Biological Sciences 300/301, Smith College | Neurophysiology
Lab 5: Computer Simulations of Membrane Potentials
http://www.science.smith.edu/departments/NeuroSci/courses/bio330/labs/L5sims.html
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This week's laboratory will involve computer simulations of experiments on action potentials. We will use the NEURALSIM package on the iMacs in our lab, which are in the folder "OS 9 Applications." There are two applications in the NEURALSIM package: apSim, which reproduces the properties that Hodgkin and Huxley worked out for the squid giant axon; and pspSim, which simulates excitatory and inhibitory postsynaptic potentials. Tody we will use only the action potential simulation, apSim.
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A note on screen capture: The simulation programs do not incorporate commands to print or save the graphs that you plot, but you can accomplish this through the Mac's own routines. Pressing Since it is easy to forget what each screen picture includes, it is good practice to change the name of each file from "Picture X" to something more informative. In addition, to keep your pictures together, create a new folder on the desktop with the names of you and your lab partner(s) as its title, and drag all screen pictures into it. (Ask a Mac friend in the class if you need help in doing this.) Finally, you can easily paste screen pictures into documents using AppleWorks, which is available on our iMacs. If this interests you, see the brief instructions at the end of this lab. |
-SHIFT-4
(together, briefly) changes the mouse cursor into a
cross-hair cursor. Place this cursor in the upper
left corner of any figure that you wish to copy,
and drag down to the opposite corner. You will see
a rectangle that defines the region that will be
copied. Release the mouse button, and you will hear
a click. A "picture" has been taken of the region
in the rectangle. It appears as a file in the
iMac's top-level folder labelled "Picture 1",
"Picture 2," etc. Double-clicking a picture will
open it in SimpleText, from which it can be
printed. You can capture more than one window in a
single picture by grouping the windows together and
then capturing that region of the screen.
Click the apSim icon in the dock to launch the program
apSim
1. Run the apSim DEMO program.
Launch the apSim program. You will see a choice to RUN, DEMO or Quit. Click the DEMO button to launch a demonstration of intracellular recording from a non-clamped squid axon in which spikes are electrically stimulated. In addition to the intracellular recording, there are plots of the conductances that Hodgkin and Huxley calculated. Read the text boxes (which you should drag to the bottom of the screen) and follow the instructions.
At the end of the DEMO program three windows will be open. Reopen these windows if you need to ("Plot" menu) for later sections of this lab. Two of the windows are already familiar to us: the main window that shows the action potential, and the window that plots membrane conductances for Na and K channels.
The third window, Channel Gates, is new. It shows the state (open = up, closed = down) of the Na-channel activation gates (red) and inactivation gates (green). The K-channel activation gates (blue) are also shown. These gates are given the labels m, h, and n, respectively, which are the variables that Hodgkin and Huxley used in their original equations.
The sodium inactivation gate (green) may be slightly counter-intuitive. Like the other gates, it is plotted with open = upward on the graph. Since most of the inactivation gates are open at rest, the graph starts high and then dips down to indicate that inactivation gates close (green curve) after activation gates open (red curve). As the spike repolarizes, the inactivation gates slowly open again. (The inactivation gates are the main cause of the refractory period -- it is not until those gates have reopened that the threshold for a second spike returns to normal.) Also note that the total sodium conductance shown in the Membrane Conductances plot (GNa, red curve) reflects the state of both the activation and inactivation gates. During a spike, sodium conductance decreases rapidly because inactivation gates close. The activation gates remain open longer than the total conductance does.
2. Run the Threshold demonstration.
When the main demo is over, you can see two more demonstrations by going to the Experiments menu. Select "Threshold" and watch the demonstration.
When it is concluded, you will be left with the same set of windows. Erase the existing curves. Press Run to stimulate the axon with the default stimulus strength. Then drag the stimulus slider slightly to the right to increase the stimulus strength. (This is equivalent to turning up the stimulus voltage on our lab stimulators.) Press Run again and note the time course of the spike compared to the previous spike. Explore several settings of the stimulus strength between the default and the maximum. (You can re-establish the default setting at any time by selecting "Reset all params" from the Params [parameters] menu.
When you have a sense of what is going to happen, erase the windows and create a tidy family of three or four curves. Arrange the windows on the screen (if you have not already done so), and capture a screen picture of them. Question: Why does a bigger stimulus make the spike reach its peak sooner?
