Download lab - Zoology, UBC

LAB EXERCISE 3
HEART FUNCTION IN THE FROG
(Seeley 681-683, 685-689, 694-695)
Work in groups of 4
Objectives
 Describe the structure of the frog heart, and the events of the cardiac cycle in the frog;
 compare with human
 Outline the conducting system of the heart in human and frog and relate to heart function
 Describe and explain the pacemaker of the heart
 State and account for the relative duration of each phase in the cardiac cycle
 Define and explain: refractory period, extra systole, compensatory pause
 Describe and explain the effects of autonomic drugs (acetylcholine, atropine, epinephrine) on frog
heart beat
SAFETY

Students handling the frog must wear gloves.
At the end of the lab:
 Discard the frogs in the heavy black plastic bags labelled "biological waste"
 Wash your dissecting instruments and rinse them with a 10% bleach solution
 Wipe your bench with a 10% bleach solution
 Wash your hands.
Equipment:
 double-pithed frogs
 diagrams and/or models of frog heart
 frog board or tray, pins
 Ringer's solution (13g of NaCl, 0.28g of KCl, 0.3g of CaCl22H2O, 0.4g of NaHCO3 for 2L of distilled water) at
room temperature.
 thermometers
 apparatus for recording heartbeat: recorder with 2 pens, amplifier, timer, transducer,
 thread, plasticine
 stimulator
 acetylcholine, 2 g/100 ml
 atropine sulfate, 1 g/100 ml.
 epinephrine solution, 1 g/100 ml.
 brown glass bottles with eyedropper (for pharmacological solution)
 watch glass
 timer
 Figures 1, 2 & 3.
Muscle cells of the heart contract as they perform their function: pumping blood. There are a
number of ways to examine this activity. One way is to look at the rate and strength of contraction
in situ by monitoring the actual movements of the ventricle itself. Another is to look at the
electrical activity generated by the heart each time it beats (ElectroCardioGram). Lastly, one can
observe the effects of heartbeat in the body by monitoring the pulse and blood pressure.
In this lab, you will monitor the rate and the strength of contraction of a frog heart in situ.
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I. STRUCTURE OF THE FROG HEART
Examine Figure 1 and familiarize yourself with the structure of the frog heart, and the vessels
entering and leaving it. Locate the single ventricle and the two atria. Blood enters the right
atrium from the sinus venosus, a thin-walled chamber on the dorsal side of the heart. The sinus
venosus receives a mixture of deoxygenated blood from the body and oxygenated blood from the
skin and mouth. Oxygenated blood from the lungs enters the left atrium. Both atria pump blood
into the single muscular ventricle.
Ventricular contraction forces blood into the tubular truncus arteriosus which divides into left and
right branches at the anterior border of the heart. Each of these branches divides into three
branches to (l) the head, (2) the body and (3) the skin, mouth and lungs. Oxygenation takes
place in the latter three organs.
Total mixing of blood from the two atria does not occur in the ventricle. A spiral valve in the
truncus arteriosus maintains separation so that oxygenated blood from the lungs is prevented
from entering the pulmocutaneous artery to the lungs, skin and mouth. The latter receives only
mixed blood from the right atrium, whereas the branches to the head and body carry both
oxygenated blood from the left atrium and mixed blood from the right atrium. Details of extent of
mixing and mechanism of separation of the two streams are still controversial.
Frogs are "cold-blooded". Their body temperature fluctuates with that of the environment. Hence
they expend less energy, and therefore utilize less oxygen than "warm-blooded" animals which
must expend energy to maintain body temperature. As a result inactive frogs can exist indefinitely without using their lungs to breathe. They can pick up enough oxygen from the skin surface
which is well-vascularized. Many frogs spend the entire winter without breathing, buried in mud
at the bottom of a pond.
The conducting system of the frog heart is essentially similar to that of the human, except that the
pacemaker of the frog is located in the wall of the sinus venosus.
NOTE: The entire central nervous system of the frogs used in this experiment has been destroyed by pithing. As a result they cannot feel pain or respond to stimuli. But their tissues
remain alive because the heart continues to beat, distributing blood with oxygen and nutrients to
them.
Perform Exercise A before hooking the frog heart to the transducer (Exercises B, C, D).
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Figure 1: Anatomy of the frog's heart: (a) ventral view showing the single truncus arteriosus
leaving the undivided ventricle; (b) longitudinal section showing the two atrial and single
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ventricular chambers; (c) dorsal view showing the sinus venosus (pacemaker).
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II. OBSERVATION OF THE CARDIAC CYCLE
EXERCISE A
1. Watch a demonstration of the technique required to expose the frog's heart.
2. Obtain a double-pithed frog and fasten it on its back in the tray provided. Lift the skin over the abdomen and cut
through it with scissors in the mid ventral line from the lower end of the abdomen to the lower jaw. Then cut
through the body wall a little to one side of the mid-line. Cut through the shoulder girdle. Be careful to avoid the
vessels entering and leaving the heart. Carefully open the pericardium and expose the beating heart. Keep the
heart moist at all times with Ringer's solution applied with an eyedropper.
3. Determine heart rate per minute (count ventricular contractions for 30 seconds and multiply by 2).
Heart rate =
4. Bathe the heart copiously with cold (5 oC to 10oC) Ringer's solution until the rate slows sufficiently to study the
sequence of contraction. Maintain the cold temperature while making the following observations.

