Laboratory 2: HHsim: Graphical Hodgkin-Huxley Simulator

(Source: http://www.cs.cmu.edu/~dst/HHsim/ )

INTRODUCTION

HHsim is a computer program intended for use in teaching neurophysiology, specifically the concepts
associated with the resting membrane potential and the action potential. Within the program of HHsim is a
simulation entitled HH which is based on the equations developed by English physiologists A. L. Hodgkin and A.
F. Huxley (J. Physiol. Lond. 117: 500-542, 1952). Much of our current knowledge about the mechanisms of
resting membrane potentials and action potentials comes from experiments performed by Hodgkin and Huxley
on a giant nerve fiber found in a species of squid called Loligo forbesi (adult squid weight 3.5 lb, adult squid
length 14 in). Because of the large diameter of this axon (up to 0.8 mm), it became a convenient model for
elecrophysiological research using small intracellular microelectrodes (tip diameter < 0.5 μm). With two
microelectrodes, researchers were able to detect an electrical potential difference between an electrode
inserted inside the nerve cell and one placed outside the cell. The internal electrode measured a potential of
near -70 mV with respect to the external electrode. This -70 mV potential difference was called the resting
membrane potential. Membrane potentials are therefore expressed as the intracellular potential minus the
extracellular potential and the extra cellular potential is set to be zero (or ground). A negative value denotes
that the cytoplasm is electrically negative relative to the extracellular fluid.

Resting membrane potential

The resting membrane potential is due to specific ions, mainly potassium, and their tendency to diffuse down
their concentration gradients until opposed by an electrical force of opposite but equal magnitude. To
illustrate, create a model of a membrane by using two chambers (A and B) separated by membrane. Assume
the membrane is permeable to cations but not to anions. If two different concentrations of a KCL solution are
placed in the chambers (0.1 M KCL in A and a 0.01 M KCL in B), K + will begin to diffuse from chamber A to
chamber B down its concentration gradient. Initially no electrical potential difference will exist, however, since
the membrane is not permeable to Cl -, side A will begin to accumulate negative charges along the membrane
thus becoming electrically negative with respect to side B. The more K + that flows from side A to side B, the
larger will be the potential difference resulting in an electrical force opposing the diffusion of K + across the
membrane. The net flow of K+ will cease when the electrical gradient (inward) equals the concentration
gradient (outward). Similarly, all cells have a negative resting membrane potential because of this selective
permeability to K+ through K+ leak channels (leak channels are channels that are usually open). The magnitude
of the electrical potential required to counterbalance the concentration gradient for a given ion can be
computed using the Nernst equation (Silverthorn pp 163, 252). The potential energy difference across the
membrane can be harnessed by the cell to do work and is the basis of the action potential.

Action Potentials

The action potential represents a rapid change in the membrane potential followed by a rapid return to the
resting state. In nerve cells the action potential is the basis of transmitting nerve signals. The action potential is
caused by changes in the plasma membrane permeability (conductance) to sodium and potassium ions that is
different from the conductance responsible for the resting membrane potential. At rest the conductance of Na +
through the membrane is extremely low. However, if the neuron receives an electrical stimulus (of sufficient
magnitude) activation gates on voltage sensitive Na + channels will open. In contrast to K +, the concentration of
Na+ is extremely high on the outside relative to the inside of the cell, therefore opening of Na + specific channels
will cause a rapid influx of Na +, down both its concentration and electrical gradient causing the charge on the
membrane to suddenly become more positive, this is referred to as depolarization of the membrane. The
membrane potential will increase rapidly in response to the increased positive charge until it approaches the
equilibrium potential for the Na+ ion ( ENa = 67 mV) thus converting the potential energy established during the
resting membrane potential to actual work. It is important to note that depolarization occurs with minimal
changes in the overall concentration of Na + or K+. (Only one out of every 100,000 Na + ions needs to enter the
cell to produce a 100 mV change in potential).

