Foundations of Oral and Systemic Disease Fall 2013 1 Foundations of Oral and Systemic Disease Laboratory Overview 1.

In the lab, students will perform virtual experiments that illustrate the physiological principles underlying membrane excitability, action potentials, and synaptic transmission. 2. Students will work in small groups to discuss and reinforce the physiological concepts covered in lecture. Faculty members will circulate through the lab to assist and answer questions.

Each student will attend two 2-hour laboratory sessions, which will be held on Monday 10/14/2013 (2-4 pm) and Wednesday 10/16/2013 (2-4 pm) or Tuesday 10/15/2013 (1-3 pm) and Thursday 10/17/2013 (1-3 pm). Students in Anatomy group A are assigned to the Monday/Wednesday sessions; students in Anatomy group B are assigned to the Tuesday/Thursday sessions. The lab sessions will be held in the Department of Physiology Computer Lab, 53-121 CHS. Attendance at the lab sessions is mandatory. Because there is limited space in the computer lab, you must attend the session to which you are assigned. You may not switch lab groups or lab times without pre-authorization from Dr. Papazian. There will be no make up lab sessions. If you are unable to attend, please contact Dr. Papazian. The laboratory programs will be available online for students who would like to use them outside the formal lab sessions. Students will complete two simulation programs: Nerve and Synapse. The laboratory exercises are described in this manual, which can be printed out and brought to the lab sessions. For maximum benefit, review the relevant lecture material and read the manual prior to your lab session. The laboratory and Dr. Papazians lectures are designed to work together to illustrate and reinforce the concepts of membrane excitability, action potential generation and propagation, and synaptic transmission. The laboratory is not graded; there are no laboratory reports to turn in. However, the lab is integral to the course and material from it will be covered on exams. The computer simulation programs used in the laboratory were created by current and former faculty members in the Department of Physiology. Drs. Francisco Bezanilla, Julio Vergara, and Nikola Jurisic created the Nerve Action Potential Simulation. Drs. Michael Letinsky, Nikola Jurisic and Julio Vergara originally created the Synaptic Transmission Simulation program, which was later modified by Drs. Jonathan Monck and Sebastian Uijtdehagge (SOM-IDTU). The manual has evolved over time with the contribution of a number of faculty members. Drs. Diane Papazian, Julio Vergara and Jonathan Monck contributed to the current version.

Foundations of Oral and Systemic Disease Fall 2013 2 Nerve & Synapse Simulation Laboratory

In these laboratory sessions you will perform a number of experiments using computer simulations to investigate the properties of the action potential and the mechanisms of synaptic transmission. In the first session you will use the Nerve program to explore the properties of excitable membranes and their ability to generate action potentials, the ionic basis of the action potential, and how action potentials propagate along a nerve. In the second session you will use the Synapse program to study the mechanisms of synaptic transmission at the neuromuscular junction. This laboratory will allow you to examine the nicotinic acetylcholine receptor current, the Ca2+ dependence and quantal nature of synaptic transmission, and the ionic basis of the postsynaptic (i.e. muscle) action potential. Table of Contents Introduction ........................................................................................................... Table of Contents .................................................................................................. Nerve: Action Potential Simulation Introduction ........................................................................................................... Program Setup ....................................................................................................... Experimental Design ............................................................................................. Experiment 1. Relationship between K+ conductance and K+ current ................. Experiment 2. Relationship between Na+ conductance and Na+ current .............. Experiment 3. Threshold for action potential firing ............................................. Experiment 4. Refractory periods ......................................................................... Experiment 5. Propagated action potential ........................................................... Synapse: Synaptic Transmission Simulation ....................................................... Introduction ........................................................................................................... Program Set Up ..................................................................................................... Experiment 1. The end plate potential and end plate current ............................... Experiment 2. The postsynaptic current and reversal potential ............................ Experiment 3. Quantal nature of synaptic transmission ....................................... Experiment 4. Presynaptic calcium current and synaptic transmission ................