Identify the sinus venosus (tip the ventricle forward and look on its dorsal surface), left and right atria, ventricle,
truncus arteriosus. Observe carefully. Do all parts of the heart beat at the same rate?

What is the sequence in which these parts contract?

Describe the changes in the ventricle during systole and diastole. Observe its size, and shape.
III. RECORDING THE CARDIAC CYCLE
EXERCISE B
1. Attach the thread to the transducer lever with plasticine. Arrange the bath so that the thread is vertical. Do not
pull the thread too tight; it should be taut, but should not stretch the heart excessively. Balance the lever with
plasticine as demonstrated. Keep the heart moist at all times with Ringer's solution.
2. Adjust the gain and offset controls on the amplifier so that a deflection of at least 2.0 cm is obtained with each
ventricular contraction.
3. Record a series of at least l0 contractions with the chart running at moderately high speed (note speed). You
should be able to obtain a trace similar to that shown in figure 2. Ventricular contractions should be spaced at
intervals of not less than 2 cm. Identify atrial and ventricular contractions on the chart. Atrial contraction
precedes ventricular contraction and is much weaker. If your record is unclear try adjusting the set-up. The
tension on the thread attaching the heart to the transducer lever is often at fault.
4. Make sure that you have a clean trace showing 5 contractions. You will have to include them or a photocopy of
them in your lab report. Label the chart clearly, and record chart speed and direction. Be sure that you can
identify which part of the trace results from contraction, and which from relaxation; label before you dismantle the
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apparatus.
You should obtain a trace similar to (1). As you can see on this figure,
this trace is a combination of the contractions of the atria and the
ventricle.
Figure 2: Cardiac cycle of frog's heart using one transducer attached
to the ventricle (1) and using two transducers (2) one recording only
the atrium contraction (top) the other recording only the ventricle
contraction (bottom).
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IV. THE REFRACTORY PERIOD AND EXTRA SYSTOLE
EXERCISE C
1. Be sure that the event-marker pen (which records electrical stimuli) is exactly aligned with the amplifier pen
(which records contraction). Set the speed of the chart recorder at 2.5 cm/sec or 5 cm/sec.
2. Set the stimulator: the intensity of the stimulus should be 10-20 volts and the frequency of stimulation should be
set at 100 stimuli/sec.
3. Before doing the stimulation, the heart should be slowed down with cold ringer (this will make it easier for you to
stimulate the ventricle precisely during the systole or the diastole as you wish it). Start the chart running. Switch
on the stimulator to repeat and touch the ventricle at different phases of its cycles. This will require a number of
tests - repeat until you have records of stimuli that coincide with all phases of the cardiac cycle. Watch for the
appearance of extra systoles - beats induced by the electrical stimulus (Figure 3). Your objective is to determine
when, during the cycle, the heart is refractory (i.e. when the electrical stimulus does not result in an extra
contraction).
4. Labelled chart records (or copies of them) of this experiment will be included in your lab report. Indicate stimuli
that failed to induce contractions as well as those that induced extra systoles. Correlate these with stages in the
cardiac cycle. Note whether the pause between beats (diastole) is of normal duration after an extra systole
