Bryan Hummel

                                                                                                            Vert. Physiology 99

                                                                                                            October 5, 1999.

Stimulation to the Point of Exhaustion.

 

Introduction:   An American bullfrog was used to extracellularly stimulate and measure the contraction of skeletal muscle.  The muscle is stimulated by action potentials from the attached nerves.  When the muscle membranes depolarize, they become permeable to Ca2+ ions which flow into the cell and cause muscle contraction.  The muscle can also be stimulated directly by an electrode.  The force of the contractions was measured under different stimuli to determine the effects of acetylcholine and curare on the latent period, contraction period and relaxation period.

 

Materials and Methods:       An American bullfrog was dissected to expose one of the sciatic nerves between the spine and the “knee” joint.  At the hip joint, a suture was tied around the nerve and the nerve was moistened with Ringer’s solution.  Next the gastrocnemius muscle was detached from the surrounding tissues, cut at the Achilles’ tendon, and tied to a force transducer.   A stimulator was touched to the nerve in order to stimulate it into producing an action potential or directly to the muscle in order to get a contraction.  A computer was used to both record the magnitude of the force put on the force transducer, and the time it took for the muscle to go through its phases of contraction.

 

Results:          When the stimulating electrode was in contact with the sciatic nerve, the threshold voltage was 0.2 Volts.  The maximal contraction was recorded at 0.6 Volts.  When the muscle was directly stimulated, the threshold voltage was 0.8 Volts, and the maximal contraction was recorded at 1.7 Volts.  The average latent period was .02 seconds, the average contraction lasted .05 seconds, and the average relaxation period was .085 seconds.  After the injection of acetylcholine, the muscle showed no response, but with the addition of the curare (an acetylcholine receptor antagonist), there were a few muscle twitches without any electrical stimulation.  The length of time it took to cause tetanus at 15 pulses per second was 44.4 seconds.

 

Discussion:     The direct stimulation of the muscle required a higher voltage in order to get a contraction.  This most likely occurs for two reasons.  First is the fact that once a nerve reaches its threshold voltage, it perpetuates the action potential down the nerve to the muscle.  This brings us to the second reason, the nerve branches out and reaches many thousand parts of the muscle, which all get depolarized.  When stimulating directly, only the cells surrounding the electrode will be depolarized to the point of releasing calcium, thus the contraction is on a part of the muscle rather than the whole thing.  With nerve stimulation, even after the nerve reached its threshold, increased voltage would still increase contraction to a point.  This was due to the electrode being able to depolarize more of the individual nerves in the compound sciatic nerve until the whole compound nerve was depolarized.  As the wave of depolarization moved down the sciatic nerve towards the muscle, a latent period was observed.  During this latent period, the muscle membranes are becoming depolarized and allowing calcium to flow into the cell from the lumen of the sarcoplasmic reticulum.  The calcium causes the troponin to change conformation, which in turn removes tropomyosin and allows for the formation of cross-bridges.  The next stage was actual muscle contraction, which involves interaction between actin (thin) and myosin (thick) filaments.  The myosin extends cross-bridges that attach to actin and pull the two filaments past one another by the movement of the myosin head.  The myosin head rotates and pulls on the actin filaments which result in an overall shortening of the sarcomere.  This happens in a cyclic fashion so that there are always cross bridges attached to maintain muscle tension. Using ATP as an energy source, the myosin head detaches from the actin and undergoes a conformational change in the myosin fiber until the next open binding site. During the period of relaxation, the intracellular calcium is actively transported to the sarcoplasmic reticulum; one ATP transports two calcium molecules.  In the S.R. a large protein (calsequestrin) binds to calcium and essentially takes it out of the fluid in the S. R., which allows a smaller concentration gradient for active transport to act against.  When the intracellular calcium levels get low enough, the calcium that was bound to troponin dissociates and flows down the concentration gradient into the intracellular fluid where it can be transported to the sarcoplasmic reticulum.  Upon dissociation, troponin returns to its initial shape and the tropomyosin returns to the actin binding site and will no longer allow for the myosin to establish cross-bridges.  Because acetylcholine is the neurotransmitter that stimulates muscle cell depolarization, and curare is an acetylcholine receptor antagonist, one would expect the addition of curare to prevent the depolarization, calcium influx and muscle contraction. This was unfortunately not what happened in lab, the muscle showed no response upon the addition of the acetylcholine, and was still able to be stimulated after the addition of curare.  This was probably due to the fact that the syringe only allows the chemical to be inserted in a localized region, and the rest of the muscle remains unaffected.  There is mechanical summation in the muscle cells due to the arrangement of the sarcomeres.  Although each sarcomere doesn’t shorten a large amount, the series of contracting sarcomeres can shorten the muscle considerably.  The stimulated skeletal muscle cannot stay stimulated forever.  A state of tetanus occurs after the muscle is re-stimulated before the muscle had time to deplete its internal calcium levels, thus the active state of the muscle contraction is prolonged until the muscle can not shorten any further. A graph of tetanus would look similar to Figure 1.  Some muscles such as cardiac muscles will not undergo tetanus.

 

 

 

 

 

Figure 1.  A graph of stimulation frequency versus muscle contraction.

            Tetanus is different than fatigue.  Tetanus can and often does cause fatigue, but fatigue cannot cause tetanus.  Fatigue is caused by the muscle running out of energy sources.  This happened in lab much more quickly than it would happen in vivo.  This is because the frog would have kept a constant supply of the precursors to ATP flowing to the muscle, and in lab only ringer’s solution was provided.  Also the frog would not stimulate its muscle as forcefully or as often as we could accomplish in lab.  Because the frog muscle was allowed to pull on the force transducer, its contractions would be considered isotonic.  If the muscle were attached between two unmoving objects, it would be considered isometric.  My final conclusion is that I am finished writing this lab report, and our frog is finished as well.

 

 

Stimulation to the Point of Exhaustion.

  

Bryan Hummel

Vert. Physiology 99

October 5, 1999.

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