1 2

3 3 4 5 6 7 8 9

11 12 13 15 16 17

Foundations of Oral and Systemic Disease Fall 2013 3

NERVE Action Potential Simulation

INTRODUCTION The computer simulation experiments described below explore the properties of excitable membranes and their ability to generate and propagate action potentials. The experiments are based on the work of Hodgkin and Huxley, who used the squid giant axon preparation to determine the ionic basis of a simple action potential. The computer program relies on the mathematical model developed by Hodgkin and Huxley in which action potentials are described using only the functional properties of the underlying voltage-gated Na+ and K+ channels. Although the squid axon action potential is relatively simple, the principles illustrated by this lab and the associated lectures are directly applicable to more complex action potentials found in the mammalian nervous system and skeletal, cardiac and smooth muscle. The relationships between ionic currents, conductances, channel activity, and membrane voltage are also vital for understanding synaptic function. Please note that subsequent courses in Dental School will build upon these concepts so it is important to master them now. Each lab exercise starts with an experimental setup associated with several specific questions. You will perform virtual experiments to answer these questions. Be sure to keep track of what you do experimentally, as the results from earlier experiments may help with the more complicated later experiments.

Figure 1. Main Window of the Nerve Program. PROGRAM SETUP Start the Nerve program. If the program was running, some of the parameters may be different from the default values. To correct this, press F10 (or click on RESET parameters) which will revert most of the parameters to their default values. Different simulation modes are accessed using the buttons on the left side of the main screen (Figure 1). For example, to initiate an action potential simulation, just click on the Membrane AP button. This action opens a window that has a plotting area and several buttons to perform different operations. Some of these operations can also be done by using the keyboard shortcuts listed below.

Foundations of Oral and Systemic Disease Fall 2013 4 EXPERIMENTAL DESIGN Shortcut keys for Menus and Commands F3: Run Propagated AP simulation (from the Propagated AP window) F6: Set axon parameters F7: Set stimulus properties F8: Change the ionic conditions F9: Control what you see on the screen using the Variables to Plot menu F10: Reset parameters to default values (see main screen, Figure 1) ESC: Close the active window

Current Clamp vs. Voltage Clamp Axon properties can be studied using two different experimental approaches: voltage clamp or current clamp. In voltage clamp, the experimenter changes the voltage and measures the resulting current(s). This technique was used by Hodgkin and Huxley, and it remains the most effective way to characterize the functional properties of voltage-gated ion channels. The laboratory experiments, however, use current clamp mode, in which the experimenter applies current pulses (stimuli) to the interior of the axon and observes how the membrane voltage changes in response. This simulates physiological conditions to provide more direct insight into how the normal axon works. In current clamp mode, the applied stimulus causes a proportional linear change in membrane voltage, which in turn activates voltage-gated ion channels. If threshold is reached, an action potential ensues. The program is set up to let you see how changes in channel activity affect membrane voltage, the approach to threshold, and the generation (or not) of action potentials. The purpose of the lab is to understand the complexities of the Hodgkin cycle, namely, how changes in voltage control channel activity, thereby allowing ionic currents to flow through the membrane and change the membrane voltage. Membrane vs. Propagated Action Potential In a real axon, changes of membrane voltage at one location differ from changes in other locations because the spread of the voltage change is not instantaneous. In the Membrane Action Potential simulations (Membrane AP button in Figure 1) this problem is eliminated by inserting an axial wire to make the interior of the axon isopotential (equal in voltage) along its entire length. Experiments 14 use the axial wire. In experiment 5, the Propagated Action Potential simulation (V vs. t Propagated AP button in Figure 1), the axon does not contain an axial wire. The stimulus is applied at a discrete location and the action potential is measured using electrodes inserted at different positions along the length of the axon.

Foundations of Oral and Systemic Disease Fall 2013 5 Experiment 1. Relationship between K conductance and K current To start, click on the Membrane AP button. You will run the simulation program using "ideal Na+ and K+ channel properties" where only sodium and potassium ions go through their respective channels (i.e. in an ideal Na+ channel, only Na+ can permeate). Therefore, click on the Axon button (or press F6) and select Ideal channels. Now close the Axon window and look at the AP simulation, which should look like Figure 2. This is a plot of the membrane voltage as a function of time (black trace, see Figure 2) along with the current pulse used to stimulate the axon, also as a function of time (red trace, see Figure 2). In this experiment, you have injected a current pulse (10 A in amplitude and 0.25 ms duration) into the axon and recorded the membrane potential. These are the default values, but in later experiments you will be able to change them.
+ +

Figure 2. Membrane Action Potential.

