Principles of voltammetry and microelectrode surface states

Journal of Neuroscience Methods, 48 (1993) 225-240

225

© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-0270/93/$06.00 NSM 01514

Principles of voltammetry and microelectrode surface states Kirk T. Kawagoe, Jayne B. Zimmerman and R. Mark Wightman Department of Chemistry, UniL,ersityof North Carolina, Chapel Hill, NC 27599 (USA) (Received 16 November 1992) (Accepted 16 February 1993)

Key words: C y c l i c v o l t a m m e t r y ; C h r o n o a m p e r o m e t r y ;

Differential

pulse

voltammetry;

Microdialysis;

E x t r a c e l l u l a r fluid; E l e c t r o d e surface; D o p a m i n e

Contents i. II.

III.

IV. V.

VI. VII.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vivo voltammetric techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Cyclic voltammetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Chronoamperometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Differential pulse voltammetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Requirements for a voltammetric work station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Electrode fabrication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Electrode calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Selectivity of in vivo voltammetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electrode modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Carbon surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Electrochemical modification of electrode surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . C. Electrodes coated with cation exchange membranes . . . . . . . . . . . . . . . . . . . . . . . . . Implantation techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vivo experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VIII. Interpretation of stimulated release of dopamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

225 226 227 228 229 230 230 231 232 233 234 234 235 235 236 236 237 238 238 238

I. Introduction * This review was originally written for The Society for Neuroscience Short Course on Measuring the Chemical Microenvironment held in 1991. Correspondence: R. Mark Wightman, Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, USA.

T h e u s e o f v o l t a m m e t r y f o r t h e in vivo d e t e c t i o n o f b i o g e n i c a m i n e s a n d r e l a t e d s u b s t a n c e s in the extracellular fluid (ECF) of the rat brain o r i g i n a t e d in t h e l a b o r a t o r y o f R a l p h A d a m s

in

t h e e a r l y 1970s. S i n c e t h a t t i m e , e l e c t r o c h e m i c a l

226

methods have shown the ability to provide unique information both in vivo, in the mammalian central nervous system (CNS) (Crespi et al., 1988; Stamford et al., 1988) and in vitro, with single-cell preparations (Wightman et al., 1991) or brain slices (Bull and Sheehan, 1991; Kennedy et al., 1992). Recent studies have focused primarily on the biogenic amine neurotransmitters, dopamine (Wightman et al., 1988a), norepinephrine (Mermet et al., 1990; Suaud-Chagny et al., 1990), serotonin (Crespi et al., 1990), metabolites of these neurotransmitters (Cespuglio et al., 1981a,b), and ascorbic acid (Yount et al., 1991). Although voltammetry has gained acceptance as a valid neurochemical technique, the most widely used method for sampling the ECF is microdialysis (Lunte et al., 1991). The microdialysis probe consists of two concentric tubes (typically stainless steel a n d / o r fused silica) with an outer diameter greater than 200 ~m. The tip of this assembly is covered with a low molecular weight cut-off dialysis membrane to isolate the dialysate (the fluid which is pumped through the dialysis probe) from the ECF. Small molecules, such as neurotransmitters, are removed from the ECF by diffusion across the dialysis membrane and are subsequently analyzed. Voltammetry and microdialysis have been used extensively to study the neurochemical composition of the brain. However, because of differences in how they sample, these techniques provide distinctive information on the composition of the extracellular fluid. Microdialysis removes sample from the ECF via transport of the analyte across a dialysis membrane. The sample is transported out of the brain in the dialysate for subsequent on- or off-line analysis. This has several benefits including chemical specificity via HPLC, and as a result, simultaneous detection of several compounds is readily accomplished. However, because the diffusion of molecules across the dialysis membrane is slow, sample collection is typically restricted to 5-10 rain intervals. Furthermore, sample degradation during the transport period may occur. In contrast, voltammetric techniques analyze the sample at the surface of the implanted electrode. Molecules detected by voltammetry are subject only to diffusion in the

ECF and the response time of the electrode. Voltammetry can be used to rapidly measure concentration changes in vivo and samples are analyzed over millisecond to minute time intervals. Thus it is clear that these two methods of sampling provide very complimentary information. Dialysis with its excellent selectivity and sensitivity can be used to simultaneously determine slow concentration changes and basal levels of neurotransmitters and their metabolites, while voltammetry provides dynamic information of neurotransmitter kinetics. Another distinction of in vivo voltammetric techniques is the extremely small size of the recording electrode. This enables the discrete sampling of small nuclei within the brain with spatial resolution of 10-100 ~m (May and Wightman, 1989a). Most electrodes used in vivo have diameters of 10-20/~m (compared to microdialysis probes of 200 p,m diameter) and sample from a region of comparable size. This allows for the exploration of heterogeneity of different brain regions while the microdialysis probe integrates chemical changes from the surrounding tissue. In this review the benefits and limitations of the electrochemical methods which have been developed for in vivo measurements are discussed, and the subject of electrode surface modification, because of its vital role in electrode sensitivity, time response and selectivity, is also addressed. The paper concludes with an overview of the neurochemical model developed in this laboratory for the quantitative description of the regulation of dopamine levels in the brain. The use of carbon microelectrodes for detection of compounds of neurochemical interest in vivo has been the subject of review, especially with regard to practical aspects of implementation (Stamford, 1986; Justice, 1987; Marsden et al., 1988; Wightman et al., 1988b; Adams, 1990). A description of the use of potentiometric methods for the selective detection of intra- and extracellular ions can be found elsewhere (Nicholson and Rice. 1988). II. In vivo voltammetric techniques

