Lab 5.1

Objectives - after completing this lab you should be able to:1) explain why we can use the frog gastrocnemius muscle to study skeletal muscle contraction2) perform the dissection to prepare the nerve-muscle preparation3) describe a basic muscle twitch and the physiological events that cause it or occur during it4) describe the effect of varying stimulus intensities5) describe the effect of varying stimulus frequencies6) differentiate between subthreshold, threshold, maximal and supermaximal stimuli7) interpret data collected from frog muscle contractions8) describe the process and result of pithing9) describe the function and composition of Ringers solution

BACKGROUND:

A skeletal muscle cell will contract only when adequately stimulated. The basic response of skeletal muscle to adequate stimulation is the muscle twitch. The twitch is modified by changing the intensity (volts) or frequency (pulses per second, pps) of stimulation or by changing the condition of the muscle tissue (temperature, O2 level, ion concentration).

In a whole muscle, weak stimuli excite only those motor units which are most sensitive. More intense stimuli excite more motor units. Thus, as the intensity of the stimulus is increased, the strength of contraction increases. Strength of contractionis also increased by increasing the frequency of stimulation so that the muscle cannot fully relax before it contracts again. These phenomena are explained furtherin the textbook.=

We are using the gastrocnemius muscle of a frog to observe skeletal muscle contraction because even though the frog is an amphibian, its skeletal muscle tissueworks the same way as mammalian muscle. The main difference is that mammaliantissue requires a high level of oxygen and glucose and a controlled temperature environment. The frog’s muscle will work at room temperature because its enzymesare not as temperature-dependent as mammalian enzymes.

Before the frog is used it will be double pithed by the instructor. Pithing is a procedure in which the central nervous system of the frog is destroyed, leaving the peripheral nerves intact. A dissecting needle is inserted into the cranium throughthe foramen magnum and used to disrupt the brain tissue. As soon as the needle goes through the foramen magnum, the spinal cord is severed and the frog feels nopain (remember, there are no sensory receptors in CNS tissue). In addition, destruction of the cortex prevents perception of pain or other sensations via the cranial nerves. What has just been described is called “single-pithing”, and it is not

LAB 6: Skeletal Muscle Function

Lab 5.2

sufficient for testing muscle responses. The spinal cord must also be destroyed, and a frog with both brain and spinal cord destroyed is “double-pithed”. At this point,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 frog’s muscle or nerve tissue is exposed, it should be frequently (once every 1 or 2 min) bathed with Ringer’s solution, an isotonic solution that has approximately the same ion concentrations as does amphibian ECF. Just a few dropsare enough to make sure the tissues don’t 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/hardwaredouble-pithed frogdissecting traydissecting kitRingers solution in dropper bottle2 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, don’t 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. Don’t 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.

Lab 5.3

8. Tie the other piece of thread firmly around the Achilles tendon (using a square knot), close to the muscle.

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

C.

varying stimulus intensity to demonstrate recruitment

1. Make sure preferences are set for a single 10 ms stimulus of amplitude 0V. Change if necessary.

2. Type 0V in the marks window, click Start on the screen, then hit the enter key.

3. Change stimulus amplitude to 0.25 V and repeat step 2.

4. Repeat, each time increasing stimulus voltage as indicated in the data chart.

5. Save file to desktop as “your name muscle”

6. Scroll back to the first response or use the Marks window to find the first mark.

7. Click Autoscale.

8. Click the two cursor button.

9. Place one cursor at the beginning of the contraction and the other at the peak.

10. Measure and record amplitude and duration of contraction and relaxation phases.

11. Repeat measurement for all other responses.

12. Print threshold and maximum response responses and label with your names and

Lab 5.5

the stimulus intensity if it isn’t already on the screen.

stimulus (V) amplitude duration of contraction

duration of relaxation

0.0250.0500.0750.10.150.20.250.3

continue only if at this point the amplitude is still increasing

D. varying stimulus frequency to demonstrate twitch summation and tetanus

1. Rinse muscle with Ringers.

2. Edit, preferences, count = 0, pps (frequency) 1 (this will change-see table below)

3. In the marks window, enter 1pps, click on start, hit the enter key, wait until the muscle stops contracting and click on stop.

4. Record the response of the muscle at each stimulus frequency as one of the follwoing: no summation, twitch summation, unfused tetanus, complete (fused) tetanus

frequency of stimulation (Hz) response15101520

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 dH2O and gently dry

Lab 5.6

5. close LabScribe and AdobeReader, shut down computerQuestions

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:

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 impairedb. muscle relaxation would be impairedc. 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 frog’s __________________________________, but leaves the ______________________ intact.

9. Is the frog used in today’s lab single- or double-pithed?

10. How long are the frog’s tissues viable after pithing?

Lab 5.7

11. What are the characteristics of Ringer’s solution?

12. What two functions does Ringer’s solution perform?

13. What two factors allow oxygen to be supplied to a pithed frog’s tissues?

14. Recruitment increases the strength of muscle contraction by adding more and more _____________________________. This is done by increasing the stimulus ______________________________.

15. How are the frog’s enzymes different from mammalian enzymes?

16. Draw a muscle twitch and label the stimulus and the three phases. Label the x and y axes.