Animal Physiology Biology 3920

Name               Lab Sec: Tue Aft Thur Aft

Partners:

Today you will be working with a turtle heart as a typical vertebrate heart. The turtle heart has the advantage that it is extremely stable and the heart will continue to beat for a very long time. The turtle heart is less advanced in structure than the mammalian heart: the right and left atria are more distinct units rather than being integrated fully with the heart and the ventricle is primarily a single chambered unit rather than two distinct chambers. What is the consequence of these differences?

The turtle is brain pithed and 'dead'. You can check this by touching the eye with a probe and you should get no blink. The turtle is not dead in the sense that the muscle biochemistry is still very much intact and sudden movement by the animal may commonly occur (spontaneous or accidental stimulation of a motor neuron).

It is important to realize that each heart cell can independently beat without any nervous input. Thus, the isolated heart can beat independently, the rate being determined by those that beat the quickest at the sino-atrial node (pacemaker). The intact heart, although it can beat independently, does have its rate and strength affected by neurotransmitters via nerves and by hormones.

First develop an understanding of your movement transducer apparatus and the electrical stimulator.

Heart Setup: You will be presented with the turtle on its back and its plastron (lower shell) is removed. You should see the heart beating within its pericardium. Carefully remove the pericardial sac. Keep the tissues moistened with Ringer's solution throughout the laboratory period. (Ringer's solution is a mixture of salts that helps keep the proper osmotic, ionic, and pH balance in the cells of the animal.) Put the hooked pin hanging from the lever through the frenulum (the piece of tissue at the base of the heart that anchors the heart down). Cut the frenulum and adjust the clamps so that the movement of the heart changes the amount of light that is striking the photocell (or whatever movement transducer you are using). Can you distinguish the atrial beat that precedes the ventricular beat on your recording? Why do the atria need to beat before the ventricle? Remember to write down all appropriate information (when things are done to the heart, chemical concentrations and amounts, etc.). You will be required to label your final computerized graphical printout for the times that additions were made or things were changed. DO NOT TRUST YOUR MEMORY OR MY ESP.

Vagus Nerve Stimulation: You want to do this part of the experiment as soon as you can in the laboratory period because the nervous tissue degenerates quickly. The success rate of this part of the experiment is only about 50%, so do not be frustrated if it does not work. Check and see if it worked for any other group.

The vagus nerve is the parasympathetic nerve that goes from the brain to the right atrium of the heart, the location of the pacemaker. (This parasympathetic nerve comes from the medulla area of the brain. When stimulated, the action potentials that terminate at the right atrium of the heart release the chemical acetylcholine. The acetylcholine binds to the muscarinic receptors that hyperpolarize the heart cells of the right atrium and thus makes them harder to generate an action potential. The overall effect is that the heart rate slows down.)

Locate the vagus nerve in the provided lab manual. Carefully slit the skin along the right side of the neck of the turtle. Separate the muscles with minimal or no cutting and locate the vagus nerve that runs lateral to the trachea and esophagus. Some major blood vessels and the sympathetic nerve run parallel with the vagus nerve. (What would be the function of the sympathetic nerve?). Nerves have a whitish, stringy appearance. When you locate the vagus nerve (I don't know how to distinguish the parasympathetic from the sympathetic either; do the best you can) carefully slide a thread under it (try to avoid stretching the nerve).

Carefully lift the nerve with the thread and place it on the two stimulator electrodes supported by a ring stand. Apply voltage pulses of about 5 volts (duration: 2 ms; frequency: 30 per second) for 10 seconds. Did the heart rate decrease? How long after the stimulation does it stay decreased? If you never got a slowing of the heart, try some higher voltages. If it still does not work, move on to the next experiment.

If you got a decrease, then stimulate the nerve again and continue to stimulate. A phenomenon known as vagal escape should eventually occur where the rate eventually comes back to normal.(You may have to wait a few minutes and you may want to slow down the recorder.)

Remember to keep your heart moist with Ringer's.

