Laboratory 08

Vertebrate skelatal muscle and work

this is the subtitle

Objectives

• To familiarize students with the methodology of muscle physiology investigation.
• To investigate the relationship between muscle length and tensin and the amount of work the muscle can perform
• To interpret the data produced in each of the investigations and integrate them into a report discussing the functions of muscles.

Introduction

Work is measured in units of force, applied through a distance (force X distance). Power is measured in terms of work divided by time (work/time). The ability of a muscle to generate power, that is, to apply force through distance in a certain time, determines how fast an animal can jump, run, swim, fly, gather and store food, and so forth. The power output of a muscle is influenced by many environmental factors, such as temperature, hormonal balance, nutritional status, and rate of oxygen delivery. However, certain purely mechanical factors within the muscle can affect its ability to perform work, and thus generate power. These include the initial length of the muscle, and the tension in the muscle's elastic parts before and during the contraction.

In this laboratory, we investigate the effects of initial tension and length on the work output during the twitch of an isolated frog gastrocnemius muscle. We then analyze the results in respect to the condition of the muscle's elastic and contractile components. In measuring work, any convenient units of force and distance could be used. We will measure force in grams, and distance in mm. To illustrate the concept of work, suppose your car died, and you had to move it out of the traffic lane to the side of the road by pushing it. You pushed with an average force of 30 kg, and managed thus to move the car 10 meters. You had to do 300 kg-meters of work. Now, suppose you did this in 1 minute. In that case, you generated 300 kg-meters min^-1 of power. If a seedy professor had to do the same, it might take 4 minutes, so the professor did the same work but with a power output of only 75 kg-meters min^-1. In this study, the time factor will be eliminated because all the muscle work will occur in the same amount of time, namely, the time for the muscle to complete a single twitch.

Equipment check out procedure

It will be difficult to interpret the results of your experiments unless you have a basic understanding of the equipment that you will be using to gather the data. In addition it will confirm that the equipment is operational and eliminate equipment malfunction as a potential source of difficulty during your experiments.

The preamplifier

The DA100 transducer amplifier section of the PB3 amplifies small changes in electrical potential produced by a wide variety of transducers, including the Harvard isotonic muscle transducer, to usable levels. It also allows for filtration of extraneous signals. It should be set as follows as an initial setup and modified as required. Gain 10 - Filter off - Signle coupling DC. The channel output switch should be set to channel 2

Acknowledge setup

Start by selecting Setting up the channel menu from the MP100 drop down menu. The evoked potential amplifier should have been set up to output on channel 2 so you should set up A1 by checking acquire and plot boxes and filling in the channel label with an appropriate description.

In Acquisition Setup, select "Autosave," or "Append" to be able to save data from several trials automatically without writing over any. In the former, each trial will be saved to a separate, numbered file. In the latter, each new data acquisition will be appended to the end of the previous, to give a continuous file of data, with markers at each stop and start. This would be analogous to a continuous strip of chart paper. Choose a Sample Rate of at least 100/sec. A total length of a 5 seconds will suffice for most of your trials.

You will also have to set up the stimulator (STM 100) to deliver the strength and frequency of stimulating shocks required. To do this, click on "MP100" and select "Setup Stimulator" from the menu. The Setup window will appear.

1. Select the first Waveform button, for square-wave pulses
2. To get a single positive square-wave pulse to be delivered at 0.5 seconds after starting the data acquisition, you would select values as follows:

  "Seg #1 Ampl," select 0 Volts

  "Seg #1 Width," select 500 msec

  "Seg #2 Ampl," 10V

  "Seg #2 Width," select the duration (msec) you want

  "Seg #3 through 5 Ampl," select 0 Volts

  "Repeats" button on "1X."

3. Select A0 in the out put section and trigger off in the trigger setion

This should let you get started in controlling stimulator output. Note also that you can copy the stimulus waveform to the data window by clicking on the "Copy" icon button in the Stimulator Setup window, which will save it to a clipboard. You can then use the Edit: Paste command for the data window to show it there along with the resulting data. Another way you can record the stimulus pulses as they are delivered, is to setup acquisition on another channel, say channel 4, and use a patch cord to feed the analog signal from the UIM 100 "Analog output" channel 0 into Analog Input Channel 4.The stimulus pulse will then be recorded simultaneously with the muscle response. This will simplify examining latency if you should chose to.

Harvard isotonic muscle lever

Attach the muscle lever to the transducer using the Allen wrench provided and attach the transducer with the venire adjustable clamp to the ring stand. Connect the output of the transducer to the DA100 using a duel banna plug patch cord. Check to insure the polarity of the connections is correct ( black to ground).