Once activated, the inactivation gates on the voltage sensitive Na + channels close after a ~0.5 msec delay
resulting in Na+ channel inactivation (the Na+ channels have two gates, an activation gate and an inactivation
gate, both of which are stimulated by a threshold stimulus). Simultaneously, voltage regulated K + channels
open, resulting in a net efflux of potassium. This net efflux is in addition to the efflux resulting from K + leak
channels that are always open. The additional efflux of potassium in combination with the termination of Na +
influx reverses the initial depolarization and the membrane potential moves back towards the resting potential
(repolarization) and even beyond (hyperpolarization), after which the K+ voltage channels close and the
resting membrane is reestablished (the potassium channels have only one gate which is activated by
depolarization and inactivated by repolarization). The small depletions that occur in the K + and Na+
concentrations following each action potential are then reestablished by Na +/K+ ATPase. It is important to note
that in order for activation of the Na+ channels to occur there needs to be a stimulus of sufficient strength to
exceed the threshold value. For example, the threshold value for the squid giant axon is near -55 mV, while the
resting membrane potential is near -70 mV. If a stimulus is not sufficient to bring the membrane up to the
threshold value (-55 mV) then an action potential cannot be initiated. Although there is an observable
potential change at the site of stimulus, it does not result in an action potential and is referred to as a local or
graded potential. However, if the stimulus is sufficient to reach the threshold value, an action potential is
triggered. The action potential differs from the local potential in two important ways: (1) it is all-or-nothing
while size of the graded potential depends on the strength of the stimulus and (2) the action potential is
propagated down the entire length of the nerve.

Guide to Using HHsim

HHsim is a graphical simulation of a section of excitable neuronal membrane using the
HodgkinHuxley equations. It provides full access to the Hodgkin-Huxley parameters, membrane
parameters, stimulus parameters, and ion concentrations, and can be used for many sorts of
exercises.

Starting HHsim
If you've downloaded and installed the executable version of HHsim for Windows, you can start
HHsim via a shortcut on the desktop or an entry in the Start menu. Or you can go to the HHsim folder
and click on the icon for hhsim.exe.

Main Window
The main window consists of two plots. The large plot displays membrane voltage in red and external
stimuli (current pulses) in blue. The smaller plot displays three quantites as yellow, green, and cyan
lines. By default these are the Hodgkin-Huxley variables m, h, and n, but other information can be
selected, such as currents or conductances. The yellow, green, and cyan pulldown menus select the
variables to be plotted.

Click on the purple Stim1 or Stim2 buttons to inject a depolarizing or hyperpolarizing current stimulus.
Click on the Membrane, Channels, or Stimuli buttons to view and modify the simulation parameters.

Radio buttons at the top of the display will allow you to use three different modes: cursor, zoom and
pan. In cursor mode, clicking on any of the plot lines causes an elliptical cursor to appear, and the
value of the selected variable is displayed in the cursor subpanel in the bottom right corner of the
main window. Cursor control buttons move the cursor left or right, either by one timestep or to the
next local maximum or minimum of the graph. In zoom mode, left-clicking on a plot will zoom in;
Shiftleft click will Zoom out. Right-clicking will bring up more options. Clicking and dragging your
cursor in zoom mode will give a zoom box for a more controlled zoom. In pan mode, clicking and
dragging on a plot will allow you to pan in any axis direction.

The simulation runs whenever a stimulus is applied or a parameter value is changed. It stops when
the membrane voltage appears to have reached an asymptote. If it stops too soon, click the yellow
Nudge button to nudge the simulation along for a few more time steps. Click Run (green button) to
enter continuous run mode, and Stop (red button) to leave this mode.

