Introduction

Electrochemical methods comprise a collection of extremely useful measurement tools for neuroscience. A central feature of these methods is an electrode that provides a surface or interface where some form of a charge-transfer process occurs. This charge-transfer process gives rise to potentials and/or currents that can be measured and related either by theory or by calibration to the concentration of substances in the solution that bathes the electrode. Broadly speaking, these methods can be divided into two groups: measurements that do not involve current, known generally as potentiometric methods, and measurements that involve current flow at an electrode under potential control, known as amperometry, voltammetry, or polarography, depending upon the details of the experimental design. Amperometric and voltammetric methods are the main, but not exclusive, focus of this book. It is completely natural that neuroscientists should find electrochemical methods of value because the nervous system contains numerous electrochemically detectable targets including simple inorganic ions, catecholamine and indolamine neurotransmitters and their metabolites, glutamate, nitric oxide (NO), glucose, lactate, ascorbate, urate, hydrogen peroxide (H₂O₂), oxygen (O₂), and pH. Moreover, the high ionic strength of biological environments creates a perfect milieu for electrochemistry, which requires conductive media.

This chapter briefly introduces the history of amperometry and voltammetry in the neurosciences and briefly surveys their applications. Although not intended as a thorough explanation of the methods themselves, the chapter provides a tutorial and primer on their basic operating principles and points the interested reader to sources of more detailed information.

In Vivo Electrochemistry: The Benefits of Size and Speed

The challenges associated with establishing the selectivity of in vivo electrochemical recordings encouraged many neuroscience laboratories to focus their efforts on the technique of microdialysis. Microdialysis is a sampling technique whereby small molecules are recovered from the extracellular fluid of the brain by passing a perfusion fluid through hollow fiber of dialysis membrane. Samples of the dialysate are analyzed by methods that involve chromatography or, more recently, capillary electrophoresis with a variety of detection strategies based on electrochemistry, fluorescence spectroscopy, or mass spectrometry, to name a few. The selectivity of these analytical tools is tremendous: no one has ever had reason to doubt the identification of a peak in the chromatogram or electropherogram of a dialysate sample. Given the availability of this alternative, superbly selective approach to in vivo neurochemical analysis, it is certainly appropriate to question the merits of the electrochemical techniques.

As the subsequent chapters in this volume will demonstrate, the merits of the electrochemical techniques lie in their ability to perform measurements that provide answers that are not presently accessible by microdialysis or any other measurement technique. Although microdialysis offers superior selectivity, electrochemical recording techniques offer high spatial and temporal resolution. Carbon fibers are available for the construction of electrodes with single-digit micrometer dimensions, which are more then twentyfold smaller than typical microdialysis probes. Due to their small dimensions, microelectrodes inflict minimal damage when they are implanted into living brain tissue (Peters et al. 2004), meaning they can be placed within micrometer distances of neuronal terminals (Venton et al. 2003; Peters et al. 2004). For this reason, electrochemical recordings suffer less diffusional distortion in the recording of dynamic events (Wightman et al. 1988) such as those associated with electrical stimulation procedures. High-speed, highly localized recordings have permitted, for example, the evaluation of the kinetics of a variety of transporters such as the dopamine transporter (Wightman and Zimmerman 1990; Garris et al. 1994; Wu et al. 2001, 2002; Garris et al. 2003; Michael et al. 2005) crucial in terminating the actions of dopamine on presynaptic and postsynaptic dopamine receptors in a number of brain regions. Other transporters have been the targets of electrochemical assays in recent years (Daws et al. 1998; Frazer and Daws 1998; Perez and Andrews 2005).

The skills honed in the refinement of techniques for monitoring electrically evoked dopamine release in the striatum and accumbens of the rat have evolved very recently into the ability to detect spontaneous, i.e., non-evoked, dopamine transients presumably associated with the naturally occurring electrical activity of dopaminergic fibers (Robinson, Heien, and Wightman 2002; Robinson et al. 2003; Cheer et al. 2004; Robinson and Wightman 2004; Roitman et al. 2004). These transients are virtually invisible to microdialysis measurements because of the temporal blurring associated with diffusion across the dialysis membrane. Hence, it is becoming increasingly apparent that the high spatiotemporal resolution of the electrochemical recordings provides unique information regarding the operational characteristics of intact neuronal systems in the living brain.

