Chromaffin cells from the adrenal gland are a widely used model system to study neuronal exocytosis, and the fusion pore properties in these cells have been studied in great detail (3-6). Release of single vesicles (quanta) of epinephrine or norepinephrine can be detected electrochemically with a carbon fiber electrode (CFE) as amperometric spikes (7,8). These spikes are often preceded by a so-called foot signal (8), which is due to slow discharge of catecholamines through a narrow fusion pore (3) and indicates the time of fusion pore formation and expansion.To obtain not only temporal but also spatial resolution of opening and expansion of single fusion pores in chromaffin cells, we fabricated electrochemical detector (ECD) arrays of four Pt electrodes patterned on glass coverslips (9). For quantal events, the oxidation currents recorded from the four electrodes allow the desired determination of fusion pore dynamics with millisecond time resolution as well as localization of the fusion event.Simultaneous observation of the cell by fluorescence microscopy can be performed. Here we show that the spatiotemporal assignments of single exocytotic events obtained by electrochemical imaging of fusion events correlate with release of a fluorescent vesicle marker during exocytosis in chromaffin cells. Materials and MethodsECD Array. Pt conductors insulated by photoresist were fabricated photolithographically on standard microscope coverslips No. 1.5 (160-190 m thick) as described in ref. 9, forming a fourelectrode ECD array. Each ECD array consists of a square window Ϸ10 ϫ 10 m 2 in size in the insulating 0.5-m-thick photoresist. The active electrodes are formed by the tips of the four Pt conductors, Ϸ3 m wide and 150 nm thick, that protrude into the corners of the square. The area and shape of the electrode arrays were imaged and quantified by using atomic force microscopy and optical microscopy.Cell Preparation and Recording Conditions. Bovine adrenal glands were obtained from a local slaughterhouse and prepared as described in ref. 10. Cells were cultured on 12-mm coverslips that were coated with 0.02% poly(D)-lysine and used on days 1-10. Before the experiment, a small coverslip with cells attached was placed in a 35-mm dish with 1 ml of bath solution containing 150 mM NaCl, 10 mM Hepes, 5 mM CaCl 2 , 5 mM KCl, 2 mM MgCl 2 (pH 7.25) (318 mosM) and supplemented with 3 M acridine orange (Molecular Probes) for 15 min in the dark. An ECD array coverslip was mounted on a custom-built stage on a Zeiss 135TV microscope, and the coverslip with the cells was placed at some distance from the ECD array. Bath solution (150-200 l) was then added to cover the cells and the active electrode array. By using a patch pipette, a cell was lifted off the coverslip and manipulated over the center of an ECD array. Some cells were stimulated by including ionomycin in the pipette or adding ionomycin to the bath. Experiments were performed at room temperature.Electrochemical Recording and Analysis. The ECD electrodes were connected via their...

The roles of nonmuscle myosin II and cortical actin filaments in chromaffin granule exocytosis were studied by confocal fluorescence microscopy, amperometry, and cell-attached capacitance measurements. Fluorescence imaging indicated decreased mobility of granules near the plasma membrane following inhibition of myosin II function with blebbistatin. Slower fusion pore expansion rates and longer fusion pore lifetimes were observed after inhibition of actin polymerization using cytochalasin D. Amperometric recordings revealed increased amperometric spike half-widths without change in quantal size after either myosin II inhibition or actin disruption. These results suggest that actin and myosin II facilitate release from individual chromaffin granules by accelerating dissociation of catecholamines from the intragranular matrix possibly through generation of mechanical forces.

Formation of a fusion pore between a vesicle and its target membrane is thought to involve the so-called SNARE protein complex. However, there is no mechanistic model explaining how the fusion pore is opened by conformational changes in the SNARE complex. It has been suggested that C-terminal zipping triggers fusion pore opening. A SNAP-25 mutant named SNAP-25⌬9 (lacking the last nine C-terminal residues) should lead to a less-tight C-terminal zipping. Single exocytotic events in chromaffin cells expressing this mutant were characterized by carbon fiber amperometry and cell-attached patch capacitance measurements. Cells expressing SNAP-25⌬9 displayed smaller amperometric ''foot-current'' currents, reduced fusion pore conductances, and lower fusion pore expansion rates. We propose that SNARE/lipid complexes form proteolipid fusion pores. Fusion pores involving the SNAP-25⌬9 mutant will be less tightly zipped and may lead to a longer fusion pore structure, consistent with the observed decrease of fusion pore conductance.amperometry ͉ capacitance measurement ͉ chromaffin cell ͉ exocytosis ͉ patch clamp