The Clear button clears the display and resets the time, but does not affect any other simulation
parameters. The Recall button resets the Hodgkin-Huxley variables to a previously-stored state.
Initially this is the resting state.
HHsim Cursor Controls
The cursor provides a way to measure the value of any plotted simulation parameter, and find maxima
and minima. Clicking on any of the plot lines in the main window sets the cursor to that point.
The cursor is displayed as a colored lozenge matching the color of the plot variable being measured.
Setting the cursor also activates the cursor panel in the lower right corner.
In the figure above, the cursor is measuring the resting potential (red line in the upper plot) just before
an inhibitory stimulus was applied. The cursor panel in the lower right indicates that the membrane
voltage is -61.1 mV at 21.74 msec from the start of the simulation.
Moving the Cursor
The cursor can be moved to a new location by clicking there. The cursor can be hidden (and the

cursor panel deactivated) by clicking at any empty point in the plot windows, i.e., where there is no
line. The cursor can also be hidden by clicking the close box (marked "X") in the cursor panel. Once
the cursor is set at a point, it can be moved left or right using the cursor control buttons in the cursor
panel. The < and > buttons nudge the cursor one time-point to the left or right, respectively. The <<
and >> buttons move the cursor left or right to the nearest local maximum or minimum value. For
example, suppose we want to find the greatest amount of closure of the sodium inactivation gate (h)
after a spike. Start by clicking on the green line to activate the cursor:
We see that the value of the inactivation gate parameter, h, is 0.575 at 29.94 msec from the start of
the simulation. Next, click on the >> button to move the cursor right to the nearest extreme value:

The cursor panel shows that the local minimum value for h is 0.0703, and it occurs at 32.34 msec.
The var button is used to move the cursor vertically from one plot line to another, to change the
variable being measured at that point in time. For example, suppose we wanted to know the value of
the potassium activation gate (n) at the peak of a spike. To measure this, set the cursor on the red
line (membrane voltage) and use the << or >> button to find the peak. Then press the varbutton until
the cursor is on the cyan line (n), and read the value in the cursor panel.
Clicking on the Save button in the cursor panel stores the Hodgkin-Huxley values at the current
cursor position. The simulator can later be reset to these values by clicking on the Recall button. This
feature is useful for resetting the system to a known resting state after each of a series of
manipulations, so that fair comparisons can be made.
Membrane Window
The Membrane window provides for manipulation of internal and external ion concentrations, and
adjustment of a few membrane parameters.

Channels Window
The Channels window provides access to parameters for each of the active or passive channel types.
Channels can be temporarily disabled by clicking the associated toggle to the left of the channel
name.
Stimuli Window
The Stimuli window controls the parameters for two external stimuli, Stim1 and Stim2, each of which
consists of either a single current pulse or a sequence of two independently-adjustable pulses.

Drugs Window
The Drugs window allows application of any of three drugs: TTX, which inhibits the sodium current;
TEA, which inhibits the potassium current; and pronase, which eliminates sodium channel
inactivation. Any time a drug is being applied, the Drugs button in the main window will be yellow
instead of the usual gray.
 Manipulation of Resting Membrane Potentials

Exercise 1: Increase/decrease extracellular potassium

Based on information from the introduction, there are two factors that help determine the membrane
potential of a cell: concentration gradients of ions (mainly potassium) and conductance (or leakiness) of the ion
across the membrane. To illustrate how the RMP is determined by these factors, we will manipulate ion
concentrations both inside and outside the cell.

Return to the Membrane Window. Notice the ion concentrations. For reference, here are those figures;

[K+] outside: 20 mM [K+] inside: 400 mM

[Na+] outside: 440 mM [Na+] inside: 50 mM
[Cl+] outside: 560 mM [Cl+] inside: 52 mM

We want to be able to see the changes in Na+ and K+ permeability through these experiments, so go to the
Yellow, Green and Blue pull down menus as the bottom of the screen:

Pull down the yellow menu and change the selection to g_Na (uS). This is a quantitative measurement of how
permeable the membrane is at any given point to Na+.