The high spatiotemporal resolution of electrochemical recording techniques are also proving powerful in a variety of measurement contexts outside the living brain. Neurochemical studies in tissue slices and synaptosomal suspensions generally lack temporal resolution due to the super-fusion methods employed in these preparations; however, direct insertion of a microelectrode into a slice or suspension restores the ability to record dynamic events otherwise lost in conventional measurements (Schenk et al. 1983; Jones, Garris, and Wightman 1995; Jones et al. 1996; Avshalumov and Rice 2003; Avshalumov et al. 2003). Microelectrodes with dimensions in the single micron range are compatible with measurements at the cellular and subcellular domains. Microelectrodes and microsensors have been used in conjunction with numerous electrochemical techniques to monitor the release of substances from single cells (Boudko et al. 2001; Kumar et al. 2001; Smith and Trimarchi 2001). Quantal size and vesicular volume have also been investigated using microelectrodes (Kozminski et al. 1998; Pothos et al. 1998, 2000; Edwards and Sulzer 2000; Colliver et al. 2000). High-speed recordings of adrenaline provided the first direct observation of exocytosis (Leszczyszyn et al. 1991; Mosharov et al. 2003). The value of the high spatiotemporal resolution offered by these electrochemical techniques is unquestionable.

New electrochemical methods and designs continue to be developed, producing better, smaller, and faster electrodes, improving this already versatile technique, while simultaneously discovering new features and functions of the brain and other neurochemical environments. Nearly 30 years after Adams and Gonon first began their in vivo experiments, thousands of papers demonstrate the versatility and functionality of electrochemistry for in vivo detection, with the field continuing to grow, expand, and explore new neurochemical arenas.

The Scope of Electrochemistry in the Neurosciences

Many neurochemical species have been studied using electrochemistry. The electrochemically active catecholamines, including dopamine, norepinephrine, and their metabolites, are perhaps the most-studied neurochemicals. In physiological media (pH 7.4) or in vivo, the catechol moiety of the catecholamines is oxidized to an ortho -quinone, as shown in Figure 1.1. Serotonin and its tryptamine-derived metabolites are also electro-active and have been studied in vivo with voltammetry and microelectrodes (Daws et al. 1998). The phenollic portion of serotonin and its metabolites is also oxidized, to form a ketone, as shown in Figure 1.1. Numerous other electrochemically detectable substances encountered in brain tissues, including ascorbic acid, NO, O₂, and H₂O₂, are easily measured with microelectrodes, with their electrochemical schemes represented in Figure 1.2.

FIGURE 1.1

Electroactive neurotransmitters and their metabolites.

FIGURE 1.2

Additional electroactive species found in the brain—ascorbic acid, nitric oxide, oxygen, and hydrogen peroxide.

The ascorbate anion is a particularly interesting neurochemical. Levels of ascorbate in the brain range from 200 to 400 μM , making it one of the most prevalent molecules in the brain. Ascorbate is oxidized at potentials close to that of dopamine and norepinephrine, so avoiding the detection of ascorbate was one of the early selectivity challenges mentioned earlier. The interferant effects of ascorbate on the electrochemical detection of dopamine can be controlled by increasing the scan rate of the applied potential. Interferant signals from the oxidation of ascorbate are decreased by increasing the scan rate because of the slow rate of charge transfer of ascorbate at the electrode surface. Ascorbate can also interfere with the reduction of dopamine-o -quinone back to dopamine, resulting in an increase in the dopamine signal at the electrode, but this effect can also be diminished by using a fast scan rate. Despite its reputation among electrochemists as an interferent in the in vivo detection of monoamines, electrochemical measurements have illuminated ascorbate’s role as a neuromodulator in brain function (Christensen, Wang, and Rebec 2000; Rebec and Wang 2001).