Let us now explore the time course of some of the variables involved in generating an action potential. The program allows you to select and plot many of the important variables on the same screen. For example, let us look at what is happening to the potassium current amplitude (IK) and the potassium conductance (gK), during the action potential. Remember that the K+ conductance is directly related to the number of open K+ channels. l. Click on Var. to plot, (or press F9), which will open the Variables to Plot window, and click on the potassium conductance (gK) and the potassium current (IK). Then close the Variables to Plot window. Now you should see plots of the membrane voltage, gK, and IK (Figure 3). In which direction Figure 3. Membrane AP, IK and gK. is the potassium current flowing, into or out of the cell? By clicking on the right hand axis you can sequentially display the scales for Im, gK and IK. You can also vary the range for the gK and IK plots by expanding or compressing the plotted scales using the E and C buttons in the upper right hand corner. For example, after you have clicked to display gK, the default full scale will be 20 mS/cm2 and if you press the E button, the maximum scale will expand to 10 mS/cm2 and the gK trace will be twice as big, allowing you to see more

Foundations of Oral and Systemic Disease Fall 2013 6 details. Press the C button to go back to the original scale. You can use the same steps to select a different plotting scale for IK. Remember, you can view the scale for each variable by repeatedly clicking on the right hand axis. In addition, by clicking the mouse in the plotting area, a vertical cursor appears and the values of time, voltage, and selected variable (on the right hand axis) are displayed at the time signaled by the cursor. Figure 3 shows the screen that you will see. Notice that the time courses for IK and gK are different. Why does the IK curve reach its peak before gK? [Hint: What two factors determine the amplitude of the current?] Display the equilibrium potentials for Na+ and K+ (ENa and EK, respectively, by clicking on E's button in the upper left hand corner. Compare EK (blue line) and the membrane voltage (black trace) at different time points during the action potential. The numeric value of EK can be found be clicking on CONC (Concentrations, F8). Does this value seem reasonable? Remember, the value of EK depends on the concentration gradient of K+ across the membrane (Nernst equation). Observe the falling phase of the potassium current. Why does the potassium current (IK) fall to nearly its resting level while gK is still much greater than 0? Is IK equal to 0? You can check this more accurately by using a higher gain (higher magnification using the E button) for the IK plot. Once you understand the reasons for the different time courses of IK and gK, you can add the n curve to your plot. The screen should look like Figure 4. n is the probability that an individual voltage sensor is in its activated (up) state. (Each voltage sensor has one n gate). In the tetrameric K+ channel, there are Figure 4. Membrane AP, IK, gK and n. four voltage sensors (and therefore four n gates) per channel, one per subunit. Remember that all four n gates must activate in order for the pore to open and conduct K+. The probability of opening of the potassium channel is: Po = n4. Click on the right hand axis until you display the scale for the n curve. Note that the n curve does not start at 0. Why might that be? Note that there is a small K+ current when the resting value of n is ~0.25. Why? Also, why is IK nearly 0 at the end of the action potential even though at this time n is ~0.7, which is significantly larger than its resting value? Experiment 2. Relationship between Na+ conductance and Na+ current Now you will consider how Na+ ions are involved in the action potential. Click on Var. to plot and deselect the K+ variables and check the Na+ variables, INa and gNa. You should see plots of the membrane voltage, gNa, and INa. Remember, as before, you can display the scales for gNa and INa by repeatedly clicking on the

Figure 5. Membrane AP, INa and gNa

Foundations of Oral and Systemic Disease Fall 2013 7 right hand axis. As before, each scale can be expanded or contracted using the E and C buttons in the upper right hand corner. In Figure 5, gNa, INa, and membrane voltage are plotted on the screen. Notice that the time courses of INa and gNa are different. Several interesting and important features the Na current trace can be revealed by answering the following questions: -In which direction does the sodium current flow during the action potential? -Why is there an upward notch in the INa trace? What is happening during the notch? [Hint: display ENa and consider the relationship of ENa to the membrane voltage Vm.] -What does the existence of a notch tell you about the relation between INa and gNa? Also, notice that the peak INa curve occurs later than the peak of the gNa curve. Why? Figure 6. Membrane AP, INa, gNa, m and h. Once you understand the reasons for the different time courses of INa and gNa, add the m and h curves to your plot. The screen should look like Figure 6. You may find it useful to simplify your screen by removing the sodium current (deselect INa in Var. to plot). Compare the time course of the action potential with the m, h, and gNa curves. Click on the right hand axis to display the scale for the m or h curves. Remember that m represents the probability that one of the Na+ channel voltage sensors is in the activated (up) position. In the Hodgkin & Huxley model, three m gates (m3) must be activated for the pore to open and conduct Na+. Remember too that h represents the probability that the channel is NOT inactivated. The inactivation gate must be in its open position for the channel to conduct Na+. Therefore, the probability that the Na+ channel is in the open (conducting) state is: Po=m3h. (Po is of course directly proportional to gNa.) Also note that the probability that the channel is inactivated (i.e. inactivation gate closed) is given by 1-h. Thus, from these two curves you can get an appreciation of how the two gates of the Na+ channel act to regulate its permeability. Note that the m curve does not start at 0 (what does this mean?) and that the h curve does not start at 1 (what does this mean?). How are these features consistent with the negligible Na+ current at the resting membrane potential? In addition, why does the sodium conductance (gNa) begin to fall while the channel's m gate is still open? What would occur if you tried to stimulate the nerve with a second current pulse delivered at the point in time when of the sodium conductance reaches the peak? Don't do this experiment now, just think about it; you have enough information to make a definitive statement. The logic here will help you to understand the ionic mechanisms responsible for the absolute and relative refractory periods (more on this later). Experiment 3. Threshold for action potential firing You will now examine threshold, which defines whether the action potential will or will not occur. Restore the action potential parameters to normal, making sure that you maintain "ideal"

