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High-speed recording of neural spikes in awake mice and flies with a fluorescent voltage sensor

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Science  11 Dec 2015:
Vol. 350, Issue 6266, pp. 1361-1366
DOI: 10.1126/science.aab0810
  • Fig. 1 Ace FRET-opsin sensors report membrane voltage with ~1-ms response times.

    (A) Linker sequences bridging Ace1Q and Ace2N to mNeonGreen. Endoplasmic reticulum (ER) export sequence and Golgi export trafficking signal (TS) at the construct’s C terminus improve the sensor’s membrane localization and hence the signaling dynamic range. (B) Fluorescence signals from neurons expressing Ace1Q-mNeon or Ace2N-mNeon. (Left) Baseline fluorescence emissions from mNeonGreen. (Right) Spatial maps of the fluorescence response (ΔF/F) to a voltage step of approximately 100 mV. Areas of fluorescence and voltage response were generally colocalized. Scale bar, 20 μm. Illumination intensity, 15 mW ⋅ mm−2. (C) Step responses of the Ace sensors, ASAP1 and MacQ-mCitrine to +100 mV command voltage steps, normalized to each sensor’s maximum (or steady state) ΔF/F response to the command voltage. The initial rise of the Ace2N-mNeon sensor was more than six times as fast as that of ASAP1 and MacQ-mCitrine (table S2). (Inset) The full step response of all sensors. The trace for Ace2N-mNeon exhibits hysteresis ~40 to 200 ms after the voltage step, outside the interval shown in the main plot. Illumination intensity, 15 to 50 mW ⋅ mm−2. Data acquisition rate, 5 kHz. Inset traces were down-sampled to 250 Hz. (D) Steady-state responses of FRET-opsin sensors as a function of membrane voltage in cultured neurons (N = 10 cells per trace). Because Ace2N-mNeon exhibited hysteric responses to voltage steps (fig. S2), we also plot its peak initial responses. Illumination intensity, 15 mW ⋅ mm−2. Error bars, SEM.

  • Fig. 2 Ace sensors provide about threefold to tenfold better spike detection fidelity (d′) than previous GEVIs.

    (A) Fluorescence signals from cultured neurons expressing Ace2N-mNeon (blue trace) had sharp peaks closely matching action potentials in concurrent electrophysiological recordings (black trace). Red arrows mark a ~5 mV depolarization apparent in both traces. (B) Optical and electrical waveforms of single action potentials in example neurons expressing Ace2N-mNeon in cell culture (top), Ace2N-mNeon in mouse brain slice (middle), and Ace2N-4AA-mNeon in anesthetized mouse brain (bottom). Data (gray points) acquired optically from different spikes were temporally aligned to the corresponding peaks in the electrical traces. Mean waveforms of the optical data (blue traces; averaged over N = 30 spikes; resampled to 2 kHz) are aligned with the mean electrical waveforms (black traces; whole-cell patch recordings for cells in culture and brain slice; loose patch recording for live mouse). (C) Peak ΔF/F responses to action potentials, as a function of the total number of photons detected per spike in cultured neurons. Isocontours (dashed lines) of spike-detection fidelity, d′, were determined from measured brightness and optical waveforms, as in (B). Error bars, SEM. (D) Concurrent optical (colored) and juxtacellular electrical (black) recordings in an anesthetized mouse from V1 cells expressing Ace2N-4AA-mNeon. Magenta trace (lower right) shows the two magenta spikes in adjacent trace. Across 837 spikes, the electrical and optical traces were in perfect accord. (Inset) Two different neocortical neurons imaged by two-photon microscopy. A maximum projection of a dual-color image stack (top) acquired in a live mouse shows an Ace2N-4AA-mNeon–labeled cell and a pipette, filled with red dye, that recorded somatic electrical activity. An image acquired in a brain slice (bottom) after in vivo experimentation shows the membrane localization of Ace2N-4AA-mNeon. Scale bars, 40 μm. (E) Histogram of timing errors for spikes detected optically as in (D), using the electrical trace to provide the actual spike time (N = 837 spikes from three cells). Red line, Gaussian fit. Error bars, SD, estimated as counting errors. Illumination: 15, 25, and 25 mW ⋅ mm−2, respectively, for studies in culture, brain slice, and live mice. Image acquisition rates: 440 Hz, 440 Hz, and 1000 Hz, respectively.

