We describe an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and we validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus–evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted postsynaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.
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We thank J. Macklin for two-photon spectrophotometry and two-photon lifetime measurements of purified proteins; H. White and S. Winfried for tissue culture; B. Shields and A. Hu for mouse brain dissection and neuronal culture; Molecular Biology and Media Prep Shared Resources for DNA preparation and sequencing and for media preparation; M. Ramirez and K. Ritola for virus production; D. Kim and the GECI Project for advice and use of the neuronal culture rig; and K. Svoboda, J. Magee and A. Hantman for helpful conversations. All affiliations are HHMI Janelia Farm. HHMI supported this work.
B.G.B. owns Borghuis Instruments, which manufactures and sells the specialized syringe that was used for intraocular virus injections in this study.
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Marvin, J., Borghuis, B., Tian, L. et al. An optimized fluorescent probe for visualizing glutamate neurotransmission. Nat Methods 10, 162–170 (2013). https://doi.org/10.1038/nmeth.2333
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