Electrical Properties of Biological Systems

Laboratory 3 Electrical Properties of Biological Systems All cells are capable of maintaining a charge separation, or POTENTIAL DIFFERENCE, across their membranes. This is due to the diffusion of sodium (Na + ) and potassium (K + ) ions down their concentration gradients. Net diffusion of Na + occurs down a concentration gradient from outside to inside the cell with net diffusion of K + occurring simultaneously, but in the opposite direction, from inside to outside the cell. When there is NO NET DIFFUSION of charge across the membrane the cell maintains a steady potential difference called the RESTING MEMBRANE POTENTIAL. In neurons, as in most cells, the CONDUCTANCE (permeability) of the membrane to K + is much greater than its conductance to Na + (K+ channel is leaky ). This means more K+ moves across the membrane than Na+. This sets up a potential difference across the membrane with the inside of the cell being negative with respect to the outside. The magnitude of this potential difference is mainly dependent on the K + concentration gradient. As the K + concentration gradient between the inside and outside of the cell increases (causing K + to leave the cell), the resting membrane potential becomes more negative. Remember: The outside of the cell is ALWAYS considered to be 0 mv, thus if more K+ moves out than Na+ in it will make the inside of the cell more negative!. As the neuron is an excitable cell, it is capable of generating and propagating ACTION POTENTIALS. Action potentials are localized, rapid reversals of the membrane potential from negative inside to positive inside and back to the negative resting membrane potential within 1 msec. The AMPLITUDE (height) of the action potential is determined by the Na + concentration gradient across the membrane. As the Na + concentration gradient increases, so does the amplitude of the action potential. OBJECTIVES This week you will experience working in a neurophysiology laboratory using computer simulations. You will use three different simulations to investigate how excitable membranes work. The objectives of this week s lab are specific to the computer program you will use: Program 1 MemPot The effect of ion concentrations on neuronal function During these experiments, you will study: a) the effects of extracellular potassium concentration on the resting membrane potential. b) the effects of extracellular sodium concentration on action potential amplitude Program 2 NEUROSIM The ionic basis of action potentials During these experiments, you will observe: a) the constancy of the duration and amplitude of action potentials, known as the all-ornone principle. 1

b) that Na + flowing into the neuron makes the inside of the neuron more positive (depolarization) and how this can initiate action potentials while K +, as it flows out of the neuron, initiates repolarization followed by hyperpolarization. c) the changes in membrane conductance (g) to Na + and K + that occur during every action potential. Conductance is the measurement of the permeability of an ion to the cell membrane. d) the effects of channel blocking drugs on action potentials. Neuron refractory periods During these experiments, you will determine: a) the duration of the refractory period, the period of time during which the neuron does not respond to a second threshold stimulus. b) the duration of the absolute refractory period, the period of time during which the neuron does not respond to a suprathreshold stimulus. c) the duration of the relative refractory period, the period of time during which the neuron does respond to a suprathreshold stimulus. Program 3 - physioex: During these experiments you will observe: a) that different types of stimuli can generate action potentials. b) how different agents block neural transmission. c) how axon size and myelination affect action potential conduction velocity. Program 1: MemPot With this program you will simulate the use of microelectrodes to penetrate living neurons. While changing the extracellular concentration of K + or Na +, you will be able to observe: 1. the effects of extracellular potassium, [K + ] o, on the resting membrane potential. 2. the effects of extracellular sodium concentration, [Na + ] o, on the amplitude of the action potential. 3. that resting membrane potentials and action potential amplitudes vary from neuron to neuron. MATERIAL Use grey laptops (not the black ones that we used the first two labs). 1. Turn on computer. 2. Double-click on the MemPot icon. 2