Next, pull down the green menu and select g_K (uS) which is a measure of permeability for K+.

Finally, pull down the blue menu and choose “blank” so that our graph is not encumbered with other lines.

1. Click on the “Drugs” button at the top of the screen and apply 100% inhibition for TTX (tetrodotoxin).
2. Go back to the “Membrane” window and alter the extracellular potassium concentrations by raising the
mM values in 10 mM increments. Continue doing this incrementally to 100 mM.
4. Use the cursor option to view Vm values and write down the results
5. Return all values back to setup values by hitting “reset”
6. Now reduce the extracellular concentrations of K+ in 5 mM increments.
7. Use the cursor option to record Vm values and write down the results.

Questions (Exercise 1)

1. Do some research and explain the mechanism of TTX (tetrodotoxin).

- TTX is a sodium channel blocker. TTX allows control of the flow of sodium, so when the inhibition
rate is higher, the mV of the cell is higher.
2. Did you ever see an action potential? Why or Why not?
- Yes, when TTX was inhibited, nothing was there to inhibit the Na, which allowed the action
potential to greatly increase.
3. During this experiment, what happened to the RMP and why? (Hint: Consider the Nernst Equation)
- The RMP depolarized as we added more K+ and hyperpolarized when we reduced the amount of
K+. It did this slowly because TTX helped prevent an action potential.
4. What would be the physiological consequence of hyperkalemia or hypokalemia on cell membranes.
- Having either a high or low level of Potassium would cause the cell to hyperpolarize or depolarize,
and create a large action potential.

Exercise 2. Increase/decrease intracellular Potassium

1. In the “Membrane” window return all values to default settings by using the reset button. Sometimes
hitting reset will cause the program to crash. We don’t know why?? If this happens to you, just restart
the program and then “reset” from now on by using the arrows in the window to move the values back
to original.
2. Continue to leave the TTX on.
3. Raise the intracellular potassium concentration stepwise to 500 nM.
4. Lower the extracellular potassium concentration stepwise from 400 nM to 200 nM.
5. Use the cursor to measure Vm as these changes are made and write down the results

Questions (Exercise 2)

1. Explain the trends that you see with your results, what happened to the RMP and why?
- As we decrease the intercellular we saw very little change, that change being a slight decrease in
the mV, but when we changed the extracellular potassium the mV sharply dropped each time we
reduced the amount of potassium.
2. What appears to have a greater impact on the resting membrane potential, changing intracellular [K +] or
changing the extracellular [K+]? Explain why?
- Change in the extracellular K+ has a higher impact. This is because when there is an abundance of
K+ outside the cell it can continually be pumped into the cell, and changed the gradient more
drastically. Because of the increase of K+ outside, K+ inside was unable to leave the cell, but when
K+ was decreased in the ECF, K+ was able to leave the cell more freely.

Exercise 3. Increase/decrease extracellular sodium:

1. Return to default settings for the membrane window.

2. Continue to leave the TTX on.
3. Use the Clear button at the bottom of the screen a few times to get a clean start.
4. Raise the extracellular sodium concentrations incrementally to about 800 mM.
5. Lower the extracellular sodium concentrations incrementally to about 5 mM.

Questions (Exercise 3)
1. What happened to the RMP and why?
- Pretty much nothing. The RMP slightly went up, then bent back down. Changing the extracellular
sodium only slightly affects the RMP Because sodium does not flow through the cell, it doesn’t
have very much effect on the cell.
2. How do these changes compare to what happened when you altered extracellular potassium? How do
explain the differences?
- K+ is flowing throughout eh ECT and the ICT, so it can greatly change the RMP, while sodium is not.
Though sodium can affect pressure, it cannot change the gradient.

