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. 2019 May;569(7756):413-417.
doi: 10.1038/s41586-019-1166-7. Epub 2019 May 1.

Voltage Imaging and Optogenetics Reveal Behaviour-Dependent Changes in Hippocampal Dynamics

Free PMC article

Voltage Imaging and Optogenetics Reveal Behaviour-Dependent Changes in Hippocampal Dynamics

Yoav Adam et al. Nature. .
Free PMC article


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 vivo3-5. 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. Fig. 1 PHOTOACTIVATED QUASAR3 (PAQUASAR3) REPORTS NEURONAL ACTIVITY IN VIVO.: 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 k-m were repeated in n = 2 cells from n = 2 mice.


Extended Data Fig 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 (IUE) 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 fused to CheRiff, an opsin with excellent membrane trafficking. Middle: Fluorescence of QuasAr mutants. Scale bar 10 μm. Right: QuasAr fluorescence transients in response to optogenetically induced spikes (10 ms blue light stimulation at 1 mW/mm2). Each construct was tested on at least 5 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 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 QuasAr expression level, quantified by normalizing QuasAr fluorescence by the fluorescence of the co-expressed CheRiff-GFP. (d-g) all error bars are mean ± s.e.m., 1-tail 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 slice. 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 sub-threshold voltage overlaid. See Extended Data Fig. 3 for statistics. Scale bar 10 μm.
Extended Data Fig. 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 ages 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 homozygous for Optopatch3. Right: slices heterozygous for Optopatch3. (g) Mean firing rate, during a 500 ms stimulus as a function of stimulus intensity, I, calculated from the data in (f). Error bars show mean ± s.e.m. In the mice with Optopatch3 expression driven by CamKII-Cre, the F vs I curve for the Optopatch3+/+ mice is compressed along the x-axis relative to the Optopatch3+/− mice, indicating a stronger optogenetic drive for a given optical stimulation strength in the mice homozygous for CheRiff. The decrease in firing rate at strong stimulus in these mice is a signature of depolarization block. Data are from 128 cells from 5 slices and 2 mice for SST-Cre+/−; Optopatch3+/+; 25 cells from 1 slice and 1 mouse for CamKII-Cre+/−; Optopatch3+/+; 152 cells from 6 slices and 4 mice for Rbp4-Cre+/−; Optopatch3+/−; 89 cells from 2 slices and 2 mice for CamKII-Cre+/−; Optopatch3+/−.
Extended Data Fig. 3.
Extended Data Fig. 3.. Photophysical properties, kinetics and electrical properties of photoactivated QuasAr3 (paQuasAr3).
(a) Photoactivation by blue light. Red lines: fluorescence of HEK cells expressing paQuasAr3 during voltage steps under constant red illumination (10 W/mm2) and variable blue illumination (average of n = 8 cells). Grey lines: same experiment in HEK cells expressing QuasAr3 (average of n = 7 cells). (b) PaQuasAr3 fluorescence vs. blue light intensity at two membrane voltages (n = 8 cells, mean ± s.e.m.). Photoactivation showed saturation behavior, with 50% maximum enhancement at I488 = 27 mW/mm2. (c) Voltage-dependent near infrared fluorescence of paQuasAr3 and QuasAr3 with and without blue light (150 mW/mm2). 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 ANOVA). (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 panel a). Response transients were fit to a double exponential. QuasAr3, n = 5 cells, paQuasAr3, n = 9 cells. All values are mean ± s.e.m. Red intensity, 10 W/mm2, blue intensity, 150 mW/mm2. (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 ~2-fold 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 nm – 740 nm. The activation wavelength was scanned from λact = 450 – 650 nm. Peak activation was at λact = 470 nm. (h) Fluorescence excitation spectra ± blue sensitization (40 mW/mm2). 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 only or red and blue illumination (mean ± s.d. paQuasAr3 ± blue, paired t-test, paQuasAr3 red and blue vs. QuasAr3 red only, 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/mm2), blue light (488 nm, 90 mW/mm2) or combination of the two, 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 CA1 pyramidal cell layer and compared with non-expressing cells in that layer (n = 8 cells, 6 slices, 5 mice). Error bars are mean ± s.d.
Extended Data Figure 4.
Extended Data Figure 4.. Patterned illumination improves brain imaging and minimizes brain heating.