Three voltammetric techniques have been used extensively in brain tissue and are described in

227

the sections following: cyclic voltammetry, chronoamperometry, and differential pulse voltammetry. The discussion begins with cyclic voltammetry because it provides insights into many of the processes occurring at and near the surface of the working electrode.

II.A. Cyclic t~oltammetry The theory for cyclic voltammetry has been developed for some time (Nicholson and Shain, 1964). Its use for neurochemical studies is illustrated by the cyclic voltammogram of 1 /xM dopamine measured with a carbon-fiber microelectrode shown in Fig. 1. During the anodic (oxidizing) scan the current begins increasing at 250 mV at which point the electrode has sufficient oxidizing power for the oxidation of dopamine. The current continues to increase with the applied potential and reaches a maximum at 600 mV when the rate of the electrode reaction exceeds the rate at which molecules can diffuse to the electrode. If the electrode was maintained at this potential, the current would decay toward the predicted steady-state value (Wightman and Wipf, 1989). During the cathodic (negative going) scan, the current increases as the dopamine-oquinone (DOQ) generated during the anodic scan is reduced at the electrode. The current peaks at - 2 0 0 mV and decays toward zero at which point 1.0

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the quinone adjacent to the electrode has been consumed. The total amount of quinone reduced is less than dopamine oxidized because of diffusional losses. During the oxidative scan, the dopamine is sampled from a region of less than 10/xm from the electrode surface. Several features of the cyclic voltammogram are used to obtain information concerning the chemical nature of the substance detected. The location of the peaks on the voltage axis (denoted Ep,a and Ev,c for the position of the anodic and cathodic peaks, respectively) are useful for identification of the compound. In many cases the midpoint between these two values is identical to the E {I, a fundamental thermodynamic quantity. While this information does not provide absolute identification, because many compounds have similar E°s, it does allow a large number of molecules to be eliminated from consideration. The ratio of the cathodic peak c u r r e n t (ip, c) t o the anodic peak current (ip,,) also provides information concerning the identity of the detected substance. For example, the product of the oxidation of ascorbate, a substance present at 200/xM in the ECF of the brain, is chemically unstable, and its cyclic voltammogram does not exhibit any current on the reverse scan. The magnitude of ip,a provides information on the concentration of the species detected. Current from repetitive cyclic voltammograms can be sampled to obtain the time-dependent composition of the surrounding solution. Fig. 2 is an example of such data collected by flow injection analysis of 1 /xM dopamine using a carbonfiber electrode. As a rule of thumb, the interval between cyclic voltammograms is usually 10 times the duration of a voltage scan to allow the diffusion layer to relax to its original state. For example, when cyclic voltammetry is performed with high scan rates (300 V / s ) a complete voltammogram is obtained in less than 10 ms and can be repeated every 50-100 ms. The limits of detection in cyclic voltammetry arise from interference by current other than the faradaic processes of interest. This residual current is comprised of at least two components: double-layer capacitance associated with the layer of ions which forms at the solution/electrode

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interface and the oxidation and reduction of surface-bound species. The residual current, because of its association with surface processes, increases linearly with the scan rate, whereas the faradaic current (arising from the oxidation and reduction of solution species) increases with the square root of scan rate because of its association with diffusion. The relationship between residual and faradaic currents suggests that very slow scan rates be employed for optimum detection limits, However, this is unsatisfactory when monitoring rapid concentration changes such as those found for neuBackgrounds

ll.B. Chronoamperometry

Subtraction

B i o.2

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rotransmitters in the brain• However, even at high scan rates, the detection limit for the in vivo m e a s u r e m e n t of dopamine is approximately 50 nM (Rice and Nicholson, 1989)• Fig. 3 indicates the large difference between the faradaic and residual currents in a fast-scan voltammetric experiment. Electrochemical verification is done via a background-subtracted voltammogram. A background voltammogram is obtained before the substance of interest undergoes a concentration change• The 'signal' voltammogram is collected during a concentration change of the analyte of interest, which can be induced in vivo either by chemical (e.g., K * ) or electrical stimulation, and a background-subtracted voltammogram is digitally generated (Fig. 3B). One further consideration is the length of the diffusion layer extending from the electrode surface. First, the possible toxic effects of D O Q on brain tissue make it desirable to minimize the quantity of the ortho-quinone generated and reduce the distance it is allowed to travel into the brain tissue• Fortunately, the amount of D O Q produced at the microelectrode during rapid scans does not a p p e a r to affect the reproducibility of measurements during in vivo experiments• A second consideration is the close proximity of the brain tissue to the implanted electrode (ca. 8 /zm). This is most important for calibration purposes and is discussed in a separate section below.

