Skeletal Muscle Lab Report
Frog Skeletal Muscle
The aim of this experiment is to explore the basic physiological principles of skeletal muscle using the isolated frog (Rana pipiens or Xenopus laevis) gastrocnemius muscle. Students will dissect a double-pithed frog. Then, they will connect the muscle to the Force Transducer to measure twitch recruitment, effect of stretch, muscle summation, muscle tetanus, and muscle fatigue.
Written by staff of ADInstruments.
Experiment Contents
1. Instructor’s Reference (this document)
This document contains information about modifying the presented experiment depending on the level of your course and proficiency of your students with LabChart software. It also contains information about troubleshooting, analyzing data, equipment alternatives, answers to the report questions, answers to the study questions, …show more content…
and LabChart settings.
2. Student Protocol
This document is the student protocol for this experiment; it provides step-by-step instructions for performing the experiment as well as how to analyze the data sets. The Student Protocol includes a Data Notebook and Study Questions. In this document, certain words are printed in color, in reference to topic entries in the Student Quick Reference Guide (available from your ADInstruments representative). References to acquisition settings are in blue, analysis topics are in green, and troubleshooting tips are in red, and Cyclic Measurements in violet. By informing your students that this resource is available to them, you can encourage students to guide themselves and improve their understanding of the software.
3. Laboratory Report
This document provides instructors with a ready-to-use format for student reports.
4. LabChart Settings Files
Settings Files contain configuration information for each exercise. The settings used are also listed in the Appendix at the end of this document.
5. LabChart Sample Data Files
These files show sample data from each exercise.
6. Multimedia
Summation and Tetanus.mov
This instructional video introduces students to the basic principles of summation and tetanus with definitions and examples of the physiology as seen in LabChart. This file is in QuickTime format, and requires the latest QuickTime player software for viewing. QuickTime is available free for Macintosh and Windows operating systems from http://www.apple.com/quicktime/download/.
General Information
This experiment was written and developed for LabChart 7.1 or later. You can check your version number by going to the Help menu and selecting Configuration information.
Make sure you have the latest version of your software by visiting www.ADInstruments.com or using the Check for Updates feature in the LabChart menu of the software.
There may be instances when you want to modify the experimental protocols for your students. You are welcome to modify this experiment to fit the needs of your classroom laboratory.
The Instructor’s Reference [this document] is now divided into exercises, so you can easily select the experiments for your laboratory class.
General Equipment to Complete Each Exercise
LabChart 7 or later
Any PowerLab with one general input
Bridge Pod [ML301]
Force Transducer [MLT0210/A] or [MLT050/A] or [MLT500/A]
Small weight between 5-50 grams (Note: This weight needs to be appropriate for your specific Force Transducer.)
Ring Stand [MLA40]
Manipulator and clamps (comes with the purchase of MLA40; you can also use the Ring Stand/Micropositioner supplied with the Organ Bath ML1110)
Strong thread
Petri dish
Pasteur pipette
One frog (Rana pipiens or Xenopus laevis)
Normal Frog Ringer’s solution (see “Frog Ringer’s Solution Recipe” below)
Cold (10 oC) Frog Ringer’s solution
Small millimeter ruler
Tape
Medium-sized beaker
Dissection tools (see “Frog Dissection” below)
Muscle Stimulation Equipment – You need one of the lists below:
LIST 1:
Stimulator Cable (BNC to Alligator Clips) [MLA250]
Muscle Holder [MLA013] (This is included in the PTK 19 Nerve and Muscle Kit II)
In this setup, the Stimulator Cable connects to the metal wires on the top of the Muscle Holder. The muscle sits inside the Muscle Holder. (Figure 2)
LIST 2:
Animal Nerve Stimulating Electrode [MLA0320]
Ring Stand clamp for positioning the electrode
Femur clamp to hold the muscle in place by the attached femur bone (for example, a Ring Stand clamp with an adjustable screw clamp)
In this setup, the Animal Nerve Stimulating Electrode touches the middle or ‘belly’ of the muscle directly. It is held in position with a Ring Stand clamp, and the muscle is held in place by a femur clamp. (Figure 3)
Frog Dissection
Dissection Tools:
Glass fingerbowl
Sharp scissors or scalpel
Blunt probe
Bone shears
Dissection tray with wax or pad
Dissection pins
The frog dissection is covered in the Student Protocol. If you want to dissect the frogs before the students enter the room, follow those instructions.
Frogs will need to be double-pithed prior to dissection. Most instructors find it easier to pith the frogs first to avoid disturbing their students. If you want the students to pith their own frogs, copy-and-paste these instructions into the Student Protocol.
Frog Pithing Procedure
Additional Equipment:
Dissection needle
Procedure:
1. Place the frog in the refrigerator for 2-3 hours to induce a state of torpor (hibernation). This will make pithing a little easier. This is especially recommended if you are using a toad.
2. Grasp the frog in your left hand, and bend the head ventrally over your middle finger with your index finger (Figure 1).
Figure 1. Pithing Procedure
3. Using a dissection needle, find the depression at the base of the skull; this is the foramen magnum.
4. Insert the dissection needle into the foramen magnum, and push it cranially to destroy the brain.
5. After the brain is destroyed, partly remove the needle and direct it caudally down the spinal column to destroy the spinal cord. The legs of the animal should stiffen at first and then become completely flaccid and unresponsive.
6. Place the frog in the dissection tray ventral side up. The rest of the dissection instructions are in the Student Protocol.
Frog Ringer’s Solution Recipe
This recipe will make one liter of frog Ringer’s.
Component
Amount (g)
NaCl
6.50
KCl
0.14
CaCl2
0.12
NaHCO3
0.20
Dissolve all components in one liter of distilled water.
Equipment Calibration
Prior to the first exercise, students will calibrate the Force Transducer.
Equipment List
An appropriate weight for the Force transducer
Equipment Setup
There are two alternative equipment configurations for this experiment. The experiment can be performed with either:
a Muscle Holder and Stimulator Cable (Figure 2) - see Page 10 of the Student Protocol;
or a femur clamp, together with the Animal Nerve Stimulating Electrode (Figure 3) - see Page 11 of Student Protocol.
In Page 7 of the Student Protocol, students are directed to choose their equipment to complete the setup. Make sure students are following the correct set of instructions. If you wish to set up the equipment beforehand, follow the instructions in the Student Protocol.
Figure 2. Equipment Setup with Muscle Holder
Figure 3. Equipment Setup with Femur Clamp
Exercise 1: Twitch Recruitment
In this exercise, students will run a macro to examine the effects of stimulus strength on contractile force. A macro is a recorded set of commands and operations that can be executed with a single command.
Equipment List
No additional equipment required
Equipment Setup
Students will set up the equipment as part of the Student Protocol.
Settings File to Complete Exercise 1
Frog Muscle Settings
Sample Data of Exercise 1
Frog Muscle Recruitment
Troubleshooting
Use the zeroing knob on the front of the Bridge Pod while viewing the waveform in the Bridge Pod dialog.
Make sure the offset button is checked in the Bridge Pod dialog. If not, the pod will not zero.
