Action Potential Worksheet PDF

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1st class graded
action potential software exercise assignment...

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2020 Brain and Behaviour:

Practical II Worksheet

Action Potential Simulation This practical class uses a software package called ‘HHSim’ which is available on the computers in the Psychology Teaching Cluster, or can be downloaded from http://www.cs.cmu.edu/~dst/HHsim/ on to your own machine (assuming compatibility). The aim of this practical is to help you understand how nerve fibres can fire action potentials. By exploring various virtual experiments you will (hopefully!) get a feel for the ionic basis of action potential generation by neurons. Work through the experiments outlined below and answer the questions on this worksheet. If you do not finish the worksheet in the practical class you should try to finish it in your own time.

Experiment 1 – the Equilibrium Potential Open HHsim which can be found on the T drive in the Psychology folder. You will see two graphs. The top graph shows the membrane potential (in red) and the external stimulus (in blue). On the bottom graph you can change the display to show what is happening at the different types of channels. Don’t worry about the bottom graph for now. Q1. Click on the red line on the top graph (membrane potential). The current membrane potential is given in the box at the bottom of the screen. What is the value of the membrane potential? A1: (2 marks) -62.6mV We are now going to explore the equilibrium potential of a cell in which only one type of channel is permeable. Open the Channels window by clicking the box in the top menu bar. In the Channels window turn off all of the channels (by clicking on the left hand button which will turn grey when the channel is deactivated) except the passive potassium (K+) channel (passive gK). Now open the Membrane window by clicking the box in the top menu bar. The Membrane window allows you to manipulate the internal and external ion concentrations and the temperature of the preparation (TC). Increase the temperature to normal body temperature (37C). Return to the main menu and click on the green ‘Run’ button at the bottom of the screen. When the recording looks stable press the red ‘Stop’ button and measure the new membrane potential. Q2. What is the membrane potential? A2: (2 marks) -79.5mV

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2020 Brain and Behaviour:

Practical II Worksheet

As only the K+ channels are active the membrane potential is the equilibrium potential for K +. Return to the Membrane window. In the current experiment the intracellular K+ concentration (Cin) is 400 mM and the extracellular concentration (Cout) 20 mM. Change these values and see what effect this has on the membrane potential. Remember to “Run” until the recording is stable. Q3. With an extracellular concentration of 20 mM, what intracellular concentration of potassium do you need to achieve a membrane potential of –58mV? A3: (2 marks) 175mM The normal physiological K+ concentrations are about 140 mM intracellular and 5 mM extracellular. Type in the values. Q4. What is the K+ equilibrium potential for these concentrations at body temperature? A4: (2 marks) -89.1mV

Experiment 2 – The membrane potential In the Channels window turn on all of the passive channels. Now the cell membrane is permeable to K+, Na+ and Cl- ions. The membrane potential is a weighted average of the equilibrium potentials for all three ionic species. The relative permeabilities for K+, Na+ and Cl- of 100%, 4% and 45% are typical of the situation at the resting potential. In the Membrane window change the default ionic concentrations to values more like those found in the human body: Intracellular: 10 mM Na+, 140 mM K+, 5 mM ClExtracellular: 140 mM Na+, 5 mM K+, 115 mM ClQ5. What is the resting membrane potential now? A5: (2 marks) -64.9mV During the action potential the Na+ permeability of the membrane rapidly increases. The permeability of the passive channels can be altered by changing the channel conductance, given on the right hand side of the Channel window. Gradually increase the conductance of the Na+ channels using the top arrow by the value. Watch the membrane potential in the main window and the equilibrium potential for each of the ions in the Membrane Window. Q6. What effect does this have on the equilibrium potentials for the three ionic species and the membrane potential?

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2020 Brain and Behaviour:

Practical II Worksheet

A6: (2 marks) Membrane potential increases to -46.9mV when sodium conductance is increased to 0.0565. Equilibrium potential remained stable for all ionic species. Continue to increase the Na+ channel conductance until you reach a level where the membrane potential is equivalent to the maximum membrane potential seen during an action potential (approx +30 mV) Q7. What is the conductance of the Na+ channel? A7: (2 marks) 0.4865µS

Experiment 3 – Current and conductance changes during an action potential Now we change to a ‘current clamp’ experiment, in which a short pulse of current is given to a nerve fibre to elicit an action potential. In the Channels window activate the Fast Sodium and Delayed Rectifier channels and click the ‘Reset’ button to return the conductance of the passive Na + channel to control levels. In the Membrane Window lower the preparation temperature to 10 C (this is necessary for the simulation to run correctly). Return to the Main window and ‘Run’ until a steady membrane potential of approximately –68 to -69 mV is seen. Open the Stimuli window by clicking the box in the top menu bar. Set stimulus 1 to an intensity of 20 nA and a duration of 1 msec Stimulus intensity = 20 nA

Stimulus duration = 1 msec

Return to the Main Window and click on the ‘Stim1’button on the lower left hand side of screen. The top graph

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2020 Brain and Behaviour:

