Muscles and Movement                                                                             Frog Skeletal Muscle: Properties of the Single Twitch, Summation, Tetanus and Fatigue

Overview

Vertebrate skeletal muscle is a type of muscle specialized for balance and movement. The basic unit is the motor unit, which consists of a single motor neuron and the skeletal muscle fiber(s) it innervates.  In vertebrates, motor neurons project from the central nervous system (CNS) to make synapses with skeletal muscle fibers at neuromuscular junctions.  These synapses are always excitatory -- an action potential in a motor neuron should always results in the contraction of one or more skeletal muscle fibers.

 A typical skeletal muscle is composed of many motor units.  Each motor neuron tends to synapse onto only one type of muscle fiber.  Stimulation of a single motor neuron tends to create contraction of only one muscle fiber type.  Motor neurons that supply weak, slow, oxidative fibers have the lowest threshold; those innervating fast, intermediate-strength oxidative fibers have higher thresholds; and those that supply the fast, strong, glycolytic fibers have the highest thresholds. The CNS modulates the overall force of skeletal muscle contraction by altering the total number and type of motor units activated at any moment.  As activity descending from the brain increases, progressively more motor neurons, and more of those innervating the stronger muscle fiber type, are activated into a response.

Force of skeletal muscle contraction is also augmented when the CNS increases the rate of electrical signaling to individual motor units.  A brief skeletal muscle contraction, otherwise known as a twitch, is usually the result of a single, supra-threshold stimulus of short duration. If multiple, supra-threshold stimuli (APs) are delivered to a motor unit at high frequency, individual twitches may sum.  The force of the summed muscle contraction should be greater than the force produced by a single twitch.  If stimuli are delivered at high enough frequency, such that the muscle cannot relax between contractions, a sustained contraction will result.  This state is known as tetanus.  A muscle experiencing tetanus generates maximum force, relative to the force produced by single or summed twitches.  Some muscles, especially those composed of the weak fiber type, cease to respond to repeated stimuli over a long duration.  These muscles cannot sustain long periods of contraction and will relax (“fatigue”), even in the presence of supra-threshold stimuli.    

In today’s exercise, you will observe the stimulus-response characteristics of the sciatic nerve-gastrocnemius muscle system of the leopard frog (Rana pipiens).  You will investigate the activation threshold of neurons and simulate motor unit recruitment by increasing the voltage applied to the sciatic nerve and measuring the response in the gastrocnemius muscle.  You will also observe the relationship between stimulus frequency and force of muscle contraction by applying a supra-threshold stimulus to the nerve at increasing frequency.  As part of the exercise, you will have the opportunity to observe and distinguish differences between a single twitch, summed twitches, tetanus and fatigue.

Equipment Required

Computer

iWorx/214 data acquisition unit and USB cable

FT-100 force transducer

Stimulating electrodes and BNC-Banana adapter

Ring stand and clamps

Femur clamp

Suture thread

6" Ruler

Frog Ringer's solution

             

The equipment used to evoke and record contractions from the frog Gastrocnemius muscle using the iWorx/214.

Equipment Setup

  Note:  the following may have already been done for you:

1.   Plug the end of the FT-100 Force Transducer into Channel 3 of the iWorx/214 unit.

2.   Plug the BNC-double banana adapter into the positive (red) and negative (black) sockets of the iWorx 214 stimulator. Check the side of the double banana adapter for a tab, often embossed with the letters GND. This is the side of the adapter that goes into the negative (black) socket of the stimulator.

3.   Attach the end of the stimulator cable to the dual banana adapter.

4.   Arrange the clamps on the ring stand so the muscle clamp is on top, the clamp holding the stimulating electrode is in the middle, and the clamp holding the transducer is on the bottom.

Start the Software

1.       Click the Windows Start menu, move the cursor to Programs and then to the iWorx folder and select LabScribe; or click on the LabScribe icon on the Desktop.

2.       When the program opens, select Load Group from the Settings menu.

3.       When the dialog box appears, select AK214.iws and then click Load.

4.       Click on the Settings menu again and select the Muscle2 settings file.

5.       After a short time, LabScribe will appear on the computer screen as configured by the Muscle2 settings.

6.       The Muscle2 settings file adjusts:

·          the stimulus amplitude to 0.00V, with adjustable increments of 0.10V.

·          The stimulus delay to 50ms, with increments of 1ms.

·          the stimulus duration to 10.0ms, with adjustable increments of 1.0ms.

·          the sampling rate to 200 per second.

·          the frequency to 0.5Hz.

·          the number of pulses to 1.

Note: These settings can also be changed by selecting Preferences from the Edit menu.

