Anal. Chem. 2002, 74, 539-546
Response Times of Carbon Fiber Microelectrodes to Dynamic Changes in Catecholamine Concentration B. Jill Venton, Kevin P. Troyer, and R. Mark Wightman*
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290
The electrode response time and the measured concentrations during dynamic catecholamine changes were compared using constant potential amperometry and fastscan cyclic voltammetry. The amperometric response to a rectangular pulse of catecholamine is more rectangular than the cyclic voltammetric response; however, the response times are very similar when, during cyclic voltammetry, the temporal lag due to adsorption and desorption of catecholamine to the electrode is removed by deconvolution. Deconvolution of cyclic voltammetry data was applied to stimulated dopamine release in vivo, allowing for modeling of release and uptake kinetics and to measure catecholamine release from single cells, resulting in better resolution of peaks from single vesicles. In vitro postcalibrations were performed to calculate concentrations of catecholamine measured with cyclic voltammetry and amperometry. The addition of 600 µM ascorbic acid to the postcalibration buffer, allowing a catalytic reaction to regenerate dopamine, resulted in similar calculated concentrations for stimulated release of dopamine using amperometry and cyclic voltammetry. Using deconvoluted cyclic voltammetry to remove the response time lag and adding ascorbic acid to the calibration buffer, the shape and concentration of dynamic catecholamine changes are very similar when measured with constant potential amperometry and cyclic voltammetry. Catecholamines act as chemical messengers that transport information between cells. The release and uptake of catecholamines at biological cells occurs on a subsecond time scale, so fast sensors must be used to accurately monitor these changes. For example, peaks resulting from the release of single vesicles from cells are 10-50 ms wide.1 In addition, dopamine transients as short as 200 ms have been measured in reaction to strong stimuli in freely moving rats.2 The sensors used to monitor these changes, therefore, must have a rapid time response to a change in catecholamine concentration. * Corresponding author. Phone: 919-962-1470. Fax: 919-962-2388. E-mail:
[email protected]. (1) Wightman, R. M.; Schroeder, T. J.; Finnegan, J. M.; Ciolkowski, E. L.; Pihel, K. Biophys. J. 1995, 68, 383-90. (2) Robinson, D. L.; Phillips, P. E. M.; Budygin, E. A.; Trafton, B. J.; Garris, P. A.; Wightman, R. M. NeuroReports 2001, 12, 2549-52. 10.1021/ac010819a CCC: $22.00 Published on Web 12/22/2001
© 2002 American Chemical Society
The carbon fiber microelectrode has been used as an electrochemical sensor for changes in catecholamine concentration. Carbon fiber microelectrodes are advantageous because of their small size and high sensitivity to catecholamines. They can be used with a variety of electrochemical techniques, including constant potential amperometry and cyclic voltammetry (CV). With amperometry, a constant potential sufficient to oxidize a catecholamine is applied, and electroactive species are immediately consumed. It provides a true measure of the local flux of electroactive species, but there is no chemical information about the species being detected. Amperometry has been used to determine the catecholamine content of single vesicles and to probe the effects of pharmacological agents in vivo;1,3,4 however, conversion of amperometric currents to concentrations is complicated, because the diffusion layer in solution extends quite far into solution during a simple electrolysis.5 It is anticipated that the diffusion layer could be quite different in the brain, and for this reason, calibrations often are not attempted;3,5,6 however, concentrations are required to interpret catecholamine changes with Michaelis-Menten-based models. Concentrations can be sampled with cyclic voltammetry by repeating scans at regular intervals. Typically, scan rates of 300 V/s are used with 100 ms between scans. Each scan provides a chemical fingerprint of the species detected. The amplitude of the peak current for the detected species provides an index of its instantaneous concentration. This technique, termed fast-scan cyclic voltammetry, has been shown to be very useful for the detection of catecholamine release from cells, in brain slices, and in vivo;7 however, we recently showed that the response time of the peak current from successive cyclic voltammograms at carbon fiber microelectrodes is not instantaneous for catecholamines.8 The slow time response is due to the kinetics of adsorption and desorption of the catecholamine. This problem does not occur (3) Dugast, C.; Suaud-Chagny, M. F.; Gonon, F. Neuroscience 1994, 62, 64754. (4) Suaud-Chagny, M. F.; Dugast, C.; Chergui, K.; Msghina, M.; Gonon, F. J. Neurochem. 1995, 65, 2603-11. (5) Wightman, R. M.; Wipf, D. O. Electroanalytical Chemistry, 15th ed.; Bard, A. J., Ed.; Marcel Dekker: New York, 1989. (6) Nicholson, C.; Rice, M. E. Neuromethods: The Neuronal Microenvironment, 9th ed.; Boulton, A. A., Baker, G. B.Walz, W., Eds..; Humana Press: Clifton, NJ, 1988. (7) Wightman, R. M.; Hochstetler, S. E.; Michael, D. J.; Travis, E. R. Interface Sci. 1996, 5, 22-26. (8) Bath, B. D.; Michael, D. J.; Trafton, B. J.; Joseph, J. D.; Runnels, P. L.; Wightman, R. M. Anal. Chem. 2000, 72, 5994-6002.
