Abstract

Genetically encoded voltage indicators (GEVIs) are emerging optical tools for acquiring brain-wide cell-type-specific functional data at unparalleled temporal resolution. To broaden the application of GEVIs in high-speed multispectral imaging, we used a high-throughput strategy to develop voltage-activated red neuronal activity monitor (VARNAM), a fusion of the fast Acetabularia opsin and the bright red fluorophore mRuby3. Imageable under the modest illumination intensities required by bright green probes (<50 mW mm−2), VARNAM is readily usable in vivo. VARNAM can be combined with blue-shifted optical tools to enable cell-type-specific all-optical electrophysiology and dual-color spike imaging in acute brain slices and live Drosophila. With enhanced sensitivity to subthreshold voltages, VARNAM resolves postsynaptic potentials in slices and cortical and hippocampal rhythms in freely behaving mice. Together, VARNAM lends a new hue to the optical toolbox, opening the door to high-speed in vivo multispectral functional imaging.

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Data availability

The DNA sequence for VARNAM is available in GenBank under submission number MH763646. CMV-VARNAM (#115552) and pAAV-Syn-VARNAM (#115554) will be available from Addgene once the paper is published. Fly stocks and viruses will be provided by the authors upon reasonable request.

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Acknowledgements

The authors thank J. Cardin (Yale University) for the PV-Cre mouse line. We thank X. Liu and D. Berman from the Pieribone laboratory for technical assistance with molecular biology, P. O’Brien for assistance with optical instrumentation for in vitro imaging and all other members for extended support during the study. We thank T. Kim from the Schnitzer laboratory for help with two-photon imaging of fly brains. We are grateful to M. Higley for valuable comments during the preparation of the manuscript. We thank the John B. Pierce Laboratory machine shop for technical support during development of the voltage screening platform. This study was supported by NIH grants U01 NS103517 and U01 NS090565 awarded to V.A.P.

Author information

Author notes

  1. These authors contributed equally: Madhuvanthi Kannan and Ganesh Vasan.

Affiliations

  1. The John B. Pierce Laboratory, New Haven, CT, USA

    • Madhuvanthi Kannan
    • , Ganesh Vasan
    •  & Vincent A. Pieribone
  2. Department of Cellular and Molecular Physiology, Yale University, New Haven, CT, USA

    • Madhuvanthi Kannan
    • , Ganesh Vasan
    •  & Vincent A. Pieribone
  3. Department of Neuroscience, Yale University, New Haven, CT, USA

    • Madhuvanthi Kannan
    • , Ganesh Vasan
    •  & Vincent A. Pieribone
  4. James H. Clark Center, Stanford University, Stanford, CA, USA

    • Cheng Huang
    • , Simon Haziza
    • , Jin Zhong Li
    • , Hakan Inan
    •  & Mark J. Schnitzer
  5. CNC Program, Stanford University, Stanford, CA, USA

    • Jin Zhong Li
    •  & Mark J. Schnitzer
  6. Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA

    • Mark J. Schnitzer

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Contributions

G.V. and V.A.P. conceived and built the hardware for the screening platform. G.V. wrote the software and database. M.K. and G.V. conceived the development of FlicR and VARNAM. M.K. performed molecular biology, mouse surgery and voltage-imaging in cultures and acute slices. G.V. and M.K. analyzed the data from cultures and slices. C.H. and J.Z.L. made transgenic flies. C.H. performed voltage-imaging in flies. H.I. wrote the algorithm for fly data analyses. J.Z.L. made viruses for TEMPO studies. S.H. performed TEMPO studies. M.K., G.V. and V.A.P. wrote the manuscript with input from S.H. (TEMPO), C.H. (fly imaging) and M.J.S. V.A.P. and M.J.S. oversaw the work.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Vincent A. Pieribone.

