LESSON 2.2 WORKBOOK How does brain structure impact its function? This lesson introduces you to the action potential, which is the method that axons use to transmit electrical signals. In this lesson you will learn how our axons make an action potential and then use the energy stored in their membranes to send the action potential down to the presynaptic terminal.

DEFINITIONS OF TERMS Resting membrane potential – the steady membrane potential of a neuron at rest, usually about -70 mV Potential energy – the energy a body has because of its position relative to others, electric charge and other factors Diffusion – net movement of molecules from areas of high concentration to areas of low concentration Electrostatic pressure – repulsion of like charges and the attraction of opposite charges Sodium-potassium pump – active transport mechanisms that pump sodium (Na+) ions out of neurons and potassium (K+) ions into neurons Voltage-gated channels – channels that open or close in response to changes in voltage across the membrane

Wo r k b o o k Lesson 2.2

Signaling is organized in the same way in all neurons To produce a behavior, each participating neuron produces, in the same sequence, four types of signals at different sites:

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The dendrites generate electrical input signals.

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The axon hillock (or initial segment) integrates the input signals into a single electrical signal, the action potential.

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The axon transmits the electrical action potential down to the presynaptic terminal.

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The presynaptic terminals convert the electrical action potential into a chemical output signal.

We will discuss each of these signals, but it’s easiest to understand if we start with the action potential, even though it comes in the middle. Before we discuss any of the signals though, we need to review the electrical properties of the cell membrane that are important to understand how these signals are generated.

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What are the four types of signals generated within neurons and where are they generated? ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________

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LESSON MATERIALS Neuronal membranes store energy in the form of membrane potentials

DEFINITIONS OF TERMS

Local potentials – small changes in voltage (membrane potential) due to dendritic signaling Depolarize – to decrease the resting membrane potential. Decreasing membrane potential means that the membrane potential is becoming more positive. Threshold – the level of depolarization needed to generate an action potential

For a complete list of defined terms, see the Glossary.

Wo r k b o o k Lesson 2.2

Neuronal membranes are electrically charged. This means there is a difference in electrical charge across their cell membranes of about 70 millivolts (mV). As we shall see in a minute this difference in charge occurs because sodium (Na+), and potassium (K+) ions and organic anions (A-) are unevenly distributed across the membrane so that the inside of the axon is negatively charged relative to the outside (Figure 7). This electrical charge is called the resting membrane potential. The term potential refers to the energy stored in the membrane or its potential energy. Because the outside of the axon is arbitrarily defined as zero, we say that the resting membrane potential of the axon is -70 mV. The resting membrane potential is produced as a result of the forces of diffusion and electrostatic pressure that the ions inside and outside the membrane experience. Remember that: •

Diffusion is the net movement of molecules (such as ions) down a concentration gradient

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Electrostatic pressure is the repulsion of like charges (positive is repulsed by positive and negative with negative) and the attraction of opposite charges

Understanding what produces the membrane potential therefore requires that we know the concentration of various ions inside and outside the axon and what forces of diffusion and electrostatic pressure they are experiencing.

Figure 7: Membrane potential. (A) When both electrodes are applied to the exterior of the axon in the extracellular fluid, no difference in potential is recorded. (B) When one electrode is inserted into the axon, a voltage difference between the inside and the outside is recorded. The graphs show the voltage change when one electrode is inside the axon.

The force of diffusion, molecules moving down their concentration gradient, predicts the result of adding one drop of blue food coloring to a glass of water. Immediately after adding one drop of blue food coloring to a glass of water, that drop sits on the top of the water in an area of high concentration. What happens if you let the water sit for 5 minutes? ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________

The force of electrostatic pressure, attraction of opposite charges and repulsion of like charges, predicts what would happen if you had negatively charged ions at the top of a cup and positively charged ions at the bottom of a cup. Where do you predict the negatively charged ions will go? ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________

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LESSON MATERIALS Athough there are many types of ions inside and outside the axon, three are particularly important for the membrane potential (Figure 8): • • • DEFINITIONS OF TERMS Absolute refractory period – a brief period after the initiation of an action potential during which it is impossible to elicit another action potential in the same neuron Hyperpolarize – to increase the resting membrane potential. Increasing membrane potential means that the membrane potential is becoming more negative. Relative refractory period – period after the absolute refractory period during which a higher-thannormal amount of stimulation is necessary to make a neuron fire Conduction of the action potential – movement of the action potential down the length of the axon

Wo r k b o o k Lesson 2.2

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Organic anions (symbolized as A-) Potassium ions (K+) Sodium ions (Na+)

Let’s now consider how each of these important ions experiences the forces of diffusion and electrostatic pressure. Once we know this we will understand why each ion is located where it is when the axon membrane is at rest. Organic anions are negatively charged proteins and intermediary products of a cell’s metabolism. They are unable to pass through neuron’s membrane and so they are only found inside the axon. Therefore, they make the interior of the axon more negative and contribute to the negative membrane potential.

