Neuronal Physiology Modeling
Neurobiology, BI 378
This week you will be using a computer program (Neuron Model) that allows you to simulate a series of physiological experiments on an individual neuron. The program allows you to stimulate the neuron electrically and to observe the induced voltage changes across the cell membrane on the computer screen. In addition you can also perform some of the classic voltage clamp experiments performed by Hodgkin and Huxley back in the 1950's. They received a Nobel Prize for this work and the results are still valid today. The reactions of the neuron are based ("modeled") on the Hodgkin-Huxley model as well as the Nernst and Goldman Equation.
I. The Ionic Basis of the Resting Potential
The presence of a potential difference (voltage) between the inside and the outside of axons and neurons at rest (the resting potential) is essential for the generation of electrical activity. Consequently, understanding the mechanisms of the generation of the resting potential is also fundamental to the understanding the mechanisms of electrophysiological excitability of axons and neurons. The first part of the exercise allows you to perform experiments on the cellular mechanisms of the generation and maintenance of the resting potential.
Procedure:
a) Double click on the “Applications” folder then the “NatSci Applications” folder then the “Animal Physiology” then the “Neuron” folder then the “C-Clamp” folder and then the application icon labeled "C-Clamp".
b) Under file pull down and release on "Open", then double click on "rest.ccs".
Under "Run", pull down and release on "begin".
- This simulation demonstrates the resting potential of the squid giant axon. Note that the resting potential is -65mV. This resting potential is determined by the "leakage" (permeabilities), at rest, of ions across the membrane. In this exercise I want you to investigate the importance of two ions (K+ and Na+ ) to the resting potential.
1) Pull down under "parameters" and release on "conductances". Write down the normal values for "pNa+ leak" and "pK+ leak" ("p" stands for "permeability"). Change the pNa+ leak from 0.06 to 0. Now pull down under "Run" and release on "overlay". Compare the results to those from above. The cell membrane, when it is leaky to only K+, will become more negative.
•••Explain why this might be so in a sentence or two. Be sure to consider the Nernst and Goldman equations.
2) Now change the pNa+ leak back to 0.06 and the pK+ leak to 0 from 0.1. Pull down on Run and release on overlay. Compare the results.
•••Explain this result in a sentence or two. Be sure to consider the Nernst and Goldman equations
3) •••Now explain how each of these two ions contribute to the resting potential. (HINT - numbers are important here; think about the Goldman equation).
Incorporate the three bulletted (•••) explanations above into a well constructed paragraph (see below for further instructions).
II. The Effects of Variable Current Injections on Membrane Potential
Neurons and their axons actively generate and propagate action potentials through their membrane, allowing communication from one part of the cell to another and, subsequent to transmitter release, communicate to other cells. The mechanisms of generation of action potentials are essential to the understanding of the electrophysiological properties of excitable cells.
Procedure:
Close the previous window (click in box in upper right corner). Do not save changes.
Under "file" pull down and release on "Open", then double click on "Active.ccs".
1) Under "Run", pull down and release on "begin". The bottom trace shows you how much current you injected and over what period of time. The top trace shows how the neuron responds to this injected current.
•••Describe what you observe (be sure to note the X and Y scales).
Procedure (cont.):
2) Now change the amount of stimulation given the neuron by:
Under "Parameters", pull/release on "Protocol".
Change Injected Current from 2.0 to 1.0 nA, hit "OK"
Under "Run", pull/release on "Overlay".
•••Now what do you observe? Explain the basis for these differences.
3) Now increase the injected current to 3.8 nA (use above steps).
•••What do you now observe? Explain.
Incorporate the three bulletted (•••) explanations above into another well constructed paragraph (see below for further instructions).
III. The Effects of Different Ions on Action Potential Generation
To investigate which ions are important in the generation of the action potential, you can change the concentration of different ions in the artificial "seawater"(which bathes the outside of the axon) one at a time and observe the effects. Among the various ions, there are significant amounts of Na+, K+, Ca++, Mg++, and Cl-.
Procedure:
- You need to take some notes on what you observe here for each ion.
- Under "Run" pull down and release on "begin".
- Now, in the "Parameters", Ions menu change the extrracellular concentration of Mg++ from 1.0 to 0.1 mM.
- Run the experiment and compare to your last experiment (use "begin" each time here).
- Now do the same with the internal concentrations of each ion and record your observations.
You will quickly recognize that the two ions that are of particular importance are Na+ and K+.
