Population synchrony during novel exploration.

(A) Schematic of the behavioral setup for voltage imaging. Mice explored a novel one-way track while fluorescence images were captured at the CA1 pyramidal layer. Left: side view; right: top view of the setup.

(B) Example time-averaged image of CA1 pyramidal cells expressing Voltron2-ST labeled with JF552-HaloTag ligand. Scale bar, 100 ìm.

(C) Fluorescence traces of cells in an example session with identified synchronous ensembles (labeled with gray vertical bars). Right: an example of magnified fluorescence traces within the bracket, pinpointing the occurrence of the identified synchronous ensemble. Spikes are indicated by dots on the top.

(D) The grand average cross-correlogram (CCG) averaged across all 71 cells. The gray line represents the mean grand average CCG between reference cells and randomly selected cells from different sessions.

(E) Distribution of the grand average CCG peak lags of all cells (n=71).

(F) Distribution of the full width at half maximum (FWHM) of the grand average CCGs (n=71 cells).

(G) Pairwise comparison of the event rates of population synchrony between original and jittered data. Bar heights indicate group means. **p<0.01

(H) Histogram of the ensemble sizes as percentages of cells participating in the synchronous ensembles.

Comparison of synchronous ensembles between immobility and locomotion.

(A) Example traces of cells in a session with identified synchronous ensembles (gray rectangles) during periods of immobility (red rectangular frame) and locomotion (blue rectangular frame). Spikes are indicated by dots at the top of individual traces. Top: the simultaneously recorded LFP trace. Bottom: simultaneously recorded speed of the animal. Inset: the band-pass filtered LFP trace at the theta frequency range (black line) overlaid on the raw LFP trace (gray line).

(B) The grand average cross-correlograms (CCGs) during immobility (red) and locomotion (blue) averaged across all cells (n=71). The gray lines represent the mean grand average CCGs calculated from jittered data.

(C) Pairwise comparison of peak widths (FWHM) in the grand average CCGs between immobility (red) and locomotion (blue). Vertical bars represent means and standard deviations for both groups (FWHM: 27±12 ms for immobility, 18±9 ms for locomotion, mean±s.d., p<0.001, paired Student’s t-test, n=71 cells). ***p<0.001

(D) Pairwise comparison of event rates of population synchrony during immobility and locomotion. Bar heights indicate group means (immobility: 1.4±0.7 Hz, locomotion: 0.6±0.5 Hz, mean±s.d., n=10 segments, p=0.002, Wilcoxon signed-rank test). **p<0.01

(E) Boxplot of ensemble sizes for synchronous ensembles occurring during immobility (red) and locomotion (blue) (median ensemble size: 47% for immobility, n=446 events, 32% for locomotion, n=313 events, p<0.001, Wilcoxon rank-sum test). ***p<0.001

Synchronous ensembles occur outside ripple episodes.

(A) Example traces with ripples and synchronous ensembles. Top: simultaneously recorded LFP displaying an episode of ripple oscillation. A red vertical line indicates the timing of the peak ripple power in the LFP trace. A yellow rectangle marks the duration of the ripple episode. Inset at the top: a magnified view of the ripple oscillation. Middle: fluorescence traces of 9 CA1PCs. Gray rectangles mark the timings of the synchronous ensembles on all traces. Bottom: animals’ speed recorded simultaneously with voltage imaging.

(B) LFP ripple power and spiking probability aligned to synchrony ensembles and ripple events. Upper panel: color-coded LFP power at ripple frequency (120-240Hz) aligned to the timings of 64 synchronous ensembles (left) and 12 ripple events (right). Middle panel: Average LFP power of ripple frequency aligned to the timings of the synchronous ensembles (left) and the ripple events (right). Lower panel: Average spike counts of all cells aligned to the timings of the synchronous ensembles (left) and ripple events (right).

(C) Percentages of co-occurrences between population synchrony and ripples. The co-occurrences in the “sync to ripples” group represent the percentages of ripple episodes co-occurring with synchronous ensembles in the same episodes. The co-occurrences in the “ripples to sync” group indicate the percentages of synchronous ensembles co-occurring with ripple episodes. Each gray circle represents an individual session.

(D) Distribution of the ripple modulation indexes (n=41 cells). Gray bar: modulation indexes with significant p values (n=35 cells); white bars: modulation indexes without significance (n=6 cells).

Theta oscillations in the LFP and subthreshold membrane potential were linked to population synchrony.

(A) LFP waveforms triggered by timings of synchronous ensembles. Red: triggered by timings of immobility synchrony; blue: triggered by timings of locomotion synchrony.

(B) Power spectral densities of the triggered LFP waveforms. Red: immobility; blue: locomotion.

(C) Distributions of the differences between LFP theta phases of individual synchronous ensembles and their preferred phases of the session. Note that most ensembles show minor differences within ±30 degrees. Red: immobility; blue: locomotion.