Experiment 1: Effects of extracellular potassium on the resting membrane potential As potassium is very permeable to the neuron membrane at rest (high conductance), the potassium concentration gradient across the membrane is the major determinant with respect to the RESTING MEMBRANE POTENTIAL. Homeostatic mechanisms maintain [K + ]o at close to 5.5 mm/l. Variations above or below 5.5 mm/l cause major changes in the resting membrane potential and therefore cell excitability. By adding 10 different extracellular potassium concentrations to your experimental neurons, you can experimentally determine the effects of [K + ]o on the resting membrane potential. Procedure 1. Select RESTING POTENTIAL EXPERIMENT 2. Select START EXPERIMENT 3. Enter values for [K + ] o. The initial value entered will be 1.0. (You need not enter mm/l). 4. The controls for moving and stopping electrode movement are in the upper right and the control for microelectrode speed is on the upper toolbar. Press the down arrow ONCE and an electrode moves towards the cells 5. Stop the electrode movement as soon as the electrode penetrates the cell. Aim for the middle of the cell to reduce the chance of killing the cell. If you do not STOP the electrode you WILL kill the cell. Kill too many cells and you have the redo the experiment from the start!!!! 6. Withdraw the electrode using the up arrow 7. Move electrode over the center of the second cell by using the horizontal arrows. 8. Repeat steps 5-8 until all cells have been examined. 9. Record the average resting membrane potential in mv in the table below. 10. Repeat steps 4-10 with solutions of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 mm/l 11. Use your results to determine the effects of [K + ] o on the resting membrane potential. Graph and examine the relationship between the extracellular concentration of K+ and the resting membrane potential. Label axes. 12. Select finish experiment. Vav [K + ] o mm/l 1 2 3 4 5 6 7 8 9 10 Resting Membrane Potential (mv) 3

Experiment 2: Extracellular sodium concentration and the amplitude of the action potential The peak amplitude of the action potential is determined to some extent by the sodium concentration gradient between the outside and inside of the cell. Increasing or decreasing [Na + ] o from 150 mm/l, its normal value, changes the sodium concentration gradient. By adding 10 different values of [Na + ] o you can experimentally determine the effects of [Na + ] o on the amplitude of the action potential. 1. Select MENU 2. Select ACTION POTENTIAL EXPERIMENT 3. Select START EXPERIMENT 4. Select 60.0 as the initial [Na + ]o in mm/l 5. Advance the electrode into the first cell then stop the advance as in MEMPOT EXPERIMENT 1 6. Once the electrode is stopped within the cell press select OK. This will stimulate the generation of an action potential within the cell. 7. Withdraw the electrode using the up arrow 8. Repeat steps 5 and 7 for the remaining cells 9. Record the average peak amplitude (in mv) of the action potential. 10. Repeat steps 4-8 with [Na + ] o values of 70.0, 80.0, 90.0, 100.0, 110.0, 120.0, 130.0, 140.0, and 150.0 mm/l. 11. Use your results to determine the effects of altering [Na + ] o on the amplitude of the action potential. Plot the concentration of Na+ vs. the amplitude of the action potential. Label axes. Vav [Na + ] o mm/l 60 70 80 90 100 110 120 130 140 150 Resting Membrane Potential (mv) 4

