The electrode response time and the measured concentrations during dynamic catecholamine changes were compared using constant potential amperometry and fast-scan cyclic voltammetry. The amperometric response to a rectangular pulse of catecholamine is more rectangular than the cyclic voltammetric response; however, the response times are very similar when, during cyclic voltammetry, the temporal lag due to adsorption and desorption of catecholamine to the electrode is removed by deconvolution. Deconvolution of cyclic voltammetry data was applied to stimulated dopamine release in vivo, allowing for modeling of release and uptake kinetics and to measure catecholamine release from single cells, resulting in better resolution of peaks from single vesicles. In vitro postcalibrations were performed to calculate concentrations of catecholamine measured with cyclic voltammetry and amperometry. The addition of 600 microM ascorbic acid to the postcalibration buffer, allowing a catalytic reaction to regenerate dopamine, resulted in similar calculated concentrations for stimulated release of dopamine using amperometry and cyclic voltammetry. Using deconvoluted cyclic voltammetry to remove the response time lag and adding ascorbic acid to the calibration buffer, the shape and concentration of dynamic catecholamine changes are very similar when measured with constant potential amperometry and cyclic voltammetry.

During exocytosis, vesicles in secretory cells fuse with the cellular membrane and release their contents in a Ca 2؉ -dependent process. Release occurs initially through a fusion pore, and its rate is limited by the dissociation of the matrix-associated contents. To determine whether this dissociation is promoted by osmotic forces, we have examined the effects of elevated osmotic pressure on release and extrusion from vesicles at mast and chromaffin cells. The identity of the molecules released and the time course of extrusion were measured with fast scan cyclic voltammetry at carbon fiber microelectrodes. In external solutions of high osmolarity, release events following entry of divalent ions (Ba 2؉ or Ca 2؉ ) were less frequent. However, the vesicles appeared to be fused to the membrane without extruding their contents, since the maximal observed concentrations of events were less than 7% of those evoked in isotonic media. Such an isolated, intermediate fusion state, which we term "kiss-and-hold," was confirmed by immunohistochemistry at chromaffin cells. Transient exposure of cells in the kiss and hold state to isotonic solutions evoked massive release. These results demonstrate that an osmotic gradient across the fusion pore is an important driving force for exocytotic extrusion of granule contents from secretory cells following fusion pore formation.

Effects of vesicular monoamine transporter inhibitors on catecholamine release from bovine chromaffin cells have been examined at the level of individual exocytotic events. As expected for a depletion of vesicular stores, release evoked by depolarizing agents was decreased following 15-min incubations with reserpine and tetrabenazine, as evidenced by a decrease in exocytotic frequency and amount released per event. In contrast, two reserpine derivatives, methyl reserpate and reserpic acid, were much less effective. Surprisingly, the incubations also decreased the accompanying rise in intracel-

No abstract

The analysis of chemical events in small volumes requires careful manipulation of samples and sensitive detection methods. Here, we describe the measurement of the neurotransmitter dopamine in a picoliter vial with electrochemical techniques. The vials were fabricated from fused-silica capillaries that provided a transparent container suitable for the observation and manipulation of a biological cell, sample solutions, and electrodes. Evaporation of the sample was prevented with a mineral oil layer, allowing for experiments lasting for several minutes. The small volume of these vials (100-200 pL) allows rapid mixing of all of the solution reagents. Similarly, the small volume allows exhaustive electrolysis of the vial contents with a 3-microm radius, disk-shaped carbon fiber microelectrode within 60 s. Fast-scan cyclic voltammetry at carbon fiber microelectrodes was used to monitor the concentration of analyte in the vial without depleting its contents. The concentration of dopamine introduced by pneumatic injection remained stable when sampled by cyclic voltammetry, and no evidence for adsorption to the walls was observed. However, when the vial contained a single HEK-293 cell transfected to express the dopamine transporter, the dopamine concentration decreased with time at a rate consistent with the uptake kinetics mediated by the transporter located on the cell membrane.