At  rest   Na+  

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What are organic anions, and where are they in highest concentration in our nervous systems?

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Figure 8: Distribution of ions at resting membrane potential. Na+ ions (represented by blue circles) are more concentrated outside the neuron. K+ ions (represented by red circles) and negatively charged proteins (represented by black stars) are more concentrated inside the neuron.axon, a voltage difference between the inside and the outside is recorded.

Potassium ions (K+) are also concentrated within the axon, however they can move to the outside through special channels in the cell membrane. Thus, the force of diffusion will tend to push them out of the cell. However the high concentration of negative organic anions makes the inside of the cell more negative relative to the outside. Because of this negative charge, electrostatic pressure tends to keep the potassium ions back inside the cell. In the case of potassium ions the two forces of diffusion and electrostatic pressure oppose each other and balance each other out. As a result, potassium ions tend to remain where they are – at high concentrations inside the axon. Sodium ions (Na+) are concentrated outside the axon, in the extracellular fluid. Like for potassium, there are sodium ion channels in the membrane and so the force of diffusion pushes them inwards. Unlike potassium though, the organic anions (A-) in the axon creates a negative environment that also attracts the positively charged sodium ions into the axon. In this case, and unlike potassium, the diffusion and electrostatic pressure work together to attract the sodium ions into the axon. But if the sodium ions did enter the axon the charge difference across the membrane would break down and the potential energy in the membrane would be lost.

Where are potassium ions in highest concentration in our nervous systems? ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________

Where are sodium ions in highest concentration in our nervous systems? ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________

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LESSON MATERIALS How then can Na+ overcome the two forces of diffusion and electrostatic pressure and stay on the outside the axon, preserving the resting membrane potential? The answer is this: there is another force provided by a pump that continuously pushes Na+ out of the axon, swapping an Na+ ion that might have leaked inside for a K+ ion that might have leaked outside. Because the membrane is not very permeable to Na+ (there are fewer Na+ channels) the sodium-potassium pump (Na+/K+ pump) is very effective at keeping the intracellular concentration of Na+ very low when the membrane is at rest. DEFINITIONS OF TERMS

Just a quick side note: Sodium-potassium pumps use enormous amounts of energy – up to 40% of a neuron’s energy is used to operate them. The Action Potential

For a complete list of defined terms, see the Glossary.

We just saw that the forces of both diffusion and electrostatic pressure tend to attract Na+ into the axon. However, we also saw that the membrane is not very permeable to Na+ ions, and that the sodium/potassium pump continuously pumps Na+ out of the axon, keeping intracellular Na+ concentrations low. But imagine what would happen if the membrane suddenly became permeable to Na+. The forces of diffusion and electrostatic pressure would cause Na+ to rush into the cell. This sudden influx of positively charged ions would drastically reduce the membrane potential. This is precisely what happens to cause the action potential: A brief increase in the permeability of the membrane to Na+ (which allows Na+ to enter the cell), is immediately followed by a transient increase in permeability of the membrane to K+ (allowing K+ to exit the cell). The question now is – what is responsible for these transient increases in permeability?

Wo r k b o o k Lesson 2.2

We already saw that there are two ways to move ions across the membrane, either through channels in the membrane or by hooking them up to pumps, like the sodium-potassium pump. Sometimes the passages or pores in the ion channels are always open, but usually they are closed and only open under specific conditions. When the channel pores are open they are only permeable to a particular type of ion, which can flow through the pore and thus enter or exit the cell. Some ion channels open or close depending on the cell’s membrane potential. They are referred to as voltage-gated ion channels.

Figure 9: The membrane’s ion channels and pumps. Two ion channels are critical in the axon’s conduction of the action potential: the voltage-gated Na+ channel and the voltage-gated K+ channel. Additionally the Na+/K+ pump plays a critical role as well. between the inside and the outside is recorded.

How does sodium remain in highest concentration outside the axon? ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________

How do sodium channels open in response to changes in the cell’s membrane potential? ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________

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LESSON MATERIALS

Describe the different types of voltage-gated ion channel

Two voltage-gated channels are critical in the action potential (Figure 9) Voltage-gated Na+ channels open when the membrane potential reaches -50 mV, and close when the membrane potential reaches +40 mV. DEFINITIONS OF TERMS

For a complete list of defined terms, see the Glossary.

Voltage-gated K+ channels open when the membrane potential reaches +40 mV, and close when the membrane potential reaches – 70 mV. The following numbered paragraphs describe how the movement of ions across the membrane using channels and pumps makes the action potential. The numbers in Figure 10 correspond to the numbers in the paragraphs below.

