experiment am-6: frog · pdf fileexperiment am-6: frog electrocardiogram equipment required pc...

Experiment AM-6: Frog Electrocardiogram

Background

Unlike a mammalian heart with four chambers, a frog heart (Figure AM-6-B1) has three chambers, two

atria and one ventricle, and two other structures that function to bring blood into and out of the heart:

• The ventricle is the single chamber at the bottom of the heart. It looks dark red when filled with

blood and pink when the chamber contracts and forces blood into the major arteries.

• The atria are the two thinned-walled chambers located just above the ventricle. They look

darker red in color than the ventricle.

• The sinus venosus is a thin-walled sac lying behind the atria. It receives blood from the major

vein draining the tissues of the body and delivers the blood to the right atria. The sinus venosus

looks dark blue in color. It is absent in mammals.

• The aortic trunk, which arises from the right side of the base of the ventricle. This trunk divides

into two major branches, and each branch divides into three large arteries: the carotid; the aorta;

and, the pulmocutaneous. The trunk contains a spiral valve which directs the more oxygenated

blood into the aorta and carotid arteries and the less oxygenated blood into the pulmocutaneous

arteries.

Figure AM-6-B1: Anatomical diagram of the frog heart showing the spiral valve in the aortic trunk.

The sinus venosus is not visible because it is on the back of the heart. The vein on the right side of the

heart enters the sinus venosus, and the vein on the left side of the heart enters the left atrium.

Just like the mammalian heart, the frog has a set of specialized myocardial cells that function as a

pacemaker. These cells, which are located in the sinus venosus, contract automatically in a rhythmic

manner. The electrical signal from the pacemaker region travels across the sinus venosus causing cells

in the sinus to contract. The signal continues to spread like a wave toward the myocardial cells in the

adjacent atria. Those cells contract in sequence after the cells in the sinus venosus. From the atria, the

Animal Muscle – FrogECG-Background AM-6-1

electrical signal spreads toward the ventricle causing the contraction of the cells in that chamber. Thus,

the repeated, sequential contraction of the parts of the heart move the blood through the heart, into the

large arteries that supply oxygen and nutrients to other organs, muscles, and skin, and back again,

continuously.

The electrical activity of the heart can be recorded and displayed in an electrocardiogram (ECG). Each

peak in an ECG corresponds to voltage changes in specific regions of the heart (Figure AM-6-B2 ):

• P wave - atrial depolarization

• QRS complex - atrial repolarization and ventricular depolarization

• T wave - ventricular repolarization.

Figure AM-6-B2: Diagram of a typical frog ECG with P and T, and the QRS complex identified.

If the beginning of the cardiac cycle is taken as the contraction of the sinus venosus, the contraction of

the various sections of the heart follows the path of the electrical signal that spreads over the heart. The

atria contract after the sinus venosus, the ventricle contracts after the atria, and the aortic trunk

contracts after the ventricle.

The sinus venosus receives blood from the venae cavae, the major vein that collects blood from all the

other veins that drain from the systemic circulation. When the sinus venosus contracts, the blood is

pumped into the right atrium. At the same time the right atrium is receiving blood from the sinus

venosus, the left atrium is receiving highly oxygenated blood from the lungs through the pulmonary

veins. During the P wave, both atria contract and empty their blood into the single ventricle. The less

oxygenated venous blood, which enters the right side of the ventricle from the right atria, is nearest the

outlet of the ventricle and enters the arterial trunk first. With the partial separation of the arterial trunk

by the spiral valve, the venous blood moves into the pulmocutaneous arteries to the lungs and the skin

during the QRS complex. The more oxygenated blood, which enters the left side of the ventricle from

the left atrium travels a longer path to the outlet of the ventricle. This blood moves into the aortic

arches and the carotid arteries during the QRS complex. As the ventricle repolarizes during the T wave,

more blood is entering the ventricle in preparation for its ejection into the arteries.

In this laboratory exercise, students will record the effects of temperature on the amplitude and

frequency of electrical activity in the heart, as well as the effects of the synaptic transmitters,

epinephrine and acetylcholine on these same parameters.

Warning: The frog heart preparation has a limited life span, so before you start the dissection set up

all the equipment needed. Chill one beaker of 100ml of Ringer's solution to 15oC. Warm a second

beaker of 100ml of Ringer’s solution to 30oC. Keep a third beaker of Ringer’s solution at room

temperature.



