Optical Analysis of Synaptic Vesicle Recycling at the Frog Neuromuscular Junction

Betz, William J.; Bewick, Guy S.

doi:10.1126/science.1553547

Cited by 710 publications

(476 citation statements)

References 23 publications

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“…Because the endocytic process also results in the uptake of a volume of fluid, even fluidphase tracers can be used. Indeed, various markers have been used, such as sulforhodamine (Lichtman et al 1985), fluorescently labeled antibodies (Kraszewski et al 1996;Westphal et al 2008), styryl dyes (i.e., FM dyes; Betz and Bewick 1992), and fluorescent dextrans (Holt et al 2003). Among these markers, FM dyes have proved to be valuable in a number of different preparations, including neuromuscular junctions (NMJ) from the frog (Betz and Bewick 1992), snake (Teng et al 1999), lizard (Lindgren et al 1997), and larval Drosophila (Ramaswami et al 1994); hippocampal neurons in culture (Ryan et al 1993) and acute slice (Zakharenko et al 2001); goldfish retinal bipolar cells (Zenisek et al 2000); Caenorhabditis elegans neurons (Kay et al 1999); rat calyx of Held (de Lange et al 2003); inner and outer hair cells (Griesinger et al 2002;Kaneko et al 2006); and a variety of nonneuronal preparations (for a review, see Cochilla et al 1999).…”

Section: Discussionmentioning

confidence: 99%

Imaging Synaptic Vesicle Recycling by Staining and Destaining Vesicles with FM Dyes

Hoopmann¹,

Rizzoli²,

Betz³

2011

Cold Spring Harb Protoc

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The synaptic vesicle is the essential organelle of the synapse. Many approaches for studying synaptic vesicle recycling have been devised, one of which, the styryl (FM) dye, is well suited for this purpose. FM dyes reversibly stain, but do not permeate, membranes; hence they can specifically label membrane-bound organelles. Their quantum yield is drastically higher when bound to membranes than when in aqueous solution. This protocol describes the imaging of synaptic vesicle recycling by staining and destaining vesicles with FM dyes. Nerve terminals are stimulated (electrically or by depolarization with high K + ) in the presence of dye, their vesicles are then allowed to recycle, and finally dye is washed from the chamber. In neuromuscular junction (NMJ) preparations, movements of the muscle must be inhibited if imaging during stimulation is desired (e.g., by application of curare, a potent acetylcholine receptor inhibitor). The main characteristics of FM dyes are also reviewed here, as are recent FM dye monitoring techniques that have been used to investigate the kinetics of synaptic vesicle fusion. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. Reagents FM dye(s)Dye concentrations are usually 2-10 µM for FM1-43 or FM4-64, and 25-40 µM for FM2-10. The dyes readily dissolve in water; stocks of 1-5 mM concentration should be prepared and can be stored at 4˚C. Neuronal tissue or cell sample of interest Equipment Imaging setupAn upright epifluorescence microscope equipped with a 40×-60× water-immersion objective is optimal. When working with cell cultures, the advantages of high-resolution oil-immersion objectives (e.g., 100×/1.4 numerical aperture [NA]) can be exploited by using an inverted setup. For FM1-43, maximal fluorescence is at 465-nm excitation/560-nm emission (Henkel et al. 1996); a conventional fluorescein filter set (435-nm excitation) can also be used. Because the major concern in FM imaging is phototoxicity (which appears usually before any clear signs of photobleaching and alters the ability of the preparations to recycle vesicles), keep the illumination to a minimum by including neutral-density filters. For example, when using a 40×/0.75-NA water-immersion objective with a 100-W mercury lamp, 5%-15% transmission is sufficient. On confocal setups, an argon 488-nm laser line is typically used for excitation, but special care has to be taken to avoid phototoxicity. Images are typically acquired by charge-coupled device (CCD) cameras or photomultiplier tubes, which allows for fast acquisition rates and software-based quantitative image analysis.Neuronal stimulator (see Step 3)

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Section: Discussionmentioning

confidence: 99%

Imaging Synaptic Vesicle Recycling by Staining and Destaining Vesicles with FM Dyes

Hoopmann¹,

Rizzoli²,

Betz³

2011

Cold Spring Harb Protoc

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“…An alternative approach is to combine a functional measure of vesicle pools with subsequent ultrastructural investigation. One of the most widelyused methods exploits FM-dyes, fluorescent reporters that readily bind to lipid membrane and are taken up into recycling vesicles during endocytosis to provide a functional readout of vesicle turnover [21][22][23] . One dye variant, FM1-43FX, also efficiently drives the polymerization of diaminobenzidine (DAB) when photoactivated, leading to the formation of an osmiophilic precipitate [24][25][26][27][28][29][30][31] .…”