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with constant potential amperometry, because the catecholamine is oxidized immediately. Adsorption during cyclic voltammetry was evidenced by a linear increase in peak currents with scan rate at low micromolar dopamine concentrations, the range of concentrations normally measured in the brain.8 Symmetrical voltammogram peaks were also evidence for catecholamine adsorption. Previously, disk-shaped, carbon fiber microelectrodes coated with a thin film of the polymer Nafion were used to monitor in vivo changes.9 Although the Nafion film offered advantages, such as preconcentration of, and increased selectivity for, positively charged catecholamines, the slow diffusion of catecholamines through the coating slowed the time response of the electrode. To account for this, a deconvolution approach was used to remove the effect of the Nafion.10 More recently, it was shown that uncoated, cylindrical electrodes are better for use in vivo, because they provide superior sensitivity.11 In this paper, we show that deconvolution also can be used to correct for the temporal distortion at uncoated electrodes during cyclic voltammetry caused by catecholamine adsorption. In addition, we show that when physiological amounts of ascorbic acid are added to the in vitro calibration buffer, the diffusion layer is reduced for amperometry. The result is that deconvoluted cyclic voltammetry and amperometry have similar responses to changes in catecholamine concentration, with respect to both concentration amplitude and time course. EXPERIMENTAL SECTION Chemicals. Dopamine, epinephrine, norepinephrine, NaCl, HEPES, ascorbic acid, urethane, and CaCl2 were used as received from Sigma-Aldrich. A 20 mM HEPES, 150 mM NaCl, 2.4 mM CaCl2, 600 µM ascorbic acid buffer adjusted to pH 7.4 was used in flow cell experiments unless otherwise indicated. All aqueous solutions were made using doubly distilled deionized water (Megapure System, Corning model D2). Flow Injection Analysis. The experimental setup has been described previously.12 Briefly, an electrode was positioned at the output of a flow injection apparatus consisting of a six-port HPLC loop injector mounted on a two-position actuator. The apparatus allowed for the introduction of a rectangular pulse of analyte to the electrode surface. The flow rate used was 3 mL/min (1.6 cm/ sec) when not specified. Electrochemistry. Carbon fiber microelectrodes were constructed as previously described.11,13 For in vivo experiments, cylinder microelectrodes were fabricated by sealing a 2.5-µmradius T-650 carbon fiber (Thornel, Amoco Co.) in a pulled glass capillary. The fiber was cut so that 40-50 µm was protruding from the end. Electrodes were epoxied (Miller-Stevenson) to ensure a good seal and dipped immediately in acetone for a few seconds to remove any epoxy from the carbon fiber. For cultured cell experiments, disk microelectrodes were fabricated by sealing a (9) Engstrom, R. C.; Wightman, R. M.; Kristensen, E. W. Anal. Chem. 1988, 60, 652-56. (10) Garris, P. A.; Wightman, R. M. Voltammetric Methods in Brain Systems; Boulton, A. A., Baker, G. B., Adams, R. N., Eds.; Humana Press: Tottowa, NJ, 1995. (11) Cahill, P. S.; Walker, Q. D.; Finnegan, J. M.; Mickelson, G. E.; Travis, E. R.; Wightman, R. M. Anal. Chem. 1996, 68, 3180-86. (12) Kristensen, E. W.; Wilson, R. W.; Wightman, R. M. Anal.Chem. 1986, 58, 986-88. (13) Kawagoe, K. T.; Zimmerman, J. B.; Wightman, R. M. J. Neurosci. Methods 1993, 48, 225-40.
