The dopamine-containing projection from the ventral tegmental area of the midbrain to the nucleus accumbens is critically involved in mediating the reinforcing properties of cocaine. Although neurons in this area respond to rewards on a subsecond timescale, neurochemical studies have only addressed the role of dopamine in drug addiction by examining changes in the tonic (minute-to-minute) levels of extracellular dopamine. To investigate the role of phasic (subsecond) dopamine signalling, we measured dopamine every 100 ms in the nucleus accumbens using electrochemical technology. Rapid changes in extracellular dopamine concentration were observed at key aspects of drug-taking behaviour in rats. Before lever presses for cocaine, there was an increase in dopamine that coincided with the initiation of drug-seeking behaviours. Notably, these behaviours could be reproduced by electrically evoking dopamine release on this timescale. After lever presses, there were further increases in dopamine concentration at the concurrent presentation of cocaine-related cues. These cues alone also elicited similar, rapid dopamine signalling, but only in animals where they had previously been paired to cocaine delivery. These findings reveal an unprecedented role for dopamine in the regulation of drug taking in real time.

Secretion of catecholamines from single bovine chromaffin cells in culture was elicited by brief pressure ejections from a micropipette containing nicotine, carbamoylcholine, or potassium ions or by mechanical stimulation. Release was monitored electrochemically with a carbon-fiber microelectrode placed adjacent to the cell. Cyclic voltammetry was used to identify secreted species, whereas constant potential amperometry was used for improved temporal resolution (millisecond range) of catecholamine detection. During secretion, brief current spikes were observed, which were shown to be due to detection of catecholamines by electrooxidation. The spikes have the physical characteristics of multimolecular packets of catecholamines released at random times and locations from the surface of the single cell. The half-width of the spikes was found to increase with an increase in cell-electrode spacing. The properties of the catecholamine spikes correlate well with expectations based on secretion from individual storage vesicles. Spikes do not occur in the absence of Ca2+ in the buffer, and the majority of spikes are found to be distributed between 0.2 and 2 picocoulombs, corresponding to 1-10 attomoles of catecholamine detected. The frequency of the spikes increases with the intensity of the stimulus, but the average quantity of catecholamine in each spike is independent of the stimulus. Thus, these measurements represent timeresolved observation of quantal secretion of catecholamines and provide direct evidence for the exocytotic hypothesis.

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.

Abstract5-HT is an important molecule in the brain that is implicated in mood and emotional processes. In vivo, its dynamic release and uptake kinetics are poorly understood due to a lack of analytical techniques for its rapid measurement. Whereas fast-scan cyclic voltammetry with carbon fiber microelectrodes is used frequently to monitor sub-second dopamine release in freely-moving and anesthetized rats, the electrooxidation of 5-HT forms products that quickly polymerize and irreversibly coat the carbon electrode surface. Previously described modifications of the electrochemical waveform allow stable and sensitive 5-HT measurements in mammalian tissue slice preparations and in the brain of fruit fly larvae. For in vivo applications in mammals, however, the problem of electrode deterioration persists. We identify the root of this problem to be fouling by extracellular metabolites such as 5-HIAA, which is present in 200-1000 times the concentration of 5-HT and displays similar electrochemical properties, including filming of the electrode surface. To impede access of the 5-HIAA to the electrode surface, a thin layer of Nafion®, a cation exchange polymer, has been electrodeposited onto cylindrical carbon-fiber microelectrodes. The presence of the Nafion® film was confirmed with environmental scanning electron microscopy and was demonstrated by the diminution of the voltammetric signals for 5-HIAA as well as other common anionic species. The modified microelectrodes also display increased sensitivity to 5-HT, yielding a characteristic cyclic voltammogram that is easily distinguishable from other common electroactive brain species. The thickness of the Nafion® coating and a diffusion coefficient (D) in the film for 5-HT were evaluated by measuring permeation through Nafion®. In vivo, we used physiological, anatomical and pharmacological evidence to validate the signal as 5-HT. Using Nafion®-modified microelectrodes, we present the first endogenous recording of 5-HT in the mammalian brain.