(a) A single Oriens IN was illuminated with red and blue light projected precisely onto the cell (1x) or with an oval mask whose area was either twice (2x) or 10 times bigger (10x) than the cell (scale bar 20 μm). Illumination intensity was held constant across measurements. (b) Spontaneous spiking activity of the Oriens IN (a) in an awake resting mouse. (c) Mean spike SNR was similar with 2x mask compared with 1x mask (p = 0.2, two-sided paired t-test) and significantly reduced in the 10x mask (p = 0.001, two-sided paired t-test, n = 10 cells from N = 2 mice, error bars ± s.e.m.). (d) Cross section of NIR fluorescence of Oriens or PCL cells imaged in anesthetized mice and illuminated with either wide-field red illumination, patterned red illumination or patterned red and patterned blue illuminations. 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 signal but not background. In the PCL, blue illumination modestly increased background, a consequence of light scattering between neighboring cells. SBR values were: PCL: wide field: 0.27 ± 0.02, Red only: 0.95 ± 0.063, Red + Blue: 1.31 ± 0.08, n = 64 cells. Oriens: wide field: 0.27 ± 0.05, Red only: 2.9 ± 0.19, Red + Blue: 4.5 ± 0.27, n = 7 cells, mean ± s.e.m. Shading represents s.e.m. 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 to 34 mW into the tissue, n = 10 cells from 2 mice, error bars show mean ± s.d., p = 0.22, two-sided paired t-test). (f) FOVs with single Oriens INs were imaged while illuminating with increasing number of masks around the cell to simulate the multicell 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, error bars show mean ± s.d., p = 0.05, two-sided paired t-test). Subsequent experiments were restricted to 10 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.
Extended Data Fig 5.
Extended Data Fig 5.. Photostability of paQuasAr3-s in vivo during ‘all-optical’ excitability measurements.
(a) 10-minute recording of fluorescence in mouse hippocampus. Oriens SST cells expressed paQuasAr3-s and CheRiff-s and were illuminated with red light at 12 W/mm2 and stimulated with blue light (up to 10 mW/mm2) using the stimulation protocol presented at the top, during quiet and walking periods. The trace shows the fluorescence from two user-defined ROIs after subtraction of the background from a cell-free region. The baseline signal photobleached in both cells by ~50% during this interval. Top: total acquisition. Dashed lines denote separate movies. Bottom: Close-up views of the indicated regions from the top graph. Similar recordings were performed in 5 FOVs. (b) The same two cells were imaged 3 weeks later. While at the end of the initial 10-minute recording the SNR was low due to photobleaching, 3 weeks later the signal had recovered. Images show wide-field epi-fluorescence images of the cells in the two imaging sessions. Scale bar 20 μm. (c) Spike SNR was on average stable in repeated recordings over 3 weeks (p = 0.34, two-sided paired t-test).
Extended data Fig. 6.
Extended data Fig. 6.. Validation of the pipeline for signal extraction from dense PCL movies using penalized matrix decomposition denoising followed by non-negative matrix factorization demixing (PMD-NMF).
(a) Illustration of the segmentation pipeline: raw movies were first corrected for motion, followed by photobleaching correction, penalized matrix decomposition denoising, manual removal of blood vessels, and demixing using non-negative matrix factorization. The pipeline produced waveforms corresponding 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 sub-threshold voltages and with the background. Poisson-distributed shot noise was added to each pixel to mimic experimental noise. Left: image of the input movie pixel-wise s.d. and of the output cell footprints. Middle: input waveforms and the pipeline output waveforms. Right top: correlation matrix of input signals (cells C1, C2, and background B). Right bottom: cross-correlation of output waveforms with input waveforms, 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. (mean ± s.e.m.). (d) Testing the pipeline with composite movies 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 movies were first processed with the pipeline to extract the ground-truth input signals and then the movies were summed such that the focused and defocused cells were ~50% overlapping. We then ran the blended movies through the pipeline and compared outputs to the input traces using cross-correlation analysis. (e) Mean cross-correlograms of 5 FOVs processed as described in (d) showing that the segmentation pipeline accurately reproduced the correlational structure of the inputs even under these challenging conditions. Shading represents s.e.m. (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 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 ROI around the cell (blue), and two PMD-NMF units (magenta and red). The flat average ROI trace showed fluctuations 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: zoom-in on the indicated inset. This experiment was performed once.
