biostudies-literatureUnknownAggarwal ANIBIB NIH HHSNIMH NIH HHSNINDS NIH HHSIdentifying the input-output operations of neurons requires measurements of synaptic transmission simultaneously at many of a neuron's thousands of inputs in the intact brain. To facilitate this goal, we engineered and screened 3365 variants of the fluorescent protein glutamate indicator iGluSnFR3 in neuron culture, and selected variants in the mouse visual cortex. Two variants have high sensitivity, fast activation (< 2 ms) and deactivation times tailored for recording large populations of synapses (iGluSnFR4s, 153 ms) or rapid dynamics (iGluSnFR4f, 26 ms). By imaging action-potential evoked signals on axons and visually-evoked signals on dendritic spines, we show that iGluSnFR4s/4f primarily detect local synaptic glutamate with single-vesicle sensitivity. The indicators detect a wide ranbioRxiv : the preprint server for biology2025.03.20.643984https://www.ebi.ac.uk/biostudies/studies/S-EPMC11974752biostudies-literatureGlutamate indicators with increased sensitivity and tailored deactivation rates.PMC11974752U24 EB028942R01 NS143141U01 NS126054U19 NS137920F30 MH138009UM1 MH136462DP2 NS136990MacLennan BJTuraga SCIyer RVevea JDFederico Rossi LHasseman JCarandini MKinsey LWReep DArthur BJKleinfeld DPodgorski KZheng JTebo AGSvoboda KHiblot JChen YLeippe PPalutla APatel RHXie MEYao PNegrean ATurner GCTsang AMarvin JSTsegaye GTarnawski MSun JLKonnerth AHagihara KMZhang YAggarwal ALiu AfalseGlutamate indicators with increased sensitivity and tailored deactivation rates.Identifying the input-output operations of neurons requires measurements of synaptic transmission simultaneously at many of a neuron's thousands of inputs in the intact brain. To facilitate this goal, we engineered and screened 3365 variants of the fluorescent protein glutamate indicator iGluSnFR3 in neuron culture, and selected variants in the mouse visual cortex. Two variants have high sensitivity, fast activation (< 2 ms) and deactivation times tailored for recording large populations of synapses (iGluSnFR4s, 153 ms) or rapid dynamics (iGluSnFR4f, 26 ms). By imaging action-potential evoked signals on axons and visually-evoked signals on dendritic spines, we show that iGluSnFR4s/4f primarily detect local synaptic glutamate with single-vesicle sensitivity. The indicators detect a wide ran2025-01-01T00:00:00Z2025 Mar2026-07-09T10:24:33.004Z2025-07-08T03:10:58.687ZS-EPMC119747524019659010.1101/2025.03.20.643984