The electrochemical methods used in the neurosciences are not exclusive to the detection of neurotransmitters and their metabolites but include the detection and measurement of small molecules and biologically significant ions as well. Ion-selective electrodes, such as pH electrodes, are often used in vitro but can be miniaturized for use in various in vivo neurochemical applications (Boudko et al. 2001). The Clark O₂ sensor is a small-molecule, rather than an ion-selective, electrode that has applicability in vivo. O₂ is reduced to produce a measurable current at the electrode surface. The O₂ sensor is constructed simply of a membrane-coated platinum wire electrode; however carbon-fiber cylinder electrodes have also been used, in place of platinum, to detect O₂ (Kennedy, Jones, and Wightman 1992). NO is another small molecule with relevance in vivo. NO sensors, which strongly resemble O₂ sensors, have been constructed to study and measure NO in a number of different environments (Zhang et al. 2000, 2002; Kumar et al. 2001). H₂O₂ is another small molecule of interest to neuroscientists. This molecule is easily detected at carbon-fiber or platinum electrodes; however, it is often oxidized at the electrode in the same potential window as many other electroactive species. Consequently, a more selective electrode or sensor is required to detect and measure H₂O₂ in vivo. Luckily, naturally occurring enzymes selective for peroxide are available and can be coupled with electrodes to make a H₂O₂ sensor (Kulagina and Michael 2003).

Electrode selectivity for electroactive compounds like peroxide and ascorbic acid can also be improved with the addition of electrode coatings and additives. Electrode coatings include electro-deposited polymers (1, 2 diaminobenzene), electronically conducting polymers (polypyrrole), and polyelectrolytes (Nafion). Polymers and polyelectrolyte films, along with a number of different conducting and nonconducting electropolymerizable coatings, can be used to capture or immobilize enzymes on an electrode surface, creating a species-selective sensor (Kulagina and Michael 2003). Enzyme-based electrochemical microsensors are also used to increase selectivity for electroactive analytes, like peroxide and AA, and to create novel detection schemes for non-electroactive compounds, like glucose, glutamate, choline, and lactate (Gregg and Heller 1990; Heller 1992; Barlett and Cooper 1993; Kulagina, Shankar, and Michael 1999; Cui, Kulagina, and Michael 2001; Kulagina and Michael 2003; Wilson and Gifford 2005; Day et al. 2006; McMahon et al. 2006).

Electrochemical measurements of non-electroactive species, such as glucose, glutamate, lactate, and choline, are conducted using electrodes constructed with specialized coatings and additives, which selectively generate electrochemical signals in response to a particular analyte. Oxidase enzymes are the most commonly encapsulated or immobilized enzymes for use in electrochemical sensors, particularly in the detection of glucose, glutamate, choline, and lactate. These enzymes catalyze the conversion of a particular substrate between the reduced or oxidized state, which can then be detected at the electrode surface (Bartlett and Cooper 1993). These redox enzymes typically undergo the following reaction scheme to generate an electrochemically sensitive species, which is monitored at the electrode surface:

Oxidase enzymes use O₂ as the primary cosubstrate, generating H₂O₂ as the final co-product, which can be electrochemically oxidized at the electrode surface. Ideally, the rate of H₂O₂ production is directly related to the concentration of the analyte detected by the enzymes at the electrode. For example, the oxidation of glutamate and subsequent oxidation of H₂O₂ is as follows:

In this type of electrochemical sensor, the redox enzymes are immobilized in thin films to decrease the distance that H₂O₂ has to travel to the electrode surface to be oxidized. Thin films assist in limiting peroxide diffusion or superfluous reaction of H₂O₂, which would result in low sensor sensitivity. The response time of the electrode also decreases with decreasing film thickness, as the diffusion distance of the substrate into the film to react with the enzyme decreases (Day et al. 2006). Thin films of encapsulated or immobilized redox enzymes are often-used electrochemical methods of detection that could be easily and readily extended for use in the neurosciences.

Another type of electrochemical sensor utilizes enzymes for the detection of different species but does not require immobilization of the enzyme on the electrode surface. Instead, this type of sensor “wires” enzymes to the electrode using a cross-linked polymeric redox macromolecule composed of a poly(vinylpyridine) complexed to Os(bipyridine)₂Cl (Gregg and Heller 1990; Heller 1992; Kulagina, Shankar, and Michael 1999; Kulagina and Michael 2003). The redox polymer forms a three-dimensional gel that incorporates, but does not immobilize, enzymes, while at the same time allowing for rapid in-and-out diffusion of substrates and products. Electrons transferred during the reduction of the enzyme are rapidly wired through the redox centers of the polymer to the electrode surface and are detected as changes in the current response. Electrochemical sensors of this nature can be constructed to detect and measure different non-electroactive species simply by changing the functional enzyme from glutamate oxidase to choline oxidase. One type of enzyme-wired electrode for the detection of glutamate utilizes a cascade consisting of a glutamate oxidase and horseradish peroxidase (HRP). HRP is used in this sensor to reduce the H₂O₂ produced by the glutamate oxidase reaction with glutamate. The HRP subsequently oxidizes the osmium redox polymer, generating a current change in response to glutamate detection (Kulagina, Shankar, and Michael 1999).