  • Fig. 3 Imaging single action potentials and subthreshold membrane voltage fluctuations in layer 2/3 visual cortical neurons of awake mice.

    (A) Optical voltage trace acquired in an awake mouse, showing spiking by a V1 layer 2/3 neuron expressing Ace2N-4AA-mNeon via the SAD-Ace2N-4AA-mNeon-RGECO1 virus. Dashed boxes indicate intervals shown at successively expanded time scales. (Insets) Image acquired in vivo of the cell that provided the optical trace and a pair of fluorescence images taken in a fixed tissue slice of an identically labeled V1 neuron from the same preparation expressing Ace2N-4AA-mNeon (green) and RGECO1 (red). Scale bar, 40 μm. (B) Periods of reduced spiking arose when Ace2N-4AA-mNeon reported a hyperpolarization. To track each cell’s membrane voltage apart from its spikes, we applied a median filter (50-ms window) to the trace. For nonparametric comparisons, we matched each spike to the voltage at which the spike occurred, quantified as a percentile of the cell’s full range of membrane voltages over the full recording. For each cell, the plot shows the spike rate (normalized to each cell’s peak rate) at voltages across all percentiles. (C) (Top) Spike-triggered average image frames, for the cell in (A), showing the mean dendritic activation before and after firing an action potential. Times are relative to the spike peak at the soma. The left two dendrites activated before the right two dendrites. We calculated mean time traces for the soma and the left and right dendrite pairs using the spatial masks shown. (Bottom) Mean fluorescence time courses (ΔF/F) for each of the three masks, normalized to the same maximum. The traces confirm the left-to-right activation pattern; the right dendrites exhibit a voltage peak 0.25 to 0.5 ms after the left dendrites. The ΔF/F image series and time traces were sampled in 0.25-ms time bins, using data from 1900 spikes mutually aligned to their peaks. Shaded regions on the time traces denote SEM and are barely discernible. (D) Example optical traces from a cortical V1→LM neuron in an awake mouse, showing visually evoked responses to moving gratings (orientations and motion directions marked above each trace). Spiking responses to the cell’s preferred grating orientation (second column) differed from the activity suppression in response to the orthogonal orientation (fourth column). (E) Mean spike rates of the cell in (D), determined from the optical voltage trace in response to gratings moving in different orientations (10 trials per stimulus). Solid black and dashed gray circles indicate spike rates of 4 s−1 and 2 s−1, respectively. Ca2+ imaging using RGECO1 in the same cell yielded similar orientation tuning. Evoked Ca2+ signals were integrated over 1 s and normalized so that Ca2+ and spiking responses to the preferred orientation (225°) were plotted at the same radius on the polar plot. Shaded areas indicate SEM. (F) Mean ± SEM spike rates in response to gratings at the preferred orientation were higher than to those oriented orthogonally (P < 0.01 for each of seven cells; permutation test; 105 permutations). Frame acquisition rate: 1 kHz for voltage imaging and 20 Hz for Ca2+ imaging. Illumination intensity: 20 and 10 mW ⋅ mm−2, respectively, for voltage and Ca2+ imaging. Labeled neurons were ~150 μm below the brain surface.

  • Fig. 4 Odor-evoked spiking, dendritic dynamics, and voltage propagation delays in fly olfactory neurons.