Equipment Required

PC or Mac Computer

IXTA data acquisition unit

USB cable

IXTA power supply

iWire-B3G ECG cable

C-ISO-F3 lead wires with flexible silver wire electrodes

Dissection tray, instruments and pins

Suture thread

Amphibian Ringer’s Solution (See Appendix)

Reagent Solutions (See Appendix)

IXTA Setup

1. Place the IXTA on the bench, close to the computer.

2. Check Figure T-1-1 in the Tutorial chapter for the location of the USB port and the power

socket on the IXTA.

3. Check Figure T-1-2 in the Tutorial chapter for a picture of the IXTA power supply.

4. Use the USB cable to connect the computer to the USB port on the rear panel of the IXTA.

5. Plug the power supply for the IXTA into the electrical outlet. Insert the plug on the end of the

power supply cable into the labeled socket on the rear of the IXTA. Use the power switch to

turn on the unit. Confirm that the red power light is on.

Start the Software

1. Click on the LabScribe shortcut on the computer’s desktop to open the program. If a shortcut is

not available, click on the Windows Start menu, move the cursor to All Programs and then to

the listing for iWorx. Select LabScribe from the iWorx submenu. The LabScribe Main window

will appear as the program opens.

2. On the Main window, pull down the Settings menu and select Load Group.

3. Locate the settings folder that contains the settings group, IPLMv4Complete.iwxgrp. Select this

group and click Open.

4. Pull down the Settings menu again. Select the FrogECG-LS2 settings file from Animal Muscle.

5. After a short time, LabScribe will appear on the computer screen as configured by the

FrogECG-LS2 settings.


6. For your information, the settings used to configure the recording channels in the LabScribe

software and IXTA for this experiment are programmed on the Channel window of the

Preferences Dialog, which can be viewed by selecting Preferences from the Edit menu on the

LabScribe Main window.

7. Once the settings file has been loaded, click the Experiment button on the toolbar to open any

of the following documents:

• Appendix

• Background

• Labs

• Setup (opens automatically)

ECG Recording Cables and Stimulus Electrode Setup

1. Locate the following items in the iWorx kit: C-ISO-B3G ECG cable (Figure AM-6-S1) and the

C-ISO-F3 lead wires with flexible silver electrodes (Figure AM-6-S2).

2. Plug the C-ISO-B3G cable into the iWire 1 input on the front of the IXTA (Figure AM-6-S3).

Attach the color coded C-ISO-F3 lead wires into the appropriate sockets.

Figure AM-6-S1: The C-ISO-B3G ECG cable.


Figure AM-6-S2: The C-ISO-F3 lead wires with flexible silver electrodes.

Figure AM-6-S3: The C-ISO-B3G and male double banana-female BNC adapter attached to the IXTA.

The Dissection

1. Place a frog in ice water for 15 minutes. Double pith the frog as soon as it is removed from the

ice water

2. Place the frog ventral surface up, in the dissection tray. Use forceps to grasp the skin over the

center of the pectoral girdle and use sharp scissors to make a cut to the skin. Use the scissors

and forceps to remove the skin over the left (the frog’s left) half of the pectoral girdle.

3. Use the scissors to cut through the pectoral girdle: first, in the mid-line; second, under the left

arm pit. Cut with the tips of the scissors up.

4. Carefully cut the girdle away from the belly area. Lift the flap of the girdle to expose the

(beating) heart. Flush the area with room temperature Ringer's solution.


5. While lifting the flap of pectoral girdle, cut it away from the throat region and remove the girdle

from the opening. Again, moisten the heart with room temperature Ringer's solution.

6. Examine the heart. Notice that it may still be covered by a white pericardial sac. Use forceps to

grasp the pericardial sac, not the heart. Cut the pericardial membrane.

7. Grasp a cut edge of the pericardial membrane with forceps and pull it to one side. Dissect away

the pericardial membrane from the heart.

The Preparation

1. Attach the ground “green” electrode to the upper limb just underneath the skin. Carefully insert

the needle electrode into the muscles of the upper forelimb. Do not go through the muscles into

the tray below.