The Effects of [Na+]o: When you reduce Na+ to 0.1 mM, you notice two effects. 1) The membrane potential of the cell becomes significantly more negative with the reduction of [Na+]o. This finding indicates that the passive influx of Na+ into the cell normally contributes to the resting potential (as you observed above). This causes a significant amount of hyperpolarization which you will need to compensate for in order to observe the effects of changing [Na+]o on the action potential only. To compensate for the hyperpolarization, you must inject current through your microelectrode to move the membrane potential back to -65 mV. To do this in the computer model, go to the "Parameters" pull down menu and release on "Protocol". Now change the "Base Current" from 0 to 2.25 nA (this will inject a constant base current into the cell and depolarize the cell back to -65 mV). Again run your program and compare it to the results that you get with [Na+]o set to 0.1 mM and without any base current. Now you notice that action potentials are blocked, indicating that external Na+ is important for the generation of action potentials. Even if you double the amount of stimulation current to 4 nA, the cell will not generate action potentials, thus confirming that Na is critical for the action potential.
The Effects of [K+]o: Now lets investigate K+ ions. First reload and run active.ccs. Now reduce [K+]o to 0.1mM, under the Parameters, Ions menu. Now run another simulation using overlay. Again you notice that this manipulation results in the resting potential becoming substantially more hyperpolarized, indication that a constant leak of K+ across the membrane helps to determine the resting potential as you discovered above. To compensate for this resting potential change, you need to inject a steady current into the cell by changing the "Base Current" (under parameters, protocol menu) to 0.8 nA and again choosing Begin in the Run menu. Now you see that, unlike reducing [Na+]o, reduction of [K+]o does not abolish the action potential, although you notice that the hyperpolarizing potentials that occur after each action potential are now larger than before. This can be observed directly by reloading active.ccs and choosing overlay in the Run menu. The increase in hyperpolarization occurring after each action potential with reduced[K+]o should suggest to you that the movement of K+ across the membrane must be important for this. In fact, you now consider two findings: reduction of [K+]o results in an increase in the hyperpolarization after the action potential, and this hyperpolarization now undershoots baseline membrane potential. Together these events suggest to you that K+ is in higher concentration inside than out, and that K+ ions move down this concentration gradient during the repolarizing phase of an action potential. To test this possibility, you need to also change the intracellular concentration of K+ (next experiment)
••• Which ions are most important for the generation of the action potential?
IV. The Effects of Intracellular K+ (the "Toothpaste Experiment")
In 1962, Baker, Hodgkin, and Shaw took advantage of the large size of the squid Giant axon to squeeze out the axoplasm, as if the axon were a tube of toothpaste, and replace the contents with an artificial one containing different ion concentrations. This enabled them to test the importance of different ion concentrations inside the axon. You can replicate their experiments by loading and running K_intra.ccs. You can reduce [K+]i by changing this value in the model to 0.1 mM in the Parameters, Ions menu. Now choose Begin, and you find that the membrane potential depolarizes and does not repolarize.
••• What do these results suggest? (HINT - First remind yourself of what you changed to get these results).
V. Measurement of Ionic Currents During Action Potentials: The Voltage Clamp
So now you have found that Na+ and K+ --- but not Cl-, Mg++, or Ca++ --- are critical to the generation of action potentials in the squid axon and you reason that Na+ moves into the cell to depolarize it and K+ moves out to repolarize it. However you have a number of questions: Why is there generation of action potentials at all? Why isn't the flow of Na+ into the cell just compensated by the flow of K+ out of the cell? Is the K+ current perhaps delayed?
To investigate these questions Hodgkin and Huxley devised a method known as the voltage clamp method. This method allows for the voltage across the membrane to be held constant so that one can measure the current flow (one can do this because of the relationship between voltage (V), current (I) and resistance (R): V=IR). Hodgkin and Huxley again made use of the large size of the squid axon (they had to since the electronics of that day were much larger) to measure currents during an action potential. By measuring currents they could deduce what was happening in the membrane since the currents carried by the ions have to flow through channels that either open or close.
To replicate Hodgkin and Huxley's experiments, first quit from the C-Clamp program by choosing quit from the file menu. To perform Voltage Clamp experiments, double click on the V-Clamp icon in the V-Clamp files folder. Open Na_K.vcs by choosing "open" under the "file" menu and opening the V-Clamp folder. Now choose "Begin" in the run menu. In this experiment you have moved the membrane potential from -100 mV to 0 mV and measured the amount of current you had to inject into the axon in order to keep the membrane constant (which in this system is equal to the current flowing through the channels in the membrane). Notice that there is first an inward flowing current (downward deflection) followed by an outward movement of ions (upward deflection).
••• What ions are flowing? When are they flowing? Be sure that you can explain this figure.
Assignment: Answer each of the above bulletted questions (•••) in a paragraph. Be sure that each paragraph has a topic (introductory) sentence and that subsequent sentences pertain directly to the topic. As with every assignment for Neurobiology, use a word processor, 12 pt times font and double space. The paragraphs are due by the beginning of lab next week. LATE PAPERS WILL NOT BE ACCEPTED. I AM willing to read over early papers.