog sciatic and gastrocnemius will be exposed to study basic physiological properties of muscle contraction. Initially, you will determine Threshold and examine the components of muscular contraction: latent, contraction <\/strong>and relaxation phases<\/strong> or periods. Also, you will study motor unit summation<\/strong> and temporal summation<\/strong> by independently changing stimulus amplitude and frequency while observing the effect on muscle responses. Within the vertebrate body, fine motor control is a product of the simultaneous integration<\/strong> of temporal and spatial summation (Figure 3).<\/p>\n

\"\"<\/p>\n

Experimental setup:<\/strong><\/h5>\n

\"\"<\/p>\n

Experiment AM-10: Summation, Tetanus, and Fatigue in an Intact Nerve-Muscle Prep<\/strong>
\nBy: RJ Cooper, Ph.D., Margaret A. Weck, D.A., and Dayton J. Ford, Ph.D.: at the St. Louis College of <\/em>Pharmacy. Adapted from: Hoff, H.E. and L.A. Geddes, Experimental Physiology (1965)<\/em><\/p>\n

Equipment Required<\/strong><\/p>\n

PC or Mac Computer
\nIWX\/214, USB cable, IWX\/214 power supply
\nFT-104 Force transducer
\nC-STIM-BNC-N2 Needle-type stimulating electrodes
\nC-BNC-SE Sleeve-type stimulating electrodes
\n<\/span>Female BNC to Dual Banana Adapter
\nRingstand and clamps
\nMuscle tension adjuster
\nThread
\nFrog board
\nDissection tray
\nGlass dissection hooks
\n1cc tuberculin syringe with needle
\nAmphibian Ringer\u2019s solution (see Appendix)
\n1% Tubocurarine solution in Ringer\u2019s<\/p>\n

For details of the setup please see the \u201cFrogNerveMuscle_Setup214\u201d PDF document.<\/strong><\/p>\n

Procedure:<\/strong><\/h5>\n

Start the Software<\/p>\n

1. Click on LabScribe
\n2. Click Settings _ Animal Muscle _ FrogNerveMuscle<\/p>\n

Exercise 1: Twitch Threshold Determined by Direct Stimulation<\/strong><\/h3>\n

Aim:<\/strong> To determine the threshold stimulus of the muscle when it is stimulated directly with single pulses of constant duration.<\/p>\n

Approximate Time: 15 minutes<\/p>\n

\u2022 Plug in the Pin stimulator into the SI port, Leave the sleeve stimulator on the nerve but not plugged in to the IWORX box<\/strong>
\n\u2022 DO NOT PIN THE FOOT, Pin skin around the knee<\/strong><\/p>\n

1. Click the Stimulator Preferences icon on the LabScribe toolbar (Figure AM-10-L1) to open the stimulator control panel (Figure AM-10-L2) on the Main window.<\/p>\n

\"\"<\/p>\n

Figure AM-10-L1: The <\/em>LabScribe<\/em> toolbar.<\/em><\/p>\n

2. Check the values for the stimulus parameters that are listed in the stimulator control panel on the Main window:<\/p>\n

\u2022 the pulse amplitude (Amp) should be set to 0.000 V;
\n\u2022 the number of pulses (#pulses) to 1;
\n\u2022 the frequency (F(Hz)) to 1;
\n\u2022 and, the pulse width (W) to 5 ms.
\n\u2022 Click APPLY in the upper left of the control panel<\/p>\n

3. The value for a stimulus parameter can be changed by either of two methods: click on the arrow buttons to the right of the window that displays the value of the parameter to increase or decrease the value; or, type the value of the parameter in the window next to the label of the parameter. Click the Apply button to finalize the change in any stimulus parameter.<\/p>\n

\"\"<\/p>\n

Figure AM-10-L2: The stimulator control panel<\/em><\/p>\n

4. Type 0.000V in the Mark box to the right of the Mark button. Click Record to stimulate the nerve with 0.000V and press the mark button to attach the comment to the recording.<\/p>\n

5. Apply a light load to the muscle by raising the force transducer with the tension adjuster until the trace moves a few mV above the baseline. If you are using a FT-302 force transducer, the initial baseline can be adjusted to 0 mV by rotating the offset knob on top of the FT-302 transducer.<\/p>\n

6. Once a light load is applied to the muscle, Click on the Stop.<\/p>\n

7. Change the stimulus amplitude (Amp) to 0.1V using one of the techniques described in Step 2. Click the Apply button on the Stimulator control panel to effect the change in amplitude.<\/p>\n