2. Carefully adjust the position of the positive “red” flexible silver recording electrode so that it is

resting on top of the ventricle.

3. Adjust the position of the negative “black” flexible recording electrode so that it rests just

below the A-V groove slightly to the frog’s left.

Warning: The heart preparation used in this experiment is functional for a limited period of time. If

the muscle is bathed periodically in Ringer’s solution, it will work for about four hours. To conserve

time, complete all the exercises in the experiment before analyzing the data.



Exercise 1: The Frog ECG and Heart Rate

Aim: To record the electrical trace (ECG) produced by the contraction of a resting heart, and to

determine the resting heart rate.

Procedure

1. Type Resting in the Mark box to the right of the Mark button.

2. Click the Record button and press the Enter key on the keyboard to attach the comment to the

record. Click AutoScale to increase the size of the deflection on the Main window.

3. Record the resting ECG and heart rate for one minute.

4. Click Stop to halt the recording.

5. Select Save As in the File menu, type a name for the file. Choose a destination on the computer

in which to save the file, like your lab group folder). Designate the file type as *.iwxdata. Click

on the Save button to save the data file.

6. Moisten the chest cavity with room temperature Ringer's solution.

Exercise 2: Effects of Cold Temperature on the ECG and Heart Rate

Aim: To record changes in the ECG and heart rate after the heart is bathed in cold Ringer’s solution.

Procedure

1. Type Room Temp Ringer’s in the Mark box to the right of the Mark button.

2. Click the Record button. Click AutoScale to increase the size of the deflection on the Main

window.

3. Record the ECG and heart rate for thirty seconds.

4. Apply ten drops of Ringer's solution (at room temperature) to the heart. Press the Enter key on

the keyboard when the Ringer’s solution is dropped on the heart.

5. Place the beaker with chilled Ringer's solution near the preparation.

6. Type Cold Ringer's in the Mark box.

7. Twenty seconds after the addition of room temperature Ringer’s to the heart, apply five drops of

cold Ringer's solution to the heart. Press the Enter key on the keyboard when the cold Ringer’s

solution is dropped on the heart.

8. Record until the heart has recovered from the effects of cold Ringer’s solution.

Note: Recovery is when the ECG amplitudes and rate of the heart contraction have returned to the

resting values.



10. Select Save in the File menu.


Exercise 3: Effects of Warm Temperature on the ECG and Heart Rate

Aim: To record changes in the ECG and heart rate after the heart is bathed in warm Ringer’s solution.

Procedure

1. Type Room Temp Ringer’s in the Mark box to the right of the Mark button.


window.


4. Apply ten drops of Ringer's solution (at room temperature) to the heart. Press the Enter key on

the keyboard when the Ringer’s solution is dropped on the heart.

5. Place the beaker with warm Ringer's solution near the preparation.

6. Type Warm Ringer's in the Mark box.

7. Twenty seconds after the addition of room temperature Ringer’s to the heart, apply five drops of

warm Ringer's solution to the heart. Press the Enter key on the keyboard when the warm

Ringer’s solution is dropped on the heart.

8. Record until the heart has recovered from the effects of warm Ringer’s solution.

Note: Recovery is when the ECG amplitudes and rate of the heart contraction have returned to the

resting values.




Exercise 4: Effects of Drugs on the Frog Heart

Aim: To monitor the effects of Epinephrine and Acetylcholine on the ECG amplitudes and rate of heart

contraction.

Procedure-Epinephrine

1. Type Resting in the Mark box to the right of the Mark button.

2. Click the Record button. Press the Enter key on the keyboard to mark the recording. Click

AutoScale to increase the size of the deflection on the Main window.



4. Type Epinephrine in the Mark box to the right of the Mark button.

5. Apply two drops of Epinephrine solution (at room temperature) to the heart. Press the Enter key

on the keyboard when the Epinephrine solution is dropped on the heart. Continue recording.

6. After recording the effects of Epinephrine for sixty seconds, rinse the heart with room

temperature Ringer’s solution until the ECG and heart rate return to the resting state.



9. Moisten the chest cavity with Ringer's solution.

Procedure-Acetylcholine

1. Type Acetylcholine in the Mark box to the right of the Mark button.


window.


4. Apply one drop of Acetylcholine solution (at room temperature) to the heart. Press the Enter

key on the keyboard when the Acetylcholine solution is dropped on the heart. Continue

recording.

