Laboratory 2: Neurosim: Membrane potentials and Action potentials INTRODUCTION NeuroSim 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 NeuroSim program 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 the squid species Loligo forbesi (adult squid weight 3.5 lb, adult squid length 14 in). Because of the large diameter of their axons (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 the inside and the outside the nerve cell. The internal electrode measured a potential of negative 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. 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 cell 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 develop a negative charge 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 electrochemical potential 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 plasma membrane permeabilities (conductance) for sodium and potassium ions that are different from the conductances 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 gradients 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 (voltage-­‐gated 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). Simultaneous with the closing of the inactivation gates on the Na+ channels, voltage-­‐gated 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. Understanding Neurosim To begin this laboratory, open the Neurosim program and click on the HH simulation. The first step is to set up the experimental conditions. In order to experimentally manipulate the resting membrane potential without generating an action potential, we must "turn off" the voltage-­‐activated Na+ channels. This is accomplished by the addition of the drug tetrodotoxin (TTX), which blocks the activation gates on the voltage activated Na+ channels. Click on the box next to TTX in the drug window. Next, turn off the pre-­‐set stimulus by changing the amps (in the current clamp window) to 0.0 for both the first and second stimulations. Click on the Results window icon on the top of the screen. The results window is currently set up to record several different tracings. Click on the Traces button. Notice that each display is generated when an "x" is placed in the box labeled "show graph". Remove the "x" from the stimulus and current boxes by clicking once on the "x". Click once on the Model button. This shows a model of the voltage activated sodium and potassium channels, and indicates where drugs bind. Finally, change the X-­‐axis scale (time) to 100 ms by left clicking on the 25 ms label on the X axis. Click once on the Start button on the Results screen. The observed tracing represents the RMP of the cell. What is the value of the RMP? To measure the RMP, click on the Measure button, move the cross hair to any point on the graph, and click once. With the cross hair you can measure voltage (vertical scale) and time (horizontal scale). These data are recorded in a box that will appear on the screen. This is how you collect and interpret data during the lab. 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. For exercises 1-­‐4 keep the TTX turned on. Return to the Setup window by clicking once on it (or use the icon on the top of the screen). Notice that the green cell has ion concentrations indicated for the extracellular and intracellular environments. For reference, here are the control (default) settings; [K+] outside: 10 mM [K+] inside: 302 mM [Na+] outside: 418 mM [Na+] inside: 64 mM 1. Click the start button to generate a control tracing. 2. Alter the extracellular potassium concentration from 10 to 20 mM by simply clicking on the values and retyping new ones. 3. Continue doing this in increments of 10 until you reach 100 mM. 4. Record the results 5. Return all values back to control values 6. Reduce the extracellular concentrations of K+ from 10 mM to 5 mM. 7. Record the results
Questions (Exercise 1) What happened to the RMP? Why? (Hint: Consider the Nearnst Equation)
When TTX present it blocks na channel. , and as extra cellular potassium concentration increases RMP becomes more positive. Have greater driving force according to the nearnst equation. Decrease K membrane becomes more negative. 1. What would be the physiological consequence of increased (hyperkalemia) or decreased (hypokalemia) extracellular K+ concentrations? Increase respiration decrease in contraction. 2. Exercise 2. Increase/decrease intracellular Potassium 1. Return all values to default settings. 2. Raise the intracellular potassium concentration stepwise in 20 mM increments from 302 mM to
402 mM. 3. Lower the intracellular potassium concentration stepwise in 20 mM increments from 302 nM to 202 nM. Questions (Exercise 2) 1. Explain the results; what happened to the RMP and why?
Intracellular potassium increase makes rmp more negative. Decreasing intracellular potassium will make rmp more positive. 2. What had a greater impact on the resting membrane potential, changing intracellular [K+] or changing the extracellular [K+]? Why?
Extracellular has greater impact. Nerst equation. Small change in outside makes bigger change in inside. Change in ratio between inside and outside concentration. Exercise 3. Increase/decrease extracellular sodium: 1. Return all values to default settings. 2. Click the start button to generate a control tracing 3. Raise the extracellular sodium concentrations incrementally by 10 mM but do not exceed 500 mM. Beyond 500 mM the system becomes artificial and the results become difficult to explain. 4. Lower the extracellular sodium concentrations incrementally by 10 mM to 200 mM. Questions (Exercise 3) 1. What happened to the RMP and why?