Extended Data Fig. 7.
Extended Data Fig. 7.. Simultaneous optical recording from 7 spiking cells in the PCL of an anesthetized mouse.
The mouse expressed paQuasAr3-s. (a) Left: wide-field epifluorescence image of Citrine fluorescence. Middle: same field of view with patterned blue illumination. Right: same field of view with patterned red illumination. (b) Simultaneous fluorescence recordings from 7 cells. Close-ups show synchronized complex spikes and action potentials riding atop sub-threshold oscillations. This experiment was performed once.
Extended Data Fig. 8.
Extended Data Fig. 8.. Chronic recordings of Oriens neurons in hippocampus.
(a) Top left: wide-field epifluorescence and two-photon (2P) images showing two Oriens interneurons. Scale bar 50 μm. Middle: fluorescence recordings from the two cells during three consecutive repeats of a 65 s protocol. The protocol comprised of 10 s of rest, followed by 15 s epochs of walking at speeds of 5, 7.5, and 10 cm/s, successively, followed by 10 s of rest. Inset shows the optical traces with clearly resolved spikes and sub-threshold events. Bottom: average spike rate across three trials of the two cells (mean ± s.d.). (b) Same two neurons and same protocol as in (a), recorded 7 days later. Trial 1 was only spontaneous activity without locomotion.
Extended Data Fig. 9.
Extended Data Fig. 9.. Examples of brain state-dependent intercellular correlations and spike triggered averages for single pairs of cells (same analysis as in Fig. 3).
(a,b,c) Left: Magnified sections of recordings from trios of cells in (a,b) the PCL and (c) Oriens. 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 behavioral 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 spike-triggered average fluorescence during quiet (left) and walking (right) in the PCL (top) and Oriens (bottom).
Extended Data Fig. 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 vs. quiet) with optogenetic stimulation vs. 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).
Figure 1.
Figure 1.. Photo-activated QuasAr3 (paQuasAr3) reports neuronal activity in vivo.
(a) PaQuasAr3 construct. (b) Blue light photoactivation enhanced red light-excited voltage signals in cultured neurons expressing paQuasAr3 (representative example of 4 repeats). (c) Model of the photocycle of paQuasAr3. (d) Confocal images of sparsely expressed paQuasAr3 in brain slices. Scale bar 50 μm. Representative images, repeated in N = 3 mice. (e) Simultaneous fluorescence and patch clamp recordings from a neuron expressing paQuasAr3 in acute brain slice. Inset shows boxed regions. (f) Simultaneous fluorescence and patch clamp recordings of IPSPs in an L2/3 neuron induced via electrical stimulation of L5–6 in acute slice. (g) ΔF/F and SNR of optically recorded PSPs as a function of the PSP amplitude. The voltage sensitivity was ΔF/F = 40 ± 1.7%/100 mV. The SNR was 0.93 ± 0.07/mV in a 1 kHz bandwidth (n = 42 PSPs from 5 cells, mean ± s.d.). (h) Optical measurements of paQuasAr3 fluorescence in CA1 region of the hippocampus (top) and glomerular layer of the olfactory bulb (bottom) of anesthetized mice (representative traces from n = 7 CA1 cells and n = 13 OB cells, N = 3 mice). (i) Spike-triggered average fluorescence from 88 spikes in a CA1 Oriens neuron. (j) Frames from the spike-triggered average movie 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 show electrical compartmentalization in the dendrites. Experiment in k–m repeated in n = 2 cells from N = 2 mice.
Figure 2.
Figure 2.. Optical recording of neuronal activity in hippocampus of walking mice.