Foundations of Oral and Systemic Disease Fall 2013 8 channel properties (click on RESET parameters, or press F10 but make sure that ideal channels is selected in the Axon parameters window). In the pulses (F7) window set the amplitude to 10 A and the total duration to 10 ms. Now, experimentally adjust the amplitude of the current pulse to identify the amplitude that is required to just exceed threshold (that is, to generate an action potential). To see the relationship between different stimulus intensities you can save each screen by clicking in SAV ALL and checking super to superimpose the traces as you change the stimulus strength.

Figure 7. Superimposed APs obtained with 6.6 and 6.7 A stimulus pulses, respectively.

Figure 7 shows one example of two superimposed traces. A stimulus of 6.7 A initiated an action potential (the stimulus was suprathreshold). In contrast, a 6.6 A stimulus did not (the stimulus was subthreshold). Experiment with the amplitude and observe how sharp the threshold is. Why does the progressive reduction of the stimulus intensity cause the onset of the action potential to occur at progressively longer times after the cessation of the stimulus? [Hint: It might help to examine the total ionic current at high magnification, together with the Na+ and K+ currents. Observe the direction of the currents just when the action potential is taking off]. Also, why does the membrane potential remain almost flat past the turn off of the current pulse when the stimulus is just suprathreshold? Try now to correlate the threshold events with the conductances (and also plot the h variable). Once you see how the membrane voltage is related to the membrane conductances you should be able to explain why the stimulus that is just suprathreshold generates a smaller than normal action potential (i.e., compared with the action potential resulting from a stimulus of 10 A). Experiment 4. Absolute and relative refractory periods So far you have examined the properties of a single action potential; now you will study how one action potential can affect the generation of a second action potential. Start by restoring the default parameters (RESET parameters). In the pulses window, increase the Total Time to 20 ms and add a second stimulus pulse (set PULSE 3 to 10 A Amplitude, and 0.25 ms Duration. Then modify the duration of PULSE 2 (which operates as an interval during which no stimulus is applied) to 12 ms. Make sure that the

Figure 8. Two paired stimuli separated by 12 ms generate similar action potentials.

Foundations of Oral and Systemic Disease Fall 2013 9 amplitudes of the two stimuli (first and third pulses) are the same. You should see two action potentials as shown in Figure 8. Save this screen (click on SAV THIS). Now you will change the first stimulus, PULSE 1, and see how this affects the second action potential. First, progressively decrease the amplitude of PULSE 1 and observe the second action potential waveform. When you decrease PULSE 1 to an amplitude that is near threshold (i.e., the amplitude of the first stimulus set between 6.7 -7 A), the second action potential is abolished. Try different amplitudes for PULSE 1. Why does PULSE 3, which was previously suprathreshold, become so much less effective in eliciting an action potential and even become subthreshold if PULSE 1 is made small enough? If you change PULSE 3, can you elicit a second action potential? If a second action potential can be generated by changing PULSE 3, the axon is in the relative refractory period. Now let us explore the generation of a second action potential as a function of the time between the two stimuli. In the Stimulus window (press F7) reset the first stimulus to 10 A and 0.25 ms and change the Interval to 6 ms. Keep changing the stimulus amplitude of the second pulse until you have an action potential (e.g. 50 A, Figure 9). Then repeat this process by making the interval even shorter and modifying the amplitude of the second pulse to recover the second action potential. Why can you not obtain a second action potential regardless of the amplitude of the second pulse when you decrease the interval below a critical level? If a second action potential cannot be generated regardless of the properties of the second stimulus, the axon is in the absolute refractory period. What do these results tell you about refractory periods and threshold? Be sure that you understand the mechanisms underlying these processes. Try to explain these results remembering what you know about Na+ channel inactivation and K+ channel activation and deactivation. A simultaneous plot of h and gK is very useful. There are many physiological factors that can affect the excitability of a cell. Dramatic changes in excitability can occur, for example, in response to small changes in ionic concentrations or when cells are exposed to low concentrations of drugs and toxins. You may want to use the program on your own to study some of these effects. Experiment 5. Propagated Action Potentials Close the Membrane AP window and click on the Stimulus button to set up the standard 0.25 ms duration pulse with a 10 A pulse amplitude stimulus and a Total Time of 10 ms. Press the RESET parameters button (or press F10 to reset). Then click on the V vs. t-Propagated AP

