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Lecture 6
The Action Potential
Lecture Outline
Lecture Objectives Return to lecture outline
After studying this lecture, you will be able to:
Required Mastery of Previous Materials Return to lecture outline
In order to fully understand this lecture, you need to have already mastered the following topics:
Key Terms Return to lecture outline
Introduction Return to lecture outline
In the previous lecture, we learned how the membrane potential (Vm) is established. We also learned about the factors that govern the value of the membrane potential. You recall that in most cells, the resting membrane potential is governed by the relative permeability to potassium (K+), sodium (Na+), and chloride (Cl-) ions. In fact, in a typical neuron at rest, the relative permeabilites are approximately 1 : 0.05 : 0.45 (pK : pNa : pCl). We also mentioned that the relative permeability to a given ion is a function of the number of channels specific for that ion that are open at rest. The resting membrane potential in a typical neuron is near -70 mV. Therefore, because at rest pK is greater than either pNa or pCl, the resting membrane potential in most cells is close to the equilibrium potential for K+ (VK ≈ -90 mV). We used the Nernst equation to determine the equilibrium potential for any given ion, and we used the Goldman-Hodgkin-Katz (GHK) equation to determine the resting membrane potential in cells. In this lecture, we will see that rapid changes in permeability to Na+ and K+ ions are responsible for electrical impulses generated by neurons. The resulting electrical impulses, in turn, form the basis for information transmission in the nervous system. As we will see below, the changes in Na+ and K+ permeability are due to the opening of voltage-gated Na+ channels and voltage-gated K+ channels.
Figure_neuronal_action_potential.gif
Figure 6-1. A typical neuronal action potential.
The action potential is a rapid reversal of the membrane potential brought about by rapid changes in plasma membrane permeability to Na+ and K+. VNa and VK are the equilibrium potentials for Na+ and K+, respectively. See text for details.
Now that we understand the mechanisms responsible for the generation and manipulation of the membrane potential, we can tackle the question of how cells of the nervous system (in particular neurons) generate nervous impulses. Nervous impulses are the electrical signals by which neurons talk to one another and also to other cells of the body. The nervous impulse is referred to as the action potential. An action potential is a brief reversal of the membrane potential (Fig. 6-1). At rest, the Vm of a neuron is around -70 mV (close to VK), but during an action potential, Vm transiently approaches +50 mV (close to VNa). The Vm then rapidly returns to the resting potential and even briefly goes beyond the resting potential to approach VK before finally returning to the resting value of about –70 mV. The entire process takes about 3–5 ms. This potential reversal of more than 100 mV is responsible for electrical signaling in the nervous system, and is the basis of information transmission in the nervous system. In this lecture, we will learn the mechanisms that give rise to the action potential. In the next lecture, we will see how this electrical signal can travel along axonal projections of neurons to reach other neurons, or other cells in the body.
To understand the uniqueness of neurons as excitable cells, we can examine the response of non-excitable cells to artificial electrical stimulation (Fig. 6-2) and compare this response to that of excitable cells such as neurons (Fig. 6-3). Recall from the previous lecture that we can impale a cell with a glass microelectrode. The microelectrode could then be used to inject positive or negative charge into the cell. The applied stimuli are in general in the form of square-wave pulses, in that at a given point in time a predefined amount of charge is introduced into the cell and maintained until the end of the pulse. Introduction of positive charge into the cell leads to membrane depolarization, and introduction of negative charge into the cell leads to membrane hyperpolarization.
Figure_stimulation_nonexcitable_cell.gif
Figure 6-2. Electrical stimulation of non-excitable cells.
In non-excitable cells, both hyperpolarizing and depolarizing electrical stimulation result in graded potentials that are proportional in magnitude to the amplitude of the pulse. The upper trace (red) shows the square-wave pulses applied to a cell, and the bottom trace (blue) shows the membrane potential of the cell being stimulated. The two traces are on the same time scale. Hyperpolarizing stimuli lead to membrane hyperpolarization, and depolarizing stimuli lead to membrane depolarization.
In non-excitable cells, depolarizing or hyperpolarizing stimulation only temporarily alters the membrane potential, but does not lead to "excitation" of the cell (Fig. 6-2). At the end of the depolarizing or hyperpolarizing pulse, the membrane potential simply returns to the resting value. This behavior is independent of the strength of the stimuli. The amplitude of the depolarization or hyperpolarization is directly proportional to the amplitude of the stimulus; the larger the amplitude of the stimulus, the larger the change in the membrane potential. These changes in the membrane potential are referred to as graded potentials because they are proportional to the magnitude of the stimulus (Fig. 6-2). These graded potentials represent the passive property of the membrane to electrical stimulation.
Figure_stimulation_excitable_cell.gif
Figure 6-3. Electrical stimulation of neurons.
In excitable cells such as neurons, hyperpolarizing electrical stimulation results in hyperpolarizing graded potentials that are proportional in magnitude to the amplitude of the pulse. Sub-threshold depolarizing stimuli cause graded potentials, but supra-threshold depolarizing stimuli elicit all-or-nothing action potentials. The upper trace (red) shows the square-wave pulses applied to a neuron, and the bottom trace (blue) shows the membrane potential of the neuron being stimulated. The two traces are on the same time scale. The dashed line represents the threshold voltage (Vthreshold) of approximately -50 mV.
