Neurophysiology of Nerve Impulses

M52_MARI0000_00_SE_EX03.qxd 8/22/11 2:47 PM Page 358 3 E X E R C I S E Neurophysiology of Nerve Impulses Advance Preparation/Comments Consider doing a short introductory presentation with the following elements: Explain how the resting membrane potential is established and maintained. Clearly distinguish between graded potentials and action potentials. Explain the importance of threshold and why an action potential is considered all or none. Describe the relationship between stimulus strength and action potential frequency. Explain the factors that affect conduction velocity. Answers to Questions/Experimental Data Pre-lab Quiz in the Lab Manual 1. Conductivity 2. a. depolarization 3. b. K 4. absolute refractory period 5. a. gastrocnemius and sciatic Activity 1: The Resting Membrane Potential (pp. PEx-36 PEx-39) Predict Question 1: If the extracellular K + concentration is increased, the resting membrane will become less negative. Chart 1: Resting Membrane Potential Extracellular fluid (ECF) Microelectrode position Voltage (mv) Control Cell body, extracellular 0 Control Cell body, intracellular 70 Control Axon, extracellular 0 Control Axon, intracellular 70 High K + Axon, intracellular 40 High K + Axon, extracellular 0 High K + Cell body, extracellular 0 High K + Cell body, intracellular 40 Low Na + Cell body, intracellular 72 Low Na + Cell body, extracellular 0 Low Na + Axon, extracellular 0 Low Na + Axon, intracellular 72 358

M52_MARI0000_00_SE_EX03.qxd 8/22/11 2:47 PM Page 359 1. The resting membrane potential is the same because the permeability of the ions is the same in both locations. 2. If the sodium-potassium pump is blocked, sodium will flow in, depolarizing the membrane. 3. The resting membrane potential would become more negative because there would be more negative anions inside the cell. Activity 2: Receptor Potential (pp. PEx-39 PEx-41) Predict Question 1: The moderate intensity pressure modality will induce a receptor potential of the largest amplitude. Predict Question 2: The moderate intensity chemical modality will induce a receptor potential of the largest amplitude. Chart 2: Receptor Potential Receptor potential (mv) Stimulus modality Pacinian (lamellar) corpuscle Olfactory receptor Free nerve ending None 70 70 70 Pressure Low 60 70 70 Moderate 45 70 70 High 30 70 65 Chemical Low 70 64 70 Moderate 70 58 70 High 70 45 70 Heat Low 70 70 60 Moderate 70 70 40 High 70 70 20 Light Low 70 70 70 Moderate 70 70 70 High 70 70 70 1. Graded receptor potentials can be depolarizing or hyperpolarizing. Since graded receptor potentials can be hyperpolarizing, they can make it more difficult to induce an action potential. 2. Membrane hyperpolarization is when the membrane becomes more negative than the resting potential. 3. The adequate stimulus for sensory receptors in the ear is a moderate intensity pressure stimulus. Intense pressure could inappropriately activate the sensory receptors in the ear. Activity 3: The Action Potential: Threshold (pp. PEx-41 PEx-42) Predict Question 1: The action potential will not change when the stimulus voltage is increased. 359

