Whether amphetamine acts principally at the plasma membrane or at synaptic vesicles is controversial. We find that d-amphetamine injection into the Planorbis giant dopamine neuron causes robust dopamine release, demonstrating that specific amphetamine uptake is not required. Arguing for action at vesicles, whole-cell capillary electrophoresis of single Planorbis dopamine neurons shows that amphetamine reduces vesicular dopamine, while amphetamine reduces quantal dopamine release from PC12 cells by > 50% per vesicle. Intracellular injection of dopamine into the Planorbis dopamine neuron produces rapid nomifensine-sensitive release, showing that an increased substrate concentration gradient is sufficient to induce release. These experiments indicate that amphetamine acts at the vesicular level where it redistributes dopamine to the cytosol, promoting reverse transport, and dopamine release.

Catecholamine release from rat pheochromocytoma (PC12) cells has been observed at zeptomole levels using dc-amperometric detection with carbon fiber microelectrodes. Time-resolved individual exocytic events from PC12 cells have been recorded and analyzed with 1.2 ms time resolution. The average area under 1912 current transients from 13 PC12 cells corresponds to 190 zmol (114,300 molecules per release event). The average width at half-height of these current transients is 9.3 ms, in agreement with the time frame of exocytosis. The detection limit of the method reported here is as low as 31 zmol. This is the first report of direct electrochemical observation of quantal release from PC12 cells. The successful application of this electrochemical scheme to monitor catecholamines released from small vesicles also suggests that it may be possible to apply this technique to monitor quantal release from synaptic vesicles.

Multiple pulse voltammetry at platinum microelectrodes Is described for Intracellular measurements. In this technique, a sequence of three potential pulses Is used for each current point measured. This pulee sequence provides a fixed cathodic activation potential and a fixed anodic cleaning potential before a varying detection potential, at which the current Is measured. Voltammetric Information Is obtained by ramping the detection potential stepwise through the potential range of Interest. The multiple pulse voltammetry technique has been applied to the study of the oxidation of potassium ferrocyanide, glucose, and several catechols at platinum microdisk electrodes. In addition, this technique has been applied at ultrasmall platinum ring electrodes to reduce electrode fouling during Intracellular voltammetry. In these In vivo experiments, the electrode response Is degraded by only 30% after 40 min of Intracellular voltammetry.