neuromuscular physiology experiment

BIOL 362: Mammalian Physiology

EXPERIMENT 7Neuromuscular Physiology and Muscle Mechanics

(revised Jul/12)

LEARNING GOALS:Be able to: Explain the steps involved in inducing a single muscle contraction, naming the structures and types of signaling involved. Set up a frog neuromuscular preparation and explain the function of the instrumentation used. Properly use the PowerLab stimulator and Chart functions for collecting, displaying, and analyzing data. Demonstrate recordings of twitch, summation, treppe, tetany, and fatigue and know the difference between what is observed in the laboratory and what occurs in the body. Analyze records to measure latency, threshold, recruitment, twitch duration, summation, tetany, fatigue, and the effects of various pharmacological agents. Develop and test hypotheses (with a scientific rationale) to determine what a specific unknown drug is (from among several possibilities), based on its effects on nerve, muscle and neuromuscular junction.

Before coming to class: This is a complicated set of experiments, so come well prepared with a flow chart that simplifies all the instructions you will carry out. Think of all the sites or steps, from the activation of the motor neuron to muscle fiber contraction that may be affected by a given drug, and their effects on muscle contraction. How could you test where the site of action may be for a particular pharmacological agent? In this lab, you will test an unknown (to you) compound to determine its site of action.

The possible drugs to be used are listed below (they will be randomly labeled A, B, C and D): Eserine (= physostigmine), an acetylcholine esterase inhibitor Curare, an antagonist of the nicotinic acetylcholine receptor Tetraethylammonium, a blocker of voltage-gated potassium channels

You will be exploring the nature of neuromuscular transmission. The isolated frog neuromuscular preparation – the sciatic nerve and the gastrocnemius muscle – will allow you to investigate basic neuromuscular physiology. Although under normal conditions a nerve AP originates within the central nervous system (CNS), in the laboratory you will mimic CNS stimulation by electrically stimulating the sciatic nerve, thus producing typical action potentials on motor neurons within the sciatic nerve, which will in turn generate APs that trigger contraction of the innervated muscle fibers. Be sure you understand the consequences of this difference (between what you do in lab and what occurs in vivo). Note that the electrical stimulation will be applied to the nerve, which is a bundle of sensory and motor neuron axons. Those closest to the site of stimulus will be depolarized the most, and the stimulus depolarization will spread out to other neurons within the nerve. Thus, those closest to the stimulating electrodes will most likely be first to depolarize.

OBJECTIVES: Using the frog sciatic/gastrocnemius preparation and electrical stimuli, the following characteristics of neuromuscular function will be demonstrated and quantified: latency,

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threshold, recruitment, twitch duration, summation, tetany, and fatigue. In addition, the effect of certain unknown drugs will be examined.

INSTRUMENTS:In order to demonstrate some of the physiology of motor units, especially muscular contraction, instruments must (1) produce an electrical stimulus, (2) record the strength of a contraction, and (3) record the time sequence between stimulation and response. The first of these we will accomplish by using an electronic stimulator that is part of the PowerLab interface box. This is your first introduction to the use of this component of the PowerLab system (refer to page D-29 of Appendix D) so a short explanation is provided below. The strength of contractions will be detected by a force transducer, which you have used in previous experiments. Again, you will use the PowerLab Chart program and interface to capture data digitally.

Use of the PowerLab Electronic Stimulator:Select the SET UP menu from the tool bar in Chart. Select STIMULATOR to open the Stimulator window. Refer to the descriptions on pages D-29 to 30 to learn the various functions of the stimulator components, which you will have to alter during the course of today’s experiment. Select the STIMULATOR PANEL from the Set Up menu to activate a window that will allow you to change the frequency, pulse duration, and pulse amplitude (stimulus strength) during an experiment. You should do this AFTER setting up the stimulator parameters as instructed in specific parts of the experiment.

MATERIALS:Electronic stimulator Stimulating electrodesFrog nerve and muscle Computer and PowerLab interface, Chart

softwareForce transducer Frog RingersCalibration weightsUnknown drugs – handle these with care and wash your hands after handling!