The Scaling for the transducer amplifier will have to reflect its gain and the electrical characteristics of each individual transducer. The easiest way to accomplish this is to use the scaling features built in to the software as follows

1. Measure the total linear deflection at the last attachment of the lever, in mm as you move the lever from upper to lower stop. One half that value is the total distance the lever can move up or down.
2. Bring up the set up channels screen and select the scaling button. While in the scaling dialog box, move the lever to the upper stop and press cal 1 then move it to the lower stop and press cal 2. The voltages generated by the transducer at its limits of travel will appear in the volts box. Entering the distance the lever moved up or down in the map value boxes as well as the units of measurement (mm) will complete the calibration procedure. Run a short data acquisition and confirm that the distance moved by the lever is reflected by the trace on screen. If upward movement of the trace is not produced when the lever is lifted either the transducer is mounted upside down or it is wired backward.

Finally thread the nylon afterloading screw into the threaded socket so that it will limit the downward movement of the arm and attach .

The stimulator output should be input to channel 4 on the MP100 so you need to check acquire and plot for analog chanel A4 as well. The scaling for this channel should be 1 to 1. Once the channels have been set up close the dialog box and select set up acquisitions from the MP100 drop down menue.

Set the acquisitions line to Record and save to MP100.

The sample rate to 50000 samples/sec and the total length to 20 ms. The acknowledge will examinte your hardware and change these values to the maximum that it can handel so dont wory if the values change as you enter them.

Ignor the repeat line as long as the repeat box is not checked. If an x appears in the box remove it.

Close down the Setup acquisitions box and open the Setup Stimulator dialog box.

Start by selecting A0 as the output channel then double check to be certain that your stimulator module has its input selector set to A0 also.

Next select the Squair wave from the waveform menu.

Set the Repeats to 1x.

The Stimulus wave form is divided into segments. As we are going to use only a single pulse of DC current you should enter 0 for all of the Seg Amp voltages except the Seg 2 Amp segment which shoud be set for 10V temperoraly.

The Seg# Widths shoud be established in the absolute mode by selecting its button from the poarameters line. It is the second from the right with arows pointing in both directions.

Set #1 to 1ms #2 to 0.1ms #3 to 0ms and #3 to 0ms. Seg#5 will be set by the softwater to fill the rest of the time in the 10ms time window.

Close out these windows and acquire a few runs to see how things are working. If the stimulus timing and amplitude General Procedures

You will remove the gastrocnemius muscle, that is the "calf" muscle that extends the ankle, from a double-pithed frog, and mount the muscle on an isotonic muscle lever. An isotonic muscle lever puts constant tension on the muscle, so that when the muscle contracts, the transducer attached to the lever measures the shortening of the muscle without any change in its tension. This is an apparatus, therefore, for measuring the dynamics of muscle shortening during contraction, when no change in tension is allowed to occur. In the body, a working muscle usually contracts with some shortening, and some change in tension at the same time. Muscle levers, therefore, allow the physiologist to isolate one or the other function of the muscle for controlled study.

You will stimulate the muscle with electrical shocks of varying voltage, to determine the threshold of stimulation, and the voltage that produces a maximal response. Duration of the shock is an important variable besides voltage, so you will investigate the relationship between duration and voltage of the shock needed to stimulate muscle activity. At first you will stimulate the muscle through its nerve. Actually, you will be stimulating the nerve fibers leading to the muscle, and the nerve fibers in turn will stimulate the muscle to contract, so in this case, you will study the response of the nerve to the duration and voltage of stimulating shock. Later, you will stimulate the muscle directly, bypassing the nerve and all the variability in its responses to the stimuli.