3. Run the Refractory Period demonstration.
Select "Refractory Period" from the Experiments menu and watch a demo of twin-pulse stimulation. In the squid giant axon, is the second spike's amplitude smaller during the refractory period of the first spike?
Question: Which ion's conductance is changing the most and seems to account for the decrease in the second spike's size? To investigate this, open the Membrane Conductances window ("Plot" menu) and re-run the refractory period demonstration.
If you wish to explore this phenomenon more closely, keep the twin-pulse setting ("Mode" menu/No. Pulses/2 Pulses). In the main APSIM window, click on the "Pulse #" control to cycle through the sliders for the strength and duration for Pulse 1 and Pulse 2, and the interpulse interval. Examine the conductances for Na and K in the Membrane Conductances window as you change the interpulse interval.
4. Explore the effect of increasing external K+ concentration.
From an earlier lab, we know that increasing external potassium leads to a depolarized resting potential. We can explore here what effect this will have on the action potential. Reset all parameters and re-establish single pulses (Mode/# Pulses). From the Params menu, select "Ion concentrations." In the dialog box that appears, click the button for "Potassium." You can now adjust the external concentration for potassium on the upper control. Run one stimulus to get a normal spike with the default concentration, 10 mM. Then, using the right arrow (not the slider), increase the external potassium concentration to 15 mM and stimulate another spike. How has the resting potential changed? Has this had any effect on the magnitude (height) of the conductances? Confirm your observations by increasing external K+ to 20 mM and stimulating a third spike.
NOTE: The simulation becomes unpredictable if you raise the external K+ beyond 20 mM.
Question: Why does increasing external potassium cause the spike to change? [Hint: if you are not sure why, open the "Channel Gates" window (Plots menu) and repeat the experiment.]
5. Explore the effect of the number of channels in the membrane.
The channel density (the total number of channels per area of membrane) is reflected in the Maximal Conductances setting (Params menu). Test what happens to the spike as you decrease the number of Na channels, G(Na), and K channels, G(K). The units of conductance are milliSiemens. Decreasing the maximal conductance is equivalent to having fewer channels of that type in the membrane.
6. Simulation mini-project.
With your partner, select an aspect of action potentials to explore further. Try to choose something that the simulation is particularly good at, such as an observation of the conductances accompanying some manipulation. Carry out a series of tests on the question you selected.
A copy of the NeuralSim manual is in your NeuralSim folder if you need to learn more about the program's functions.
Capture screen pictures, and compile a one-page "poster" of your mini-project. Your poster should state what the question was, show captioned examples of your experimental records, and have a conclusion that says what you found. Put everyone's full names on the poster, and tack it to the bulletin board. When the entire class is ready, we'll have brief presentations of each group's project.
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Using AppleWorks to make posters: • Launch AppleWorks by clicking its
icon • Make the page appear smaller if it
is not all visible on the screen by clicking on the
small hills • Insert your pictures by dragging their file icons from your folder to the new page. The pictures will open on the page. [If dragging does not work, double-click the icon to open the picture in its native application, Select all (Edit menu), Copy (Edit menu), reactivate the AppleWorks page by clicking it, and Paste the picture (Edit menu). You can close the window with the original picture in it.] • You can move pictures by selecting
the arrow tool • Insert blocks of text by selecting
the text tool • Lock pictures and text in place after you have arranged them on the page. Select each one (or all together if you prefer) and choose "Lock" from the Arrange menu. The black handles at the corners of each locked object will turn gray. You can later unlock any selected item if you need to move it. • Print your page. Save its file in the same folder as your pictures. |
in the dock. Select Drawing in the dialog
box. A blank page with grid lines will open.
at the bottom left of the window. Seeing the whole
page is useful in arranging your pictures.
,
clicking on a picture to activate it, and dragging
the picture to its new location. You can
resize pictures by dragging one corner, or
by using the "Scale by Percent..." option in the
Arrange menu.
,
dragging a rectangle where you want the text to be,
and typing your text. Magnify the page to see what
you are typing by clicking the large hills
at the bottom left of the window. The text's font,
size and style can be set from menus. Text blocks
can be moved with the arrow tool, just like
pictures.Appendix: Screen shots and AppleWorks posters
Updated: February 23, 2009
© 2003, 2004, 2009 by Richard F. Olivo. Permission is granted to non-profit educational institutions to reproduce or adapt this Web page for internal use provided that the original source and copyright are acknowledged.