Mosharov, E.V. & Sulzer, D. Analysis of exocytotic events recorded by amperometry. Nat. Methods 2, 651-658

Department of Neurology, Black Building 305, 650 W 168th Street, Columbia University, New York, New York 10032, USA.
Nature Methods (Impact Factor: 32.07). 10/2005; 2(9):651-8. DOI: 10.1038/nmeth782
Source: PubMed


Amperometry is widely used to study exocytosis of neurotransmitters and hormones in various cell types. Analysis of the shape of the amperometric spikes that originate from the oxidation of monoamine molecules released during the fusion of individual secretory vesicles provides information about molecular steps involved in stimulation-dependent transmitter release. Here we present an overview of the methodology of amperometric signal processing, including (i) amperometric signal acquisition and filtering, (ii) detection of exocytotic events and determining spike shape characteristics, and (iii) data manipulation and statistical analysis. The purpose of this review is to provide practical guidelines for performing amperometric recordings of exocytotic activity and interpreting the results based on shape characteristics of individual release events.

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Available from: David Sulzer, Jun 18, 2014
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    • "The amperometric data were analyzed in Igor Pro 6 (Version; WaveMetrics, Lake Oswego, OR) using an Igor Procedure File designed for analysis of quantal release by the group of David Sulzer30. Vesicle size measurements were performed with the NIH developed ImageJ software where the diameter of each vesicle was measured 5 times and an average was calculated. Data were tested for significant differences using two-tailed t-test assuming equal variances. "
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    ABSTRACT: The details of exocytosis, the vital cell process of neuronal communication, are still under debate with two generally accepted scenarios. The first mode of release involves secretory vesicles distending into the cell membrane to release the complete vesicle contents. The second involves partial release of the vesicle content through an intermittent fusion pore, or an opened or partially distended fusion pore. Here we show that both full and partial release can be mimicked with a single large-scale cell model for exocytosis composed of material from blebbing cell plasma membrane. The apparent switching mechanism for determining the mode of release is demonstrated to be related to membrane tension that can be differentially induced during artificial exocytosis. These results suggest that the partial distension mode might correspond to an extended kiss-and-run mechanism of release from secretory cells, which has been proposed as a major pathway of exocytosis in neurons and neuroendocrine cells.
    Full-text · Article · Jan 2014 · Scientific Reports
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    • "(Ai) Current spike parameters were calculated between the times when current exceeded five s.d. of the baseline (Tbkg1) and subsequently returned to the baseline (Tbkg2) and included maximum current amplitude (Imax), area (Q), width of the spike at half the amplitude (t1/2), duration of the rise (trise) and decay (tdecay) phases between 25% and 75% of the Imax and rate of rise (pA/ms). (Aii) Pre-spike foot signal parameters were tfoot [which corresponds to the time between Tbkg1 and the pre-spike foot signal end as defined by the interception of the rise slope with the baseline (Mosharov and Sulzer)], Qfoot (shaded area) and Ifoot [the average current amplitude within tfoot (Mosharov and Sulzer, 2005)]. HUVEC parameters (mean±s.e.m.) are shown for each in parenthesis (n = 762 spikes and 542 pre-spike foot signals from 97 cells). "
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    ABSTRACT: Regulated secretion from endothelial cells is mediated by Weibel-Palade body (WPB) exocytosis. Plasma membrane cholesterol is implicated in regulating secretory granule exocytosis and fusion pore dynamics; however, its role in modulating WPB exocytosis is not clear. To address this we combined high-resolution electrochemical analysis of WPB fusion pore dynamics, by amperometry, with high-speed optical imaging of WPB exocytosis following cholesterol depletion or supplementation in human umbilical vein endothelial cells. We identified serotonin (5-HT) immunoreactivity in WPBs and VMAT1 expression allowing detection of secreted 5-HT as discrete current spikes during exocytosis. A high proportion of spikes (∼75%) had pre-spike foot signals, indicating that WPB fusion proceeds via an initial narrow pore. Cholesterol depletion significantly reduced pre-spike foot signal duration and increased the rate of fusion pore expansion, while cholesterol supplementation had broadly the reverse effect. Cholesterol depletion slowed the onset of hormone-evoked WPB exocytosis, while supplementation increased the rate of WPB exocytosis and hormone-evoked proregion secretion. Our results provide the first analysis of WPB fusion pore dynamics, and highlight an important role for cholesterol in the regulation of WPB exocytosis.
    Full-text · Article · Oct 2013 · Journal of Cell Science
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    • "For the analysis of fusion events in acridine orange labelled vesicles, the green channel images taken at 20 ms intervals that showed fusion flashes were subjected to maximal intensity determination (see Figure 3) and transferred to Igor Pro. Fusion events were analyzed using software developed for amperometric detection of exocytotic events (Quanta analysis [22]). Kinetic parameters such as the time at the half-height amplitude (t1/2) were obtained for hundreds of fusion events and are represented as distributions. "
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    ABSTRACT: Lipid molecules such as arachidonic acid (AA) and sphingolipid metabolites have been implicated in modulation of neuronal and endocrine secretion. Here we compare the effects of these lipids on secretion from cultured bovine chromaffin cells. First, we demonstrate that exogenous sphingosine and AA interact with the secretory apparatus as confirmed by FRET experiments. Examination of plasma membrane SNARE microdomains and chromaffin granule dynamics using total internal reflection fluorescent microscopy (TIRFM) suggests that sphingosine production promotes granule tethering while arachidonic acid promotes full docking. Our analysis of single granule release kinetics by amperometry demonstrated that both sphingomyelinase and AA treatments enhanced drastically the amount of catecholamines released per individual event by either altering the onset phase of or by prolonging the off phase of single granule catecholamine release kinetics. Together these results demonstrate that the kinetics and extent of the exocytotic fusion pore formation can be modulated by specific signalling lipids through related functional mechanisms.
    Full-text · Article · Sep 2013 · PLoS ONE
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