1. Set up and connect your apparatus in the manner shown in the diagram of Figure 1. When everything is set up and operating satisfactorily it is time to dissect the gastrocnemius muscles of the frog as follows:
2. Double-pith a frog and remove the skin from the lower trunk and legs by cutting the skin around the trunk and pulling the skin off the lower body and legs inside out like the fingers from a rubber glove. These operation removes the skin without letting poison secreted by the skin contact the muscle and its nerve.
3. Now wash the dissecting instruments you used to remove the skin, and wash your hands so that none of the poisonous mucus from the skin will contaminate the muscle and nerve during the next operations.
4. Push a glass probe between the gastrocnemius muscle and the tibiofibula bone of the shank, and separate the muscle from the bone along its length.
5. Tie a strong thread tightly around the tendon of Achilles that connects the gastrocnemius muscle to the heel of the foot at the ankle, and then cut the tendon free of the foot distal to (toward the far end from) the thread. Now you will have the gastrocnemius muscle free at its distal (= far) end with a length of thread tied securely to its tendon.
6. With your scissors cut the tibiofibula bone just below the knee, leaving the gastrocnemius muscle attached to the femur (see Figure 2).
7. With scissors, cut off the femur and the attached muscles of the thigh near its joint with the hips (Figure 2).
8. Separate all the muscles of the thigh from the femur by sliding a glass probe between the muscles and moving it back and forth.
9. Expose the cream-colored, string-like sciatic nerve, and carefully separate it from the surrounding tissues. Lay it against the gastrocnemius muscle so that it will not be injured, and remove the thigh muscles from the femur and discard them. You should now have a preparation as shown in Figure 2.
10. Put your preparation in a beaker of frog Ringer's solution and prepare the other gastrocnemius in the same way for a spare.
11. Attach one of the nerve-muscle preparations to the apparatus and prepare to record the muscle contractions as shown in Figure 1. During the following steps, be sure to keep the muscle and nerve moist with Ringer's solution, but take care not to get the solution on the transducer body and lever shaft.

Attach the electrode leads to the stimulator, and set the stimulator duration to 2 milliseconds. Allow the nerve to lie against the muscle, and stimulate with the electrodes against the nerve and muscle together, at the proximal end of the muscle where the nerve enters it. This should give the most reliable response to stimulus. Be sure to keep muscle and nerve moist with Ringer's solution. Take care to keep Ringer's solution away from the fulcrum and shaft of the lever transducer.

1. Adjust the afterloading screw so that it supports the weight of the lever and pan, and the muscle is stretched to a moderate degree. The muscle needs to have some tension, to keep the attachment to the lever taught, but the muscle should not support the whole weight of the empty pan. In this way, the muscle will first have to build up tension equal to the force of the load in the pan before it can begin to shorten and lift the load. It must do work on its own elastic components before it can start to work on the load.
2. With weight pan empty, and with the stimulator set to deliver a stimulus after data acquisition is started, adjust stimulus voltage to produce a maximal twitch, and select a voltage a little above this value (10%) so as to assure a uniform, maximal twitch each time the muscle is stimulated. The height of the twitch registered for the empty weight pan will arbitrarily be designated the twitch response for a load of zero grams. Obviously, the muscle is doing work by lifting the empty pan and lever, but your calculation of work will have a value of 0 to start with. (You are going to subtract the work done on the empty pan from all tests and only measure work done on the weight added to the pan.) Indicate with your record that the load is "0 grams." Save this, together with your complete record of each experiment.
3. Put a 10-gram weight in the pan, and record the twitch. Note on the record "10 grams." And save the recording
4. Repeat step 3, adding 10 g. each time, until the muscle fails to move the lever.

1. Back out the afterloading screw so that the muscle supports the full weight of the pan and its contents. In this way the muscle will always be under tension equal to the force of the load. When it contracts, the muscle will do so isotonically, that is, "with constant (iso-) tension (tonus)." Note that as you increase the load, the muscle will stretch, so you will have to readjust the position of the transducer so that its output is 0 mm (0 volts).
2. Record twitch heights with the weight pan empty, and indicate "0 grams" with your record.
3. Repeat step 2, adding a 10 gram weight to the pan each time, recording the results until the muscle fails to move the load, or until the attachments of the muscle to the lever break. Strong thread and good knots are going to be essential to successful completion of this part of the experiment! Note that as the load increases, twitch height decreases. Does the work output decrease also? Save your records.

Prepare your report by completing the report form on the following pages.

1.Attach your chart records to separate page(s) and include it with your report.A Xerographic copy is acceptable. (Please, make copies at your personal expense.)

2.On the attached data table, fill in your calculations from the record of afterloaded contractions. (Be sure to state the conversion factor you used to make your calculations.)

1. Attach your chart recordings to a separate page, and include it with your report.

2.On the attached data table, fill in your calculations from the record of the free-loaded contractions.

1. Plot the data from the data table on two graphs as follows:

a. The first graph shows distance (mm) on the ordinate, load (g) on the abscissa.Use different symbols for the after-loaded and freeloaded conditions. Make the symbols (data points) big enough to see clearly if a line gets drawn through them!Label the graph appropriately.

b. The second graph shows work (g-mm) on the ordinate, load on the abscissa. Label the graph appropriately.

c. Fit curves to each set of data points by eye, estimating the best fit to the data.

