Download BSL PRO Frog Lessons

Updated 02.05.14
BSL PRO Lesson A17: Frog Heart - Intracellular Action Potentials
Developed in conjunction with Department of Biology, University of Northern Iowa, Cedar Falls
This PRO Lesson explains how to prepare the frog heart and describes the hardware and software setup
necessary to record intracellular action potentials.
Objectives:
1)
2)
3)
4)
To make an intracellular electrical recording in cardiac tissue.
To observe and understand the electrical properties of myocardial cells in the vertebrate heart.
To develop an understanding of the ionic basis of the electrical signals in cardiac tissue.
To study the effects of pharmacological probes on the cardiac action potential.
Equipment:

Biopac Student Lab System:
o MP36 or MP35 hardware
o BSL 4.0 or greater software
o
SS9LA BNC Adapter cable (10)

Compound microscope

Micromanipulator (MANIPULATOR-R or
MANIPULATOR-L)

Goggles Examination/surgical gloves

Dissection Pan

1000 to 5000 µL autopipette

BSL PRO template file: “a17.gtl”

Microelectrode amplifier

Tissue bath (with Sylgard®)


Perfusion system (gravity-fed), connected to
vacuum line
Dissection Kit: Scalpel, Forceps, Scissors,
Dissection pins, Insect pin


Faraday cage
Ringer's solution (dissolve 6 g NaCl, 0.075 g KCl,
0.10 g CaCl2, and 0.10g NaHCO3 (pH 7.4) in liter
of water)

Microelectrode puller


Large iron grounding plate with soft rubber feet to
suppress vibration
Drug preparations: acetylcholine (10µM),
epinephrine (adrenalin chloride) (5 µM), nifedipine
(50 µM) dissolved in dimethyl sulfoxide (DMSO)

