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Voltage imaging and optogenetics reveal behaviour-dependent changes in hippocampal dynamics


A technology that simultaneously records membrane potential from multiple neurons in behaving animals will have a transformative effect on neuroscience research1,2. Genetically encoded voltage indicators are a promising tool for these purposes; however, these have so far been limited to single-cell recordings with a marginal signal-to-noise ratio in vivo3,4,5. Here we developed improved near-infrared voltage indicators, high-speed microscopes and targeted gene expression schemes that enabled simultaneous in vivo recordings of supra- and subthreshold voltage dynamics in multiple neurons in the hippocampus of behaving mice. The reporters revealed subcellular details of back-propagating action potentials and correlations in subthreshold voltage between multiple cells. In combination with stimulation using optogenetics, the reporters revealed changes in neuronal excitability that were dependent on the behavioural state, reflecting the interplay of excitatory and inhibitory synaptic inputs. These tools open the possibility for detailed explorations of network dynamics in the context of behaviour.

a, Schematic of the paQuasAr3 construct. b, Photoactivation by blue light enhanced voltage signals excited by red light in cultured neurons that expressed paQuasAr3 (representative example of n = 4 cells). c, Model of the photocycle of paQuasAr3. d, Confocal images of sparsely expressed paQuasAr3 in brain slices. Scale bars, 50 μm. Representative images, experiments were repeated in n = 3 mice. e, Simultaneous fluorescence and patch-clamp recordings from a neuron expressing paQuasAr3 in acute brain slice. Top, magnification of boxed regions. Schematic shows brain slice, patch pipette and microscope objective. f, Simultaneous fluorescence and patch-clamp recordings of inhibitory post synaptic potentials in an L2–3 neuron induced by electrical stimulation of L5–6 in acute slice. g, Normalized change in fluorescence (ΔF/F) and SNR of optically recorded post-synaptic potentials (PSPs) as a function of the amplitude of the post-synaptic potentials. The voltage sensitivity was ΔF/F = 40 ± 1.7% per 100 mV. The SNR was 0.93 ± 0.07 per 1 mV in a 1-kHz bandwidth (n = 42 post-synaptic potentials from 5 cells, data are mean ± s.d.). Schematic shows brain slice, patch pipette, field stimulation electrodes and microscope objective. h, Optical measurements of paQuasAr3 fluorescence in the CA1 region of the hippocampus (top) and glomerular layer of the olfactory bulb (bottom) of anaesthetized mice (representative traces from n = 7 CA1 cells and n = 13 olfactory bulb cells, n = 3 mice). Schematics show microscope objective and the imaged brain region. i, STA fluorescence from 88 spikes in a CA1 oriens neuron. j, Frames from the STA video showing the delay in the back-propagating action potential in the dendrites relative to the soma. k, Sub-Nyquist fitting of the action potential delay and width shows electrical compartmentalization in the dendrites. Experiments in km were repeated in n = 2 cells from n = 2 mice.

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Data availability

The datasets generated during the current study are available from the corresponding author upon reasonable request.

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Custom data analysis codes are available from the corresponding author upon reasonable request.

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We thank M. S. Lee and V. Joshi for help with tissue culture, A. H. Gheorghe for help with spectroscopy experiments, N. Rollins and S. Brownsberger for technical help, L. Yapp for advice on hippocampal surgeries, A. Ruangkittisakul and K. Ballanyi for neuronal cultures, K. Podgorski for heating simulation code, G. Buzsaki and members of the Cohen laboratory for helpful discussions and D. Hochbaum, C. Straub, J. L. Saulnier, B. L. Sabatini, V. Kapoor and V. Murthy for help at early stages of the project. Y.A. was supported by fellowships from the Human Frontiers Science Program and the Edmund and Lili Safra Center for Brain Sciences. This work was supported by the Howard Hughes Medical Institute.