Program 2: NEUROSIM More on Action Potentials An action potential is the rapid reversal of membrane potential occurring when an excitable cell has been depolarized to its threshold potential. Excitable cells in the body include neurons and muscle cells (skeletal, smooth, and cardiac). Action potentials are an ALL-OR-NONE phenomenon The ALL-OR-NONE principle means that, in a given neuron, under the same conditions, the duration (typically 1 ms) and amplitude of the action potential is identical each time an action potential is generated in that neuron. The shape of the action potential is formed by sodium and potassium flowing passively across the cell membrane, down their respective concentration gradients (Na + from out to in; K + from in to out). The Na + and K + concentration gradients are maintained by the Na + /K + ATPase active pump. This pump has NO role in the generation of an action potential but maintains the high K + intracellular / high Na+ extracellular concentrations while the neuron is at rest! The Na + /K + ATPase active pump also helps reset the concentrations of Na + and K + necessary for generating another action potential. To generate an action potential, a stimulus (or multiple stimuli) must DEPOLARIZE the neuron to its THRESHOLD POTENTIAL. Once reaching threshold, four sequential events generate the action potential: 1. VOLTAGE SENSITIVE Na + GATES open and Na + passively rushes into the neuron, down its concentration gradient, and the neuron becomes positive on the inside relative to the outside. 2. After the reversal of polarity, Na + gates close and are inactivated for a short time and Na + no longer enters the neuron. 3. At the peak of action potential, VOLTAGE SENSITIVE K + gates open and K + flows out of the cell, initiating REPOLARIZATION. The repolarization overshoots, resulting in the HYPERPOLARIZATION period. 4. The Na + /K + ATPase pump then actively transports Na + out and K + into the neuron to reset the Na + and K + concentration gradients for the NEXT action potential. NEUROSIM OBJECTIVES During these experiments, you will observe: a. the sequential events that generate the action potential discussed above. b. the changes in membrane conductance (g) to Na + and K + that occur during every action potential. REMEMBER: Conductance is the measurement of the permeability of an ion to the neuron membrane. c. the effects of channel-blocking drugs on action potentials. d. the constancy of the duration and amplitude of action potentials generated by the THRESHOLD and SUPRATHRESHOLD stimuli. 5

Experiment 3: The Ionic Basis of Action Potentials 1. Select NEUROSIM from the Start Menu and Press 1 (Hodgkin-Huxley Model) 2. A screen will be displayed showing various parameters that can be altered during the experiment 3. a. Change Ion Concs to Na + 150mM out and 15mM in K + 5mM out and 150mM in These are physiologically real values for a neuron Do not alter the default membrane properties. Simply realize that these vary between different types of neurons and other excitable cells (i.e. muscle) b. Select Current Clamp (not voltage clamp) c. Under Current Clamp a. Pulse 1: Amp = 33, dur = 0.25, ramp = 0, delay = 0.5 b. Pulse 2: Amp = 200, dur = 0.25, ramp = 0, delay = 15 d. Start the experiment, which will take you to the graph screen 4. The experimental results are depicted on four graphs i. the top graph shows the change in membrane potential (mv) in red ii. the next graph shows stimulus strength in green (square waves) iii. the next graph shows the current iv. the bottom graph depicts the conductance (permeability) of the cell membrane to Na + (pink) and K + (blue) and conductance changes during the course of the action potential. Conclusions from Experiment 3 Top Graph-Changes in membrane potential Stimulus 1 is a THRESHOLD stimulus. Stimulus 2 is a SUPRATHRESHOLD stimulus. There is no difference in the duration or amplitude of the action potentials produced by either stimulus, demonstrating the ALL-OR-NONE principle of action potentials. Bottom Graph-Changes in Na + and K + conductance The first conductance to change was that of Na +. As threshold potential was reached, a rapid, but short lived, increase in Na + conductance was initiated. Shortly after the onset of the increase in Na + conductance, K + conductance began to increase. In contrast to Na +, the increase in K + conduction is relatively slow and longer lived. Press Clear Button to Clear out graphs, make sure to do this after each experiment or your simulations will overlap on the graph screen! EXPERIMENTS 4-6: Add Experimental Drugs. Use the next three experiments to discover the mechanism by which each of these neurotoxins alter normal action potentials. Use the same setting in the experimental set-up for experiment 3 and then add the appropriate neurotoxin. 6

Experiment 4: Effects of Tetrodotoxin (TTX) on membrane conductance 1. Add TTX to the experimental set-up and run the simulation. The experimental results are displayed in the same graphical format used in the prior experiment. Examine the graphs and answer the following questions: What happened to the action potentials? Are they still present? Was there a Na + influx? Was there a K + efflux? Did the conductance of the membrane to either Na + or K + increase? DRAW THE RESULTS OF EXPERIMENT 4 Conclusions from experiment 4: TTX only blocks Na + channels, inhibiting any influx of Na +, and therefore depolarization. As there was NO ensuing increase in K + conductance, the activation of K + channels is dependent on prior activation of Na + channels and depolarization of the neuron 7

