Features:iGluSnFR3.v857 improves sensitivity, signal-to-noise ratio, linearity, and spatial specificity both in vitro and in vivo, showing postsynaptic localization. The GPI and SGZ sequences significantly enhance the probe’s signal-to-noise ratio, with the SGZ background fluorescence being weaker and the functional response being less pronounced. The PDGFR transmembrane domain leads to poorer postsynaptic localization of the probe.