Electrochemical imaging of fusion pore openings by electrochemical detector arrays

Abstract

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...

Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore

“…It is possible that ion exchange mediating dissociation from the intravesicular matrix in chromaffin granules may involve Ca 2+ ions, which could not be omitted in our experiments because they are essential to stimulate exocytosis. Experiments with electrode arrays revealed a reduced diffusion coefficient of catecholamines near the cell surface 20 suggesting that the amperometric spike kinetics may also be limited by slow diffusion near the cell membrane.…”

Role of the synaptobrevin C terminus in fusion pore formation

“…To determine if the vesicles carrying these constructs are fusion competent, simultaneous TIRF microscopy and recording of catecholamine release from the cell membrane in contact with the coverslip was performed using microfabricated electrochemical detector arrays (19). This method allows determination of time and location of a fusion event by amperometric recordings with multiple electrodes.…”

“…Amperometric currents recorded with the ECD array during fluorescence imaging ( Fig. 1 A-C) were low pass filtered at 500 Hz, acquired with 5-kHz sampling rate, and analyzed as described (19). For analysis, a 10-pA minimum current peak height was used as threshold, with a minimum of four analyzable events per cell.…”

“…The four electrodes of an ECD reside at the corners of a square sized such that a cell can be placed atop the array and viewed through the glass coverslip while amperometric currents are recorded from all four electrodes simultaneously. The ECD arrays were fabricated as described (19,40) except for an improved insulation layer, which was created by chemical vapor deposition of approximately 300 nm of SiO 2 onto the electrodes (IPE 1000 PECVD), and subsequent etching of the SiO 2 layer via reactive ion etching (PlasmaLab 80Plus, Oxford Instruments) to expose the active areas of the electrodes and contact pads. described above.…”

Nanocrystalline diamond microelectrode arrays fabricated on sapphire technology for high-time resolution of quantal catecholamine secretion from chromaffin cells

“…Recently, other planar amperometric devices based on var-ious technologies, e.g. platinum, gold, indium-doped tin oxide (ITO) and nitrogen-doped diamond-like carbon (DLC:N) deposited on glass and silicon layers have been shown to monitor well-resolved quantal amperometric events (Dias et al, 2002;Chen et al, 2003Chen et al, , 2008Hafez et al, 2005;Parpura, 2005;Cui et al, 2006;. Among these technologies, ITO is the most interesting one for producing transparent electrodes and wires (Guillen and Herrero, 2005).…”