Teaching Experiment

Physiology of the in situ Amphibian Heart

This experiment explores the basic principles of cardiac muscle physiology, including
contraction force, electrocardiogram (ECG) and the effect of neurotransmitters on the heart.
Written by staff of ADInstruments.

Background
Studies of isolated organs were pioneered in the late 19th century when scientists such as
Sidney Ringer (18351910) developed a perfusion solution (Ringers solution) that could sustain
an isolated organ from a pithed animal. A classic example of this phenomenon is the frog heart,
which will continue to beat in situ for several hours allowing for the study of basic cardiac
functions. The heart is made up of specialized tissue called cardiac muscle. Cardiac muscle is
similar to skeletal (striated) muscle, but exhibits some special properties, the most important of
which is rhythmicity. Specialized heart muscle cells called pacemakers spontaneously
depolarize and repolarize; the depolarization spreads to the entire heart via electrical
connections between cardiac muscle cells called gap junctions. This process occurs in rhythmic
fashion, giving rise to an intrinsic, regular heartbeat. While no external stimulation is required to
maintain the heartbeat, the heart receives continuous input from the sympathetic and
parasympathetic nervous systems. Cardiac muscle responds to a variety of
neurotransmitters, which can increase or decrease the heart rate. These molecules are able
to influence heart rate by changing the rate of spontaneous depolarization of the hearts
pacemaker cells, located in the sinoatrial (SA) and atrioventricular (AV) nodes of the
mammalian heart. In the frog, the sinus venosus is similar to the SA node.

Required Equipment
A computer system
Chart 5 software
PowerLab 4/20T, /4ST
ML301 Bridge Pod
MLT500 Force Transducer
MLA1605 Shielded Lead Wires/Alligator Clips
MLA40 Mounting stand with micropositioner
Suture thread
Straight pins
Barb-less hook
Eyedropper
Frog Ringers solution at room temperature
Frog Ringers solution in 40 C water bath
Frog Ringers solution in 10 C ice bath
Acetylcholine (0.1 mg/mL)
Epinephrine (1% solution)
Pilocarpine (2.5% solution)
Atropine sulfate (5% solution)
Figure 1. MLA40 Mounting
Stand and Micropositioner set
up with MLT500 Force
Transducer.

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Procedures
A. Setup and calibration of equipment
1. Set up your mounting stand with the MLT500 Force Transducer mounted on the
micropositioner (Figure 1).
2. Connect the force transducer cable to the back of the ML301 Bridge Pod.
3. Tie a piece of strong thread about 18 inches in length to the force transducer. Attach a small,
barb-less hook to the other end of the thread.
4. Attach the patient cable to the Bio Amp socket on
the PowerLab.
5. Attach three MLA1605 Lead Wires to the patient
cable: Channel 1 positive and negative, and Earth
(Figure 2).

Figure 2. Bio Amp patient cable

connected to MLA1605 Lead Wires.

6. Plug the Bridge Pod into the Pod Port on Input 1 of

the PowerLab (Figure 3). Make sure the PowerLab is
plugged in and attached to the computer via the
USB cable.
7. Turn on the PowerLab using the power switch on the back of the unit.
8. Launch Chart 5 from your computer.
9. Open the settings file called Frog Heart Settings.
10. From the Force Channel Function pop-up menu, select Bridge Pod.
11. Turn the zeroing knob on the front of the Bridge Pod until you get a reading of zero in the
dialog box.
12. Click OK.

Zeroing knob

ML301 Bridge
Pod

Output from force

transducer

MLA1605 Lead
Wires

Figure 3. Equipment setup for frog heart recording.

