Ultrasensitive fluorescent proteins for imaging neuronal activity

Journal name:
Nature
Volume:
499,
Pages:
295–300
Date published:
DOI:
doi:10.1038/nature12354
Received
Accepted
Published online

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.

At a glance

Figures

  1. GCaMP mutagenesis and screening in dissociated neurons.
    Figure 1: GCaMP mutagenesis and screening in dissociated neurons.

    a, GCaMP structure27, 51 and mutations in different GCaMP variants relative to GCaMP5G. b, Responses averaged across multiple neurons and wells for GCaMP3, 5G, 6f, 6m, 6s, and OGB1-AM. Top, fluorescence changes in response to 1 action potential. Bottom, 10 action potentials. c, Screening results, 447 GCaMPs. Top, fluorescence change in response to 1 action potential (vertical bars, ΔF/F0; green bar, OGB1-AM, left; black bars, single GCaMP mutations; red bars, combinatorial mutations; blue, GCaMP6 indicators) and significance values for different action potential stimuli (colour plot). Middle, half decay time after 10 action potentials. Bottom, resting fluorescence, F0 normalized to nuclear mCherry fluorescence. Red line, GCaMP3 level; green line, GCaMP5G level; blue line, OGB1-AM level. AP, action potential. d–g, Comparison of GCaMP sensors and OGB1-AM as a function of stimulus strength (colours as in b). d, Response amplitude. e, Signal-to-noise ratio (SNR). f, Half decay time. g, Time to peak (after stimulus offset). Error bars correspond to s.e.m (n = 300, 16, 8, 11, 13 and 11 wells for GCaMP3, GCaMP5G, OGB1-AM, 6f, 6m and 6s, respectively).

  2. GCaMP6 performance in the mouse visual cortex.
    Figure 2: GCaMP6 performance in the mouse visual cortex.

    a, Top, schematic of the experiment. Bottom, field of view showing neurons colour-coded according to their preferred orientation (hue) and response amplitude (brightness) for GCaMP5G (left) and GCaMP6s (right). b, Example traces from three neurons expressing GCaMP6s. Single sweeps (grey) and averages of 5 sweeps (black) are overlaid. Directions of grating motion (8 directions) are shown above traces (arrows). c, Example traces from three neurons expressing GCaMP6f. Single sweeps (grey) and averages of 5 sweeps (cyan) are overlaid. d, Left, high magnification view of fluorescence changes corresponding to the red boxes in b (black) and c (cyan), normalized to the peak of the response. Right, Fourier spectra normalized to the response amplitude at 0 Hz for neurons driven with 1Hz drifting gratings, transduced with GCaMP5G, OGB1-AM, 6f or 6s. e, The fraction of cells scored as responding to visual stimulation when loaded with different calcium indicators. Error bars correspond to s.e.m. (n = 70, 39, 23, 38, 21 and 34 fields of view (FOVs) for GCaMP3, 5G, OGB1-AM, 6f, 6m and 6s, respectively). GCaMP3, 5G, and OGB1-AM data are from ref. 16. f, The distribution of fluorescence changes across cells at the preferred orientation.

  3. Combined imaging and electrophysiology in the visual cortex.
    Figure 3: Combined imaging and electrophysiology in the visual cortex.

    a, Simultaneous fluorescence dynamics and spikes in a GCaMP6s (top) and a GCaMP6f (bottom) expressing neuron. The number of spikes for each burst is indicated below the trace (single spikes are indicated by asterisks). Left inset, a GCaMP6s-expressing neuron with the recording pipette indicated schematically. b, Zoomed-in view of bursts of action potentials. Top, GCaMP6s; bottom, GCaMP6f. c, Fluorescence change in response to one action potential. Top, GCaMP6s; bottom, GCaMP6f. d, Median fluorescence change in response to one action potential for different calcium indicators. Shading corresponds to s.e.m., n = 9 (GCaMP5K, data from ref. 16), 11 (GCaMP6f), 10 (GCaMP6m), 9 (GCaMP6s) cells. GCaMP5K and GCaMP5G have similar properties16. e, Peak fluorescence change as a function of number of action potentials in a 250ms bin (5K: n = 161, 65, 22, 4 events for 1, 2, 3, 4 action potentials; 6f: n = 366, 120, 50, 15, 7 events for 1, 2, 3, 4, 5 action potentials; 6m: n = 354, 105, 31, 11, 7 events for 1, 2, 3, 4, 5 action potential; 6s: n = 250, 60, 20, 5, 4 events for 1, 2, 3, 4, 5 action potentials). Error bars correspond to s.e.m. f, Comparison of GCaMP indicators. Left, fraction of isolated spikes detected at 1% false positive rate. Middle, half decay time. Right, rise time to peak. Error bars correspond to s.e.m.