    (A) (Top) Fluorescence images of a local neuron (left) and a projection neuron (right) expressing Ace2N-2AA-mNeon in the fly antenna lobe. (Bottom) Spatial maps of the fluorescence response (ΔF/F) at the peak of an odor-evoked transient. Scale bar, 40 μm. (B) (Top) Optical voltage traces (blue traces) from the local neuron in (A) reveal increased spiking during 5% benzaldehyde odor presentation (purple bar). (Bottom) Odor-evoked spike rate as a function of time, averaged over five trials (shaded area denotes SEM). (Inset) Mean peak spike rate during odor presentation was higher than baseline rates without odor [P < 10−3; Wilcoxon signed-rank test; N = 8 neurons and 8 flies (20 × UAS-Ace2N-2AA-mNeon/+; R55D11-GAL4/+)]. (C) (Left) Concurrent optical voltage and whole-cell patch electrical recordings in whole brain explants, from the same cell type as in (B). Spikes were evoked by current injection. (Middle) Paired optical and electrical traces, taken from periods enclosed by dashed boxes in the left panel. Spikes are clearly distinguished in the optical traces from both the plateau of subthreshold depolarization and each rise to spike threshold. (Right) Histogram of timing errors for the spikes detected optically, relative to the spike times in the whole-cell patch recordings (N = 18,141 spikes from 4 flies). Red line is a Gaussian fit. Standard deviation of the timing errors was 0.19 ± 0.002 ms. Error bars estimating the SD as counting errors are too small to be seen. (D) (Left) Spike-triggered average image frames for the cell in (C), showing the mean activation in the neural processes before and after firing a somatic spike, as determined from spike times in the electrical recording. Depolarization started at about –1.0 ms in the soma (located in the upper right of each image) and propagated right to left across the dendritic tree during a spike. We calculated mean time traces for the soma and two subportions of the dendrites using the spatial masks in the lower right panel (cyan line encloses the soma; red and gold lines enclose dendritic regions). Scale bar, 40 μm. (Right) ΔF/F for each of the three spatial masks, normalized to the same maximum. The traces confirm the right-to-left activation pattern; the left dendrites exhibited a voltage peak 0.5 to 0.7 ms after the right dendrites. Mean ΔF/F image series and traces were sampled in 0.25-ms bins, using data from 1300 spikes, temporally aligned to the spike peaks. Shaded regions denote SEM and are only discernible for the soma. (E) Odor-evoked, optical voltage traces from the DL3 projection neuron dendrites of (A), with significant rises in activity over baseline for some [10% ethyl acetate (EA) and 3-octanol (1O3O); P < 0.04 for both EA and 1O3O), but not other odors (10% benzaldehyde (BEN) and isopropanol (IPA); P = 1 and 0.4 for BEN and IPA, respectively; Wilcoxon signed rank tests; N = 6 flies (20 × UAS-Ace2N-2AA-mNeon/+; R26B04-GAL4/+)]. (F) Odor-evoked dendritic activation levels, relative to baseline levels. (G) (Top) Example fly expressing Ace2N-2AA-mNeon in DL3 cells in which we concurrently imaged dendrites and axons. (Bottom) Optical traces from both regions show concurrent activity. (Inset) Averaged traces aligned to onset of dendritic activity reveal a ~4-ms propagation delay between dendrites and axons (N = 30 transients; shaded regions denote SEM). Scale bar, 50 μm. Illumination, 20 mW ⋅ mm−2. Frame acquisition rate, 1 kHz. For display only, each optical trace in (B) was high-pass filtered by subtracting a median-filtered (50-ms window) version of the trace. Traces in (E) were processed the same way for display, but with a 1-s filter window.

Supplementary Materials

  • High-speed recording of neural spikes in awake mice and flies with a fluorescent voltage sensor

    Yiyang Gong, Cheng Huang, Jin Zhong Li, Benjamin F. Grewe, Yanping Zhang, Stephan Eismann, Mark J. Schnitzer

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S9
    • Tables S1 and S2
    • References

    Images, Video, and Other Other Media

    Movie S1
    Action potential dynamics in a visual cortical neuron of an awake mouse. A movie of the spike-triggered average fluorescence response (ΔF/F) before, during and after firing of an action potential, for the layer 2/3 visual cortical neuron of Fig. 3C as imaged using Ace2N-4AA-mNeonGreen in an awake mouse. The movie depicts a time interval of 6.5 ms and represents an average over 1,900 spikes, mutually aligned to the times of action potential peaks at the soma. The left two dendrites activate before the right two dendrites. The time increment between successive image frames is 0.25 ms, and the playback speed is 10 frames · s-1. Spatial scale bar: 40 μm.
    Movie S2
    Action potential dynamics of a neuron in the antenna lobe of an intact fly brain. A movie of the spike-triggered average fluorescence response (ΔF/F) before, during and after firing of an action potential, for the antenna lobe neuron of Fig. 4C, D as imaged using Ace2N- 2AA-mNeonGreen in an intact fly brain explant. The movie depicts a time interval of 9.25 ms and represents an average over 1,300 spikes, mutually aligned to the times of action potential peaks as recorded electrically at the soma (Fig. 4C). Depolarization started at about –1.0 ms in the soma (located in upper right of each image) and propagated right to left across the dendritic tree during a spike. The time increment between successive image frames is 0.25 ms. The playback speed is 10 frames · s-1. Spatial scale bar: 40 μm.

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