Integrated supplementary information

  1. Supplementary Figure 1 Voltage screening, data analysis and management on the high-throughput platform.

    (a) Schematic of optical and electrical setup. Each well of the sample plate is sequentially aligned along the light path and cells are stimulated by a single field electrode. A white light source is used for sample illumination and fluorescence emission is collected by a high-resolution camera. Ex and Em denote excitation and emission filters; DM, dichroic mirror. (b) Schematic of image acquisition. In each well, images (reporter channel) are captured in 16 fields of view (FOVs) in a 4 × 4 grid, and ranked by transfection density. Time-series images in the GEVI channel are acquired in the top four FOVs following field stimulation. (c) Fluorescence responses of ArcLight-transfected HEK cells to 0.5-ms electric field pulses of intensities 1, 5 and 10–80 V were recorded on the high-throughput platform. Values represent mean ± s.e.m., n = 24 wells. Responses steadily increased with the intensity of field stimulation at the lower voltages and plateaued between 50 and 60 V. 60 V was chosen as the standard stimulus intensity. (d) Software-controlled data acquisition, analysis and storage. Left, acquisition control panel written in LabView. The software allows the user to control microscope hardware, start, stop or resume acquisition and view the current FOV and screening progress. Center, data are extracted using a mask image (top) and time-series plots of fluorescence change are generated for all identified cells in a FOV (bottom). Red trace indicates the maximum fluorescence response. Right, the maximum responses from individual wells are entered into a FileMaker database, which also has information on the plate, libraries and transfection. All raw traces are stored and accessed via centrally located server.

  2. Supplementary Figure 2 Improving the voltage sensitivity of FlicR1 on the screening platform.

    (a) Alignment of amino acid sequence of the FlicR1 fluorophore cp-mApple with mApple sequence1 and the sequence of R-GECO FP2, which is a cp-mApple variant. (b) Alignment of amino acid sequences of cp-mApple in FlicR1 with the sequences of mApple, mRuby3 and RCaMP FP3, which is a cp-mRuby variant. Boxes indicate residues in the FP barrel which were targeted for saturation mutagenesis in FlicR1 based on sequence and/or structural homology with key residues that improved performance of R-GECO or RCaMP2,3. (b) Top, Schematic of the FlicR2 construct fused to a nuclear localization signal (NLS)-tagged blue fluorescent protein (BFP) reporter. FlicR2 and NLS-BFP were interspersed by a T2A peptide (scissor). Mutations in the FP that improved voltage sensitivity are shown. Bottom, crystal structures of CiVSD (PDB 4G80) and cp-mApple in R-GECO1 (PDB 4I2Y) in a fusion configuration. S1-4 indicate transmembrane helices of the VSD. (c) Epifluorescence image of HEK cells expressing FlicR2 (n = 12 wells, 3 cultures). (d) Fluorescence responses from a FlicR2-transfected HEK cell held at –70 mV to depolarizing and hyperpolarizing voltage steps (∆20 mV). (e) Fluorescence-voltage (∆F/∆V) plots for FlicR1 and FlicR2 in HEK cells (n = 4 and 6 cells, respectively). Values represent mean ± s.e.m. (f) Epifluorescence images of FlicR2-electroporated pyramidal neurons (left) and magnified images of a soma (right) in an acute brain slice (n = 8 neurons). 1. Shaner, N. C. et al. Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat. Methods 5, 545–551 (2008). 2. Zhao, Y. et al. An expanded palette of genetically encoded Ca2+ indicators. Science 333, 1888–1891 (2011). 3. Akerboom, J. et al. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front. Mol. Neurosci. 6, 2 (2013).

  3. Supplementary Figure 3 Membrane localization and voltage sensitivities of Ace-FP fusions.

    (a) Schematic of spectral overlap of the absorption of Ace and emission of indicated fluorophores4–7. (b) Epifluorescence images of HEK cells transfected with the Ace-FP fusion constructs. While Ace-SEP227D, Ace-mNeon and Ace-mRuby3 expressed brightly and showed excellent membrane localization, Ace-mCitrine and Ace-mCardinal2 exhibited dim fluorescence and large intracellular aggregates (n = 4 wells per condition, 500 cells per well). (c) ∆F/∆V curves for Ace-mCitrine, Ace-SEP227D and Ace-mCardinal2 in HEK cells acquired during concurrent optical and electrical recordings in voltage clamp (n = 6 cells for Ace-mCitrine and 4 cells each for Ace-SEP227D and Ace-mCardinal2). Values represent mean ± s.e.m. Illumination intensities were 15–20 mW mm–2 except for Ace-mCardinal2, which was imaged at 40 mW mm–2 at the specimen plane. 4. Bajar, B. T. et al. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. Sci. Rep. 6, 20889 (2016). 5. Chu, J. et al. Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein. Nat. Methods 11, 572–578 (2014). 6. Gong, Y. et al. High-speed recording of neural spikes in awake mice and flies with a fluorescent voltage sensor. Science 350, 1361–1366 (2015). 7. Gong, Y., Wagner, M. J., Zhong Li, J. & Schnitzer, M. J. Imaging neural spiking in brain tissue using FRET-opsin protein voltage sensors. Nat. Commun. 5, 3674 (2014).

