Michael Galloway
BIOL 3810.506
Compound Action Potential of the Bullfrog Sciatic Nerve
Date Performed: February 7, 2013
Date Due: February 29, 2013
TA: Vinoo Urity
Introduction
Neurons and nerve impulses form the basis of life on earth. Sense of the environment and
internal processes of the body all involve a complex process of input and output involving nerve
cells. Neurons are specialized cells that communicate with each other, as well as muscle and
organs, through electrical synapses throughout the body. The neuron consists of the soma (cell
body), axon, and dendrites. Nerves are made up of a bundle of nerve fibers or axons. Each axon
located in the nerve can produce a phenomenon called an action potential. An action potential is
an all or nothing response that does not deteriorate as it travels down the axon. The action
potential is governed by the sodium/potassium pump. At first, the sodium channels open up to
allow an outflow of sodium ions into the cell causing depolarization. As more and more sodium
ions leave, the potential rises to about 35mV causing the sodium pump to close. Then the
potassium pump opens and potassium ions leave the axon bringing the potential back to resting
state. However, the potassium pump often does not close quickly enough which causes an
overshoot. This overshoot or hyperpolarization makes a brief drop beneath the resting potential.
As noted by many people who work with action potentials, “most models of electrical activity of
cells show that only the motion of positive ions, and specially those of potassium, calcium and
sodium, influence the membrane potential” (Endresen, 2000). The constant change from
depolarization and hyperpolarization states allows action potentials to self-propagate.
One of the unique characteristics of an action potential is that it can be “elicited in each
axon simultaneously by electrical stimulation” (Brink, 1983). This phenomenon is called a
compound action potential (CAP) which is a collective, artificial response of a nerve. The
amplitude of the action potential for each individual axon doesn’t change with stimulus intensity,
but increases in the amplitude of a CAP at higher stimulus voltages which incorporates other
axons within the same nerve reaching their own threshold.
During this experiment, the sciatic nerve, the largest nerve in the human being, of a frog
will be dissected out and examined. This nerve will be taken and measured based on extracellular
readings. Using the nerve, we can test “threshold phenomenon, temporal summation, refractory
periods, strength-duration curves, and conduction velocity” (Eckert, 2002). Conducting the
experiment, should allow us to get a better understanding on how to interpret compound action
potentials. Also, it should allow us to see that the sodium/potassium pump clearly is the reason
for causing action potentials. The purpose of this investigation was to test the effects of a
stimulus voltage on a frog’s sciatic nerve to observe the threshold, refractory periods, and
conduction velocity of the resulting compound action potential.
Materials and Methods
In this experiment, the sciatic nerve from a frog was removed to test for compound action
potentials. Along with the nerve, the Lab Tutor software and PowerLab 400 hardware were used
in order to test the nerve for data and oscillations.
I. Frog Sciatic Nerve Dissection
A double-pithed bullfrog frog was obtained and secured to a dissecting board with
straight pins. The frog was double-pithed and already dead which prevented it from feeling pain
or any external sensations during the dissection. Starting from the mid-abdomen, a small and
careful incision was made in the frog’s body. Then, the skin was carefully pulled off from the
abdomen to one part of the leg, so that the internal muscles and nerves were exposed. The sciatic
nerve is located in the thighs underneath the striated muscle. The frog was cut at the urostyle
(near the base of the spine) and the hemostat was used to hold it up as the muscles were severed
on both sides of the bone. To easily identify the nerve, it was ideal to start at the vertebrae
column because it is located beneath the organs and above the column. By removing or
displacing the leg muscle, the nerve should of been easily visible. Then, string was carefully
inserted around the nerve to be used to tie a knot at that point and the nerve was cut as close to
the spinal cord as possible. Great care was taken not to stretch, compress, or touch the nerve with
metal or a finger to maintain its vitality. Ringer’s solution, an electrolyte solution containing
glucose, was constantly applied to the tissue to prevent it from drying out and to provide a source
of glucose for the mitochondria in the axons to make ATP (since repeated APs deplete ATP).
Following the nerve down to the knee, a second knot was tied around the end of the nerve and
then removed from the body of the frog. Moving quickly, we then placed the nerve in a small
beaker of Ringer’s solution after it was totally freed.
