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

Abstract

(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.

Dedicated setup for the photoconversion of fluorescent dyes for functional electron microscopy

“…Fixation and photoconversion were performed reducing the samples’ exposure to light and following the protocol of Marra et al (2014). Slices were carefully removed from the recording chamber and fixed in 6% glutaraldehyde/2% paraformaldehyde in PBS using microwave-assisted fixation.…”

“…Once ready for photoconversion, the fixed tissue is placed in a 35 mm petri dish containing a plastic insert (Figure 2C) and held down with a glass O-ring (19 mm O.D.) with nylon strings (Marra et al, 2014). Once the user has identified the region of interest, PBS can be replaced with the DAB solution and oxygenated using Carbogen (95% O 2 and 5% CO 2 ) bubbled using a glass capillary with an opening of >150 µm and held by a 3D-printed holder (Figure 2C) with a magnet glued to its base for positioning.…”

“…In spite of its usefulness, DAB photoconversion in thick neuronal tissue has only been employed by a relatively small number of research groups. A number of different protocols have been published to describe the technique in a range of different preparations and using different modes of light delivery to drive the photoconversion (Opazo and Rizzoli, 2010; Marra et al, 2014; Sabeva and Bykhovskaia, 2017).…”

“…Here, we present a low-cost system to drive targeted photoconversion of DAB in thick neuronal tissue. The intent of this article is not to describe the entire photoconversion procedure (already presented by Marra et al, 2014), but to provide instructions to build a photoconversion setup to easily identify and illuminate a region of interest in a controlled manner.…”

Probes for monitoring regulated exocytosis

“…In addition to synaptic vesicles, FM1–43 has been applied to label different types of secretory granules in non-neuronal cells, though it has been noted that certain secretory granules do not take up FM1–43 reliably [9]. Besides fluorescence imaging, electron microscopy (EM) can also be applied to map the location of FM dyes at nanoscale dimension after photo-conversion in the presence of diaminobenzidine [10, 11]. When combined with serial-section EM, this offers the possibility of characterizing the ultrastructure of secretory vesicles and their 3D organization and function relationships.…”