  4. Supplementary Figure 4 In utero delivery of GEVI constructs and performance of Ace-mNeon and Ace-mRuby3 in acute, electroporated slices.

    (a) Schematic of the timeline for in utero electroporation and postnatal optical electrophysiology in acute slices. (b) Epifluorescence image of an Ace-mNeon-electroporated slice showing indicator expression in the visual cortex (n = 3 mice). (c) Left, 80 x 80 pixel CCD image of a pair of pyramidal neurons expressing Ace-mNeon in acute slice. Right, optical and electrical (black) responses from select regions of interest (color-coded) to depolarizing current injections delivered to the green soma via the recording pipette. Large optical signals were observed in the soma, corresponding to APs, while smaller amplitude signals were noted in the blue apical dendrite corresponding to backpropagating APs. Even smaller optical EPSPs were conspicuous in the neighboring yellow cell and its apical dendrite (n = 3 experiments). (d) Single-trial optical (red) and electrical (black) signals to APs elicited by somatic current injections at 10 Hz (left) and 50 Hz (right). (e) Optical and electrical responses to 200-ms somatic current injections of increasing amplitudes (starting at 100 pA, 5 cycles, ∆100 pA). (f) Optical recordings of spontaneous APs. Gray spikes are shown at an expanded time scale to the right. (g) Mean waveform (red) of the optical and electrical spikes (n = 43 spikes). Gray shading represents all spikes combined.

  5. Supplementary Figure 5 Platform-based screening of linker mutations in Ace-mRuby3.

    (a) Schematic of deletional and insertional mutations in the Ace-mRuby3 linker. Residues in the linker and N-terminus of the FP (unstructured region) were sequentially deleted to bring mRuby3 closer to Ace. Single, double or triple AA insertions were achieved by introducing one, two or three NNK codons, respectively. Of all 288 variants screened on the platform, only the double AA insertions showed improvements in voltage sensitivity. (b) Screening results obtained on the platform for the double insertion library (96 variants). Fluorescence responses in individual wells are indicated. Responses are normalized to control (Ace-mRuby3, wells A12 and H12). Red and blue wells represent variants that performed better or worse than control, respectively. Some of the top performing wells were sequenced. The best performing well F7 contained a tryptophan/arginine insertion. 7 of 17 sequenced wells (41%) had at least one arginine insertion.

  6. Supplementary Figure 6 High-throughput voltage screening of Ace-mRuby3 RBP mutants.

    (a) Alignment of amino acid sequences of Ace, bacteriorhodopsin (BR) and Gloeobacter rhodopsin (GR). Boxes indicate conserved residues constituting the RBP, mutations of which have been shown to modulate the opsin’s absorption spectrum in BR and GR8–-10. The corresponding sites in Ace were targeted for saturation mutagenesis in Ace-mRuby3 and again in Ace-WR-mRuby3. (b) Screening results on the platform for the Ace-WR-mRuby3 N81X library, which was part of a 96-well plate that also contained the Ace-WR-mRuby3 T85X library. Fluorescence responses in individual wells are indicated. Responses are normalized to control (Ace-mRuby3, wells A12 and H12). Red and blue wells represent variants that performed better or worse than control, respectively. The wells indicate individual mutations. N81S, which was represented seven times in the plate, was on average twice as sensitive as control in quadruplicate screens. Contrary to N81X, T85X was mostly detrimental to voltage sensitivity. 8. Engqvist, M. K. et al. Directed evolution of Gloeobacter violaceus rhodopsin spectral properties. J. Mol. Biol. 427, 205–220 (2015). 9. Greenhalgh, D. A., Farrens, D. L., Subramaniam, S. & Khorana, H. G. Hydrophobic amino acids in the retinal-binding pocket of bacteriorhodopsin. J. Biol. Chem. 268, 20305–20311 (1993). 10. Russell, T. S., Coleman, M., Rath, P., Nilsson, A. & Rothschild, K. J. Threonine-89 participates in the active site of bacteriorhodopsin: evidence for a role in color regulation and Schiff base proton transfer. Biochemistry 36, 7490–7497 (1997).

