Several approaches can be used to analyze the structure and function of the nervous system in health and disease. Many of these techniques—for example, the biochemical analysis of neurotransmitter and metabolite levels, anatomical studies of axonal projection sites or neurotransmitter enzymes, and molecular biological studies of messenger levels and turnover—examine the nervous system at the level of groups or populations of neurons. In contrast, by its very nature, electrophysiology is oriented toward the physiological analysis of individual neurons. In this chapter, we describe preparations and techniques that are in a general sense applicable to many systems, with specific examples from the dopaminergic system to draw on our field of expertise.

The use of electrophysiological techniques for the analysis of neuronal physiology depends on the unique properties of the neuronal membrane. Like many other cell types, the neuron possesses an electrochemical gradient across its membrane. The electrochemical gradient itself is a product of two forces: 1) an electrical potential force derived from the voltage difference between the inside and the outside of the cell and 2) a chemical potential force resulting from the unequal distribution of ions across the membrane. Cells set up and maintain this electrochemical gradient because of the selective permeability of their membranes to particular ionic species. Thus, the membrane has a rather high degree of permeability to ions such as potassium but is relatively impermeable to ions such as sodium and calcium.

In the resting state, cells have a very low internal concentration of sodium and calcium. To achieve this state, the cell must expend energy (in the form of adenosine triphosphate [ATP] hydrolysis) to extrude sodium from the intracellular space in exchange for potassium ions. The extrusion of sodium sets up both a chemical gradient (because sodium attempts to exist in equal concentrations across the membrane) and an electrical gradient (because sodium is positively charged and is not freely permeable across the membrane; thus, net positive charges are being removed from inside the cell). To partially counter this electrical gradient, potassium—which is more permeable—flows down the electrical gradient to become concentrated inside the cell. However, during this process, the cell is also setting up an opposing chemical gradient, because potassium is achieving higher concentrations within the cell in comparison with the extracellular environment. When the electrical force drawing potassium into the cell balances the chemical force of the concentration gradient forcing potassium out of the cell, the membrane is at equilibrium—with high extracellular sodium concentrations, relatively high intracellular potassium concentrations, and a transmembrane potential causing the inside of the cell to be negatively charged with respect to its environment.

A typical resting membrane potential for a neuron is rather small, being on the order of -70 mV with respect to the extracellular fluid. In actuality, potassium itself is not freely permeable. A small electrochemical gradient exists in most neurons that attempts to force potassium out of the cell and draw the membrane potential to more negative values. Although the scenario is somewhat more complicated than this (e.g., involving charged proteins and other ionic species with selective permeabilities), this description approximates how a cell gains an electrochemical gradient via the energy-dependent extrusion of sodium.

Note that neurons are not the only cells that have transmembrane potentials. In fact, all living cells have an electrochemical gradient across their membranes that they use for transporting glucose and other essential materials and accumulating them against a concentration gradient. Such energy-dependent processes are usually coupled to other gradients from which they derive this energy. For example, a compound may be taken up and concentrated by linking its transport to sodium, which itself has a large electrochemical gradient in the opposite direction. What makes the neuron unique is its ability to rapidly change the permeability of its membrane to one or more ion species in a regenerative manner. This process underlies the generation of an action potential, sets up active propagation of an action potential down an axon, and triggers the procedure that ultimately results in neurotransmitter release. It also provides the electrophysiologist with a measure of neuronal activity that can be assayed by recording the electrical activity generated by the neuron.

The action potential is an active regenerative phenomenon, which means that the events that initiate the action potential also serve as the force that drives this event to completion. Normally, a given neuron receives information in the form of synaptic potentials. For example, an axon terminal synapsing on the neuron releases a neurotransmitter, which binds to the neuron and selectively alters the permeability of its membrane by opening ion channels linked to its binding site. An ion channel that opens in response to a neurotransmitter is referred to as a ligand-gated channel. If the neurotransmitter activates a channel that increases the permeability of the membrane to a negatively charged ion present in high concentrations in the extracellular fluid (e.g., chloride), the influx of chloride down its electrochemical gradient causes a negative shift in the membrane potential of the cell, thereby increasing the potential difference across the membrane, or a hyperpolarization of the cell. If activation of this channel causes a positively charged ion such as sodium to flow down its electrochemical gradient and into the cell, it will cause a brief decrease in the membrane potential (i.e., a depolarization) of the neuron.

