Real-time monitoring of electrically stimulated norepinephrine release in rat thalamus: II. Modeling of release and reuptake characteristics of stimulated norepinephrine overflow.

As in the preceding study, electrical stimulation was used to effect release overflow of norepinephrine in the rat thalamus. Using a weak electrochemical pretreatment of a carbon fiber electrode, it was possible to "tune in" the electrochemical response signal for norepinephrine without metabolite interference. This reasonably selective signal was then used to study the degradation of norepinephrine release ability caused by prolonged stimulation. Further, the signals were modeled by the method used successfully for stimulated dopamine overflow, providing hitherto unavailable information on the temporal and spatial characteristics of norepinephrine release overflow. Pertinent comparisons between the release characteristics of the dopamine and norepinephrine systems show that the half-life for norepinephrine in the extracellular fluid space is approximately 1 s in thalamus compared with 33 ms for dopamine in caudate.

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.

Effect of chronic haloperidol treatment on stimulated synaptic overflow of dopamine in the rat striatum.

In vivo voltammetry was used to assess the change in stimulated striatal dopamine overflow in response to various treatments with the dopamine receptor antagonist haloperidol. Dopamine overflow was induced with stimulating electrodes implanted in the medial forebrain bundle of anesthetized rats while dopamine concentrations were monitored with Nafion-coated, carbon-fiber microelectrodes implanted in the striatum. An acute challenge of haloperidol (0.5 mg kg-1, i.p.) given to naive animals caused stimulated overflow to increase at all stimulation frequencies (10-60 Hz), with the greatest change, 5-fold, occurring at 30 Hz. These results have been compared to those obtained in a different group of rats given daily injections of haloperidol (0.5 mg kg-1, s.c.) for 30 consecutive days. On the 30th day, dopamine striatal tissue levels and uptake kinetics were not altered by this treatment, but 3,4-dihydroxyphenylacetic acid tissue levels were elevated almost 2-fold. A challenge dose of haloperidol (0.5 mg kg-1, i.p.) administered to the animals treated with chronic haloperidol did not elicit a change in stimulated dopamine overflow. In two other groups, rats were withdrawn from 30-day haloperidol treatment for 3 days or 14 days before experimentation. Stimulated dopamine overflow concentrations in both groups were not significantly different from naive animals. When the withdrawn animals were given a haloperidol challenge (0.5 mg kg-1, i.p.), 15- and 12-fold increases in overflow for 3-day and 14-day withdrawal groups, respectively, were observed at a stimulation frequency of 30 Hz. Thus, chronic treatment with haloperidol induces long-lasting effects on the capacity of dopamine receptors to modulate dopamine release.

Strategies for low detection limit measurements with cyclic voltammetry.

Cyclic voltammetry of Nafion-coated, carbon-fiber electrodes is used to detect trace concentrations of dopamine, both in a flow injection apparatus and in the brain of an anaesthetized rat. To improve signal-to-noise ratios, the sources of noise during cyclic voltammetry have been determined and strategies have been developed to decrease the noise. With the potentiostat employed, the measured noise is comparable to that expected for Johnson noise from the feedback resistor of the current transducer. Additional noise arises from the waveform generator employed and, in some cases, line noise. Line noise is discriminated against by starting each cyclic voltammogram either in phase or 180 degrees out of phase with the line frequency. When used in vivo, additional noise also arises from the physiological activity of the animal. Detection limits are found to closely correspond to those predicted on the basis of simulation of the voltammetric shape and the measured noise. Detection limits are improved by the use of appropriate analog and digital filtering, ensemble averaging, and appropriate timing of repetitive cyclic voltammograms. The combined use of these techniques enables the in vivo detection of approximately 100 nM of dopamine with a signal-to-noise ratio of 25.

Interference by DOPAC and ascorbate during attempts to measure drug-induced changes in neostriatal dopamine with Nafion-coated, carbon-fiber electrodes.

The selectivity of Nafion-coated, carbon-fiber electrodes was evaluated for the voltammetric detection of dopamine in the presence of 3,4-dihydroxyphenylacetic acid (DOPAC) and ascorbate, which are interferant anions in the brain. Nafion coating was applied to both polished carbon-disk electrodes and electrochemically modified electrodes. During in vitro testing, polished disk electrodes showed the greatest selectivity for dopamine at the fastest scan rate tested (300 V s-1), but the drift in signal made slow changes in dopamine difficult to determine. Electrochemically modified electrodes already provide an ascorbate wave distinct from that for dopamine, but Nafion coating actually decreased dopamine selectivity with respect to DOPAC. In vivo testing was carried out in the neostriatum of urethane-anesthetized rats in response to drug- or stimulation-induced increases in dopamine transmission. Administration of haloperidol (0.5 mg kg-1) followed by GBR 12909 (20 mg kg-1), which is known to cause a 10-fold increase in extracellular dopamine, failed to produce a selective signal for dopamine when measured voltammetrically. Comparable results were obtained following administration of amphetamine (2.5 mg kg-1), which also increases dopamine overflow. Voltammetric detection of dopamine was possible, however, during electrical stimulation of dopaminergic afferents in the medial forebrain bundle. Thus, voltammetry with Nafion-coated electrodes is best suited to the measurement of transient changes in extracellular dopamine rather than the relatively prolonged changes in dopamine overflow produced by various drugs.