  7. Supplementary Figure 7 Photobleaching characteristics of VARNAM.

    (a) Photobleaching curves of VARNAM, Ace-mNeon and Ace-mRuby3 in HEK cells under continuous illumination (n = 9, 7 and 9 cells, respectively). Imaging conditions: 565-nm LED (VARNAM and Ace-mRuby3) and 505-nm LED (Ace-mNeon); 15 mW mm–2. Photobleaching time constants were 256 s for VARNAM, 304 s for Ace-mRuby3 and 142 s for Ace-mNeon. (b) SNR at photobleaching rates in cultured cells under continuous illumination as above. Optical responses to ∆120 mV/5 Hz voltage steps were acquired for 1 s after every 19 s and averaged across multiple cells. The noise was determined as the s.d. of baseline fluorescence during a 100-ms interval preceding the first spike in every time bin. Values represent mean ± s.e.m. n = 6 cells per condition.

  8. Supplementary Figure 8 Passive membrane properties and intrinsic excitabilities of control and indicator-expressing neurons.

    (a) AP FWHM and amplitude in GFP and VARNAM-transfected primary neurons. P = 0.47 and P = 0.35, respectively, two-tailed Mann–Whitney U test; n = 4 neurons (control), 6 neurons (VARNAM). (b) FWHM and amplitudes of spike waveforms from neurons electroporated with indicated constructs and control cells in acute brain slices. P > 0.99, Kruskal–Wallis test with Dunn’s multiple comparisons correction; n = 4 neurons per condition (control, Ace-mNeon and VARNAM+CheRiff-EGFP), 7 neurons (VARNAM) and 8 neurons per condition (Ace-mRuby3 and VARNAM+Ace-mNeon). (c) Input resistance, (d) membrane capacitance and (e) holding current (VH = –65 mV) values of neurons electroporated with indicated constructs and control cells in acute slices. P > 0.99, Kruskal–Wallis test with Dunn’s multiple comparisons correction; n = 4 neurons per condition (control, Ace-mNeon and VARNAM+CheRiff-EGFP), 7 neurons (VARNAM) and 8 neurons per condition (Ace-mRuby3 and VARNAM+Ace-mNeon). (f) Mean ± s.e.m. spike rate during a 300-ms epoch plotted as a function of the amplitude of current injection for control, VARNAM and Ace-mNeon-electroporated neurons. (g) Rheobase values of control, VARNAM and Ace-mNeon-neurons in (f). P > 0.99, Kruskal–Wallis test with Dunn’s multiple comparisons correction; n = 7, 5 and 6 neurons for control, VARNAM and Ace-mNeon conditions, respectively. (a-e), (g), Values represent mean ± s.e.m.

  9. Supplementary Figure 9 VARNAM expression in electroporated neurons and optical recordings in acute slices.

    (a) High-resolution confocal images of VARNAM-expressing superficial layer pyramidal neurons in fixed brain slice. Vivid membrane localization with minimal intracellular aggregation was noted. (b) Concurrent optical (red) and electrical (black) recordings of high-speed AP bursts in a VARNAM-expressing neuron in acute brain slice. Bursts were triggered by current injections of 300-ms duration at increasing amplitudes (starting at 200 pA, 5 cycles, ∆100 pA). Dashed lines indicate trial shown at an expanded time scale below, overlaid with the optical trace. (c) Concurrent optical and electrical recordings of spontaneous APs in a VARNAM-expressing neuron in acute brain slice. Optical APs were observed in the soma (orange) and proximal apical dendrite (pink). Dashed box indicates time frame expanded to the right. Arrows indicate afterhyperpolarizations recapitulated in the optical trace.

  10. Supplementary Figure 10 VARNAM-positive neurons exhibit negligible steady-state photocurrents.

    (a) Left, representative electrical recordings of photocurrents in VARNAM-positive neurons during a 1-s illumination with wavelengths of 565, 505 and 450 nm in acute slice. Right, mean peak and steady-state photocurrents at the different wavelengths. Error bars represent s.e.m. Neurons exhibited peak photocurrents of 107.4 ± 15.5, 53.24 ± 10.73 and 16.41 ± 4.3 pA and steady-state currents of 1.86 ± 0.9, 1.24 ± 0.14 and 2.4 ± 1.0 pA under 565-nm, 505-nm and 450-nm light, respectively. n = 6, 5 and 5 neurons, respectively. The latter was computed by subtracting the average current across the last 10 ms of illumination from the mean current preceding light onset. (b) To compute the refractory period of the transient photocurrent, we modulated the frequencies of 50-ms-long 561-nm light pulses, as indicated. The currents began to disappear at interpulse intervals of 2 ms and were undetected at higher frequencies. This refractory period is well above the laser modulation frequencies used in TEMPO recordings (>5 kHz). (c) To determine the reversal potential of the current, we voltage-clamped HEK cells and measured the peak photocurrent during 1 s of illumination with 561-nm light at indicated VH. The current did not dissipate in the physiological range of depolarizing potentials but exhibited progressively smaller amplitudes. n = 5 cells. Values represent mean ± s.e.m.