Make sure the Bridge Pod is zeroed before the muscle is connected to the Force Transducer.
Make sure students keep the frog muscle moist throughout the experiment. Use a Pasteur pipette to bathe the muscle with fresh room temperature Ringer’s solution after each exercise. If the muscle dries out, it will no longer function, and the experiment cannot be continued.
If the muscle is not contracting, make sure the muscle is in contact with the silver wires on the Muscle Holder or the Animal Nerve Stimulating Electrode.
Make sure students allow the muscle to recover between each exercise.
If you find this exercise is not working, you can have the students change the Sampling and Stimulator settings:
Select Sampling from the Setup menu. Ensure the Trigger checkbox is unchecked, and uncheck the Fixed Duration checkbox. Close the dialog.
Select Stimulator from the Setup menu. Set Stimulator mode to Step, and adjust the output amplitude (End Level) to the desired value.
Follow the rest of the settings as shown in Figure 4. When the student clicks Start, the Stimulator should now deliver 20 pulses of increasing amplitude, each 2 seconds apart.
Figure 4. Optional Stimulator Settings
Analysis Tips
Examine the entire data trace and Autoscale, if necessary. The Marker needs to be dragged to its position on the data trace.
Make sure students start analyzing the data from the last peak, moving backward. Also make sure the students fill out the data table in the correct order. The value from the last peak should go in the last row.
Exercise 2: Effect of Stretch on Contractile Force
In this exercise, students will examine the effect of stretch (also called preload) on the muscle by raising the Manipulator/Micropositioner. This exercise also uses a macro to stimulate the muscle for the students.
Equipment List
No additional equipment required
Equipment Setup
The same setup is used as in Exercise 1. Students will be adjusting the Manipulator/Micropositioner by turning the knob. Make sure students do not try to move the whole unit.
Settings File to Complete Exercise 2
Frog Muscle Settings
Sample Data of Exercise 2
Frog Muscle Tension
Troubleshooting
Refer to the tips from Exercise 1. Make sure students zero the Bridge Pod again.
This experiment is set up for the muscle to be stretched 10 times, for a total of 10 millimeters. However, if there is no increase in contractile force for two consecutive recordings before 10 millimeters is reached, students should exit the macro by clicking Cancel in the on-screen dialog for the macro. They should then return the equipment to its original position and move on to the next exercise.
It is very important to return the Manipulator/Micropositioner to its original position as quickly as possible to avoid damaging the muscle.
Analysis Tips
Make sure the Marker is not on the data trace when doing the analysis for this exercise.
Make sure the students complete the analysis for each data block in this exercise.
Make sure students keep track of the amount of stretch, and record these values in the Data Notebook.
Exercise 3: Muscle Summation
In this exercise, students will investigate muscle summation. Students will stimulate the muscle with twin pulses at different pulse intervals; the stimulation is done through a macro. There is a QuickTime movie included with this experiment that explains summation (and tetanus).
Equipment List
No additional equipment required
Equipment Setup
The same setup is used as in Exercise 1.
Settings File to Complete Exercise 3
Frog Muscle Settings
Sample Data of Exercise 3
Frog Muscle Summation
Troubleshooting
Refer to the tips from Exercise 1.
Make sure students zero the Bridge Pod again.
Analysis Tips
Refer to the Analysis Tips from Exercise 1.
Make sure students determine the force at each peak. When the peaks merge, students should put the value for contractile force into the column for the second peak.
Exercise 4: Muscle Tetanus
In this exercise, students will examine the muscle’s response to repetitive stimuli at different frequencies by using a macro. There is a QuickTime movie included with this experiment that explains tetanus (and summation).
Equipment List
No additional equipment required
Equipment Setup
The same setup is used as in Exercise 1.
Settings File to Complete Exercise 4
Frog Muscle Settings
Sample Data of Exercise 4
Frog Muscle Tetanus
Troubleshooting
Refer to the tips from Exercise 1.
Make sure students zero the Bridge Pod again.
Analysis Tips
Refer to the Analysis Tips from Exercise 1.
Exercise 5: Muscle Fatigue
In this exercise, students will examine the effect of muscle fatigue by stimulating the muscle repeatedly through the use of a macro.
Equipment List
No additional equipment required
Equipment Setup
The same setup is used as in Exercise 1.
Settings File to Complete Exercise 5
Frog Muscle Settings
Sample Data of Exercise 5
Frog Muscle Fatigue
Troubleshooting
Refer to the tips from Exercise 1.
Make sure students zero the Bridge Pod again.
Analysis Tips
Refer to the Analysis Tips from Exercise 1.
Optional Exercise
It is possible to explore the effects of temperature on the muscle. You can copy-and-paste the procedure into the Student Protocol. Use the settings described below to create a new settings file for this exercise.
Additional Equipment:
Warm Frog Ringer’s solution (40 oC)
Additional Settings:
Use the Settings file to begin. Add these settings for the Stimulator (Setup > Stimulator) to the file.
Stimulator:
Isolated Stimulator checkbox: unchecked
Stimulator Mode: Pulse
Marker channel: 2
Output: fixed number of pulses
Number of pulses: 1
Start: Manually
Range/Frequency: 20 Hz
Pulse duration: 1 ms
Output range: 2 V
Amplitude: Use the supramaximal stimulus voltage value from Exercise 1
Baseline 0.0 V
Close the Stimulator dialog and open the Stimulator Panel.
Procedure:
1. Zero the Bridge Pod as before.
2. Perfuse the muscle with cold Ringer’s for 10 minutes.
3. Start recording. Click Stimulate in the Stimulator Panel to stimulate the muscle.
4. When finished, Stop recording.
5. Repeat steps 2-4 with warm Ringer’s.
Student Exercises: Study Questions and Lab Reports
The Student Protocol includes a Data Notebook section for students to record results and calculations and Study Questions. In addition, this experiment kit includes a preformatted Laboratory Report. Instructors wishing to modify these documents are free to do so. Both documents are optional materials, which can be used at the instructor’s discretion.
Answers to 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)?
The all or none law refers to a single muscle fiber. However, a while muscle is a bunch of muscle fibers. At higher stimulation amplitudes, more fibers are recruited, resulting in a stronger contractile force.
2. What effect does stretching the muscle have on contractile strength? Is this effect linear? What preload force resulted in the highest contractile force?
A stretched muscle responds with a stronger contractile force to a supramaximal stimulus than a flaccid muscle. Stretching causes the myofibrils to be positioned optimally for contraction. However, at the highest loads, the myofibrils can be stretched too far; the lack of overlap between actin and myosin will reduce contractile strength.
3. What effect does varying the stimulation frequency have on contractile force? Which stimulus interval caused the greatest contractile force?
Muscle force increases with increased stimulation frequency. The highest frequency should cause the greatest result, but this depends on student data.
4. Define tetanus. At which stimulus interval did you observe tetanus?
Tetanus is defined as a smooth muscle contraction of a whole muscle that generates maximum force. It occurs when the stimulus frequency is high; the muscle does not have time to relax between stimulus pulses. Tetanus should be observed with a stimulus interval of 20-50 ms.