The SNARE complex consists of the three proteins synaptobrevin-2, syntaxin, and synaptosomal-associated protein 25 (SNAP25) and is thought to execute a large conformational change as it drives membrane fusion and exocytosis. The relation between changes in the SNARE complex and fusion pore opening is, however, still unknown. We report here a direct measurement relating a change in the SNARE complex to vesicle fusion on the millisecond time scale. In individual chromaffin cells, we tracked conformational changes in SNAP25 by total internal reflection fluorescence resonance energy transfer (FRET) microscopy while exocytotic catecholamine release from single vesicles was simultaneously recorded using a microfabricated electrochemical detector array. A local rapid and transient FRET change occurred precisely where individual vesicles released catecholamine. To overcome the low time resolution of the imaging frames needed to collect sufficient signal intensity, a method named event correlation microscopy was developed, which revealed that the FRET change was abrupt and preceded the opening of an exocytotic fusion pore by ∼90 ms. The FRET change correlated temporally with the opening of the fusion pore and not with its dilation.TIR-FRET imaging | electrochemical imaging | time superresolution microscopy | image analysis | transmitter release N eurotransmitters, hormones, and many other mediators are stored in secretory vesicles, and their release occurs by the mechanism of exocytosis that begins with formation of a narrow fusion pore (1). Fusion-pore formation in neurosecretory vesicles is stimulated by an increase of intracellular [Ca 2+ ] and is thought to be induced by a large conformational change in the SNARE complex (2-4). Such changes may be involved in various steps from preparing vesicles for fusion (5) to fusion-pore dilation (6). To determine whether a conformational change in SNAREs is linked to fusion, the synaptosomal-associated protein 25 (SNAP25) mutant SCORE (SNARE COmplex REporter) has been developed (7), which contains two fluorescent proteins, CFP as a fluorescence resonance energy transfer (FRET) donor and Venus as a FRET acceptor. SCORE and constructs like it (5, 7-9) have the advantage that donor and acceptor exist at fixed stoichiometry, facilitating the analysis and interpretation of the measurements. Like SNAP25, SCORE forms SNARE complexes with syntaxin and vesicle-associated membrane protein (VAMP)/synaptobrevin (7,8), and when endogenous SNAP25 in chromaffin cells is cleaved by botulinum toxin E, a toxin-resistant SCORE rescues exocytosis (8). In PC12 cells expressing SCORE, a FRET change was evoked by high [K + ] stimulation, but with this method the time scale was tens of seconds (7). This FRET change was abolished in the absence of extracellular Ca 2+ , indicating that it is dependent on Ca 2+ entry. In contrast, the FRET change was not affected by tetanus neurotoxin treatment (7), which causes the blockade of exocytosis (10). In beta cells, a SCORE-like construct indicated a FRET chan...

Surface patterned platinum microelectrodes (PtEs) insulated with 300 nm thick fused silica were fabricated using contact photolithography. These electrodes exhibit low noise and were used for monitoring single vesicle exocytosis from chromaffin cells by constant potential amperometry as well as fast scan cyclic voltammetry. Amperometric spike parameters were consistent with those obtained with conventional carbon fiber electrodes (CFEs). Catecholamine voltammograms acquired with PtEs exhibited redox peaks with full width at half maximum of ~45 mV, much sharper than those of CFE recordings. The time course of voltammetrically measured release events was similar for PtEs and CFEs. The fused silica insulated PtEs allowed incorporation of micrometer precision surface patterned poly-D-lysine (PDL). PDL-functionalized devices were applied to stimulate mast cells and record single release events without serotonin pre-loading. Microfabricated PtEs are thus able to record single exocytotic events with high resolution and should be suitable for highly parallel electrode arrays allowing simultaneous measurements of single events from multiple cells.