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Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators

Nature Methods volume 6, pages 875–881 (2009)Cite this article

Abstract

Genetically encoded calcium indicators (GECIs) can be used to image activity in defined neuronal populations. However, current GECIs produce inferior signals compared to synthetic indicators and recording electrodes, precluding detection of low firing rates. We developed a single-wavelength GCaMP2-based GECI (GCaMP3), with increased baseline fluorescence (3-fold), increased dynamic range (3-fold) and higher affinity for calcium (1.3-fold). We detected GCaMP3 fluorescence changes triggered by single action potentials in pyramidal cell dendrites, with signal-to-noise ratio and photostability substantially better than those of GCaMP2, D3cpVenus and TN-XXL. In Caenorhabditis elegans chemosensory neurons and the Drosophila melanogaster antennal lobe, sensory stimulation–evoked fluorescence responses were significantly enhanced with GCaMP3 (4–6-fold). In somatosensory and motor cortical neurons in the intact mouse, GCaMP3 detected calcium transients with amplitudes linearly dependent on action potential number. Long-term imaging in the motor cortex of behaving mice revealed large fluorescence changes in imaged neurons over months.

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Figure 1: In vitro characterization of GCaMP3.
Figure 2: Action potential–evoked response of GCaMP3 in hippocampal pyramidal and layer 2/3 cortical neurons.
Figure 3: Comparison of GECI responses in pyramidal cell principal dendrite in acute cortical slice to back-propagating APs.
Figure 4: In vivo imaging of sensory-evoked Ca2+ transients with GCaMPs in C. elegans.
Figure 5: In vivo imaging of sensory-evoked Ca2+ transients with GCaMPs in Drosophila.
Figure 6: In vivo Ca2+ imaging of evoked and spontaneous activity with GCaMP3 in awake, behaving mice.

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Acknowledgements

We thank J. Simpson and S. Hampel for cloning GCaMPs into pMUH, and members of the Fly Core for making fly crosses. pMUH was a generous gift from B. Pfeiffer and G. Rubin. We thank J. Seelig, who contributed to the Drosophila imaging experiments, S. Viswanathan and S. Sternson for assistance in screening mutants in HEK293 cells, J. Marvin for assistance in screening mutants in Escherichia coli, B. Zemelman for assistance in virus production, H. Zhong, T. Sato and B. Borghuis for developing image analysis software, D.K. O'Connor for assistance with in utero electroporation, H. White and S. Winfrey for cell culture, B. Shields and A. Hu for immunostaining, A. Arnold for helping with imaging experiments, members of the Molecular Biology Shared Resource for plasmid preparation and sequencing, and J. Marvin, J. Simpson and G. Tervo for critical reading of the manuscript. All affiliations are Howard Hughes Medical Institute, Janelia Farm Research Campus. GCaMP2 plasmid was a gift from M. Kotlikoff (Cornell University); TN-XXL plasmid was a gift from O. Griesbeck (Max Planck Institute of Neurobiology); D3cpVenus plasmid was a gift from A. Palmer (University of Colorado); GH146-Gal4 flies were a gift from L. Luo (Stanford University); UAS-GCaMP1.6 flies were a gift from D. Rieff and A. Borst (Max Planck Institute of Neurobiology).

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Author notes

  1. Sean A McKinney

    Present address: Present address: The Stowers Institute, Kansas City, Missouri, USA.,

Authors and Affiliations

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

    Lin Tian, S Andrew Hires, Tianyi Mao, Daniel Huber, M Eugenia Chiappe, Leopoldo Petreanu, Jasper Akerboom, Sean A McKinney, Vivek Jayaraman, Karel Svoboda & Loren L Looger

  2. Howard Hughes Medical Institute, Laboratory of Neural Circuits and Behavior, The Rockefeller University, New York, New York, USA

    Sreekanth H Chalasani & Cornelia I Bargmann

  3. Department of Chemistry, University of Puerto Rico, Río Piedras, San Juan, Puerto Rico

    Eric R Schreiter

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Contributions

L.L.L., K.S., L.T., S.A.H. and T.M. designed the project; L.T. and L.L.L. designed the sensor and screen in E. coli and HEK293 cells; L.T., T.M., S.A.H. and L.P. tested the GCaMP variant in brain slice; S.A.H. tested FRET sensor and GCaMP2 in brain slice; S.A.H., L.T. and D.H. analyzed imaging data of brain slice and in vivo; D.H. performed in vivo mouse brain imaging; S.H.C. and C.I.B. performed worm imaging and data analysis; V.J. and M.E.C. performed calcium imaging and data analysis in the fly; S.A.M. and S.A.H. analyzed data for AP detection probability; J.A., L.L.L. and E.R.S. analyzed the structure; L.L.L. and L.T. led the project; L.L.L., K.S., L.T. and S.A.H. wrote the paper.

Corresponding author

Correspondence to Loren L Looger.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Tables 1–3 (PDF 7089 kb)

Supplementary Movie 1

Spontaneous neural activity visualized by GCaMP2 in cultured hippocampal brain slice (3× real time) (MOV 7960 kb)

Supplementary Movie 2

Spontaneous neural activity visualized by GCaMP3 in cultured hippocampal brain slice (3× real time) (MOV 9095 kb)

Supplementary Movie 3

Naturally occurring activity of populations of layer 2/3 neurons expressing GCaMP3 in the primary motor cortex (M1) of awake, behaving mouse during 140 s sequence of two-photon images (10× real time) (AVI 7773 kb)

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Tian, L., Hires, S., Mao, T. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods 6, 875–881 (2009). https://doi.org/10.1038/nmeth.1398

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