It should be mentioned that electrochemistry is used in the neurosciences in many capacities, frequently as an online detector for sampling and separation devices like microdialysis and high performance liquid chromatography (HPLC); however, this introduction will focus on the utility of this technique as an in vivo analytical device. The benefits of using microelectrodes coupled to voltammetry in vivo include real-time, microsecond recording and improved spatial resolution. The small size of microelectrodes also limits implantation-induced damage to the brain tissue, while still allowing for extremely fast recording of analytes in the nanomolar concentration range. The numerous features that make microelectrodes excellent tools for in vivo electrochemical applications in neuroscience will be described in more detail later in this introduction. This introduction will concentrate on the applications of electrochemical methods in vivo, but with the awareness that microelectrodes and electrochemical detection are also widely used in other capacities in the neurosciences.

Electrochemistry Fundamentals

This section provides a very brief introduction to the principles of the electrochemical methods of analysis by way of introduction and orientation to the subsequent chapters in this volume. The fundamental aspects of the numerous electrochemical methods of analysis are fully explained in a number of excellent resources, which the interested reader is encouraged to review for a deeper understanding. It should be emphasized that these methods are not mysterious. Instead, they have evolved steadily from the pioneering and Nobel Prize-winning work of Heyrovsky, and they find innumerable applications in many fields of science and technology. In fact, the measurement of pH, which is most often performed electrochemically, is reputed to be the most frequently performed analytical measurement, outnumbering all others combined. At present, sales of home glucose testers generate more revenue than the sales of all other devices for analytical determinations combined. The home glucose tester is a gold microelectrode that detects H₂O₂ generated by glucose oxidase immobilized in a thin polymer film attached to the electrode.

The best sources of more detailed information regarding these techniques are the textbooks used in courses on analytical chemistry. Virtually all of these textbooks (Skoog, Holler, and Nieman 1998, Harris 2003; Christian 2004) have numerous chapters devoted to electroanalytical methods. One particularly noteworthy textbook is entirely devoted to electrochemical methods and is more advanced, intended for use in semester-long, graduate-level chemistry courses (Bard and Faulkner 2001). The remainder of this chapter introduces the principles of amperometry and voltammetry, i.e., methods that involve the flow of current at electrodes under potential control. Most of the subsequent chapters in this book discuss the fundamental aspects of the particular electrochemical technique used in the authors’ hands.

Chronoamperometry

During an electrochemical measurement, the current that provides information about the composition of the solution bathing the electrode is derived from the oxidation and reduction of solution components at the surface of the electrode. When a molecule undergoes oxidation at the electrode surface, electrons flow from the molecule, into the electrode material, and up a contact wire into the terminal of an ammeter, a device that measures and reports the magnitude of the current. When a molecule undergoes reduction, the electrons flow in the opposite direction, i.e., from the electrode material to the molecule. Thus, it is completely appropriate to view the electrode as analogous to either an oxidizing agent or a reducing agent, depending on the direction of current flow. The deciding factor in whether the electrode behaves as an oxidant or reductant at any point in time is the electrode potential, which is under the control of the experimenter. When the electrode potential is adjusted towards negative values, electrons accumulate on the surface of the electrode where they experience coulombic repulsion. One way to escape this repulsion is to leave the electrode, i.e., to enter the adjacent solution. Thus, negative potentials make the electrode a stronger reducing agent. If the potential is adjusted to more positive values, then the electrode begins to attract electrons from its surroundings so that it acts as an oxidizing agent.