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Figure 7-1. Graded response of whole muscle due to recruitment of motor units. Strong stimuli lead to recruitment of fibers (A). As the stimulating voltage is diminished, fewer fibers are stimulated until a threshold stimulus is achieved (B). Stimuli with lower stimulus strength than the threshold stimulus fail to elicit a contraction (C).

Figure 7-2. Summation, tetany, and fatigue in while muscle. With increasing frequency individual twitches add or sum, leading to treppe, incomplete tetany, and complete tetany. Continued stimulation leads to fatigue.

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A B C

Summation begins

Treppe (Staircase effect)

Complete Tetany

Fatigue

Stim

ulus

Tens

ion


.

Figure 7-3. Computer display of measurable parameters of a single twitch.

METHODS:You will have 2 lab meetings to complete this experiment. However, frogs will be available for experiments only during the first meeting. The second lab meeting will be devoted to completing the analysis and graphing of the data.Dissect the frog to remove the nerve/muscle preparation (Figs. 7-4, 7-5, 7-6) and attach it to the apparatus (Fig. 7-7). Perform the 7 steps described below.

Dissection: Caution: handle preparation with care. Do not stretch or touch exposed nerve with metal instruments and keep tissues (especially the nerve) moist with Ringer's solution at all times.

1. You will receive a skinned frog hind leg with the area where the sciatic nerve can be located cleared of connective tissue (refer to Fig. 7-4, 7-5).

2. Carefully expose the sciatic nerve within the thigh (see Figure 7-4, 7-5) by separating the thigh musculature from the dorsal aspect using a saline filled fire-polished glass pipette (Fig. 7-6A ). Do not cut the major leg artery. Do not stretch the nerve. Handle the nerve only with the glass hook.

3. Loosen but DO NOT CUT the Achilles tendon from its attachment at the bone (Fig. 7-5, 7-6A). Pass a length of thread (~6-8" long) between the tendon and the bone. Tie the thread securely around the tendon. Carefully free the entire gastrocnemius from the bone using the fire-polished glass pipette (step 4 in Fig. 7-5). Cut the Achilles tendon as indicated by the pointer in Fig. 7-4, leaving the gastrocnemius still connected at the knee and a length of thread attached to the tendon. Tie the thread around the tendon to the force transducer.

4. Position the frog leg so it is parallel and about 2” from the edge of the dish and so that the leg is positioned perpendicular to the force transducer, not at an oblique angle. Test whether the nerve is still viable by touching the sciatic nerve with the stimulating electrode

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and stimulate the nerve using a 0.1 V (100 mV) pulse and see if it activates the muscle. If nothing happens you may have damaged or disconnected the nerve from the muscle. Start another dissection immediately.

5. Pin the upper leg and the webbed foot securely to the base of the dish and near the dish edge so the clamps and electrode can reach the preparation (Fig. 7-6B and Fig. 7-7). Be sure the pins securely hold the leg to the pan and that the leg (especially the knee area) does not move up off the pan when the gastrocnemius contracts.

6. Mount the string attached to the Achilles tendon to the transducer hook as in Fig. 7-7 with a small amount of tension. Be sure the electrodes are not immersed in saline and are not touching one another or they will short out. Place a small strip of Kimwipe on the muscle to serve as a wick and moisten it with saline (Ringers). Using the glass hooks, carefully place the nerve over both wires of the stimulating electrode. KEEP THE NERVE AND MUSCLE MOIST WITH PHYSIOLOGICAL SALINE AT ALL TIMES OR THEY WILL NOT WORK!

7. Use a plastic pipette to suck out fluids that accumulate in the dish.

Figure 7-4. Anatomy of the frog hindleg.

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Figure 7-5. Steps for dissecting the frog leg. Step one will have been completed for you.

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Figure 7-7. Overview of set-up for studying the frog nerve-muscle preparation. Normally, you will use two ring stands and attach the stimulating electrode to one and the rest of the set-up to the other ring stand.