Increase in extracellular sodium does not make any change because of TTX. It blocks channel that allows na to across membrane. 2. How do these changes compare to what happened when you altered extracellular potassium? How do explain the differences?
Extra cellular potassium has channels that allows to go through membrane. Exercise 4. Increase /decrease Intracellular sodium: 1. Return all values to default settings. 2. Click the start button to generate a control tracing. 3. Raise and lower the intracellular sodium concentrations incrementally. Questions (Exercise 4) 1. Describe and explain the results?

It should not change rmp much because channels are still blocked with TTX.

Exercise 5. Changes in potassium conductance: Return all values to default settings and turn off TTX. On the setup window, find the box in the lower right corner that indicates Membrane properties. Capacitance is a measure of how much current must flow (in this case how many ions must move) to produce a change in the potential. A greater capacitance means it takes more current to generate potential difference changes. (Changing capacitance will have no effect on RMP). The leak conductance gives an indication of overall ionic leakiness, or non-­‐specific ion movement across the membrane. Maximum Na+ conductance and maximum K+ conductance indicate the maximum conductance that can occur for each ion. In other words, it is a measure of the number of channels available for each ion including both voltage and leak channels. Record the default conductance settings so that when asked to return to the default state you can easily make the change. 1. Generate a control tracing. 2. Gradually increase the maximum potassium conductance in 10 mS increments and record the changes in RMP. 3. Return all values and setting to control levels and gradually decrease the maximum K+ conductance in 10 mS increments and record the changes in RMP. Questions (Exercise 5) 1. What did you observe? Explain the results?

Rmp becomes more negative. Because it increases leakiness. More holes to goes through. It stayed but when it decreased at a certain point, action potential was fired. It depolarized enough to fire it. More potassium channel than sodium. It has to be relatvley hight. To fire p. 198 it fires.

Exercise 6. Changes in sodium conductance: 1. Return all values to default settings. 2. Obtain a control tracing. 3. Alter the maximum sodium conductance by increasing gradually from 120 to 200 in 20 mS increments. 4. Return all values and setting to control levels. 5. Gradually decrease the maximum Na+ conductance from 120 to 40 in 20 mS increments. Questions (Exercise 6) 1. What happened to RMP? What do these two experiments (exercise 5 and 6) tell you about the relative conductance of these ions at rest?
Did not change much. Because action potential caused mostly by potassium. Potassium has relatively more channels than sodiums. Thus relative increase must be greater tofire action potential.

2. What is the physiological relevance of decreased K+ conductance and increased Na+ conductance? (For example, are there any physiological functions that could benefit from the response you saw when the Na+ conductance was increased?) The Action Potential Exercise 7. Effect of stimulus strength
Return all values and settings to default conditions. In the results window, turn on the voltage, stimulus and conductance tracings and change the x-­‐axis to 25 msec. *Note, you will not be able to change the axes unless all tracings are cleared. 1. In the setup window, gradually increase the stimulus strength for Pulse 1 in 5μA increments beginning at 0μA (current clamp window, pulse 1). Leave Pulse 2 at 0μA throughout the exercise. 2. Leave the duration of the impulse at the default setting (0.25 msec). 3. Observe the tracings for each trial.