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.
Depends on student data.
6. In your own words, explain a possible mechanism for why the muscle was unable to maintain a prolonged contraction in Exercise 5.
While muscle fatigue is not completely understood, it most likely results from a depletion of ATP stores, buildup of lactic acid, or both.
Laboratory Report: Instructor’s Notes
Format
Each Laboratory Report has the following sections: Introduction, Results, and Conclusions. The Introduction provides space for students to write a paragraph introducing the lab topic, define relevant terms, and explain what they accomplished in the experiment.
The Results section is where students can place figures from their data files and graphs from spreadsheet programs. Therefore, these exercises are written with the assumption that students have access to a graphing/statistics package and/or a spreadsheet program. Alternatively, instructors may wish to have their introductory students make plots by hand on millimeter graph paper.
The Conclusions section is formatted as a series of questions that the students should answer in complete sentences.
When asked to provide a scatter plot, students should supply something similar to Figure 5 and Figure 5 below.
Figure 5. Sample Graph of Twitch Recruitment
Figure 6. Sample Graph of the Effect of Load (Stretch)
Answers to Conclusion Questions
1. Describe why you observe twitch recruitment during increasing stimulus intensity in light of the “all or none” law governing muscle contraction.
The all or none law applies to a single muscle fiber. Recruitment of more muscle fibers occurs when the stimulus amplitude is increases, causing the apparent graded response.
2. Explain why muscle contracts more forcefully when stretched.
At the cellular level, actin and myosin are arranged so they slide past each other. These protein molecules must overlap in order to interact with each other. Placing tension on the muscle causes the molecular arrangement of actin and myosin to overlap properly so they are optimally aligned. Too little tension and the muscle contracts sub-optimally, because the actin and myosin are not aligned for maximal interaction.
3. What happens to the muscle at the highest preload force?
At high tensions, the actin and myosin can be pulled too far apart, thus reducing the amount of overlap between them that is necessary for an optimal interaction. Therefore, contractile force decreases at high tensions.
4. When the muscle was stimulated repeatedly, what happened to the force of contraction? Explain the mechanism behind this phenomenon.
When the frequency of stimulation increased, the second contraction started before the first contraction was completed. The force of contraction increased in part because intracellular stores of Ca2+ are readily available immediately following a twitch. Eventually, the calcium ions are sequestered back into the sarcoplasmic reticulum.
5. How long was your preparation able to maintain a maximum contraction? Would your results have differed if you were measuring from smooth muscle tissue? Why or why not?
Depends on student data; smooth muscle differs from skeletal muscle in that it does not fatigue. It is able to maintain slow, forceful contractions for extended periods and does not typically utilize anaerobic metabolic pathways.
Appendix 1: LabChart Settings
These are the settings used in the pre-made LabChart Settings Files for this experiment. Connect the equipment to the PowerLab and turn it on before opening LabChart.
Software Compatibility
This experiment was written and developed for LabChart 7.1 or later. Make sure you have the latest version of your software by visiting www.ADInstruments.com or using the Check for Updates feature in the software.
Frog Muscle Settings
Sampling Rate: 4k/s
View: 20:1 compression
Number of Channels: 2
Channels 1 and 2 should be “on.” Check the boxes to do this, if necessary.
Channel 1: Title: Force
Range: 20 mV
Input Settings: Bridge Pod
Low Pass: 300 Hz
Select x10 Gain
Select Offset (Note: The Bridge Pod will not zero if this is not selected.)
Select Anti-alias
Calculation: No Calculation
Channel 2: Title: Stim Mark.
Range: 2 V
Input Settings: Input Amplifier
Calculation: No Calculation
DVM Windows: Time, Channel 1
Macros:
There are five macros included with this experiment, one for each exercise. These macros control the stimuli being applied to the muscle.
Note: Check to make sure the hot keys assigned to each macro in the Settings File works with your computer. You may need to change the hot keys, depending on how you have your keyboard setup. This is possible in LabChart 6 and LabChart 7 (but not in Chart 5) by selecting the Macro menu and choosing Manage.
Appendix 2: Recording Macros
The following description of how each macro was recorded is for your information only. You will not need to carry out the following steps.
Recruitment
1. Go to the Macro menu and select Start Recording. Macro now appears in the LabChart Toolbar.
2. Go to Stimulator in the Setup menu. Make the following settings in the dialog:
Isolated Stimulator checkbox: unchecked
Stimulator Mode: Pulse
Output: Fixed number of pulses
Number of Pulses: 1
Marker Channel: Channel 2 (Stim Mark.)
Start: When recording starts
Delay: 250 ms
Range: 2 Hz
Frequency: 1 Hz
Pulse Duration: 1 ms
Range: 2 V
Amplitude: 0.05 V
Baseline 0.0 V
3. Go to Sampling in the Setup menu. Make the following settings in the dialog:
Start: Ensure the Trigger checkbox is unchecked.
Stop: Check the Fixed duration checkbox, and enter 1 s.
4. Click Start. In the dialog, select “Finish sampling before doing other macro steps” and Stop sampling “Fixed duration” and enter 1 second. Click OK.
5. Now go back to Stimulator. Change the Amplitude ONLY. Change it to 100 mV.
6. Click Start again. Use the same settings from step 4.
7. Go back to Stimulator. You will keep repeating the pattern in steps 5 and 6. You want to change the Amplitude by 50 mV each time.
8. Once you have changed the Amplitude to 1 V and clicked Start and OK, do not continue the pattern. 9. Press Stop Recording Macro from the LabChart Toolbar. Give the macro a name, select where you want the file to appear in the Menu bar, and assign it a hotkey. In the Settings File, the macro is called Recruitment and is assigned the hotkey Command+1. (If you change these settings, you also need to change the Student Protocol.)
Note: For all remaining macros, students should enter the stimulus amplitude in the Stimulator Panel based on the muscle supramaximal stimulus voltage calculated in Exercise 1.
Muscle Tension
As the students will be setting the amplitude on their own, it is important to follow the steps regarding the Stimulator carefully. If you set the Stimulator Amplitude after you start recording the macro, it will not work for the students.
1. Go to the Macro menu and select Start Recording.
2. Go to Stimulator in the Setup menu. Make the following settings in the dialog (the text in bold denotes the changes from the previous macro):
Isolated Stimulator checkbox: unchecked
Stimulator Mode: Pulse
Output: Fixed number of pulses
Number of pulses: 1
Marker Channel: Channel 2 (Stim Mark.)
Start: When recording starts
Delay: 250 ms
Frequency: 1 Hz
Pulse duration: 1 ms
Range: 500 mV
Amplitude: DO NOT TOUCH THIS VALUE – students will set it in the Protocol
Baseline 0.0 V
3. Go to Sampling in the Setup menu. Make the following settings in the dialog:
Start: Ensure the Trigger checkbox is unchecked.
Stop: Check the Fixed duration checkbox, and enter 1 s.
4. In the Macro menu, go to Macro Commands and select Message. In the text box that appears, write “Make sure the muscle tension is adjusted so the thread is slack. When you are ready to begin, click OK.” Select the white information bubble as the message icon. Check the checkbox for Show cancel button.