2. Answer these questions:

a.The optimum load is that on which the muscle does the most work per twitch. Estimate the optimum loads based on your data and graphs.Enter these estimates in the blanks below:

after-loaded free-loaded

Optimum load = _____________ ____________

b.Interpret your results in terms of the mechanical models of a muscle illustrated in Figure 2 below. Address these questions:

i. To what extent are your results consistent with the model shown? Be specific.

look ok the you should proceed to the string experiment

The nerve chamber

It is considerably easier to record the weak electrical potentials associated with action potentials if the nerve is completely removed from the organism and placed in a controlled environment chamber.

Set up

Set up the nerve chamber and connect the bioamplifier cables and stimulator output cables as shown in Appendix D Add a few mm of Ringer's solution to the bottom. Set up the nerve chamber and connect the bioamplifier cables and stim-ulator output cables as shown. Add a few mm of Ringer's solution to the bottom. Place a thread soaked in Ringer's over the electrodes. Cover the chamber with a microscope slide to retard evaporation.

Trial run

The following will allow you to make a trial run of the nerve chamber using electrical noise from the class room rather than a live nerve. Touch the recording electrode furthest from the stimulator with a finger. Have your lab partner click the start button. If all is well a sixty cycle electrical noise signal should appear on the screen. It will appear as a slightly distorted sine wave. It represents AC voltages that you (acting as an antenna) are picking up from the lights.

The experiments

If all of the preceding procedures make sense , you are finally in a position to begin your experiments The response of a non-living conductor to stimuli The moist string that you placed in the chamber has the same resistance to electric current as a nerve. Remember you soaked it in a conductive Ringer's solution. It differs from the nerve in that it ,of course, is not living. Using the techniques mastered above stimulate the string with single pulses of increasing voltage and observe the trace produced. Be certain that you understand what you are observing.

Nerve preparation

Your success with this experiment depends on your ability to remove an undamaged nerve from a living crab. Work quickly and carefully. With a sharp scissors cut through the ischium of the swimming leg of a large crab. Remove a walking leg from the crab by cutting through the coxa. With a sturdy scissors cut the apodemes (muscle attachments) at each of the joints in the leg. Firmly grasp the propodus and carpus and gently pull off the merus. The nerve should remain attached to the next leg segment. Continue to pull off the remaining segments until only the nerve remains attached to the dactyl (last segment). Place the nerve in very shallow pool of ringers. If it is kept cool the nerve should remain functional for many hours. Caution The nerve is extremely delicate. Avoid excess stretching during the removal process. Do not touch it with your fingers or anything else; use the attached dactyl as a handle. Avoid excessive warming. Mounting the nerve preparation Fill the bottom of the nerve chamber with some of the crab plasma,but not so deep that the fluid contacts the electrodes. Lay the nerve across the electrodes with the proximal end across the stimulating electrodes, and the rest extending across as many recording electrodes as possible, but without being unduly stretched. Place the cover on the chamber to keep the preparation moist. Determination of threshold and maximal voltages

Two of the interesting questions that we can ask about a nerve are what is the minimum stimulation required to produce a response and at what point does the nerve fail to produce a greater response to increased stimulation. With the stimulator delivering 0.05ms impulses, begin stimulating with a voltage setting of 0 volts by delivering a single pulse with the stimulator. Slowly increase the voltage (0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, and 10) and repeat the stimulation. Save the experiment as a sequence of files. You will probably note a stimulus artifact which appears as a positive spike or wave at the beginning of the trace. This is not the action potential, but the stimulus impulse that is conducted passively along the nerve to the recording electrodes. Note any similarity to the response of the string? At some voltage, usually less than 10, a new deflection in the beam should appear to the right of the stimulus artifact. This is the action potential. It should look somewhat like the idealized recording shown in Figure 3. The stimulus voltage at which a discernible action potential can just be seen is the threshold voltage. Now continue to increase the stimulus voltage, noting that the action potential gets larger as you do so. The increase in the action potential voltage may occur somewhat irregularly, because it results from the activation of more and more nerve fibers, various classes of which have different stimulus thresholds as well as different conduction velocities. As more fibers fire, a greater action potential voltage is recorded by the oscilloscope. At the point where an increase in stimulus voltage fails to cause a further increase in the action potential voltage, a maximal stimulus has been delivered. Record the maximal stimulus voltage.