(premature contraction) is induced. Measure intervals for several normal beats and compare with that after a
premature contraction.
Figure 3: Refractory period and compensatory pause of the frog heart when stimulated at various
times during the cardiac cycle.
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V. EFFECTS OF AUTONOMIC DRUGS
EXERCISE D
1. Record a series of l2 beats on a chart moving at moderate speed (note speed), then bathe the heart copiously
with epinephrine solution using an eyedropper. Indicate the time. Continue recording until you notice an effect.
If no effect is observed within 3 to 5 minutes, apply more epinephrine. When an effect has been recorded, rinse
the heart thoroughly with Ringer's solution.
2. When the heart has returned to its normal rate, again record a sequence of normal beats, then, using a different
eyedropper, bathe the heart with acetylcholine solution, noting time on chart. Continue recording until an effect is
observed. If no effect is observed within 3 to 5 minutes add more acetylcholine.
When a definite effect has been obtained, or if the heart stops beating, bathe the heart freely with atropine
sulfate, indicating time on the chart. Continue recording until recovery is observed, noting time on chart. Wash
copiously with Ringer's solution.
3. Be sure to label all activities on your chart as you proceed. You will include labeled charts (or copies of them) in
your lab report.
4. Calculate the following heart rates:
- Control heart rate and highest heart rate reached with epinephrine
- Control heart rate, lowest value reached with acetylcholine, highest value reached with atropine.
Record your results in Table 2.
Heart rate (HR) is calculated with the following equation:
HR = speed of the chart recorder x 60) / heart beat interval.
HR: heat rate in beats/min. = speed of chart recorder: in cm/sec x 60 / heart beat interval: in cm
Note: Atropine is a competitive inhibitor of acetylcholine which acts at receptor sites in smooth and cardiac muscle,
but not in skeletal muscle. It is not produced by the body, but is sometimes used as a drug to inhibit the effects of the
parasympathetic nerves.
Table 2: Effects of autonomic drugs on cardiac cycles.
Control
Epinephrine
New Control
Acetylcholine
Atropine
HR (beats/min)
VI. AUTOMATICITY OF THE HEART
EXERCISE E
1. Using the pin and string, lift the ventricle, carefully cut through the arteries and veins and remove the heart. Be
careful to keep the sinus venosus intact; cut the veins which drain into it. Place the heart in a watch-glass filled
with Ringer's solution. Observe. Count the rate after l minute.
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2. Using scissors carefully cut through the junction between the sinus venosus and the right atrium. Wait several
minutes. Both parts of the heart should continue to beat. Count the rate for each part.
3. Cut the atria free from the ventricle. Describe the activity of each segment. Count the rate (if present) for each
segment. Observe for l0 minutes. If the ventricle still shows no activity, stimulate it by pricking it with a pin.
Determine its rate.
4. Record your results in Table 3 and tabulate heart rate for each chamber under each condition.
Note: This procedure is not always successful. If your frog's heart fails to beat after separating the atria from the
ventricle, observe and describe one that does or predict what you would expect to see.
Table 3: Automaticity of the heart.
EXPERIMENTAL CONDITIONS
PART OF THE HEART
UNDER OBSERVATION
Isolated heart
Whole heart
Section between the sinus
venosus and the right atrium
Sinus venosus
RATE OF BEATING (beats/min
or predicted rate)
Rest of the heart
Section between the atria and the
ventricle
Atria
Ventricles
CLEAN UP:

