A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses
Summary: Elucidating the molecular mechanisms underlying the functional diversity of synapses requires a high-resolution, sensitive, diffusion-free, quantitative localization method that allows the determination of many proteins in functionally characterized individual synapses. Array tomography per...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2020-07-01
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| Series: | Cell Reports |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2211124720309499 |
| _version_ | 1828854824279474176 |
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| author | Noemi Holderith Judit Heredi Viktor Kis Zoltan Nusser |
| author_facet | Noemi Holderith Judit Heredi Viktor Kis Zoltan Nusser |
| author_sort | Noemi Holderith |
| collection | DOAJ |
| description | Summary: Elucidating the molecular mechanisms underlying the functional diversity of synapses requires a high-resolution, sensitive, diffusion-free, quantitative localization method that allows the determination of many proteins in functionally characterized individual synapses. Array tomography permits the quantitative analysis of single synapses but has limited sensitivity, and its application to functionally characterized synapses is challenging. Here, we aim to overcome these limitations by searching the parameter space of different fixation, resin, embedding, etching, retrieval, and elution conditions. Our optimizations reveal that etching epoxy-resin-embedded ultrathin sections with Na-ethanolate and treating them with SDS dramatically increase the labeling efficiency of synaptic proteins. We also demonstrate that this method is ideal for the molecular characterization of individual synapses following paired recordings, two-photon [Ca2+] or glutamate-sensor (iGluSnFR) imaging. This method fills a missing gap in the toolbox of molecular and cellular neuroscience, helping us to reveal how molecular heterogeneity leads to diversity in function. |
| first_indexed | 2024-12-13T00:41:01Z |
| format | Article |
| id | doaj.art-3dc56d918c2140058ebe0bed068d4fcc |
| institution | Directory Open Access Journal |
| issn | 2211-1247 |
| language | English |
| last_indexed | 2024-12-13T00:41:01Z |
| publishDate | 2020-07-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cell Reports |
| spelling | doaj.art-3dc56d918c2140058ebe0bed068d4fcc2022-12-22T00:05:07ZengElsevierCell Reports2211-12472020-07-01324107968A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual SynapsesNoemi Holderith0Judit Heredi1Viktor Kis2Zoltan Nusser3Laboratory of Cellular Neurophysiology, Institute of Experimental Medicine, Budapest 1083, HungaryLaboratory of Cellular Neurophysiology, Institute of Experimental Medicine, Budapest 1083, HungaryLaboratory of Cellular Neurophysiology, Institute of Experimental Medicine, Budapest 1083, HungaryLaboratory of Cellular Neurophysiology, Institute of Experimental Medicine, Budapest 1083, Hungary; Corresponding authorSummary: Elucidating the molecular mechanisms underlying the functional diversity of synapses requires a high-resolution, sensitive, diffusion-free, quantitative localization method that allows the determination of many proteins in functionally characterized individual synapses. Array tomography permits the quantitative analysis of single synapses but has limited sensitivity, and its application to functionally characterized synapses is challenging. Here, we aim to overcome these limitations by searching the parameter space of different fixation, resin, embedding, etching, retrieval, and elution conditions. Our optimizations reveal that etching epoxy-resin-embedded ultrathin sections with Na-ethanolate and treating them with SDS dramatically increase the labeling efficiency of synaptic proteins. We also demonstrate that this method is ideal for the molecular characterization of individual synapses following paired recordings, two-photon [Ca2+] or glutamate-sensor (iGluSnFR) imaging. This method fills a missing gap in the toolbox of molecular and cellular neuroscience, helping us to reveal how molecular heterogeneity leads to diversity in function.http://www.sciencedirect.com/science/article/pii/S2211124720309499synapse diversityactive zonemolecular composition of the synapsemultiplexed immunolabelingconfocal imagingSTED imaging |
| spellingShingle | Noemi Holderith Judit Heredi Viktor Kis Zoltan Nusser A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses Cell Reports synapse diversity active zone molecular composition of the synapse multiplexed immunolabeling confocal imaging STED imaging |
| title | A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses |
| title_full | A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses |
| title_fullStr | A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses |
| title_full_unstemmed | A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses |
| title_short | A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses |
| title_sort | high resolution method for quantitative molecular analysis of functionally characterized individual synapses |
| topic | synapse diversity active zone molecular composition of the synapse multiplexed immunolabeling confocal imaging STED imaging |
| url | http://www.sciencedirect.com/science/article/pii/S2211124720309499 |
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