In excitable cells, hyperpolarizing stimuli lead to the same graded responses that are seen in non-excitable cells (Fig. 6-3). However, the nature of the response of excitable cells to depolarizing stimuli depends on the strength of the applied stimulus. If weak stimuli are given, the response is graded and is similar to that of a non-excitable cell (Fig. 6-3). If, however, a strong enough stimulus is given such that the resulting depolarization surpasses a certain critical voltage, an action potential is generated. The voltage that must be surpassed in order to get an action potential is referred to as threshold. In most neurons threshold is around -40 to -50 mV. If a stimulus leads to a membrane depolarization that is more negative than the threshold value, the stimulus is said to be sub-threshold. Sub-threshold stimuli do not lead to action potentials. If the stimulus leads to a membrane depolarization that is less negative (more positive) than the threshold value, it is said to be supra-threshold. In general, supra-threshold stimuli lead to action potentials. This is almost always true if the supra-threshold stimulus is applied to a neuron at rest (i.e., a neuron that is not undergoing excitation). However, if a neuron is undergoing excitation, there are times when even supra-threshold stimuli do not lead to excitation of neurons. This is because during excitation, there is a period during which the neuron is refractory to subsequent stimulation (see Refractory Periods below). In this lecture, we will consider these scenarios in detail.
It is important to note that while in this lecture we discuss artificial electrical stimulation of neurons, physiologically, neurons become stimulated by other neurons or by environmental stimuli (in the case of sensory receptors). The mechanisms by which neurons become stimulated under physiological conditions will be discussed later when we consider how electrical information is transmitted from one neuron to another neuron (see Neuronal Signaling: Synaptic Neurotransmission). Nevertheless, once a neuron is stimulated such that its membrane potential reaches threshold, a neuronal action potential will be generated. Thus, whether a neuron is stimulated by another neuron, environmental stimuli, or by an intracellular microelectrode, the resulting action potential will be the same. Therefore, electrophysiological stimulation of neurons provides a reliable and convenient method by which to study neuronal physiology. Although we will focus on neurons in this lecture, keep in mind that neurons are not the only excitable cells in the body. Muscle cells (skeletal, cardiac, and smooth muscle), as well as some endocrine cells are also excitable.
Figure_all_or_nothing.gif
Figure 6-4. Action potentials are all-or-nothing.
So long as threshold is reached, further increases in the amplitude of the stimulus (top, red trace) do not cause increases in the size of the voltage deflection of the action potential (bottom, blue trace). The two traces are on the same time scale. The dashed line represents the threshold voltage (Vthreshold) of approximately -50 mV. Here, a threshold stimulus refers to that which is just strong enough to bring a resting neuron to threshold.
 
Important Features of the Neuronal Action Potential Return to lecture outline
When the stimulus given to a resting neuron is supra-threshold, it results in an action potential. As long as the depolarization caused by the stimulus is above threshold, the size of the resulting action potential will be the same (Fig. 6-4). In other words, once the membrane potential surpasses threshold, it will uncontrollably go to the peak of the action potential at around +50 mV (near the Na+ equilibrium potential, VNa). So long as threshold is surpassed, additional increases in stimulus strength do not lead to increases in the magnitude of the voltage deflection of the action potential. This is referred to as the all-or-nothing law, and refers to the fact that there is no "in-between" action potential (Fig. 6-4). The neuron either does not respond (in the case of sub-threshold stimuli), or it will generate a full-fledged, all-or-nothing action potential (in the case of supra-threshold stimuli). See the section on Frequency Coding in the Nervous System below for how the nervous system encodes varying strengths of supra-threshold stimuli.
Figure_action_potential_phases.gif
Figure 6-5. Important features of the neuronal action potential.
0, resting state; 1, depolarization to threshold and beyond; 2, overshoot; 3, peak of the action potential; 4, repolarization; and 5, hyperpolarization.
Now let us summarize the events that take place during an action potential. The action potential can be divided into five phases: (1) depolarization to and beyond threshold, (2) overshoot, (3) peak, (4) repolarization, and (5) hyperpolarizing afterpotential (or hyperpolarization) (Fig. 6-5). Let's consider these events.
As mentioned above, if the initial depolarization does not reach the threshold voltage, no action potential will result. However, if depolarization is large enough such that the membrane potential reaches -50 mV, a complete all-or-nothing action potential will result. As will be discussed below, unique voltage-gated ion channels, namely voltage-gated Na+ and K+ channels, respond to membrane depolarization to or beyond the threshold voltage. Remarkably, all of the features of the action potential can be explained by understanding the molecular properties of these voltage-gated ion channels, and the electrochemical gradients of Na+ and K+.
Figure_hodgkin_cycle.gif
Figure 6-6. The Hodgkin cycle.