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T-650 carbon fiber in glass and polishing at a 45° angle on a diamond-dust-embedded micropipet-beveling wheel (Sutter Instrument Co.). Prior to use, all electrodes were soaked overnight in 2-propanol purified with activated carbon (Norit A, ICN).8 For dopamine detection using cyclic voltammetry, the electrode was held at -0.4 V and scanned from -0.4 to 1.0 V at 300 V/s every 100 ms. To discriminate between norepinephrine and epinephrine, the electrode was scanned from -0.2 to 1.6 V at 2000 V/s, and cyclic voltammograms were repeated at 60 Hz. Currentvs-time traces were obtained from cyclic voltammetry by integrating the current in a 100 mV window centered around the oxidation peak for each cyclic voltammogram. Currents were converted to concentrations using postcalibration values from flow injection experiments. For amperometry, the electrode was held at +0.3 V vs Ag/AgCl, and the collection frequency was 60 Hz. Data Acquisition. The data acquisition software and hardware have been described elsewhere.14 A computer-generated waveform was input into a patch clamp amplifier (Axopatch 200B, Axon instruments) modified for used in electrochemical measurements. Data were collected through an acquisition board (National Instruments) interfaced through a PC. A timing board was used to synchronize electrical stimulations with data acquisition. Deconvolution was performed using locally written software in LabVIEW (National Instruments). Animals. Male Sprague-Dawley rats (275-350 g) were anesthetized with urethane (1.5 g/kg rat weight). Holes were drilled in the skull for the placement of the carbon fiber microelectrode in the caudate-putamen (stereotaxic coordinates AP, +1.2; ML, +2.0; DV, -4.5) and the bipolar stimulating electrode in the ventral tegmental area (AP -5.6; ML, +1.0; DV, -7.5).15 The dorsoventral placement of the stimulating electrode was adjusted to maximize dopamine efflux. A Ag/AgCl wire was also implanted into the brain as a reference electrode. Biphasic stimulating pulses, 4 ms long, 120 µA each phase, were applied at 60 Hz for one second to evoke dopamine release. Cultured Chromaffin Cells. Primary cultures of bovine adrenal chromaffin cells were prepared as described previously.16 Briefly, chromaffin cells were isolated from several bovine adrenal glands by digestion with collagenase and Renografin density gradient centrifugation to obtain the epinephrine- and norepinephrine-enriched fractions. Cells were plated on 35 mm tissue culture plates at a density of 3 × 105 cells per plate and maintained in a controlled atmosphere of air with 5% CO2 at 37 °C. Experiments were performed during days 3 through 7 of culture. Release was induced by applying a 5-s pulse of 5 mM Ba2+ via a pulled glass capillary. THEORY Kinetic Model of Adsorption. Recently, we showed that the peak current during detection of dynamic changes of dopamine with repeated cyclic voltammograms arises primarily from dopamine adsorbed to the electrode surface.8 The data for surface coverage of dopamine (ΓDA) as a function of bulk dopamine (14) Michael, D. J.; Joseph, J. D.; Kilpatrick, M. R.; Travis, E. R.; Wightman, R. M. Anal. Chem. 1999, 71, 3941-47. (15) Paxinos, G.; Watson, C. The Rat Brain in Stereotaxic Coordinates; Academic: New York, 1986. (16) Leszczyszyn, D. J.; Jankowski, J. A.; Viveros, O. H.; Diliberto, E. J., Jr.; Near, J. A.; Wightman, R. M. J. Neurochem. 1991, 56, 1855-63.
concentration were fit with a Langmuir isotherm. Typical dopamine concentrations measured in an in vivo experiment are