5. In the Macro menu, go to Macro Commands and select Repeat (or Begin Repeat). Type “9” into the Repeat dialog.
6. Click Start. In the dialog, select “Finish sampling before doing other macro steps” and Stop sampling “Fixed duration” and enter 1 second. Click OK.
7. In the Macro menu, go to Macro Commands and select Message. In the text box that appears, write “Raise the Manipulator/Micropositioner by 1 mm using the adjustment knob. Wait 30 seconds, then click OK.” Select the white information bubble as the message icon. Check the checkbox for Show cancel button.
8. In the Macro menu, go to Macro Commands and select End Repeat.
9. Click Start. Follow the settings in step 6.
10. Stop Recording the macro. In the Settings File, the macro is called Muscle Tension and is assigned the hotkey Command+2. (If you change these settings, you also need to change the Student Protocol.)
Summation
As the students will be setting the amplitude on their own, it is important to follow the steps regarding the Stimulator carefully. If you set the Stimulator Amplitude after you start recording the macro, it will not work for the students.
1. Go to the Macro menu and select Start Recording.
2. Go to Stimulator in the Setup menu. Make the following settings in the dialog (the text in bold denotes the changes from the previous macro):
Isolated Stimulator checkbox: unchecked
Stimulator Mode: Pulse
Output: Fixed number of pulses
Number of pulses: 2
Marker Channel: Channel 2 (Stim Mark.)
Start: When recording starts
Delay: 250 ms
Range: 200 Hz
Frequency: 2.5 Hz
Pulse duration: 1 ms
Output range: 500 mV
Amplitude: DO NOT TOUCH THIS VALUE – students will set it in the Protocol
Baseline 0.0 V
3. Go to Sampling in the Setup menu. Make the following settings in the dialog:
Start: Ensure the Trigger checkbox is unchecked.
Stop: Check the Fixed duration checkbox, and enter 1 s.
4. In the Macro menu, go to Macro Commands and select Message. In the text box that appears, write “Make sure the muscle is moistened with Ringer’s solution. When you are ready to begin, click OK.” Select the white information bubble as the message icon. Check the checkbox for Show cancel button.
5. Click Start. In the dialog, select “Finish sampling before doing other macro steps” and Stop sampling “Fixed duration” and enter 1 second. Click OK.
6. Go back to the Stimulator. Change the Frequency to 5 Hz. Do not change any of the other settings.
7. In the Macro menu, go to Macro Commands and select Message. In the text box that appears, write “Wait 30 seconds, then click OK.” Select the white information bubble as the message icon. Check the checkbox for Show cancel button.
8. Click Start. Follow the settings from step 5.
9. Repeat steps 6-8. Change the Stimulator Frequency to 10 Hz.
10. Repeat steps 6-8 again. Change the Stimulator Frequency to 20 Hz.
11. Repeat steps 6-8 again. Change the Stimulator Frequency to 50 Hz.
12. After you have clicked Start and OK when the Frequency is 50 Hz, Stop Recording the macro. In the Settings File, the macro is called Muscle Summation and is assigned the hotkey Command+3. (If you change these settings, you also need to change the Student Protocol.)
Tetanus
As the students will be setting the amplitude on their own, it is important to follow the steps regarding the Stimulator carefully. If you set the Stimulator Amplitude after you start recording the macro, it will not work for the students.
1. Go to the Macro menu and select Start Recording.
2. Go to Stimulator in the Setup menu. Make the following settings in the dialog:
Isolated Stimulator checkbox: unchecked
Stimulator Mode: Pulse
Output: Fixed number of pulses
Number of pulses: 50
Marker Channel: Channel 2 (Stim Mark.)
Start: When recording starts
Delay: 250 ms
Range: 500 Hz
Frequency: 2.5 Hz
Pulse duration: 1 ms
Output range: 500 mV
Amplitude: DO NOT TOUCH THIS VALUE – students will set it in the Protocol
Baseline 0.0 V
3. Go to Sampling in the Setup menu. Make the following settings in the dialog:
Start: Ensure the Trigger checkbox is unchecked.
Stop: Check the Fixed duration checkbox, and enter 1 s.
4. In the Macro menu, go to Macro Commands and select Message. In the text box that appears, write “Make sure the muscle is moistened with Ringer’s solution. When you are ready to begin, click OK.” Select the white information bubble as the message icon. Check the checkbox for Show cancel button.
5. Click Start. In the dialog, select “Finish sampling before doing other macro steps” and Stop sampling “Fixed duration” and enter 1 second. Click OK.
6. Go back to the Stimulator. Change the Frequency to 5 Hz. Do not change any of the other settings.
7. In the Macro menu, go to Macro Commands and select Message. In the text box that appears, write “Wait 30 seconds, then click OK.” Select the white information bubble as the message icon. Check the checkbox for Show cancel button.
8.
Click Start. Follow the settings from step 5.
9. Repeat steps 6-8. Change the Stimulator Frequency to 10 Hz.
10. Repeat steps 6-8 again. Change the Stimulator Frequency to 20 Hz.
11. Repeat steps 6-8 again. Change the Stimulator Frequency to 50 Hz.
12. After you have clicked Start and OK when the Frequency is 50 Hz, Stop Recording the macro. In the Settings File, the macro is called Muscle Tetanus and is assigned the hotkey Command+4. (If you change these settings, you also need to change the Student Protocol.)
Fatigue
As the students will be setting the amplitude on their own, it is important to follow the steps regarding the Stimulator carefully. If you set the Stimulator Amplitude after you start recording the macro, it will not work for the students.
1. Go to the Macro menu and select Start Recording.
2. Go to Stimulator in the Setup menu. Make the following settings in the dialog:
Isolated Stimulator checkbox: unchecked
Stimulator Mode: Pulse
Output: Fixed number of pulses
Number of pulses: 1500
Marker Channel: Channel 2 (Stim Mark.)
Start: When recording starts
Delay: 250 ms
Frequency: 50
Hz
Pulse duration: 1 ms
Output range: 2 V
Amplitude: DO NOT TOUCH THIS VALUE – students will set it in the Protocol
Baseline 0.0 V
3. Go to Sampling in the Setup menu. Make the following settings in the dialog:
Start: Ensure the Trigger checkbox is unchecked.
Stop: Check the Fixed duration checkbox, and enter 45 s.
4. In the Macro menu, go to Macro Commands and select Message. In the text box that appears, write “Make sure the muscle is moistened with Ringer’s solution. When you are ready to begin, click OK.” Select the white information bubble as the message icon. Check the checkbox for Show cancel button.
5. Click Start. In the dialog, select “Finish sampling before doing other macro steps” and Stop sampling “Fixed duration” and enter 45 seconds. Click OK.
6. Stop Recording the macro. In the Settings File, the macro is called Muscle Fatigue and is assigned the hotkey Command+5. (If you change these settings, you also need to change the Student Protocol.)
Note: Check to make sure the Settings File works with your computer. You may need to change the hot keys, depending on how you have your keyboard setup.