8. Type 0.1V in the Mark box to the right of the Mark button. Click Record to stimulate the nerve with 0.1V and press the mark button to attach the comment to the recording. Stop the recording as soon as the muscle twitch is finished, or after a couple of seconds if no twitch is detected.<\/p>\n

Note: Be sure to have at least one member of your lab group <\/em>LOOKING at the specimen at all times<\/em>.<\/em> It is possible for contractions to occur and not be recorded. Click <\/em>AutoScale<\/em> to make sure the<\/em> recording is displayed properly. Check the tautness of the thread going from the tendon to the<\/em> transducer, the transducer itself, and check the connection from the transducer to the <\/em>iWorx<\/em> recorder.<\/em><\/strong><\/p>\n

9. Determine the threshold voltage for direct muscle stimulation by increasing the stimulus voltage in 0.1V increments. DON\u2019T SKIP A VALUE and DO NOT EXCEED 1V.<\/span> Remember to click the Apply button on the Stimulator control panel each time it is changed. Record and mark the muscle response as described in Steps 3 and 7.<\/p>\n

10. Once the threshold voltage has been determined, test the muscle\u2019s response to direct stimulation at additional stimulus voltages (Figure AM-10-L3). Increase the stimulus amplitude in 1.0V increments, up to 5 V. Record the muscle response and mark the recording as described in Steps 3 and 6.<\/p>\n

11. Select Save As in the File menu, type a name for the file. Click on the Save button to save the data file.<\/p>\n

\"\"<\/p>\n

Figure AM-10-L3: Muscle twitches resulting from direct stimulation of the muscles.<\/em><\/p>\n

Exercise 2: Twitch Threshold Determined by Nerve Stimulation<\/strong><\/h3>\n

Aim<\/strong>: In this exercise, the muscle will be stimulated by action potentials generated in the nerve attached to the muscle. The nerve will be stimulated by single pulses of constant duration.<\/p>\n

Approximate Time: 30 minutes<\/p>\n

1. On the iWorx box, disconnect the BNC connector of the pin electrodes used for direct<\/p>\n

stimulation of the muscle. Leave the pin electrodes in place in the muscle.<\/p>\n

2. On the iWorx box, connect the BNC connector of the sleeve electrodes to the BNC-banana adapter on the stimulator output of the IWX\/214 or to the BNC output of the stimulator on the front of the IXTA.<\/p>\n

3. Reset the pulse amplitude (Amp) to 0.000V. Remember to click the Apply button on the stimulator control panel to finalize the change.<\/p>\n

4. Repeat Exercise 1 while stimulating the gastrocnemius muscle through the sciatic nerve with single stimulus pulses of increasing amplitude (Figure AM-10-L4). Label the recording at each voltage attempted.<\/p>\n

5. Determine the threshold voltage for nerve stimulation by increasing the stimulus voltage in 0.1V increments. Above threshold, test the muscle\u2019s response to nerve stimulation in 1.0V increments.<\/p>\n

6. Click the Save button to save the file.<\/p>\n

\"\"<\/p>\n

Figure AM-10-L4: Muscle twitches resulting from nerve stimulation.<\/em><\/p>\n

Exercise 3: Effect of Stimulus Frequency on Twitch Amplitude<\/strong><\/h3>\n

Aim<\/strong>: In this exercise, the muscle will be stimulated by action potentials generated in the nerve attached to the muscle. The nerve will be stimulated for short periods at different stimulus frequencies.<\/p>\n

Approximate Time: 30 minutes<\/p>\n

1. On the iWorx box, leave the BNC connector of the sleeve electrodes attached to the BNC banana adapter on the stimulator output of the IWX\/214 or on the stimulator output on the front of the IXTA.<\/p>\n

2. Set the stimulus frequency (F(Hz)) to 2 Hz, the number of pulses (#pulses) to zero (0), and the pulse amplitude (Amp) to the lowest value that still gave strong contractions in the previous exercise. This voltage will usually be in the range of 0.400V to 1.000 V, but your frog may be different. Keep the pulse width (W) at 5 ms. Remember to click the Apply button on the Stimulator control panel with each change.<\/p>\n

Warning: Since the number of <\/em>pulses (#<\/em>pulses) is set to zero (0), the stimulus pulses will be delivered<\/em> continuously at the frequency selected. It is important to stop recording once you see the effect of<\/em> stimulating at a certain frequency.<\/em><\/strong><\/p>\n