Figure 9. Two paired stimuli separated by 6 ms. The second stimulus requires a larger amplitude than the first one.

Foundations of Oral and Systemic Disease Fall 2013 10 button and observe the time course of the propagated action potential as detected at three points along the axon, denoted by three electrodes in Figure 10. In this experiment, there is no axial wire. The stimulus is injected locally at the spot marked Stim on the depiction of the axon at the top of the screen (Figure 10). The voltage is measured at different locations indicated by the black, blue, and red recording electrodes. Compute the conduction velocity. This can be easily accomplished using the cursor feature. Click with the mouse at the peak of the blue action potential and you will get the time and the value of the voltage at this point: take note of the time and the position of the electrode. Next, click on the peak of the red action potential and take note of the time and position of the electrode. Knowing the time difference and the distance between the electrodes you can compute the conduction velocity. Note the radius of the axon during the previous simulations and now change it to 400 m. Run the simulation again. What happened? How would you cause an action potential in the axon with the larger radius? [Hint: Vary the stimulus intensity.] Make sure you understand how the effectiveness of the stimulus is related to axon radius. Once you obtain an action potential, calculate the new conduction velocity for this larger diameter cell. Did it increase, decrease or remain the same? Did the shape of the action potential waveform change? Now change the radius of the axon to 100 m, but before running the simulation think about the magnitude of the stimulus pulse. What type of a voltage response do you expect in the nerve if you use the same stimulus parameters as you used with the 400 m radius axon? After you adjust the stimulus, calculate the conduction velocity. After completing the measurements at several radii, what can you conclude about the effect of fiber diameter on conduction velocity? Be sure that you can give an explanation of the mechanisms responsible for the conduction velocity in an axon.

Figure 10. The propagated action potential recorded at three different sites

NOTE: In the propagated action potential window, to see the effect of any change in parameters, you must rerun the simulation by clicking on the START button (or press F3).

Foundations of Oral and Systemic Disease Fall 2013 11 SYNAPSE Synaptic Transmission Simulation INTRODUCTION The experiments described below use the Synapse program which simulates synaptic transmission at the amphibian neuromuscular junction (or motor end plate). This experimental preparation was used to work out most of the basic features of synaptic transmission. The mechanisms you will investigate are applicable to most mammalian synapses. The experiments are designed to enhance your understanding of both presynaptic and postsynaptic mechanisms involved in synaptic transmission. Representative examples of the types of traces you will observe are provided in each experiment. However, because the stochastic nature of synaptic transmission is realistically modeled in Synapse, the responses that you actually observe will be slightly different than those in the laboratory manual. Synapse is a typical Windows application that is controlled by clicking menu options. For example, click the Run menu to run a simulation under various conditions. Choose the Presynaptic Terminal, Synaptic Cleft, and Postsynaptic Cell menus to change conditions inside the presynaptic terminal, at the synaptic cleft and in the postsynaptic motor end plate, respectively. Hot key combinations for many of these operations are available.

