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Sharp wave ripples during learning stabilize the hippocampal spatial map

Nature Neuroscience volume 20, pages 845–853 (2017)Cite this article

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Abstract

Cognitive representation of the environment requires a stable hippocampal map, but the mechanisms maintaining a given map are unknown. Because sharp wave-ripples (SPW-R) orchestrate both retrospective and prospective spatial information, we hypothesized that disrupting neuronal activity during SPW-Rs affects spatial representation. Mice learned new sets of three goal locations daily in a multiwell maze. We used closed-loop SPW-R detection at goal locations to trigger optogenetic silencing of a subset of CA1 pyramidal neurons. Control place cells (nonsilenced or silenced outside SPW-Rs) largely maintained the location of their place fields after learning and showed increased spatial information content. In contrast, the place fields of SPW-R-silenced place cells remapped, and their spatial information remained unaltered. SPW-R silencing did not impact the firing rates or proportions of place cells. These results suggest that interference with SPW-R-associated activity during learning prevents stabilization and refinement of hippocampal maps.

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Figure 1: Daily spatial learning of hidden reward locations on the cheeseboard maze.
Figure 2: Closed-loop focal optogenetic silencing of pyramidal cells contingent upon SPW-R detection at goal locations.
Figure 3: Silencing pyramidal neurons during SPW-Rs impairs place map stability of place cells.
Figure 4: SPW-R silencing impact information measures of place cells.
Figure 5: Silencing neurons during SPW-Rs impairs place map stability of place cell ensembles.
Figure 6: SPW-R Silenced ensembles of place cells show destabilized spatial representation as compared to simultaneously recorded Control ensembles.

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Acknowledgements

We thank N. Chenouard, G. Girardeau, L. Sjulson, A. Peyrache and all members of the lab for invaluable discussions, advice and comments on the manuscript. This work was supported by NIH grants MH107396, MH54671, U01NS090583, the Simons Foundation, and the G. Harold and Leila Y. Mathers Foundation. L.R. was supported by the NIH grant K99NS094735 and the Bettencourt Schueller Foundation. E.S. was supported by the Rothschild Foundation, Human Frontiers in Science Program LT-000346/2009-l, Machiah Foundation 20090098 and ERC-2015-StG 679253. B.H. was supported by the National Natural Science Foundation of China (grant no. 31471050).

Author information

Author notes

  1. Bo Hu & Ronny Eichler

    Present address: Present addresses: Third Military Medical University, College of Basic Medical Sciences, Department of Physiology, Chongqing, China (B.H.) and Radboud University Nijmegen, Donders Centre for Neuroscience, Departments of Neuroinformatics and Neurophysiology, Nijmegen, Netherlands (R.E.).,

Authors and Affiliations

  1. New York University Neuroscience Institute, New York University, New York, New York, USA

    Lisa Roux, Bo Hu, Ronny Eichler, Eran Stark & György Buzsáki

  2. Department of Physiology and Pharmacology, Tel Aviv University, Sackler Faculty of Medicine and Sagol School of Neuroscience, Tel Aviv, Israel

    Eran Stark

  3. Department of Neurology, Medical Center, New York University, New York, New York, USA

    György Buzsáki

  4. Center for Neural Science, New York University, New York, New York, USA

    György Buzsáki

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Contributions

L.R. and G.B. designed the experiments and wrote the manuscript; L.R. performed the experiments and analyzed the data; B.H. performed experiments; R.E. provided the software for online tracking of mouse position; and E.S. provided assistance for data analysis.

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Correspondence to György Buzsáki.

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Integrated supplementary information

Supplementary Figure 1 Information on transgenic mouse models and their recording and optogenetic equipment.

