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Frog Heart

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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.

[pic]

Background

Studies of isolated organs were pioneered in the late 19th century when scientists such as Sidney Ringer (1835–1910) developed a perfusion solution (Ringer’s 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 heart’s “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 Ringer’s solution at room temperature
Frog Ringer’s solution in 40° C water bath
Frog Ringer’s solution in 10° C ice bath
Acetylcholine (0.1 mg/mL)
Epinephrine (1% solution)
Pilocarpine (2.5% solution)
Atropine sulfate (5% solution)

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).

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.

[pic]

Figure 3. Equipment setup for frog heart recording.

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 Ringer’s 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 animal’s 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.

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.

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 Ringer’s. Add a comment to your data trace called “warm”. Record for 30 seconds.

4. Bathe the heart in cold (10 °C) Frog Ringer’s. Add a comment to your data trace called “cold”. Record for 30 seconds.

5. Click Stop. Bathe the heart in room temperature Frog Ringer’s before continuing to Exercise 3.

Exercise 3: Starling’s 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 Ringer’s 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 Ringer’s and allow the heart two minutes to recover.

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 Ringer’s 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 Ringer’s 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 Ringer’s and allow the heart two minutes to recover.

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 (Δt) 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 1–3 for the 10 °C data trace.

Exercise 3: Starling’s 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.

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.

13. Calculate and record the percent change in heart rate for each drug using the following equation:

[pic]

Data Notebook

Table 1. Determination of resting heart rate.

|Number of beats in selection |Time differential between first and last|Calculated Heart Rate (BPM) |Heart Rate from Data Pad (BPM) |
| |beat (sec) | | |
| | | | |

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 | | | |

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 Starling’s 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

[pic]
Copyright © 2004 ADInstruments. All rights reserved.

MacLab and PowerLab are registered trademarks, and Chart and Scope are trademarks, of ADInstruments. Windows and the Windows logo are either trademarks or registered trademarks of Microsoft Corporation. Macintosh and the Mac logo are either trademarks or registered trademarks of Apple Computer, Inc. Other trademarks are the properties of their respective owners.

www.ADInstruments.com

-----------------------

Figure 4. Setup of dissected frog and MLT500 Force Transducer.

Output from force transducer

Figure 1. MLA40 Mounting Stand and Micropositioner set up with MLT500 Force Transducer.

Figure 2. Bio Amp patient cable connected to MLA1605 Lead Wires.

MLA1605 Lead Wires

Zeroing knob

ML301 Bridge Pod

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...Tikhomirova 1 Tatiana Tikhomirova Marcio Pre-Degree I 3 March 2014 Life Cycle of a Frog Despite the fact that the frog is a small animal, in terms of symbolism, it is of some interest. In ancient Egypt, because of its fertility, as well as a striking transformation from egg to a tadpole, and then in the quadruped animal, the frog was considered strange creature and was a symbol of emerging and ever-renewing life. Often, the ancient gods of care arising out of the mire, depicted with frog heads. Goddess of child-birth, who was a good helper of the popular religion, looked like a frog. (“Heqet”) This essay will describe frog’s transformation from egg to adult frog by three steps. First of all, frogs lay eggs. When multiple eggs stick together, they are collectively known as frogspawn. After fertilization, the innermost portion liquefies to allow free movement of the developing embryo. Most eggs are black or dark brown, and it has the advantage of absorbing heat from the sun, which holds the insulating capsule. Frogs lay a lot of eggs because there are many dangers between fertilization and grown frog. Those eggs that die turn white. Life starts right as the central yolk splits in two. It then divides into four, then eight, etc. Soon, the embryo starts to look more and more like a tadpole, getting longer and moving in egg. Usually, about 6-21 days after being fertilized, the egg will hatch. Most eggs are found in calm or static waters. When the rain comes along, after......

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