Kirk T. Kawagoe

Principles of voltammetry and microelectrode surface states

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.

Regulation of transient dopamine concentration gradients in the microenvironment surrounding nerve terminals in the rat striatum.

Synaptic overflow of dopamine in the striatum has been investigated during electrical stimulation of the medial forebrain bundle in anesthetized rats. Dopamine has been detected with Nafion-coated, carbon-fiber electrodes used with fast-scan voltammetry. In accordance with previous results, dopamine synaptic overflow is a function of the stimulation frequency and the anatomical position of the carbon-fiber electrode. In some positions the concentration of dopamine is found to respond instantaneously to the stimulus when the time-delay for diffusion through the Nafion film is accounted for. In these locations the measured rates of change of dopamine are sufficiently rapid such that extracellular diffusion is not apparent. The rate of dopamine overflow can be described by a model in which each stimulus pulse causes instantaneous release, and cellular uptake decreases the concentration between stimulus pulses. Uptake is found to be described by a constant set of Michaelis-Menten kinetics at each location for concentrations of dopamine from 100 nM to 15 microM. The concentration of dopamine released per stimulus pulse is found to be greatest at low frequency (< or = 10 Hz) with stimulus trains, and with single-pulse stimulations in nomifensine-treated animals. The frequency dependence of release is not an effect of dopamine receptor activation; haloperidol (2.5 mg/kg) causes a uniform increase in release at all frequencies. The absence of diffusional effects in the measurement locations means that the constants determined with the electrode are those operant inside intact striatal tissue during stimulated overflow. These values are then extrapolated to the case where a single neuron fires alone. The extrapolation shows that while the transient concentration of dopamine may be high (200 nM) at the interface of the synapse and the extrasynaptic region, it is normally very low (< 6 nM) in the bulk of extracellular fluid.

Characterization of amperometry for in vivo measurement of dopamine dynamics in the rat brain

Constant potential amperometry with Nafion-coated carbon-fiber electrodes has been evaluated as a technique for in vivo detection of the neurotransmitter dopamine. The results of this technique have been compared to results obtained with fast-scan cyclic voltammetry at the same electrode during release of dopamine into the extracellular space of the brain during electrical stimulation of neurons. The data indicate that constant potential amperometry is a viable technique for detecting low concentrations of dopamine. Dopamine permeates the film more quickly with constant-potential amperometry than with repeated fast-scan cyclic voltammetry as predicted by diffusion equations. For the case of cyclic voltammetry, it is demonstrated that the temporal delay caused by diffusion through Nafion film can be removed by deconvolution procedures. Despite the suitability of constant potential amperometry as an in vivo monitoring technique, it does have several disadvantages when compared to fast-scan cyclic voltammetry. The diffusion layer extends outside of the Nafion film making determination of concentration based on in vitro calibrations more difficult to interpret. The reported concentrations are larger than obtained by cyclic voltammetry, a technique with the diffusion layer restricted to the Nafion film, and this result is likely an underestimation of the effect of the catalytic reaction between the o-quinone of dopamine and ascorbate. Amperometry was found to provide only slightly improved signal-to-noise ratios than cyclic voltammetry despite the use of greater filtering. This was because the advantage of dopamine accumulation in the film was lost. In addition, the small magnitude of the amperometric signal makes it more susceptible to electrical interference.

Interference by pH and Ca2+ ions during measurements of catecholamine release in slices of rat amygdala with fast-scan cyclic voltammetry

Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes was used to investigate catecholamine release and uptake induced by local electrical stimulation of rat brain slices containing the basolateral amygdaloid nucleus. The amygdala contains less catecholamine than the striatum, and the observed release is proportionately smaller. Stimulus trains of long duration were required to obtain a well-resolved concentration change in the basolateral amygdala. Voltammetric detection of catecholamines under these conditions was complicated by interference from two extracellular ions, H+ and Ca2+. Ion-selective microelectrodes were used in conjunction with carbon-fiber microelectrodes to monitor pH and Ca2+. The magnitude of the pH changes was correlated with stimulation length and followed the pattern of a brief alkaline shift followed by a longer acidic shift. Extracellular Ca2+ concentration decreased during stimulation and returned fairly rapidly to baseline after the stimulation was over. Because it was not possible to account for all of the ionic interferences using information in the voltammograms, other strategies were employed. Exposure of amygdala slices to L-DOPA or DA increased electrically evoked release of catecholamine, but the effect was transient, and uptake rates decreased during continued exposure to these agents. The most successful approach to remove the interferences was to subtract the response obtained after exposure of the slice to the catecholamine depleter, Ro 4-1284. This agent eliminates the catecholamine response but does not appear to alter the ionic changes.

pH-dependent processes at Nafion®-coated carbon-fiber microelectrodes

Cyclic voltammetry has been used to examine pH-dependent processes in aqueous solution at carbon-fiber electrodes with scan rates above 100 V s−1. Specifically, the reduction of p-benzoquinone and the oxidation of dopamine have been investigated. The mechanism of electrolysis of quinoidal compounds has been extensively characterized at slow scan rates with conventional electrodes in prior work. Digital simulations based on the reported rates and E° values show that the electrode reaction mechanisms can be diagnosed at high rates of scan by examination of the pH dependence of the peak potentials of cyclic voltammograms. However, the experimentally determined pH dependences of the voltammetric peak potentials for these compounds differ from those obtained by simulation suggesting the electrode mechanism differs at carbon-fiber electrodes. The altered mechanism may reflect surface catalysis since the data show a correlation between the position of the surface voltammetric waves found at carbon-fiber electrodes and the voltammetric peak positions of the compounds. When the electrodes are coated with Nafion®, the pH dependence of the voltammetric waves is further altered. The surface waves are found to shift linearly with pH, and can be used to diagnose alterations in the pH of the solution.

Microelectrodes in Biological Systems

The small size of microelectrodes enables their use as sensors in biological systems of easily oxidized chemical substances. Carbon-fiber electrodes implanted in the brain can be used to examine the dynamic concentration changes of the neurotransmitter dopamine. In thin slices of brain tissue, the electrodes can be used to simultaneously detect neurotransmitter secretion and oxygen use. At single cells secretion processes can be resolve in real time.