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The aim of this experiment is to explore the basic physiological principles of skeletal muscle using the isolated frog (Rana pipiens or Xenopus laevis) gastrocnemius muscle. Students will dissect a double-pithed frog. Then, they will connect the muscle to the Force Transducer to measure twitch recruitment, effect of stretch, muscle summation, muscle tetanus, and muscle fatigue.
Written by staff of ADInstruments.
Experiment Contents
1. Instructor’s Reference (this document)
This document contains information about modifying the presented experiment depending on the level of your course and proficiency of your students with LabChart software. It also contains information about troubleshooting, analyzing data, equipment alternatives, answers to the report questions, answers to the study questions, …show more content…
and LabChart settings.
2. Student Protocol
This document is the student protocol for this experiment; it provides step-by-step instructions for performing the experiment as well as how to analyze the data sets. The Student Protocol includes a Data Notebook and Study Questions. In this document, certain words are printed in color, in reference to topic entries in the Student Quick Reference Guide (available from your ADInstruments representative). References to acquisition settings are in blue, analysis topics are in green, and troubleshooting tips are in red, and Cyclic Measurements in violet. By informing your students that this resource is available to them, you can encourage students to guide themselves and improve their understanding of the software.
3. Laboratory Report
This document provides instructors with a ready-to-use format for student reports.
4. LabChart Settings Files
Settings Files contain configuration information for each exercise. The settings used are also listed in the Appendix at the end of this document.
5. LabChart Sample Data Files
These files show sample data from each exercise.
6. Multimedia
Summation and Tetanus.mov
This instructional video introduces students to the basic principles of summation and tetanus with definitions and examples of the physiology as seen in LabChart. This file is in QuickTime format, and requires the latest QuickTime player software for viewing. QuickTime is available free for Macintosh and Windows operating systems from http://www.apple.com/quicktime/download/.
General Information
This experiment was written and developed for LabChart 7.1 or later. You can check your version number by going to the Help menu and selecting Configuration information.
Make sure you have the latest version of your software by visiting www.ADInstruments.com or using the Check for Updates feature in the LabChart menu of the software.
There may be instances when you want to modify the experimental protocols for your students. You are welcome to modify this experiment to fit the needs of your classroom laboratory.
The Instructor’s Reference [this document] is now divided into exercises, so you can easily select the experiments for your laboratory class.
General Equipment to Complete Each Exercise
LabChart 7 or later
Any PowerLab with one general input
Bridge Pod [ML301]
Force Transducer [MLT0210/A] or [MLT050/A] or [MLT500/A]
Small weight between 5-50 grams (Note: This weight needs to be appropriate for your specific Force Transducer.)
Ring Stand [MLA40]
Manipulator and clamps (comes with the purchase of MLA40; you can also use the Ring Stand/Micropositioner supplied with the Organ Bath ML1110)
Strong thread
Petri dish
Pasteur pipette
One frog (Rana pipiens or Xenopus laevis)
Normal Frog Ringer’s solution (see “Frog Ringer’s Solution Recipe” below)
Cold (10 oC) Frog Ringer’s solution
Small millimeter ruler
Tape
Medium-sized beaker
Dissection tools (see “Frog Dissection” below)
Muscle Stimulation Equipment – You need one of the lists below:
LIST 1:
Stimulator Cable (BNC to Alligator Clips) [MLA250]
Muscle Holder [MLA013] (This is included in the PTK 19 Nerve and Muscle Kit II)
In this setup, the Stimulator Cable connects to the metal wires on the top of the Muscle Holder. The muscle sits inside the Muscle Holder. (Figure 2)
LIST 2:
Animal Nerve Stimulating Electrode [MLA0320]
Ring Stand clamp for positioning the electrode
Femur clamp to hold the muscle in place by the attached femur bone (for example, a Ring Stand clamp with an adjustable screw clamp)
In this setup, the Animal Nerve Stimulating Electrode touches the middle or ‘belly’ of the muscle directly. It is held in position with a Ring Stand clamp, and the muscle is held in place by a femur clamp. (Figure 3)
Frog Dissection
Dissection Tools:
Glass fingerbowl
Sharp scissors or scalpel
Blunt probe
Bone shears
Dissection tray with wax or pad
Dissection pins
The frog dissection is covered in the Student Protocol. If you want to dissect the frogs before the students enter the room, follow those instructions.
Frogs will need to be double-pithed prior to dissection. Most instructors find it easier to pith the frogs first to avoid disturbing their students. If you want the students to pith their own frogs, copy-and-paste these instructions into the Student Protocol.
Frog Pithing Procedure
Additional Equipment:
Dissection needle
Procedure:
1. Place the frog in the refrigerator for 2-3 hours to induce a state of torpor (hibernation). This will make pithing a little easier. This is especially recommended if you are using a toad.
2. Grasp the frog in your left hand, and bend the head ventrally over your middle finger with your index finger (Figure 1).
Figure 1. Pithing Procedure
3. Using a dissection needle, find the depression at the base of the skull; this is the foramen magnum.
4. Insert the dissection needle into the foramen magnum, and push it cranially to destroy the brain.
5. After the brain is destroyed, partly remove the needle and direct it caudally down the spinal column to destroy the spinal cord. The legs of the animal should stiffen at first and then become completely flaccid and unresponsive.
6. Place the frog in the dissection tray ventral side up. The rest of the dissection instructions are in the Student Protocol.
Frog Ringer’s Solution Recipe
This recipe will make one liter of frog Ringer’s.
Component
Amount (g)
NaCl
6.50
KCl
0.14
CaCl2
0.12
NaHCO3
0.20
Dissolve all components in one liter of distilled water.
Equipment Calibration
Prior to the first exercise, students will calibrate the Force Transducer.
Equipment List
An appropriate weight for the Force transducer
Equipment Setup
There are two alternative equipment configurations for this experiment. The experiment can be performed with either:
a Muscle Holder and Stimulator Cable (Figure 2) - see Page 10 of the Student Protocol;
or a femur clamp, together with the Animal Nerve Stimulating Electrode (Figure 3) - see Page 11 of Student Protocol.
In Page 7 of the Student Protocol, students are directed to choose their equipment to complete the setup. Make sure students are following the correct set of instructions. If you wish to set up the equipment beforehand, follow the instructions in the Student Protocol.
Figure 2. Equipment Setup with Muscle Holder
Figure 3. Equipment Setup with Femur Clamp
Exercise 1: Twitch Recruitment
In this exercise, students will run a macro to examine the effects of stimulus strength on contractile force. A macro is a recorded set of commands and operations that can be executed with a single command.
Equipment List
No additional equipment required
Equipment Setup
Students will set up the equipment as part of the Student Protocol.
Settings File to Complete Exercise 1
Frog Muscle Settings
Sample Data of Exercise 1
Frog Muscle Recruitment
Troubleshooting
Use the zeroing knob on the front of the Bridge Pod while viewing the waveform in the Bridge Pod dialog.
Make sure the offset button is checked in the Bridge Pod dialog. If not, the pod will not zero.