Questions (Exercise 7) 1. What is the underlying physiological basis for threshold.
Preventing to much signals. Prevents too often production of action potential. Prote

2. At what point was an action potential generated (threshold)?
60mv

Exercise 8. Effect of stimulus duration: 1. Return to the setup window and set the stimulus strength 35μA. Again we will be using only
Pulse 1, leave Pulse 2 at 0. 2. Obtain a control tracing. 3. Decrease the duration of the stimulus (default is 0.25 msec) in 0.05 msec increments. 4. Record the minimum duration necessary to elicit an action potential. Once you hit the duration at which there is no action potential refine you results by altering the duration in 0.01 msec increments. Record the strength and duration.
35amp, 0.19 duration 5. Decrease the duration by another 0.05 msec and then gradually increase the stimulus strength until an action potential is again generated. Record the strength and duration for this point. 6. Continue lowering the duration and increasing the strength until you reach a duration at which it is not possible to generate and action potential regardless of the stimulus strength. Record each data point as you go. 7. Reset strength and duration to the conditions recorded in step 4 above. 8. Decrease the strength of the stimulus by 5 μA and gradually increase the duration until an action potential is generated. Record the data. 9. Continue decreasing strength in 5 μA increments and increasing the duration until it is not possible to elicit an action potential regardless of the duration of the stimulus. Record each data point. 10. Plot your data points with duration on the y axis and stimulus strength on the x axis. Questions (Exercise 8) 1. What is the relationship between stimulus strength and duration in the generation of action potentials?
Stimulus must stronger than threshold to fire action potential. Once it reaches threshold, doesn’t matter how strong the stimuli is it will fire same strength of action potential but more longer. Duration will affect how long action potential will fire. Stronger the stimuli is number of action potential firing per time gets lager.

2. How do stimulus strength and duration relate to how action potentials are generated in living systems?
Strength : how many action potentials per unit time
Duration : how long entire action potential was fired.

Exercise 9. Effects of various drugs tetrodotoxin (TTX). TEA and Scorpion Toxin 1. Set the Pulse 1 stimulus strength to 30uA and the duration to 0.25 msec. 2. Obtain a tracing of a single action potential . 3. Use the model button to see how the drug affects the various membrane channels. 4. Add TTX to the preparation and obtain a tracing. 5. Turn off TTX and add TEA to the preparation and obtain a tracing. 6. Turn off Tea and add Scorpion Toxin to the preparation and obtain a tracing. Questions (Exercise 9) 1. What effects do these drugs have on action potentials and why? How do they differ?
TTX blocks Na channel, scorp tx opens na channel , Tea blocks potassium

2. Based on the results, which ion (K+ or Na+) has the greater effect on membrane repolarization?
Na has geater result on something 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? 1. Return all values to default settings. 2. Turn on the current axis. 3. Set the x-­‐axis to 50 msec. 4. In the set up set the delay for Pulse 2 to 30 msec. This is the delay between the first and second stimuli. Leave the first pulse at the default setting (0.5 msec). 5. Set the first and second pulses to 35 mV. 6. Obtain a control tracing. 7. Gradually decrease the interval for pulse 2 in increments of 3-­‐5 msec at a time. At some point you will fail to obtain an action potential from the second pulse. This failure to obtain an action potential even though the voltage was sufficient previously means the nerve is refractory. There are two kinds of refractory states (or periods) for nerve cells: relative refractory period and absolute refractory period. If the nerve is in a relative refractory period you should be able to obtain a second action potential by either (1) increasing the duration of pulse 2, or (2) increasing the stimulus strength of pulse 2. When the increment in time is decreased to the point that neither duration nor strength of the second pulse is sufficient to generate an action potential, the membrane is in the absolute refractory period. 8. determine the length of the absolute refractory period by decrease the delay between pulses and increasing the strength/duration of the pulsed until you find the minimum time between pulses that still allows you to generate and action potential Question (Exercise 10) 1. Explain, physiologically (ie. membrane channels) the difference between relative and absolute refractory periods. 2. Based on the data you collected what would be the maximum possible frequency of action potentials in the squid giant axon?

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 the stimulus strength to 27 μA and the temperature to 6 oC 3. Record the tracing. 5. Repeat at 3 oC, at 10 oC. 6. Increase the stimulus strength to 50 μA and repeat at the three different temperatures. Questions (Exercise 11) 1. What effect does temperature have on the action potential? Explain your results.