Article | Published:

Ultrasensitive fluorescent proteins for imaging neuronal activity

Nature volume 499, pages 295300 (18 July 2013) | Download Citation

Abstract

Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5–40-µm long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.

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Author information

Author notes

    • Trevor J. Wardill

    Present address: Marine Biological Laboratory, Program in Sensory Physiology and Behavior, 7 MBL Street, Woods Hole, Massachusetts 02543, USA.

Affiliations

  1. Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia 20147, USA

    • Tsai-Wen Chen
    • , Trevor J. Wardill
    • , Yi Sun
    • , Stefan R. Pulver
    • , Amy Baohan
    • , Eric R. Schreiter
    • , Rex A. Kerr
    • , Vivek Jayaraman
    • , Loren L. Looger
    • , Karel Svoboda
    •  & Douglas S. Kim
  2. Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, Avenida Brasília, Doca de Pedrouços, 1400-038, Lisbon, Portugal

    • Sabine L. Renninger
    •  & Michael B. Orger
  3. Department of Neurobiology, University of California Los Angeles, Los Angeles, California 90095, USA

    • Amy Baohan

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Contributions

V.J., R.A.K., L.L.L. and K.S. initiated the project. T.-W.C., T.J.W. and D.S.K. conducted neuronal culture screening. T.-W.C. performed mouse visual cortical experiments on pyramidal neurons. T.-W.C. and A.B. performed experiments on inhibitory neurons. Y.S., T.J.W. and S.R.P. carried out fly larval neuromuscular junction studies. Y.S. and V.J. carried out adult fly antennal lobe imaging. S.L.R. and M.B.O. conducted zebrafish tectal imaging. E.R.S. performed protein assays. All authors analysed data. T.-W.C., R.A.K., M.B.O., V.J., L.L.L., K.S. and D.S.K. wrote the paper with comments from all authors.

Competing interests

The authors have applied for a patent on materials and methods related to the GCaMP6 variants.

Corresponding authors

Correspondence to Michael B. Orger or Vivek Jayaraman or Loren L. Looger or Karel Svoboda or Douglas S. Kim.

Supplementary information

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  1. 1.

    Supplementary Information

    Supplementary Information

Videos

  1. 1.

    Simultaneous recording of spikes and GCaMP6s fluorescence signal of a L2/3 neuron in the visual cortex in vivo

    This video shows simultaneous recording of spikes and GCaMP6s fluorescence signal of a L2/3 neuron in the visual cortex in vivo. The soundtrack of the video encodes the recorded spikes.

  2. 2.

    Calcium imaging of dendritic spines of GCaMP6s-expressing V1 neurons in response to drifting grating stimulation

    This video shows 320 s of continuous calcium imaging of dendritic spines of GCaMP6s-expressing V1 neurons in response to drifting grating stimulation (2 s duration, 0.05 cycles per degree, 1 Hz temporal frequency). The direction of grating movement is indicated by the arrow in the upper right.

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DOI

https://doi.org/10.1038/nature12354

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