Reviewer information

Nature thanks Kenneth Harris, Michael Hausser, Samuel Andrew Hires and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Y.A. performed the patch-clamp and imaging experiments in acute slices, cultured neurons and in vivo. J.J.K. performed patch-clamp measurements in HEK293T cells. Y.A. and Y.Z. performed protein engineering, with supervision by A.E.C. and R.E.C., respectively. Y.A. and D.B. performed spectroscopy experiments. Y.A. developed the imaging system with help from H.W., J.J.K., S.K. and V.P. S.L., L.M. and H.Z. developed and characterized the Ai155 Optopatch3 mice. M.A.M.-R. performed in utero electroporation, supervised by P.A. M.E.X. optimized and validated the penalized matrix decomposition–non-negative matrix factorization algorithm in collaboration with E.K.B., I.K., D.Z and L.P. S.C. and C.D.H. shared unpublished reagents for soma targeting of opsins. K.J.W. helped with molecular biology. B.G. performed the heating simulation. S.L.F. designed the CheRiff–HA construct. Y.A. and A.E.C. designed the project, analysed data and wrote the manuscript. A.E.C. supervised all aspects of the project.

Competing interests

A.E.C. is a founder of Q-State Biosciences. The other authors declare no competing interests.

Correspondence to Adam E. Cohen.

Extended data figures and tables

  1. Extended Data Fig. 1 Development and characterization of QuasAr3.

    a, Screening pipeline. Rationally designed constructs were cloned in the Optopatch configuration, expressed in primary neurons and tested for spike SNR using light-induced spikes. Constructs with improved SNR were then expressed in vivo using in utero electroporation and tested for spike SNR in acute slices. The process was repeated iteratively. b, Examples of SNR measurements in cultured neurons. Left, wide-field epifluorescence images of GFP (green) fused to CheRiff, an opsin with excellent membrane trafficking. Middle, fluorescence of QuasAr (red) mutants. Scale bar, 10 μm. Right, QuasAr fluorescence transients in response to optogenetically induced spikes (10 ms blue-light stimulation at 1 mW mm−2). Each construct was tested on at least five cultured neurons. c, Hierarchical screen for improved membrane trafficking of QuasAr variants (see Methods for details). Diagram, schematic of the FCK_DuEx1.0 construct and overview of the screening pipeline. E. coli colonies were transformed with libraries in FCK_DuEx1.0. The colonies with the brightest fluorescence were picked for lentivirus production and secondary screening in primary neuronal culture. Images, example images of the fluorescent protein (FP) channel of QuasAr2–FP fusions: (i) mOrange; (ii) mRuby2; (iii) mKate2; (iv) citrine. Scale bar, 10 μm. d, SNR of N-terminal modifications compared with QuasAr2. All constructs showed reduced SNR (see Methods for details). e, Replacing mOrange2 with Citrine as a fusion protein improved the trafficking only with two specific linkers. f, Adding additional TS sequences at the linker and C-terminal improved the spike SNR. g, The mutation K171R increased the expression level of QuasAr, quantified by normalizing QuasAr fluorescence by the fluorescence of the co-expressed CheRiff–GFP. dg, Data are mean ± s.e.m., one-tailed Student’s t-test. h, Top, diagram of the QuasAr2 and QuasAr3 constructs. Bottom, confocal images of brain slices expressing QuasAr2 and QuasAr3. Scale bar, 500 μm. Insets, single-cell bodies; scale bar, 10 μm. Representative images from n = 2 mice (QuasAr2) and n = 3 mice (QuasAr3). i, Confocal images of brain slices expressing Cre-dependent QuasAr3 with sparsity controlled by co-expression of hSyn-Cre. Scale bar, 50 μm. j, Simultaneous fluorescence and patch-clamp recordings from two neurons expressing QuasAr3 using AAV virus in acute brain slices. Left, image of QuasAr3 fluorescence in the soma. Scale bar, 10 μm. Middle, spiking during ramp current injection. Right, mean spike, overlay of fluorescence and voltage. Inset, boxed regions showing correspondence of optical and electrical recordings of subthreshold voltage overlaid. See Extended Data Fig. 3 for statistics.