(a) In four mice, cre-dependent expression of the hyperpolarizing opsin Arch in pyramidal cells was achieved via the expression of Cre recombinase under the CamKIIalpha promoter. In one mouse, cre-dependent expression of the depolarizing opsin ChR2 (Ai32) in inhibitory PV positive cells was achieved via the expression of Cre recombinase under the PV promoter. Four or 8-shank silicon probes equipped with laser diode (LD) coupled optical fibers were implanted uni- or bilaterally (right and/or left hemisphere) (Stark, E., Koos, T. & Buzsaki, G. Diode probes for spatiotemporal optical control of multiple neurons in freely moving animals. J Neurophysiol 108, 349-363, 2012). (b-c) Illustration of the tracks of the silicon probe shanks and opsin distribution in a CAMKII-cre::Arch mouse (mouse #5). Left: DAPI staining of the hippocampus (gray) with lesion sites in CA1 pyramidal layer (red arrows). Right: DAPI staining (blue) with superimposed EYFP (reporter for Arch expression; green). (c) is a higher magnification of (b) highlighting the CA1 implantation site. Scale bars: 400 (b) and 200μm (c). (d) Left: Distribution of EYFP in CA1 of a CAMKII-cre::Arch-EYFP mouse. The fusion protein Arch-EYFP is targeted to the cell membrane in the mouse line. Middle and right panels: the anti-PV immunostaining (red) shows no overlap with EYFP. Scale bar: 30μm. (e) High magnification image of EYFP expression and PV immunoreactivity in CA1 of a CAMKII-cre::Arch-EYFP mouse. Scale bar: 15μm. (f) Distribution of EYFP in CA1 of a PV-cre::ChR2-EYFP mouse. Middle and right panels: the anti-PV immunostaining (red) shows striking overlap with EYFP. Scale bar: 100μm. (g) High magnification image of EYFP expression and PV immunoreactivity in CA1 of a PV-cre::ChR2-EYFP mouse. Scale bar: 15μm.

Supplementary Figure 2 Results for the four CamKII-cre::Arch mice without the PV-cre::ChR2 mouse.

(a) Cumulative distributions of the correlation coefficients, calculated by comparing firing rate maps in Pre and Post exploration epochs, obtained for individual place cells in the three groups. Kruskall Wallis test: *P = 0.01; Tukey’s post-hoc tests: *P = 0.038 (Control vs Silenced), *P = 0.028 (Delayed vs Silenced), P = 0.703 (Control vs Delayed); n = 264 Control, n = 81 Delayed and n = 135 SPW-R Silenced place cells. (b) Proportions of place cells with shifting fields (no overlapping place fields, black) or overlapping place fields (white) in the three groups of place cells. The number of cells in each category is indicated on the bars. χ 2 test: **P = 0.002; post-hoc two-sided Fisher’s exact tests followed by Bonferroni correction: *P = 0.02 (Control vs Silenced), P = 0.06 (Delayed vs Silenced), P = 1 (Control vs Delayed). (c-e) Distributions of “information content” values carried by place cells during pre- and post-learning exploration epochs. The information content of Silenced place cells (c) remained similar across pre- and post-learning exploration epochs (0.70 ± 0.05 and 0.69 ± 0.05 bit/spk for Pre and Post, respectively; Wilcoxon’s paired signed rank test: P = 0.91; n = 135 SPW-R Silenced place cells) while Control place cells (a) showed an increased information content (Control group: 0.75 ± 0.03 and 0.82 ± 0.03 bit/spk for Pre and Post, respectively; Delayed group: 0.70 ± 0.05 and 0.75 ± 0.05 bit/spk; **P = 0.009, P = 0.13 for Control and Delayed groups, respectively; n = 264 Control, n = 81 Delayed place cells). (f) Stability scores for the individual ensembles of place cells. Kruskall Wallis test: *P = 0.024; Tukey’s post-hoc tests: *P = 0.027 (Control vs Silenced), P = 0.99 (Control vs Delayed), P = 0.12 (Delayed vs Silenced). n = 21, n = 6 and n = 13 ensembles of Control, Delayed and Silenced place cells. Bars indicate s.e.m. (g) Within-session differences between the stability scores of optogenetically manipulated ensembles and their matched Control ensembles (0.01 ± 0.02 for Delayed-Control pairs, n = 6, and -0.14 ± 0.04 for Silenced-Control pairs, n = 12). Mann-Whitney U test: *P = 0.032. Dashed grey line indicates zero level (no difference). Bars indicate s.e.m.