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EXPERIMENTAL PROCEDURE:

Be sure that the power to the PowerLab box is turned on before launching the Chart program. Open the folder Lab 7 NMJ of the Experiments Gallery and click on Lab 7NM1 settings. This will launch Chart with Stimulator and Recording Channel settings preset for you. You should see 2 channels on the screen, the top channel will display the stimulator record and the bottom channel will display the transducer recording of muscle contractions. If at any time, the tops of your stimulus pulse or responses are being cut off (are flat) or if the stimulus response does not show up, change the time and amplitude settings during the experiment to best display the data. BE SURE YOUR TRACES ARE NOT PEGGED OR YOU WILL NOT BE ABLE TO OBTAIN ACCURATE MEASUREMENTS!

Save Part 1 in your lab 7 folder.

PART I. THE EFFECTS OF SINGLE STIMULI: in this section you will study the effects of increasing the strength of individual stimuli on the muscle.

A. Threshold and graded responses due to recruitment of motor units (See Fig. 7-1) Open the Stimulator in the Setup toolbar and adjust the stimulator settings to produce individual stimuli of different strengths or amplitudes (V). You will begin at 0 V and increase by 0.125 volt with each pulse, continuing until you reach supramaximal stimulus (see below), and record all responses. As you increase stimulus strength, the muscle contractile response should increase (why?), until it reaches a maximum (what happens at this maximum?). Try to increase the voltage relatively rapidly (but do not initiate a second stimulus until the muscle twitch is complete) and steadily until you reach maximum contractile response. Record all data as you collect it.BE SURE TO SAVE YOUR DATA! Keep the preparation moist!

B. Supramaximal stimulus: If your muscle contractile response plateaus (levels off), select the stimulator voltage of the second stimulus that produced a maximal response. This will be defined as the supramaximal stimulus. Go to Part II.

If your muscle contraction amplitudes DO NOT plateau (level off) you will need to repeat this step with a greater maximal (end level) stimulus strength.

1. Open the Setup menu and select Stimulator. Change the End Level (V) to 5 V.2. Repeat the 20 stimuli and hopefully you will attain a maximal response.3. BE SURE TO SAVE YOUR DATA. You will use this data to graph the stimulus strength vs.

contractile force.

C. Time measurements of muscle contraction phases and latency (see Fig. 7-3):This part can be done on one of the records taken in Part B using the zoom window, and will be done after all raw data has been collected during Data Analysis (see below). These data will also be used to estimate the stimulus frequency at which summation and tetany will occur (see Part II). If a second stimulus occurs before the muscle has completely relaxed, summation will occur. If a second stimulus occurs before the first muscle twitch has peaked, tetany will occur.PART II. EFFECTS OF MULTIPLE STIMULI: in this section you will study the effects of increasing frequency of stimulus of the muscle, with stimulus kept constant.

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Summation, Tetany and Fatigue (see Fig. 7-2):1. Close the Chart file you have been using.2. Open the file named Lab 7NM2.4 settings in the Experiments Gallery.3. SAVE the new FILE under another filename into the appropriate folder for your lab section.

Read steps 5 and 6 before you attempt to go on.4. You will be making a recording like that in Fig. 7-2. Go to SetUp and select STIMULATOR.

Change the setting from Set Number of Pulses to CONTINUOUS pulses and change the Range to 200 Hz. Set the voltage in the stimulator panel to that which you determined above to elicit the supramaximal response.

5. In the channel that records muscle tension (contractile force), set the y-axis scale so that a single twitch takes up only 1/4 of the entire scale (about 500 mV to 1 V setting on the Muscle channel). The muscle contractions can get very large so be sure there is enough room for the response to increase (as in Fig. 7-2), or you will have to repeat this part of the lab, possibly with another frog leg. Review what you must do next before you begin.