Warning: If the heart goes into cardiac arrest, rinse the Acetylcholine solution off the heart with

fresh, room temperature Ringer’s solution. If the heart is still in cardiac arrest after 10 seconds, add

two drops of Epinephrine solution to the heart.

5. After recording the effects of Acetylcholine for sixty seconds, rinse the heart with room

temperature Ringer’s solution until the ECG and heart rate return to the resting rate.



8. Moisten the chest cavity with Ringer's solution.

Data Analysis

Exercise 2: Effect of Cold Temperature

1. Scroll to the data recorded from the heart fifteen seconds before cold Ringer’s solution was

added to the heart. Click the AutoScale button to maximize the size of the ECG and heart rate

channels on the window (Figure AM-6-L1).

2. Use the Display Time icons to adjust the Display Time of the Main window to show ten

complete cardiac cycles on the Main window. The cycles can be selected by:


• Placing a cursor before the first P Wave, and a cursor after the tenth T Wave; and

• Clicking the Zoom between Cursors button on the LabScribe toolbar to expand the ten

selected cardiac cycles to the width of the Main window.

Figure AM-6-L1: Frog ECG and heart rate displayed on the Main window.

Figure AM-6-L2: The LabScribe toolbar.

3. Click on the Analysis window icon in the toolbar or select Analysis from the Windows menu to

transfer the data displayed in the Main window to the Analysis window (Figure AM-6-L3).

4. Look at the Function Table that is above the uppermost channel displayed in the Analysis

window. The mathematical functions, V2-V1, T2-T1, and Mean should appear in this table.

Values for V2-V1, T2-T1, and Mean on each channel are seen in the table across the top margin

of each channel.

5. Once the cursors are placed in the correct positions for determining the amplitude and period of

each cardiac cycle, the values of the parameters in the Function Table can be recorded in the on-

line notebook of LabScribe by typing their names and values directly into the Journal, or on a

separate data table.


6. The functions in the channel pull-down menus of the Analysis window can also be used to enter

the names and values of the parameters from the recording to the Journal. To use these

functions:

• Place the cursors at the locations used to measure the amplitude and period of each heart

contraction.

• Transfer the names of the mathematical functions used to determine the amplitude and

times to the Journal using the Add Title to Journal function in the Frog ECG Channel

pull-down menu.

• Transfer the values for the amplitude and period to the Journal using the Add Ch. Data to

Journal function in the Frog ECG Channel pull-down menu.

7. On the Frog ECG Channel, use the mouse to click on and drag the cursors to specific points on

the recording to measure the following parameters:

• The R wave amplitude. To measure the R wave amplitude, place one cursor on the Q

wave that precedes the R wave and the second cursor on the peak of the R wave. The

value for V2-V1 on the ECG channel is this amplitude. Measure the amplitudes of four

additional R waves.

• The P wave amplitude, To measure the P wave amplitude, place one cursor on the

baseline before the P wave and the second cursor on the peak of the P wave. The value

for V2-V1 on the ECG channel is this amplitude. Measure the amplitudes of four

additional P waves.

• The T wave amplitude, To measure the T wave amplitude, place one cursor on the

baseline after the T wave and the second cursor on the peak of the T wave. The value for

V2-V1 on the ECG channel is this amplitude. Measure the amplitudes of four additional

T waves.

• Beat Period, which is the time between the peaks of two adjacent R waves. To measure

this parameter, place one cursor on the peak of an R Wave, and the other cursor on the

peak of an adjacent R Wave. The value for the T2-T1 function on the Frog ECG Channel

is the beat period.

• Mean Heart Rate, is the average heart rate calculated from the ECG Channel. To

measure this parameter, place one cursor at the beginning of the first of 10 cardiac cycles

selected and the second cursors at the end of the cycles selected. The value for Mean on

the Heart Rate (ECG) channel is this mean heart rate for that 10 cycle period.

8. Record the values in the Journal using the one of the techniques described in Steps 6 or 7, and

on Table AM-6-L1.

9. Scroll to the section of data recorded when cold Ringer’s solution was added to the heart. Click

AutoScale to maximize the size of the response on the window.