In c h r o n o a m p e r o m e t r y , the potential is stepped and t h e current measured as a function of time. For in vivo chronoamperometry, the values of the potentials employed in the square wave are approximately 200 mV beyond the anodic and cathodic peaks found in a cyclic voltammogram of the species of interest. The chronoamperometric current for the oxidation of d o p a m i n e is shown in Fig. 4B. During the forward step, the current arises from the oxidation of dopamine, whereas the current on the reverse step arises from the reduction of the D O Q generated during the initial step. At the beginning of each step the current is a sharp spike that then relaxes towards the

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Fig. 4. Potential waveforms for (A) chronoamperometry and (C) differential pulse voltammetry, and their corresponding current outputs (B and D, respectively), d E is the pulse height in differential pulse voltammetry, and dt is the time between current measurements.

zero current line. (The residual current has been digitally subtracted from Fig. 4B.) The time-dependent behavior of the current is once again a function of the diffusion processes that occur because of the depletion of the electroactive species at the surface of the electrode by electrolysis. One advantage of chronoamperometry over cyclic voltammetry is that the current throughout the entire interval of the potential step is proportional to the concentration of the species electrolyzed. Thus, more data can be averaged and, in theory, signal-to-noise ratios can be improved. The chief disadvantage is that one can no longer tell the origin of the current. Any species that is electrolyzed in the potential range of the step as well as the residual current will contribute to the observed current. Because the residual current decays exponentially with time, whereas the faradaic current decays with the square root of time (again, because of diffusion), current measurements are usually made at times near the end of the step. Like cyclic voltammetry, the measurements can be repeated rapidly to obtain good temporal resolution of concentration. The ratios of the integrated oxidative and reductive currents (charge) obtained by fast chronoamperometry can be used

as a method for the identification of the substance detected (Gratton et al., 1989). Ascorbic acid, for example, has no current on the return step and can be distinguished from any of the catechol-containing neurotransmitters or their metabolites. The charge ratios of dopamine and serotonin have been reported to be ca. 0.5 and 0.25, respectively, and can be distinguished from each other (Gratton et al., 1989). However, although this technique has shown some utility, any concentration dependencies, effects of mixtures, or the chemical basis for these results have not been reported. The detection limits for dopamine by fast chronoamperometry appear to be approximately that of cyclic voltammetry.

II. C. Differential pulse uoltammetry This technique was originally developed to provide high discrimination against residual current and is a hybrid of the above two techniques. While it is the most sensitive of the three techniques discussed in this paper (ca. 5 nM detection limits), the sensitivity is obtained at the expense of time resolution. The applied potential consists of a ramp on which are superimposed small amplitude potential pulses (Fig. 4C). The scan rate is very slow (typically 5 m V / s , requiring 80 s for a complete scan), which keeps residual current to a low value, and scans can be repeated at 2-3 rain intervals. Further discrimination against residual current is obtained by the use of sample-and-hold techniques in the measurement of the current. The current is sampled directly before each pulse, and this value is subtracted from the current measured at the end of the pulse (the current sampling times are indicated in Fig. 4C). Thus, the current is given in differential form, and the peaks for each species electrolyzed are much sharper than found in cyclic voltammetry. This is advantageous since species that are electrolyzed at adjacent potentials can be resolved more easily. Shown in Fig. 3D is a differential pulse voltammogram of D O P A C and ascorbate at a carbon-fiber electrode. Much of the sensitivity of the differential pulse method, as it is used in vivo, results from electrochemical modification of the electrode surface (see below). This surface modification greatly in-

23t) creases the sensitivity of the electrode to dopamine and shifts the peak for ascorbate oxidation towards it thermodynamic value. This treatment is beneficial because it allows for the simultaneous monitoring of catechols and ascorbic acid, and it removes the potential for catalytic regeneration of the catechols by ascorbic acid. However, this greatly increases the response time of the electrode. In addition to the techniques described here, many other voltammetric techniques exist. Some, such as semidifferential techniques, have been employed in vivo, but are less used today. A subset of the differential pulse technique developed by Gonon, differential pulse amperometry (Marcenac and Gonon, 1985), is able to obtain rapid measurements at the expense of electrochemical resolution. Millar has proposed a variant of cyclic voltammetry, fast differential ramp voltammetry, for the rapid measurement of endogenous dopamine and ascorbate levels (Millar and Williams, 1990). Other techniques such as square-wave voltammetry remain to be explored for use in the brain. However, in all cases the central processes of diffusion and control of electrode potential dictate the appearance of the current, and the ability to discriminate against the residual current determines the sensitivity.