Make sure the Bridge Pod is zeroed before the muscle is connected to the Force Transducer.
Make sure students keep the frog muscle moist throughout the experiment. Use a Pasteur pipette to bathe the muscle with fresh room temperature Ringer’s solution after each exercise. If the muscle dries out, it will no longer function, and the experiment cannot be continued.
If the muscle is not contracting, make sure the muscle is in contact with the silver wires on the Muscle Holder or the Animal Nerve Stimulating Electrode.
Make sure students allow the muscle to recover between each exercise.
If you find this exercise is not working, you can have the students change the Sampling and Stimulator settings:
Select Sampling from the Setup menu. Ensure the Trigger checkbox is unchecked, and uncheck the Fixed Duration checkbox. Close the dialog.
Select Stimulator from the Setup menu. Set Stimulator mode to Step, and adjust the output amplitude (End Level) to the desired value.
Follow the rest of the settings as shown in Figure 4. When the student clicks Start, the Stimulator should now deliver 20 pulses of increasing amplitude, each 2 seconds apart.
Figure 4. Optional Stimulator Settings
Analysis Tips
Examine the entire data trace and Autoscale, if necessary. The Marker needs to be dragged to its position on the data trace.
Make sure students start analyzing the data from the last peak, moving backward. Also make sure the students fill out the data table in the correct order. The value from the last peak should go in the last row.
Exercise 2: Effect of Stretch on Contractile Force
In this exercise, students will examine the effect of stretch (also called preload) on the muscle by raising the Manipulator/Micropositioner. This exercise also uses a macro to stimulate the muscle for the students.
Equipment List
No additional equipment required
Equipment Setup
The same setup is used as in Exercise 1. Students will be adjusting the Manipulator/Micropositioner by turning the knob. Make sure students do not try to move the whole unit.
Settings File to Complete Exercise 2
Frog Muscle Settings
Sample Data of Exercise 2
Frog Muscle Tension
Troubleshooting
Refer to the tips from Exercise 1. Make sure students zero the Bridge Pod again.
This experiment is set up for the muscle to be stretched 10 times, for a total of 10 millimeters. However, if there is no increase in contractile force for two consecutive recordings before 10 millimeters is reached, students should exit the macro by clicking Cancel in the on-screen dialog for the macro. They should then return the equipment to its original position and move on to the next exercise.
It is very important to return the Manipulator/Micropositioner to its original position as quickly as possible to avoid damaging the muscle.
Analysis Tips
Make sure the Marker is not on the data trace when doing the analysis for this exercise.
Make sure the students complete the analysis for each data block in this exercise.
Make sure students keep track of the amount of stretch, and record these values in the Data Notebook.
Exercise 3: Muscle Summation
In this exercise, students will investigate muscle summation. Students will stimulate the muscle with twin pulses at different pulse intervals; the stimulation is done through a macro. There is a QuickTime movie included with this experiment that explains summation (and tetanus).
Equipment List
No additional equipment required
Equipment Setup
The same setup is used as in Exercise 1.
Settings File to Complete Exercise 3
Frog Muscle Settings
Sample Data of Exercise 3
Frog Muscle Summation
Troubleshooting
Refer to the tips from Exercise 1.
Make sure students zero the Bridge Pod again.
Analysis Tips
Refer to the Analysis Tips from Exercise 1.
Make sure students determine the force at each peak. When the peaks merge, students should put the value for contractile force into the column for the second peak.
Exercise 4: Muscle Tetanus
In this exercise, students will examine the muscle’s response to repetitive stimuli at different frequencies by using a macro. There is a QuickTime movie included with this experiment that explains tetanus (and summation).
Equipment List
No additional equipment required
Equipment Setup
The same setup is used as in Exercise 1.
Settings File to Complete Exercise 4
Frog Muscle Settings
Sample Data of Exercise 4
Frog Muscle Tetanus
Troubleshooting
Refer to the tips from Exercise 1.
Make sure students zero the Bridge Pod again.
Analysis Tips
Refer to the Analysis Tips from Exercise 1.
Exercise 5: Muscle Fatigue
In this exercise, students will examine the effect of muscle fatigue by stimulating the muscle repeatedly through the use of a macro.
Equipment List
No additional equipment required
Equipment Setup
The same setup is used as in Exercise 1.
Settings File to Complete Exercise 5
Frog Muscle Settings
Sample Data of Exercise 5
Frog Muscle Fatigue
Troubleshooting
Refer to the tips from Exercise 1.
Make sure students zero the Bridge Pod again.
Analysis Tips
Refer to the Analysis Tips from Exercise 1.
Optional Exercise
It is possible to explore the effects of temperature on the muscle. You can copy-and-paste the procedure into the Student Protocol. Use the settings described below to create a new settings file for this exercise.
Additional Equipment:
Warm Frog Ringer’s solution (40 oC)
Additional Settings:
Use the Settings file to begin. Add these settings for the Stimulator (Setup > Stimulator) to the file.
Stimulator:
Isolated Stimulator checkbox: unchecked
Stimulator Mode: Pulse
Marker channel: 2
Output: fixed number of pulses
Number of pulses: 1
Start: Manually
Range/Frequency: 20 Hz
Pulse duration: 1 ms
Output range: 2 V
Amplitude: Use the supramaximal stimulus voltage value from Exercise 1
Baseline 0.0 V
Close the Stimulator dialog and open the Stimulator Panel.
Procedure:
1. Zero the Bridge Pod as before.
2. Perfuse the muscle with cold Ringer’s for 10 minutes.
3. Start recording. Click Stimulate in the Stimulator Panel to stimulate the muscle.
4. When finished, Stop recording.
5. Repeat steps 2-4 with warm Ringer’s.
Student Exercises: Study Questions and Lab Reports
The Student Protocol includes a Data Notebook section for students to record results and calculations and Study Questions. In addition, this experiment kit includes a preformatted Laboratory Report. Instructors wishing to modify these documents are free to do so. Both documents are optional materials, which can be used at the instructor’s discretion.
Answers to 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)?
The all or none law refers to a single muscle fiber. However, a while muscle is a bunch of muscle fibers. At higher stimulation amplitudes, more fibers are recruited, resulting in a stronger contractile force.
2. What effect does stretching the muscle have on contractile strength? Is this effect linear? What preload force resulted in the highest contractile force?
A stretched muscle responds with a stronger contractile force to a supramaximal stimulus than a flaccid muscle. Stretching causes the myofibrils to be positioned optimally for contraction. However, at the highest loads, the myofibrils can be stretched too far; the lack of overlap between actin and myosin will reduce contractile strength.
3. What effect does varying the stimulation frequency have on contractile force? Which stimulus interval caused the greatest contractile force?
Muscle force increases with increased stimulation frequency. The highest frequency should cause the greatest result, but this depends on student data.
4. Define tetanus. At which stimulus interval did you observe tetanus?
Tetanus is defined as a smooth muscle contraction of a whole muscle that generates maximum force. It occurs when the stimulus frequency is high; the muscle does not have time to relax between stimulus pulses. Tetanus should be observed with a stimulus interval of 20-50 ms.