M52_MARI0000_00_SE_EX03.qxd 8/22/11 2:47 PM Page 360 Chart 3: Threshold Stimulus voltage (mv) Peak value at R1 (µv) Peak value at R2 (µv) Action potential 10 0 0 No 20 100 100 Yes 30 100 100 Yes 40 100 100 Yes 50 100 100 Yes 1. Each region of the neuron contains distinct membrane proteins that provide the basis for the threshold differences. 2. The action potential is regenerated by the influx of sodium which establishes local currents that depolarize adjacent sections of the membrane to threshold. The action potential must be regenerated at adjacent sections of the membrane. 3. The action potential is not graded. It is all or none, so the peak value of the action potential doesnít change. Activity 4: The Action Potential: Importance of Voltage-Gated Na Channels (pp. PEx-42 PEx-44) Predict Question 1: If you apply TTX between recording electrodes R1 and R2, TTX will block the response at R2 but have no effect at R1. Predict Question 2: If you apply lidocaine between recording electrodes R1 and R2, lidocaine will block the response at R2 but have no effect at R1. Chart 4: Effects of Tetrodotoxin and Lidocaine Peak value of response (µv) Condition Stimulus voltage (mv) Electrodes 2 sec 4 sec 6 sec 8 sec 10 sec Control 30 R1 100 100 100 100 100 Control 30 R2 100 100 100 100 100 TTX 30 R1 100 100 100 100 100 TTX 30 R2 100 100 0 0 0 Lidocaine 30 R1 100 100 100 100 100 Lidocaine 30 R2 100 100 100 0 0 1. The sodium channels are voltage-gated sodium channels. Closure of the inactivation gate is a delayed response to the initial depolarization to threshold which closes the inactivation gate about 1 msec after sodium channels open. 2. Careful preparation of the pufferfish requires removal of the toxic portions that contain tetrodotoxin. The poison is most concentrated in the liver, ovaries and skin of the fish. 3. Calcium channels could possibly substitute for sodium channels to provide an influx of cation. Activity 5: The Action Potential: Measuring Its Absolute and Relative Refractory Periods (pp. PEx-44 PEx-45) Predict Question 1: If you further decrease the interval between the stimuli, the threshold for the second action potential will be higher (requiring a larger depolarization). 360

M52_MARI0000_00_SE_EX03.qxd 8/22/11 2:48 PM Page 361 Chart 5: Absolute and Relative Refractory Periods Interval between stimuli (msec) Stimulus voltage (mv) Second action potential? 250 20 Yes 125 20 Yes 60 20 No 60 25 No 60 30 Yes 30 30 No 30 35 No 30 40 No 30 45 Yes 15 60 Yes 7.5 60 Yes 3.75 60 No 1. A refractory period is when the membrane is less excitable. When local currents develop, the action potential must move forward because the previous section is in the absolute refractory period. 2. A long absolute refractory period ensures that the muscle contracts fully. 3. The benefit of a relative refractory period in an axon of a sensory neuron is that it is easier to modify the intensity of the sensation by altering the frequency of stimulation. Activity 6: The Action Potential: Coding for Stimulus Intensity (pp. PEx-46 PEx-47) Predict Question 1: Increased stimulus intensity will increase the frequency of action potentials. Chart 6: Frequency of Action Potentials Stimulus voltage (mv) Stimulus duration (msec) ISI (msec) Action potential frequency (Hz) 20 0.5 20 500 100* 10* 30 500 60* 16.6* 45 500 30* 33.3* * The data in these columns are populated by student calculations. 1. The action potential frequency would be increased in the hot water when compared to the warm water. 2. The two determinants that are being overcome are the frequency of stimulation required to overcome the relative refractory period, and the greater than threshold stimulus required during the relative refractory period. 3. Two ways to overcome the relative refractory period are to increase the stimulus frequency and the strength of the stimulus. Pharmacologically, this would require timed release of the medication in bursts of increasing amounts. 361

