Lab #6: Neurophysiology Simulation Background Neurons (Fig 6.1) are cells in the nervous system that are used conduct signals at high speed from one part of the body to another. This enables rapid, precise responses to occur in order to compensate for changes in the environment. Neurons are able to send signals at high speed due to their ability to generate and conduct an electrical signal called an action potential down the length of their axons. An action potential is a brief reversal of the membrane potential, so that for a brief interval at a segment of the axon the intracellular fluid just inside of the plasma membrane is more positive than is the extracellular fluid just outside the plasma membrane. This signal is typically generated at the axon hillock of the neuron, and requires the opening of voltage-gated ion channels specialized pore-like transmembrane proteins that open to allow ion passage in response to changes in the relative charge difference across the plasma membrane. There are two different types of voltage-gated ion channels important for the generation action potentials: those specific for sodium ion ( ), and those specific for potassium ion (K + ). In the intervals between action potentials (i.e., when the neuron is resting ) the two types of ions are kept at different concentrations across the plasma membrane (Fig 6.2). is maintained at higher concentrations outside the cell than inside the cell. Conversely, K + tends to be accumulated at higher concentrations inside the cell than outside the cell. The potential for movement of these ions across the cell membrane is thus influenced by the Nucleus Dendrites Cell Body Schwann Cells Axon Hillock Axon Axon Terminals Fig 6.1. Illustration of a neuron and its major associated structures Figure 6.2. Distribution of ions across the plasma membrane during resting potential. Different font sizes for and K + indicate differences in relative concentration. concentration gradients for each ion. Moreover, charge differences across the cell membrane affect the potential for diffusion of these ions. The interior of cells is typically more negatively charged than is the outside of the cell, due to negative charges on certain side-chains of the amino acids of proteins inside the cell, phosphorylated compounds (e.g., ATP), etc. As a result, under resting conditions, there is a strong electrochemical gradient favoring the flow of into the cell, and a weak electrochemical gradient favoring the flow of K + out of the cell. Ion concentrations are maintained at relatively constant levels, however, due to the normally low permeability of the plasma membrane to and low-level activity of the /K + pump, which pumps back out into the extracellular fluid and K + back into the intracellular fluid. The distribution of charged particles across the cell membrane at rest generates the resting potential of the cell membrane, which is variable among different neurons, but typically around -70 mv. The membrane potential (the difference in overall charge across the plasma membrane) of the neuron can change if the relative difference in charges across the membrane is changed. The action potential is generated by just such a redistribution of charged particles across the membrane. By opening large numbers of voltage-gated channels, the permeability of the membrane to and K + is increased markedly, allowing the ions to flow along their respective electrochemical gradients from one side of the membrane to the other.
Figure 6.3. Change in voltage-gated ion channels and redistribution of ions during the depolarization phase of the action potential However, in order for voltage-gated ion channels to open and allow this redistribution of ions across the plasma membrane, the membrane potential itself needs to be changed from resting level by a minimum amount (threshold). Changing the membrane potential to the threshold level causes a redistribution of charged areas within the protein itself, causing a shape change in the channel and opening the passage for the ion. The changes in membrane potential needed to induce the voltage-gated ion channels to open are typically due to the binding of chemical signals (e.g., neurotransmitters) in the extracellular environment to chemically-gated ion channels in the dendrites and cell body of the cell, which increase the permeability of the membrane to certain ions. Physical factors such as mechanical distortion of the plasma membrane or extreme temperature changes, as well as other chemical changes that may affect the shape of proteins in the plasma membrane (e.g. ph), can also alter the permeability of the plasma membrane to certain ions. Moreover, changes in the concentration gradients of the ions themselves across the cell membrane can alter the membrane potential, as the movement of the ion across the membrane through fixed open channels may be changed. In some cases, the resultant change in charge distribution depolarizes the membrane (moves the membrane potential closer to 0 mv), and thus moves the membrane potential towards the threshold value. In other cases, the membrane may become hyperpolarized (more negative, further away from 0 mv), which typically moves the membrane potential away from the threshold value needed to open the voltage-gated ion channels. Figure 6.4. Changes in membrane potential during an action potential. An action potential begins when the plasma membrane at the axon hillock is depolarized to threshold. This induces the opening of the voltage-gated ion channels (Figs 6.3 and 6.4). The channels specific for open very quickly, thus there is a rapid increase in the permeability of the plasma membrane to. rapidly flows into the interior of the cell along its electrochemical gradient, and drives