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Analysis of synaptic vesicle endocytosis in synaptosomes by high-content screening

Nature Protocols volume 7, pages 1439–1455 (2012)Cite this article

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Abstract

Small molecules modulating synaptic vesicle endocytosis (SVE) may ultimately be useful for diseases where pathological neurotransmission is implicated. Only a small number of specific SVE modulators have been identified to date. Slow progress is due to the laborious nature of traditional approaches to study SVE, in which nerve terminals are identified and studied in cultured neurons, typically yielding data from 10–20 synapses per experiment. We provide a protocol for a quantitative, high-throughput method for studying SVE in thousands of nerve terminals. Rat forebrain synaptosomes are attached to 96-well microplates and depolarized; SVE is then quantified by uptake of the dye FM4-64, which is imaged by high-content screening. Synaptosomes that have been frozen and stored can be used in place of fresh synaptosomes, reducing the experimental time and animal numbers required. With a supply of frozen synaptosomes, the assay can be performed within a day, including data analysis.

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Figure 1: The high-throughput SVE assay.
Figure 2: Frozen-thawed synaptosomes after labeling with FM4-64 and calcein blue–AM.
Figure 3: Polystyrene foam box for freezing synaptosomes.
Figure 4: Morphological comparison of fresh and frozen-thawed synaptosomes.
Figure 5: Plate layout for screening.
Figure 6: Stimulus-dependent uptake of FM4-64 in synaptosomes.
Figure 7: Application of the SVE assay to screening of dynamin inhibitors Dyngo-4a and dynasore.

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Acknowledgements

This work was supported by grants from the National Health and Medical Research Council, Australia, and a grant from a private foundation. We thank R. Boutros for commenting on this manuscript. Ultrastructural studies were performed in the Electron Microscope Laboratory, Westmead—a joint facility of the Institute for Clinical Pathology and Medical Research, the University of Sydney and the Westmead Research Hub.

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Authors and Affiliations

  1. Cell Signalling Unit, Children's Medical Research Institute, University of Sydney, Westmead, New South Wales, Australia

    James A Daniel, Emma Kettle & Phillip J Robinson

  2. Department of Molecular Physiology, Molecular Medicine Research Group, School of Medicine, University of Western Sydney, Penrith, New South Wales, Australia

    Chandra S Malladi

  3. Department of Chemistry, School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia

    Adam McCluskey

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Contributions

J.A.D. performed all experiments, developed analytical strategies and analyzed data. J.A.D. and P.J.R. conceived experiments and wrote the manuscript. C.S.M., E.K. and A.M. provided comments on the manuscript. C.S.M. performed essential laboratory work in the development of this assay. E.K. prepared samples for electron microscopy and assisted in image acquisition. A.M. provided small molecules for screening.

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Supplementary information

Supplementary Fig. 1

Clumping of synaptosomes induced by resuspension in isotonic buffer Shown is a preparation of synaptosomes that were rapidly thawed and resuspended in HBK, rather than the standard SET buffer (used in Fig. 2). Synaptosomes were attached by centrifugation (protein concentration = 20 µg/ml), labelled with calcein blue-AM and then depolarized in the presence of FM4-64 (as described in PROCEDURE). FM4-64 (A) and calcein blue (B) labelling are shown, along with an overlay of the two colour channels (C). In contrast to synaptosomes in Fig. 2, after resuspension and attachment in isotonic buffer, synaptosomes formed large aggregates (arrows) that were not observed when synaptosomes were suspended in SET. Scale bar = 40 µm. (PDF 107 kb)

Supplementary Fig. 2

An example of a small molecule that induces loss of synaptosomes: JZNC-7 The effects of JZNC-7 on FM4-64 uptake and synaptosomes attachment, using calcein blue as a cytosolic marker, were investigated. The SVE assay was carried out as described in PROCEDURE. A dose-dependent decrease in both FM4-64 fluorescence (B) and calcein blue puncta (B) was observed in synaptosomes following treatment with increasing concentrations of JZNC-7 (n = 3 independent experiments). The IC50 for the reduction of calcein blue pit count, which is an indicator of synaptosomal attachment, was similar to the IC50 for the reduction FM4-64 fluorescence. Thus, we cannot interpret the reduction in FM4-64 fluorescence as SVE inhibition. Error bars represent SEM. (PDF 25 kb)