M52_MARI0000_00_SE_EX03.qxd 8/22/11 2:48 PM Page 362 Activity 7: The Action Potential: Conduction Velocity (pp. PEx-47 PEx-49) Predict Question 1: The conduction velocity in the B fiber will be slower because the B fiber has a smaller diameter and less myelination. Predict Question 2: The conduction velocity in the C fiber will be slower because the C fiber has a smaller diameter and less myelination. Chart 7: Conduction Velocity Axon type Myelination Stimulus voltage (mv) Distance from R1 to R2 (m) Time between action potentials at R1 and R2 (msec) (sec) Conduction velocity (m/sec) A fiber Heavy 30.1 2.002* 50* B fiber Light 30.1 10.01* 10* C fiber None 30.1 100.1* 1* * The data in these columns are populated by student calculations. 1. The conduction velocity is dependent upon both myelination and the diameter of the axon. The large diameter of the squid axon contributes to its fast reaction. 2. The sharp immediate pain is probably carried by an A fiber with large diameter and heavy myelination. The slower dull pain is probably carried by a C fiber with small diameter and no myelination. 3. The mixture of axon types contributes to the variety of responses generated. Specifically, the rate with which a response is made can vary. Activity 8: Chemical Synaptic Transmission and Neurotransmitter Release (pp. PEx-49 PEx-50) Predict Question 1: There will be no neurotransmitter release if the extracellular calcium is removed. Predict Question 2: When low amounts of calcium are added back to the extracellular solution, neurotransmitter release will increase a small amount. Predict Question 3: There will be less neurotransmitter released when magnesium is added. 1. It is unlikely that sodium could substitute for calcium since the addition of magnesium blocked the calcium channels. 2. Botulinum toxin blocks the release of acetylcholine from the axon terminal. It is used in cosmetic procedures because it results in paralysis of the muscles that are contracting and causing the wrinkles. Activity 9: The Action Potential: Putting It All Together (pp. PEx-50 PEx-52) Predict Question 1: When you apply a very weak stimulus to the sensory receptor, small, depolarizing response will occur at R1, and no responses will occur at R2, R3, and R4. Predict Question 2: When you apply a moderate stimulus to the sensory receptor, a larger, depolarizing response will occur at R1, and an action potential will be generated at R2 and maybe at R4. Predict Question 3: When you apply a strong stimulus to the sensory receptor, a large, depolarizing response will occur at R1 and R3, and action potentials will occur at R2 and R4. 362

M52_MARI0000_00_SE_EX03.qxd 8/22/11 2:48 PM Page 363 Chart 9: Putting It All Together Peak value of response (mv) Stimulus Sensory neuron Interneuron Receptor Axon Axon terminal Receptor Axon None 70 70 Weak 60 0 0 70 0 Moderate 40 16.6 4 50 5 Strong 25 33.3 6 40 10 1. All action potentials are all or none. Threshold must be met, but once it is met, all action potentials are the same. 2. If the axons were unmyelinated, the peak value of the action potential wouldn t change. 363

M52_MARI0000_00_SE_EX03.qxd 8/22/11 2:48 PM Page 364 3 R E V I E W S H E E T EXERCISE Neurophysiology of Nerve Impulses NAME LAB TIME/DATE A C T I V I T Y 1 The Resting Membrane Potential 1. Explain why increasing extracellular K reduces the net diffusion of K out of the neuron through the K leak channels. Increasing the extracellular potassium reduces the steepness of the concentration gradient and so less potassium diffuses out of the neuron. 2. Explain why increasing extracellular K causes the membrane potential to change to a less negative value. How well did the results compare with your prediction? The membrane potential became less negative because less potassium diffused out. If more potassium stays in, it is more positive or less negative. 3. Explain why a change in extracellular Na did not alter the membrane potential in the resting neuron. There are less leakage sodium channels that leakage potassium channels, and more of the potassium channels are open. 4. Discuss the relative permeability of the membrane to Na and K in a resting neuron. The resting neuron is (4 5) times more permeable to potassium because of the increased number of leakage channels. 5. Discuss how a change in Na or K conductance would affect the resting membrane potential. A change in the potassium conductance would have a greater effect on the resting membrane potential than a change in sodium would. A C T I V I T Y 2 Receptor Potential 1. Sensory neurons have a resting potential based on the efflux of potassium ions (as demonstrated in Activity 1). What passive channels are likely found in the membrane of the olfactory receptor, in the membrane of the Pacinian corpuscle, and in the membrane of the free nerve ending? The efflux of potassium ions is maintained by passive potassium channels. 2. What is meant by the term graded potential? Graded potentials are brief, localized changes in the membrane potential that can be either depolarizing or hyperpolarizing. 3. Identify which of the stimulus modalities induced the largest amplitude receptor potential in the Pacinian corpuscle. How well did the results compare with your prediction? The moderate intensity pressure modality induced a receptor potential of the largest amplitude in the Pacinian corpuscle. 364