Fast-scan cyclic voltammetry, a demonstrated analytical method for the in vivo detection of dopamine, is extended to the detection of in vitro and in vivo 5-hydroxytryptamine (5-HT) with the use of a specific potential wave form applied at 1000 V/s. The wave form, 0.2 to 1.0 to -0.1 to 0.2 V, is employed to accelerate electrode response times which are significantly slower with other wave forms due to the adsorption of 5-HT. The scan rate of 1000 V/s enables follow-up reactions which lead to the formation of strongly adsorptive products to be outrun. The peak current at a carbon fiber disk microelectrode exposed to 1 microM 5-HT in flow injection experiments is 1 nA, with a half-rise time of less than 200 ms. The peak current of Nafion-coated electrodes exposed to the same concentration of 5-HT is 5 nA, with a half-rise time on the order of 400 ms. The rate of adsorption of 5-HT was determined to be 4.22 +/- 0.33 s-1. Several compounds present in brain tissue as well as the pharmacological agents used to elicit 5-HT release in the caudate of the rat were evaluated. Those which gave a response could be differentiated from 5-HT on the basis of respective oxidative and reductive peak potentials. Nafion-coated electrodes were used to monitor transient increases in both dopamine and exogenous 5-HT in the caudate of the anesthetized rat in response to electrical stimulation. The rate of cellular uptake of 5-HT was shown to be 3-fold slower than dopamine uptake. NS-15841

The voltammetric responses of carbon-fiber microelectrodes with a 1.0 V and a 1.4 V anodic limit were compared in this study. Fast-scan cyclic voltammetry was used to characterize the response to dopamine and several other neurochemicals. An increase in the adsorption properties of the carbon fiber leads to an increase in sensitivity of 9 fold in vivo. However the temporal response of the sensor is slower with the more positive anodic limit. Increased electron transfer kinetics also causes a decrease in the relative sensitivity for dopamine vs. other neurochemicals, and a change in their cyclic voltammograms. Stimulated release in the caudate-putamen was pharmacologically characterized in vivo using Ro-04-1284 and pargyline, and was consistent with that expected for dopamine.

Rewarding and aversive stimuli evoke very different patterns of behavior and are rapidly discriminated. Here taste stimuli of opposite hedonic valence evoked opposite patterns of dopamine and metabolic activity within milliseconds in the nucleus accumbens. This rapid encoding may serve to guide ongoing behavioral responses and promote plastic changes in underlying circuitry.

The regulation of extracellular dopamine (DA) concentrations was examined and compared in vivo in four projection fields of mesotelencephalic dopaminergic neurons with fast-scan cyclic voltammetry at carbon-fiber microelectrodes. Transient electrical stimulation of ascending DA fibers in a near physiological range of frequencies (10-20 Hz) elicited similar levels of extracellular DA in the medial prefrontal cortex (MPFC), basal lateral amygdaloid nucleus (BAN), caudate-putamen (CP), and nucleus accumbens (NAc) despite the documented 90-fold disparity in DA tissue levels and terminal density. However, marked differences were observed in the dynamics and overall frequency dependence of the evoked synaptic overflow of DA. These differences are due to the significantly different rates of release and uptake found in each of the four regions. For example, rate constants for the release of the four regions. For example, rate constants for the release and uptake of DA were similar in the MPFC and BAN but approximately 8 and 50 times less, respectively, than that in the CP and NAc. When the parameters were normalized to endogenous DA tissue content, a unique picture emerged: compared to all other regions, relative release was 10-fold greater in the MPFC while relative uptake was at least 10 times less in the BAN. The results further differentiate the functional characteristics of mesotelencephalic dopaminergic systems and demonstrate the regiospecific nature of DA neural transmission in the brain. In addition, the regulation of extracellular DA levels in the MPFC and BAN is suitable for the "long-range" transfer of chemical information in the brain and is consistent with a hypothesis of extrasynaptic neurotransmission.