Silver ground wire (for tissue bath)
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Background:
Across many animal phyla, the circulation of body fluids is important for the distribution of gases, nutrients and
waste products. In many cases, the well-being of the organism is contingent on the production and precise
regulation of internal fluid flow. Contractions by muscular regions in the vascular system maintain and control
the flow and pressure of internal fluids. Among most organisms with a circulatory system, there are basically
two types of hearts. In the neurogenic heart, beat and strength of cardiac contractions are determined
extrinsically by the nervous system of the organism.
If the nervous system is destroyed the heart stops beating. Other organisms have myogenic hearts. In these,
the fundamental signal regulating cardiac contraction, or beat, is incipient or generated within the cardiac
tissue. When this type of heart is removed from the organism, it continues to beat for some time in isolation.
Regions of the cardiac tissue known as the pacemaker rhythmically produce action potentials. The signal is
transmitted to myocytes which then contract. Consequently, the pacemaker produces a cardiac action
potential that is propagated and eventually stimulates myocytes to generate a force. The force propels fluids
through the circulatory system. The signal produced by the pacemaker is, however, up and down regulated by
the nervous system. So when the organism needs a faster heart beat and greater cardiac output, neurons or
hormones will increase the rate of signal production by the pacemaker. Alternatively, other neurons may
antagonize the pacemaker, slowing it down thus decreasing the beat-frequency and cardiac output. The
myogenic heart is common among all higher vertebrates. However, it is not uniquely vertebrate in nature.
Myogenic hearts also occur in many invertebrates and protochordates. In fish and amphibians, the pacemaker
is located in the sinus venosus (SV). In reptiles, birds and mammals, it is located in the sinoatrial node (SA).
This exercise involves recording action potential from cardiac tissue in an amphibian. Here, we will use the frog
heart as a physiological model to understand basic cardiac activity because they are small, economical, and
readily accessible. The frog is suggested because its myogenic heart survives prolonged exposure to room
temperature and reduced oxygen levels (hypoxia).To experiment on a mammalian heart, the tissue must be
maintained at 37˚C and aerated vigorously with a special gas mixture. To be sure, there are differences
between the mammalian and amphibian heart.
The frog heart is three chambered (Figures 1 – 3) compared to four in the mammalian heart. The frog’s
pacemaker is located in the SV tissue while the SA node is found in the mammal’s right atrium. Otherwise, the
two are remarkably similar in physiology.
Figure 1
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Figure 2
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Figure 3
For coordinating myocardial contractions, the sequence is controlled by an orderly propagation of the cardiac
impulse from the pacemaker to the myocyte. This signal spreads rapidly from the pacemaker to the atrial and
ventricular myocardium via intercellular pathways established by cellular couplings. Connexin proteins
establish gap junction connections (also called intercalated disks) between myocytes permitting the spread
of the impulse from cell to cell. The cardiac potential, in fact, assumes different forms as it travels from the
pacemaker to the contractile myocytes. Depending upon placement of the microelectrode, it is possible to
record any of the three types of signals shown in Figure 4. It is most likely you will record an atrial or ventricular
signal rather than a “nodal” signal during this exercise.
Figure 4a Sinoatrial Node
Figure 4b Atrium
Figure 4c Ventricle
As electrical impulses are propagated across cardiac muscles, the myocyte contracts. Unlike skeletal muscle,
calcium (Ca++) for actinomyosin interaction and cellular shortening comes from outside the cell. The
endoplasmic reticulum (i.e. sarcoplasmic reticulum) of cardiac myocytes is poorly developed when compared
to skeletal myocyte fibers. In skeletal myocytes, an impulse initialed at the neuromuscular junction travels
along the cell membrane until it reaches a transverse tubule (t-tubue). Here the impulse is diverted to the
interior of the myocyte and is eventually transmitted to the sarcoplasmic reticulum (SR). As the signal passes
over the SR, voltage-gated Ca++ channels open releasing the divalent cation into the sarcoplasm.
After interacting with troponin covering the thin filament, muscle tension develops as Ca++ increases in the
sarcoplasm. During relaxation, Ca++ is actively pumped by a Ca++-ATPase back into the sarcoplasmic
reticulum and awaits another impulse. In cardiac myocytes, on the other hand, the impulse opens voltagegated Ca++ channels as it moves across the cell membrane and the divalent cation enters from the exterior