Supplementary Fig. 3

Selection of optimal FM4-64 concentration (A) For a range of FM4-64 concentrations, we compared FM4-64 fluorescence under unstimulated (white bars) and depolarized conditions (black bars). 1 µM FM4-64 showed the greatest increase in FM4-64 fluorescence when depolarized. On this basis, 1 µM FM4-64 was used for all other experiments described. (B) As the concentration of FM4-64 present in the extracellular buffer increased, so too did the total FM4-64 fluorescence in the synaptosomes. n = 3 independent experiments. Error bars represent SEM. (PDF 31 kb)

Supplementary Fig. 4

An example of a small molecule that exhibits fluorescence: Dyngo-4a Many small molecules exhibit fluorescence. Synaptosomes were labelled with calcein blue-AM and then incubated with either 1% DMSO (0 µM Dyngo-4a) or 30 µM Dyngo-4a. DMSO and Dyngo-4a were washed out prior to imaging. Calcein blue was imaged using a fluorescence filter set optimized for the fluorophore DAPI and showed no change with Dyngo-4a treatment (upper panels). However, when synaptosomes were imaged with a filter set optimized for imaging the green fluorophore FITC (lower panels), synaptosomes that had been treated with Dyngo-4a appeared as fluorescent puncta. We recommend that small molecules be checked for fluorescence as a part of the screening process. Since Dyngo-4a does not exhibit excitation/emission properties that overlap with those of FM4-64, its fluorescence properties did not interfere with the SVE assay. Scale bar = 40 µm. (PDF 112 kb)

Supplementary Fig. 5

Segmentation of fluorescence images and impact of thresholding parameters (A) To quantify synaptosomal uptake of FM4-64, images are subjected to segmentation by thresholding in ImageXpress software. Shown is an image of synapsotomes depolarized in the presence of FM4-64 (left panel). The image is then subjected to thresholding by ImageXpress software, identifying objects that are 1-2 µm in width and 60 grey levels above background. An image showing the identified objects is shown in the right panel. Integrated fluorescence intensity from frozen synaptosomes in Fig. 6 was recalculated using three different sizes for image thresholding: 0.88-1.76 µm (B), 1-2 µm (C) and 1-4 µm (D). All three exhibited similar responses to increasing depolarization strength. When all three are plotted on the same graph and normalized to the initial fluorescence (E), the three different size thresholds exhibited differences in their relative response to increasing depolarization. Scale bar = 40 µm. Error bars represent SEM. (PDF 114 kb)

Supplementary Fig. 6

Impact of different quantification parameters Data from frozen synaptosomes in Fig. 6 was quantified using four different parameters – integrated pit intensity (A), total pit area (B), pit count (C) and average pit fluorescence intensity (D). All plots exhibited increases in FM4-64 uptake with increasing depolarization strength. When all four data sets are normalized against initial fluorescence and plotted on the same graph (E), the three different size thresholds exhibited differences in their relative response to increasing depolarization. Error bars represent SEM. (PDF 28 kb)

Supplementary Fig. 7

Exocytic loss of FM4-64 in synaptosomes Exocytosis in response to depolarization was examined in synaptosomes. Freeze-thawed synaptosomes were attached to PEI-coated microtiter plates. Synaptosomes were then exposed to FM4-64, either with no depolarization (unstimulated, white bar) or depolarized by the addition of 40 mM KCl. This depolarization is referred to as S1 and FM4-64 labelling by S1 depolarization is shown in the black bar. After S1 labelling, FM4-64 was washed away using Advasep-4. Synaptosomes that were then subjected to a second round of depolarization (S2), this time in the absence of FM4-64, exhibited a loss of fluorescence of approximately 20% (grey bar). This suggests that depolarization induced a release of FM4-64 from vesicles undergoing exocytosis. Data was normalized against fluorescence of S1-loaded synaptosomes. Data is from three independent experiments (n = 3). Error bars represent SEM. (PDF 13 kb)

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Daniel, J., Malladi, C., Kettle, E. et al. Analysis of synaptic vesicle endocytosis in synaptosomes by high-content screening. Nat Protoc 7, 1439–1455 (2012). https://doi.org/10.1038/nprot.2012.070

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