II. Electrophysiology Setup
After the dissection had taken place, and the nerve was successful removed, the setup of
LabTutor and the nerve chamber was preceded. First, Ringer’s solution was added up to the top
of the smaller, inner trough of the nerve chamber making sure not to touch the wires which could
possibly short circuit the experiment or cause erroneous results. To setup LabTutor, the first and
second sets of electrodes were connected to the nerve chamber as specified. Then, a piece of
moist filter paper by Ringer’s solution was placed across all the electrodes. The positive and
negative BNC connectors of the stimulating electrode leads were connected to the analog outputs
on the PowerLab. After, the first recording electrodes were connected to Input 1 on the
PowerLab, and the second set to Input 2. To test the setup, LabTutor automatically stimulated
and recorded data for one second, after Start was clicked the “Test Channel” which matched up
to stimuli shown in the “Stimulus Channel” showed that the connections were working. Addition
of the sciatic nerve followed the test run by placing it length-wise in the chamber using forceps
and making sure one end touched the first electrode wire. Ringer’s solution was continually
added and the cover was placed over the chamber to prevent drying.
A. Eliciting the Compound Action Potential
The first exercise utilized the sciatic nerve to find the threshold voltage and maximal
CAP amplitude. In the Stimulator panel of LabTutor, the stimulus voltage was first set to 10mV.
After clicking Start, LabTutor stimulated the nerve and recorded for 5ms. Then the stimulus
voltage was increased by 10mV by clicking the up arrow in the Simulator Panel Amplitude Box
and then recorded. The voltage was increased by 10mV and tested until there were three
successive responses that didn’t increase in amplitude.
B. Refractory Periods
Our second exercise, two stimuli were sent to the nerve, progressively decreasing the
interval between them each time, to determine the absolute refractory period. The stimulus
voltage was supposed to be set to the minimum voltage that was required to give a maximal CAP
in the first exercise. We finally got a good reading at 200mV. The stimulus interval was set to
4.0ms. After clicking start, LabTutor stimulated the nerve twice at the interval and recorded for a
total time of 10ms. Then, the interval was decreased in the stimulator panel to 3.5ms and started.
These steps were repeated decreasing the interval to 3.0 ms, then 2.5 ms, 2.0 ms, 1.9 ms, and
then by steps of 0.1 ms until the interval between the stimuli reached 1.0ms.
C. Conduction Velocity
The third exercise was used to determine the conduction velocity of the nerve. A voltage
which was twice the amount used in Exercise 2 (200mV) was entered into the stimulator panel.
LabTutor was set up so that two channels of data were recorded for 5ms after clicking start. The
upper channel represented the CAP from the nearest recording electrode and the lower channel
showed the CAP from the further electrode. The distance between the recording electrodes was
determined by measuring the distance in millimeters between the negative leads (black) of each
of the two electrodes. The time interval for the CAP to travel between the two recording
electrodes was figured out by measuring the distance between the nearest and further CAPs.
Results
I.
Eliciting the Compound Action Potential
The frog sciatic nerve’s threshold stimulus voltage was observed at 10mV. With each
increase in the stimulus voltage, a subsequent increase was observed in the amplitude of the
CAP. At a stimulus voltage of 40mV the maximal stimulus, or suprathreshold, was established at
1.96mV (the amplitude of the CAP stopped increasing). A summary of the stimulus voltages
used and resulting amplitudes of the CAPs are shown in Table 1. Figure 1 shows the relationship
between the stimulus voltage and responding amplitude of the CAP.
Table 1. Stimulus and Action
Potential Amplitude
Figure 1. Stimulus and Action Potential Amplitude
II.
Refractory Periods
The stimulus voltage used in the second exercise was 200mV. The first decrease, which
was significantly lower, was detected at the magnitude of the 2nd CAP was observed at the 3.5ms
stimulus interval. The stimulus interval at which the 2nd CAP first disappeared was 1.7ms. The
relation of the stimulus intervals to the amplitude of the 2nd CAPs is illustrated in Figure 2. See
Table 2 for a record of all of the intervals tested and resulting amplitudes of the
CAP.
Table 2. Stimulus
Interval and 2nd CAP
Amplitude
Figure 2. Stimulus Interval and 2nd CAP Amplitude
III.
Conduction velocity
In the third exercise, a stimulus voltage of 400mV was used. The distance between the
recording electrodes measured 21mm. A single CAP was generated at the nearest electrode and
took 1.11ms to reach the further electrode. Using this data and the velocity formula (V= Δd/Δt),
the average fiber velocity, or conduction velocity, in the frog sciatic nerve was found to be
18.9m/s. The summary of data used for the calculation of the velocity can be seen in Table 3. See
Figure 3 for amplitudes of the CAPs elicited in the proximal and distal electrodes with respect to
time.