The Dissection

1.       Obtain a frog from your TA and place it in your dissecting pan.

2.       Before you begin dissection, view the movie clips on your computer by selecting the Movie Folder icon on the desktop.  Watch the movies in the order specified by the lab instructor.

3.       Remove the skin from the legs by making an incision through the skin around the entire abdomen. Cut the connections between the skin and the body--especially around the base of the pelvic girdle. Use stout forceps to pull the skin off the frog in one piece (like a pair of pants).

4.       Place the frog in the dissection tray with its dorsal side up (belly down).

       

Moisten the exposed limbs of the frog with Ringer's solution every five minutes or so.

5.       Identify the Gastrocnemius muscle (calf muscle) on the lower leg.

6.       Use a glass hook to separate the Gastrocnemius muscle from the bone and other muscles of the lower leg.

7.       Tie a knot around the Achilles tendon using suture thread.  Make sure the suture is secure and will not slip off the muscle!  Leave one end of the thread long enough to attach the muscle to the force transducer (see figure below).

8.       Cut the Achilles tendon as close to the bottom of the foot as possible, so the suture thread is still attached to the Gastrocnemius muscle.

       

Isolate as much tendon as possible, since it will be used to attach the muscle to the transducer.

9.       Move the Gastrocnemius muscle away from the rest of the lower leg.  Cut the tibia just below the knee to separate the rest of the lower leg from the preparation. Rinse the preparation with Ringer's solution to moisten the tissue and rinse away any blood.

10.    Dissect the muscles of the upper leg to expose both the sciatic nerve and the femur. Free the nerve and the bone from as much tissue as possible. Use a stout pair of scissors to cut the femur close to the pelvis. Free the nerve from the knee region to the trunk of the body.  Cut the nerve as high as you can, preferably above the pelvic girdle.  Rinse the preparation with Ringer's solution to moisten the tissue and rinse away any blood.  When you have completed the above, you should be left with the femur bone, the sciatic nerve (from knee to trunk) and the attached gastrocnemius muscle.

 

Gastrocnemius muscle separated from the remainder of the lower leg.

The Preparation

1.       Place the pelvic end of the femur bone between the jaws of the muscle clamp and tighten until the bone is held securely.  The gastrocnemius muscle should hang from the bone at a 90 ° angle (see figure below).  Do NOT place the nerve in the muscle clamp as it will become damaged!

2.       Once the preparation is mounted securely, attach the suture hanging from the gastrocnemius muscle to the hole in the blade of the force transducer.

3.       Adjust the muscle clamp and the force transducer so the thread hangs vertically (perpendicular to the floor) between the end of the muscle and the transducer blade. There should be no slack in the thread, but do not stretch the muscle past it’s its resting length.

4.       Position the stimulating electrode close to the knee joint without making contact with any of the frog’s tissue.  GENTLY lay the sciatic nerve across both leads (+ and --) of the stimulating electrode.  Wet the nerve with Ringer’s solution.

 

Gastrocnemius muscle prep attached to the femur clamp.

Exercise 1: Stimulus-Response

Aim: To measure the force of muscle contraction as a function of stimulus amplitude. 

Procedure

1.       Check values listed in the stimulator panel, which is below the LabScribe toolbar. The stimulus amplitude should be 0.00V and the pulse width should be 10ms.

2.       Type "0V" on the comment line to the right of the Marks button in the Main window. Click Start and press the Enter key on the keyboard to annotate the stimulus.

3.       Click Stop to halt recording.

4.       Use the arrow buttons in the stimulator panel to change the stimulus amplitude to 0.25V. Click the Apply1 button on the right side of the stimulator panel to effect the change in the stimulus.

5.       Type "0.25V" on the comment line. Click Start and press the Enter key on the keyboard to annotate the record. Click Stop.

6.       Increase the stimulus amplitude in 0.25 Volt increments until the muscle reaches a maximum response or the stimulus amplitude is 5V. 

7.       Moisten the nerve and the muscle with frog Ringer's solution.

8.       Select Save As in the File menu.  Save the file within the Class Folder (ex. Monday’s Lab) in My Documents.

Data Analysis

1.       Scroll to the beginning of this section of data. Click AutoScale to maximize the size of the response in the window. Remember that at low stimulus voltages, the amplitude of the muscle response may be zero.

2.       Use the Display Time icons (Single or Double Mountains) in the LabScribe toolbar to adjust the Main window so that each twitch response spans about 50% of the width of the window.

3.       Click the 2-Cursor icon so that two blue vertical lines appear over the Muscle channel on the Main window. Drag the cursors left and right so that one is on the baseline (before the twitch) and the other is on the peak of the twitch.