1. The Resting Membrane potential: At rest the voltage-gated Na+ channels and voltage-gated K+ channels are closed and the Na+/K+ pump is working hard, using ATP to sustain the resting membrane potential to move three Na+ ions out of the axon for every two K+ ions moved into the axon. As a result the concentration of Na+ outside the axon is high, and the concentration of K+ inside the axon is high (Figure 11). Because of the contribution of the organic cations (A-) the inside of the axon is more negative than the outside, even though the K+ is there. As a result the membrane potential at rest is -70 mV, as we saw before.

Wo r k b o o k Lesson 2.2

Figure 10: Stage of the action potential. The opening and closing of voltage-gated Na+ and K+ channels is responsible for the characteristic shape of the action potential. Refer to figures 13-17 to see what is happening at each stage of the action potential..

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Describe where the Na+ and K+ ions are when the axon’s membrane is at rest. Why are they located there?

Na+   Na+  

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Figure 11: Resting membrane potential (Stage 1 in Figure 12). The resting membrane potential is maintained by the Na+/K+ pump. At rest, there is a slow leak of K+ ions out of the cell, which the Na+/K+ pump corrects by pumping 3 Na+ ions out of the cell for every 2 K+ ions it pumps into the cell.

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LESSON MATERIALS 2. Reaching Threshold: When the dendrites reNa ceive a signal just a few voltage-gated Na+ chanNa Na Na Na Na nels open and the charge across the dendritic Na membranes drops briefly causing small changes in voltage or local potentials. The forces of diffusion and electrostatic pressure then pull Na+ ions K Na K into the cell through the open channels (Figure K K Na K K 12). This inward flow of positive sodium ions starts to reduce or depolarize the membrane potential meaning that the inside of the cell is becoming more positive relative to the outside. If enough Figure 12: Reaching threshold (Stage 2 in Figure 9). Na+ channels open and enough Na+ ions enter the Local potentials open a few voltage-gated Na+ chancell, then the membrane potential will decrease to nels, allowing Na+ ions to enter the axon. the threshold (-50 mV) at which all the Na+ channels will open and large quantities of Na+ ions will enter the cell. Threshold is the critical level of membrane depolarization at which the cell can actively generate an action potential. As we see, whether threshold is reached depends on the strength of the dendritic signal. If the dendritic signal is strong then we are more likely to reach threshold. +  

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DEFINITIONS OF TERMS

For a complete list of defined terms, see the Glossary.

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3. Depolarization: When a threshold of -50 mV is reached, many more voltage-gated Na+ channels open allowing even more Na+ ions to quickly flow into the axon (Figure 13). This inward flow of Na+ Na into the axon further depolarizes the membrane, Na reducing the membrane potential even more so that eventually the inside of the axon becomes positive relative to the outside. +  

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K 4. Hyperpolarization: When so many Na K Na Na K Na K ions have entered the axon that the interior has reached +40 mV (relative to the external value of 0mV) the voltage-gated Na+ channels close. This inactivates them they cannot open for a period of time. This is called the absolute refractory peFigure 13: Depolarization (Stage 3 in Figure 9). Once riod.

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Wo r k b o o k Lesson 2.2

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the membrane reaches threshold (-50 mV), many more voltage-gated Na+ channels open, allowing even more Na+ ions to enter the axon.

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Describe where the Na+ and K+ ions are when the axon’s membrane is reaching threshold. Why are they there? ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________

Describe where the Na+ and K+ ions are when the axon’s membrane is depolarizing. Why are they there? ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________

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LESSON MATERIALS Remember that voltage-gated K+ channels also open at +40 mV. This opening of K+ channels alNa K K K lows K+ ions to flow out of the axon (Figure 14). K K Na + The K ions flow out of the axon because the prior passage of Na+ ions into the cell has altered the forces of diffusion and electrostatic pressure that Basal  ganglia   the K+ ions now experience. Cingulate   First of   all,Corpus   K+ cisallosum   in Na Na Na Na cortex   higher concentrations inside the axon than outThalamus   Na Na Na K + side the axon, so with the K channel open the Cerebral     K+ ion is forced down its concentration gradient cortex   and out of the cell by diffusion. Furthermore, with Na+ ions now inside the axon, the outside is more Figure 14: Hyperpolarization (Stage 4 in Figure 9). At Hypothalamus   negative than it was, so electrostatic pressure +40 mV the voltage-gated Na+ channels close and Amygdala   the voltage-gated K+ channels open, allowing K+ ions also attracts the positive K+ ion to theHippocampus   outside. to exit the axon. This flow of K+ outside of the axon decreases the positive charge on the inside, and has the effect of hyperpolarizing the axon membrane – meaning that the inside of the membrane becomes more negative relative to the outside.