III. Requirements for a voltammetric work station

The components required to establish a voltammetric work station include a potentiostat, the appropriate electrodes, and a data acquisition and processing system. Several options are available in each of these areas; unfortunately, the instrumentation typically used by electroanalytical chemists is not suitable for in vivo electrochemical studies. These systems have been optimized for current levels in the milli- to microampere range, whereas, the currents measured at microelectrodes typically range from femto- to nanoamps. As a result, system noise is far too large for precise and accurate measurement of transmitter levels. The typical approach for im-

provement of signal-to-noise is reduction of instrumental bandwidth. This is not suitable for fast electrochemical measurements since a high bandpass (2-3 kHz) must be maintained for accurate collection of voltammograms. Other considerations also play a role in the selection of instrumentation. For example, if neurotransmitter kinetics arc the target of the proposed studies, the primary requirement of the system is that it allows rapid measurements. The necessary speed is not available with many commercial potentiostats designed for general voltammetric experiments. Another critical component is the size of the electrode that will be used. To minimize injury to the brain, the working electrode should be as small as possible. However, the faradaic current is proportional to the surface area of the electrode. For this reason, the ammeter used with small electrodes must have very large amplification. Unfortunately, no single commercial system is capable of executing all of the vo[tammetric experiments that may be of interest to neuroscientists. For these reasons, one first needs to decide on the questions to be asked with voltammetry before the equipment is purchased. In the following paragraphs some of the options that are available will be discussed to aid in these decisions. III.A. Instrumentation General purpose potentiostats are available from several companies, which include E G & G Princeton Applied Research (CN 5206, Princeton, N J), Bioanalytical Systems (West Lafayette, IN), and Tacussel (Villeurbanne, France). The BAS CV-37 is capable of the low-current measurements necessary for use with the small electrodes used in in vivo experiments. However, it lacks a data acquisition system and is restricted to relatively slow voltammetric measurements. The Tacussel 'Biopuls' instrument is suitable for in vivo experiments with differential pulse voltammetry and other techniques that involve application of potential pulses. Its chief limitation is that one is restricted to the techniques that have been designed into the instrument. Several small companies have built voltammetric systems specifically designed for in vivo

231

voltammetric experiments. In our laboratory we use a potentiostat from Ensman Instrumentation (EI-400, Bloomington, IN). This instrument excels at low-current, high-speed measurements, and can be used as a stand-alone instrument for cyclic voltammetry (Wiedemann et al., 1991). The use of other techniques requires an external waveform generator. Although this instrument does not come with a computer system, it has all of the necessary input and output connections for computer interfacing and can be operated with locally written software. A particularly useful feature of the EI-400 is that it can simultaneously control two working electrodes, which allows direct comparison of voltammetric results in different brain regions. An instrument specifically designed for in vivo cyclic vohammetry is sold by PD Systems, Ltd. (Surrey House, Pool Close, West Molesey, Surrey, UK), and is designed around a memory circuit that allows some data processing. The IVEC5 (Medical Systems, Greenvale, NY) is a computer-based potentiostat that was specifically designed for chronoamperometric experiments. The system is very user friendly, which enables one to quickly master the system and begin experiments. A computer-based voltammetric system is also sold by Cypress Systems (Lawrence, KS). This system is suitable for use with small electrodes and is programmed to execute several different types of voltammetric experiments. However, its use with in vivo applications has been restricted to date. Because the currents are typically small, extreme care must be taken to avoid electrical noise. The preparation is typically placed in a Faraday cage to prevent pick-up of electrical noise. Noise from the waveform generator is differentiated by the electrochemical cell and can serve as a major noise source in an improperly designed system (Howell et al., 1986). Current amplification is usually done with a 2-stage amplifier circuit: a preamplifier, situated near the working electrode to prevent noise pick-up, and a second amplifier within the potentiostat. Appropriate low-pass filtering is essential for optimum signal-to-noise. The bandpass depends on the technique and can range from 15 kHz for cyclic

voltammograms obtained at 1000 V / s to less than 1 Hz for slower techniques. The temporal changes of substances adjacent to the electrode come from successive voltammograms. This information can be sampled from each successive run with a sample-and-hold amplifier or with computer processing. The data extracted from each voltammogram need to be filtered at a lower bandpass than used during each voltammogram and can be done digitally if the data are stored in the computer.