Hot key combinations for Menus and Commands F3 F4 F7 F8 F9 Ctrl+F8 Ctrl+F9 Shift+F7 Shift+F8 Shift+F9 Shift+F5 Run > Run Simulation Run > Check to Superimpose Traces Presynaptic Terminal > Stimulus Presynaptic Terminal > Physiological Parameters Presynaptic Terminal > Curves to Plot Synaptic Cleft > Physiological Parameters Synaptic Cleft > Curves to Plot Postsynaptic Cell > Stimulus Postsynaptic Cell > Physiological Parameters Postsynaptic Cell > Curves to Plot Postsynaptic Cell > Postsynaptic Cell Parameters

Foundations of Oral and Systemic Disease Fall 2013 12 PROGRAM SETUP Start by examining a typical response recorded at the neuromuscular junction. Once Synapse is running you will observe the main menu. Begin by clicking Run, then click on Run Simulation (i.e., Run>Run Simulation) to start the simulation. Your screen should look like Figure 1.

Figure 1 Note: If the screen does not closely resemble Figure 1, then check the Experimental Preparations menu to see that the Amphibian Neuromuscular Junction is checked. Also, reset the default conditions by clicking Experimental Preparations>Default Experimental Settings. Note that pressing the hot key combination, Ctrl+D, will also reset the default conditions.

Hint: you can display the equilibrium potentials for K+ and Na+ in the presynaptic motor neuron and the postsynaptic muscle cell by clicking View>Equilibrium Potentials and checking the appropriate options.

Examine the voltage traces displayed in the window. The black trace is the presynaptic action potential as recorded intracellularly at the presynaptic nerve terminal. The color trace is the postsynaptic membrane potential recorded in the muscle fiber. Notice that there are scale displays for the pre- and the postsynaptic signals on the left and right sides of the screen, respectively. Compare the shapes of the presynaptic and postsynaptic action potentials. What differences do you see? Note that the presynaptic action potential is similar to the action potential

Foundations of Oral and Systemic Disease Fall 2013 13 you studied using the Nerve program. Why does the presynaptic action potential have an undershoot (afterhyperpolarization) whereas the postsynaptic action potential does not? Why is there a slow component of repolarization at the end of the muscle action potential? The membrane potential waveform in the postsynaptic muscle cell is complex because several types of channels contribute to current flow across the membrane: 1) Current flow through the nicotinic acetylcholine receptor is called the end plate current (EPC). The EPC changes the membrane potential of the muscle cellthis component of the voltage response is called the end plate potential or EPP. 2) The muscle membrane also contains a variety of voltage-gated channels, including voltage-gated Na+, K+, and Cl- channels. Current flow through these channels sums electrically with the EPC to generate changes in the postsynaptic membrane potential. To better study synaptic transmission and the effect of acetylcholine on the postsynaptic muscle cell, it is helpful to examine the EPP in the absence of the action potential. Note that the Synapse program displays the postsynaptic membrane potential and does not plot the EPP in isolation. However, if the action potential does not occur, most of the voltage response will correspond to the EPP. EXPERIMENTAL DESIGN Experiment 1. The end plate potential and the end plate current To study the time course of the EPP in isolation, you must eliminate the action potential. To do this, you must reduce the magnitude of the local depolarization so that it is below threshold for the initiation of the muscle action potential. This can be achieved by using low doses of a postsynaptic blocking agent like curare, which is a competitive inhibitor of nicotinic acetylcholine receptors. As you will see later, similar results can be achieved by reducing the external Ca2+ concentration. Initially, you will use curare to reduce the amplitude of the EPC. Click on Synaptic Cleft>Physiological Parameters and add some curare to the external solution bathing the preparation. To change values you can either use the spin bars or directly enter values into the field. Begin with approximately 1.0 M curare. Once you have entered a curare concentration click Run>Run Simulation to start the simulation. Gradually increase the curare concentration to see how the postsynaptic response changes. You can change the amplification of the displayed traces to better see changes in the value of the EPP by clicking Postsynaptic Cell>Curves to

Figure 2

Foundations of Oral and Systemic Disease Fall 2013 14 Plot. Then modify the amplitude scale value by clicking the spin bar. Once you have a good understanding of the effect of curare on the amplitude of the postsynaptic membrane potential, set the curare concentration to 4 M. To best see the EPP, adjust the amplitude gain of the Postsynaptic Membrane Potential to 5 mV/div. Your screen should look like Figure 2. You have now attained a situation in which the local depolarization of the muscle membrane primarily reflects the EPP because the EPC (i.e. ion flow through the acetylcholine receptor channels) constitutes most of the current flowing across the postsynaptic membrane. To display the current through the AChR channels, click Postsynaptic Cell>Curves to Plot and check Synaptic Current. Here synaptic current refers to the end plate current (EPC). Adjust the current scale to 0.1 mA/cm2 and run the simulation (your display should look similar to Figure 3). Notice the shape and time course of the synaptic current trace. This trace shows only the current through the synaptic channels opened by the neurotransmitter. The scale values for each trace can be selected by clicking in the space between the scale values (see arrowhead on the right side in Figure 3). What is the direction of EPC flow? What ions carry this current? Note: you can save experimental parameters by clicking File>Save Experimental Protocols.

