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High-speed imaging of glutamate release with genetically encoded sensors

Dürst, Céline D., Wiegert, J. Simon, Helassa, Nordine, Kerruth, Silke, Coates, Catherine, Schulze, Christian, Geeves, Michael A., Török, Katalin, Oertner, Thomas G. (2019) High-speed imaging of glutamate release with genetically encoded sensors. Nature Protocols, 14 . pp. 1401-1424. ISSN 1754-2189. (doi:10.1038/s41596-019-0143-9) (Access to this publication is currently restricted. You may be able to access a copy if URLs are provided)

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Abstract

The strength of an excitatory synapse depends on its ability to release glutamate and on the density of postsynaptic receptors. Genetically-encoded glutamate indicators (GEGIs) allow eavesdropping on synaptic transmission at the level of cleft glutamate to investigate properties of the release machinery in detail. Based on the sensor iGluSnFR, we recently developed accelerated versions that allow investigating synaptic release during 100 Hz trains. Here we describe the detailed procedures for design and characterization of fast iGluSnFR variants in vitro, transfection of pyramidal cells in organotypic hippocampal cultures, and imaging of evoked glutamate transients with two-photon laser scanning microscopy. As the released glutamate spreads from a point source - the fusing vesicle - it is possible to localize the vesicle fusion site with a precision exceeding the optical resolution of the microscope. By using a spiral scan path, the temporal resolution can be increased to 1 kHz to capture the peak of fast iGluSnFR transients. The typical time frame for these experiments is 30 min per synapse.

Item Type: Article
DOI/Identification number: 10.1038/s41596-019-0143-9
Uncontrolled keywords: genetically-encoded glutamate indicator, GEGI, glutamate, two-photon imaging, two-photon microscopy, synaptic transmission, stopped-flow, iGluSnFR, hippocampal culture, rat, pyramidal cell, CA1, excitatory synapse, multivesicular release, organotypic culture, single-cell electroporation
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Michael Geeves
Date Deposited: 25 Mar 2019 15:53 UTC
Last Modified: 03 Jun 2019 09:36 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/73197 (The current URI for this page, for reference purposes)
Geeves, Michael A.: https://orcid.org/0000-0002-9364-8898
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