(D) Fluorescence traces triggered by timings of synchronous ensembles. Traces with spikes are excluded. Left panel: traces triggered by timings of immobility synchrony. Right panel: traces triggered by timings of locomotion synchrony. Thin gray lines: mean triggered fluorescence traces averaged across triggers for representative cells. Thick red and blue lines: mean triggered fluorescence traces averaged across cells.

(E) Polar plot illustrating theta modulation of spikes participating in the immobility synchrony (red dots) and of spikes participating in the locomotion synchrony (blue dots). The angle of the dots indicates the preferred phase, and the distance from the center of the circle represents the modulation strength. Each dot represents the averages from a neuron.

(F) Pairwise cross-correlation of the subthreshold membrane voltages (subVm). Top: color-coded cross-correlation of subVm between cell pairs during immobility and locomotion periods. Bottom: averaged cross-correlation over all cell pairs for immobility (red) and locomotion (blue) subVm. Both show a central peak at zero lag flanked by theta oscillation.

(G) Scatter plots of theta coherence against soma distances of immobility subVm (red) and locomotion subVm (blue) between cell pairs.

Negative correlation between spatial tuning similarity and synchronization strength during novel exploration.

(A) Spatially binned rate maps of place cells. High firing rates are indicated in yellow, and low firing rates are noted in blue. Bottom: the mean firing rates along spatial bins averaged across laps.

(B) Spatial tunings of 10 place cells from an example session. Cells are sorted based on the locations of their peak firing rates.

(C) Two example pairs of neurons with cross correlograms during locomotion and spatially binned firing rate maps.

(D) A scatter plot of correlation coefficients of spatial tunings against synchronization strengths for place cell pairs. Each dot represents a pair of place cells. Synchronization strengths are negatively correlated with similarities of spatial tunings (Spearman correlation coefficient=-0.4, p<0.001, n=73 pairs).

Mean firing rate distribution and correlation with animals’ speed.

(A) Cumulative distribution of mean firing rates averaged throughout the entire 180-second recording period.

(B) Mean firing rates during locomotion plotted against mean firing rates during immobility for individual cells. Each dot represents a cell. Mean firing rates are higher during immobility than during locomotion (4.2±0.2 Hz for immobility, 2.8±0.2 Hz for locomotion, n=71 cells, p<0.001, Wilcoxon signed-rank test).

(C) Histogram of correlation coefficients between instantaneous firing rates and the animals’ speed for each neuron.

Pairwise cross-correlogram (CCG) between CA1 pyramidal cells.

(A) CCGs of two representative cell pairs exhibiting significant peaks compared to the jittered spike trains of the target cells (gray lines).

(B) Distribution of the CCG peaks (n=497 pairs). Black: CCG peaks with significance (p<0.05). White: CCG peaks without significance (p>=0.05).

(C) Histogram of peak lags of significant CCG peaks (n=315 pairs).

(D) Histogram of FWHMs of significant CCG peaks (n=315 pairs).

LFP ripples recorded in the first and subsequent sessions in a novel maze.

(A) An example LFP trace with ripples recorded in a subsequent session following the first session of novel exploration, where no ripple was detected.

(B) Ripple rates in the first and subsequent sessions recorded in the same animals (n=5 mice) during novel exploration.

Theta modulation of spikes during immobility and locomotion periods of novel exploration.

(A) Polar plot comparing theta modulation between spikes participating in synchronous ensembles (sync spikes) and spikes not participating in synchronous ensembles (other spikes) during immobility. Each dot represents the averaged modulation of a cell. The modulation strengths of spikes participating in synchrony are significantly higher than those of spikes not participating in synchrony (modulation strength for spikes participating in synchronous ensembles: 0.81±0.08, for spikes not participating in synchronous ensembles: 0.63±0.12, mean±s.d., n=71 cells, p<0.001, pair Student’s t-test).

(B) Same as in (A), but during locomotion (modulation strength for spikes participating in synchronous ensembles: 0.76±0.18, for spikes not participating in synchronous ensembles: 0.60± 0.12, mean±s.d., n=71 cells, p<0.001, pair Student’s t-test).

(C) Average fluorescence triggered by cells’ spikes participating in synchronous ensembles (red, sync spikes) and not participating in synchronous ensembles during immobility (black, other spikes; n=59 cells). Inset: Pairwise comparison of average fluorescence 50±5 ms before time of spikes (t=0) between the two triggered groups (n=59 cells).

(D) Same as in (C), but during locomotion.

Synchronization strengths during immobility show little correlation with the similarity of spatial tunings between place cells.

Synchronization strengths during immobility plotted against correlation coefficients of spatial tunings for pairs of place cells. Synchronization strengths show little correlation with similarities of spatial tunings (Spearman correlation coefficient=-0.003, p=0.98, n=88 cell pairs).