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.
Depends on student data.
6. In your own words, explain a possible mechanism for why the muscle was unable to maintain a prolonged contraction in Exercise 5.
While muscle fatigue is not completely understood, it most likely results from a depletion of ATP stores, buildup of lactic acid, or both.
Laboratory Report: Instructor’s Notes
Format
Each Laboratory Report has the following sections: Introduction, Results, and Conclusions. The Introduction provides space for students to write a paragraph introducing the lab topic, define relevant terms, and explain what they accomplished in the experiment.
The Results section is where students can place figures from their data files and graphs from spreadsheet programs. Therefore, these exercises are written with the assumption that students have access to a graphing/statistics package and/or a spreadsheet program. Alternatively, instructors may wish to have their introductory students make plots by hand on millimeter graph paper.
The Conclusions section is formatted as a series of questions that the students should answer in complete sentences.
When asked to provide a scatter plot, students should supply something similar to Figure 5 and Figure 5 below.
Figure 5. Sample Graph of Twitch Recruitment
Figure 6. Sample Graph of the Effect of Load (Stretch)
Answers to Conclusion Questions
1. Describe why you observe twitch recruitment during increasing stimulus intensity in light of the “all or none” law governing muscle contraction.
The all or none law applies to a single muscle fiber. Recruitment of more muscle fibers occurs when the stimulus amplitude is increases, causing the apparent graded response.
2. Explain why muscle contracts more forcefully when stretched.
At the cellular level, actin and myosin are arranged so they slide past each other. These protein molecules must overlap in order to interact with each other. Placing tension on the muscle causes the molecular arrangement of actin and myosin to overlap properly so they are optimally aligned. Too little tension and the muscle contracts sub-optimally, because the actin and myosin are not aligned for maximal interaction.
3. What happens to the muscle at the highest preload force?
At high tensions, the actin and myosin can be pulled too far apart, thus reducing the amount of overlap between them that is necessary for an optimal interaction. Therefore, contractile force decreases at high tensions.
4. When the muscle was stimulated repeatedly, what happened to the force of contraction? Explain the mechanism behind this phenomenon.
When the frequency of stimulation increased, the second contraction started before the first contraction was completed. The force of contraction increased in part because intracellular stores of Ca2+ are readily available immediately following a twitch. Eventually, the calcium ions are sequestered back into the sarcoplasmic reticulum.
5. How long was your preparation able to maintain a maximum contraction? Would your results have differed if you were measuring from smooth muscle tissue? Why or why not?
Depends on student data; smooth muscle differs from skeletal muscle in that it does not fatigue. It is able to maintain slow, forceful contractions for extended periods and does not typically utilize anaerobic metabolic pathways.
Appendix 1: LabChart Settings
These are the settings used in the pre-made LabChart Settings Files for this experiment. Connect the equipment to the PowerLab and turn it on before opening LabChart.
Software Compatibility
This experiment was written and developed for LabChart 7.1 or later. Make sure you have the latest version of your software by visiting www.ADInstruments.com or using the Check for Updates feature in the software.
Frog Muscle Settings
Sampling Rate: 4k/s
View: 20:1 compression
Number of Channels: 2
Channels 1 and 2 should be “on.” Check the boxes to do this, if necessary.
Channel 1: Title: Force
Range: 20 mV
Input Settings: Bridge Pod
Low Pass: 300 Hz
Select x10 Gain
Select Offset (Note: The Bridge Pod will not zero if this is not selected.)
Select Anti-alias
Calculation: No Calculation
Channel 2: Title: Stim Mark.
Range: 2 V
Input Settings: Input Amplifier
Calculation: No Calculation
DVM Windows: Time, Channel 1
Macros:
There are five macros included with this experiment, one for each exercise. These macros control the stimuli being applied to the muscle.
Note: Check to make sure the hot keys assigned to each macro in the Settings File works with your computer. You may need to change the hot keys, depending on how you have your keyboard setup. This is possible in LabChart 6 and LabChart 7 (but not in Chart 5) by selecting the Macro menu and choosing Manage.
Appendix 2: Recording Macros
The following description of how each macro was recorded is for your information only. You will not need to carry out the following steps.
Recruitment
1. Go to the Macro menu and select Start Recording. Macro now appears in the LabChart Toolbar.
2. Go to Stimulator in the Setup menu. Make the following settings in the dialog:
Isolated Stimulator checkbox: unchecked
Stimulator Mode: Pulse
Output: Fixed number of pulses
Number of Pulses: 1
Marker Channel: Channel 2 (Stim Mark.)
Start: When recording starts
Delay: 250 ms
Range: 2 Hz
Frequency: 1 Hz
Pulse Duration: 1 ms
Range: 2 V
Amplitude: 0.05 V
Baseline 0.0 V
3. Go to Sampling in the Setup menu. Make the following settings in the dialog:
Start: Ensure the Trigger checkbox is unchecked.
Stop: Check the Fixed duration checkbox, and enter 1 s.
4. Click Start. In the dialog, select “Finish sampling before doing other macro steps” and Stop sampling “Fixed duration” and enter 1 second. Click OK.
5. Now go back to Stimulator. Change the Amplitude ONLY. Change it to 100 mV.
6. Click Start again. Use the same settings from step 4.
7. Go back to Stimulator. You will keep repeating the pattern in steps 5 and 6. You want to change the Amplitude by 50 mV each time.
8. Once you have changed the Amplitude to 1 V and clicked Start and OK, do not continue the pattern. 9. Press Stop Recording Macro from the LabChart Toolbar. Give the macro a name, select where you want the file to appear in the Menu bar, and assign it a hotkey. In the Settings File, the macro is called Recruitment and is assigned the hotkey Command+1. (If you change these settings, you also need to change the Student Protocol.)
Note: For all remaining macros, students should enter the stimulus amplitude in the Stimulator Panel based on the muscle supramaximal stimulus voltage calculated in Exercise 1.
Muscle Tension
As the students will be setting the amplitude on their own, it is important to follow the steps regarding the Stimulator carefully. If you set the Stimulator Amplitude after you start recording the macro, it will not work for the students.
1. Go to the Macro menu and select Start Recording.
2. Go to Stimulator in the Setup menu. Make the following settings in the dialog (the text in bold denotes the changes from the previous macro):
Isolated Stimulator checkbox: unchecked
Stimulator Mode: Pulse
Output: Fixed number of pulses
Number of pulses: 1
Marker Channel: Channel 2 (Stim Mark.)
Start: When recording starts
Delay: 250 ms
Frequency: 1 Hz
Pulse duration: 1 ms
Range: 500 mV
Amplitude: DO NOT TOUCH THIS VALUE – students will set it in the Protocol
Baseline 0.0 V
3. Go to Sampling in the Setup menu. Make the following settings in the dialog:
Start: Ensure the Trigger checkbox is unchecked.
Stop: Check the Fixed duration checkbox, and enter 1 s.
4. In the Macro menu, go to Macro Commands and select Message. In the text box that appears, write “Make sure the muscle tension is adjusted so the thread is slack. When you are ready to begin, click OK.” Select the white information bubble as the message icon. Check the checkbox for Show cancel button.