The Hodgkin cycle represents a positive feedback loop in which an initial membrane depolarization leads to uncontrolled deflection of the membrane potential to near VNa. The initial depolarization must reach or surpass threshold in order to activate voltage-gated Na+ channels. Opening of Na+ channels allows Na+ inflow which, in turn, further depolarizes the membrane. Additional depolarization activates additional Na+ channels. This cycle leads to a very rapid rise in Na+ conductance (gNa), which moves the membrane potential close to VNa.
The uncontrolled depolarization that takes place (also referred to as the spike phase of the action potential; 1 in Fig. 6-5) is strictly a function of voltage-gated Na+ channels in neurons. At rest (-70 mV), the voltage-gated Na+ channels are closed, but begin to open at membrane potentials ranging from -40 to -50 mV (threshold voltage, Vth). Opening of Na+ channels leads to the entry of a large amount of Na+ ions into the cell. Remember that a very large driving force (~100 mV) acts on Na+ ions favoring their movement into the cell through Na+ channels. This is brought about both by the Na+ concentration gradient, and the inside negative membrane potential. Entry of Na+ into the cell brings about further depolarization. Membrane depolarization further activates additional Na+ channels which, in turn, leads to the entry of more Na+ into the cell. Therefore, a positive feedback loop is established, which leads to increasing entry of Na+ into the cell. This positive feedback loop is called the Hodgkin cycle (Fig. 6-6), so named because of the investigator who pioneered most of what we know today about electrical activity in neurons. Thus, the Hodgkin cycle is responsible for the spike phase of the action potential.
The continued entry of Na+ into the cell leads to rapid depolarization of the cell (< 1 ms). Because rapid opening of Na+ channels leads to a rapid rise in membrane permeability to Na+, the membrane potential reverses its sign (goes from negative to positive) and approaches the equilibrium potential for Na+ (about +50 mV). Remember from the previous lecture on the membrane potential that the movement of an ion down its electrochemical gradient tends to move the membrane potential towards the equilibrium potential for that ion. In this case, Na+ entry into the cell through voltage-gated Na+ channels takes the membrane potentials close to the Na+ equilibrium potential (VNa). Reversal of the sign where the membrane potential becomes positive is referred to as overshoot (2 in Fig. 6-5). Whereas at rest, the relative permeabilities of K+ (pK) and Na+ (pNa) are 1 : 0.05 (pK : pNa), at the peak of the action potential, the pK : pNa ratio is about 1 : 12. However, keep in mind that at the peak of the action potential the absolute pK is also larger than its value at rest (see Fig. 6-8 below). At the peak of the action potential pNa is about 600 times greater than its resting value, whereas pK is about 3 times its resting value. If you use the GHK equation and the permeability values at the peak of the action potential (pK : pNa ratio of 1 : 12), you can convince yourself that Vm approaches VNa. While the maximum pNa is observed at the peak of the action potential, maximum pK is observed shortly after the peak of the action potential (see Fig. 6-8 below).
Figure_na_channel_inactivation.gif
Figure 6-7. Na+ channel activation, inactivation, and recovery from inactivation.
Neuronal voltage-gated Na+ channels can exist in one of three different conformations: close state, open state, and inactive state. At the resting membrane potential, Na+ channels are closed (non-conducting). Membrane depolarization to the threshold voltage leads to the opening (activation) of voltage-gated Na+ channels (channels conduct ions). The channels then spontaneously enter an inactive state (non-conducting state). Recovery from inactivation is a time- and voltage-dependent process and takes about 3–5 ms. Channels in the open state do not directly return to the closed state without entering the inactive state first. Inactive channels cannot enter the open state without returning to the closed state first. Even though the channel does not conduct Na+ ions in either the closed or inactive state, the two states represent separate and distinct conformations of the channel.
At the peak of the action potential, the membrane potential is close to VNa, but it never reaches VNa (3 in Fig. 6-5). There are two reasons for this. First, the voltage-gated Na+ channels begin to inactivate spontaneously very rapidly after opening. Channel inactivation "plugs" the pore of the channel so that Na+ ions can no longer pass through. A cytosolic region of the Na+ channel actually blocks the Na+ permeation pathway of the channel. This has been referred to as the ball-and-chain model of inactivation (Fig. 6-7).
The second reason for the fact that the peak of the action potential does not reach VNa is that neurons also have voltage-gated K+ channels that become activated by membrane depolarization (also at around the threshold voltage of -40 to -50 mV). Activation of the voltage-gated K+ channels, however, is much slower than that of voltage-gated Na+ channels (Fig. 6-8). For this reason, these K+ channels are referred to as delayed rectifiers. Therefore, at the peak of the action potential, pK is greater than its value when the neuron is at rest, and movement of K+ out of the cell opposes the depolarization caused by the movement of Na+ into the cell (Fig. 6-8).
Figure_na_k_permeability.gif
Figure 6-8. Changes in pNa and pK during the action potential.
Changes in pNa and pK are shown during the action potential. The relative permeabilities are shown (right vertical axis). The relative permeabilites for Na+ and K+ are not drawn to scale. At the peak of the action potential, pNa is ~12 times larger than pK. See text for details.