III.B. Electrode fabrication A variety of materials can be used to fabricate voltammetric electrodes. Carbon fibers are often employed because of their resistance to drift when exposed to biological tissue and are attractive for in vivo use because of their small size (7-35 ~m). Carbon fibers can be obtained from several manufacturers and constructed electrodes can be obtained from Bioanalytical Systems (West Lafayette, IN). Noble metal electrodes have also been reported; however, they are too unstable for practical use in vivo. The graphpoxy electrode is a carbon-based electrode which has found use for in vivo electrochemical recording (Conti et al., 1978); however, these electrodes are larger (150 /xm diameter) than carbon-fiber electrodes. Proper electrode fabrication is essential to minimize residual currents. To prepare a carbon-fiber electrode (Fig. 5), a single carbon fiber (type P-55, Amoco Greenville, SC) is inserted Asplrote

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232

into a microfillament glass capillary (0.6 mm inner diameter, 1.2 mm outer diameter, A.M. Systems, Everett, WA). This can be done by using a small amount of suction on one end of the capillary to pull the fiber into the other end. The capillary is then placed in a micropipette puller (Model PE-2, Narashige, Tokyo, Japan) and pulled to a sharp tip. The pulled ends of the capillary are trimmed with a scalpel to a diameter slightly larger than the fiber itself ( < 15p.m), and the tips are placed in an epoxy to obtain a seal between the glass and the fiber. The epoxy (Epon 828) is heated to approximately 80°C and 14% (w/w) of the hardener (m-phenylenediamine, both from Miller-Stevenson, Danbury, CT) is added. The electrodes are dipped in this mixture for sufficient time to allow the epoxy to enter the capillary tip (approximately 20 s). If the electrodes are to be used as cylinders, the electrode is rinsed in hot acetone immediately after dipping to remove excess epoxy. The epoxy is allowed to gel overnight (or longer if possible) at room temperature, and is cured at 100°C for 2 h or more followed by 150°C for 2 h. If the curing procedure is not properly performed, gaps between the carbon fiber and epoxy will occur, resulting in high residual currents. The epoxy and hardener have limited shelf lives and should be replaced every 6-12 months. At this point the carbon fiber protrudes from the glass to give an active surface area with the geometry of a cylinder. An elliptical surface is obtained by polishing the electrode with a micropipette polishing wheel (KT Brown Type, Sutter Instruments, Novato, CA) at an angle of 2545 °. To make electrical connection with the fiber, colloidal graphite (Bio-Rad, Cambridge, MA) is injected into the glass capillary with a 2 - 3 in. hypodermic needle and a wire is inserted into the capillary. A number of suitable reference electrode materials are available. The most commonly used in vivo are the A g / A g C I and sodium-saturated calomel reference electrodes. Reference electrodes can be made in-house; however, electrodes for experiments with anesthetized animals can be purchased from BAS. Ag/AgCI electrodes can be constructed by anodizing ( + 1 V) 0.25 mm

silver wire in 0.1 N HCI for 5-11) rain. Good reference electrodes should be dark grey to black in appearance and should be re-anodized before each experiment. If an auxiliary electrode is employed, a platinum or chromalloy wire attached to the skull with a mounting screw will suffice.

III.C. Electrode calibration Calibration of the electrode provides a way to relate the current and concentration of the species detected and to determine the temporal response of the electrode. Calibration before an experiment is used to determine whether the electrode has appropriate sensitivity and response time. The sensitivity of the electrode may deteriorate by as much as 40% in the brain; thus, calibration after in vivo use is necessary. We typically calibrate the electrodes in a flowing stream so that the electrode can be quickly exposed to the transmitter of interest. Concentrations employed should be in the range of those found in vivo and should replicate conditions found in the brain. For example, the concentration of dopamine in the striatum during electrical stimulation experiments is typically 0.1-2 tzM. Calibrations should be made with potential interferants as well as the substance of interest to determine the selectivity of the measurement. We typically examine the effect of AA (200 tzM) with dopamine (4 #M). Other compounds that should be tested include DOPAC, uric acid, and glutathione (Hafizi et al., 1990). Quantitation and identification of detected substances have been major issues in in vivo electrochemistry. As a result, a number of studies have appeared in the literature assessing the reliability of electrochemical measurements a n d / o r exploring new techniques to enhance selectivity of the methods (Cespuglio et al., 1981; Baur et al., 1988; Hafizi et al., 1990). Quantitative approaches to improvement of signal-to-noise has received little attention but remains a key issue in the field (Rice and Nicholson, 1989; Wiedemann et al., 1991). With all in vivo techniques, the question can be raised as to the validity of the use of calibration curves obtained in free solution to determine in vivo concentrations, Fast techniques are least

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problematic in this regard. Electrolysis for 10 ms results in a diffusion layer, the region where concentrations are perturbed by the measurement, that extends approximately 4 /~m from the electrode surface. Thus, the diffusion layer in the brain will be similar to the calibration experiment, which gives validity to the conversion of the measured currents to concentration.