  11. Supplementary Figure 11 Optical cross-talk in all-optical experiments with VARNAM in acute slices.

    (a) Overlay of the action spectrum of CheRiff and excitation spectrum of mRuby3. Blue and green lines denote wavelengths used for excitation of CheRiff and VARNAM, respectively. (b) Burst firing in CheRiff-expressing neurons in acute slice during 300 ms of blue light stimulation (455-nm LED, 2.4 mW mm–2). (c) Blue-light artifact in VARNAM-positive but CheRiff-negative neurons during 0.5-ms light pulses (455-nm LED, 2.4 mW mm–2). Gray shaded box denotes interval shown at an expanded time scale. Note the polarity, kinetics and size of the transient, which are distinct from voltage signals. (d) Representative electrical recordings in voltage clamp (top) and current clamp (bottom) showing depolarization induced in CheRiff-electroporated neurons during 561-nm illumination. Yellow lines indicate illuminated time intervals. (e) 561-nm light generated an average inward current of 35.21 ± 6.4 pA (left) resulting in an average voltage depolarization of 6.4 ± 0.9 mV (right). n = 5 neurons from 2 mice. (f) AP bursts induced in CheRiff-positive neurons by 300-ms current injections of increasing amplitudes (starting at 100 pA, 5 cycles, ∆50 pA) in the absence (top) and presence (bottom) of 1-s illumination with 561-nm light. (g) Average spike rate in the control and illuminated conditions plotted as a function of the amplitude of current injection (n = 6 neurons from 3 mice). (h) Optical cross-talk in cell-type-specific all-optical recordings in PV-Cre+ mice. Left, representative eIPSPs induced by blue light pulses in the absence (top) and presence (bottom) of 561-nm light. Vertical blue lines indicate incidence of 455-nm light (2 mW mm–2). Right, quantifications of the amplitude of the first eIPSP (top) and paired-pulse ratios (PPR) (bottom) during optogenetic activation in the absence and presence of 561-nm light. n = 14 trials from the same cell, epochs without and with 561-nm illumination were interleaved, P = 0.8 and 0.6, respectively, two-sided Wilcoxon test. (e), (g), (h), Values represent mean ± s.e.m.

  12. Supplementary Figure 12 Dual-color voltage imaging in acute slices.

    (a-c) Left, 80 x 80 pixel image of a neuron in slice electroporated with (a) VARNAM, (b) Ace-mNeon and (c) VARNAM and Ace-mNeon imaged in the red and green channels. Recording pipette is shown in yellow. (a-b) Right, optical (colored) and electrical (black) recordings of 10-Hz current-induced APs, sequentially recorded in the two channels. (c) Optical (colored) and electrical (black) recordings of 10-Hz (center) or 50-Hz (right) current-induced APs, sequentially recorded in the two channels. Illumination: 561-nm laser (VARNAM), 488-nm laser (Ace-mNeon), 15 mW mm–2.

  13. Supplementary Figure 13 VARNAM reports oscillatory dynamics in cortical pyramidal neurons in anesthetized mice.

    (a) Optical sketch of the TEMPO apparatus for VARNAM recordings. Two single-mode fiber pigtailed lasers emitting at 488 nm and 561 nm were used for the green reference channel (YFP) and the red voltage channel (VARNAM), respectively. Photodiodes monitored the fluctuations in the beams’ intensities. Only the 561-nm laser showed prominent fluctuations that impeded the red voltage channel recording. This signal was systematically extracted to unmix the red signal. The beams were combined and focused onto a polymer-clad multi-mode optical fiber, which delivered light to the brain via implanted fiber. Fluorescence was collected via the same fiber, split into green and red emissions, and detected by transimpedance photoreceivers. (FP: fiber pigtailed; PD: photo detector; tiPR: transimpedance photoreceiver; DM: dichroic mirror; BPF: band-pass filter; BS: beam splitter; AL: astigmatic lens). (b) Normalized integrated signal in the indicated frequency bands in VARNAM and Ace-mNeon recordings. The hemodynamic frequency band (11-14 Hz) showed significantly reduced normalized integrated signal in VARNAM recordings compared to Ace-mNeon, suggesting that VARNAM may provide a better SNR in the biologically-relevant theta band (13.5 ± 1% (VARNAM) versus 18 ± 0.5% (Ace-mNeon), mean ± s.e.m., *P = 0.034, two-tailed t-test, n = 3 mice per condition). The integrated power was comparable between the indicators in the delta (1-4 Hz), theta (6-10 Hz) and gamma bands (25-50 Hz). (c-d) Left, unfiltered time traces of TEMPO and (c) LFP or (d) EEG during anesthesia, showing closely matched oscillations. Right, normalized power spectral density. Note the prominent delta rhythm in both cases (0.5 Hz-4 Hz). (e) To assess the coherence of TEMPO and LFP (right) or EEG (left) oscillations, we computed the cross-correlation in 10-s increments across a 20-min recording just after KX injection. Consistent slow wave synchrony appeared a few tens of seconds after KX injection and reached steady-state in <5 min. (f) Average cross-correlation coefficient in the delta band.

