High-speed imaging of glutamate release with genetically encoded sensors
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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.
in: NAT PROTOC, Jahrgang 14, Nr. 5, 05.2019, S. 1401-1424.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - High-speed imaging of glutamate release with genetically encoded sensors
AU - Dürst, Céline D
AU - Wiegert, J Simon
AU - Helassa, Nordine
AU - Kerruth, Silke
AU - Coates, Catherine
AU - Schulze, Christian
AU - Geeves, Michael A
AU - Török, Katalin
AU - Oertner, Thomas G
PY - 2019/5
Y1 - 2019/5
N2 - 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.
AB - 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.
KW - Biosensing Techniques/methods
KW - CA3 Region, Hippocampal/cytology
KW - Cells, Cultured
KW - Glutamic Acid/analysis
KW - Humans
KW - Microscopy, Confocal
KW - Molecular Probes/analysis
KW - Optical Imaging
KW - Synaptic Transmission/genetics
KW - Transfection
U2 - 10.1038/s41596-019-0143-9
DO - 10.1038/s41596-019-0143-9
M3 - SCORING: Journal article
C2 - 30988508
VL - 14
SP - 1401
EP - 1424
JO - NAT PROTOC
JF - NAT PROTOC
SN - 1754-2189
IS - 5
ER -