the depolarization phase of the action potential. The membrane is fully depolarized to 0 mv, but even then continues to flow into the interior of the cell, so the fluid inside the cell becomes more positive than the adjacent extracellular fluid, and the membrane polarity is reversed from normal resting levels. The membrane potential rises to ~ +30 mv, but then the flow of into the cell effectively stops not because has reached equilibrium, but because the voltage-gated channels close at that potential, cutting off the flow of. At approximately the same time the flow of stops, the voltage-gated K + channels, which began opening at threshold but require more time to open than do the voltage-gated channels, begin to open in earnest (Figs. 6.4 and 6.5). Since it is now more positive inside the cell than outside the cell, there is a strong gradient favoring the flow of K + out of the cell. Thus K + flows out of the cell, driving the repolarization phase of the action potential. As the positively charged K + leaves the cell, the interior of the cell becomes progressively more
Figure 6.5. Change in voltage-gated ion channels and redistribution of ions during the repolarization phase of the action potential. negative, and the membrane potential moves back towards the resting potential. Once the membrane potential is repolarized below threshold, the voltage-gated K + channels close. Although the resting potential has been restored, the concentration gradients for and K + are now different from resting levels, with large amounts of inside the cell and high amounts of K + outside the cell. The /K + pump restores the local concentration gradients back to resting levels by pumping out of the cell and K + back into the cell. Note that the action potential is an all-ornone response (Fig 6.6). The action potential can occur only if the membrane is depolarized enough to reach threshold and induce the opening of voltage-gated ion channels. Therefore, if the stimulus is not strong enough to reach threshold, no action potential will occur. If threshold is reached, however, a positive feedback loop ensues that quickly leads to the opening of all of the voltage-gated ion channels. Thus, regardless of whether the membrane is depolarized just to threshold or above threshold, maximum permeability of the membrane to and K + will be achieved. Moreover, since the voltage-gated close at a specific potential as well, the amplitude of the action potential is always going to be the same the difference in voltage between the threshold and the potential at which the channels close. Once an action potential has started at the axon hillock it quickly travels down the length of the axon. Remarkably, the strength of the action potential is maintained along the entire length of the axon. This is because once sodium enters the cell during the depolarization stage of the action potential it quickly diffuses though the intracellular fluid along its electrochemical Figure 6.6. The all or none action potential of a neuron. The plot at the top illustrates a series of stimuli of progressively increasing intensity applied to the neuron, whereas the plot at the top illustrates corresponding electrical responses (action potentials) from the neuron. Notice that the neuron only undergoes an action potential when stimulus intensity is high enough to reach the threshold membrane potential for the neuron, and that the amplitude of the action potential (i.e., how much it depolarizes) does not differ with stimulus intensity as long as the stimulus is intense enough to reach threshold. gradient. Lateral flow of inside the cell down the length of the axon triggers the next segment of the axon to be depolarized to threshold, causing it to undergo an action potential. This effect is repeated down the length of the axon, with one segment of the axon undergoing an action potential stimulating the next to depolarize to threshold. The speed at which the action potential travels down the length of the axon is referred to as its conduction velocity, and can be calculated by dividing the distance an action potential travels by the time it takes the action potential to travel that distance. Two major factors influence conduction velocity: 1) Myelination neurons with axons surrounded by Schwann cells or oligodendrocytes conduct action potentials in a manner called saltatory conduction (Fig. 6.7). Since myelination prevents ion exchange between the inside and outside of the cell to occur
A B C Na+ Figure 6.7. Saltatory conduction in myelinated axons. A) The axon hillock reaches threshold, and flows into the cell through voltage gated ion channels. B)On inside the cell, rapidly diffuses to the next node. Moreover, the flux of from the outside of the cell to the inside of the node creates an electrochemical gradient that draws away from the second node towards the first, which also depolarizes the membrane at the second node. The combined effect of these two actions caused the next node to quickly depolarize to threshold and undergo an action potential. C) Once voltage gated ion channels at the second node open, the process repeats with the third node. over most of the length of the axon, the only places where this ion exchange can occur are in the nodes of Ranvier between myelinated segments, where there are particularly high concentrations of voltagegated ion channels. When the axon segment is depolarized to threshold, rapidly flows into the cell and diffused quickly through the cytoplasm to the next node. Moreover as flows into the cell at one node, it creates a gradient favoring in the extracellular fluid near the next node to flow back to the previous node. This reduces the charge difference between the outside and inside of the cell at the second node