Because a change in the membrane potential alters the electrochemical gradient of potassium across the membrane, potassium ions will flow through their respective channels to restore the membrane to its resting level. Thus, a neurotransmitter that depolarizes the membrane causes an efflux of potassium ions and a return of the membrane potential to resting levels. However, if the depolarization is large enough, another type of channel is activated—the voltage-gated or voltage-dependent sodium channel. In response to a given level of depolarization, this channel increases its permeability to sodium to allow more of this ion to enter the cell. The result is a further depolarization of the membrane and consequently an increased activation of this voltage-dependent channel. Because of the positivefeedback nature of this event, it is referred to as regenerative, because the depolarization augments the very factor that causes the cell to be depolarized. The membrane potential at which this regenerative process is initiated is thus the threshold potential for action potential generation, with a hyperpolarization of the cell causing a decrease in its excitability and a depolarization increasing the likelihood that it will generate an action potential.

The regenerative depolarization of the membrane has limits, however. One limit is the equilibrium potential for sodium. The equilibrium potential is the membrane potential at which the electrochemical gradient for a particular ion is zero, with no net flux of the ion across a membrane. This would occur when the membrane potential is sufficiently positive to oppose the further influx of the positively charged sodium ion across its concentration gradient. Although this potential is usually about +40 mV in many cells, the action potential does not actually reach this value. Instead, another voltage-activated channel that is selectively permeable to potassium is activated. The resultant massive increase in potassium permeability starts to return the membrane potential to its original state, thereby inactivating the regenerative sodium conductance. The increased potassium permeability is sufficient to drive the membrane potential negative to the resting potential and toward the equilibrium potential for potassium (i.e., approximately -80 to -90 mV) before the subsequent decrease in voltage-dependent potassium conductance returns the membrane to its original resting state.

The equilibrium potential of an ion determines the net effect that opening its associated ion channels will have on the neuron. The equilibrium potential occurs when the membrane is depolarized or hyperpolarized sufficiently to offset exactly the effects of the concentration gradient on the ion; as a result, no net flux of this ion crosses the membrane. For example, because of the very high concentration of sodium outside the neuron compared with inside the cell, the large concentration gradient for sodium across the membrane attempts to force sodium into the neuron. Therefore, to oppose this concentration gradient, the membrane potential of the neuron would have to be highly positive to provide an electrical gradient of equivalent force. This occurs at approximately +40 mV for sodium. However, potassium's equilibrium potential is about 10-20 mV more negative than its resting potential, which is partly the result of ATP hydrolysis that exchanges extruded sodium for potassium. As a result, increasing potassium permeability causes a hyperpolarization of the neuron because of an efflux of potassium down its concentration gradient.

Chloride is another common ionic species. This ion is negatively charged and therefore has an electrical gradient that would act against its entering the cell. However, the concentration of chloride is so much higher in the extracellular fluid that the chemical gradient predominates. As a result, opening chloride ion channels causes chloride to flow into the cell, hyperpolarizing the membrane (Figure 5-1). In fact, the opening of chloride ion channels is the mechanism through which the primary inhibitory neurotransmitter in the brain (i.e., 1-aminobutyric acid [GABA]) decreases neuronal activity.

FIGURE 5-1. Determining the ionic nature of a synaptic event.

25 msec

IPSP reversal

Membrane potential (mV)

-60 -50 -40 - 30 -20 -10 i-1-1-1-1-r r - 0.98 V = -69.2 mV

Copyright © American Psychiatric Publishing, Inc,.. or American Psychiatric Association, unless otherwise indicated in figure legend, All rights reserved,