Once the peak of the action potential is reached, Na+ channels inactivate, and as a result pNa falls rapidly with time, and approaches its value at rest (Fig. 6-8). At this time, however, because of the delayed response of the voltage-gated K+ channels to membrane depolarization, pK is still becoming larger. Now the balance of ion flow across the membrane is in favor of K+ moving out of the cell. Movement of K+ out of the cell brings about rapid repolarization of the membrane back to the resting value (4 in Fig. 6-5). However, pK remains elevated for some time even after Vm has reached the resting value (Fig. 6-8). Therefore, continued movement of K+ out of the cell causes a membrane hyperpolarization (i.e., more negative than Vrest). This phase is commonly referred to as the hyperpolarizing afterpotential or simply hyperpolarization (5 in Fig. 6-5). This is also sometimes referred to as undershoot. This occurs because during this time pNa is at its resting value, but pK is higher than its resting value. Therefore, K+ movement out of the cell will tend to move the Vm closer to VK. Finally, pK returns to its value at rest, and at this time the membrane potential also returns to baseline at its resting value of about -70 mV. It is important to note that K+ channels do not inactivate. They close simply because the membrane potential becomes more negative than the threshold potential (the potential at which Na+ and K+ channels become activated). Thus, the repolarization and hyperpolarization that is caused by movement of K+ out of the cell through the voltage-gated K+ channels, also causes the closing of the same voltage-gated K+ channels.
Refractory Periods Return to lecture outline
Figure_refractory_periods.gif
Figure 6-9. Absolute and relative refractory periods.
During the absolute refractory period, a second stimulus (no matter how strong) will not excite the neuron. During the relative refractory period, a stronger than normal stimulus is needed to elicit neuronal excitation.
As mentioned above, after opening, Na+ channels spontaneously and rapidly enter the inactivation state. At the peak of the action potential, all Na+ channels become inactivated. When Na+ channels are inactivated, they cannot be immediately opened again (Fig. 6-7). Recovery from inactivation is a time- and voltage-dependent process, and full recovery usually takes about 3–4 ms. Therefore, it takes about 3–4 ms for all Na+ channels to come out of inactivation in order to be ready for activation (opening) again. The period from the initiation of the action potential to immediately after the peak is referred to as the absolute refractory period (ARP) (see Figs. 6-9 and 6-10). This is the time during which another stimulus given to the neuron (no matter how strong) will not lead to a second action potential. Thus, because Na+ channels are inactivated during this time, additional depolarizing stimuli do not lead to new action potentials. The absolute refractory period takes about 1-2 ms. After this period, Na+ channels begin to recover from inactivation and if strong enough stimuli are given to the neuron, it may respond again by generating action potentials. However, during this time, the stimuli given must be stronger than was originally needed when the neuron was at rest. This situation will continue until all Na+ channels have come out of inactivation. The period during which a stronger than normal stimulus is needed in order to elicit an action potential is referred to as the relative refractory period (RRP). During the relative refractory period, since pK remains above its resting value (Fig. 6-8), continued K+ flow out of the cell would tend to oppose any depolarization caused by opening of Na+ channels that have recovered from inactivation.
Figure_excitability_recovery.gif
Figure 6-10. Recovery of excitability.
During the absolute refractory period, the neuron cannot be excited to generate a second action potential (no matter how intense the stimulus). As Na+ channels begin to recover from inactivation, excitability is gradually restored. This recovery period is the relative refractory period during which a stronger than normal stimulus is needed to initiate a new action potential.
In summary, inactivation of Na+ channels is solely responsible for the absolute refractory period. Both Na+ channel inactivation and the greater than resting pK value are responsible for the relative refractory period.
The absolute refractory period is responsible for setting the upper limit on the maximum number of action potentials that can be generated during any given time period. In other words, the absolute refractory period determines the maximum frequency of action potentials that can be generated at any point along the axon plasma membrane. This frequency, in turn, has important physiological implications for how the nervous system can respond to high-frequency stimuli, and also for the ability of the nervous system to send high-frequency signals to effector organs when needed (see Frequency Coding in the Nervous System below).
One final note about the refractory period is in order. As mentioned before, the numbers reported in these lectures for various physiological processes correspond to what has been established to be the "norm" or the best-studied example of the process. Although we have reported the refractory period to be 3-4 ms long, it should be noted that the hyperpolarization phase can last up to 15 ms in some neurons. In these neurons, therefore, the relative refractory period is much longer.
Na+ and K+ Concentrations Do Not Change during an Action Potential Return to lecture outline
It is important to note that although large changes take place in the membrane potential as a result of Na+ entry into the cell and K+ exit from the cell, the actual Na+ and K+ concentrations inside and outside of the cell do not change. This is because compared to the total number of Na+ and K+ ions in the intracellular and extracellular solutions, only a small number moves across the neuronal plasma membrane during the action potential. This can be shown by performing a simple calculation (see the following paragraph if you are interested). Under rare conditions of continued excitability, the concentrations may change a little. It is especially possible that continued excitation along small diameter axons may lead to concentration changes. However, it most in most cases ion concentrations do not change.