depolarizing the cell membrane towards threshold and evoking an action potential at the next node. Therefore, the action potential jumps quickly from one segment to the next, and occurs more quickly than if the entire length of the axon was involved in ion exchange across its membrane. Fig 6.8 Plot of experimentally measured conduction velocity as a function of axon diameter in mammalian neurons. After Ruch TC, Patton HD (eds.) (1982): Physiology and Biophysics, 20th ed., 1242 pp. W. B. Saunders, Philadelphia 2) Axon diameter Action potential propagation requires the ability for, once it enters the cell, to be able to diffuse laterally down the length of the axon. Thus, factors that influence the movement of material within a space with restricted diameter (i.e., current), influence how quickly can move from one axon segment to the next, and thus the conduction velocity. The diameter of the axon, thus, influences the ease by which can move from one axon segment to the next. Small-diameter axons create more resistance to current, as the area over which can flow is restricted. Thus less can move per unit time from one segment to the next, and it takes longer to accumulate enough positive charges at the next segment to reach threshold and evoke the action potential. Conversely, large-diameter axons facilitate the lateral flow of. Conduction velocity of a neuron, therefore, tends to be proportional to the diameter of that axon (Fig 6.8). Effects of Drugs on Neurophysiology. Many chemicals have neurological effects. Although the specific effects of chemicals on neurophysiology can differ considerably, there area two primary ways in which neuron are affected (Fig 6.9):
Synaptic communication Chemically-gated channels Action potential propagation Voltage-gated channels Synaptic communication Neurotransmitter Release Fig 6.9. Regions of a neuron where neurotoxins may have an effect. Some neurotoxins affect action potential propagation by influencing the voltage-gated ion channels along the length of the axon (between the dashed lines), whereas other toxins might affect synaptic transmission of signals from one neuron to the next by influencing either neurotransmitter release from the synaptic terminal or by altering the ability of the dendrites and cell body to bind neurotransmitter and transduce it into an electrical signal. 1) Function of voltage-gated ion channels some chemicals influence action potential generation by binding to portions of voltagegated ion channels and preventing their function. For example, tetradotoxin, a poison produced by puffer fish, prevents voltage-gated channels from opening when threshold is reached. Another poison, dendrotoxin (a component in the venom of black mambas) prevents voltage-gated K + channels from opening, preventing repolarization of an axon during an action potential and thus greatly increasing the refractory period for an action potential and slowing conduction velocity. 2) Synaptic transmission of signals the primary means by which most neurons are stimulated to undergo action potentials is through the receipt of chemical signals (neurotransmitters) that activate specific pathways in the cell that lead to a change in membrane potential. Different chemicals can influence the signaling interactions between one cell and another. Some chemicals alter the amount of neurotransmitter released from a presynaptic cell into the synaptic cleft (e.g., amphetamines stimulate dopamine release) or the degradation of the neurotransmitter in the synaptic cleft (e.g., cocaine blocks norepinephrine and serotonin re-uptake by presynaptic cells). Other chemicals bind to the receptor proteins on the postsynaptic cell that are responsible for triggering the graded potentials that would normally determine whether the neuron would undergo an action potential. Some of these act as agonists they bind to a particular receptor protein on the surface of the postsynaptic cell and stimulate the metabolic pathway connected to that receptor. Since they may supplement the normal neurotransmitter that normally activates that pathway, and may not be degraded as easily as the normal neurotransmitter, they amplify the effects of the neurotransmitter (e.g., nicotine mimics the effects of acetylcholine for certain ion channels). Conversely, others act as antagonists they bind to a receptor protein but do not activate the metabolic pathway connected to that receptor and prevent the normal neurotransmitter from binding that receptor, suppressing the normal effects of the neurotransmitter (e.g., cobratoxin, found in cobra venom, prevents acetylcholine from binding to particular receptors). Observing Action Potentials in Whole Nerves The computer simulation we will run in this lab mimics experiments that are conducted on segments of whole nerves, not on individual neurons. Importantly, this means that the action potentials we will record are not action potentials from a single neuron, but a compound action potential, showing the cumulative action potentials of the neurons within the nerve. This is important because although many of the neurons in this nerve have the same threshold, some neurons have thresholds that are slightly higher than others. Also, because of the way we are stimulating the nerve (applying an electrical shock to the outer surface of the nerve), not all of the neurons in the nerve will receive she same amount of stimulus. Thus, it is possible to vary the amplitude of the action potential by varying the strength of the stimulus. This does not violate the all-or-none principle! Each neuron is undergoing an action potential in an all or none fashion. What is changing is the number of neurons undergoing action potentials in response to a stimulus of a particular strength.