Exercise 4. Increase /decrease Intracellular sodium:

1. Return to default settings for the membrane window.

2. Continue to leave the TTX on.
3. Raise and lower the intracellular sodium concentrations incrementally.

Questions (Exercise 4)

1. What are your results? Explain what you see.

- Basically the effect of the IC sodium has minimal. We saw a very slight decrease in RMP as sodium
increased, and a slight increase as sodium decreased.

Exercise 5. Changes in potassium conductance:

Fix the TTX to be 0% inhibition in the Drugs window at the top of the screen. Open the Channels window.
Notice that you have the option to uncheck and change values for several channels. Gradually increase the
conductance for the passive gK (us) channel – this is the K+ leak channel.

1. You are no longer using TTX.

2. Incrementally increase and decrease the potassium leak channel conductance and record how this
changes the RMP.

Questions (Exercise 5)

1. What did you observe and why?

- Again, very little changed happened, just micro amounts of change was observed, because K+ already had
reached its gradient.

Exercise 6. Changes in sodium conductance:

1. Reset the channels window to original values.

2. Incrementally increase and decrease the gNa (US) channel – this changes how “leaky” the membrane is
to Na+.
 Questions (Exercise 6)

1. What did you observe and why?

- When we decreased the channel the RMB flat-lined and stayed constant, while when we barely raised past
the norm it set of an action potential pattern, which seemed to be unable to stabilize. This probably
happened because if sodium is able to freely flow into the cell, the cell must work hard to compensate for
the change in charge.

The Action Potential

Exercise 7. Effect of stimulus strength

Hit the “clear” button at the bottom of the screen several times to clear all tracings for a fresh new screen.
Open the “Stimuli” window by clicking the box at the top of the screen. You will notice that this screen allows
you to set values for two stim buttons. We will be using just the first stim button for this exercise. The first
horizontal slider should be set at 0 (this sets how much time elapses before the stim is applied). The vertical
slider is how strong the stimulus is (measured in a very small increment of amps). The second set of sliders is
to establish a second stimulus so that we can do experiments where we control how two stimuli occur in
succession (We do this in Experiment 10) . For this experiment, we only want one stimulus.

Start by trying a stimulus strength of 1 (vertical slider on stim window). Increase by increments of one and
notice when you get an action potential. To apply the stimulus that you have set up, use the button in the
bottom left hand side of the screen that is titled “Stim1”. It is probably purple in color.

Questions (Exercise 7)

2. What was the minimal stimulus strength to initiate an action potential?

- Anything above or below -7 to 4 caused an action potential
3. Did you notice that when you first got an action potential, you did not get one every time? But, when
the stimulus got stronger you did get one every time. What could explain this?
- When a stimulus isn’t a large change or shock to the system, the cell can easy counter act the
stimulus, while once a certain threshold is reached, the cell can no longer control what is
happening and loses control and causes and action potential.
Exercise 8. Effect of stimulus duration:

1. Hit the clear button at the bottom several times and return to a nice clean screen.
2. Use the stim window and set the stim strength to be 1 and the duration of this stim to be 1. Use these
stim settings and apply the stim with the “Stim 1” button and see if you get an action potential or a
graded potential.
3. Leave the stim strength at 1 and gradually increase the stim duration. Try to find the lowest duration
where you get an action potential.
4. Leave the duration at the lowest value that you could get at least one action potential and then continue
to increase the stim strength to the lowest value that you consistently get an action potential every time.
5. Now open the membrane window again and notice that you can change the membrane capacitance.
Raise the capacitance and then use the same stim button with the previous settings (duration from
number 3 above and stim strength from number 4 above).

Questions (Exercise 8)

1. What was the lowest stim duration where you could get an action potential? Did you get one every time
at this duration? Explain why the action potential was skipped some times.
- 2, and no we didn’t see an action potential every time. The action potential was skipped
sometimes because the stim was not strong enough, and only occasionally could through the cell
out of whack.
2. How did the action potentials change when you raised the capacitance. Explain what capacitance is on a
cell membrane and how capacitance affects the membrane to cause the results you found.
- When we increased the capacitance, action potentials stopped showing up. A cell’s capacitance
determines how quickly the membrane potential can adapt to a stimulus change. A higher
capacitance allows the cell to resist action potentials.

