Modeling the Axonal Action Potential

Using the NEUROSYS Program

(Note that this exercise previously used in ZOOL 211 is provided for your use as a review and may be used in conjunction with the Membrane Potentials Made Easy review page. You must complete the experiment described in #2 under Membrane Characteristics below. You may safely ignore all other instructions for generating reports. However the concepts are crucial!)

The NEUROSYS computer program was written by Mr. Zayd Eldadah (a former Zoology major and ZOOL 422 alumnus) and Dr. Herbert Levitan, former Professor of Zoology at UMCP. This simulation allows the experimenter to record several properties of an action potential after delivering stimuli of variable intensities (strength and duration) under different environmental conditions. You will use parts of this program to conduct experiments and thus gain a better understanding of nerve cell membrane properties.

Turn on the computer and select the NEUROSYS option from the Main Menu. Answer the question about the color monitor by pressing C and then press B to begin the program.

From this first menu, select the constant current stimulus by pressing C. Before changing any of the preset values (called default values), strike the Function 2 (F2) key and then P for Plot. Notice that you are watching a stimulus being delivered to a nerve on the middle plot. The x axis is time; the y axis is the current delivered in microamperes (uA) per cm2 of axonal membrane area. The top plot is a representation of the membrane potential (Vm) in mV. The bottom graph traces the changes in sodium and potassium conductances (gNa+ and gK+) as expressed in mS/cm.

You may terminate a trace at any time by pressing the ESCAPE (Esc) key. The F2 key will always return you to the main menu. Return to the main menu after you are familiar with the data recordings.

You may obtain a hard copy of your data at any time by reviewing the printer section (page 29) and pressing the PRINT SCREEN key. First, put the printer "on line" and load it with paper.

Stimulus Parameters

For your first experiment, examine the effects of changing the stimulus intensity and frequency. Begin this section by selecting (S)timulus parameters from the main menu. The program asks you to accept the preset, default values. Record these values for later reference before pressing Y for "yes". Observe the resulting action potential by pressing (P)lot. Return to the stimulus menu and systematically change the intensity, i.e., the strength and duration of the first stimulus. To more easily visualize the effects of each change, plot the results by pressing the (O)verlay key. This will superimpose successive plots. Be certain to record the summary of the results and some representative traces in your lab notebook.

Notice that the second stimulus has a 0% strength (amplitude). You may increase that to deliver a second pulse. Changing the delay value (start time) will enable you to deliver this second pulse at any time after the first.

Use this portion of the program to determine:

• threshold stimulus intensity (uA of current applied/cm2 of axon membrane) to the nearest µA at a constant stimulus duration (default value) for the first stimulus:

• threshold stimulus values at decreased and increased stimulus durations (a plot of stimulus duration vs. threshold stimulus intensity might be a nice touch for your report!):

• length of the refractory period (to nearest 0.5 msec) with both stimuli set at the same intensity (default or original value of the first stimulus):

(Before leaving this section of the program, be certain to return all values to their default settings. It may be easier to exit the program and restart it before moving on to the next section.)

Membrane Characteristics

From the main menu, examine the (M)embrane Characteristics option. This section of the program allows you to change:

A. Initial membrane potential (Vm)

B. Maximum voltage-dependent sodium conductance (a property of sodium channel density and sodium equilibrium potential)

C. Maximum voltage-dependent potassium conductance

D. Voltage-independent leakage conditions

E. Membrane capacitance

You will conduct and report on one of the following investigations:

1. Construct a plot of voltage-dependent sodium conductance vs. a relevant dependent variable of your choice. Examine the effect of potassium conductance on this same dependent variable.

2. YOU MUST PERFORM THIS EXPERIMENT AND SUBMIT YOUR RESULTS!

Vary one or more of the listed menu items (A - E above) to make the axon spontaneously active, i. e., a pacemaker. Remove the electrical stimuli (set amplitude = 0) and lengthen the recording screen trace to 50 msec (use the Range option in the main menu to change the duration of the plot) before you begin. Once you have a pacemaker, change one of the variables to increase the rate of firing. Restore the original rate and change another variable to decrease the firing rate. A printout of the three rates on one plot (use the "overlay" function) would make a nice addition to your report.

Your final report should contain a complete description of the parameters you altered to yield a spontaneously active neuron or pacemaker and the factors you altered to increase and decrease the rate of activity. Please include a printed copy of your data screen.

3. If you elect to look at capacitance (choice E), remember that capacitance is a measure of the amount of charges that is stored across a membrane. The charges (current) moving across a membrane first discharge/charge the capacitor (the membrane) before causing a change in Vm. A myelinated nerve membrane has a very low capacitance. Explain why this is the case. Use the capacitance variable to construct a plot of stimulus current vs. threshold. Note any differences in the onset times of the action potential. Explain the results. {NOTE: if you get spurious results, you may have to change the iteration time (interval between each calculation) by calling up the Range screen from the main menu.}

In each of the above experiments, record all default values!

Environmental Conditions

After restoring all values to their default settings, return to the main menu and examine the (E)nvironmental conditions option. Select choice b (ion concentrations) from this menu. Notice that you may now alter sodium, potassium, or chloride concentrations (the program automatically calculates the resulting equilibrium potentials for you!). You may also apply two drugs to the nerve, tetrodotoxin and tetraethylammonium. Use the program to determine the:

• relationships between the external sodium concentration and the resting membrane potential and the height of the action potential;

• relationships between the external potassium concentration and the resting membrane potential and the shape of the action potential;

• the mechanisms of action of the two drugs.

For simplicity, change only one variable at a time (e.g., external sodium concentration), and return that to its default value before moving on to explore the mysteries of potassium concentrations.

In your final report, include the data summaries of the above experiments and a brief written discussion of the results. Your data summaries will take the form of composite graphs of the results of each experiment and/or representative data tracings taken from the computer screen.

To EXIT the Neurosys program, return to the main menu, select option B(egin) and then select E(nd). Answer Y(es) to the last question and you are finished!