10. Repeat Steps 8, 9 and 10 to measure and record the various amplitudes and beat period of the

heart at the time the cold Ringer’s solution was added to the heart and at 10 second intervals for

the first minute after the addition of the cold Ringer’s.


Figure AM-6-L3: Frog ECG and heart rate displayed in the Analysis window. the cursors are placed to

measure the mean hear rate over a period of 8.963 seconds.

11. Repeat Steps 8, 9 and 10 to measure and record the ECG amplitudes and beat period of the

heart at the end of the recovery period from the effects of cold Ringer’s.

12. Determine the mean heart rate for each of the 10 cardiac cycles selected in the Journal and on

Table AM-6-S1.

13. The mean heart rate can also be calculated by converting the beat periods to heart rates using

the following equation:


Exercise 3: Effect of Warm Temperature

1. Scroll to the data recorded from the heart fifteen seconds before warm Ringer’s solution was

added to the heart. Click the AutoScale button to maximize the size of the ECG and heart rate

channels on the window.

2. Use the same techniques used in Exercise 2 to measure the ECG amplitudes and beat period for

the heart during the rest, treatment, and recovery periods when warm Ringer’s was applied to

the heart.


3. Measure and calculate the mean heart rate during each period.

4. Record your data in Table AM-6-S1.

Exercise 4: Drug Effects

1. Scroll to the beginning of the data from Exercise 4 and find the normal heart contractions that

occurred before the first drug treatment.

2. Use the same techniques used in Exercise 2 to measure the ECG amplitudes and beat period for

the heart during the rest, treatment, and recovery periods for the two drugs applied to the heart.

3. Measure and calculate the mean heart rate during each period.

4. Record the values for the amplitudes and heart rates from this exercise in the Journal and on

Table AM-6-S2 for Epinephrine and Table AM-6-S3 for Acetylcholine.

Table AM-6-S1:Amplitudes, Periods, and Rate of Heart Contractions at Different Temperatures.

Frog ECG

Treatment R Wave (V) P Wave (V) T Wave (V)Mean Heart Rate

(BPM)

Room Temp Ringer’s

Cold Ringer’s

10 sec after Cold Ringer’s






Recovered from Cold

Room Temp Ringer’s

Warm Ringer’s


10sec after Warm Ringer’s






Recovered from Heat

Table AM-6-S2: Amplitudes, Periods, and Rates of Heart Contraction with Epinephrine

Treatment.

Frog ECG

Treatment R Wave (V) P Wave (V) T Wave (V)Heart Rate

(BPM)

Resting

Epinephrine

10 sec after Epinephrine






Recovered


Questions

1. What is the effect of cold Ringer's solution on the rate and the amplitude of the ventricular

contraction (R wave amplitude and duration)? What mechanism is responsible for this effect?

2. How does warm Ringer’s affect the heart? How are the various wave amplitudes different from

when cold Ringer’s was applied to the heart?

3. What effect does Epinephrine have on the heart rate and the amplitudes of the ECG waves?

4. How does Epinephrine produce its effects on the heart rate and the amplitude of the ventricular

contraction specifically?

5. What effect does Acetylcholine have on the heart rate and the amplitude of the ECG waves?

6. How does Acetylcholine produce its effects on the heart rate and the amplitude of the

ventricular contraction specifically?

7. Do the time courses for the effect of each drug on the amplitude and the rate of ventricular

contraction differ?

Table AM-6-S3: Amplitudes, Periods, and Rate of Heart Contraction with Acetylcholine

Treatment.

Frog ECG

Treatment R Wave (V) P Wave (V) T Wave (V)Heart Rate

(BPM)

Resting

Acetylcholine

10 sec after Acetylcholine






Recovered



Appendix

Recipe for Amphibian Ringer’s Solution.

Concentration (mMolar) Salt Grams/Liter DI H20

111.0 Sodium Chloride 6.49

1.9 Potassium Chloride 0.142

1.06 ∗Calcium Chloride 2H2O 0.156

1.0 Tris 0.121

5.55 Glucose 1.00

Adjust pH to 7.6 with 6N HCl

Concentrations of Reagents in Ringer’s that Alter Heart Activity

Concentration (mMolar) Reagent mg/ml in Ringer’s

1 L-Epinephrine 0.20

1 Acetylcholine Chloride 0.16


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