Acetylcholine Stimulation: What do you expect if we directly apply acetylcholine to the heart? Using a Pasteur pipette, remove and discard the fluid in the body cavity. While recording the heart beats, squirt several drops of 10 mg/ml acetylcholine onto the heart using a Pasteur pipette. Is there a change in rate? A change in strength? Eventually rinse with Ringer's and wait until you get a normal rate before you start the next part of the experiment.

If the heart completely stops, apply a similar amount of 5mg/ml atropine which should counteract the effects of acetylcholine. Atropine is not a natural substance in the animal body, but its ability to block muscarinic receptors has a very important medical use. Can you imagine conditions where atropine should be used on a human patient?

Epinephrine Stimulation: Epinephrine and adrenaline are two names for the same chemical. Very similar in structure and function to epinephrine is a second chemical called norepinephrine (or noradrenaline). Both epinephrine and norepinephrine are released from the adrenal gland (what effect does it have on the heart?). In addition, noradrenaline is also released from certain sympathetic nerves such as the sympathetic nerve that goes to the S-A node of the heart from the medulla. Artificially add 0.3 ml of epinephrine (10 µg/ml) by injecting it into the heart. Is there a rate change? (It may be subtle.) Wait until you get a normal rate before you start the next part of the experiment.

Temperature Effects on the Heart Rate: Apply large amounts of the available cold Ringer's solution to the heart and measure the rate. Once this is done, get the heart back to a normal rate by applying room temperature Ringer's. Apply large amounts of warm Ringer's solution to the heart and record the rate. What are the implications of these results considering a turtle? considering your own heart?

Stannius Ligature: Use the string to tightly tie off the ventricle from the atria. You actually want to actually prevent the flow of action potentials between the two so you will need to damage tissue. Can you get the atria to beat independently from the ventricle. With your knowledge of SA and AV nodes, what should happen by this action?:

Report the observations you have visually made:

Heart Removal : Remove the heart and place in Ringers solution in a Petri dish. Does the beating continue? Cut the heart into pieces. Do the pieces continue to beat? Do they beat at the same rate? Does this meet your expectations? Record your observations and answer the questions:

Try some of the drugs that are available (acetylcholine, atropine, epinephrine, nicotine) and see if there are effects :

Lungs: This has nothing to do with the heart, but as long as the turtle has sacrificed his life for us, let us make full use of him (or her - can you tell the sex?). Cut the trachea close to the mouth. Carefully slide an available plastic pipette down the trachea; try to achieve a snug fit (you may want to tie a string around it to get the snug fit). Carefully inflate the lungs using the large orange bulb fitted on the other end of the pipette. Make sure you find the lungs (and not just the liver). The reptile lungs are crude and have far fewer alveoli than mammalian lungs. How does a turtle ventilate? Does it have a diaphragm?

Muscle stimulation: Stimulate (using the stimulator set at 50 ms and 1 event/sec) a skeletal muscle of the turtle in one of the legs and watch what happens. Stimulate the smooth muscle of the small intestines and observe what happens.

Recorder

Lever system, photocell, etc

Stimulator

Turtle lab manual (p45)

Ring stand for stimulator electrodes.

Pasteur pipette

syringe, 1 ml

cold Ringer's solution

warm Ringer's solution

string

thread

Petri dish

plastic Pasteur pipette

orange bulb

Ringer's solution

10 mg/ml acetylcholine (Freezer 406 R3B5)

0.1 g dissolved in 10 ml of DI

5mg/ml atropine (Refrig 401 R2B7)

0.1 g dissolved in 20 ml

epinephrine (10 µg/ml) (Refrig 401 R2B7)

0.01 g dissolved in 100 ml DI then dilute 10 ml in 100 ml Ringer's

Nicotine . 1 ml to 100 ml

Physostigmine (Eserine) (1mg/ml) 10 mg in 10 ml H2O

Pilocarpine (0.5mg/ml)

See chap 11 of Ottis

Had trouble (0 success) in finding vagal nerve - not sure why. Reread Shirley. Vagal nerve does not lie in trachel-esophageal crevice, but rather is more peripheral.