Below, we will perform a simple calculation in order to assess the extent to which Na+ and K+ concentrations may change during an action potential. The charge separated across the two plates of a capacitor can be described by the following relationship:
Q = CV
where Q is the charge (Coulombs, Coul.), C is the capacitance (Farads, F), and V is the voltage across the capacitor (Volts, V). In the same fashion, the charge separated across the plasma membrane is given by:
Q = CmVm
where Cm is the specific membrane capacitance and Vm is the membrane voltage. Experiments on many cells and also on artificial lipid bilayers have shown that Cm is approximately 1 μF/cm2 (10-6 F/cm2) in most cells. Although estimates as low as 0.7 μF/cm2 have been proposed, it is usually very convenient to use 1 μF/cm2. Therefore, most investigators use 1 μF/cm2 as the specific membrane capacitance for biological membranes.
If we now assume that during an action potential, approximately a 100-mV change occurs in the membrane potential (actually more than 100 mV from –70 mV to about +40 mV), we can easily calculate the total number of charges that must be separated (moved across the plasma membrane) to cause this 100-mV shift in Vm. Therefore,
Q = (1 × 10-6 F/cm2) × (0.1 V) = 1 × 10-7 Coul./cm2
We now need to convert this charge into the total number of ions. Remember that in solutions, charge is carried by ions and that when we are talking about monovalent ions such as Na+ and K+, the charge on one ion is equivalent to 1.6 × 10-19 Coul. (elementary charge). Thus,
equation_1.gif
This number refers to the number of ions that must be translocated across 1 μm2 of plasma membrane in order to bring about a 100-mV change in the transmembrane potential. It is better to convert this number to correspond to an area that is more compatible with that of a typical cell. For a cell of about 10 μm in diameter, the surface area (4πr2) of the plasma membrane would be ≈314 μm2 (assume that the cell is a smooth sphere devoid of microvilli). The volume enclosed by this cell (i.e., the cytoplasmic volume; 4πr3/3) is ≈524 μm3. Assuming intracellular Na+ and K+ concentrations of 10 mM and 150 mM, respectively, the cytoplasm of this cell contains 3.2 × 109 Na+ ions and 4.7 × 1010 K+ ions.
With a total surface area of ≈314 μm2, approximately 2,000,000 Na+ ions (314 μm2 × 6250 ions/μm2 = 1,963,495 ions) enter the cell (spike phase) during a single action potential and, likewise, approximately 2,000,000 K+ ions leave the cell (repolarization phase). A simple comparison of this number with the total number of Na+ ions in the cell shows that the total number of Na+ ions is increased by about 0.06%. This increase is not enough to lead to a significant increase in the macroscopic (bulk) concentration of Na+. Following a similar argument, it can be seen that the relative decrease in the intracellular K+ concentration is even smaller (because the intracellular K+ concentration is much higher).
In summary, during action potentials, Na+ and K+ concentrations do not change. The Na+ ions that enter the cell are too few in number to change the overall (macroscopic) Na+ concentration. Likewise, the K+ ions that leave the cell are few compared to the total number of K+ ions in the cell. Because of the larger volume of the extracellular space, the Na+ and K+ concentrations in this compartment are affected even to a lesser extent. Given enough time, an equal number of Na+ and K+ ions that are translocated during action potentials are moved in opposite directions by the action of the Na+/K+ ATPase. Remember that the turnover rate of this pump is much slower than that of Na+ and K+ channels. Thus, it takes longer for the pump to return an equal number of ions to their appropriate fluid compartment.
Having considered the above argument, it is possible to envision circumstances in which the Na+ and K+ concentrations may change. When small diameter axons (with small cytoplasmic volume) fire repetitively at high frequency, it is likely that the Na+ and K+ concentrations may change. This final point does not alter the argument put forth against changes in ionic concentrations. It only highlights a special case in which small volumes and rapid firing may lead to changes in Na+ and K+ concentrations.
Figure_frequency_coding_threshold_stimulus.gif
Figure 6-11. Frequency coding in the nervous system: Threshold stimulus.
If a threshold stimulus is applied to a neuron and maintained (top, red trace), action potentials occur at a maximum frequency that is limited by the sum of the absolute and relative refractory periods (bottom, blue trace). Here, a threshold stimulus refers to that which is just strong enough to bring a resting neuron to threshold. Thus, with maintained threshold stimulus, subsequent action potentials occur only at the end of the relative refractory period of the preceding action potential. The top and bottom traces are on the same time scale. The dashed line represents the threshold voltage (Vthreshold) of approximately -50 mV. ARP, absolute refractory period; RRP, relative refractory period.
 
Frequency Coding in the Nervous System Return to lecture outline
We have said that once the depolarization caused by the stimulus is above threshold, the action potential is a complete action potential (i.e., it is all-or-nothing). If the stimulus strength is increased, the size of the action potential does not get larger (see Fig. 6-4). If the size of the action potential is always the same, how then does the nervous system code the intensity of the stimulus? The trick that the nervous system uses is that the strength of the stimulus is coded into the frequency of the action potentials that are generated. Thus, the stronger the stimulus, the higher the frequency at which action potentials are generated (see Figs. 6-11 and 6-12). Therefore, we say that our nervous system is frequency-modulated and not amplitude-modulated. If this does not make any sense to you, you will have a chance to test it for yourself during the next computer-based laboratory exercise on the Electrical Activity of the Neuron. Because of the absolute refractory period, there is a limit to the highest frequency at which neurons can respond to strong stimuli. Because the absolute refractory period can last between 1-2 ms, the maximum frequency response is 500-1000 s-1 (Hz). A sample calculation is shown below with the assumption that the absolute refractory period is 1 ms in duration.
