Aniphys Lab Report 1

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

Figure 3. The CAPs from the Proximal and Distal Electrodes

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 244, Issue 3 198-C204. 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