High-speed imaging of glutamate release with genetically encoded sensors

Duerst, Celine D, Wiegert, J Simon, Helassa, Nordine ORCID: 0000-0003-3743-1886, Kerruth, Silke, Coates, Catherine, Schulze, Christian, Geeves, Michael A, Torok, Katalin and Oertner, Thomas G (2019) High-speed imaging of glutamate release with genetically encoded sensors. NATURE PROTOCOLS, 14 (5). pp. 1401-1424.

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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 of GEGIs that allow investigation of 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 amplitude of fast iGluSnFR transients. The typical time frame for these experiments is 30 min per synapse.

Item Type:	Article
Uncontrolled Keywords:	Cells, Cultured, Humans, Glutamic Acid, Molecular Probes, Microscopy, Confocal, Transfection, Biosensing Techniques, Synaptic Transmission, CA3 Region, Hippocampal, Optical Imaging
Depositing User:	Symplectic Admin
Date Deposited:	25 Apr 2019 07:33
Last Modified:	19 Jan 2023 00:53
DOI:	10.1038/s41596-019-0143-9
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3038205