How positive does the electrode potential have to be in order to carry out an oxidation reaction? This depends on the nature of the substance undergoing the reaction. Each substance has its own formal potential, the potential at which the substance would be 50% oxidized and 50% reduced if sufficient time is allowed for the reaction to reach equilibrium. Electrochemical methods do not bother to wait for equilibrium to be reached, and instead, measure the current that flows as the reaction moves toward equilibrium; Therefore, faster reactions produce more current than slower ones. Differences between the formal potential values and the kinetics of different reactions give rise to unique electrochemical responses from different substances that turn out to be very useful for confirming the identity of detected substances.

Diffusion of molecules in the solution adjacent to the electrode plays a central role in determining the magnitude of the current observed during electrochemical measurements. In order to undergo electrochemical oxidation or reduction reactions, molecules in the bathing solution must be in molecular-scale contact with the electrode material. Free electrons have virtually no existence in aqueous solutions, so transfer of electrons between a molecule and electrode requires physical contact between them. Hence, the speed at which molecules diffuse to the electrode is also an important determinant in the rate of electrochemical oxidation and reduction reactions. Diffusion is a mode of mass transport that occurs as a result of the tendency of molecules in fluids to undergo continuous random motion, roughly equivalent to the Brownian motion of small particles. In the presence of a concentration gradient, random motion carries more molecules from regions of high concentration to low concentration than vice versa, resulting in net-mass transport down the concentration gradient. In electrochemical systems, the concentration gradient of interest is that caused by oxidation and reduction reactions at the electrode surface. The reaction lowers the concentration of the substance at the surface of the electrode, which initiates diffusion from the surroundings towards the electrode. Ultimately, the rate of diffusion and the rate of the electrochemical reaction are mutually interdependent.

Diffusion is described by Fick’s laws. Fick’s first law relates the diffusion flux, J , to the steepness of the concentration gradient:

$$J=-D\frac{\partial C}{\partial x},$$

1.1

where D is the diffusion coefficient, C is the concentration, and x is distance. In Equation 1.1, the units of J are moles cm⁻² s⁻¹. Converting this quantity to the corresponding current, coulombs per second, requires multiplication by the area of the electrode and a factor for the number of coulombs per mole of reactant:

$${i=nFAD\frac{\partial C}{\partial x}|}_{x=0},$$

1.2

where i is the current, n is the number of electrons transferred per molecule of reactant, F is Faraday’s constant, and A is the surface area of the electrode. In Equation 1.2, the subscript x = 0 specifies that it is the concentration gradient at the surface of the electrode that determines the current.

Perhaps the best way to appreciate Equation 1.2 is to consider its application to an example of an electrochemical measurement. Chronoamperometry is one of the simplest methods to describe, and do. In this method, the electrode is initially held at some rest potential that does not cause oxidation or reduction of molecules in the bathing solution. To perform the measurement, the potential is suddenly stepped to a new potential to initiate the electrochemical reaction (Figure 1.3a). Because of the step-wise change in the electrode potential, chronoamperometry is a member of the so called potential pulse methods. In some cases, it is possible to step the potential to a value that causes the immediate and complete reaction of any molecule of the target substance in contact with the electrode. Hence, the concentration of the substance at the electrode surface goes immediately to zero, creating a concentration gradient between the electrode surface and the bathing solution that initiates diffusion of the substance towards the electrode (Figure 1.3b).

FIGURE 1.3

(a) Diagram of the step potential used in chronoamperometry. (b) Current vs. time response generated in a chronoamperometric experiment. (c) Concentration profile for times after the start of the chronoamperometric experiment (Circled Inset). The decrease (more...)

The concentration gradient evolves over time according to Fick’s second law of diffusion:

$$\frac{\partial C}{\partial t}=\frac{\partial J}{\partial x}=D\frac{{\partial }^{2}C}{\partial x^2}.$$

1.3

As differential equations go, Equation 1.3 is quite simple to solve, and solutions under many conditions are known. In the case of the chronoamperometry experiment under discussion, the solution is very well known (Figure 1.3c). As the reaction at the electrode continues, the solution next to the electrode becomes further depleted, meaning that with the passage of time, the concentration gradient at the electrode surface becomes less and less steep. Hence, the current decreases, as it turns out, with the reciprocal of the square root of time. The current-time relationship for this experiment is known as the Cottrell equation:

$$i(t)=\frac{nFA\sqrt{D}C}{\sqrt{\pi t}},$$

1.4

where all the variables have the same meaning as before.

