See Supplement pages 173-176, and PowerPoint slides for Hole Ch. 9. 

For details, use the Physiogrip lab manual (available on \\valshare\biology).

Physiogrip Lab 1 = Threshold and spatial summation. 

Physiogrip Lab 2 = Temporal summation. 

Physiogrip Lab 3 = Twitch. 

Physiogrip tips (improve muscle response):
  Warm the muscle with exercises and stretching.
  Keep duration of stimulus as short as possible.
  Scrub the skin vigorously with alcohol during skin preparation.
  Keep forearm in supine position.

Experiment 1. Effect of Stimulus Strength: Threshold stimulus, and maximal response (Spatial Summation):

1. Select "Experiment menu" from the "Physiogrip Menu" then select "Alter sweep mode" and choose 15 seconds per frame.
2. Select "continuous experiment mode" from the "experiment menu."  Press the A key to turn Autostop OFF. Have the subject grasp the pistol grip (no stimulus) with the third finger on the trigger in such a way as to put slight pressure on the trigger which will lift the screen plot up off the bottom of the screen.
3. Keep the frequency at 1 stimulus per second  (Stimulus frequency: events/sec dial); duration 0.2 ms; turn the voltage to 40 volts (Stimulus strength: amplitude/volts dial) ; apply the stimulus probe to the motor point and ask your partner to gradually increase the voltage until you get a slight response that can be seen on the screen.  Record this threshold voltage.
4. Don't stop!  Keep increasing the stimulus voltage until the strength of the muscle contraction does not increase.  Keep a record of the voltage.  Record the highest stimulus that increased the size of muscle contraction.  Now your partner increases the voltage even more gradually, until you find no increase in the strength of the response; record this measurement. 
Threshold Stimulus: ____________ Volts       Maximum Stimulus: ____________ Volts 
5. Return to the "physiogrip menu" by pressing ESC twice; select "Review/Analyze menu" and then select "displacement/time analysis." 
Measure the height of the minimal contraction and the maximal contraction. 
Minimum Displacement: ___________ mm  Maximum Displacement: __________ mm 
Stimulus strength: _________________ volts      Stimulus strength: ___________ volts
 6.  Plot the data you recorded on a graph of diplacement vs. time.  Annotate your graph to note the voltage. 

Muscles are made up of functional units called motor units.  Each motor unit consists of a motor neuron and all the muscle fibers it branches to and communicates with via neuromuscular junctions.  When any motor neuron is stimulated (sends a nerve impulse) all the muscle fibers it communicates with (i.e. all the muscle fibers in the motor unit) will contract with all the force they can generate or they will not contract at all (all or none principle).  In a given muscle there are many motor units.  The motor units vary in their excitability.  Some motor units can be excited by a weak stimulus while others require a strong stimulus. 
At the lowest stimulus strength that results in some contraction (i.e. threshold), only a few motor units are stimulated.  By increasing the strength of stimulus, an increased number of motor units can be "recruited" to increase contraction force, and a greater displacement (muscle contraction) is recorded on the graph.  The strength of the contraction (displacement) is the sum of the force exerted by all the motor units that are excited.  So this experiment demonstrates the phenomenon called multiple motor unit summation (or recruitment). 

Experiment 2. Effect of Stimulus Frequency  (Temporal Summation)

1. Adjust Intellitool: Set the sweep speed to 5 seconds (Stay in Continuous Experiment Mode with the Autostop OFF) (refer to the detailed instructions) 
2. Adjust the stimulator so that the stimulus strength is enough to produce a good twitch that fills about 25% of the vertical space on the graph, and the frequency to 1 stimulus per second. 
3. Apply the stimulus to the motor point and steadily increase the frequency until the muscle contraction does not increase in force (displacement).   (You must increase the frequency fast enough to active maximal displacement within 1-2 screens.) 
As you increase the frequency of stimulus; the individual responses (waves) of the muscle begin to fuse and later merge to show a continuous line indicating tetanic contraction of the muscle. Record the stimulus frequencies at which partial and complete tetanus occurred.
4. Use the Time / Displacement Menu in Intellitool to measure and record the time interval between stimuli where partial tetany first began and the time interval between stimuli where complete tetany began. Also measure and record the displacement of a single twitch (before tetanus) and the displacement at complete tetanus. 

shortest stimulus interval without tetany: _________ sec. 
stimulus interval for complete (fused) tetany: ________ sec. 
displacement before tetany: _________ mm 
displacement at complete (fused) tetany: _______ mm 

5. Record the data from the myogram.
6.  Plot the data you recorded on a graph of diplacement vs. time.  Annotate your graph to note the frequency of stimulation.

