High-speed imaging of glutamate release with genetically encoded sensors

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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.