 

 The LabScribe toolbar

 

4.       The amplitude of the muscle twitch is displayed as the value for V2-V1 in the upper right corner of the Muscle channel (CH3).

5.       Enter amplitude data into your laboratory notebook in the form of a table (stimulus amplitude versus response amplitude).

6.       Repeat the measurement for all responses.

7.       Make a graph in Excel that relates the amplitude of the muscle response (force of contraction) to the amplitude of the stimulus.  Be sure to include an appropriate title and label the axes.

 

      

A recording of a single muscle twitch (upper trace) and the stimulus pulse (lower trace). The cursors were placed on the baseline (left) and the peak of the twitch (right).

 

Exercise 2: Summation and Tetanus

     Aim: To measure the force of contraction in a muscle stimulated with repeated pulses delivered at progressively higher frequencies.

Procedure

1.       Begin a new file by selecting New in the File pull-down menu.

2.       Check the values listed in the stimulator panel, which is below the LabScribe toolbar. Use the arrow buttons in the stimulator panel to change:

·           the stimulus amplitude (Amp) to the value that will create a maximum muscle response (determined in Exercise 1)

·           the stimulus frequency (F) to 500mHz (which is also 0.5Hz)

·          the number of pulses (N) to 15

·          Click the Apply1 button on the right of the stimulator panel to effect the change in the stimulus.

3.       Type" 0.5Hz" on the comment line to the right of the Marks button. Click Start and press the Enter key on the keyboard to mark your record. Record at this frequency for about 15 twitches. Click Stop.

4.       Stimulate the muscle at higher frequencies. Use the arrow buttons in the stimulator panel to increase the frequency to 1, 2, 3, 4, 5, 10, 20, and then 30Hz. Click the Apply1 button on the right side of the stimulator panel each time the stimulus is altered. Type the value of each new frequency on the comment line.  Record 15 twitches at each frequency.

5.       Notice that at a certain frequency:

·          the muscle does not have sufficient time to relax fully between contractions.

·          the muscle does not return to baseline between contractions.

·          the amount of tension produced by the muscle is greater than during a single twitch.

6.       Select Save As in the File menu.  Save the file within the Class Folder (Ex. Monday’s Lab) in My Documents.

7.       Moisten the nerve and the muscle with Ringer's solution.

 

A recording showing mechanical summation, where the muscle does not have time to return to "baseline" (resting length) between contractions.

                                                                                                                                                                                       A recording showing muscle stimulation with a short burst of high frequency stimuli to produce complete tetanus.

Data Analysis

Summation

1.       Scroll through the data to find where mechanical summation first appears.

2.       Click the 2 -Cursor icon so that two blue vertical lines appear over the Muscle channel in the Main window. Drag the cursors left and right so that one is on the peak of a twitch and the other is on the peak of an adjacent twitch. The value for T2-T1 is the period between twitches. Record this value in your lab notebook.

3.       Calculate the frequency at which mechanical summation first appears. Remember that frequency is the inverse of the period:

Frequency (Hz) = 1/ (#sec/1cycle); (1000 msec/sec).  Record this value in your lab notebook.

 

Tetanus

1.       Scroll through the data to locate where complete tetanus first appears.

2.       Use the two cursors to measure the maximum amplitude of the persistent muscle contraction. Compare the amplitude of the tetanic contraction with that of a single twitch (from earlier exercise). Record these amplitudes in your laboratory notebook.

Clean Up Instructions

1.       Check with your TA for clean up requirements.

Thought Questions for Exercises 1 & 2:

Questions for Exercise 1

1.       Why does electrical stimulation of the sciatic nerve produce contractions in the Gastrocnemius muscle? Explain.

 

2.       Did you observe a response in the muscle from every stimulus applied to the nerve?  Why or why not?

 

3.       Why does the response amplitude increase as the stimulus voltage increases?  Explain in terms of both nerve and muscle using terms such as threshold and recruitment. 

 

4.       At some stimulus, the maximum response is reached.  Explain what is taking place in the muscle and nerve at this point.  Why won’t the response amplitude increase with further increases in the stimulus amplitude? 

Questions for Exercise 2

  1. Within a single muscle fiber, force of contraction (response amplitude) is dependent upon the elevation and persistence of intracellular calcium ions (Ca2+  in the cytoplasm (“sarcoplasm”) of individual muscle cells).  Apply what you know about excitation-contraction coupling to describe what is taking place (biochemically) in a muscle cell during summation and tetanus. 

 

  1. Can a muscle sustain contraction indefinitely if exposed to high frequency, suprathreshold stimuli over a prolonged period of time?  Why or Why not?  If your answer is no, describe a realistic, alternative response.