Section: Labelling and Visualizing Functional Vesiclesmentioning

confidence: 99%

Ultrastructural readout of functional synaptic vesicle pools in hippocampal slices based on FM dye labeling and photoconversion

et al. 2014

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(2014) Ultrastructural readout of functional synaptic vesicle pools in hippocampal slices based on FM dye labeling and photoconversion. Nature Protocols, 9 (6). pp. 1337 -1347 . ISSN 1754 -2189 This version is available from Sussex Research Online: http://sro.sussex.ac.uk/60182/ This document is made available in accordance with publisher policies and may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher's version. Please see the URL above for details on accessing the published version. Copyright and reuse:Sussex Research Online is a digital repository of the research output of the University.Copyright and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable, the material made available in SRO has been checked for eligibility before being made available.Copies of full text items generally can be reproduced, displayed or performed and given to third parties in any format or medium for personal research or study, educational, or not-for-profit purposes without prior permission or charge, provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way.

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“…Despite many similarities of the exocytotic machinery in (neuro-) endocrine cells and that in neurotransmitter-releasing fast synapses, there are important differences: 1) synaptic vesicles are small (diameter Ͻ30 nm; von Gersdorff 1996) compared with peptide containing vesicles (hundreds of nm; Plattner et al 1997;Olofsson et al 2002); 2) synaptic exocytosis occurs without delay after the influx of Ca 2π and is at least one order-of magnitude faster than peptide secretion (Ämmälä et al 1993;Mennerick & Matthews 1996); 3) synaptic vesicles may stay largely intact during exocytosis and are refilled with new transmitter molecules immediately after endocytosis, whereas peptide-containing granules are assembled and filled with secretory peptides in the Golgi apparatus (Betz & Bewick 1992;Hutton 1994); 4) synaptic exocytosis generally requires higher concentrations of Ca 2π (Ämmälä et al 1993;Mennerick & Matthews 1996, Chow et al 1996Bollmann et al 2000); and 5) although most secretory cells contain more than one type of voltage-gated Ca 2π -channel, synaptic transmitter exocytosis depends on the faster N-or P-type (Hirning et al 1988;Uchitel et al 1992), whereas endocrine cells preferentially use the L-type (Lopez et al 1994;Barg et al 2001a). Taken together, the findings in neurones suggest that the site of Ca 2π -influx is within minimal distance of the Ca 2π -sensor at the synaptic vesicle and functionally associated within release sites.…”

Section: Coupling Of Ca 2π -Influx and Exocytosismentioning

confidence: 99%

Mechanisms of Exocytosis in Insulin‐Secreting B‐Cells and Glucagon‐Secreting A‐Cells

Barg

2003

Pharmacology & Toxicology

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In pancreatic B-and A-cells, metabolic stimuli regulate biochemical and electrical processes that culminate in Ca 2π -influx and release of insulin or glucagon, respectively. Like in other (neuro)endocrine cells, Ca 2π -influx triggers the rapid exocytosis of hormone-containing secretory granules. Only a small fraction of granules (Ͻ1% in insulin-secreting B-cells) can be released immediately, while the remainder requires translocation to the plasma membrane and further ''priming'' for release by several ATP-and Ca 2π -dependent reactions. Such functional organization may account for systemic features such as the biphasic time course of glucose-stimulated insulin secretion. Since this release pattern is altered in type-2 diabetes mellitus, it is conceivable that disturbances in the exocytotic machinery underlie the disease. Here I will review recent data from our laboratory relevant for the understanding of these processes in insulin-secreting B-cells and glucagon-secreting A-cells and for the identification of novel targets for antidiabetic drug action. Two aspects are discussed in detail: 1) The importance of a tight interaction between L-type Ca 2π -channels and the exocytotic machinery for efficient secretion; and 2) the role of intragranular acidification for the priming of secretory granules and its regulation by a granular 65-kDa sulfonylurea-binding protein.

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Optical Analysis of Synaptic Vesicle Recycling at the Frog Neuromuscular Junction

Cited by 710 publications

References 23 publications

Imaging Synaptic Vesicle Recycling by Staining and Destaining Vesicles with FM Dyes

Imaging Synaptic Vesicle Recycling by Staining and Destaining Vesicles with FM Dyes

Ultrastructural readout of functional synaptic vesicle pools in hippocampal slices based on FM dye labeling and photoconversion

Mechanisms of Exocytosis in Insulin‐Secreting B‐Cells and Glucagon‐Secreting A‐Cells

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