Note

It is extraordinarily easy to produce a fried preparation here. Do not increase the voltage past the point where you are certain that you have reached the maximal voltage. Determine the amplitude of the compound action potentials elicited above by measuring the height of the upward deflection from the baseline to the peak. Record your parameters, and using your knowledge of the equipment, calculate the maximal action potential voltage. Strength duration curves By examining the relationship between the strength of a threshold stimulus and its duration the values for rheobase and chronaxie can be determined You are to examine the effect of varying the stimulus duration on the threshold voltage of your nerve preparation. Repeat the threshold determination experiments over the following range of stimulus durations: 0.025, 0.05, 0.075, 0.10, 0.25, 0.50, 0.75, 1.0ms Rrecord the stimulus voltage required at each stimulus duration to elicit an discernible action potential as a sequential file. From the curve determine the reobase voltage (voltage required to elicit a response from an infinitely long stimulus) From the curve determine the chronaxie time (stimulus duration required to elicit a response at a stimulus intensity twice the reobase voltage).

Conduction Velocity

You should have noticed that the nerve chamber has several additional electrodes located down from the stimulating electrodes at one centimeter intervals. This arrangement will allow you to make incremental changes in the distance that an action potential must travel in order to be recorded and allow you to determine the speed of conduction. Move the recording leads an additional centimeter further from the stimulating electrodes. The action potential will now travel 2cm before reaching the detection electrodes. Stimulate with single pulses at the maximal voltage determined above. You may note that the action potential is not as strong at this distal point as it is proximally. How can you rationalize this in light of the all or nothing law of action potential propagation? You may also note that the action potential has become wider. How can you explain this phenomenon. Rrecord the results in a sequential file.

Monophasic action potentials

All of the traces that you have produced thus far are biphasic action potentials. As the wave of depolarization reaches the first electrode that electrode becomes more negative than the more distant electrode. As the depolarization reaches the second electrode it is the one that becomes more negative than the first. Note that this is very different from a monophasic re cording produced from one electrode within the nerve and one on the outside. Monophasic action potentials can be produced with the following techniques Completely deactivate the portion of the nerve lying over the most distal recording electrode by crushing the nerve with a forceps and the placing a drop of isotonic KCl (0.16M) on the crushed portion of the nerve. Equalizing the potassium concentration inside and outside of the axonal membrane in this fashion will inhibit the ability of the cell membrane to depolarize and conduct an action potential past this point. Connect the second recording electrode lead wire (red) to the most distal recording electrode (under the deactivated portion of the nerve). Connect the first recording electrode lead wire to the recording electrode 1cm from the stimulating electrode. Repeat the maximal voltage experiment and note that new peaks appear as the voltage increases. Move the first recording electrode lead wire to the next most distal electrode and again stimulate at increasing voltages. Obtain a trace that most clearly demonstrates these additional peaks (by varying the voltage and electrode position) and produce a high speed hard copy by increasing the chart speed to 25mm/second. What is the source of these additional peaks? Why do they spread out as the distance from the stimulus increases?

Report

Your report should incude your data reported as duration and magnitude of each response to stimulation for each of the experiments that you performed. Be certain to include the response of a non-living conductor, the threshold and maximal voltages, the strength duration relationship, the conduction velocity and the various classes of fibers that you can demon-strate in the monophasic action potential. Include the data in a well organized table where applicable. Use your own experience and judgement as to the appropriatness of a graph. You should discuss how your data compres to what would be expected from a vertebrate (your text is a reasonable reference for this).

References

Bullock, T. H., A. Grinnell, and R. Orkland, 1977. Introduction to the Nervous System; W.H. Freeman and Co., San Francisco. pp298.

Erlanger, J. and H.S. Gasser. 1968. Electrical Signs of Nervous Activity; University of Pennsylvania Press, Philadelphia, PA.

Hodgkin, A. L. 1964. Conduction of the Nervous Impulse; Liverpool University Press. Katz, B., 1952 The nerve impulse, Scientific American Vol 187 #5.

Katz, B., 1966. Nerve Muscle and Synapse. McGraw-Hill, New York. Mountcastle, V. B. 1974. Medical Physiology, Vol 1. C. V. Mosby, St Louis.

Ruch, T. C., H. D. Patton, J. W. Woodbury,, and A. L. Towe. 1965. Neurophysiology; W. B. Saunders Co., Philadelphis. Silverman, R. M. and Brunett,

0h.D. The Compound Action Potential, An Introduction to Principles of Neurophysiology. Richmond, VA., Phipps and Bird, Inc. (1978)