IV. Selectivity of in vivo v o i t a m m e t r y

The chief limitation of in vivo voltammetry is limited chemical resolution. Compounds that are electrolyzed at similar potentials all give very similar responses. For example, the fast-scan cyclic voltammogram of dopamine is identical to that of norepinephrine (Fig. 6). Furthermore, the voltammograms of ascorbate and DOPAC, while different in shape, occur in the same voltage range and can obscure the signals obtained for catecholamines. This problem is particularly aggravating because ascorbate and D O P A C are present in the extracellular fluid in at least 1000 times greater concentration than dopamine in the unstimulated animal. Similarly, 5-HIAA and uric acid can interfere in the detection of serotonin.

As will be discussed in Section V, selectivity can be improved by modification of the surface of the electrode. Nevertheless, it is prudent to undertake a series of experiments to ensure the identity of the detected substances. We have established the following guidelines for identification (Wightman et al., 1987). As previously mentioned, voltage scan techniques can reveal differences in shapes and positions of the voltammetric waves that can serve as a signature of the compound detected (cf., dopamine and ascorbate voltammograms in Fig. 6); however, this should be verified by the use of other methods of chemical analysis. This can be done by a combination of voltammetry with microdialysis techniques or other approaches such as local injection of enzymes which can remove likely interfering species in the tissue near the electrode (Wightman et al., 1988b). A third way to provide confirmation of identity is to take advantage of the anatomical complexity of the brain. The voltammetric results must correlate with the regional distribution of the identified compound. For example, the striatal dopaminergic system must respond only when specific stimulation of the nigrostriatal pathway is performed. Some caution must be taken for biogenic amines; however, since their presence is not necessarily indicative of their functional importance in a particular region. Detection of the species in question must also correspond to the known physiology of a given system (e.g., loss of response due to specific lesion of a pathway). Concurrent recording of electrical activity of neurons during electrochemically detected neurotransmitter overflow has also been performed using a separate physiological electrode or at the same working electrode (Millar and Armstrong-James, 1984; Kuhr et al., 1987; Williams and Millar, 1990a,b) and can be used to show loss or enhancement of electrical and chemical activity. Pharmacology can also be used to provide supporting evidence for identification. The changes in the voltammetric signal must reflect the known pharmacology of the drug employed. These experiments are best performed with drugs that have well-established and simple actions (i.e.,

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synthesis inhibitors or synthetic precursors) so that the results can be easily interpreted.

A

V. Electrode modifications

The most successful approaches to selectivity of voltammetric techniques for in viva use have involved modification of electrode surfaces. For faradaic electrochemistry to occur, the molecule of interest must be able to come into contact with the electrode surface. Furthermore, during the approach to the surface, the molecule will interact with chemical sites on the electrode. Thus, manipulation of the surface can have profound effects on the observed electrochemistry.

KA. Carbon surfaces The surfaces of graphitic materials such as carbon fibers are very complex (McCreery, 1991). A cartoon of the structure of graphite is shown in Fig. 7. At the atomic level, graphite has a 'chicken-wire' appearance. The surface of the chicken wire, referred to as the basal plane, is almost electrochemically inert in aqueous solution. It appears that most electron transfer reactions occur at the edges of the basal plane. Note that this surface is often covered with a large number of oxygen-containing functional groups. Carbon fibers are structurally well defined as a result of a manufacturing process. Thus, the cylindrical surface tends to contain more basal SurFace Carbon

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Fig. 8. Background-subtracted vottammograms at a Nafioncoated carbon-fiber microelectrode in pH 7.4 HEPES buffer at 100 V / s . A: - 0 . 4 unit pH change. B: 5 /~M DA with a I).4 unit pH change.

plane character than the cross-sectional region, and the surface of bevelled carbon fibers contains many surface functional groups. An important consequence of this surface chemistry is that compounds that are cations at physiological pH give voltammograms of different shape than those of anions. Carboxyl groups on the surface will be deprotonated at physiological pH, resulting in a negative charge on the electrode surface. The fast-scan voltammograms of D O P A C and dopamine (Fig. 6B and D) show the effects of this charge: While the voltammogram of dopamine is well defined, that for D O P A C is smaller in amplitude and the peak potentials show a greater separation, an indication of a stow rate of electron transfer with the electrode surface. Experiments in which the surface states are altered suggest that surface functional groups can directly result in this type of behavior (McCreery, 1991). Furthermore, cationic substances such as dopamine appear to adsorb on carbon surfaces (Baur et al., 1988), which again suggests strong interactions with the surface functional groups. Surface oxides also influence the magnitude of the residual current at carbon electrodes. Functionalities such as quinones and phenolic groups are electroactive and result in the waves seen in voltammograms recorded at carbon electrodes in buffer solutions at approximately 0.15 V and - 0 . 3 V (Fig. 3A). The voltage at which these waves occur depends on the pH of the solution because the electrolysis process involves the transfer of hydrogen ions as well as electrons. For this reason, background subtraction of fast-scan vottam-

235

mograms can reveal the presence of a pH shift of 0.4 units (peak at 0.1 V, Fig. 8A). This signal does not obscure that for dopamine, but is additive (Fig. 8B).