3. Type 2 Hz in the Mark box to the right of the Mark button. Click Record to stimulate the nerve and click the mark button to mark the recording. Stop the recording after 5 seconds<\/p>\n

4. Change the stimulus frequency to 4 Hz. Repeat Step 3. Continue the recording for stimulus frequencies of 5, 10, 25, 50, and 100 Hz (Figure AM-10-L5).<\/p>\n

5. Click the Save button to save the file.<\/p>\n

6. When you analyze the data for this exercise, note the frequency at which wave summation begins, the frequencies at which incomplete and complete tetanus occur, and the shape of the contraction waves and any indication of fatigue.<\/p>\n

\"\"<\/p>\n

Figure AM-10-L5: Muscle response from nerve stimulation with increased stimulus frequencies.<\/em><\/p>\n

Exercise 4: Fatigue with Repeated Stimuli<\/strong><\/h3>\n

Approximate Time: 45 minutes<\/p>\n

1. On the iWorx box, disconnect the sleeve electrodes from the stimulator output, and connect the pin electrodes to the stimulator. Leave the sleeve electrodes in place on the nerve.<\/p>\n

2. Set the Display Time to 20 sec, the stimulus frequency to 4 Hz, the number of pulses (#pulses) to zero (0), and pulse amplitude (Amp) to an adequate value between 0.4V and 1 V. Remember to click the Apply button.<\/p>\n

3. Type 4Hz-Direct in the Mark box to the right of the Mark button. Click Record to stimulate the nerve with 4 Hz and press the mark button to attach the comment to the recording. Stop the recording after 5 seconds<\/p>\n

4. After recording at 4Hz with the pin electrodes, switch to the sleeve electrodes.<\/p>\n

5. Type 4Hz-Nerve in the Mark box to the right of the Mark button. Click on the Record button to stimulate the nerve with 4 Hz and press the mark button to attach the comment to the recording. Stop the recording after 5 seconds.<\/p>\n

6. Change the stimulus frequency to 50 Hz. Remember to click the Apply button on the Stimulator control panel.<\/p>\n

7. Type 50Hz-Nerve in the Mark box to the right of the Mark button. Click Record to stimulate the nerve with 50Hz and press the mark button to attach the comment to the recording. Continue to stimulate through the nerve until the force of the muscle contraction drops to an amplitude that is only 25% of the maximum force at the beginning of this tetanic contraction.<\/p>\n

8. Continue to record after the contraction force of the muscle has dropped below 25% of maximum. Quickly disconnect the BNC connector of the sleeve electrodes from stimulator, and reconnect the BNC connector of the pin electrodes to the stimulator.<\/p>\n

9. Stimulate the muscle directly for 3 seconds, with the same voltage, duration, and frequency that was used to fatigue the muscle.<\/p>\n

Note: If no contraction is seen while using these stimulus parameters, increase the stimulus amplitude<\/em> until a contraction is measured.<\/em><\/strong><\/p>\n

10. Continue to record after the direct stimulation of the muscle. Quickly disconnect the BNC connector of the pin electrodes from the stimulator, and reconnect the BNC connector of the sleeve electrodes to the stimulator.<\/p>\n

11. Apply the same stimulus used in Step 9 to the nerve for a period of 3 seconds.<\/p>\n

12. After you stop the recording, label the recording with marks and notations to indicate the site of stimulation (muscle or nerve) and the stimulus voltage that generated each response.<\/p>\n

13. Click the Save button to save the file.<\/p>\n

14. When you analyze the data for this exercise:<\/p>\n

\u2022 Note the shape of the contraction waves and any indication of fatigue.
\n\u2022 Note whether the muscle contraction was stronger when the stimulus was applied directly to the muscle or when the stimulus was applied to the nerve. What does this indicate about the location of the fatigue observed during nerve stimulation?<\/p>\n

\"\"<\/p>\n

Figure AM-10-L6: Decrease in muscle force (fatigue) with continuous, high frequency stimulation<\/em> through the nerve innervating the muscle. The period of fatigue occurring during nerve stimulation is<\/em> followed by a large response by the muscle when it is stimulated directly.<\/em><\/p>\n

Note: As you complete each exercise, make sure to record your data and answer the questions in the lab report document.<\/strong><\/span><\/p>\n

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