OBJECTIVE
To record the twitch threshold, maximal twitch response, summation, tetanus and fatigue for frog skeletal muscle and its
motor nerve.
EQUIPMENT
n Stimulator (BSLSTM; includes AC100A power)
n Force Transducer Assembly (SS12LA includes S-hooks)
n Ring stand
n Tension adjuster (HDW100A)
n Weights for calibration (must attach to S-hook)
n Needle Electrodes (ELSTM2)
n Thread (non-stretch nylon or equivalent)
n BSL PRO template file: FrogMuscle.gtl
n Live frog
n Amphibian Ringer’s solution
n Goggles
n Rubber Gloves
n Dissection Pan
n Dissection Kit: Scalpel, Forceps,
Scissors, Dissection Pins, Tape
n Glass probes (can create your own)
n Acrylic Board (for dissection)
SETUP
If you are setting up the hardware and software from scratch, then you may want to perform these steps prior to prepping
the frog since they may take some time to complete.
1. Connect the Hardware as shown here:
BSLSTM Stimulator
Back:
Trigger to Analog Out on back of MP30
AC100A to Mains (wall) power
Reference Output to CH1 on front of MP30
Front:
ELSTM2 to Input port
MP30
Back:
CBLSERA to serial port on host computer
AC100A to Mains (wall) power
Front:
SS12LA to CH2
2. Turn on the MP30, then turn on the BSLSTM stimulator.
3. Establish the stimulator settings.
n If you are using the older, manual stimulator (model BSLSTM), click here for details.
a. Turn the Level knob counterclockwise until it stops at 0 Volts (full counter-clockwise).
b. Turn the key to the right to set the Range at "10V."
c. Flip the Reference switch to Fixed (15ms); this setting is not required on the older BSLSTM.
d.
4. Launch the BSL PRO software on the host computer. The program should create a new "Untitled1" window.
5. Choose MP30 menu > Show Stimulator to bring up the stimulator window.
6. Make sure the stimulator is working.
a. Use the On/Off button in the Stimulator window to turn the stimulator ON.
b. Check to make sure the Pulse light on the front of the BSLSTM is blinking.
c. Use the On/Off button in the Stimulator window to turn the stimulator OFF.
7. Bring up the Frog Muscle template by choosing
File menu > Open > choose Files of type: GraphTemplate (*GTL) > File Name: FrogMuscle.gtl.
IMPORTANT: DO NOT CLOSE THE STIMULATOR WINDOW!
The Stimulator window must remain open during acquisition. You may need
to re-position the windows such that the stimulator and data windows are in
full view with the stimulator window lying to the right of the data window.
8. Put the tension adjuster (BIOPAC HDW100A or equivalent) on the ring stand, and attach the BIOPAC SS12LA
Force transducer such that the hook holes are pointing down. The SS12LA should be roughly set so that it is level
both horizontally and vertically.
9. Set the tension adjuster such that it is approximately ¼ the distance from the lowest setting. This will allow the
majority of the range to be used for adding tension (raising the adjuster).
n Do not firmly tighten any of the thumb-screws at this stage.
10. Determine the optimal force range for this experiment (0 to 50, 0 to 100, 0 to 200, 0 to 500, or 0 to 1000 grams).
n Pick the lowest range to optimize the output resolution. If you have a small or medium frog, there is a good
chance that the 0 to 50 gram range will be optimal.
n The default BIOPAC template assumes that your experiment will work under a force transducer range of 0
to 50 grams because this range should be appropriate for the majority of users. However, if large frogs are
used, the range may need to be increased. See Change Force on PC or Change Force on Macintosh for
directions to change the template settings.
11. Select and attach the proper S-hook.
S-hooks
The SS12LA includes two "S-hooks" for attachment of line or weight. Use the smaller S-hook for 0 to 50, 0 to 100
or 0 to 200 grams; use the larger S-hook for 0 to 500 or 0 to 1000 grams. Place the appropriate S-hook in the proper
hook hole on the SS12LA. The larger S-hook will have a tight fit, but this is necessary for accurate force readings.
Note: To calibrate the transducer for the full range, you will need to have a weight representing the upper limit of
the range (50 grams, 100 grams, etc.). It must be capable of attaching to the S-hook.
Hints for minimizing measurement error:
A. The SS12LA Force transducer must be level on the horizontal and vertical planes.
B. Set up the Tension Adjuster and Force Transducer in positions that will minimize movement when
tension is applied. In other words, try to keep the point of S-hook attachment as close as possible to the
Ring Stand support (see assembly picture).
C. Position the Tension Adjuster such that the Adjustment knob is easily accessible so you will not bump
cables or the tray during adjustment.
D. Position the Stimulator and Force transducer cables such that they cannot be easily pulled or bumped. If
necessary, use tape to adhere cables to the tray or ring stand to relieve strain or pull.
E. Set up the dissection tray such that it is very stable and will not wobble or rock.