It is necessary to bear in mind that there might be more than one contribution to the total current observed during an electrochemical measurement. Equation 1.4 describes only that contribution to the current associated with the oxidation or reduction reaction, sometimes called the Faradaic current in deference to Michael Faraday, who first related electrical charge to mass. However, when the potential is suddenly stepped to a new value, there is a second source of current associated with the process of actually changing the potential of the electrode surface. This component is called the charging current and usually decays exponentially after a potential step. A quick glance at Figure 1.3c might lead one to suspect that measuring the current at short times after the potential step would provide the greatest sensitivity. However, this is not so because at very short times the Faradaic current is overwhelmed by the charging current, so typically one waits for the charging current to decay before making the measurement. With the very small electrodes used in most neuroscience applications, the charging current decays within a few milliseconds of the potential step, which is one of the reasons that fast electrochemical recordings are possible.

Other Potential Step Methods

An extension of the chronoamperometric technique is normal pulse voltammetry (NPV), which involves the application of a series of potential pulses each to sequentially different values (Figure 1.4). As in chronoamperometry, the current produced during each step is proportional to the analyte concentration, but it also reflects how the applied potential during each pulse controls the electrochemical reaction so that the voltammogram, i.e., a plot of the current vs. the potential applied during each pulse, is diagnostic of the substance being detected. The voltammogram associated with NPV is sigmoidal in shape, with the Faradaic current starting out at a small value and increasing to a plateau once the potential pulse reaches a value that causes complete oxidation or reduction of the target substance. The current reaches its half-maximal value at the formal potential.

FIGURE 1.4

Diagram of the potential waveform used in NPV.

Differential pulse voltammetry (DPV) is another technique that provides increased sensitivity and more efficient differentiation and resolution of different species. The pulse waveform is different from that used in NPV in that the potential between each pulse is not returned all the way back to the same rest potential (Figure 1.5a). This has the effect of diminishing the charging current between each pulse and allowing for slightly quicker recording of the voltammogram. The current is sampled immediately before each potential pulse is applied (i_a) and immediately before each pulse ends (i_b), and it is the difference between these current responses (i_b − i_a), that is used to produce the voltammogram (Figure 1.5b), hence, the term differential in the name of the technique. DPV produces a peaked voltammogram, roughly corresponding to the derivative of the NPV voltammogram; this peaked voltammogram can improve both the qualitative and quantitative aspects of the technique.

FIGURE 1.5

(a) Diagram of the potential waveform used in DPV. (b) Differential pulse voltammogram.

Cyclic Voltammetry

Chronoamperometry, NPV, and DPV are potential pulse techniques. Potential sweep methods are also available. Cyclic voltammetry (CV) is widely used in neuroscience applications. As the name implies, the potential is swept from an initial potential to a final potential and then returned to the initial potential, usually at the same sweep rate (Figure 1.6a). The current measured continuously during the sweep is reported against the applied potential (Figure 1.6b). Hence, the technique provides voltammetric information (current vs. potential) about the substance being detected. This is useful for the purposes of qualitative identification as the voltammogram of a substance is generally unique in its position along the potential axis and its shape. For example, the cyclic voltammogram of dopamine is easily distinguished from the voltammogram of ascorbate. With a small electrode that rapidly charges to the new applied potential, sweep rates of several hundred volts per second are quite feasible, so the entire CV can be recorded in a matter of milliseconds. This is interesting because the entire CV can be completed in about the same amount of time that it takes to record a single chronoamperometric pulse. Thus, CV is the fastest voltammetric recording technique available, attracting quite a bit of attention since temporal resolution is one of the performance attributes of electrochemistry compared to microdialysis.

FIGURE 1.6

(a) Diagram of a potential waveform used in linear sweep (cyclic) voltammetry. (b) A cyclic voltammogram.

Conclusion

Electrochemical methods are versatile tools for detecting, monitoring, and measuring various neurochemical species. Microelectrodes, in conjunction with the electrochemical techniques presented within this chapter, have been used to explore environments too small or too fragile to be examined in other ways. Electrochemical techniques have also been used to examine chemical events on a subsecond time scale, providing new kinetic information and real-time information about exocytosis and spontaneous neurotransmitter release. Overall, the ease of use and flexibility in application make electrochemical methods ideal for studies requiring superior temporal and spatial resolution of neurochemicals in physiological environments.

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