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PSYC 373 Lab Report 1: Action Potential Simulation http://metaneuron.org/ Background information: Neurons are able to convey information by sending rapid impulses, called action potentials, down their axons. These electrochemical messages are then received by dendrites of other neurons, forming a chain by which the message is sent throughout any given brain region, or another brain structure. When a neuron is NOT firing an action potential, it is said to be “at rest”. An axon has a potential difference (or a resting potential) of -70 mV due to the permeability of the plasma membrane and varying concentrations of ions on either side of the membrane. This means that when the neuron is at rest and is not being stimulated, the interior of the cell is relatively negative compared to the exterior of the cell. This is due to a high sodium (Na+) concentration outside the cell compared to the potassium (K+) concentration. In contrast, there is a high concentration of K+ on the inside of the cell compared to Na+. The ions’ tendency to move across the membrane (when permitted to) is influenced by the concentration gradients for each ion. The permeability of the membrane creates a gradient that favors Na+ to rush into the cell while simultaneously creating a weak gradient for K+ to rush out of the cell. The membrane potential (or the difference in charge) can change due to a redistribution of ions across the membrane. This can only be done if the neuron reaches the threshold of excitation, or -55mV. When the cell is excited enough by neurotransmitter action, voltage-gated ion channels are allowed to open. Voltage-gated sodium (Na+) channels open first and Na+ rushes into the cell, producing a rapid reversal in charge and depolarization. This means that the interior of the axon is now more positive than the outside of the axon. The inside of the cell reaches +40 mV which is close to Na+ equilibrium potential. The equilibrium potential is the point at which there is no net flow of a particular ion from one side of the membrane to the other. This means that Na+ ions flow equally back and forth across the membrane. Shortly after Na+ channels open, the slower K+ channels open, allowing K+ to escape to the outside of the cell. At +40 mV, Na+ voltage-gated channels close, but K+ continues to rush out of the cell, producing repolarization. The potential reaches -90 mV which is close to K+ equilibrium potential. This means that the inward and outward flow of K+ ions are equal. At this point, the cell is said to be in a hyperpolarized state. At this time, the K+ channels close. Although the charge of the neuron is relatively negative again, the ion concentrations are redistributed such that Na+ is on the inside of the cell and K+ is on the outside of the cell. The ion concentrations are restored by passive (or leaking) channels and the Na+/K+ pump. These mechanisms permit the neuron to fire again. The speed of the action potential can be influenced by two things: (1) myelination and (2) axonal diameter. Myelination prevents action potentials from occurring at every single location where there are voltage-gated channels. Instead, the action potential occurs at nodes of Ranvier, where there are voltagegated channels, with a high concentration of voltage-gated Na+ channels. When the axon is depolarized, Na+ rushes inward and diffuses through the cytosol to the next node. This creates a new gradient at the next node, favoring Na+ to rush inward which evokes the next action potential. This mechanism is known as saltatory conduction where the action potential “jumps” from one node to the next. Axonal diameter is also important because small diameters restrict movement of substances down the length of the axons. With increased resistance to current comes decreased speed and conductance. On the other hand, large diameters increase the speed at which Na+ can flow down the axon length because there is little resistance.

1. Using slide 15 of lecture 2 and the introduction above, answer the following questions regarding concentration gradients and movement of ions at resting membrane potential. Sodium (Na+) is highly concentrated on the outside of the membrane. Its gradient causes it to rush inside the cell when the neuron is stimulated. Potassium (K+) is highly concentrated on the inside of the axon. Its gradient is very weak (weak/strong) compared to Na+, but it has a tendency to rush out the cell when the neuron is stimulated. Organic ions (A-) are trapped within the axon and carry a negative (negative/positive) charge. Chloride (hint: ion) is more concentrated outside the neuron, but despite its negative (negative/positive) charge, it will have the tendency to rush inside the axon when the neuron is stimulated.