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B. Frog dissection procedure
Refer to your frog dissection guide for diagrams of this procedure.
1. Obtain a double-pithed frog from your instructor. Secure the animal ventral side up to a
dissecting board using straight pins.
2. Using a scalpel, make a longitudinal incision from the thorax to the abdomen of the frog.
Refer to the frog dissection guide in the Appendix for a procedural diagram.
3. Peel back the skin to expose the sternum and ribs.
4. Using sharp scissors, cut through the sternum to expose
the thoracic cavity. You should see the heart in its
pericardial sac.
5. Using forceps, grasp the pericardium and carefully cut it
away, exposing the heart. Apply Frog Ringers solution to
the heart every two or three minutes to prevent
desiccation.
6. Attach the heart to the Force Transducer with the small
hook. Push the hook through the apex of the heart.
Note: Be very careful NOT to pierce the ventricular
cavity!
7. Gently lift the heart away from the animals body cavity,
and tie the other end of the thread to the Force
Transducer (Figure 4). Reduce the slack in the thread by
adjusting the micropositioner on your mounting stand.
Note: Do not over-tighten the thread! Doing so
can damage the heart.

Figure 4. Setup of dissected frog

and MLT500 Force Transducer.

8. Attach the MLA1605 Lead Wire Alligator Clips to the frog to record the ECG. Positive: Left
forelimb; Negative: Right forelimb; Earth: Right hindlimb.

Exercise 1: Recording baseline heart rate and ECG

1. In Chart, click the Start button and record for 30 seconds. You should see a heartbeat
waveform in the Force channel and an ECG signal in the ECG channel. Adjust the tension on
the heart with the micropositioner if you get a weak signal in the Force channel, but be
careful not to over-tighten the thread. If your ECG signal is poor, check the connections to
the animal, and turn off any overhead fluorescent lights. Make sure the animal is not close to
the computer monitor, as this could cause interference.
2. Click the autoscale button from the Chart toolbar to scale all channels.

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Exercise 2: Effect of temperature
1. Record room temperature in Table 2 of your Data Notebook.
2. Click Start, and record 30 seconds of baseline data.
3. Using an eyedropper, bathe the heart in warm (40 C) Frog Ringers. Add a comment to
your data trace called warm. Record for 30 seconds.
4. Bathe the heart in cold (10 C) Frog Ringers. Add a comment to your data trace called
cold. Record for 30 seconds.
5. Click Stop. Bathe the heart in room temperature Frog Ringers before continuing to Exercise
3.

Exercise 3: Starlings law of the heart

1. Click Start and record 10 seconds of baseline data.
2. While recording, slowly increase the tension on the heart by turning the micropositioner
knob. Add a comment to your data file called stretch. You do not need to turn the knob
excessively to see a result.
3. Immediately return the micropositioner to its original position to reduce the tension on the
heart.
4. Click Stop.
5. Allow the heart to recover for two minutes before proceeding to Exercise 4.

Exercise 4: Effects of drugs on the heart

Note: Be sure to apply these drugs in the order indicated. Between each step, allow the heart to
recover for two minutes and flush with fresh Frog Ringers solution.
Acetylcholine
Acetylcholine is released by the parasympathetic nervous system.
1. Click Start and record 30 seconds of baseline data.
2. Using a syringe, apply two or three drops of acetylcholine (0.1 mg/mL) to the heart. Add a
comment called ACh to your data trace. Record for two minutes.
3. Click Stop.
4. Rinse the heart with frog Ringers and allow the heart two minutes to recover.