Table 3. The Calculation of Conduction
Velocity
Discussion
I.
Eliciting the Compound Action Potential
Once each axon reached its threshold, the nerve is in a fixed, all-or-nothing action
potential. As the voltage was increased from 10mV, to 20mV, 30mV, 40mV, and so on, in which
progressively higher amplitudes were observed in the CAPs, proving that not all of the fibers
within the nerve had previously met their thresholds. The first voltage at 10mV caused a CAP
amplitude of 8.12mV proving that at least one of the fibers had reached threshold at that point.
After reaching a stimulus voltage of 110mV, the CAP amplitude no longer increased, marking
the maximal stimulus (the point at which all of the thresholds of the individual fibers within the
nerve were met). The frog sciatic nerve’s suprathreshold of 110mV resulted in the CAP not
being able to produce an amplitude above 29.96mV. In conclusion, the nerves required a
minimum voltage to elicit a compound action potential but the amplitude of that compound
action potential would not increase after the maximal stimulus voltage was met.
II.
Refractory Periods
As the time interval between the conditioning and test stimuli grew closer, the 2nd CAP
showed a significant decrease in amplitude at 2.5ms. A decrease in amplitude is significant of the
end of the relative refractory period since, at that point, only a few of the Na+ channels started to
reopen since the very recent action potential firing (Waxman, 1988). The interval of 2.5ms was
hypothesized to be the actual end of the relative refractory period since it was the shorter interval
between stimuli. The major decrease could have been due to error. When the intervals between
the stimuli reached 1.7ms, there was no 2nd CAP. This is indicative of the end of the absolute
refractory period, because after which, CAP can be propagated. The absolute refractory period,
which is the moment right after the action potential fired, the Na+ channel becomes completely
inactivated, rendering the neuronal membrane unable to be stimulated by another stimulus.
Because the axons within a nerve have different relative and absolute refractory periods, the
refractory periods found mark the axon with the shortest absolute and relative refractory periods
(since it would be the first one to be able to fire and AP again). This exercise determined how
quickly another stimulus could have been detected in the sciatic nerve of a frog after a previous
stimulus elicited an AP.
III.
Conduction velocity
The conduction velocity of the bullfrog sciatic nerve was measured to be 18.9m/s, a little
lower than the normal range for any nerve fiber, which is measured to be 50 to 60 m/s. This
result may be due to a conversion error in the program. If the measure in milliseconds was
actually in seconds, the conduction velocity would have resulted in a lower measured velocity.
Then the average fiber diameter would have been about 4µm according to the equation V=2.5D,
where D = diameter of the fiber in micrometers (Animal Physiology Lab Manual, 2011). This
diameter is indicative of a B fiber, which is 3µm on average. Another possible error could have
resulted from the Ringer’s solution coming in contact with the wires, shorting out the stimulus
electrodes.
Conclusion
This laboratory experiment demonstrated the importance and how to determine
compound action potentials. The methodology behind the experiment shows how Na+ and K+
influxes and efflux out of the cell membrane can produce an action potential. The membrane
concentration gradient is essential for all life on earth. The knowledge obtained from performing
this experiment could help better understand the same biological process in humans without
having to use them for surgery. Further studies could provide insight into the effects of damage
to the sciatic nerve in humans and how to treat it (Endresen, 2000). Knowing exactly where the
sciatic nerve is located, where it sends signals to, and how, can help scientists and researchers to
able to recognize the abnormalities in humans. Testing the sciatic nerve for action potentials with
different refractory periods, stimuli, and time intervals in frogs will expand the knowledge about
AP’s firing within the human body. Additional studies could be performed to specify and give
further details to the mechanisms responsible for the behavior of the sciatic nerve in humans.
Literature Cited
Brink Jr, F. Linear range of Na+ pump in sciatic nerve of frog. AJP - Cell Physiology. 1983; Vol
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Eckert, R., Randall, D., Burggren, W., and French, K. ANIMAL PHYSIOLOGY, 5th Edition.
2002.
Endresen, L.P., Hall, K., Hùye, J.S., Myrheim, J. A theory for the membrane potential of living
cells. Eur Biophys J. 2000; 29: 90-103.
University of North Texas Biology 3810 Animal Physiology Lab Manual, 2011
Waxman, S. Joel A. Black, Jeffery D. Kocsis, J. Murdoch Ritchie. Low Density of Sodium
Channels Supports Action Potential Conduction in Axons. 1988. Neurobiology. Vol. 86,
pp. 1406-1410