2. Hodgkin and Huxley performed various experiments on giant squid axons. These axons proved to be valuable to neuroscientific research because their large size allowed researchers to visualize the relationship between conduction, resistance, and diameter. Giant squid axons are unmyelinated, but are still fast at conducting messages down the axon. Why do you think that the giant squid axon can conduct fast messages? -

We use squid axons mainly because they are much larger than human axons. They can grow to be about 1mm in diameter. Because they are so big, it allows the experimenter to measure electrical currents that are entering and leaving the axon through its membrane. Gian squids’ axons are huge because they need to be able to spread messages fast. The bigger the diameter of the axon, the faster the axon can conduct the message/current.

3. Obviously, humans do not have axons that are the size of the giant squid axon in our brains. Our axons are much smaller. What other sort of mechanism permits fast transduction without relying on the size of the axon? Why is this mechanism just as efficient? -

Our axons are not the size of a giant squid, but we are stull able to transmit signals quickly. We do this through our myelin sheath. When we have an action potential, it jumps from the gaps that we have in the myelin sheath called the nodes of Ranvier. The action potential jumps from one node of Ranvier, to the next. The myelin sheath covers the length of the axon, except at the nodes, which contain the voltage sensitive channels. The regeneration of an action potential allows for increased conduction speed. This whole process is called Saltatory Conduction.

Experiment 1: Messing with membrane parameters at resting potential 1. Click on the MetaNeuron link provided at the top of report. Download the MetaNeuron program and go through the prompts. Make sure you are downloading the correct version for your operating system. 2. Once MetaNeuron has opened, go to the top of the window and select “lesson”. Select “lesson 1: resting membrane potential”. This lesson demonstrates how K+ and Na+ channels contribute to the generation of resting membrane potential. The concentrations of these ions, both inside and outside of the axon, can be changed. We can look at the ion equilibrium potential by using the Nernst equation:

[ ion ]∈¿ Ion equilibrium potential (mV) = 58 * log [ion ] out ¿ ¿

…. where [ion]out and [ion]in are the concentrations of the ions outside and inside the cell, respectively. Remember, ion equilibrium potential means that there is no net flow of ions across the membrane. The Goldman-Hodgkin-Katz equation models resting membrane potential which is determined by the concentrations of the aforementioned ions and the permeability of the membrane. We won’t include Cl- or temperature in our equations to make things easier to understand.

Resting membrane potential (mV) = 58 * log

+¿ ¿ K ¿ +¿ ¿ Na ¿ out ¿ +¿ ¿ K ¿ +¿ ¿ Na ¿ Pk ¿ ¿ ¿

PK = relative permeability of K+ PNa = relative permeability of Na+ [K+] and [Na+] = ion concentrations inside and outside the membrane

1. On the interface, you can alter the ion concentrations on the inside and outside of the axon. Change the Na+ concentrations to 120 (out) and 17 (in). Change the K+ concentrations to (3) out and 64 (in). Using the ion equilibrium potential formula above (the first one), calculate the ion equilibrium potentials for both Na+ and K+. Does this match the values that the simulation provided? Show your work. 120 - (Na+) 58 * log = 49.23. Yes, this number matched the values that the simulation 17 provided 3 - (K+) 58 * log = -77.09. Yes, this number matched the values that the simulation 64 provided

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2. What is the equilibrium potential when you change Na+ (out) to 100 mM and change Na+ (in) to 100 mM? Why does this occur? - When you chance Na+ (out) to 100mM and Na+ (in) to 100mM, the equilibrium potential is 0.00. This happens because there is no net flow of ions across the membrane. Both inside and outside of the cells are equal already. 3. Using the third option (to the right) on the interface, change the relative membrane permeability of Na+ to 1 and K+ to 130. Using the ion concentrations that I provided in question 1, calculate the resting membrane potential. Does this match the resting membrane potential that the simulation provided? Show your work. -

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130 x 3 ) +( 1 x 120) = 70.38mV. 130 x 64 )+( 1 x 17 ) Yes, this number matches the resting membrane potential (mV) = 58 * log

Experiment 2: Messing with action potentials Go to “lesson” and select “lesson 4: axon action potential”. This lesson demonstrates how voltage-dependent and time-dependent Na+ and K+ conductances generate action potentials. The Na+ conductance represents Na+ channels, and the K+ conductance represents the delayed K+ channels. You can alter several parameters in the MetaNeuron interface. 