M52_MARI0000_00_SE_EX03.qxd 8/22/11 2:48 PM Page 365 4. Identify which of the stimulus modalities induced the largest-amplitude receptor potential in the olfactory receptors. How well did the results compare with your prediction? The moderate intensity chemical modality induced a receptor potential of the largest amplitude in the olfactory receptor. 5. The olfactory receptor also contains a membrane protein that recognizes isoamyl acetate and, via several other molecules, transduces the odor stimulus into a receptor potential. Does the Pacinian corpuscle likely have this isoamyl acetate receptor protein? Does the free nerve ending likely have this isoamyl acetate receptor protein? The Pacinian corpuscle and the free nerve ending are not likely to have the isoamyl acetate receptor because they did not respond to chemical stimuli. 6. What type of sensory neuron would likely respond to a green light? Photosensory neurons would respond to green light. A C T I V I T Y 3 The Action Potential: Threshold 1. Define the term threshold as it applies to an action potential. Threshold is the voltage that must be reached in order to generate an action potential. 2. What change in membrane potential (depolarization or hyperpolarization) triggers an action potential? A depolarization in the membrane potential results in an action potential. The membrane potential must become less negative to generate an action potential. 3. How did the action potential at R1 (or R2) change as you increased the stimulus voltage above the threshold voltage? How well did the results compare with your prediction? The action potential didn t change as the stimulus voltage increased. This is because once threshold is met, the event is all or none, not graded. 4. An action potential is an all-or-nothing event. Explain what is meant by this phrase. This means that once threshold is met an action potential occurs. If the stimulus is too small an action potential does not occur. 5. What part of a neuron was investigated in this activity? The trigger zone was investigated. This is where the axon hillock and the initial segment come together. A C T I V I T Y 4 The Action Potential: Importance of Voltage-Gated Na Channels 1. What does TTX do to voltage-gated Na channels? TTX blocks the diffusion of sodium through the voltage-gated sodium channels. 2. What does lidocaine do to voltage-gated Na channels? How does the effect of lidocaine differ from the effect of TTX? Lidocaine blocks the diffusion of sodium through the voltage-gated sodium channels. 3. A nerve is a bundle of axons, and some nerves are less sensitive to lidocaine. If a nerve, rather than an axon, had been used in the lidocaine experiment, the responses recorded at R1 and R2 would be the sum of all the action potentials (called 365

M52_MARI0000_00_SE_EX03.qxd 8/22/11 2:48 PM Page 366 a compound action potential). Would the response at R2 after lidocaine application necessarily be zero? Why or why not? With a compound action potential, the results would not necessarily be zero because some axons could remain unaffected. 4. Why are fewer action potentials recorded at R2 when TTX is applied between R1 and R2? How well did the results compare with your prediction? TTX blocked the sodium channels, preventing the propagation of the action potential from R1 to R2. 5. Why are fewer action potentials recorded at R2 when lidocaine is applied between R1 and R2? How well did the results compare with your prediction? Lidocaine blocked the sodium channels, preventing the propagation of the action potential from R1 to R2. 6. Pain-sensitive neurons (called nociceptors) conduct action potentials from the skin or teeth to sites in the brain involved in pain perception. Where should a dentist inject the lidocaine to block pain perception? Lidocaine should be applied to the receptors to prevent the generation of an action potential that would lead to the perception of pain. A C T I V I T Y 5 The Action Potential: Measuring Its Absolute and Relative Refractory Periods 1. Define inactivation as it applies to a voltage-gated sodium channel. Voltage-gated sodium channels are inactivated when they no longer allow sodium to diffuse through. 2. Define the absolute refractory period. The absolute refractory period is the time in which no action potential can be generated regardless of the strength of the stimulus. 3. How did the threshold for the second action potential change as you further decreased the interval between the stimuli? How well did the results compare with your prediction? The threshold for the second action potential increased as the interval between the stimuli decreased as predicted. 4. Why is it harder to generate a second action potential during the relative refractory period? A greater stimulus is required because voltage gated potassium channels that oppose depolarization are open during this time. A C T I V I T Y 6 The Action Potential: Coding for Stimulus Intensity 1. Why are multiple action potentials generated in response to a long stimulus that is above threshold? The longer stimuli allow time for recovery and the above threshold allows the action potential to occur after the relative refractory period. 2. Why does the frequency of action potentials increase when the stimulus intensity increases? How well did the results compare with your prediction? Action potential can occur more frequently if there is a constant source of stimulation as long as the relative refractory period is reached. 3. How does threshold change during the relative refractory period? The threshold that must be achieved is higher than the original stimulus intensity during the relative refractory period. 366 Review Sheet 3