6. Record data as you collect it. Starting at a frequency of 1 Hz, point the cursor to the stimulus frequency and increase the frequency of the continuous stimulus by clicking on the mouse at a rate of about 2-3 clicks per second. Do not stop between changes in frequency (see Fig. 7-2). Continue to increase the frequency until you observe fatigue (a decline in the waveform), and stop immediately after fatigue occurs. BE SURE TO SAVE YOUR DATA!

7. If you did not get the response demonstrated in Fig. 7-2, allow the muscle 5 minutes rest while bathing in Ringer's before repeating Part II. Or, continue with Part III.

PART III. DRUG EFFECTS ON NEURON, MUSCLE, AND THE NEUROMUSCULAR JUNCTION:

A. Effects of unknown drugs: Before you begin, review with your group members what results you would expect for each of the possible unknown drugs, and how to distinguish among them.

1. Close the present file and open the Chart file named Lab 7 NM 3.4 in the Experiments Gallery. Go File and remove the Auto-save feature by removing the checked box.

2. SAVE the new FILE under another filename into the appropriate folder for your lab section. Include the letter of the drug you were assigned in the filename. Do not use a previously used filename.

3. Set the stimulator voltage to the voltage used in Part I that represents a suprathreshold stimulus. Set the stimulator to apply a Pulse, for 5 pulses, at 0.5 Hz. This will give you 5 stimulus pulses that can be averaged or viewed to see if there is decrement in the responses with multiple stimuli. This same stimulus protocol will be used for your control and experimental conditions. Be sure to record all data as you collect it and add comments as you go.

4. Place a small strip of Kimwipe on the muscle to act as a wick during application of your drug. This will allow the drug to penetrate into the muscle more effectively than if you simply pipette it over the muscle. Record a control contraction to be sure the wick does not affect contraction and to obtain pre-drug control responses.

5. To determine the mode of action of your unknown drug, first apply the drug to the nerve alone, to see if it affects conduction of the nerve action potential. Record and stimulate as before. Repeat this process for a total of 3 trials. If there is no response, increase stimulus intensity to 2-5 times initial (but for only one trial). If you still get no response, stimulate the muscle directly (see below).

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6. Rinse the nerve carefully with Ringers and stimulate the nerve again to see if any effect is reversible.

7. Obtain a pre-drug control response once more. 8. Then add drops of your drug to the wick in contact with the muscle. In some cases, you

may need to add the drug where the nerve enters the muscle to see an effect. Stimulate via the nerve while recording any effects of the drug on muscle contraction. Repeat this process for a total of 3 trials. If you see an effect, rinse the muscle to see if the effect is reversible. If the response is blocked stimulate the muscle directly (see below) while recording any effects.

Direct muscle stimulation:1. Use the same stimulator and recording settings as above. Set up the direct muscle

stimulating electrodes so that both pins touch the muscle instead of the nerve. 2. Start the recording and stimulate the muscle with the same voltage setting as above. You

may need to increase it further to obtain a response. However, do not go over 10 volts.3. Note the response of the muscle and compare it to that when the nerve is stimulated.

Calibrate the force transducer:Carefully disconnect the frog leg from the force transducer and dispose of it in the appropriate container (a bag in the back of the lab). Then, calibrate the force transducer. Zero the tracing of the force transducer. Then obtain a weight of known mass, and carefully hang it from the end of the force transducer. Record the voltage. Use the ratio between the known mass and the voltage recorded as a conversion factor to convert all measurements of voltage into force in mg.

DATA ANALYSIS (most analysis will be done in week 2 of this experiment) :The Data Pads should be set up to take the appropriate measurements, but check them so you do not waste your time collecting data that you do not need.Part I. Single Stimuli analyses:Open the file saved for part I. Under WINDOWS, open up the Data Pad and note the order of measurements to be made.

A. Measuring the stimulus voltage and the corresponding twitch for the smallest stimulus that produced a discernible muscle contraction, up through supramaximal stimulus [to measure recruitment (Part IB)]. The Data Pad should read: Channel 1 (stimulator) Max-Min and Channel 2 (muscle) Max-Min.