At least three techniques can be used to determine the ionic species that mediates a synaptic response: determining the reversal potential of the ion, reversing the membrane potential deflection produced by changing the concentration gradient of the ion across the membrane, and determining the reversal potential (or blocking the synaptic response) after applying a specific ion channel blocker. In this figure, three techniques are used to illustrate the involvement of a chloride ion conductance increase evoked in dopamine-containing neurons by stimulation of the striatonigral "Y-aminobutyric acid (GABA)ergic projection. (A) The reversal potential of a response may be determined by examining the amplitude of the response as the membrane potential of the neuron is varied. In this example, we superimposed several responses of the neuron evoked at increasingly hyperpolarized membrane potentials (top traces), with the membrane potential altered by injecting current through the electrode and into the neuron (bottom traces = current injection). (A1) A synaptic response in the form of an inhibitory postsynaptic potential (IPSP) is evoked in a dopamine neuron by stimulating the GABAergic striatonigral pathway (arrow). When increasing amplitudes of hyperpolarizing current (lower traces) are injected into the neuron through the electrode, a progressive hyperpolarization of the membrane occurs (top traces). As the membrane is made more negative, the IPSP diminishes in amplitude, eventually being replaced by a depolarizing response. (A2) Plotting the amplitude of the evoked response (y-axis) against the membrane potential at which it was evoked (x-axis) illustrates how the synaptic response changes with membrane potential. The membrane voltage at which the synaptic response is equal to zero (i.e., ~69.2 mV in this case) is the reversal potential of the ion mediating the synaptic response (i.e., the potential at which the electrochemical forces working on the ion are zero). Therefore, there is no net flux of ions that cross the membrane. At more negative membrane potentials, the flow of the ion is reversed, causing the chloride ion (in this case) to exit the cell and result in a depolarization of the membrane. (B) The flow of an ion across a membrane may also be altered by changing the concentration gradient of the ion across the membrane. Normally, chloride ions flow from the outside of the neuron (where they are present at a higher concentration) to the inside of the neuron (where their concentration is lower), causing the membrane potential to become more negative. In this case, the concentration of chloride ions across the membrane of the dopamine neuron is reversed by using potassium chloride as the electrolyte in the intracellular recording electrode. (B1) Soon after the neuron is impaled with the potassium chloride-containing electrode, stimulation of the striatonigral pathway (arrow) evokes an IPSP (bottom trace). However, as the recording is maintained, chloride is diffusing from the electrode into the neuron, causing the electrochemical gradient to decrease progressively over time. As a result, each subsequent stimulation pulse evokes a smaller IPSP, eventually causing the IPSP to reverse to a depolarization (top trace). The depolarization is caused by an efflux of chloride ions out of the neuron and down its new electrochemical gradient. This has caused the reversal potential of the chloride-mediated response to change from a potential that was negative to the resting potential to one that is now positive to the resting potential. (B2) After injecting chloride ions into the neuron, spontaneously occurring IPSPs that were not readily observed in the control case are now readily seen as reversed IPSPs (i.e., depolarizations) occurring in this dopamine neuron recorded in vivo. (C) Another means for determining the ionic conductance involved in a response is by using a specific ion channel blocker. This can be done in two ways: by using the drug to block an evoked response or (as shown in this example) by examining the effects of administering the drug on the neuron to determine whether the cell is receiving synaptic events that alter the conductance of the membrane to this ion. To do this, the current-voltage relationship of the cell is first established. This is done by injecting hyperpolarizing current pulses into the neuron (x-axis) and recording the membrane potential that is present during the current injection (y-axis). These values are then plotted on the graph (filled circles), with the resting membrane potential being the membrane potential at which no current is being injected into the neuron (y-intercept). The slope of the resultant regression line (solid line) is equal to the input resistance of the neuron (Rinput = 36 megohms). After administration of the chloride ion channel blocker picrotoxin (open boxes), a new current-voltage relationship is established in a similar manner. Picrotoxin caused a depolarization of the membrane (y-intercept of dashed line is more positive) and an increase in the neuron input resistance (the slope of the dashed line is larger). The intersection of the membrane current-voltage plots obtained before and after picrotoxin administration is then calculated. By definition, this point of intersection (i.e., ~75 mV) is the reversal potential of the response to picrotoxin, because a neuron at this membrane potential would show no net change in membrane potential on drug administration.

Source. Adapted from Grace AA, Bunney BS: "Opposing Effects of Striatonigral Feedback Pathways on Midbrain Dopamine Cell Activity." Brain Research 333:271-284, 1985. Copyright 1985, Elsevier. Used with permission.

Neurons within the vertebrate nervous system have additional conductances that provide them with unique functions. One of these conductances is the voltage-gated calcium conductance. Like sodium, calcium exists in higher concentrations outside the neuron compared with inside the cell. However, the gradient is even more extreme than it is for sodium. Even though much less calcium than sodium is present in the extracellular fluid, the equilibrium potential for calcium is almost +240 mV because of the extremely low intracellular concentration of this ion. The neuron maintains this low intracellular concentration so as to use this ion for specialized purposes. Thus, calcium influx causes neurotransmitter release, activates calcium-gated ion channels, and triggers second-messenger systems (e.g., calcium-regulated protein kinase). Calcium channels, like their sodium counterparts, are also voltage gated and cause calcium influx into the neuron during the action potential. Furthermore, calcium can influence the excitability of the cell by activating the calcium-activated potassium current, which then causes a large membrane hyperpolarization after the spike, known as an afterhyperpolarization, that delays the occurrence of a subsequent spike in that neuron. After entering the neuron, calcium is rapidly sequestered into intracellular organelles to terminate its action and reset the neuron before the next event. Therefore, calcium can alter the physiological activity and the biochemical properties of the neuron it affects (Llinas 1988).

In addition to the role of ion channels in setting the resting membrane potential, generating the action potential, and repolarizing the membrane potential, they can have a more subtle effect on aspects of electrophysiological function. For example, by regulating properties of the neuronal membrane, such as the amplitude and speed of the neuronal voltage response to inputs, ion channels can regulate neuronal responses to synaptic inputs. By modulation of these parameters, ion channels influence the integration of multiple synapses (temporal and spatial integration) and ultimately regulate the ability of synaptic inputs to depolarize membrane potentials and drive the generation of action potentials. Furthermore, neurotransmitters such as acetylcholine and monoamines can influence these same parameters through modulation of ion channels.

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