Let us imagine that we are stimulating a nerve segment with progressively more intensive (i.e., higher voltage) electrical shocks (Fig. 6.10). There are ranges of stimulus intensity that evoke different amounts of depolarization in the compound action potential of a nerve. A subthreshold stimulus is a stimulus with such a low intensity that none of the neurons reach threshold, therefore no compound action potential is recorded. A threshold stimulus is just intense enough to depolarize a few neurons in the nerve to threshold and cause them to undergo an action potential, so a very weak compound action potential is recorded. If stimulus intensity is increased above threshold, the amplitude of the compound action potential increases as more and more neurons undergo action potential. We refer to this range of stimulus intensities in which variable compound action potential amplitudes can be produced as submaximal stimuli. Eventually, we will apply a stimulus intense enough to induce every neuron in the nerve to undergo an action potential. That stimulus intensity is referred to as the maximal stimulus. If the intensity is increased above this maximal stimulus, no further increase in compound action potential amplitude will occur, since all neurons in the nerve are already undergoing action potential. We sometimes refer to stimuli with intensities higher than a maximal stimulus as supramaximal stimuli. Figure 6.10. Variation in the amplitude of the compound action potential of a whole nerve with varying stimulus intensity. The upper plot depicts the strength of electrical stimuli applied to the nerve, and the lower plot depicts the amplitude of the resultant electrical response by the nerve. Note that a threshold stimulus here is a stimulus with the minimum amount of intensity to generate a detectable compound action potential. Increasing stimulus intensity above this threshold value leads to the generation of bigger compound action potentials until the maximal stimulus is reached, whereupon all neurons in the nerve will be undergoing action potential and the compound action potential will reach its maximum amplitude.
Experimental Procedures We will be running a simulation of experiments conducted on whole nerve segments using PhysioEx (Benjamin Cummings). The software should be loaded for you and the screen illustrated in Fig. 6.11 should be displayed. There are three different pieces of equipment displayed on the screen which you should note. First, there is a nerve chamber. A segment of a nerve has been dissected and suspended over a series of metal bars that act as electrodes. The two electrodes at the bottom are stimulating electrodes and are connected to an electrical stimulator. The stimulator can be used to apply electrical current to the nerve at different voltages and frequencies to try to elicit an action potential. The other set of electrodes are called recording electrodes, and they are connected to an oscilloscope. Differences in charge between the two recording electrodes (such as those caused by an action potential passing by) cause the line traced on the oscilloscope screen to deflect. Thus, we can observe any action potentials forming in the nerve Experiment 1. Stimulation of an Action Potential. A. Electrical Stimulation Observation of Threshold Fig 6.11. View of the PhysioEx screen used for Experiment #1. Initially, the voltage on the electrical stimulator is set to 0.0 V. Increase the voltage to 1.0 V by clicking on the + button next to Voltage. Then click on Single Stimulus. Notice that a flat-line tracing is recorded on the oscilloscope, meaning no action potential was generated. Increase the voltage by 0.3 V using the + button next to Voltage and click on Single Stimulus again. A second tracing should appear in a different color. Repeat this procedure, increasing the voltage by 0.1 to 0.2 V increments until an action potential is recorded. Determine and record the minimum amount of voltage needed to evoke the action potential. This value is the threshold stimulus. Record this voltage on your data sheet. B. Electrical Stimulation Observation of Compound Action Potential and Determination of Maximal Stimulus Continue increasing the voltage above threshold by 0.1-0.2 V increments. Notice that as you increase the stimulus strength, the amplitude of the action potential increases slightly (Fig 6.12). This is because as you increase stimulus strength you are reaching the threshold of more individual neurons in the nerve, more neurons undergo action potentials, and thus the compound action potentials strength increases. Eventually, though, you will reach a point where no further increase in the amplitude of the compound action potential will occur. This is because the stimulus strength is now strong enough for all the neurons in the nerve to undergo action potential. The lowest stimulus strength required to cause all the neurons in the nerve to undergo action potentials is called the maximal stimulus. Determine and record this voltage.
