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Ultrasensitive fluorescent proteins for imaging neuronal activity

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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Figure 1: GCaMP mutagenesis and screening in dissociated neurons.
Figure 2: GCaMP6 performance in the mouse visual cortex.
Figure 3: Combined imaging and electrophysiology in the visual cortex.
Figure 4: Imaging activity in dendritic spines in the visual cortex.
Figure 5: The orientation preference of populations of dendritic spines predicts the orientation preference of their parent neuron.
Figure 6: Orientation-tuned domains in dendrites of GABAergic interneurons.

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Authors and Affiliations

Authors

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.

Corresponding authors

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

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Competing interests

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

Supplementary information

Supplementary Information

Supplementary Information (PDF 7370 kb)

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. (MP4 12818 kb)

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. (MP4 19513 kb)

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Chen, TW., Wardill, T., Sun, Y. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013). https://doi.org/10.1038/nature12354

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