Article

Analysis of exocytotic events recorded by amperometry. Nat Methods 2:651-8

Department of Neurology, Black Building 305, 650 W 168th Street, Columbia University, New York, New York 10032, USA.
Nature Methods (Impact Factor: 25.95). 10/2005; 2(9):651-8. DOI: 10.1038/nmeth782
Source: PubMed

ABSTRACT 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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    • "Analysis of single amperometric spike kinetics was carried out as previously described [25] using an automated macro [26] written in Igor (Wavemetrics, Oregon, USA). Median and mean values for all the spikes of each cell were obtained and then pooled together for statistical comparison; this method helps overcoming the large variability is spike number and spike kinetics by giving each cell the same weight independently of the number of spikes produced. "
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    ABSTRACT: The kinetics of single-amperometric exocytotic events has been measured in chromaffin cells of C57 mice and in an APP/PS1 mouse model of Alzheimer's disease (AD). K(+) depolarisation causes a burst of spikes that indicate the quantal release of the single-vesicle content of catecholamine. The kinetic analysis of 278 spikes from 10 control cells and 520 spikes from 18 APP/PS1 cells shows the following features of the latter compared with the former: (i) 45% lower t(1/2); (ii) 60% smaller quantal size; (iii) 50% lower decay time. Spike feet also showed 60% smaller quantal size. Immunofluorescence and thioflavin staining showed no amyloid β (Aβ) burden in adrenal medulla slices of APP/PS1 mice that however exhibited dense Aβ plaques in the cortex and hippocampus. Furthermore, acetylcholinesterase staining of adrenal medulla indicated no apparent differences in the innervation by splanchnic cholinergic nerve terminals of chromaffin cells from control and APP/PS1 mice. This is the first report identifying subtle differences in the last steps of exocytosis that could be an indication of synaptic dysfunction of the secretory machinery not linked to Aβ burden in AD.
    Biochemical and Biophysical Research Communications 10/2012; 428(4). DOI:10.1016/j.bbrc.2012.10.082 · 2.28 Impact Factor
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    • "For the analysis of fusion events in acridine orange-labeled vesicles, the green channel images taken at 20-ms intervals that showed fusion flashes were subjected to maximal intensity determination (see Fig. 3) and transferred to Igor Pro. Fusion events were analyzed using software developed for amperometric detection of exocytotic events (Quanta analysis, (Mosharov and Sulzer 2005)). Kinetic parameters such the time at the half height amplitude (t 1/2 ) were obtained for hundreds of fusion events, and they were represented as distributions. "
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    ABSTRACT: Chromaffin cell catecholamines are released when specialized secretory vesicles undergo exocytotic membrane fusion. Evidence indicates that vesicle supply and fusion are controlled by the activity of the cortical F-actin-myosin II network. To study in detail cell cortex and vesicle interactions, we use fluorescent labeling with GFP-lifeact and acidotropic dyes in confocal and evanescent wave microscopy. These techniques provide structural details and dynamic images of chromaffin granules caged in a complex cortical structure. Both the movement of cortical structures and granule motion appear to be linked, and this motion can be restricted by the myosin II-specific inhibitor, blebbistatin, and the F-actin stabilizer, jasplakinolide. These treatments also affect the position of the vesicles in relation to the plasma membrane, increasing the distance between them and the fusion sites. Consequently, we observed slower single vesicle fusion kinetics in treated cells after neutralization of acridine orange-loaded granules during exocytosis. Increasing the distance between the granules and the fusion sites appears to be linked to the retraction of the F-actin cytoskeleton when treated with jasplakinolide. Thus, F-actin-myosin II inhibitors appear to slow granule fusion kinetics by altering the position of vesicles after relaxation of the cortical network.
    Journal of Molecular Neuroscience 05/2012; 48(2):328-38. DOI:10.1007/s12031-012-9800-y · 2.76 Impact Factor
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    • "Disruption of myosin II or MARCKS function favored smaller and slower spikes characteristic of kiss-and-run exocytosis. Moreover, MARCKS or myosin II inhibition had no effect on prespike foot currents (Supplemental Table S1), a parameter that correlates to formation of the initial fusion pore (Chow et al., 1992; Mosharov and Sulzer, 2005). Thus, we conclude that MARCKS and myosin II do not alter the initial fusion event or the formation of the fusion pore. "
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    ABSTRACT: Adrenal medullary chromaffin cells are innervated by the sympathetic splanchnic nerve and translate graded sympathetic firing into a differential hormonal exocytosis. Basal sympathetic firing elicits a transient kiss-and-run mode of exocytosis and modest catecholamine release, whereas elevated firing under the sympathetic stress response results in full granule collapse to release catecholamine and peptide transmitters into the circulation. Previous studies have shown that rearrangement of the cell actin cortex regulates the mode of exocytosis. An intact cortex favors kiss-and-run exocytosis, whereas disrupting the cortex favors the full granule collapse mode. Here, we investigate the specific roles of two actin-associated proteins, myosin II and myristoylated alanine-rich C-kinase substrate (MARCKS) in this process. Our data demonstrate that MARCKS phosphorylation under elevated cell firing is required for cortical actin disruption but is not sufficient to elicit peptide transmitter exocytosis. Our data also demonstrate that myosin II is phospho-activated under high stimulation conditions. Inhibiting myosin II activity prevented disruption of the actin cortex, full granule collapse, and peptide transmitter release. These results suggest that phosphorylation of both MARCKS and myosin II lead to disruption of the actin cortex. However, myosin II, but not MARCKS, is required for the activity-dependent exocytosis of the peptide transmitters.
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