Abstract

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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Acknowledgements

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.

Author information

Author notes

    • Lin Tian

    Present address: Department of Biochemistry and Molecular Medicine, University of California Davis School of Medicine, Sacramento, California, USA.

Affiliations

  1. Howard Hughes Medical Institute (HHMI), Janelia Farm Research Campus, Ashburn, Virginia, USA.

    • Jonathan S Marvin
    • , Bart G Borghuis
    • , Lin Tian
    • , Mark T Harnett
    • , Jasper Akerboom
    • , Tsai-Wen Chen
    • , Eric R Schreiter
    • , S Andrew Hires
    •  & Loren L Looger
  2. Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Bart G Borghuis
    •  & Jonathan B Demb
  3. Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York, USA.

    • Joseph Cichon
    •  & Wen-Biao Gan
  4. HHMI, Laboratory of Neural Circuits and Behavior, The Rockefeller University, New York, New York, USA.

    • Andrew Gordus
    •  & Cornelia I Bargmann
  5. Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, Lisbon, Portugal.

    • Sabine L Renninger
    •  & Michael B Orger
  6. Department of Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Jonathan B Demb

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Contributions

J.S.M. and L.L.L. conceived of the project. J.S.M. designed iGluSnFR and performed in vitro and cultured cell characterization; L.T. characterized cultured neurons and astrocytes; M.T.H. performed glutamate uncaging experiments; B.G.B. and J.B.D. characterized retina; A.G. and C.I.B. characterized worms; S.L.R. and M.B.O. characterized zebrafish; J.C. and W.-B.G. characterized mouse motor cortex; J.A. and E.R.S. provided RCaMP1e; T.-W.C. assisted with data analysis; S.A.H. and L.L.L. provided global perspective and planning of experiments across species.

Competing interests

B.G.B. owns Borghuis Instruments, which manufactures and sells the specialized syringe that was used for intraocular virus injections in this study.

Corresponding author

Correspondence to Loren L Looger.

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DOI

https://doi.org/10.1038/nmeth.2333