High-repetition fast-scan cyclic voltammetry and chronoamperometry were used to quantify and characterize the kinetics of dopamine and dopamine-o-quinone adsorption and desorption at carbon-fiber microelectrodes. A flow injection analysis system was used for the precise introduction and removal of a bolus of electroactive substance on a sub-second time scale to the disk-shaped surface of a microelectrode that was fabricated from a single carbon fiber (Thornel type T650 or P55). Pretreatment of the electrode surfaces consisted of soaking them in purified isopropyl alcohol for a minimum of 10 min, which resulted in S/N increasing by 200-400% for dopamine above that for those that were soaked in reagent grade solvent. Because of adsorption, high scan rates (2,000 V/s) are shown to exhibit equivalent S/N ratios as compared to slower, more traditional scan rates. In addition, the steady-state response to a concentration bolus is shown to occur more rapidly when cyclic voltammetric scans are repeated at short intervals (4 ms). The new methodologies allow for more accurate determinations of the kinetics of neurotransmitter release events (10-500 ms) in biological systems. Brain slice and in vivo experiments using T650 cylinder microelectrodes show that voltammetrically measured uptake kinetics in the caudate are faster using 2,000 V/s and 240 Hz measurements, as compared to 300 V/s and 10 Hz.

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Carbon electrodes are useful for the detection of oxidizable species with cyclic voltammetry. In particular, carbon-fiber microelectrodes have been employed for the measurement of several neurotransmitters in brain tissue. However, during cyclic voltammetry with carbon-fiber electrodes the current varies with changes in concentration of some inorganic cations as a result of their interaction with surface functional groups. The electrode's response to the hydronium ion is a particular concern because its voltammetric response occurs over a broad range of potentials that overlap those of neurotransmitters of interest such as dopamine. This is especially a problem in vivo because simultaneous changes of dopamine and pH frequently occur in brain tissue. In this work, voltammetric current changes are shown to arise from pH dependent shifts in the peak potentials of background voltammetric waves that arise from species confined to the carbon-fiber electrode surface. Polishing the electrode with alumina suspended in cyclohexane in an environment containing lowered oxygen, a method previously demonstrated to remove oxides from the carbon surface, leads to a substantial reduction in the sensitivity to pH changes. However, this is accompanied by a loss in signal amplitude for dopamine. The dopamine response can be restored using the cation exchanger Nafion without significantly increasing the pH response. To investigate which oxide functional groups play a direct role in the electrode's current responses to changes in pH, surface-confined carbonyl and alcohol functionalities were chemically modified. In both cases, the modification did not affect the carbon-fiber electrode's responsiveness to changes in pH. Nonetheless, the polishing technique proved to be effective in reducing pH interferences in in vivo applications.

Abstract:The dopamine (DA) transporter (DAT) regulates DA neurotransmission by recycling DA back into neurons. Drugs that interfere with DAT function, e.g., cocaine and amphetamine, can have profound behavioral effects. The kinetics of DA transport by DAT in isolated synaptosomal or single cell preparations have been previously studied. To investigate how DA transport is regulated in intact tissue and to examine how amphetamine affects the DAT, the kinetics of DA uptake by the DAT were examined in tissue slices of the mouse caudate-putamen with fastscan cyclic voltammetry. The data demonstrate that inward DA transport is saturable and sodium-dependent. Elevated levels of cytoplasmic DA resulting from disruption of vesicular storage by incubation with 10 M Ro 4-1284 did not generate DA efflux or decrease its uptake rate. However, incubation with 10 M amphetamine reduced the net DA uptake rate and increased extracellular DA levels due to DA efflux through the DAT. In addition, a new, elevated steady-state level of extracellular DA was established after electrically stimulated DA release in the presence of amphetamine, norepinephrine, and exogenous DA. These results from intact tissue are consistent with a kinetic model of the DAT established in more purified preparations in which amphetamine and other transported substances make the inwardly facing DAT available for outward transport of intracellular DA.

Described is an improved data acquisition system for fast-scan cyclic voltammetry (FSCV). The system was designed to significantly diminish noise sources that were identified in previously recorded FSCV measurements for the detection of neurotransmitters. Minimized noise is necessary to observe the low concentrations of neurotransmitters that are physiologically important. The system was based on a high-speed, 16-bit AD/DA acquisition board that allowed high scan rates and better resolved the small faradaic currents which remained after background subtraction. Irregularities that occur when independent timing sources are used for generation of the voltage waveform and collection of the current can create large noise artifacts near the voltage limits during FSCV. These were eliminated by the use of a single acquisition board that generated the voltage waveform and collected the current. Noise from frequency drift of the power line was eliminated through the use of a phase-locked loop. To demonstrate the improved performance of the system, data were collected using carbon-fiber microelectrodes in a flow injection analysis system and in brain slices. This new data acquisition system performed significantly better than another system previously used in our laboratory without these features. The improved detection limits of the new system allowed clearly resolved current spikes featuring pre-release "feet" to be recorded adjacent to individual mast cells following chemical stimulation. When combined with false-color plots, the low-noise system facilitated identification of dopamine release in a freely moving animal.