equation_2.gif
A cycle here refers to the duration of the absolute refractory period. If the neuron absolute refractory period is 2 ms, the maximum frequency would be 500 Hz as shown below:
equation_3.gif
The above calculations correspond to the maximum frequency of action potentials, and would only be present if the applied stimulus is very large in order to overcome the relative refractory period. Thus, the maximum frequency of action potentials is ultimately limited by the duration of the absolute refractory period. On the other hand, if the applied stimulus is only large enough to bring the neuron to threshold at rest, the maximum frequency of action potentials will now be governed by the total duration of the neuron refractory period (i.e., sum of the absolute and relative refractory periods) (see Fig. 6-11). In a typical neuron, this is 1 + 4 = 5 ms. Under this condition, the maximum frequency of action potentials is 200 Hz as shown below:
equation_4.gif
Here, a cycle refers to the full duration of the action potential (absolute refractory period + relative refractory period).
Figure_frequency_coding_suprathreshold_stimulus.gif
Figure 6-12. Frequency coding in the nervous system: Supra-threshold stimulus.
If a supra-threshold stimulus is applied to a neuron and maintained (top, red trace), action potentials are not allowed to complete the relative refractory period (bottom, blue trace). Thus, with maintained supra-threshold stimulus, subsequent action potentials occur during the relative refractory period of the preceding action potential. With increasing stimulus strength, subsequent action potentials occur earlier during the relative refractory period of the preceding action potentials. With very strong stimuli, subsequent action potentials occur following the completion of the absolute refractory period of the preceding action potential. Thus, the maximum frequency of action potentials is ultimately limited by the duration of the absolute refractory period. The top and bottom traces are on the same time scale. The dashed line represents the threshold voltage (Vthreshold) of approximately -50 mV. ARP, absolute refractory period; RRP, relative refractory period.
 
Pharmacological Inhibition of Na+ and K+ Channels Return to lecture outline
From the description of the action potential it is clear that the changes in the membrane potential result from ionic movements across the cell plasma membrane. These movements are made possible by opening of voltage-gated channels specific for Na+ and K+ ions. Movement of Na+ is into the cell down its electrochemical gradient, and this Na+ entry into the cell depolarizes the membrane and moves Vm close to VNa. Movement of K+ is out of the cell down its electrochemical gradient, and this outward K+ flow repolarizes, and even hyperpolarizes the membrane. Since movement of charge leads to an electrical current, these events can also be studied by using electrophysiological methods. In physiological solutions, current is carried by ions such as Na+ and K+. Physiologists may use many different experimental strategies in order to study the transmembrane movement of ions during an action potential. One simple way is to perform ion substitution experiments. For example, the role of Na+ in the action potential can be studied by experimental elimination of this ion from the extracellular fluid. In addition, the action potential can be studied at different concentrations of external Na+. And similar experiments can be done with K+. In fact, these simple experiments provided much of the early information about action potentials.
As another experimental strategy, physiologists can carefully study the ionic currents passing through the membrane by using agents that specifically block either the voltage-gated Na+ channels or the voltage-gated K+ channels. Tetrodotoxin (TTX) is a highly potent toxin that inhibits voltage-gated Na+ channels. The source of TTX is the ovary of puffer fish. Interestingly, puffer fish is a delicacy in some countries and, therefore, the ovaries must be carefully and completely removed before puffer fish is served. If done incorrectly, even minute quantities of ingested TTX are fatal! Local anesthetics such as lidocaine (Xylocaine®) and procaine (Novacaine®) prevent the generation of action potentials by inhibiting voltage-gated Na+ channels of sensory neurons. Thus, depolarization elicited by sensory stimulation does not lead to the generation of action potentials that can travel to the central nervous system. Lidocaine and procaine are commonly referred to as nerve blocking agents and, as we have mentioned, the molecular basis of their action is inhibition of the voltage-gated Na+ channels. Tetraethyl ammonium (TEA) is a chemical agent that inhibits the voltage-gated K+ channels. Blockers such as TTX and TEA have been instrumental in revealing the workings of ion channels and their roles in neuronal function.
Graded Potentials versus Action Potentials Return to lecture outline
There are important differences between graded potentials and action potentials of neurons. Table 6-1 lists the main differences between graded potentials and action potentials. As discussed in this lecture and upcoming lectures, most of these difference are due to the fact that graded potentials result from the passive property of the neuronal membrane, whereas action potentials results from an orchestrated response involving the coordinated activity of voltage-gated ion channels. Graded potentials must occur to depolarize the neuron to threshold before action potentials can occur. In the next lecture, we will consider the propagation of action potentials and we will see that additional neuronal adaptations allow action potentials to travel over long distances without losing any strength (i.e., amplitude). In yet another later lecture, we will see how summation of graded potentials is responsible for much of information processing at specialized contact regions between neurons (synapses).