Supplementary Figure 3 Details of unit classification.

(a) Distribution of the spike waveform features used to distinguish putative pyramidal cells and interneurons (see Stark et al., Neuron 80, 1263–1276, 2013; Online Methods). (b) Distribution of the silenced (P < 0.05, Wilcoxon’s paired signed rank test) place cells used in our study (n = 167 Silenced and n = 81 Delayed place cells) according to their response to light pulses. Left: The percentage of silencing is computed by comparing the mean rate during the 100 ms light pulses of the response mapping and the mean rate measured during 100ms intervals starting 1 s before the pulses (baseline rate). The median silencing value (92.2%) is shown by a green line. Most cells show more than 50% silencing (87.5 ± 0.8 %; mean ± s.e.m.; range 40-100%). Right: Distribution of the light-response indices for the same silenced place cells (n = 248; Online Methods).

Supplementary Figure 4 Relationship between the light-response index of individual place cells and their firing rate change during the SPW-Rs between task and postexploration epochs.

Specifically, we computed the firing rates during the 60ms period that followed SPW-R detections during immobility periods of the task for each place cell at the goal locations (illumination period in ‘SPW-R-locked’ condition): Rtask. Next, we computed the rate during the 60ms period that followed SPW-R detections during immobility periods of the post-exploration epoch (i.e., when no light was applied): Rpost. The change in SPW-R rate was calculated with the following formula:

(Rtask - Rpost) / (Rtask + Rpost).

Silenced place cells showed a significant correlation (Pearson’s test; r = 0.34, ***P<0.001, n = 167) between the change in SPW-R firing rate and the light-response index, whereas Delayed cell did not (r = 0.04 and P = 0.70 for Delayed place cells, n = 81).

Supplementary Figure 5 Indirect silencing of pyramidal cells by optogenetic activation of inhibitory PV cells in the PV-cre::ChR2 mouse.

(a) Peristimulus response of 47 simultaneously recorded pyramidal cells. The position of the units on the 8-shank silicon probe is indicated on the right. Blue light was delivered on shank 3 (Sh3; blue box). Consistent with previous observations (Berenyi, A. et al., J Neurophysiol 111, 1132-1149, 2014), a weak non-local silencing is also observed for neurons recorded on the adjacent shank (200 μm from illuminated shank) but not on more distant shanks (>= 400 μm from the illuminated shank). More pyramidal cells are silenced than with direct Arch-suppression of pyramidal neurons possibly because axons collaterals of CA1 PV interneurons cover a larger region. (b) Peristimulus histogram for a light-activated PV-positive neuron. (c) Quantification of light-response indices for Control and Silenced place cells recorded in the PV-cre::ChR2 mouse. Indices: -0.25 ± 0.05 and -0.64 ± 0.03 for Control and Silenced pyramidal cells, respectively. Mann-Whitney U test: ***P = 1.7 x 10-7; n = 19 and n = 32 Control and Silenced pyramidal cells, respectively). The inclusion of the PV-cre::ChR2 mouse does not change the overall conclusions drawn from our study (Supplementary Fig. 2).

Supplementary Figure 6 Quantification of SPW-R detection and silencing.

(a) Distribution of ripple durations during the post-exploration epoch (n = 10,213 offline detected events in 29 sessions; median ± SD: 33 ± 24 ms). Similar durations were found for SPW-Rs detected during the learning task, in sessions with the delayed condition of stimulation (i.e., uninterrupted SPW-Rs; Mann and Whitney U test, P = 0.67; 33 ± 25 ms, n = 825 SPW-R events in 7 sessions). (b) Distribution of online detection times relative to ripple onset detected offline (median ± SD: 16 ± 20 ms; 1,689 offline detected events during post-exploration epochs). The negative values are due to slight differences between online and offline detection methods. Based on observations from (a) and (b), we estimate that approximately half of the (larger amplitude) ripples were disrupted by optogenetic silencing. (c) Peri-stimulus activity of Silenced pyramidal cells during immobility periods of the learning task at goal locations (n = 268).