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





Turn off and disconnect electronic equipment.
Discard the frogs in the heavy black plastic bags labeled "biological waste"
Wash glassware, rinse it thoroughly with water, and leave it to dry on paper towels on your
bench.
Rinse the frog board with water. DO NOT USE DETERGENT OR BLEACH.
Wash your dissecting instruments and rinse them with a 10% bleach solution
Wipe your bench with a 10% bleach solution
Leave your space clean and tidy.
Wash your hands.
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NAME & section #:
Biology 153 LAB REPORT (2002-2003)
EXERCISE 3: HEART FUNCTION IN THE FROG.
Do not copy word-for-word from text.
INTRODUCTION
State the purpose of this exercise
MATERIALS AND METHODS
See the lab manual.
RESULTS
Use separate pages.
A. OBSERVATION OF THE CARDIAC CYCLE (Exercise A)
- Record observations as instructed in the lab manual. Answers to questions should be brief.
B. RECORDING THE CARDIAC CYCLE (Exercise B)
- Include fully labelled (atrial systole, ventricular systole, ventricular diastole, chart speed)
section of chart paper from the recorder with your report.
- In a table show the following calculations:
a) heart rate in beats/min.
b) duration of the atrial systole
c) duration of the ventricular systole
d) duration of the ventricular diastole.
C. THE REFRACTORY PERIOD AND EXTRA SYSTOLE (Exercise C)
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- Include fully labelled chart recordings showing the effects of stimulation at different points of
the cardiac cycle. Include:
- a "normal" recording for comparison.
- a recording showing the extra systole and compensatory pause. Indicate where during the
cardiac cycle the stimulus would be applied to produce the extra systole.
- Label chart speed and pen direction
- In no more than five lines describe what you observed.
D. EFFECTS OF AUTONOMIC DRUGS (Exercise D)
- Include a fully labelled chart recording showing the effects of epinephrine, acetylcholine
and atropine sulfate. Include:
- a recording of the control heart beats and a recording following drug application until a
change in rate or contractility can be seen (indicate where the drug was added).
- cut down the tracing if the effects take too long and note the time (do not fold tracings to fit
the page).
- In a table show the changes in heart rate before and after drug application.
- Illustrate with a bar graph the effect of these autonomic drugs on the heart rate.
- In five lines describe your results.
E. AUTOMATICITY OF THE HEART (Exercise E)
- Tabulate your results.
- In 5 lines describe your results. If you are missing data, include a column for predicted
results.
DISCUSSION
Use spaces provided below. Do not use extra pages.
THE REFRACTORY PERIOD AND EXTRA SYSTOLE (Exercise D)
1) When during the cardiac cycle must the ventricle be stimulated in order to produce an extra
systole? Explain why you obtained different responses depending on when you stimulated the
ventricle? (some stimulations will cause an extra-systole, others will not)
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2) What is the physiological explanation for the compensatory pause?
3) What is the advantage for cardiac muscle to exhibit an absolute refractory period that is
nearly as long as its contraction?
4) What is the advantage for skeletal muscle to exhibit a refractory period, which is so small as
to be negligible?
EFFECTS OF AUTONOMIC DRUGS (Exercise D)
In terms of the neural regulation of the heart, explain the effects of epinephrine, acetylcholine
and atropine on heart rate. What is the effect on heart rate and contractility and how do the drugs
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act to produce these effects?
AUTOMATICITY OF THE HEART (Exercise E)
1) Explain why the sinus venosus, the atria and the ventricles beat with different rates when they
are separated from each other.
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2) Will the rate of beating of the whole heart be similar to the rate of the isolated ventricle, of the
isolated atria or of the isolated sinus venosus? Why?
The End
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