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Epinephrine
Epinephrine is released by post-ganglionic sympathetic nerves.
1. Click Start and record 30 seconds of baseline data.
2. Using a syringe, apply two or three drops of epinephrine (1 mg/mL) to the heart. Add a
comment called EPI to your data trace and record for two minutes.
3. Click Stop.
4. Rinse the heart with Frog Ringers and allow the heart two minutes to recover.
Pilocarpine
Pilocarpine stimulates muscarinic acetylcholine receptors in the heart.
1. Click Start and record 30 seconds of baseline data.
2. Using a syringe, apply two or three drops of pilocarpine (0.2 mg/mL) directly on the heart.
Add a comment to your data called pilocarpine.
3. Record for two minutes.
4. Click Stop.
5. Rinse the heart with Frog Ringers and allow the heart two minutes to recover.
Atropine and Acetylcholine
Atropine is a plant alkaloid that blocks acetylcholine receptors in the heart.
1. Click Start and record 30 seconds of baseline data.
2. Using a syringe, apply two or three drops of atropine (1 mg/mL) to the heart. Add a
comment called atropine to your recording.
3. Record for 30 seconds.
4. Apply two or three drops of acetylcholine to the heart; add a comment called ACh to the
data trace.
5. Record for two minutes.
6. Click Stop.
7. Rinse the heart with Frog Ringers and allow the heart two minutes to recover.

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Analysis
Exercise 1: Determining resting heart rate
1. Select the baseline heart data you recorded in Exercise 1 from the Force data (Channel 1).
2. Click the Zoom button from the Chart toolbar to open the Zoom window.
3. Place the Marker on the first peak.
4. Drag the Waveform Cursor to the last peak.
5. Record the time differential (t) and number of beats in Table 1 of your Data Notebook.
6. Calculate the heart rate in beats per minute. Record this value in Table 1 of your Data
Notebook.
7. In the Chart window, add the data from Heart Rate (Channel 2) to your selection by holding
down the Shift key and dragging in the Heart Rate channel.
8. Click the Data Pad button in the Chart toolbar to open the Data Pad.
Record the value for mean heart rate (BPM) in Table 1 of your Data Notebook.

Exercise 2: Effect of temperature on heart rate

1. Select the data for the 40 C trial from the Force channel (Channel 1).
2. Open the Data Pad.
3. Record the mean heart rate in Table 2 of your Data Notebook.
4. Repeat steps 13 for the 10 C data trace.

Exercise 3: Starlings Law of the Heart

1. Select the data in the Force channel for your stretch experiment.
2. Open the Zoom window.
3. Place the Marker on the data trace just prior to the first beat.
4. Use the Waveform Cursor to determine the amplitude of the first beat.
5. Repeat steps 3 and 4 for five additional beats, ending with the last beat before you released
the tension on the heart.
6. Record your results for beat amplitude in Table 3 of your Data Notebook.
7. Determine the heart rate for this experiment from the ECG channel.
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8. Record the heart rate in Table 3 of your Data Notebook.

Exercise 4: Effect of drugs on heart rate

1. For each drug administered, determine the heart rate as you did in Exercise 1 and record
your results in Table 4 of your Data Notebook.
2. Calculate and record the percent change in heart rate for each drug using the following
equation:

%change =

(ratewithdrug restingrate)
100
restingrate

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Data Notebook
Table 1. Determination of resting heart rate.
Number of beats in
selection

Time differential
between first and last
beat (sec)

Calculated Heart
Rate (BPM)

Heart Rate from

Data Pad (BPM)

Table 2. Effect of temperature on heart rate.

Condition

Heart Rate (BPM)

Room temperature: _______C

40 C
10 C

Table 3. Effect of tension on heartbeat amplitude

Condition

Heart contractile force (N)

Baseline (no stretch)

Stretched 1
Stretched 2
Stretched 3
Stretched 4
Stretched 5

Table 4. Effect of drugs on heart rate.

Drug

Heart rate before

drug given

Heart rate after

drug applied

% change in heart
rate

Acetylcholine
Epinephrine
Pilocarpine
Atropine and
acetylcholine

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Study Questions
Answer the following questions using complete sentences.
1) Describe the basis for the delay between the atrial and ventricular contractions.

2) How did temperature affect heart rate? What do you suppose is a consequence of being a
poikilotherm?

3) What is Starlings Law of the Heart? Does your data support this law?

4) Describe the mechanisms by which the following drugs affect heart rate:
a) Acetylcholine

b) Epinephrine

c) Atropine followed by acetylcholine

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