Supplementary Figure 7 Behavioral performance in sessions with ripple-locked and ripple-delayed stimulation conditions.

(a) Time spent at the goal locations during the first 10 min of the pre-learning exploration epoch compared to the first 10 min of the post-learning exploration epoch in sessions with ripple-delayed condition (8.8 ± 1.0 and 13.3 ± 3.3 % of the time for Pre and Post, respectively; *P = 0.047, Wilcoxon’s paired signed rank test, n = 7 sessions). (b) Time spent at the goal locations during the first 10 min of the pre-learning exploration epoch compared to the first 10 min of the post-learning exploration epoch in sessions with ripple-locked condition (6.2 ± 1.1 and 9.1 ± 1.4 % of the time for Pre and Post, respectively; P = 0.08, Wilcoxon’s paired signed rank test, n = 13 sessions). (c) Same graph as in Figure 1d with superimposed data from ripple-delayed (blue lines) and ripple-locked (green lines) sessions. Gray lines represent sessions where delayed and locked conditions were delivered in different brain hemispheres (7.2 ± 0.7 and 10.8 ± 1.1 % of the time for Pre and Post, respectively; ***P = 0.0006, Wilcoxon’s paired signed rank test, n = 29 sessions) (d) Comparison of memory performance between sessions with ripple-delayed and ripple-locked conditions (P = 0.70; Mann-Whitney U test on the differences Post - Pre; n = 7 ripple-delayed and n = 13 ripple-locked sessions). (e) Learning performance during the task in the two different conditions. Lines with shaded areas show mean ± s.e.m. The two learning curves are similar (P>0.5 for all trials, Mann and Whitney U tests with Bonferroni correction).

Supplementary Figure 8 Place field overlap calculation method.

(a) Schematic illustrating the method used to compute place field overlap for a place cell with overlapping place fields (top) and a place cell with non-overlapping place fields (bottom). (b-c) Examples of place cells with non-overlapping (b) and overlapping (c) place fields. Firing rate maps are shown for the pre- and post-exploration epochs (top; minimum and maximum rates are indicated below each map) with their corresponding detected place fields (white areas) below. The sum of the ‘pre’ and ‘post’ place fields, with (c) or without (b) overlap (red), is depicted on the right. (d-e) Proportions of place cells with shifting fields (no overlapping place fields, black) or overlapping place fields (white) in the three groups of place cells when different criteria of place field definition are used. The number of cells in each category is indicated on the bars (in total: n = 283, 81, and 167 place cells in the Control, Delayed and Silenced group, respectively). (d) Results obtained with a minimum in-field peak of 70% of peak rate on the maze. χ2 test: P = 0.001; post-hoc two-sided χ2 tests followed by Bonferroni correction: P = 0.01 (Control vs Silenced), P = 0.05 (Delayed vs Silenced), P = 1 (Control vs Delayed). (e) Results obtained with a minimum in-field rate of 40% of peak rate on the maze. χ2 test: P = 0.002; post-hoc two-sided χ2 tests followed by Bonferroni correction: P = 0.02 (Control vs Silenced), P = 0.04 (Delayed vs Silenced), P = 1 (Control vs Delayed).

Supplementary Figure 9 Comparison of place cells recorded in ripple-locked and ripple-delayed categories supports a role for SPW-Rs in place field stabilization and refinement.