  2. Extended Data Fig. 2 Mapping neuronal excitability in Cre-on Optopatch3 transgenic mice (line Ai155).

    a, Construct design for a Cre-dependent Optopatch3 transgenic mouse. b, Representative traces for all-optical electrophysiology recordings in acute brain slices from Optopatch3 transgenic mice crossed with different Cre driver lines. Scale bar, 10 μm. c, Confocal images showing citrine fluorescence from QuasAr3-citrine, in offspring of crosses between Optopatch3 mice and different Cre driver mice. Acute brain slices were prepared from mice aged 14 to 17 days and imaged in the cortex. Scale bar, 50 μm. d, Composite bright-field image of a coronal brain slice from an Rbp4-cre+/−;Optopatch3+/− transgenic mouse, with locations of optical recordings marked with white spots. e, Spike raster showing 94 cells recorded sequentially from a single Rbp4-cre+/−;Optopatch3+/− acute brain slice. f, Optogenetic stimulus intensity-dependent firing rates in acute slices with different Cre drivers. Left, slices that are homozygous for Optopatch3. Right, slices that are heterozygous for Optopatch3. g, Mean firing rate (F), during a 500-ms stimulus as a function of stimulus intensity (I) calculated from the data shown in f. Data are shown as mean ± s.e.m. In the mice with Optopatch3 expression driven by CamKII-Cre, the FI curve for the Optopatch3+/+ mice is compressed along the x axis relative to the Optopatch3+/− mice, which indicates a stronger optogenetic drive for a given optical stimulation strength in the mice homozygous for CheRiff. The decrease in firing rate at a strong stimulus in these mice is a signature of depolarization block. Data are from 128 cells from 5 slices from 2 SST-cre+/−;Optopatch3+/− mice; 25 cells from 1 slice from 1 CamKII-cre+/−;Optopatch3+/− mouse; 152 cells from 6 slices from 4 Rbp4-cre+/−;Optopatch3+/− mice; 89 cells from 2 slices from 2 mice for CamKII-cre+/−;Optopatch3+/− mice.

  3. Extended Data Fig. 3 Photophysical properties, kinetics and electrical properties of paQuasAr3.

    a, Photoactivation by blue light. Red lines, fluorescence of HEK293T cells that express paQuasAr3 during voltage steps under constant red illumination (10 W mm−2) and variable blue illumination (average of n = 8 cells). Grey lines, same experiment in HEK293T cells that express QuasAr3 (average of n = 7 cells). b, PaQuasAr3 fluorescence compared to blue-light intensity at two membrane voltages (n = 8 cells, mean ± s.e.m.). Photoactivation showed saturation behaviour, with 50% maximum enhancement at I488 nm = 27 mW mm−2. c, Voltage-dependent near-infrared fluorescence of paQuasAr3 and QuasAr3 with and without blue light (150 mW mm−2). All fluorescence values are normalized to fluorescence with red-only illumination at Vm = −75 mV. d, Same data as c but each fluorescence trace was normalized to its value F0 at Vm = −75 mV. Blue illumination enhanced the absolute fluorescence and the absolute voltage sensitivity, but the fractional voltage sensitivity (ΔF/F0) was the same between QuasAr3 and paQuasAr3 and was not affected by blue illumination (n = 7 cells in each condition, P = 0.91, one-way analysis of variance). e, Kinetics of QuasAr3 and paQuasAr3 measured in HEK293T cells. Cells were subjected to a square wave from −60 mV to +40 mV at 5 Hz (see a). Response transients were fitted to a double exponential. QuasAr3, n = 5 cells; paQuasAr3, n = 9 cells. Data are mean ± s.e.m. Red intensity, 10 W mm−2; blue intensity, 150 mW mm−2. f, Response of paQuasAr3 and QuasAr3 to steps of blue illumination. The blue-light enhancement arose with a 50 ± 14-ms time constant and subsided with a 167 ± 26-ms time constant (mean ± s.d.). Blue light activated paQuasAr3 was approximately twofold brighter than QuasAr3 (mean of n = 10 cells). g, Action spectrum for photosensitization, measured in E. coli expressing paQuasAr3. Fluorescence was excited at λexc = 640 nm and emission was collected from λem = 660–740 nm. The activation wavelength was scanned from λact = 450–650 nm. Peak activation was at λact = 470 nm. h, Fluorescence excitation spectra with and without blue sensitization (40 mW mm−2). The sensitized state of paQuasAr3 had a fluorescence excitation spectrum similar to QuasAr3, with peak excitation at λexc = 580 nm. i, SNR of single spikes in acute slices for QuasAr3 (n = 10 cells) and paQuasAr3 (n = 10 cells) with either red light only or red and blue illumination (mean ± s.d. paQuasAr3 with and without blue light, paired Student’s t-test, paQuasAr3 red and blue illumination versus QuasAr3 red light only, Student’s t-test). j, Voltage-clamp recordings in CA1 pyramidal cell expressing paQuasAr3-s showed no photocurrents in response to illumination with red light (640 nm, 12 W mm−2), blue light (488 nm, 90 mW mm−2) or a combination of the two lights, both when the cell was held at −70 mV and at 0 mV (repeated in n = 2 cells). k, Electrical properties measured by patch clamp in acute slices. QuasAr3 (n = 11 cells, 6 slices, 2 mice) and paQuasAr3 (n = 9 cells, 6 slices, 3 mice) were expressed in the visual cortex and compared with non-expressing cortical cells (n = 9 cells, 7 slices, 7 mice). paQuasAr3-s (n = 7 cells, 7 slices, 5 mice) was expressed in the CA1 pyramidal cell layer, and compared with non-expressing cells in that layer (n = 8 cells, 6 slices, 5 mice). Data are mean ± s.d.