Hint: try using the following method to determine the maximal stimulus: Once you reach your threshold stimulus, increase the voltage by 0.1-0.2 V and apply a stimulus. Then click on the Clear button on the lower right corner of the oscilloscope. Apply another stimulus with the same voltage as you had just applied, then increase the voltage by 0.1-0.2 V and stimulate again. You should only have two tracings on your screen one pink and the other green. You should still see two different tracings, meaning that you have not reached your maximum action potential amplitude. Repeat the procedure. Eventually, you should reach a point where one tracing completely overlaps the other. When you see this, then the lower of the two voltages you applied is your maximal stimulus strength. Fig 6.12. A pair of compound action potentials recorded on the PhysioEx oscilloscope. Notice that the green tracing (produced when the nerve was stimulated at a higher voltage) has a higher amplitude than does the pink tracing. This illustrates that through the submaximal range of stimuli strengths, action potential amplitude will increase with increased stimulus intensity. C. Chemical Stimulation. Click on the Clear button on the oscilloscope to remove previous tracings. Move your pointer over to the dropper bottle on the left labeled Sodium Chloride, left click on the dropper and hold, then drag the dropper over the nerve chamber and release the left mouse button to apply the NaCl solution onto the nerve. Notice what happens on the oscilloscope. If we assume that the NaCl concentration in this solution is greater than that normally found in extracellular fluid, explain why the application of NaCl to the nerve caused this response. Click the Clean button on the nerve chamber to wash the NaCl solution from the nerve. Then apply a dropper from the bottle labeled Hydrochloric Acid to the nerve. Again, note the response of the nerve, and provide an explanation for this response. Click on the Clean button of the nerve chamber when you are finished. D. Heat Stimulation Place your pointer over the glass rod on the lower left portion of the screen, left click, and drag it down to the heater just below it. Click on Heat to make it red hot. Left click on the heated rod, drag it over the nerve chamber, and release the left click to apply the stimulus. Note the response of the nerve on the oscilloscope, and provide an explanation for this response. Hint: For parts C and D, think about how these substances might influence a) the difference in electrical charge, ion gradients, and diffusion rates of ions across the cell membranes and b) the permeability of the cell membrane to different ions, with specific emphasis on the membrane proteins needed to allow ions to move from one side of the membrane to the other.
Experiment 2. Inhibitory effects of drugs on action potential generation. Move your mouse over the Experiment menu at the top of the screen. Select Inhibiting a Nerve Impulse by left clicking on that line in the menu. A new screen will pop up, which looks very similar to the screen used in experiment one except that on the left-hand side there are now three bottles of drugs with neurotoxic effects (see Fig. 6.13). Set the voltage on the electrical stimulator to a level that will evoke an action potential (e.g., 5 V). Fig 6.13. View of the PhysioEx screen used for Experiment #2. A. Effect of Ether (ethyl ether was the first chemical used as a general anesthetic). Click the stimulate button on the stimulator and notice the action potential displayed on the oscilloscope. Transfer a dropperful of fluid from the bottle labeled Ether. Click on the stimulate button again. What is the effect on action potential generation (does an action potential occur in the nerve once treated with ether)? Based on the effect you observe and the fact that you are using electrical stimuli to stimulate a segment of nerve containing only axons, what do you think is the target of action for this chemical (i.e., synapse or voltage-gated channels)? B. Effect of Lidocaine (a substance found in cloves used as a local anesthetic and anti-arrhythmic agent). Stop the tracing. Click on the Clear button on the oscilloscope to remove the previous tracings, and click on the Clean button above the nerve chamber to wash off the ether. Click the stimulate button on the stimulator and notice the action potential displayed on the oscilloscope. This indicates that the ether from the previous exercise has been removed. Transfer a dropperful of fluid from the bottle labeled Lidocaine. Fig 6.14. A view of compound action potential recordings associated with a nerve stimulated with maximal stimuli before treatment with a neurotoxin (pink) and after treatment with a neurotoxin (green) in Experiment 2.