Figure 3 Remember: an inward flow of positive ions is depicted in the downward direction (i.e. as a negative current).

Display the total postsynaptic membrane current by clicking Postsynaptic Cell>Curves to Plot and checking Total Current. If you have added sufficient curare, you should see that the total current and the EPC superimpose. (Compare them after setting the scales for each to the same amplification.) Why is the contribution from voltage-gated Na+ and K+ channels so small even though the postsynaptic membrane has been depolarized? Compare the time course of the synaptic current with the time course of the EPP. Why is the time course of the synaptic current (EPC) different from the time course of the EPP waveform? Why does the EPC reach its peak before the EPP? Hint: Think back to the Nerve Lab (Experiment 3). What is the relationship between membrane voltage and current?

Foundations of Oral and Systemic Disease Fall 2013 15

Examine the initial onset phase and the decay phase of the synaptic current trace. What do you think is responsible for the jagged, uneven nature of the rising phase of the synaptic current trace in this experiment? Experiment 2. The postsynaptic current and the reversal potential In this experiment you will analyze the properties of the synaptic current (EPC). Start by reinitializing the system with the default settings (Experimental Preparations>Default Experimental Settings). Run the experiment without curare (Synaptic Cleft>Physiological Parameters), at 20 mV/div (Postsynaptic Cell>Curves to Plot) and remove the histogram of quantal release (Synaptic Cleft>Curves to Plot). Display the synaptic current and set the scale to 0.5 mA/div (Postsynaptic Cell>Curves to Plot>Synaptic Current). Now you should see the presynaptic action potential, the complex postsynaptic action potential combined waveform, and the synaptic current (see Figure 4).

Figure 4 Examine the synaptic current trace. Notice that there is an inward phase (as observed in the previous experiment), followed by an outward phase (not seen before). How can you account for the outward direction of current flow in the synaptic current trace? Why didnt you see this outward flow of synaptic current in the previous experiment? You can now display the Na+ and K+ contributions to the synaptic current by selecting Postsynaptic Cell>Curves to Plot and checking Na and K (top right). Remember, the AChR channel is non-selective; Na+ and K+ can both go through. What can you say about these current components when the total synaptic current reverses? At what postsynaptic membrane potential do you predict the synaptic current to reverse direction (reversal potential)? Can you explain why the magnitude of the inward current is larger than the magnitude of the outward current?

Foundations of Oral and Systemic Disease Fall 2013 16 NOTE: There is more than one explanation for the larger inward current. Think about what experiments you could do with the program to determine which is the most important explanation. Before proceeding, make sure that you understand the salient features of the synaptic current, and how they are related to the action potential waveform. Experiment 3. The Quantal Nature of Synaptic Transmission The next series of experiments is designed to investigate the presynaptic mechanisms involved in the release of neurotransmitters. In Experiment 1 you used curare to eliminate the action potential so that the EPP could be studied in isolation. In this experiment you will study the quantal nature of synaptic transmission at the neuromuscular junction. As before, the amplitude of the EPP must be reduced to below threshold so that it can be studied in isolation from the action potential. However, in this experiment, the EPP amplitude will be reduced by blocking the presynaptic mechanism of release. This will be achieved by lowering the external Ca2+ concentration. Be sure that you understand the difference between pre- and postsynaptic methods of blocking synaptic transmission. To begin, run the default simulation parameters with the postsynaptic membrane potential gain set at 20 mV/div. You can investigate the quantal nature of synaptic transmission by displaying a histogram of the quanta released. First click Synaptic Cleft>Curves to Plot and check both the Histogram of Quantal Released box and the Do Not Superimpose Histograms box. Then click View>Check to Show Quantal Content to display a quantal content counter. This displays the number of quanta released, which is equivalent to the number of synaptic vesicles fusing with the presynaptic membrane. Rerun the simulation. Figure 5 shows a typical example in which the presynaptic response caused the release of 151 quanta (i.e., the quantal content, m = 151). Your value will probably be different. Why? Now, rerun the simulation several times. What did you notice about the synaptic current trace?