promoting contraction. During relaxation, a Ca++-ATPase extrude the Ca++ to the extracellular space.
Compounds like nifedipine (NIF) are known as dihydropyridine calcium blockers that work on the L-type
(long-time open) calcium channel. This drug will reduce the force generate during cardiac myocyte contraction.
The form of the impulse in cardiac tissue is somewhat like the action potential in the axon (see Figure 4a, b,
and c). From experiments with the giant squid axon, we know that voltage-gated channels and concentration
gradients for both sodium (Na+) and potassium (K+) are required to generate an action potential. Likewise,
these are required for the myocardial impulse. In addition, there is a role for Ca++ ions and channels.
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+
After depolarization to threshold, voltage-gate Na channels open temporally shifting the membrane voltage
to a positive value before they automatically inactivate. During this period, the delayed-rectified K+ channels
open to repolarize the membrane voltage. While the Na+ channels inactivate, the depolarized status of the
membrane activates voltage-gated Ca++ channels. After a few milliseconds, the Ca++ channels automatically
inactivate, and the outwardly-directed K+ ion-current repolarizes the membrane voltage to the resting
potential (Vm). As a result of an inwardly directed Ca++current, there is a “lagging” shoulder or plateau in the
cardiac impulse (see Fig 4c). The fast depolarizing component of the impulse in the cardiac myocyte is due to
the inwardly-directed Na+ current while the slow plateau is due to a delayed inwardly-directed Ca++ current.
Ultimately after inactivation of the two cation channels, the outwardly-directed K+ current will return the
membrane voltage to the resting potential. The membrane is then ready for another depolarization cycle. The
various ion phases in the signal can be altered easily by manipulating K+, Na+ and Ca++ concentrations in the
bathing
Ringer’s solution.
During this exercise you will record cardiac potentials from several regions of an isolated heart. Because
cardiac cells are coupled by gap junctions and form a syncytium, the electrode only needs to be placed near a
group of cells to record the potential. You will also apply different chemicals or pharmacological probes
(drugs) to the preparation. These substances we use will either alter the pacemaker’s ability to produce a
signal (to accelerate or to impede the oscillator,) or change the ability of the myocardial cells to contract.
Substances that modify the beat-frequency of the heart are called chronotropic agents while those altering
the amplitude or strength of muscular contractions are called inotropic. Common chronotropic agents
resemble the neurotransmitters norepinephrine (a sympathetic agent) and acetylcholine (a
parasympathetic agent).
Some compounds block sodium or potassium channels independently controlling the production of the
pacemaker signal. Other compounds are calcium antagonists which have a major impact on myocyte
contraction. These compounds may also influence the behavior of the pacemaker. Interestingly, there are
“designer” calcium-blocking drugs selective for either contraction or pacemaker activity. You will apply some of
these compounds to your preparation to understand how heart function is modulated.
To keep the extirpated frog heart optimal, it
should be constantly perfused with fresh,
aerated Ringer’s solution. An inexpensive
gravity/vacuum perfusion system can be
constructed for this purpose from common
laboratory hardware (Figure 5). A one or
two-liter saline reservoir jar is elevated
about 18-20” (45-60 cm) above the tissue
bath. Flow rate into the bath is regulated by
an adjustable stopcock. The flow rate
through the tissue bath should be about
1000 ml/hr. It should not disturb electrode
impalements. Exhaust from the bath is
controlled by vacuum (pump or wateraspiration). If the saline reservoir is aerated,
the heart is assured of a high level of
oxygen. Evacuation of fluids removes used
chemicals and metabolic waste.
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Figure 5
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The microelectrode measurement is very sensitive to
external vibration and electrical interference. The
tissue bath and micromanipulator can be placed on a
large (heavy) iron base plate that has soft rubber feet
to absorb vibration. The perfusion system,
micromanipulator, microelectrode amplifier and base
plate should all be contained within a Faraday cage to
minimize electrical interference. The cage and iron
base plate are both connected to earth ground at the
same point. The microelectrode amplifier reference
input (“-” or “Voltage Follower” etc.) must be in contact
with the tissue bath Ringer’s solution via a silver wire
(inserted in bath). Figure 6 shows an example of a
low-cost setup.
Figure 6
Setup:
The following assumptions are made:
 The MP36/35 is connected to the host computer and that the Biopac Student Lab software has been
installed and is known to work with the MP unit.