Table 6-1. Features of graded potentials and action potentials
Graded potentialsAction potentials
Depending on the stimulus, graded potentials can be depolarizing or hyperpolarizing.Action potentials always lead to depolarization of membrane and reversal of the membrane potential.
Amplitude is proportional to the strength of the stimulus.Amplitude is all-or-none; strength of the stimulus is coded in the frequency of all-or-none action potentials generated.
Amplitude is generally small (a few mV to tens of mV).Large amplitude of ~100 mV.
Duration of graded potentials may be a few milliseconds to seconds.Action potential duration is relatively short; 3-5 ms.
Ion channels responsible for graded potentials may be ligand-gated (extracellular ligands such as neurotransmitters), mechanosensitive, or temperature sensitive channels, or may be channels that are gated by cytoplasmic signaling molecules.Voltage-gated Na+ and voltage-gated K+ channels are responsible for the action potential.
The ions involved are usually Na+, K+, or Cl-.The ions involved are Na+ and K+.
No refractory period is associated with graded potentials.Absolute and relative refractory periods are important aspects of action potentials.
Graded potentials can be summed over time (temporal summation) and across space (spatial summation).Summation is not possible with action potentials (due to the all-or-none nature, and the presence of refractory periods).
Graded potentials travel by passive spread to neighboring membrane regions.Action potential propagation to neighboring membrane regions is characterized by regeneration of a new action potential at every point along the way.
Amplitude diminishes as graded potentials travel away from the initial site (decremental).Amplitude does not diminish as action potentials propagate along neuronal projections (non-decremental).
Graded potentials are brought about by external stimuli (in sensory neurons) or by neurotransmitters released in synapses, where they cause graded potentials in the post-synaptic cell.Action potentials are triggered by membrane depolarization to threshold. Graded potentials are responsible for the initial membrane depolarization to threshold.
In principle, graded potentials can occur in any region of the cell plasma membrane, however, in neurons, graded potentials occur in specialized regions of synaptic contact with other cells (post-synaptic plasma membrane in dendrites or soma), or membrane regions involved in receiving sensory stimuli.Occur in plasma membrane regions where voltage-gated Na+ and K+ channels are highly concentrated.
The details of action potentials noted here refer to those of neuronal action potentials. As we will see throughout the course, other action potentials (for example, in skeletal and cardiac myocytes) exhibit different features than those mentioned here.
 
Exercises Return to lecture outline
Questions Requiring Short Answers
  1. Name a toxin that inhibits voltage-gated Na+ channels of neurons.
  2. Name a reagent that inhibits voltage-gated K+ channels of neurons.
  3. This proposed model accounts for the molecular mechanism governing the inactivation process of voltage-gated Na+ channel.
  4. During this phase of the action potential, if a second stimulus is applied to the neuron (no matter how strong the stimulus), a second action potential will not be generated.
  5. During this phase of the action potential, only a stronger than normal stimulus will lead to the generation of a new potential.
  6. Name at least two agents that inhibit voltage-gated Na+ channels of neurons.
  7. Name at least one agent that inhibits voltage-gated K+ channels of neurons.
  8. In a typical neuron, what are the values for the duration of the absolute refractory period and relative refractory period?
  9. This positive feedback cycle is responsible for the spike phase of the action potential.
 
Multiple-Choice Questions
  1. A typical neuron would have a resting membrane potential of about (A) +70 mV; (B) +70 V; (C) -70 mV; (D) -70 V; (E) All of the above are observed at rest.
  2. Which of the following ions are involved in neuronal action potentials? (A) Na+; (B) K+; (C) Cl-; (D) A and B only; (E) All of the above.
  3. At the peak of the action potential, the membrane potential is: (A) exactly at the Na+ equilibrium potential; (B) close to but more positive than the Na+ equilibrium potential; (C) close to but less positive than the Na+ equilibrium potential; (D) exactly at 0 mV; (E) the same as the resting membrane potential.
  4. In the nervous system, the strength of the stimulus is coded into (A) the frequency of action potentials generated; (B) the amplitude of action potentials generated; (C) both the frequency and amplitude of action potentials generated.
  5. At what membrane voltage do neuronal voltage-gated Na+ channels become activated? (A) –70 mV; (B) –50 mV; (C) 0 mV; (D) +50 mV; (E) None of the above.
  6. At what membrane voltage do neuronal voltage-gated K+ channels become activated? (A) –90 mV; (B) –70 mV; (C) –50 mV; (D) 0 mV; (E) +50 mV.
  7. The spike phase of the action potential: (A) Is due to the opening of voltage-gated Na+ channels; (B) Is due to the opening of voltage-gated K+ channels; (C) Is due to the closure of resting K+ channels; (D) is due to the opening of voltage-gated Cl- channels; (E) None of the above.
  8. The hyperpolarization phase of the action potential: (A) Is due to the opening of voltage-gated Cl- channels; (B) Is due to the prolonged opening of voltage-gated K+ channels; (C) Is due to the closure of resting Na+ channels; (D) Is due to the closure of Cl- channels; (E) None of the above.