1. Carefully highlight the entire twitch response (including a portion of the baseline both before and after the twitch). Press and hold the shift key, then also highlight the stimulus tracing. Add this highlighted area to the Data Pad. Be sure your waveform has not pegged because the data exceeded the maximum amplitude for the window. Check your Data Pad. Both the stimulus strength and muscle contractile response will be recorded in mV (but you will be converting the muscle data to mg of contractile force, using the conversion factor obtained when you calibrate the force transducer; you will do that at the end of this experiment).

2. Repeat for all stimuli and resulting contraction traces.

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Threshold measurements.Minimum Stimulus Voltage for Twitch

Minimum Stimulus Voltage for Maximum Contraction

Measurements for Time Course of a Twitch and the Latency PeriodA. Your Data Pad should also have a value for Time and Mean for both channels (stimulator

and muscle). In the tracings from the recruitment experiment, find the stimulus that produced a muscle contractile response that was approximately 50% of the maximal response. Insert a COMMENT somewhere at the beginning of this trace to mark it. Highlight both the stimulus and entire response by holding the SHIFT key so both traces are highlighted. Use the Zoom Window to expand both traces.

B. Use the “overlay” function in the top right corner of the Zoom Box. Refer to Fig. 7-3 depicting parts of a twitch response to obtain data for the Time Course of Twitch Table in Report 7. Annotate the data pad to indicate which values are in which row. SAVE the DATA PAD as a textfile with an appropriate filename for the data.

Take the following time measurements by double clicking on the following points. (Check your data pad to make sure the measurements are added).

1. Time at the beginning of the stimulus. 2. Time at the beginning of the twitch response.3. Peak amplitude and time to reach the peak. (If your data pad is labeled correctly, both

measurements will be taken.)4. Time at the end of the twitch response (back to the baseline).

Print the Zoom Window and re-save your DATA PAD TEXT FILE!Duration of Twitch (msec)

Duration of contraction phase (msec)

Duration of relaxation phase (msec)

Latency period between the initiation of the stimulus and the initiation of contraction (msec)Force of contraction or amplitude of twitch (mg)

C. PRINTING RELEVANT TRACES: 1. If you did not yet do so, print the Zoom Window depicting the trace used for obtaining the

time course and amplitude measurements for a single twitch response. 2. In preparation for printing portions of the Chart record, delete traces that are not

necessary by highlighting the trace and, from the EDIT menu, selecting DELETE SELECTION. Use the compress function at the bottom of the Chart window to compress the traces (refer to Appendix D, Figure D-3). To print the traces demonstrating threshold stimulus and recruitment, highlight the relevant traces and PRINT THE SELECTION (use landscape setting for horizontal printing). Include the comments that are within in the selected traces.

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3. Later , when you are in Excel, open the Data Pad file and carry out the necessary arithmetic calculations between columns to obtain the measurements requested in Part I of Report 7. NOTE: be sure to convert all mV measurements for muscle response to contractile force, using the appropriate conversion factor obtained when you calibrate the force transducer. Rearrange and sort the data pad measurements in Excel for graphing stimulus strength (mV) vs. twitch muscle contractile force (mg) from the data collected in the Recruitment studies (Part IB).

4. Close this file when you have completed your analysis but be sure you have saved your analyzed and edited file.

Part II: Multiple Stimuli. Open the file you saved for Multiple Stimuli experiments.A. Obtain data taken from different regions of the tracing representing different stimulus frequencies and corresponding response of the muscle. Stimulus strength and duration were kept constant for this part of the experiment.

1. Obtain the amplitude of the contractile response (force of contraction) by highlighting the waveform, including the baseline and the region of interest. The function in the Data Pad will read Max-Mean to give you the amplitude of your waveform from the baseline to the highest peak value. When converted to mg, this value will be contractile force.

2. Determine the frequency of the two stimulus pulses by highlighting the stimuli pulses and adding to the Data Pad. A function in the Data Pad under “Cycle” will be used to automatically calculate frequency of stimulus pulses that produced the muscle response.