The glass microelectrodes filled with 3 M KCl have been made and tested to have a resistance
between 10 and 40 Megohms.
1.
Turn OFF MP unit.
2.
Connect the BIOPAC SS9LA Adapter cable (10) between CH 1
of the MP36/35 and the microelectrode amplifier (BNC) output.
3.
Turn ON the MP36/35 unit.
4.
Launch the BSL PRO software and open the template file
“a17.gtl” in one of two ways:
a. From Startup dialog, click the PRO Lessons tab and doubleclick on the lesson title in the list. If you do not see the lesson
title, try the next opening method.
b. From Startup dialog, choose the Create/Record a new
experiment option, click Open Graph template from disk
and then click OK. Navigate to the file, select it and
click Open.
Figure 7
FROG PREP:
Double-pith the frog (if necessary, see BSL PRO Lesson A01 - Frog Preparation for details). You should apply
amphibian Ringer’s solution to the frog in five-minute intervals.
1.
Carefully expose the frog heart.
a. Cut the skin from the groin to the throat of the frog.
b. Cut through the pectoral girdle to expose the heart in the pericardial sac.
c.
Use dissection pins to hold the body to the dissection tray.
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Figure 8
Figure 9
Figure 10
Figure 11
d. Carefully cut through the pericardial sac, then gently
grasp the ventricle with forceps and lift the heart out.
e. Excise the heart by cutting through all vessels
running into the atria and the sinus venosus.
f.
Place the heart in a small Petri dish and rinse with
Ringer’s solution.
g. Transfer the heart to the tissue bath container.
Figure 12
h. Use a small insect pin to secure the heart to the
Sylgard® at the base of the container. Insert the pin
through the anterior vessels to avoid damaging
the heart.
2.
Start the flow of Ringer’s through the perfusion system. After the Ringer’s has reached its fill line (above
the preparation), adjust the flow such that the saline trap is slowly filling with no visible waves in the
tissue bath.
3.
Position the Glass electrode via the micromanipulator such that it is just above the ventricle.
Recordings:
One data file containing several recordings will be saved for this lesson. Each successive recording will be
appended onto the previous one. Assign three students in the group the following roles:
a.
A Director will read aloud the recording steps and will adjust the microelectrode amplifier “Position”
control and adjust the perfusion system flow control as needed.
b.
An Operator will position the glass electrode with the aid of the microscope and micromanipulator.
c.
A Recorder will start and stop the recording, monitor the data, and provide feedback to the
Operator and Director. The Recorder should be familiar with basic software operation particularly
zooming, and autoscaling (see Appendix 1 for details).
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Zero adjustment:
1.
Operator: Move the micromanipulator, using the coarse vertical adjustment, until the tip of the glass
electrode is submerged in the Ringer’s solution BUT NOT impaling the heart.
2.
Recorder: Click Start to begin the recording.
3.
Director: Adjust the “Position” knob on the microelectrode amplifier until the baseline reads 0 mV.
4.
Operator: Use the coarse vertical adjustment to slowly move the electrode tip towards the tissue. The
Recorder must call out when they see a deflection in the data at which point the Operator backs out the
electrode just slightly.
Note: If the tip of the microelectrode contacts the tissue too quickly, it will break off!
Finding the action potential:
5.
Operator: Use the fine vertical adjustment of the micromanipulator to carefully bring the electrode tip
back down to the tissue. Carefully move the electrode up and down at the surface tissue until the
Recorder sees a downward deflection and an action potential signal.
Notes:
 Be patient and move slowly and cautiously.
 The electrode may move out of the cell due to the movement of the beating heart. Readjust the
electrode position using the fine vertical adjustment.

