Imaging Synaptic Glutamate Release with Two-Photon Microscopy in Organotypic Slice Cultures

The strength of an excitatory synapse relies on the amount of glutamate it releases and on the amount of postsynaptic receptors responding to the released glutamate. Here we describe a strategy to investigate presynaptic release independently of postsynaptic receptors, using a genetically encoded glutamate indicator (GEGI) such as iGluSnFR to measure synaptic transmission in rodent organotypic slice cultures. We express the iGluSnFR in CA3 pyramidal cells and perform two-photon glutamate imaging on individual Schaffer collateral boutons in CA1. Sparse labeling is achieved via transfection of pyramidal cells in organotypic hippocampal cultures, and imaging of evoked glutamate transients with two-photon laser scanning microscopy. A spiral scan path over an individual presynaptic bouton allows to sample at high temporal resolution the local release site in order to capture the peak of iGluSnFR transients.