Figure 5

To help see the difference, try superimposing the traces by clicking Run>Check to Superimpose Traces.

Foundations of Oral and Systemic Disease Fall 2013 17 Normal amounts of transmitter are released when the Ca2+ and Mg2+ concentrations are set to 2.0 and 0.0 mM respectively (the default settings). The effect of Ca2+ on transmitter release can be seen by progressively changing the concentrations in the external solution. To alter concentrations click on Presynaptic Terminal>Physiological Parameters and change the appropriate values. Try setting Ca2+ to 1.5 mM and run the simulation. How is synaptic transmission affected by reducing external Ca2+ concentration to 1.5 mM? How does the change in transmitter release affect the postsynaptic response? Under what conditions does the postsynaptic response rise more rapidly? Repeat this same experimental protocol using 1.0 mM Ca2+. The change in the muscle membrane potential should be significantly reduced, but still above threshold. As you rerun the simulation at these new Ca2+ concentrations notice that the muscle fiber action potential waveform changes. How does the shape and size of the muscle fiber action potential change? Why does the initial rate of the depolarization change? Continue to alter the Ca2+ concentration until only 2 - 4 quanta are released in each trial. As release is decreased, the gain of the postsynaptic membrane potential should be increased to 1 mV/div. Now the EPP constitutes all or virtually all of the voltage response in the muscle. Superimpose traces to see how release changes from trial to trial. The screen should look approximately like Figure 6. How has the postsynaptic response changed as the number of quanta released has decreased? Note: To best observe the behavior of the release process under these varying conditions each new simulation paradigm should be run several times while superimposing the traces.

Figure 6

Notice the shape of the EPP, its rise time, and its time of onset (i.e., the synaptic delay). What causes the wide fluctuations in these features of the EPP? Why does the rate of rise of the EPP seem to change in some EPPs? Why does the time of EPP onset shift in different trials? Synaptic transmission can be even further reduced so that sometimes no transmitter is released in response to a normal presynaptic action potential (i.e., presynaptic activity produces a failure). Adjust the Ca2+ value so the response fails approximately 50-70% of the time. Under these conditions, transmitter release consists mainly of single quanta. Measure the amplitude of a number of single quanta EPPs (click View>Measure Values; click the left mouse button to measure the EPP amplitude). What could account for EPPs having different quantal sizes? Rerun

Foundations of Oral and Systemic Disease Fall 2013 18 the simulation several times. You should see that the variation in the synaptic delay is even more clearly seen now, i.e. release occurs at different times in each run. Why does the time of release vary each time the presynaptic nerve terminal is depolarized?

Experiment 4. Presynaptic Calcium Current and Synaptic Transmission The results in the previous experiment clearly demonstrate that synaptic transmission is highly sensitive to the Ca2+ concentration in the external medium. However, neurotransmitter release occurs when Ca2+ enters the presynaptic nerve terminal. To further investigate this process, you will now examine how changes in presynaptic Ca2+ current result in changes in neurotransmitter release. Run the same simulation protocol as above with about 50% failures with the presynaptic Ca2+ current trace displayed (click on Presynaptic Terminal>Curves to Plot and check Calcium Current with the scale set to 0.005 mA/cm2 ). First run simulation as just described above (i.e., as the control for this experiment). Then, change the external Ca2+ concentration slightly and rerun the simulation with superimposition of traces turned on. Observe how the Ca2+ current trace changes and how this is reflected in the amount of neurotransmitter released. Figure 7 is an example of typical results you might see. Try several different Ca2+ levels, e.g. 0.5, 1.0 and 2.0 mM. How would you describe the sensitivity of the neurotransmitter release mechanism and changes in Ca2+ current to changes in external Ca2+ concentration?

Figure 7.

To finish, you will look at the effect of Ca2+ channel blockers. Restore the external Ca2+ concentration to 2 mM. Rerun the simulation. You can test the effect of nifedipine (a Ca2+ channel blocker of the dihydropyridine class; related drugs are sometimes used to treat heart disease) by selecting Synaptic Cleft>Physiological Parameters and increasing the concentration of nifedipine with the spin bar. Try 5, 10, and 50 M. What happens to the Ca2+ current? What happens to the quantal content and the voltage response in the muscle? What can you conclude about the role of Ca2+ in synaptic transmission? What is the site of action (presynaptic or postsynaptic; intracellular or extracellular)?