When the Recorder sees an action potential signal, press
the “F9” key (“Fn” + “F9” on Mac) to place an event marker.
You may click in the event marker label region and type in a
marker label. This will assist in finding the data during
analysis.

6.
If no action potential signals can be found, carefully back
the electrode out and move to a different location on the heart.
Repeat Steps 1 – 5.
Recorder: Click Stop to halt the recording.
Figure 13
Pharmacological Probes:
Discuss with your instructor a protocol for applying the pharmacological probes (drugs). Note that the
concentration indicated on the bottle will not be the actual concentration to which the tissue is exposed,
because it will become diluted in the tissue bath’s Ringer’s solution.
1. Epinephrine (EPI)
1.
Recorder: Click Start and record a few seconds of baseline data and click Stop. Change the append
marker label to “Control.”
Note: If no action potential is seen, start the recording again and have the Operator slowly move the
electrode to try and regain an action potential signal. It may be necessary to repeat Steps 1
through 5 above to obtain signal. The Rewind toolbar button can be use to erase a previous,
unwanted data segment.
2.
Director: Adjust the flow control valve to stop flow through the perfusion system.
3.
Operator: Deliver about 1 mL of EPI solution above the heart using the autopipette.
Note: Do not touch the heart, as this can dislodge or break the electrode.
4.
Let the solution bathe the heart for 30 to 40 seconds.
5.
Recorder: Click Start and record for 1 to 2 minutes, then click Stop. Label the Append marker “EPI.”
6.
Director: Turn ON flow of the perfusion system for about 2 minutes to wash out the drug.
7.
Operator: Change the tip on the autopipette.
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2. Acetylcholine (ACH)
Repeat steps 1 – 7 above, but apply 1mL of ACH in Step 3. Label the append marker for Step 5 “ACH.”
3. Nifedipine (NIF)
Repeat steps 1 – 7 above, but apply 1mL of NIF in Step 3. Label the append marker for Step 5 “NIF.”
Optional
If time permits, additional experiments using Na+ free or 10 mM K+ Frog Ringer’s solutions may
be performed.
Done
Save the data by choosing “File > Save As.”
Data Analysis Suggestions:
Figure 14 shows some basic measurements that characterize the Action Potential signal.
Figure 14
Fill in Table 1 using measurement tools in the Biopac Student Lab software. You will need to manually
calculate the slopes using the peak to peak (P-P) or Delta measurement for amplitude and the Delta T
measurement for time. The listed conditions would include the control recordings made prior to the application
of each drug.
Condition
Vm
(mV)
Heart Rate
(BPM)
Duration
(sec)
Amplitude
(mV)
Upstroke Slope
(mV/sec)
Pacemaker Slope
(mV/sec)
Table 1
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Measurement examples:
The I-beam cursor is used to select an area from the
start of the AP signal to the end. The P-P
measurement displays the amplitude and the Delta T
measurement displays the time interval of the
selected area (Figure 15).
Figure 15
An area is selected from the start of one AP signal to
start of the next AP signal. The BPM measurement
calculates and displays the “beats per minute”
between the endpoints of the selected area.
Figure 16
Note: CH 41 is a real-time calculation channel that calculates the heart rate from the AP signal on a beat-bybeat basis. Because each BPM value can only be determined at the end of the cycle, it will be out of
sync with the AP signal by one cycle. It also takes at least two cycles to lock onto the signal after the
start of the recording. Real time BPM can be a very useful view to observe changes in heart rate;
however it cannot always “lock onto” the signal due to noise or wandering baseline. CH 41 can be
toggled on or off by holding down the “Alt” button (“Option” on Mac) AND clicking on the Channel button
in the graph.
Questions:
1) When drugs are added to heart tissue, did the form of the cardiac signal change?
2) How does BPM change with EPI? ACH? Can you explain how these influence the “pacemaker?”
3) Is there a change in amplitude of the cardiac signal with EPI or ACH? Duration? Period?
4) Do ACH and EPI have a similar effect on the pacemaker slope?
5) Does NIF have an impact on amplitude and/or the shape of the cardiac signal? Does it appear to
influence BPM?
6) If you increased the K+ concentration in the Frog Ringer’s solution, how do you think that would change
the cardiac signal? What would happen if you used a Na+-free Frog Ringer’s solution?
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Appendix 1:
Autoscaling data:
Graph data can be autoscaled vertically and horizontally for enhanced viewing.

toolbar button or “Display > Autoscale Waveforms” to
Autoscale vertically by using the
optimize the vertical display and allow closer examination of the waveform.

Autoscale horizontally by using the
toolbar button or “Display > Autoscale Horizontal” to
display the entire horizontal time scale in a single graph window.
Zooming in and out of data:
Use the Zoom tool to magnify portions of the waveform for a closer look.

Zoom in by selecting the

To zoom back, use Ctrl – (minus) or “Display > Zoom Back.”
toolbar icon and click/drag over the area of interest.
Using the Event Palette to quickly locate Event Markers in the graph:
An easy way to locate event markers in the graph is by using the Event Palette, accessible by clicking the
toolbar icon or via “Display > Show > Event Palette.”
Zoom in on any section of data, simply highlight an event in the Event list, and the portion of data
corresponding to that event will be displayed in the graph. Other features in the Event Palette include
summarizing, renaming, removing or editing existing events.
To navigate quickly through consecutive event markers, use the arrows to the left of the Event Palette toolbar
icon.
Measurements used in BSL PRO Lesson A17:
Graph data measurements are taken by using the I-beam tool
basic measurements are used in this experiment:
to select an area of interest. The following
P-P (Peak-to-Peak)
Shows the difference between the maximum amplitude value and the minimum amplitude
value in the selected area.
Value
Shows the data value at the cursor position, or if an area is selected, the data value at the
end of the selection.
Delta-T
Shows the horizontal axis time value in the selected area.
For a full explanation of the features described above, see the BSL PRO Tutorial or BSL PRO Software Guide.
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