  4. Extended Data Fig. 4 Patterned illumination improves brain imaging and minimizes brain heating.

    a, A single oriens interneuron was illuminated with red and blue light projected precisely onto the cell (1×) or with an oval mask of which the area was either twice (2×) or 10 times bigger (10×) than the cell. Scale bar, 20 μm. Illumination intensity was held constant across measurements. b, Spontaneous spiking activity of the oriens interneuron from a in an awake resting mouse. c, Mean spike SNR was similar with the 2× mask compared to the 1× mask (P = 0.2, two-sided paired Student’s t-test) and significantly reduced with the 10× mask (P = 0.001, two-sided paired Student’s t-test, n = 10 cells from n = 2 mice, data are mean ± s.e.m.). d, Cross-section of near-infrared fluorescence of oriens or PCL cells imaged in anaesthetized mice and illuminated with either wide-field red illumination, patterned red illumination or patterned red and patterned blue illumination. The lower SBR in the PCL is attributed to the greater density of expressing cells. All cells visible in the focal plane were targeted with illumination, leading to higher background in the PCL. In the oriens, blue illumination increased the signal but not background. In the PCL, blue illumination modestly increased background, a consequence of light scattering between neighbouring cells. SBR values were as follows. PCL: wide field, 0.27 ± 0.02; red only, 0.95 ± 0.063; red and blue, 1.31 ± 0.08; n = 64 cells. Oriens: wide field, 0.27 ± 0.05; red only, 2.9 ± 0.19; red and blue, 4.5 ± 0.27; n = 7 cells. Data are mean ± s.e.m. (shown as line and shading). These data were used to calculate the improvement in SBR in Fig. 2d. e, Left, spontaneous activity of a representative oriens neuron in an awake, resting mouse illuminated with the indicated laser intensity. Right, population average shows no change in the firing rate in the illumination range tested (corresponding to 12.5–34 mW into the tissue, n = 10 cells from 2 mice, data are mean ± s.d., P = 0.22, two-sided paired Student’s t-test, N.S, not significant). f, FOVs with single oriens interneurons were imaged while illuminating with increasing number of masks around the cell to simulate the multicellular imaging conditions used in the PCL. Left, typical illumination pattern with the indicated number of masks. Middle, representative traces from an oriens FOV imaged in an awake, resting mouse with the indicated number of masks. Right, mean spontaneous firing rate as a function of the total laser power. Firing rates were stable with illumination of up to 10 masks (80 mW). Projection of 15 masks (120 mW) caused a modest increase in the spontaneous firing rates (n = 10 cells from 2 mice, data are mean ± s.d., P = 0.05, two-sided paired Student’s t-test). Subsequent experiments were restricted to ten or fewer masks. g, Simulated spatial temperature profile in brain tissue with an imaging cannula and immersion water. Profiles are shown for no illumination, 100-mW 640-nm illumination and 100-mW 920-nm illumination, corresponding to a two-photon imaging experiment.

