Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR

An Author Correction to this article was published on 28 February 2019

A Publisher Correction to this article was published on 02 January 2019

This article has been updated

Abstract

Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.

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Fig. 1: SF-iGluSnFR.A184S detects orientation-selective glutamate transients in ferret visual cortex.
Fig. 2: SF-iGluSnFR.S72A permits resolution of multiple glutamate-release events in cultured neurons and in single cerebellar granule cell boutons.
Fig. 3: Utility of SF-Venus-iGluSnFR in cultured neurons and in vivo.

Data availability

All data from this study are available upon request. All constructs have been deposited at Addgene (#106174–106206; hSynapsin1, FLEX-hSynapsin1, FLEX-CAG, GFAP promoters; some fusions with the red fluorescent protein mRuby3 are available). Sequences have been deposited in GenBank (MH392460, MH392461, MH392462, MH392463, MH392464, and MH392465). AAV is available from Addgene.

Change history

  • 28 February 2019

    The version of this paper originally published cited a preprint version of ref. 12 instead of the published version (Proc. Natl. Acad. Sci. USA 115, 5594–5599; 2018), which was available before this Nature Methods paper went to press. The reference information has been updated in the PDF and HTML versions of the article.

  • 02 January 2019

    In the version of this paper originally published, important figure labels in Fig. 3d were not visible. An image layer present in the authors’ original figure that included two small dashed outlines and text labels indicating ROI 1 and ROI 2, as well as a scale bar and the name of the cell label, was erroneously altered during image processing. The figure has been corrected in the HTML and PDF versions of the paper.

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Acknowledgements

We thank J. Macklin (Janelia Research Campus) for two-photon spectra, and K. Ritola and Janelia Virus Services for AAV production. E.C. was a Janelia Undergraduate Scholar.

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Authors

Contributions

J.S.M., L.L.L., A.N.T., and E.C. carried out protein engineering; B.S., D.E.W., and D.F. conducted experiments on ferret visual cortex; J.A.M., S.S., and D.D. carried out neuronal culture analysis; J.P.L. assessed brightness in somatosensory cortex; K.P. and A.K. carried out high-speed imaging of the yellow variant; H.B. and E.R.C. assessed stopped-flow kinetics; N.R., F.J.U.Q., S.S.-H.W., A.W.H., and D.A.D. conducted cerebellum experiments; V.J.D. and B.G.B. conducted retina experiments; and J.S.M. and L.L.L. wrote the manuscript.

Corresponding author

Correspondence to Loren L. Looger.

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Competing interests

J.S.M. and L.L.L. are named on US patent 9719992, which pertains to original iGluSnFR.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14

Reporting Summary

Supplementary Video 1

Example of two-photon imaging of dendritic SFiGluSnFR.A184S activity during visual stimulation. Video is shown at 4× normal speed.

Supplementary Video 2

High-speed two-photon imaging of SF-Venus-iGluSnFR.A184V using 1,030-nm fiber laser excitation. Neurons in culture were imaged by scanned line angular projection microscopy at a 1,016-Hz frame rate. Glutamate was uncaged at two locations, 10 ms apart, starting at t = 400 ms. Saturation denotes ΔF/F0. Blue-tinted regions denote areas where excitation was blocked during high-speed imaging. Recording is a single trial, without averaging. Representative example of 4 trials.

Supplementary Video 3

Example two-photon recording of dendrites in mouse visual cortex labeled with SF-Venus-iGluSnFR.A184S, at a frame rate of 3.41 Hz. Drifting grating visual stimuli of 8 different directions, lasting 2.3 s each, were presented at 5-s intervals. Field-of-view width: 90 μm.

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Marvin, J.S., Scholl, B., Wilson, D.E. et al. Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR. Nat Methods 15, 936–939 (2018). https://doi.org/10.1038/s41592-018-0171-3

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