Click on the stimulate button once again to attempt to trigger a second action potential. What is the effect on action potential generation (does an action potential occur in the nerve once treated with lidocaine)? Based on the effect you observe and the fact that you are using electrical stimuli to stimulate a segment of nerve containing only axons, what do you think is the target of action for this chemical (i.e., synapse or voltage-gated channels)? C. Effect of Curare (a potent neurotoxin produced in the skin of poison arrow frogs). Click on the Clear button on the oscilloscope to remove the previous tracings, and click on the Clean button above the nerve chamber to wash off the ether. Click the stimulate button on the stimulator and notice the action potential displayed on the oscilloscope. This indicates that the lidocaine from the previous exercise has been removed, and the nerve is once again functioning properly. Transfer a dropperful of fluid from the bottle labeled Curare. Click on the stimulate button once again to attempt to trigger a second action potential. What is the effect on action potential generation (does an action potential occur in the nerve once treated with curare)? Based on the effect you observe and the fact that you are using electrical stimuli to stimulate a segment of nerve containing only axons, what do you think is the target of action for this chemical (i.e., synapse or voltage-gated channels)? Hint: In this last experiment, what does NOT happen is as important as what does happen. Some neurotoxins may apparently have no effect on the nerve. Keep this in mind, though we are stimulating a segment of a nerve which contains only axon tissue (so no dendrites, cell bodies, or axon terminals). We are also applying an electrical stimulus to the nerve to trigger the opening of the voltage gated ion channels. Experiment 3. Nerve Conduction Velocity. Move your mouse over the Experiment menu at the top of the screen. Select Nerve Conduction Velocity by left clicking on that line in the menu. A new screen will pop up displaying a somewhat different setup than seen in the previous experiments (see Fig. 6.15). The stimulator is now at the top right of the screen, with the oscilloscope below it. In addition to the nerve chamber, there is a bioamplifier that must be switched on. To the left are three nerve segments and an earthworm. You can sedate the earthworm with the ethanol above it if you so desire. The setup on this screen will allow you to measure how long it takes for an action potential to travel a set distance along a nerve, and thus determine that nerve s conduction velocity (velocity = distance/time). The recording electrodes in the nerve chamber are set 43 mm away from the stimulating electrodes. Therefore, conduction velocity can be Fig 6.15. View of the PhysioEx screen used for Experiment #3. calculated for each nerve by the
following equation: Conduction velocity (m/sec) = 43 mm time (msec) Procedure You will be determining the conduction velocity for three different nerves: a frog nerve (thin, myelinated), rat nerve #1 (thin, unmyelinated), and rat nerve #2 (thick, myelinated). Select one of the three nerves and place it in the nerve chamber. Set the voltage on the stimulator to an amount that will give an action potential (e.g., 5 V). Click on the Pulse button to ready the stimulator, then click on the Stimulate button. An action potential will be recorded on the oscilloscope. Click on the Measure button on the stimulator, and then click and hold on the + sign next to Time. Notice that a yellow line will scroll across the oscilloscope. Using the + and buttons, position the line so that it intersects the action potential tracing at the point just when the tracing starts to move above baseline (Fig 6.16). Record the time displayed at the top right of the stimulator. Divide the distance the action potential traveled (43 mm) by this time to calculate the conduction velocity for this neuron, and record this value on your data sheet. Repeat this procedure for the other two nerves. Comparing the conduction velocity of the rat nerve #1 (thin, unmyelinated) with that of the frog nerve (thin, myelinated), what can you conclude about the effect of myelination on conduction velocity? Comparing the conduction velocity of the frog nerve (thin, myelinated) with that of the rat nerve #2 (thick, myelinated), what can you conclude about the effect of axon diameter on conduction velocity? Fig 6.16 Recording of an action potential showing the use of the measurement tool (yellow bar) in order to determine conduction time from the stimulating electrodes to the recording electrodes.