  9. Which of the following is NOT true about the refractory period? (A) It is thought that the refractory period is caused by the hyperpolarization phase of the action potential; (B) The refractory period is important in preventing the overlap of succeeding action potentials; (C) The absolute refractory period refers to that time during which a stronger stimulus will lead to the generation of a new action potential; (D) The relative refractory period refers to that time during which a stronger stimulus will lead to the generation of a new action potential; (E) The relative refractory period coincides with the hyperpolarization phase of the action potential.
  10. Which of the following is NOT true of the absolute refractory period? (A) Na+ channel inactivation is responsible for the absolute refractory period; (B) The absolute refractory period is the time during which another stimulus given to the cell (no matter how strong) will not lead to a second action potential; (C) The absolute refractory period takes about 5 ms; (D) The molecular basis of the absolute refractory period is described by the ball-and-chain model; (E) All of the above are true about the absolute refractory period.
  11. Na+ and K+ permeation through their respective ion channels represents an example of (A) Passive transport; (B) Primary active transport; (C) Secondary active transport; (D) A and C only; (E) A, B, and C.
  12. Which of the following is NOT consistent with the function of neuronal voltage-gated Na+ channels? (A) After becoming activated (opening) at the threshold voltage, these channels very rapidly enter an inactive state; (B) Na+ channels may go from the inactive state to the open state; (C) Na+ channels may go from the inactive state to the closed state; (D) Na+ channels may go from the open state to the inactive state; (E) Na+ channels cannot go from the open state to the closed state.
  13. Which of the following is NOT true about the action potential? (A) Action potentials are all-or-nothing; (B) Action potentials travel in a non-decremental fashion; (C) The spike phase of the action potential is due to the opening of voltage-gated Na+ channels; (D) Repolarization and hyperpolarization are due to the activity of K+ channels; (E) All of the above are true about action potentials.
  14. The threshold potential refers to the voltage at which (A) The axon blows up; (B) The membrane breaks down; (C) Voltage-gated Na+ and K+ channels open.
 
True / False Questions
  1. Neuronal voltage-gated Na+ channels inactivate.
  2. Neuronal voltage-gated K+ channels inactivate.
  3. At the peak of the action potential, plasma membrane permeability to K+ (pK) is higher than the permeability to Na+ (pNa).
  4. Graded potentials are all-or-nothing.
  5. Threshold voltage is approximately the same for voltage-gated Na+ and K+ channels.
  6. Influx of Na+ and K+ through their respective ion channels represents an electrogenic process.
  7. The Hodgkin cycle represents an example of a positive feedback loop.
  8. During a typical action potential, the intracellular and extracellular concentrations of K+ and Na+ change significantly.
 
Essay Questions
  1. Explain how pharmacological inhibition of Na+ and/or K+ channels can help physiologists gain a better understanding of the role of these ion channels in the action potential.
  2. Describe the inactivation mechanism for neuronal voltage-gated Na+ channels.
  3. Describe how excitable and non-excitable cells respond differently to hyperpolarizing and depolarizing electrical stimulation.
  4. List and explain a few differences between graded potentials and action potentials.
  5. Explain the molecular basis of the neuronal refractory period. Be sure to discuss both the absolute refractory period and the relative refractory period.
  6. Explain in detail the molecular and ionic basis of action potentials in neurons.
 
Calculation Problems
  1. At the peak of the action potential, Vm is approximately +50 mV. Assuming normal intracellular and extracellular K+ concentrations, (1) calculate the driving force (in mV) that acts on K+ ions, and (2) use the information obtained in part 1 to determine the direction in which K+ ions will flow (i.e., into the cell or out of cell).
  2. Assume the following concentrations of Na+ and K+ ions: [Na+]o = 120 mM, [Na+]i = 6 mM, [K+]o = 2 mM, and [K+]i = 150 mM. Assume further that at the peak of the action potential, pK : pNa is 1 : 12. Calculate Vm at the peak of the action potential.
  3. In a typical vertebrate axon, the absolute refractory period is 1.0 ms and the relative refractory period is 4.0 ms. Thus, the axon is refractory for a total of 5.0 ms. If the axon is continuously stimulated with stimuli only large enough in amplitude to ensure excitation when the neuron is at rest, what is the highest frequency of action potentials that can be generated?
  4. In a typical vertebrate axon, the absolute refractory period is 1.0 ms and the relative refractory period is 4.0 ms. If the axon is continuously stimulated with stimuli large enough in amplitude to ensure excitation, what is the highest frequency of action potentials that can be generated?
Web Resources Return to lecture outline
Please notify S. Eskandari if any of the following sites is no longer available.
Further Reading Return to lecture outline
Advanced Physiology Textbooks
  1. Berne, R.M., Levy, M.N., Koeppen, B.M., Stanton, B.A., editors. 2004. Physiology. 5th edition. Mosby, St. Louis, MO.
  2. Boron, W.F., Boulpaep, E.L., editors. 2005. Medical Physiology. Elsevier Saunders, Philadelphia, PA.
Glossary of Key Terms Return to lecture outline
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Lecture Notes on Physiology
Copyright © 2000-2009 Sepehr Eskandari, Ph.D.
Biological Sciences Department
California State Polytechnic University, Pomona