5. In the Macro menu, go to Macro Commands and select Repeat (or Begin Repeat). Type “9” into the Repeat dialog.
6. Click Start. In the dialog, select “Finish sampling before doing other macro steps” and Stop sampling “Fixed duration” and enter 1 second. Click OK.
7. In the Macro menu, go to Macro Commands and select Message. In the text box that appears, write “Raise the Manipulator/Micropositioner by 1 mm using the adjustment knob. Wait 30 seconds, then click OK.” Select the white information bubble as the message icon. Check the checkbox for Show cancel button.
8. In the Macro menu, go to Macro Commands and select End Repeat.
9. Click Start. Follow the settings in step 6.
10. Stop Recording the macro. In the Settings File, the macro is called Muscle Tension and is assigned the hotkey Command+2. (If you change these settings, you also need to change the Student Protocol.)
Summation
As the students will be setting the amplitude on their own, it is important to follow the steps regarding the Stimulator carefully. If you set the Stimulator Amplitude after you start recording the macro, it will not work for the students.
1. Go to the Macro menu and select Start Recording.
2. Go to Stimulator in the Setup menu. Make the following settings in the dialog (the text in bold denotes the changes from the previous macro):
Isolated Stimulator checkbox: unchecked
Stimulator Mode: Pulse
Output: Fixed number of pulses
Number of pulses: 2
Marker Channel: Channel 2 (Stim Mark.)
Start: When recording starts
Delay: 250 ms
Range: 200 Hz
Frequency: 2.5 Hz
Pulse duration: 1 ms
Output range: 500 mV
Amplitude: DO NOT TOUCH THIS VALUE – students will set it in the Protocol
Baseline 0.0 V
3. Go to Sampling in the Setup menu. Make the following settings in the dialog:
Start: Ensure the Trigger checkbox is unchecked.
Stop: Check the Fixed duration checkbox, and enter 1 s.
4. In the Macro menu, go to Macro Commands and select Message. In the text box that appears, write “Make sure the muscle is moistened with Ringer’s solution. When you are ready to begin, click OK.” Select the white information bubble as the message icon. Check the checkbox for Show cancel button.
5. Click Start. In the dialog, select “Finish sampling before doing other macro steps” and Stop sampling “Fixed duration” and enter 1 second. Click OK.
6. Go back to the Stimulator. Change the Frequency to 5 Hz. Do not change any of the other settings.
7. In the Macro menu, go to Macro Commands and select Message. In the text box that appears, write “Wait 30 seconds, then click OK.” Select the white information bubble as the message icon. Check the checkbox for Show cancel button.
8. Click Start. Follow the settings from step 5.
9. Repeat steps 6-8. Change the Stimulator Frequency to 10 Hz.
10. Repeat steps 6-8 again. Change the Stimulator Frequency to 20 Hz.
11. Repeat steps 6-8 again. Change the Stimulator Frequency to 50 Hz.
12. After you have clicked Start and OK when the Frequency is 50 Hz, Stop Recording the macro. In the Settings File, the macro is called Muscle Summation and is assigned the hotkey Command+3. (If you change these settings, you also need to change the Student Protocol.)
Tetanus
As the students will be setting the amplitude on their own, it is important to follow the steps regarding the Stimulator carefully. If you set the Stimulator Amplitude after you start recording the macro, it will not work for the students.
1. Go to the Macro menu and select Start Recording.
2. Go to Stimulator in the Setup menu. Make the following settings in the dialog:
Isolated Stimulator checkbox: unchecked
Stimulator Mode: Pulse
Output: Fixed number of pulses
Number of pulses: 50
Marker Channel: Channel 2 (Stim Mark.)
Start: When recording starts
Delay: 250 ms
Range: 500 Hz
Frequency: 2.5 Hz
Pulse duration: 1 ms
Output range: 500 mV
Amplitude: DO NOT TOUCH THIS VALUE – students will set it in the Protocol
Baseline 0.0 V
3. Go to Sampling in the Setup menu. Make the following settings in the dialog:
Start: Ensure the Trigger checkbox is unchecked.
Stop: Check the Fixed duration checkbox, and enter 1 s.
4. In the Macro menu, go to Macro Commands and select Message. In the text box that appears, write “Make sure the muscle is moistened with Ringer’s solution. When you are ready to begin, click OK.” Select the white information bubble as the message icon. Check the checkbox for Show cancel button.
5. Click Start. In the dialog, select “Finish sampling before doing other macro steps” and Stop sampling “Fixed duration” and enter 1 second. Click OK.
6. Go back to the Stimulator. Change the Frequency to 5 Hz. Do not change any of the other settings.
7. In the Macro menu, go to Macro Commands and select Message. In the text box that appears, write “Wait 30 seconds, then click OK.” Select the white information bubble as the message icon. Check the checkbox for Show cancel button.
8.
Click Start. Follow the settings from step 5.
9. Repeat steps 6-8. Change the Stimulator Frequency to 10 Hz.
10. Repeat steps 6-8 again. Change the Stimulator Frequency to 20 Hz.
11. Repeat steps 6-8 again. Change the Stimulator Frequency to 50 Hz.
12. After you have clicked Start and OK when the Frequency is 50 Hz, Stop Recording the macro. In the Settings File, the macro is called Muscle Tetanus and is assigned the hotkey Command+4. (If you change these settings, you also need to change the Student Protocol.)
Fatigue
As the students will be setting the amplitude on their own, it is important to follow the steps regarding the Stimulator carefully. If you set the Stimulator Amplitude after you start recording the macro, it will not work for the students.
1. Go to the Macro menu and select Start Recording.
2. Go to Stimulator in the Setup menu. Make the following settings in the dialog:
Isolated Stimulator checkbox: unchecked
Stimulator Mode: Pulse
Output: Fixed number of pulses
Number of pulses: 1500
Marker Channel: Channel 2 (Stim Mark.)
Start: When recording starts
Delay: 250 ms
Frequency: 50
Hz
Pulse duration: 1 ms
Output range: 2 V
Amplitude: DO NOT TOUCH THIS VALUE – students will set it in the Protocol
Baseline 0.0 V
3. Go to Sampling in the Setup menu. Make the following settings in the dialog:
Start: Ensure the Trigger checkbox is unchecked.
Stop: Check the Fixed duration checkbox, and enter 45 s.
4. In the Macro menu, go to Macro Commands and select Message. In the text box that appears, write “Make sure the muscle is moistened with Ringer’s solution. When you are ready to begin, click OK.” Select the white information bubble as the message icon. Check the checkbox for Show cancel button.
5. Click Start. In the dialog, select “Finish sampling before doing other macro steps” and Stop sampling “Fixed duration” and enter 45 seconds. Click OK.
6. Stop Recording the macro. In the Settings File, the macro is called Muscle Fatigue and is assigned the hotkey Command+5. (If you change these settings, you also need to change the Student Protocol.)
Note: Check to make sure the Settings File works with your computer. You may need to change the hot keys, depending on how you have your keyboard setup.
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