M52_MARI0000_00_SE_EX03.qxd 8/22/11 2:48 PM Page 367 4. What is the relationship between the interspike interval and the frequency of action potentials? The frequency of the action potentials is the reciprocal of the interspike interval with a conversion from milliseconds to seconds. A C T I V I T Y 7 The Action Potential: Conduction Velocity 1. How did the conduction velocity in the B fiber compare with that in the A fiber? How well did the results compare with your prediction? The velocity of the B fiber was slower because it had a smaller diameter and less myelinated. 2. How did the conduction velocity in the C fiber compare with that in the B fiber? How well did the results compare with your prediction? The conduction velocity of the C fiber was slower because it has no myelination and a smaller diameter. 3. What is the effect of axon diameter on conduction velocity? The larger the axon diameter, the greater the conduction velocity. 4. What is the effect of the amount of myelination on conduction velocity? The greater the myelination, the greater the conduction velocity. 5. Why did the time between the stimulation and the action potential at R1 differ for each axon? The time between the stimulation and the action potential at R1 differed for each axon because the diameter and the degree of myelination varied. 6. Why did you need to change the timescale on the oscilloscope for each axon? This is necessary in order to see the action potentials. The velocity changes so when it get very slow you need a longer time scale. A C T I V I T Y 8 Chemical Synaptic Transmission and Neurotransmitter Release 1. When the stimulus intensity is increased, what changes: the number of synaptic vesicles released or the amount of neurotransmitter per vesicle? The number of synaptic vesicles released increases when the stimulus intensity increases. 2. What happened to the amount of neurotransmitter release when you switched from the control extracellular fluid to the extracellular fluid with no Ca 2? How well did the results compare with your prediction? Without calcium present, no neurotransmitter was released because the exocytosis of the synaptic vesicles is dependent upon calcium. 3. What happened to the amount of neurotransmitter release when you switched from the extracellular fluid with no Ca 2 to the extracellular fluid with low Ca 2? How well did the results compare with your prediction? When a small amount of calcium is added back, a small amount of synaptic vesicles are released. 4. How did neurotransmitter release in the Mg 2+ extracellular fluid compare to that in the control extracellular fluid? How well did the result compare with your prediction? The neurotransmitter release was less when magnesium was added. 5. How does Mg 2 block the effect of extracellular calcium on neurotransmitter release? When magnesium is added to the extracellular fluid it blocks the calcium channels and inhibits the release of neurotransmitter. 367

M52_MARI0000_00_SE_EX03.qxd 8/22/11 2:48 PM Page 368 A C T I V I T Y 9 The Action Potential: Putting It All Together 1. Why is the resting membrane potential the same value in both the sensory neuron and the interneuron? The resting membrane potential is the same value because this is the typical resting membrane potential regardless of the type of neuron. 2. Describe what happened when you applied a very weak stimulus to the sensory receptor. How well did the results compare with your prediction? When you applied a very weak stimulus to the sensory receptor, a small, depolarizing response occurred at R1, and no responses occurred at R2, R3, and R4. 3. Describe what happened when you applied a moderate stimulus to the sensory receptor. How well did the results compare with your prediction? When you applied a moderate stimulus to the sensory receptor, a larger, depolarizing response occurred at R1, and an action potential was generated at R2 and at R4. 4. Identify the type of membrane potential (graded receptor potential or action potential) that occurred at R1, R2, R3, and R4 when you applied a moderate stimulus. (View the response to the stimulus.) Action potentials occurred at R2 and R4 and graded receptor potentials occurred at R1 and R3. 5. Describe what happened when you applied a strong stimulus to the sensory receptor. How well did the results compare with your prediction? When you applied a strong stimulus to the sensory receptor, a large, depolarizing response occurred at R1 and R3, and action potentials occurred at R2 and R4. 368