V.B. Electrochemical modification of electrode surfaces In 1980 Gonon and coworkers showed that the response of carbon fibers could be greatly altered by electrochemical oxidation of the cylindrical surface (Gonon et al., 1980). The modification employs fast-scan cyclic voltammetry over the range of 0.0-3.0 V. After this treatment, differential pulse voltammograms show sharper, resolved peaks for ascorbate and D O P A C and greatly enhanced signals for the biogenic amines with detection limits that approach the 5 nM level (Gonon et al., 1984). The voltammogram in Fig. 4D was obtained following this modification. This treatment appears to generate a thick oxide film on the surface as well as cracking of the surface evidenced by electron microscopy and increased residual current (Kovach et al., 1986). This was an important step, since the responses with this electrode showed that ascorbate concentrations in the extracellular fluid of the rat striatum could change in response to pharmacological agents that were purportedly dopaminergic. Thus, it became very clear that electrode selectivity was essential. A limitation of this approach for the detection of dopamine is that its voltammetric peak is very close to that of DOPAC. V.C. Electrodes coated with cation exchange membranes Another approach to voltammetric selectivity, developed in Adams's laboratory, is to coat the electrode with a perfluorinated ion exchange resin, Nation (Gerhardt et al., 1984; Kristensen et al., 1987). In this way anionic chemicals have their access to the electrode surface blocked. Shown in Fig. 9 are the cyclic voltammograms recorded at a Nation-coated electrode of the same substances shown in Fig. 6. This procedure greatly improves the selectivity for dopamine over ascorbate and DOPAC. Nation is applied to carbon-fiber electrodes by dipping the electrodes into a 2.5% solution of Nation (suspended in

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isopropanol, available from Sigma as a 5 ~ solution) for 15 min. The coated electrode is placed under a heat gun for 15 min and allowed to dry overnight at room temperature before use. A significant advantage of in vivo electrochemical methods is the ability to measure transient neurochemical processes in real time. However, electrode response rates are ultimately determined by the state of the electrode surface. While treatment of the electrode surface improves sensitivity and selectivity, virtually all increase the response time to concentration changes. Slowly responding electrodes may prevent rapid changes of neurotransmitter concentrations from being observed in vivo. This is especially important when measuring rapid kinetics of neurotransmitter release and inactivation. Electrochemical treatments produce electrodes with response times from seconds to minutes and would be unsuitable for the measurements of rapidly changing transmitter events. Electrodes used for fast measurements of neurotransmitter release have used Nation-coated electrodes, with half maximal response times of less than 0.1-1 s, or untreated electrodes (Stamford et al., 1988; Wightman et al., 1988a).

236

VI. Implantation techniques Application of the carbon microelectrode for acute in vivo measurements in the CNS is analogous to, and may be combined with, the use of other microelectrode or micropipet assemblies. The carbon microelectrode is implanted stereotaxically. The reference and optional auxiliary electrodes must also be in electrical contact with the preparation, a requirement carried out in practice as a moist contact with the meninges or an exposed neck muscle. To prevent protein adsorption, the reference contact is made indirectly by placing the electrode in a disposable pipet tip filled with saline. The pipet tip is then placed in contact with the tissue. (To prevent the loss of saline in the pipet, it is useful to place a small piece of moistened laboratory wipe in the pipet tip.) Electrodes are normally implanted on the day of the experiment. The limitation to chronic implantation is more a question of reaction of the tissue to the prolonged presence of the electrode than of sensor lifetime. Although a small but significant decay of sensitivity occurs upon implantation, the stability over a 4-8 h period is excellent. Chronic studies in conscious animals have been accomplished with electrode replacement by chronically implanted micromanipulators (Gonon et al., 1983).

VII. In vivo experiments The voltammetric techniques with high time resolution such as fast cyclic voltammetry, chronoamperometry, and differential pulse amperometry have been used to monitor rapid changes in neurotransmitter concentrations in response to electrical and chemical (K ÷) stimulations. When pulsed electrical stimulation is used, it is often desirable to synchronize the stimulus and the electrochemical potential scan or step such that the stimulus pulses occur only during the period between scans or steps. The stimulation circuit should be optically isolated from the electrochemical circuit to minimize electrical cross-talk (Neurolog NL-100, Medical Systems,

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TIME (s) Fig. I0. Effect of 2fl mg/kg benztropine on DA overflow, Temporal responses for 60 Hz, I s stimulation in a single animal are shown (points) before (A) and after (B) drug administration. Each curve is the average of three replicate stimulations at a single electrode position. Solid lines: simulations with kinetic model. Reproduced with permission from May et al, (1988).

