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Nat Protoc. 2019 May;14(5):1401-1424. doi: 10.1038/s41596-019-0143-9. Epub 2019 Apr 15.

High-speed imaging of glutamate release with genetically encoded sensors.

Author information

1
Institute for Synaptic Physiology, Center for Molecular Neurobiology Hamburg, Hamburg, Germany.
2
Research Group Synaptic Wiring and Information Processing, Center for Molecular Neurobiology Hamburg, Hamburg, Germany.
3
Molecular and Clinical Sciences Research Institute, St George's, University of London, London, UK.
4
Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK.
5
Department of Biophysical Chemistry, J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic.
6
School of Biosciences, University of Kent, Canterbury, UK.
7
Institute for Synaptic Physiology, Center for Molecular Neurobiology Hamburg, Hamburg, Germany. thomas.oertner@zmnh.uni-hamburg.de.

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.

PMID:
30988508
PMCID:
PMC6751072
[Available on 2020-05-01]
DOI:
10.1038/s41596-019-0143-9
[Indexed for MEDLINE]

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