Exercise 9. Effects of various drugs tetrodotoxin (TTX). TEA and Scorpion Toxin

1. Make sure Capacitance is returned to it original value and then hit the clear button at the bottom several
times and return to a nice clean screen.
2. Open the Stimulus window and set the Stim 1 button back to a stim strength of 10 and a duration of 1
3. Open the Drugs window and apply 100% TTX. Use the Stim 1 button and see what happens.
4. Hit the clear button several times again for a fresh screen. Remove the TTX drug and use the stim button
to get a control tracing. Then open the drug window and apply TEA at 100% inhibition. Use the Stim 1
button a few times and see what happens.
4. Hit the clear button several times again for a fresh screen. Remove the TEA drug and use the stim button
to get a control tracing. Then open the drug window and apply Pronase. Use the Stim 1 button a few
times and see what happens.

Questions (Exercise 9)

1. Explain how TTX affects the action potential (don’t just describe the tracings but explain how the
differences occurred compared to the control).
- The control run resulted in an action potential, so TTX prevented an action potential from
happening.
2. Explain how TEA affects the action potential (don’t just describe the tracings but explain how the
differences occurred compared to the control).
 - The control resulted in an action potential, and the TEA drug caused an action potential the first
time, then left the RMP at around 15 mV, instead of the regular -70 mV. This happened probably
because the sodium gates get closed from the drug, and K+ was unable to flow out of the cell.
3. Explain how Pronase affects the action potential (don’t just describe the tracings but explain how the
differences occurred compared to the control).
- The control resulted in an action potential, and the Pronase initially caused an action potential,
then kept the RMP very constant at around 30 mV instead of the regular -70 mV. This happened
probably because the sodium gates get closed from the drug, and K+ was unable to flow out of the
cell.

Exercise 10. Determination of refractory periods

Nerves and other excitable cells very seldom communicate using only a single action potential. Instead, “trains”
of action potentials are used to carry specific messages. The duration of the action potential trains and the
frequency of action potentials per unit of time, are important components of neural communication. Consider
the pacing of the human heart during extreme activity. Each beat is generated by action potentials, and the
rapid heart beat must necessitate a fairly high frequency of AP's to function. The flight muscles of an insect is
an even more extreme example. Ultimately, there is an upper limit to action potential frequency. The goal of
this exercise is to experimentally determine what factors control how close in time individual action potentials
can occur with respect to each other. In other words, what is the maximum number of action potentials per
unit of time?

Question (Exercise 10)

1. Now that you are familiar with the program, try to design and experiment to discover the following:
 How long is absolute refractory period
- Because absolute refractory period is about 1-2 milliseconds, we could set the stimulus to about 5
seconds and increase the strength to the max setting, then run the stimuli with a variety of drugs
added, and see what it takes to ensure a second action potential does not occur. Then measure
how much time went by between the time we added the stimuli and the RMP stabilized without a
second action potential occurring. Thus giving us the absolute refractory period.

Exercise 11. Effects of temperature on action potentials

Even mammals, which have the ability to achieve a relatively constant body temperature regardless of
external temperature, experience some differences in body temperature. Nervous tissue, indeed all
excitable tissue, is particularly sensitive to changes in temperature.

Procedures (Exercise 11)

1. Return all values to default settings.
2. Set up Stim 1 to have a strength of 6 and a duration of 1
3. Establish a control tracing.
4. Now open the membrane window and find the temperature control. Raise the temperature
incrementally and note that tracings after you use the Stim button. Do this up to about 20 degreees.

Questions (Exercise 11)

1. How does the action potential tracing change as the temperature increases?

- After increasing the temperature to over 14 degrees, action potentials no longer occurred.