(a) Place cells from both groups have similar light response indices (index = -0.42 ± 0.02 and -0.48 ± 0.02, for “ripple-delayed” and “ripple-locked” place cells with identifiable light-response; n = 240 and 377; Mann-Whitney U test, P = 0.07). (b) Proportion of place cells identified among ripple-delayed and ripple-locked pyramidal cells - PYR - (247 out of 369; 385 out of 601 PYR for ripple-delayed and ripple-locked groups, respectively). Place cells coding in the start box are included. χ2 test: P = 0.22. (c) The mean firing rates of ripple-delayed and ripple-locked place cells are similar between pre- and post-learning exploration epochs (ripple-delayed: 0.27 ± 0.02 spk/s for both Pre and Post; ripple-locked: 0.28 ± 0.01 and 0.27 ± 0.01 spk/s for Pre and Post, respectively; Kruskall Wallis test, P = 0.92; n = 247 and 385 place cells for ripple-delayed and ripple-locked groups, respectively). (d) The peak firing rates of ripple-delayed and ripple-locked place cells are similar between pre- and post-learning exploration epochs (ripple-delayed: 4.90 ± 0.20 and 5.40 ± 0.23 spk/s; ripple-locked: 5.34 ± 0.19 and 5.34 ± 0.19 spk/s for Pre and Post, respectively; Kruskall Wallis test, P = 0.59). Both mean and peak firing rates were obtained from the rate maps for each exploration epochs (pre- and post-learning). (e) The proportion of place cells identified among ripple-delayed and ripple-locked PYR cells is similar between the pre- and post-learning exploration epoch (χ2 tests: P = 0.64 and P = 0.86 for ripple-delayed and ripple-locked groups, respectively). (f-g) Distribution of information content values carried by place cells before and after learning (during pre- and post-learning exploration epochs). The information content of ripple-locked place cells remained similar in the pre- and post-learning exploration epochs (Wilcoxon’s paired signed rank test: P = 0.49; n = 385 place cells) while the control ripple-delayed place cells showed an increased information content (***P = 6.6 x 10-5, n = 247 place cells). Two outlier values in the ripple-locked group and one in the ripple-delayed group are not displayed but included in the statistical analyses (their exclusion does not affect the conclusions). (h) Cumulative distributions of the Pearson correlation coefficients (r) obtained for individual place cells in the two groups (r = 0.60 ± 0.02 and 0.53 ± 0.02 for ripple-delayed and ripple-locked groups, respectively; Mann-Whitney U test: **P = 0.007). Results were similar with Spearman's correlation coefficients (**P = 0.006). (i) No linear correlation (R) was present between the light response index and the pre-post correlation value measured for individual place cells in the ripple-locked (green) and ripple-delayed (blue) conditions (R = 0.07, P = 0.2 for ripple-locked units; R = -0.09, P = 0.2 for ripple-delayed units; Pearson’s test). (j) Proportions of place cells with shifting fields (no overlapping place fields, back) or overlapping place fields (white) in the two groups of place cells. The number of cells in each category is indicated on the bars. χ2 test: P = 0.07. (k) Both place cells with shifting fields (n = 74) and non-shifting cells (n = 166) have similar light response indices in the ripple-delayed group (indices: -0.39 ± 0.04 and -0.43 ± 0.03; Mann-Whitney U test, P = 0.58). (l) Place cells with shifting fields (n = 140) have lower light response indices than non-shifting place cells (n = 237) in the ripple-locked group (indices: -0.54 ± 0.03 and -0.44 ± 0.02; **P = 0.005). Only cells with identifiable light-responses are included. (m) Examples of correlation maps obtained for ripple-delayed and ripple-locked place cell ensembles. The population correlation value, computed for individual spatial bins (r), is color coded. The stability score is indicated on the left of each map (black). Goal locations are shown with black crosses. (n) Cumulative distributions of the correlation values accumulated for all ensembles of place cells from the two groups. (o) Ensembles of ripple-locked place cells showed reduced stability as compared to control ripple-delayed ensembles. Stability scores: 0.75 ± 0.02 and 0.64 ± 0.03 for ripple-delayed and ripple-locked ensembles, respectively; Mann-Whitney U test: **P = 0.009. n = 12 ripple-delayed and n = 19 ripple-locked place cell ensembles.

Supplementary Figure 10 Silencing pyramidal cells during SPW-Rs does not affect their firing rates or ability to code for space.