  14. Supplementary Figure 14 VARNAM reports state-dependent voltage oscillations in a freely behaving mouse.

    (a-b) Time traces of TEMPO and LFP during (a) locomotion and (b) rest. Traces showed low-matched oscillations during both states in the opposite band (delta and theta band, respectively). (c-d) Left, time traces of TEMPO and EEG during (c) locomotion and (d) rest showing raw, delta-band-filtered and theta-band-filtered traces. Right, normalized power spectral density. Note the prominent theta rhythm (6 Hz-10 Hz) during locomotion and delta rhythm (0.5 Hz-4 Hz) at rest. (e, g) Cross-correlation plot between TEMPO and EEG filtered in the (e) theta and (g) delta bands. Two-state behavior is evident with high coherence in the theta band during locomotion (beginning of recording) and delta band at rest (end of recording). (f, h) Average cross-correlation coefficient in the (f) theta and (h) delta bands during locomotion and rest, showing opposite frequency band content.

  15. Supplementary Figure 15 Imaging spontaneous and odor-evoked spiking in live Drosophila.

    (a) Optical voltage trace acquired from MB058B-GAL4>20xUAS-Ace2N-2AA-mNeon fly, showing spontaneous spiking in a PPL1-α’2α2 neuron over 10 s (left), and a 0.3-s interval shown at expanded time scale (right). Yellow dashes indicate spike detection threshold. Green arrows denote identified spikes. (b) Mean spike waveform (blue trace) from recordings in (a) (n = 172 spikes). Blue shading denotes s.d. (c) Top, example trace of evoked spiking in PPL1-α’2α2 during 5-s IAA presentation. Bottom, raster plots of 18 trials of odor-evoked spiking (n = 6 flies). (d) Time-varying mean spike rates computed from recordings in (c). Red shading denotes s.d. (e) Mean peak spike rate before and during odor presentation. Error bars represent s.e.m. *P = 0.03, two-sided Wilcoxon signed-rank test, n = 6 flies. (f) Two-photon image showing axonal region and soma of a PPL1-α’2α2 neuron in a MB058B-GAL4>20xUAS-VARNAM fly (left). Red dotted box denotes region magnified (right), showing VARNAM localization on the membrane. (g) Matched-filtering algorithm used in voltage imaging analysis in Fig. 6 (c), (e) and (i). Optical voltage trace showing spontaneous spiking in PPL1-α’2α2 over 20 s (left), and a 1-s interval shown at expanded time scale (right). Top to bottom, representative unfiltered, median-filtered and matched-filtered traces. (h-m) Imaging spontaneous spiking in three neuron types using VARNAM (n = 6 flies per condition). (a), (c) and (e) Spatial map of ΔF/F during spiking in (a) the axonal region of PPL1-γ2α’1 in a MB296B-GAL4>20xUAS-VARNAM fly, (c) the dendritic tree of MBON-γ1pedc in a MB085C-GAL4>20xUAS-VARNAM fly, and (e) the dendritic tree of olfactory LN in a R55D11-GAL4>20xUAS-VARNAM fly. (b), (d) and (f) Optical voltage traces of spontaneous spiking in (a), (c) and (e), respectively, over 10 s (left), and a 0.3-s interval shown at expanded time scale (right). Yellow dashed line indicates spike detection threshold. Green arrows denote identified spikes.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–15, Supplementary Table 1 and Supplementary Notes 1 and 2

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https://doi.org/10.1038/s41592-018-0188-7