F. Make sure the end of the frog muscle is firmly pinned to the dissection tray, such that it will not rise up
when tension is applied.
G. Position the entire setup on a solid workbench such that it will not wobble if the table vibrates.
H. Use an attachment line that will not stretch under tension (such as non-stretch nylon).
I. Use a knot that will not allow the line to lengthen or come undone under tension (such as the Bowline
knot; see the Appendix for tying instructions).
J. Always keep the line attached to the SS12LA straight up and down (perpendicular to transducer).
K. Do not have excessive pre-tension on the muscle.
CALIBRATION:
IMPORTANT! Calibration must be performed each time the FrogMuscle template is opened.
Channel 1 Stimulator Marker:
1. Bring up the Channel 1 (CH 1) Scaling window by selecting:
MP30 menu > Setup Channels > Channel 1 (wrench icon) >Scaling button
The initial scaling parameters for stimulator model BSLSTMA are:
If you are using the older model BSLSTM with knob control,
click here for details about changing the Preset.
2. With the controls on the BSL Stimulator set to a Level of 0 Volts (full counter-clockwise), and the stimulator not
running (pulse light not ON or blinking)…
Click on the Cal1 button in the Scaling window.
n The Cal1 Input Value should now reflect the actual reading on Channel 1.
3. Determine the Cal2 Input Value (Cal2 Input Value = Cal1 Input Value + 50), then manually enter it.
n For example, if Cal1 Input Value = -.12 mV, then Cal2 Input Value = -.12 mV + 50 mV = 49.88 mV, so you
would type in 49.88.
4. Click on "OK" to exit the Scaling window.
Channel 2 (Force):
1. Bring up the Channel 2 Scaling window by selecting:
Setup Channels Window > Channel 2 (wrench icon) > Scaling button
2. With only the S-hook attached to the SS12LA Force Transducer, click on the Cal1 button.
3. Attach the 50 gram weight to the S-hook and stop any swing on it.
n If not using the 0-50 gram force range settings, attach a weight corresponding to your settings.
4. Click on the Cal2 button.
5. Click on "OK" to exit the Scaling window.
6. Close out of Setup Channels.
You are now ready to prep the frog and adjust the SS12LA Force Transducer position.
FROG PREP:
Pith the frog and excise the skin from the frog leg (if necessary, see Application Note BSL-A01 Frog Prep for details). We
strongly recommend that you leave the frog intact for all segments of your experiment. Leaving the leg, muscle and
nerve attached to the frog will improve circulation and preserve the muscle and nerve. You should apply amphibian
Ringer’s solution to the frog and muscle in five-minute intervals. The size and condition of the frog can influence the
recording result.
1. Isolate the Gastrocnemius Muscle
2. Isolate the Sciatic Nerve
Use a blunt dissection to free up the nerve, being very careful not to damage the nerve.
3. Pin the knee firmly to the dissection pan
Pin the knee in place, being very careful not to penetrate or damage the sciatic nerve. (To stabilize knee
position, pin the muscles of the anterior thigh and the anterior leg.)
4. Position the force transducer
l Position the tray such that the frog's knee is beneath the transducer hook.
l Adjust the tension adjuster/force transducer assembly such that the S-hook is approximately over the knee.
5. Attach the muscle to the transducer
l Slide the tension adjuster/force transducer assembly down the ring stand to a point where you can loosely
hang the loop off the S-hook.
l Slide the tension adjuster/force transducer assembly up the ring stand to a point where the line slack is
removed but the muscle is not stretched.
l Adjust the assembly such that the thread line falls vertically.
n For a true reflection of the muscle's contractile force, the muscle must not be pulled at an angle.
l Tighten all thumb-screws to secure the positioning of the assembly.
6. Place the stimulator electrodes under the sciatic nerve
l Remove the covers from the electrode tips.
n DO NOT DISCARD THE COVERS.
n Always put the covers back on after an experiment.
l Use tape to connect the two electrode leads, making sure the needle tips do not touch.
l Insert the electrode needles underneath the nerve, being very careful not to penetrate or damage the sciatic
nerve.
l Take care in placing the electrodes to ensure that the surrounding muscle is not stimulated by needle
contact as that will distort the result.
7. Adjust transducer pretension
l Use the tension adjuster knob to make the line taught — approximately 5 grams of tension is appropriate.
DO NOT OVER-TIGHTEN!
l Let the setup sit for a minute, then re-check the tension to make sure nothing has slipped or stretched.
When completed, your setup should look similar to this:
RUNNING THE EXPERIMENTS
NOTE-- This recording is set up for the Append mode, so when the acquisition is stopped then re-started, data
will be added onto the previous data. A marker will automatically be inserted with a time stamp to indicate the
new segment start time.
To save recorded data, choose