  5. Extended Data Fig. 5 Photostability of paQuasAr3-s in vivo during ‘all-optical’ excitability measurements.

    a, Fluorescence in the mouse hippocampus was recorded for 10-min in total. Oriens SST cells expressed paQuasAr3-s and CheRiff-s and were illuminated with red light at 12 W mm−2 and stimulated with blue light (up to 10 mW mm−2) using the stimulation protocol presented at the top, during quiet and walking periods. The trace shows the fluorescence from two user-defined regions of interest after subtraction of the background from a cell-free region. The baseline signal photobleached in both cells by around 50% during this interval. Top, total acquisition. Dashed lines denote separate videos. Bottom, magnified views of the indicated regions from the top graph. Similar recordings were performed in five FOVs. b, The same two cells were imaged three weeks later. While at the end of the initial 10-min recording the SNR was low due to photobleaching, 3 weeks later the signal had recovered. Images show wide-field epifluorescence images of the cells in the two imaging sessions. Scale bars, 20 μm. c, Spike SNR was on average stable in repeated recordings over three weeks (P = 0.34, two-sided paired Student’s t-test).

  6. Extended Data Fig. 6 Validation of the pipeline for signal extraction from dense PCL videos using PMD–NMF.

    a, Illustration of the segmentation pipeline: raw videos were first corrected for motion, followed by photobleaching correction, PMD denoising, manual removal of blood vessels and demixing using NMF. The pipeline produced waveforms that correspond to individual cell traces and to the background (see Methods for details). b, Testing the pipeline using simulated data composed of two cells partially overlapping in space, and with varying levels of correlation in their subthreshold voltages and with the background. Poisson-distributed shot noise was added to each pixel to mimic experimental noise. Left, image of the input video pixel-wise s.d. and the output cell footprints. Middle, input waveforms and the pipeline output waveforms. Top right, correlation matrix of input signals (C1, C2, cells 1 and 2; B, background). Bottom right, cross-correlation of output waveforms with input waveforms, data are mean ± s.d. for n = 5 simulations. c, Performance of the pipeline as a function of input parameters (n = 5 simulations per condition). Top, output-to-input correlation and output C1-to-C2 cross-correlation as a function of the pixel noise level. Noise is scaled to the spike amplitude, input C1-to-C2 cross-correlation is 0.5 and the input correlation with the background is also 0.5. Middle, output-to-input correlation and output C1-to-C2 cross-correlation as a function of the correlation between the input and the background, at two noise levels. Input C1-to-C2 correlation is 0.5. Bottom, output-to-input correlation and output C1-to-C2 cross-correlation as a function of the cross-correlation between input C1 and C2 at two noise levels. Input correlation with background is 0.5. (data are mean ± s.e.m.). d, Testing the pipeline with composite videos composed from real data. We imaged FOVs with single oriens neurons spontaneously spiking in awake, resting mice. Each cell was imaged in the focal plane, and then at 20-μm defocus. The two videos were first processed with the pipeline to extract the ground-truth input signals and then the videos were summed such that the focused and defocused cells were about 50% overlapping. We then ran the blended videos through the pipeline and compared outputs to the input traces using cross-correlation analysis. e, Mean cross-correlograms of five FOVs processed as described in d, showing that the segmentation pipeline accurately reproduced the correlational structure of the inputs even under these challenging conditions. Data are mean ± s.e.m. (line and shading). f, Validation of the image segmentation pipeline using patch-clamp recording as the ground-truth. Top left, FOV with dense expression of paQuasAr3-s in CA1 PCL in an acute brain slice. The FOV was imaged while the voltage in the blue outlined cell was recorded by manual patch clamp. Bottom left, two of the spatial footprints identified by the PMD–NMF pipeline. Middle, ground-truth voltage recording (black), flat average region of interest around the cell (blue) and two PMD–NMF units (magenta and red). The flat average region of interest trace showed fluctuations that were not present in the patch-clamp recording, presumably from an out-of-focus cell. These events were absent in the magenta PMD–NMF demixed trace. Right, magnification of the indicated trace inset. Arrow indicates the out-of-focus event. This experiment was performed once.