___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ __________________________________ ___________________________________ ___________________________________ _________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Due to the huge flow of K+ out of the cell, the membrane potential becomes higher than it is at rest (the ___________________________________ inside of the axon is more negative relative to the outside). This period of higher membrane potential is ___________________________________ called the relative refractory period because the sodium channels are now able to open, so if enough ___________________________________ + + positive charge came along the axon could potentially reach threshold and depolarize again. However Describe where the Na and K ions are when if you think about it, because the membrane potential is higher, more positive ions would be needed to the axon’s membrane is returning to rest. reach threshold than if the cell was at rest, so depolarization during the relative refractory period is less Why are they there? ___________________________________ likely to occur. ___________________________________ Na Na Na Na Na __________________________________ Na Na K Na ___________________________________ 5. Returning to rest: To return the membrane ___________________________________ to its resting membrane potential of -70mV, the ___________________________________ Na+/K+ pump works hard and uses ATP to move ___________________________________ K K K three Na+ ions out of the cell for every two K+ ions Na ___________________________________ K K moved into the cell (Figure 15). ___________________________________ ___________________________________ ___________________________________ +  

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For a complete list of defined terms, see the Glossary.

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DEFINITIONS OF TERMS

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Wo r k b o o k Lesson 2.2

Describe where the Na+ and K+ ions are when the axon’s membrane is hyperpolarizing. Why are they there?

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Figure 15: Returning to rest (Stage 5 in Figure 9). The voltage-gated K+ channels close, and the Na+/ K+ pump returns the membrane to rest by pumping 3 Na+ ions out of the cell for every 2 K+ ions it pumps

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LESSON MATERIALS Conduction of the Action Potential along the Axon

DEFINITIONS OF TERMS

For a complete list of defined terms, see the Glossary.

Now we have a basic understanding of what the resting membrane potential is and how the action potential is produced, we can turn our attention to how this electrical message moves along the axon down to the presynaptic terminal. This movement is called the conduction Corpus  callosum   of the Basal  ganglia   Cingulate     action potential. cortex  

Thalamus  

The membrane depolarization that occurs during the acCerebral     cortex   tion potential is localized to a small area of membrane where the ions and channels are localized, and so the electrical signal does not move very far down the axon. Hypothalamus   The axons therefore need to use another method, called Amygdala   Hippocampus   active conduction, to prevent the electrical signal from de- Figure 16: Conduction of the action potential. caying. It does this by repeatedly generating action poten- An action potential is generated as Na+ ions flow in at one location along an axon. The tials along the length of the axon. depolarization spreads to the neighboring

Axons can use active conduction by stacking many volt- region of the membrane, initiating an action age-gated Na+ channels along their membranes in close potential there. The original region repolarproximity to one another. When the dendrite signal causes izes as K+ ions flow out. The depolarizationrepolarization process is repeated as the the axon hillock to reach threshold and the Na+ channels action potential is propagated down the to open, the depolarization of the membrane will cause length of the axon. adjacent Na+ channels to also open generating another action potential. This process is repeated until the action potential reaches the presynaptic terminal where it is converted to a chemical signal to cross the synaptic cleft (Figure 16). So, essentially conduction of an action potential down the length of the axon requires many individual action potentials along the length of the axon to be generated in sequence. Each individual action potential provides a depolarizing current which causes the next set of voltage-gated Na+ channels to reach threshold and trigger another action potential, causing a domino effect down the length of the axon.

Wo r k b o o k Lesson 2.2

It is important to note that in order for the action potential to be conducted efficiently it is critical that the voltage-gated Na+ channels are stacked up along the entire length of the axon. If they are not, the depolarizing current from a single action potential will get smaller as it travels down the axon, either because the current leaks or because the proteins the axon in made of offer resistance to conduction. When voltagegated Na+ channels are in close proximity, the depolarizing current does not have enough space to decline before the next set of Na+ channels open and initiate a new action potential. ■■ You can watch a video about the action potential here: http://www.youtube.com/watch?v=ifD1YG07fB8

How do axons conduct the action potential down its length? ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ __________________________________ ___________________________________ ___________________________________ _________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ __________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________

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STUDENT RESPONSES Write a summary of what is happening at each stage of the action potential diagrammed below. Step 1:_______________________________________________________________________________________________ ____________________________________________________________ ____________________________________________________________ ____________________________________________________________ __________________________________________________________

Step 2:______________________________________________________ Remember to identify your sources

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Step 3:______________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _________________________________________________________________________

Step 4:_______________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ _________________________________________________________________________________________________

Step 5:_______________________________________________________________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________

Wo r k b o o k Lesson 2.2

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