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Lab 5.2
the frog is clinically dead, but its tissues continue to be viable for minutes to hours,
depending on how well we take care of the frog.
As soon as the frogs muscle or nerve tissue is exposed, it should be frequently
(once every 1 or 2 min) bathed with Ringers solution, an isotonic solution that has
approximately the same ion concentrations as does amphibian ECF. Just a few drops
are enough to make sure the tissues dont dry out and have enough of the right ions.
Frogs absorb oxygen through their MOIST skin and through their lungs. Because the
lungs stop working when we pith the frog, we rely on the skin to allow oxygen to diffuse
into the blood. The heart keeps working and circulating the blood through the skin and
muscle, and supplies enough oxygen as long as the skin is kept moist (damp paper
towel).
PROCEDURES
A. Frog Nerve-Muscle Preparation
Equipment and materials:
Labscribe software/hardware
double-pithed frog
dissecting tray
dissecting kit
Ringers solution in dropper bottle
2 pieces thread, 12" each
1. Refer to the diagram below. Make an incision through the skin at the base of one
thigh (where it joins the trunk) and cut it all the way around.
2. Strip the skin down over the entire leg, carefully freeing it from underlying tissues at
the knee. Pull, dont cut.
3. Keep exposed tissues moist with Ringers solutions at all time.
4. Use blunt dissection to separate the muscles on the dorsal surface of the thigh to
expose the sciatic nerve. Have the instructor cut the superficial fascia if it is in the way.
5. Be careful not to touch the nerve with metal or your fingers, and also not to stretch
it. Dont pull up on it or twist it.
6. Using a glass rod or the tip of a pipette, carefully pull a thread under the nerve. This
thread will be used to position the stimulating electrode in part DO NOT TIE THIS
THREAD.
7. Insert a glass rod between the gastrocnemius muscle and the leg bones and run it
up and down to separate them.
8. Tie the other piece of thread firmly around the Achilles tendon (using a square knot),
close to the muscle.
Lab 5.3
9. Cut the Achilles tendon distal to the tie, leaving at least 1/8" of the tendon distal to
the knot.
10. Use metal clips to secure frog to board in dissecting tray.
11. Keep the rest of the body covered with a wet paper towel.
B. Apparatus Preparation
1. Start LabScribe using icon on desktop.
2. Settings menu, Load, group, choose ahk114, OK
3. Settings, choose summation-tetanus, OK.
4. Attach tendon to transducer as demonstrated by instructor.
5. Put stimulating electrode under nerve as demonstrated by instructor.
6. Have instructor check setup before proceeding.
Lab 5.4
12. Print threshold and maximum response responses and label with your names and
the stimulus intensity if it isnt already on the screen.
stimulus (V)
amplitude
duration of
contraction
duration of
relaxation
0.025
0.050
0.075
0.1
0.15
0.2
0.25
0.3
continue only if at this point the amplitude is still increasing
D. varying stimulus frequency to demonstrate twitch summation and tetanus
Lab 5.5
response
CLEANUP:
1. frog and tissues go in zip lock bag on counter
2. rinse clips, board and pan in plain water and put in drainer
3. wash dissecting instruments in soapy water, rinse, dry and return to plastic box
4. rinse stimulating electrode prongs in dH 2O and gently dry
5. close LabScribe and AdobeReader, shut down computer
Questions
1. What was the threshold?
2. What was the maximum stimulus?
3. Recruitment began at ___V and ended at ___V.
4. How long did contraction and relaxation take at the threshold stimulus?
contraction:
relaxation:
5. Wave summation leads not only to a sustained muscle contraction, but also an
increase in contraction strength EVEN AFTER RECRUITMENT STOPS. The
physiological basis for the increase in strength (not the sustained contraction) is:
Lab 5.6
6. Ca++ is pumped out of a cardiac contractile cell via cotransporter that exchanges
Ca++ for Na+. (refer to how cotransporter systems work i.e. an active transport pump
is required to set up the initial ion gradient) How would muscle activity be affected by a
higher than normal level of Na+ in the cytosol?
a. muscle contraction would be impaired
b. muscle relaxation would be impaired
c. there would be no change in muscle activity
7. Dendrodotoxin blocks voltage-gated K channels in the motor neuron axon terminal.
a. What effect would dendrodotoxin have on repolarization of the axon terminal?
enhance, prevent or impair, or none?
b. What effect would dendrodotoxin have on the release of ACh?
enhance, prevent or impair, or none?
c. What effect would dendrodotoxin have on the duration of muscle contraction?
shorten, lengthen, or none?
8. Pithing destroys the frogs __________________________________, but leaves
the ______________________ intact.
9. Is the frog used in todays lab single- or double-pithed?
10. How long are the frogs tissues viable after pithing?
11. What are the characteristics of Ringers solution?
13. What two factors allow oxygen to be supplied to a pithed frogs tissues?
14. Recruitment increases the strength of muscle contraction by adding more and
more _____________________________. This is done by increasing the stimulus
______________________________.
Lab 5.7
15. How are the frogs enzymes different from mammalian enzymes?
16. Draw a muscle twitch and label the stimulus and the three phases. Label the x and
y axes.