Experiment 5: Effects of Tetraethylammonium (TEA) on membrane conductance 1. Remove TTX from the experimental setup. 2. Add TEA to the experimental set-up and re-run the simulation The experimental results are displayed in the same graphical format used in the prior experiments. What has happened to the action potentials? Are they still there? Was there a Na + influx? Was there a K + efflux? Did the conductance of the membrane to either Na+ or K + increase? DRAW THE RESULTS OF EXPERIMENT 5 Conclusions from Experiment 5: TEA only blocks K + channels. The initial stimulus depolarized the neuron to its threshold potential initiating opening of Na + channels and depolarization. As TEA blocked the K + channels, the neuron was unable to repolarize in its normal fashion. What mechanism is causing the slow partial repolarization that you can see? (Think about any mechanisms that move Na + out of the neuron). 8

Experiment 6: Effects of Scorpion Toxin on membrane conductance 1. Remove TEA from the experimental setup. 2. Add Scorpion Toxin to the experimental set-up and re-run the simulation What has happened to the action potentials? Are they still there? Was there a Na + influx? Was there a K + efflux? Did the conductance of the membrane to either Na + or K + increase? DRAW THE RESULTS OF EXPERIMENT 6 Conclusions from Experiment 6: First, look at the top graph (membrane potential). The neuron depolarizes, reaches threshold, and initiates an action potential. Therefore, Na + channels must have opened. However, the membrane potential never decreases (i.e. there is no repolarization). Now look at the bottom conductance graph. Both Na + (pink) and K + (blue) increased after the initial stimulus. Like the membrane potential, both conductances stayed elevated. Scorpion Toxin locks the Na + channels in the "OPEN" position. The voltage-gated K + channels remain open since the membrane potential remains positive. Since both Na + and K + channels remain open, Na + and K + currents are equal (but in flowing in opposite directions) as are the concentration gradients for Na + and K + across the cell membrane. Therefore, the cell just swaps one positive charge in for one positive charge out. 9

Refractory Periods Once a THRESHOLD STIMULUS depolarized a neuron enough to reach its THRESHOLD POTENTIAL, the neuron initiates an action potential. It takes several milliseconds (ms) before a second THRESHOLD STIMULUS can initiate a second action potential in the neuron. This time period during which a neuron does not respond to a second threshold stimulus is called a REFRACTORY PERIOD. The REFRACTORY PERIOD can be subdivided into an ABSOLUTE REFRACTORY PERIOD and a RELATIVE REFRACTORY PERIOD. Absolute refractory period (ARP) The ARP is the time period during which a neuron is incapable of responding to a SUPRATHRESHOLD STIMULUS, regardless of the strength of this stimulus! Na + gates MUST be reset before the neuron can respond to any second stimulus. Resetting of the "timing gates" is voltage dependent, i.e. the cell must be almost completely repolarized before the timing gates reset. The function of the ARP is to make transmission or PROPAGATION of action potentials along an axon one way. Relative refractory period (RRP) The RRP occurs immediately after the ARP. It is defined as that period of time during which a neuron will generate a second action potential, but ONLY in response to a SUPRATHRESHOLD STIMULUS, i.e. it responds to an increase in STIMULUS INTENSITY. The AMPLITUDE and DURATION of the action potential produced in response to a suprathreshold stimulus is identical to that produced by a threshold stimulus. This is once again an example of the ALL-OR-NONE PRINCIPLE. The intensity of the suprathreshold stimulus required to initiate an action potential during the RRP decreases in intensity in proportion to the time interval between the first and second stimulus. For example, immediately following the ARP the suprathreshold stimulus intensity required to generate a second action potential may be 500 ua. 2 ms into the RRP the required stimulus may be only 300 ua. 4 ms into the RRP 150 ua may be required, etc. The stimulus intensity required will continue to decrease with time until the time period between the first and second stimulus is sufficient for the neuron to once again respond to a THRESHOLD STIMULUS. This signifies the end of the RRP. The duration of the RRP corresponds approximately to the time period during which the "K + channels" are open. When K + channels are open K + conductance is increased and the cell becomes HYPERPOLARIZED. The RRP permits the nervous system to respond to changes in STIMULUS INTENSITY by changing the rate (FREQUENCY) at which action potentials are firing. OBJECTIVES To determine: a. the duration of the REFRACTORY PERIOD: the period of time during which the neuron does NOT respond to a second THRESHOLD STIMULUS. b. the duration of the ABSOLUTE REFRACTORY PERIOD: the period of time during which the neuron does NOT respond to a SUPRATHRESHOLD STIMULUS. c. the duration of the RELATIVE REFRACTORY PERIOD: the period of time during which the neuron DOES respond to a SUPRATHRESHOLD STIMULUS. 10