In this experiment the strength of stimulus is held constant and only the frequency of stimulus is increased.  It is important to note that the time measurements you made in this experiment were durations or intervals (time between stimuli & resulting contractions). These measurements are converted to frequencies by calculating how many intervals would fit into one second (stimuli per second). 

• To calculate frequency divide 1 by the time interval.  Here's an example:  If you measured the minimum time interval between stimuli where partial tetanus began as .2 sec, then the frequency of stimulus was 1/.2 or 5 stimuli per second. 

Note that in experiment 2 the stimulus frequency was increased until the muscle fibers no longer had time to completely relax. At that point, partial tetanus (incomplete tetanus) was observed. When the stimulus frequency was increased further so that the muscle fiber could not even begin to relax, fused (complete tetanus) was observed. 

Experiment 3. Single Muscle Twitch

1. Adjust Intellitool: Set the Physiogrip Program on "Single Twitch Mode." Select the "single twitch mode" from the "experiment menu."
2. Adjust the stimulatorso that the stimulus frequency is 1 per second, and set the stimulus strength (voltage) so that it produces a good muscle contraction that occupies about 75% of the vertical space on the displacement graph.  (Duration of 2ms.).
3. Switch the stimulator Mode to Off (Manual). 
4. Press B (begin data collection) the screen does not show the response, however, the data is recorded by the computer.
5. Press the Single stimulus switch to cause a single .2 millisecond stimulus. 
6. Use the Displacement Time Analysis.  Return to "Review/Analyze menu;"  select "Displacement/ Time Analysis.  The left hand edge of the screen represents the exact moment when the stimulator is fired. 
Measure and record: 

Latent Period: ________ sec. 
Contraction Period: ________ sec. 
Relaxation Period: ________ sec. 

7. Record the data from the myogram.
8.  Plot the data you recorded on a graph of diplacement vs. time. 

When a single, brief stimulus is applied to a muscle fiber, either naturally in the form of a nerve impulse, or artificially in the form of an electrical stimulus, a type of muscle contraction called a twitch contraction occurs. This is the type of muscle contraction was also observed in experiment 2, on the part of the graph prior to the onset of tetanus (i.e temporal summation). 

The Intellitool software you used to obtain this graph is set up to begin the graph at the instant the stimulus is applied by the Stimulator.  Notice that for a small interval of time no displacement occurred.  This time interval is the latent period.  It exists because of all the things that must happen for the stimulus to reach the muscle and cause the myofilaments in the muscle fibers to begin sliding.  Consider what happens when the nerve is stimulated by the Stimulus: an action potential (nerve impulse) travels to the axon end bulb, calcium channels in the end bulb open and allow calcium ions to flow into the end bulb, this causes synaptic vesicles to fuse with the neurolemma and discharge their neurotransmitter (acetylcholine) into the synaptic cleft, the neurotransmitter diffuses across the synaptic cleft and binds to receptors on the motor end plate of the muscle fiber, this causes sodium ion (NA⁺) channels in the motor end plate to open, sodium ions diffuse into the muscle fiber causing an action potential to spread over the entire surface of the muscle fiber and down into the T-tubules, this causes calcium ions (Ca²⁺) to be released by the sarcoplasmic reticulum, the calcium ions bind with troponin on the thin filaments causing tropomyosin to move away from the myosin binding sites of actin, then myosin binds to the actin filaments causing the myofilaments to slide past each other (actin slides towards the center of the sarcomere).  Even at this point, actual shortening does not occur until the slack is removed from the elastic elements of the muscle fibers. 

Once actual shortening does begin, displacement occurs on the graph.  The time interval during which muscle shortening is occurring is called the contraction period of the twitch.  As soon as calcium ions are removed from the sarcoplasm, tropomyosin moves back into the blocking position over the myosin binding sites of the actin filaments.  Since myosin can no longer slide the actin this causes the muscle fibers to relax and the displacement graph returns to zero.  This part of the graph is called the relaxation period of the twitch. 

• You will need to be able to explain the concepts and draw sample graphs.