  7. Extended Data Fig. 7 Simultaneous optical recording from seven spiking cells in the PCL of an anaesthetized mouse.

    The mouse expressed paQuasAr3-s. a, Left, wide-field epifluorescence image of citrine fluorescence. Middle, same FOV with patterned blue illumination. Right, same FOV with patterned red illumination. Numbers indicate individual cells. b, Simultaneous fluorescence recordings from seven cells. Magnifications show synchronized complex spikes and action potentials that occur on top of subthreshold oscillations. This experiment was performed once.

  8. Extended Data Fig. 8 Chronic recordings of oriens neurons in hippocampus.

    a, Top left, wide-field epifluorescence and two-photon images showing two oriens interneurons. Scale bar, 50 μm. Middle, fluorescence recordings from the 2 cells during 3 consecutive repeats of a 65-s protocol. The protocol consisted of 10 s of rest, followed by 15-s epochs of walking at speeds of 5, 7.5 and 10 cm s−1, successively, followed by 10 s of rest. Inset (top right) shows the optical traces with clearly resolved spikes and subthreshold events. Bottom, average spike rate across three trials of the two cells (data are mean ± s.d.). b, Same two neurons and same protocol as in a, recorded seven days later. Trial 1 was only spontaneous activity without locomotion.

  9. Extended Data Fig. 9 Examples of brain-state-dependent intercellular correlations and STAs for single pairs of cells.

    ac, Left, magnified sections of recordings from trios of cells in the PCL (a, b) and oriens (c). Top, recordings during quiet. Bottom, recordings from the same cells during walking. Right, auto- and cross-correlations of the fluorescence traces, calculated from the complete 9-s recording in each brain region and behavioural state. The auto- and cross-correlations clearly show enhanced θ-rhythm in both brain regions during walking and differing cross-correlations between simultaneously recorded pairs of cells. d, e, Distribution of equal-time correlation coefficients between pairs of simultaneously recorded cells in the PCL (d) and oriens (e). f, Three examples of single-pair STA fluorescence during quiet (left) and walking (right) in the PCL (top) and oriens (bottom).

  10. Extended Data Fig. 10 Optopatch measurements of hippocampal SST neurons.

    a, Fluorescence waveforms of all 25 SST cells shown in Fig. 4. b, Cell-by-cell comparison of the change in θ-band (6.7 to 8.3 Hz) power between walking and quiet brain states. Left, without optogenetic stimulation. Middle, same cells with tonic optogenetic stimulation. Right, ratio of θ-band enhancement (walking versus quiet) with optogenetic stimulation versus without (cell-by-cell comparison for the data in Fig. 4h). c, Spike raster of 19 SST cells imaged at week 0 and week 3 and stimulated with the blue light protocol at the top (full dataset for the data in Fig. 4j–k).

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Fig. 1: Photoactivated QuasAr3 (paQuasAr3) reports neuronal activity in vivo.
Fig. 2: Optical recording of neuronal activity in hippocampus of walking mice.
Fig. 3: Behaviour-dependent intercellular correlations in the hippocampus.
Fig. 4: Simultaneous optogenetic stimulation and voltage imaging in hippocampal SST cells in walking mice.
Extended Data Fig. 1: Development and characterization of QuasAr3.
Extended Data Fig. 2: Mapping neuronal excitability in Cre-on Optopatch3 transgenic mice (line Ai155).
Extended Data Fig. 3: Photophysical properties, kinetics and electrical properties of paQuasAr3.
Extended Data Fig. 4: Patterned illumination improves brain imaging and minimizes brain heating.
Extended Data Fig. 5: Photostability of paQuasAr3-s in vivo during ‘all-optical’ excitability measurements.
Extended Data Fig. 6: Validation of the pipeline for signal extraction from dense PCL videos using PMD–NMF.
Extended Data Fig. 7: Simultaneous optical recording from seven spiking cells in the PCL of an anaesthetized mouse.
Extended Data Fig. 8: Chronic recordings of oriens neurons in hippocampus.
Extended Data Fig. 9: Examples of brain-state-dependent intercellular correlations and STAs for single pairs of cells.
Extended Data Fig. 10: Optopatch measurements of hippocampal SST neurons.


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