(a) Soma-localized paQuasAr3 (paQuasAr3-s). (b) Confocal images of brain slices. Broadly expressed paQuasAr3 filled the neuropil, preventing optical resolution of individual cells (left), while paQuasAr3-s resolved cell bodies (right). Insets: sparsely expressed constructs showing the difference in dendritic expression between paQuasAr3 and paQuasAr3-s. Scale bars 100 μm. Representative images, repeated in N = 2 mice per condition. (c) Optical system for simultaneous 2-photon imaging and patterned illumination with red and blue light. (d) Left, top: epifluorescence images with wide-field red illumination of paQuasAr3-s in the CA1 region of the hippocampus, in vivo. Middle: same fields of view with patterned red illumination. Bottom: Addition of patterned blue illumination increased the image contrast. Scale bar 50 μm. Right: Effect of patterned red and blue light on signal-to-background ratio (SBR) in the Oriens and PCL (PCL, n = 64 cells from N = 2 mice, Oriens, n = 7 cells from N = 2 mice, mean ± s.e.m). (e) Two-photon images of paQuasAr3-s expression in the Oriens (left) and the PCL (right). Scale bar 100 μm. (f) Fluorescence recordings from PCL (red) and Oriens (blue, N = 5 mice). Traces with similarly shaded backgrounds were acquired simultaneously. Right: Magnified views showing complex spikes, bursts, correlated activity between cells, and modulation of the spiking by subthreshold dynamics. (g–i) Effect of brain state (Quiet or Walking) on (g) firing rate (h) total power in the subthreshold oscillations, and (i) population-average power spectra in the PCL (red) and the Oriens (blue). (f–i) n = 48 cells in PCL, 36 cells in Oriens, N = 5 mice. g,h, mean ± s.d, paired t-test. i, Shading shows mean ± s.e.m.
Figure 3.
Figure 3.. Behavior-dependent intercellular correlations in the hippocampus.
(a,e) Left: Samples of recordings from a trio of cells in (a) the PCL and (e) Oriens. Top: during quiet. Bottom: during walking. Representative traces from 19 FOVs in the PCL and 20 FOVs in the Oriens. Right: auto- and cross-correlations of the fluorescence traces, calculated from 9 s recordings in each brain region and behavioral 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. (b,f) Grand average auto- and cross-correlations during quiet (top) and walking (bottom) in the PCL (n = 46 cells for autocorrelations, 43 pairs for cross correlations) and Oriens (n = 29 cells for autocorrelations, 19 pairs for cross correlations). Grey: Mean cross-correlation between randomly selected cells from different fields of view. (c,g) Spike-triggered grand average fluorescence during quiet (top) and walking (bottom) in the PCL (n = 86 pairs) and Oriens (n = 38 pairs). During walking, spikes in both layers occurred on average on the rising edge of the STA θ-rhythm, leading to a 22-degree phase shift in the PCL and 42-degree shift in the Oriens between the mean spike and the peak of the mean intracellular θ -rhythm. (d,h) Spike-triggered grand average spiking probability during quiet (top) and walking (bottom) in the PCL (d) (quiet: n = 4796 spikes, walking: n = 1983 spikes) and Oriens (h) (quiet: n = 2719 spikes, walking: n = 4258 spikes). Y axis is on a log scale.
Figure 4.
Figure 4.. Simultaneous optogenetic stimulation and voltage imaging in hippocampal SST cells in walking mice.
(a) Optopatch construct for viral Cre-dependent co-expression of CheRiff-s and paQuasAr3-s. (b) Top: Protocol for optogenetic stimulation during quiet and walking. Middle: fluorescence of a single SST Oriens cell showing optogenetically and behaviorally modulated firing. Bottom: Spike raster from n = 25 SST interneurons from 17 FOVs in N = 2 mice. (c) Optically recorded activity of a single Oriens interneuron during 500 ms steps of optogenetic stimulation from 0 to 10 mW/mm2. Left: Quiet. Right: Walking. (d) Spike rate as a function of optogenetic stimulus strength (F-I curve) during quiet and walking (mean ± s.e.m., n = 25 SST neurons in Oriens). (e) Spontanous spike rate (no optogenetic stimulation) during quiet and walking. (f) Cell-by-cell excitability during quiet vs. walking, measured as the slope of the F-I curve between 0 to 8 mW/mm2. (g) Subthreshold dynamics during quiet and walking, with and without optogenetic stimulation (from the dataset in b). Gray: raw fluorescence. Blue, black: subthreshold dynamics (spikes digitally removed, bandpass filtered 3–20 Hz). (h) Effect of brain state and optogenetic stimulation on population-average power spectra. (i) Grand-average spike waveforms showing increased afterhyperpolarization during optogenetic stimulation (arrow). (j) Two SST cells, recorded before and after a 3-week interval. The full dataset is presented in Extended Data Fig. 10. (k) Correlation of the firing rate and excitability (F-I slope) of n = 19 SST cells recorded before and after a 3-week interval. Population-average firing rates slightly increased after 3 weeks (Quiet: 3.2 ± 2.2 to 4.2 ± 2.5 Hz, p = 0.04. Walking 11.8 ± 5.9 to 16.6 ± 11.6 Hz, p = 0.03, mean ± s.d, paired t-test). The F-I slope was stable between sessions (quiet p = 0.82, walking p = 0.67, paired t-test).

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