Experiment 7: Determination of the Refractory Period 1. Enter 35 ua for the amplitude of both the first and second stimuli. This is the threshold stimulus. 2. Set delay for 0.5 msec for first stimulus and 25 msec for the second. 3. Start the simulation. 4. Make sure the time axis is set to 50msec. To do this clear out the graphs and then set the maximum value of the time axis (in red) to 50. 5. Now click on the Start button again. 6. You should see two action potentials. This means that the second threshold stimulus occurred after the refractory period. To determine the refractory period decrease the time of the second stimulus by 3 msec and rerun the simulation. Repeat this procedure until you determine the refractory period. Experiment 8: Determination of the relative and absolute refractory periods 1. Set the delay for stimulus #2 to 1 msec less than the Refractory Period you determined in the Experiment 7. 2. Start the simulation and note if the second stimulus produces an action potential. If not, you are inside of the refractory period and a suprathreshold stimulus is required to initiate a second action potential. 3. Go back to the set-up screen and increase the Second Stimulus (pulse 2) from 35 ua to 70 ua and start the simulation. This should produce a second action potential. You are now in the Relative Refractory Period, the time during which a suprathreshold stimulus can produce a second action potential. 4. Keep decreasing the delay until the second stimulus until the 70 ua suprathreshold stimulus does NOT produce a second action potential. 5. Then begin to increase the second stimulus amplitude by 35 ua interval. Keep decreasing the time interval and increase the second stimulus amplitude until the second stimulus DOES NOT produce a second action potential. 6. Note the time interval when you no longer observe a second action potential. This is the Absolute Refractory Period. 7. The duration of the RELATIVE REFRACTORY PERIOD is the difference in time between the REFRACTORY PERIOD and the ABSOLUTE REFRACTORY PERIOD. The membrane did NOT respond to a SUPRATHRESHOLD STIMULUS for a time interval of ms. This is the duration of the REFRACTORY PERIOD and is the time during which channels are "open". When the membrane did respond to a SUPRATHRESHOLD STIMULUS, but NOT to a THRESHOLD STIMULUS, the cell was in its REFRACTORY PERIOD corresponding to the duration of "open" channels. The STIMULUS INTENSITY required to fire an action potential during this period with time. 11

Program 3: physioex Exercise 3: Neurophysiology of Nerve Impulses The classic experiment in neurophysiology lab involves dissecting the sciatic nerve from a recently killed frog, subjecting it stimulation within a nerve chamber, and then recording the neurophysiological output via a computer (i.e. voltage changes associated with action potentials, graded potentials, etc). We will use PhysioEx to simulate this procedure. Complete Activities 1-8 (p.29-36). Answer the questions listed at the end of each activity. 12