B. To obtain data for Tetany, determine the frequency of the stimulus that first produced tetany.

Frequency (Hz)

First occurrence of summation

Range of frequencies producing treppe (staircase effect)First occurrence of complete tetany

First occurrence of fatigue

Length of time from beginning of stimulation to first occurrence of fatigue

(time in sec)

C. To obtain data for fatigue, determine the stimulus frequency that elicited fatigue and the time from the start of stimulation to when fatigue was first noted.

1. Use the SAVE AS command under FILE to save the DATA PAD as a textfile. Enter the name of the file and its folder above.

2. IN PREPARATION FOR PRINTING, delete irrelevant traces, compress, and highlight the relevant traces. PRINT your best trace showing the different phases of tetany.

D. Effects of the unknown drug will be descriptive and no data need to be analyzed but you will need to append the Chart traces that pertain to this experiment. As before, PRINT only selected traces, deleting irrelevant traces and compressing the traces to be printed.

Experiment Report 7

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Neuromuscular Physiology and Muscle Mechanics

WORK DONE BEFORE THE EXPERIMENT: List your working hypothesis or hypotheses, your predictions about the experimental results, and the rationale for your hypotheses and predictions.Hypotheses:

Predicted results and rational:

DISCUSSION QUESTIONS:1. Which of the possible drugs do you think you used? Explain, based on the results you

obtained (i.e., did the drug affect the nerve and/or the muscle) and the mode of action of the three drugs. Compare the effects of the drug when the nerve was stimulated versus when the muscle was stimulated directly (bypassing the nerve).

2. What does threshold represent in this experiment? How does this compare with threshold for action potential initiation on a typical neuron?

3. Explain your recruitment data. What is recruited (what is happening within the muscle as a whole, and within the individual muscle fibers)? What does recruitment accomplish in your bodily movements?

4. What electrical, chemical and mechanical events are occurring during the latency period (as defined by this experiment) (see Table 7-2)?

5. In the summation relationship in Graph 7-2, what is being summed? In other words, what is happening at the level of the sarcomere and at the level of the whole muscle cell during summation and tetany?

6. a) Using your value for twitch duration (the time it takes for an entire twitch, contraction and relaxation), calculate the maximum number of complete twitches which could occur in 1 sec. (Note that stimulation at a greater frequency than this will necessarily lead to minimal summation because, if a second stimulus occurs before the muscle has completely relaxed, summation will occur.) b) Using your value for contraction duration (the time it takes for just the contraction phase of a twitch), calculate the maximum number of individual, unfused contractions that could occur in 1 sec. (Note that above this frequency tetany will occur because, if a second stimulus occurs before the first muscle twitch has peaked, tetany will occur). c) Compare your results for the stimulus frequency required to achieve summation and tetany to the values calculated in a and b. Did your experimental findings correspond with those calculations? Explain.

THOUGHT QUESTIONS:1. Explain why it is important for the body to have all of the following: “pinpoint” neurogenic

control of muscles and other effectors (somatic motor system), more widespread control of functions via the endocrine system, and “involuntary, unconscious” control exerted by the autonomic nervous system.

2. Compare and contrast the somatic and autonomic motor nervous systems in terms of how control of effectors occurs, type of regulation [absolute control (off or on) versus modulated (increase or decrease)], type of effectors, type of neurotransmitters, latency and duration of response, etc.

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3. How would the force of contraction in the gastrocnemius muscle of a weight lifter and a long distance runner compare to that of a frog’s gastrocnemius? (Assume you can adjust for the size difference between human and frog muscles, such as by expressing measurements in units of force/muscle mass.) Explain.

4. Is it possible to sum the twitches of cardiac muscle to produce tetany? Why or why not?5. What would happen if you stretched the gastrocnemius muscle prior to stimulation, as in

the frog heart lab (Starling's Law of the Heart)?

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neuromuscular physiology experiment

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