  4. Imaging activity in dendritic spines in the visual cortex.
    Figure 4: Imaging activity in dendritic spines in the visual cortex.

    a, Image of an L2/3 dendritic branch expressing GCaMP6s. Regions of interest (ROIs) are indicated as dashed circles (red, spines; yellow, dendrites). b, Map of fluorescence change (ΔF = FresponseFbaseline) in response to drifting gratings of 8 different orientations. c, Pixel-based map of orientation preference. d, Responses of dendritic spines (s1–s3) and neighbouring dendritic shafts (d1–d3) to drifting gratings with different orientations (corresponding to ROIs indicated in a). e, Orientation tuning of individual spines (s1, s2, s3). Error bars correspond to s.e.m. (n = 5 trials). f, Fraction of spines that show detectable calcium transients (active) and respond to visual stimulation (responsive) (see Methods for definitions) (228 spines; 15 dendrites; 4 mice). g, Distribution of the orientation selectivity index across visually responsive spines (62 spines). h, Baseline fluorescence across individual dendritic spines over 320s of continuous imaging (228 spines; 15 dendrites; 4 mice; error bars reflect s.e.m. across spines). i, Left, the same GCaMP6s labelled spine imaged over weeks. Right, fluorescence responses to oriented drifting gratings. Insets, parent soma of imaged spines. j, Orientation selectivity of single spines measured over time (same as i). k, Top, preferred orientation for spines that responded in two imaging sessions separated by one week. Opposing stimulus directions are considered as equivalent in this analysis. Bottom, the distribution of ΔOrientation (difference in preferred orientation between two sessions).

  5. The orientation preference of populations of dendritic spines predicts the orientation preference of their parent neuron.
    Figure 5: The orientation preference of populations of dendritic spines predicts the orientation preference of their parent neuron.

    a, Somatic fluorescence responses of a GCaMP6s-expressing layer 2/3 pyramidal neuron (depth, 120µm) to oriented drifting gratings (top) and the corresponding tuning curve (bottom, normalized). b, Reconstruction of the dendritic arbor (red dendrites, dendrites shown in d; dashed squares, additional imaged regions). c, Top, fluorescence responses of visually responsive spines (84/298) sorted by their preferred orientation (averaged over 5 trials). Each row shows one spine normalized to its peak. Bottom, summed ΔF/F0 across all spines (without normalization). d, Locations of orientation selective spines on a subset of imaged dendrites (corresponding to red dendrites in b). The size of the circle corresponds to the averaged ΔF/F0 at the preferred stimulus, the colour indicates the preferred orientation, and the saturation of the colour encodes the orientation selectivity index (OSI = 1, saturated colour; OSI = 0, white). e, Top, tuning curve of somatic ΔF/F0. Bottom, summed spine ΔF/F0. Cell 1 corresponds to panels ad. f, Averaged output tuning (black) and integral spine response (grey) across the 5 neurons (same cells as in e). The turning curves were aligned to the preferred orientation of the output response (0 degrees). The average was normalized. g, The distribution of preferred orientation of dendritic spines (5 cells; number of spines sampled: 298, 166, 137, 278, 116). h, Fraction of visually responsive spines preferring orientations 0, 45 or 90 degrees away from the postsynaptic cell’s preferred orientation. Opposing stimulus directions are considered as equivalent in this analysis. Error bars correspond to s.e.m.

  6. Orientation-tuned domains in dendrites of GABAergic interneurons.
    Figure 6: Orientation-tuned domains in dendrites of GABAergic interneurons.

    a, A GCaMP6s-expressing interneuron (soma depth, 250µm), identified post hoc as a parvalbumin-positive interneuron. b, Somatic fluorescence changes to oriented drifting grating (same cell as in a). Bottom, polar plot. c, Reconstruction of the dendritic arbor based on GCaMP6s fluorescence. d, Left, a dendrite of the cell (red in c) was imaged along its entire length. Coloured squares indicate dendritic sites showing significant orientation tuning (P<0.01, ANOVA across 8 stimulus directions). The col our of each square indicates the local preferred orientation, and the saturation of the colour encodes the orientation selectivity index (OSI = 1, saturated colour; OSI = 0, white). Right, example dendritic fluorescence changes and the corresponding polar plots for four locations with distinct orientation preferences. Scale bars, 10s; 50% ΔF/F0. e, Zoomed-in view of the dendritic calcium signal corresponding to the box in d. The signal shows modulation at the frequency of the drifting grating (1Hz).

Videos

  1. Simultaneous recording of spikes and GCaMP6s fluorescence signal of a L2/3 neuron in the visual cortex in vivo
    Video 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. Calcium imaging of dendritic spines of GCaMP6s-expressing V1 neurons in response to drifting grating stimulation
    Video 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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Author information

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

    • Trevor J. Wardill

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 financial interests

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

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Supplementary information

Video

  1. Video 1: Simultaneous recording of spikes and GCaMP6s fluorescence signal of a L2/3 neuron in the visual cortex in vivo (12,818 KB, Download)
    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. Video 2: Calcium imaging of dendritic spines of GCaMP6s-expressing V1 neurons in response to drifting grating stimulation (19,513 KB, Download)
    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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