Practical II Worksheet

shows the size and time course of the action potential (in red) and the stimulation (in blue). Q8. Measure the resting potential before the start of the action potential, the maximum membrane potentiation and the maximal hyperpolarization. A8: (4 marks) Before the start of the action potential: -69.5mV The maximum membrane potentiation: -55.3mV Maximal hyperpolarisation: -80.6mV Q9. What happens to the action potential when you increase/decrease the stimulus intensity? A9: (2 marks) If the stimulus intensity is increased, the membrane potential at the peak of the action potential increases and the maximal hyperpolarisation increases. If the stimulus intensity is decreased, the membrane potential at the peak of the action potential decreases and the maximal hyperpolarisation decreases. Q10. Find the minimum current pulse (with a duration of 1 ms) that can elicit an action potential. A10: (2 marks) 8.5nA Q11. Describe the shape of the action potential at this stimulus intensity when compared with a stimulus of 30 nA. A11: (2 marks) The shape of the action potential at 30nA has a peak membrane potential of 56.2mV whereas the action potential at 8.5nA has a peak membrane potential of 45.9mV. The higher stimulus intensity (30nA) caused in a wider action potential suggesting that this action potential takes a longer period of time to reach resting potential than the action potential at the lower stimulus intensity. At 30nA the action potential shows a steeper incline towards maximum potentiation. This could show that a stronger stimulus intensity means that the action potential reaches maximum potentiation faster. Now let’s use the lower graph to look at the conductance of the Na + and K + channels. A simple way to think of conductance is just as a measure of how active the channels are. In the Main window, at the bottom left hand side of the screen, change the yellow graph line to display the Na+ channel conductance (g_Na), the green graph line to display the K+ channel conductance (g_K), and the blue graph line to be ‘blank’. The lower graph now displays the conductance of the channels during the action potential. Q12. Describe the essential differences in the changes of the Na+ and K + conductances during the action potential. A12: (2 marks)

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2020 Brain and Behaviour:

Practical II Worksheet

The graph shows that sodium conductance (27.9pS) reaches a higher peak than potassium conductance (13.6pS) during the action potential. Na+ conductance increases faster and has a smaller duration than Ka+ due to the sodium channels closing as the action potential is reached. At repolarisation, the potassium channels open which is shown by the increase of K+ conductance. Q13. What is the hyperpolarization due to? A13: (2 marks) Hyperpolarisation is where the membrane potential becomes negative as a result of the opening of voltage gated potassium channels and the movement of potassium down the concentration gradient. The membrane potential descends below the resting point because the opening of K+ channels causes potassium to leave the cell rapidly. This process means that no further action potentials will be fired and prevents the action potential from travelling back down the axon. The role of the hyperpolarization is to prevent the action potential from travelling back down the axon. To test this we will apply 2 stimulations in quick succession. In the Stimuli window set stimulus 1 to deliver 2 electrical pulses. Both should have an intensity of 20 nA and a duration of 1 msec. Alter the length of time between the 2 pulses. What happens to the action potential? [Do this as it relates to the next question]

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2020 Brain and Behaviour:

Practical II Worksheet

Stimulus intensity = 20 nA

Interval between pulses = 1 ms

Stimulus duration = 1 msec

Q14. What is the minimum delay between the pulses that allows 2 action potentials to the activated? A14: (2 marks) 4.2ms

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2020 Brain and Behaviour:

Practical II Worksheet

Experiment 4 – Na+ and K+ dependence of the action potential There are numerous drugs and toxins available that specifically block certain ion channels. You will now apply some of these drugs to study the Na + and K+ currents in isolation. In the Stimuli window set stimulus 1 to deliver a single pulse with an intensity of 10 nA and a duration of 1 msec. Open the Drugs window by clicking the box in the top menu bar. The first drug that we will apply is TEA (tetraethylammonium ions). This drug inhibits K+ channels. Click on the TEA graph to give a concentration of 10 mM (101). This will give 50% inhibition of the channels. Return to the Main window and stimulate by clicking on the Stim1 button. Q15. Describe what has happened to the conductance of the Na+ and K+ channels. A15: (3 marks) When the TEA was applied, the conductance of K+ channels decreased (9.06pS) as it has been 50% inhibited from the drug. However, the conductance of Na+ channels showed a great increase and a steeper incline indicating depolarisation suggesting that there is a faster uptake of sodium. Now increase the concentration of TEA to give 70% inhibition. Q16. Describe what happens to the ‘nerve’ and why. A16: (4 marks) The graph shows how using TEA affects potassium permeability by blocking potassium channels. Repolarisation of the cell still occurs due to the deactivation of sodium gated channels. The drug also effects the duration of the action potential due to slower repolarisation because potassium reuptake slows down. In the Drugs window click on the ‘reset’ button to remove the TEA and now apply TTX (tetrodotoxin) at a concentration that will produce 50% inhibition. TTX inhibits Na+ channels. Return to the Main window and stimulate the preparation. Q17. What happens to the shape of the action potential? A17: (1 marks) The size of the action potential is significantly reduced. Q18. Describe what has happen to the conductance of the Na+ and K+ channels. 7

2020 Brain and Behaviour:

Practical II Worksheet

A18: (1 marks) The graph demonstrates a low rate of conductance for both Na+ and k+ channels, there is no significant conductance displayed. Q19. What percentage inhibition of Na+ channels still allows an action potential to occur? A19: (1 marks) 17%

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