File menu > Save As… > file type: BSL Pro files (*.ACQ) File name: (Enter Name) > Save button
To erase all recorded data (make sure you have saved it first), and begin from Time 0, choose:
MP30 menu > Setup Acquisition > Click on "Reset" button
THRESHOLD AND MAXIMAL RESPONSE:
n If more than one person is working on this experiment, have one person watch for the muscle twitch and another
watch the screen for a recorded response.
1. Make sure the option "Specified number of pulses" is selected in the Stimulator window.
n Remember...the Stimulator window must remain open during acquisition.
2. Click on the "Start" button to begin recording.
3. Increase the stimulator voltage Level to .1 Volts then click on the On/Off button in the Stimulator window.
n You should see the stimulus pulse recorded on CH 1 Stimulator, but no response on CH 2 Force.
4. Increase the Level to .2 Volts and click on the Stimulator On/Off button again.
5. Continue increasing and stimulating in .1 V increments until you see a response on CH 2 Force.
n This is the Threshold response.
6. Continue increasing and stimulating in .1 V increments until a point is reached where further increases in
stimulator voltage produce no further increase in the response amplitude.
n This will be the Maximal Response.
7. Click on the "Stop" button to stop recording.
SUMMATION, TETANUS AND FATIGUE:
1. Check the line tension to make sure it is still taut.
n Increase tension using the fine tension adjustment knob if necessary.
2. Select the "Continuous Pulses" option in the Stimulator window.
3. Drag the Pulse Rate scroll bar to the far left so that the starting Rate is 1 Hz.
4. Click on the "Start" button to begin recording.
5. Use the On/Off button in the Stimulator window to turn the stimulator ON.
6. Click once on the right arrow of the scroll bar to increase the stimulator Pulse rate by 1 Hz.
7. Continue to increase the Pulse rate by 1 Hz approximately every 2 seconds until the recording shows that the point
of fatigue has been reached.
8. Use the On/Off button in the Stimulator window to turn the stimulator OFF.
9. Click on the "Stop" button to stop recording.
10. Save the data by selecting File menu > Save As… > file type: BSL Pro files (*.ACQ) File name: (Enter Name)
> Save button
ANALYSIS
The BSL PRO offers a wide array of measurement and analysis tools to help you isolate the data segments of interest for
your particular experiment. As one example, you might zoom in on a maximal response segment and select an area from
the start of contraction to the end of contraction, then set the delta T measurement to determine the duration of the
contraction.
Note: the BSLSTM Stimulator outputs a marker pulse which is much longer than the actual stimulator pulse, so you
always need to reference the leading edge of the stimulator pulse. The amplitude of the pulse is accurate, and the marker
information defines the settings associated with the pulse; so students do not need to manually record stimulator data.
RECORDING VARIATIONS
Stimulate the gastrocnemius muscle
If the threshold voltage is first determined for a somatic motor nerve, and then, using the same preparation and procedure,
determined for the skeletal muscle by way of direct stimulation, it will be found that the threshold voltage for the muscle
is greater than the threshold voltage for its somatic motor nerve. More connective tissue is present in skeletal muscle than
in nerve and therefore a greater voltage is required for adequate stimulating current to flow into the muscle and excite a
sufficient number of muscle fibers for a threshold response. Nerve fibers are also much more sensitive to low-voltage
stimuli than are muscle fibers. Typically, a threshold strength stimulus for a muscle activates only a small number of
component muscle fibers. However, the same strength stimulus applied to the muscle's motor nerve will usually activate
all of the component nerve fibers, resulting in maximal contraction of the muscle.
1. Position Electrode on the Muscle.
n Ground can be at the knee (proximal end of the muscle) or near the distal end of the muscle (where the
Achilles tendon is located).
2. Stimulate the muscle with a voltage adequate to excite the muscle; this will confirm that a contractile response can
be recorded (and that you don’t have too much thread slack).
n Start stimulus strength at a maximal response level (such as 5V) to generate a contractile response and
confirm that the transducer picks it up.
3. Repeat the recording procedure outlined in this Application Note but use stronger voltages.
n Voltage required to stimulate the muscle will be at least 10x greater than the voltage required to stimulate
the nerve.
The Threshold Response for directly stimulating the muscle is much smaller than the Threshold Response for stimulating
the nerve fibers.
APPENDIX
GRAPH TEMPLATE SETTINGS
Click here to open a PDF of the graph template file settings.
BSLSTM STIMULATOR SETTINGS
If you are using the older, manual stimulator (model BSLSTM),

Walter I. Hatch
wihatch@smcm.edu

August 12, 2012