Na+, K+, and leak equilibrium potentials: change the driving force through the Na+, K+, and leak channels

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gNa+ max, K+ max, gLeak: represent the number of Na+, K+, and leak channels in the membrane, respectively TTX and TEA: TTX blocks voltage-gated Na+ channels, and TEA blocks voltage-gated K+ channels (but not Leak conductance!)

1. When the interface is open, you see a standard action potential curve. Change the amplitude (µA) of stimulus 1 from 65 to 10. The curve disappears. Why does this happen? -

The action potential disappears when you change the amplitude from 65 to 10 because you are changing the strength of the stimulus, and the stimulus was not strong enough to fire an action potential. There was no threshold of excitation.

2. Set stimulus 1 back to 65 µA. Turn stimulus 2 on by clicking the box under “stimulus 2”. Set the delay to 1.5 milliseconds. Why do you not see another curve form? -

We don’t see another curve from because the neuron is still in the refractory period, which means that the Na+ channels are closed; therefore, we are not able to fire another action potential.

3. Set the delay of stimulus 2 to 2 milliseconds. Why do you see a tiny “blip” instead of a large depolarization? What do you think this “blip” represents (hint: “it’s like I have ESPN or something”). -

We see the tiny “blip” because of depolarization. Another term for this is EPSP, or gradient depolarization. There is no full-blown action potential, because we are stull in the refractory period.

4. Action potentials are initiated at the axon hillock where there is a high density of Na+ channels. Vary “gNa max” from 260 mS/cm2 to 10 mS/cm2. What happens to the action potential? Why does this happen? -

When we lowered the gNA max from 260, to 10, the action potential disappeared. This happened because there was not enough strength for the action potential to be fired.

5. Reset the file parameters by going to “file” and selecting “restore lesson to default”. Select “show ionic conductances” in the “conductances and currents” window. The green and blue traces show the time course of the inward Na+ current and the outward K+ current that generate action potential. Why does the K+ conductance turn on slower and last longer than the Na+ conductance?

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An action potential is an “all or none” thing that begins at the axon hillock. For an action potential to be generated, stimulation must produce depolarization of the membrane beyond a certain level. During the resting potential, the voltage-gated Na+ channels are closed, so, the Na+ on the outside, that really want to come inside, cannot enter the cell. As the incoming signals are received, the potential reaches the threshold of excitation, this is due to the increased permeability of the Na+ ions. Na+ is freer to enter the cell with the open channel. This brings the cell to a more positive charge. When the threshold finally reaches -55mV, the Na+ channels open, and the Na+ rush inside until the point of reverse polarity. There is so much influx of the Na+ ions, that the charge inside of the cell reaches to +40mV. Potassium channels are also voltage-gated, but they don’t react as quickly as the Na+ channels do. After the action potential has happened, the slower voltage gated channels open, and the K+ flows outward because the negative charge is gone from the inside of the cell, because it is following its concentration gradient. Because the K+ ions are leaving the cell while the K+ channels are still open and slower toc lose, it takes away some of the positive charge, bringing the charge back down. After the heightened action potential, repolarization occurs. The K+ conduction pushes membrane potential back to more negative levels. The voltagegated channels are a bit slow to close, so the excess potassium ions flow outwards, causing the neuron to hyperpolarize. The neuron can re-fire at this state, but it takes a stronger stimulus.

6. Now apply TTX and TEA. What happens to the action potential? Why does this happen? -

Some of the gates can be blocked by chemicals such as TTX, and TEA, which prevents the passage of ions through the membrane and disrupts the action potentials. At certain points during the action potential, channels are closed because they are voltage dependent....