Virtual Lab: Action Potentials

Please work through the instructions provided. At the very bottom of the page, there is a worksheet for you to download, complete, and submit for a grade. 

In this exercise you will run a model neuron to explore the concepts of action potential threshold, refractory period and the role of Na in generating action potentials.  First, you will need to install and download the simulation program MetaNeuron by clicking here. If you have any issues you can consult the manual here. 

Once you have the program running, in the Lesson menu, select Lesson 4: Action Potential. This lesson simulates what you would observe if you recorded the voltage inside a cell with one microelectrode and injected current into the cell through a second microelectrode to depolarize the membrane potential. (If you need to review the concept of intracellular recordings, go back and re-read the current module.) 

You will see several boxes with parameters that can be adjusted, and in the bottom box you will see two graphs. The top graph shows an action potential in yellow, the green line represents the equilibrium potential for Na and the blue line thew equilibrium potential for K (if you need to review the concepts of equilibrium potentials go back to the previous lesson). The bottom graph shows a red line, the red line represents the current you are experimentally injecting into your cell through your electrode. Notice that at 0.5 sec there is a little blip in the graph, this is the current you are injecting. You can see that after you inject the current, the membrane potential (yellow) is depolarized eventually triggering an action potential.

Part 1. Action potential threshold

Remember that if the membrane potential is depolarized beyond a certain point, known as threshold, then the cell will generate an action potential. Thus, if you inject a large amount of current into the cell, the faster the membrane potential will depolarize and the more likely the neuron is to fire an action potential. In contrast, with a smaller amount of current, them membrane potential will take longer to reach threshold, or maybe not even reach it at all. Let's examine the relationship between membrane depolarization and threshold.

First let's measure the action potential on the screen. You will make three measurements: the inflection point, the peak and the undershoot. The inflection point is when the action potential is triggered, the peak is the top of the action potential and the undershoot is the lowest point after the peak.

If you click and drag on the yellow graph you will see a measurement cursor. Use this cursor to measure the membrane potential (in millivolts, mV) and the time (in milliseconds, ms) for the inflection point, peak and undershoot, and write these down in a table:

Now let's see what happens if we inject increasing amounts of current to the cell.  

Find the window that says "Stimulus 1" and set the value of Amplitude to 25 and press enter (note the units are in micro amperes or µA, which are the units for electrical current). This reduces the amount current that you are injecting into your cell. You can see the reduction in current injected because the blip in the red line gets smaller.

What happens to the action potential? 

Why do you think this happens?

Now increase the amplitude of "Stimulus 1" to 45, 55, 65, 75, 100 and 200. For each value, note what happens to the action potential:

Why do you think this happens?

What is the minimum amount of current you need to inject into your cell to trigger an action potential?

Set the stimulus amplitude to 200 and perform the same set of measurements you did earlier. and enter these in a table:

How do the new values compare to the old values? What changed?

Threshold is the membrane potential value at which a depolarization triggers an action potential, and can be approximated by measuring the membrane potential at the inflection point. As you increase the amplitude of Stimulus 1, does the action potential threshold change? Why or why not?

The take home message here is that the more current you inject into your cell, the more the membrane potential is depolarized and the faster it reaches action potential threshold. 

Part 2: Refractory Period

After a neuron fires an action potential, it is followed by an absolute refractory period during which it cannot generate another action potential and a relative refractory period, during which it is possible but more difficult to generate more action potentials. Let's measure the relative and absolute refractory periods. First find the box that says graph and set the sweep duration to 10 ms. Set the amplitude of Stimulus 1 to 200. In the box that says Stimulus 2, click the On check box, set the delay to 1 ms and amplitude to 200. Notice that the red line now has two blips, each representing a current injection into the cell. 

Do both stimuli trigger an action potential?

Now set the delay value of stimulus 2 to 2, 3 and 4 ms. What is the shortest interval (delay) between stimuli that gives you a second action potential? 

This is showing you the refractory period. Let's find out if this is the absolute or relative refractory period. Set the delay to 2 ms. If this is the relative refractory period, we should still be able to evoke an action potential if we depolarize the cell more. Increase the amplitude of the second stimulus to 250, 300, 350, 400.

Do any of these values evoke a second action potential?

What is different between the second and the first action potential?

Now let's look at the absolute refractory period. Keep the amplitude of stimulus 2 at 400 and set the delay value of stimulus 2 to 1 ms. 

What happened to the second action potential?

Try increasing the amplitude of stimulus 2 as high as you want. Can you evoke an action potential?

This is the absolute refractory period because no matter how much current you inject, you cannot evoke another action potential so soon after the first one.

Part 3: Sodium and the action potential. 

When you reach threshold, you increase the chances that voltage gated Na channels open up, causing Na ions to rush into the cell and change the membrane potential toward the Na equilibrium potential (ENa). This is why the peak of the action potential is close to ENa. 

What would happen if ENa were less positive; would you predict that the peak of the action potential to be shorter or taller?

 Turn off Stimulus 2, set the amplitude of Stimulus 1 to 150. Measure and enter in the table the time and membrane potential values for the inflection point, peak and underhsoot:

Now let's change ENa. In the box labeled Membrane Parameters, set the Na equilibrium potnetial to 0 mV and perform the same measurements:

What is different? Why you think this is?

What changes could you make to the Na concentration in the extracellular solution to cause ENa to become less positive?

Bonus: The rising phase action potential is mediated by Na entry into the neuron, the falling phase is due to K ions leaving the cell. In the box labeled Membrane Parameters, set the Na equilibrium potential to 50 mV and the K equilibrium potential to -95 mV. 

What happens to the undershoot?

Why?

 Instructions:

Option 1:

1. Download the following worksheet: ActionPotentialWorksheet

2. Save the worksheet on your computer and rename it: LastName.FirstName.Membrane.doc

3. Keep the worksheet open and toggle back and forth between the virtual lab and worksheet, making sure to answer each question based on the simulator. Remember to save regularly.

Note: To add images (.jpg, .png, .tiff, etc.) to a Word document, click 'Insert' and choose photo. For more detailed instructions from Microsoft, click here.  

4. Click 'Submit Assignment' and upload your completed worksheet on this assignment page. 

Option 2:

1. Open 'Google Docs'. (You must have a google account first. To set up a Google account, click here.)

2. Create a new 'Document'.

3. Cut and paste the assignment questions found above into your new Google doc. and enter your answers. 

Note: To add images (.jpg, .png, .tiff) to a Google document, click 'Insert' and choose photo. For more detailed instructions from Google, click here. 

4. When you are finished with your work, use the 'File' menu to rename the document as LastName.FirstName.Membrane and download it as a .pdf. 

5.  Click 'Submit Assignment' and upload your completed worksheet on this assignment page as .pdf.