Greenvale, NY). Local chemical stimulation is most commonly implemented by attachment of a micro-ejection pipet with adhesive to the separately fabricated carbon electrode (Gerhardt and Palmer, 1987), and compounds are typically ejected by pressure or iontophoresis. A particularly fruitful line of investigation associated with the pharmacology and kinetics of the nigrostriatal and mesolimbic dopaminergic pathways has been the use of voltammetric measurements in the striatum during electrical stimulation of the medial forebrain bundle (Miltar et al., 1985; Kuhr and Wightman, 1986; Michael et al., 1987; Stamford et al., 1988; Gratton et al., 1988; May and Wightman, 1989b). Fig. 10 shows fast-scan cyclic vottammetry data taken in the caudate nucleus of the rat with a Nation-coated electrode during a 60 Hz electrical stimulus of the medial forebrain bundle before and after inhibition of uptake with benztropine. A voltammogram was collected every 0.1 s. The dramatic effects of uptake inhibition clearly illustrate the important role that uptake normally plays in the regulation of extracellular fluid concentrations. Differential pulse amperometry has been used for investigations of dopamine dynamics in the

237

striatum and olfactory tubercle during medial forebrain bundle stimulation in pargyline-treated rats (Gonon, 1988) and more recently has been employed for measurement of norepinephrine overflow in hypothalamus paraventricular nucleus during stimulation of ventrolateral medulla (Suaud-Chagny et al., 1990). A simple extension of the potential waveform employed for fast-scan cyclic voltammetry provides the capability for measurement of dopamine and 02 during each scan (Zimmerman and Wightman, 1991). In this way a chemical index of neurotransmission and the combined effects of local blood flow and energy production are obtained simultaneously. Voltammetric data obtained in vivo are shown in Fig. 11. Although measured simultaneously at a single electrode, the 0 2 (A) and dopamine (C) responses to the electrical stimulation are clearly independent and not due to a change in background current (B). Such rapid, simultaneous changes would be very difficult to obtain with other methods.

VIII. Interpretation of stimulated release of dopamine The concentration of dopamine, as observed by in vivo voltammetry, provides valuable infor-

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Fig. 12. Mathematical modeling of stimulated dopamine release. A: schematic representation of the nerve terminal region of a neuron; SYN, synthesis of dopamine; DA, dopamine; V, synaptic vesicle; R, receptor site; M, mitochondrion; U, neuronal uptake; O overflow: [DA]r, amount of dopamine released per impulse. W h e n the neurons are stimulated with constant frequency, the model predicts that extracellular dopamine levels will increase as shown in B and C for 7 pulses at 10 Hz and 40 Hz, respectively. With each pulse is an associated increase in the concentration of dopamine by an a m o u n t designated as [DA]p. The rate of uptake of dopamine during and following the stimulation is described by the Michaelis-Menten equation. Reproduced with permission from Wightman and Z i m m e r m a n (1990).

mation concerning the effects of drugs on stimulated overflow (e.g., electrical, pharmacological, or an event such as hypoxic insult or seizure) and reflects the balance, or imbalance, between the opposing influences of exocytosis and uptake (see Fig. 10). We employ a simple kinetic model to separate the kinetics of release from those of uptake. Fig. 12A illustrates the presumed origin of overflow (O), and Fig. 12B and C indicates the net effect on overflow of release triggered by a stimulus pulse train and uptake (U). It involves no adjustable parameters and some simple, though arguable, assumptions: (1) dopamine uptake is a continual process and can be described with homogeneous Michaelis-Menten kinetics, and (2) each stimulus pulse would produce, in the

238

absence of uptake, an incremental increase in the concentration of dopamine in the extracellular fluid (designated IDA]p) that is independent of its position in the stimulus pulse train and the stimulus frequency (Wightman and Zimmerman. 1990), Consider Fig. 10 as an example. Whereas the two simulated curves shown were both obtained with the same value for [DA]p, curve B represents the effect of a 10-fold increase in the value of K m for uptake following benztropine, a competitive uptake inhibitor, with no change in VmaXWe interpret the time lag between termination of the stimulus and the maximum dopamine concentration as a manifestation of diffusional barriers between the electrode and release sites. This simple kinetic model has also predicted some remarkable observations concerning the different effects of D-2 antagonists and uptake inhibitors on dopamine overflow in different brain regions (Wightman and Zimmerman, 1990).

IX. Summary In vivo voltammetry is approaching the end of its second decade as a technique to explore extracellular concentrations in the brain. The issues of selectivity and sensitivity, which caused considerable discussion and confusion in the early 1980s, are now resolved. It is clear that in vivo voltammetry and dialysis are complimentary tools to understand neurotransmitter dynamics. The two chief advantages of voltammetry compared to dialysis, improved temporal resolution and reduced tissue damage, make this technique exceptionally well suited for providing information which is complementary to that obtained by single-unit recording and is uniquely capable of providing information on the short-term regulation of extracellular levels of biogenic amines.

Acknowledgement This research was supported by NSF (BNS) and N1H (R01NS15841).

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