(a) Proportion of place cells identified among Control, Delayed and Silenced pyramidal cells - PYR - (308 out of 460; 90 out of 129; 176 out of 273 PYR for Control, Delayed and Silenced groups, respectively). Inclusion of place cell had to meet our criteria (see Online Methods) in at least one of the exploration epochs (pre- or post-learning). Place cells in the start box are included. χ2 test: P = 0.56. (b) The mean firing rates of Control, Delayed and Silenced place cells are similar between pre- or post-learning exploration epochs (Control: 0.27 ± 0.01 and 0.27 ± 0.02 spk/s; Delayed: 0.28 ± 0.03 and 0.29 ± 0.03 spk/s; Silenced: 0.30 ± 0.02 and 0.29 ± 0.02 spk/s for Pre and Post, respectively; Kruskall Wallis test, P = 0.67; n = 283; 81; 167 place cells for Control, Delayed and Silenced groups, respectively). (c) The peak firing rates of Control, Delayed and Silenced place cells are similar between pre- or post-learning exploration epochs (Control: 5.01 ± 0.21 and 5.28 ± 0.22 spk/s; Delayed: 5.45 ± 0.35 and 5.88 ± 0.43 spk/s; Silenced: 5.43 ± 0.29 and 5.35 ± 0.28 spk/s for Pre and Post, respectively; Kruskall Wallis test, P = 0.41). Both mean and peak firing rates were obtained from the rate maps for each exploration epochs (pre and post-learning). (d) The proportion of place cells identified among Control, Delayed and Silenced pyramidal cells is similar between the pre- and post-learning exploration epochs. n = 460, 129 and 273 PYR for Control, Delayed and Silenced groups, respectively. χ2 tests: P = 0.84, P = 0.90 and p = 0.61 for Control, Delayed and Silenced groups, respectively. The fact that the proportion of place cells is identical between the pre- and post-learning exploration epochs in the Silenced group indicates that SPW-R silencing does not impact the ability of pyramidal cells to code for their environment. (e) Some neurons identified as place cells in the pre-learning exploration did not meet the criteria for place cell in the post-learning exploration epoch. The proportion of these neurons was similar among the three groups indicating that SPW-R silencing does not induce place cell disappearance (χ2 test, P = 0.76; n = 276, 79 and 149 place cells identified in the pre-learning exploration epoch for Control, Delayed and Silenced groups, respectively). (f) Some of the neurons identified as place cells in the post-exploration were not place cells in the pre-learning exploration epoch. The proportion of these neurons was similar in the three groups indicating that SPW-R-silencing does not result in additional place cell formation (χ2 test, p = 0.13; n = 273, 80 and 155 place cells identified in the post-learning exploration epoch for Control, Delayed and Silenced groups, respectively; place cells with place field in start box are included).

Supplementary Figure 11 Place cell remapping is not related to measures of recording instability or to the probability of firing at goal locations before learning.

(a-b) Spike waveform amplitude and cluster quality (as assessed by the Mahalanobis distance or L-ratio) (Schmitzer-Torbert, N., Jackson, J., Henze, D., Harris, K. & Redish, A. D. Quantitative measures of cluster quality for use in extracellular recordings. Neuroscience 131, 1-11, 2005) was calculated for the pre- and post- exploration epochs. Changes in these measures ((Post-Pre)/(Post+Pre)) was similar across experimental groups (Kruskall Wallis test, P = 0.90 for spike amplitude changes; P = 0.17 for L-ratio changes; n = 283, 81 and 167 place cells in Control, Delayed and Silenced groups, respectively). (c-d) None of these measures was correlated with place cell remapping (correlation coefficient between the pre- and post-exploration rate maps). Spike amplitude changes (c): r = - 0.06, P = 0.16 (all place cells, n = 531, Pearson’s test); r = 0.01, P = 0.85 (Control place cells, black, n = 283); r = - 0.15, P = 0.17 (Delayed place cells, blue, n = 81); r = - 0.15, P = 0.051 (Silenced place cells, green, n = 167). L-ratio changes (d): r = 0.005, P = 0.91 (all place cells, n = 531); r = 0, P = 0.99 (Control place cells, black, n = 283); r = - 0.02, P = 0.86 (Delayed place cells, blue, n = 81); r = 0.05, P = 0.51 (Silenced place cells, green, n = 167). (e) Relationship between place field stability and the probability of firing at goal locations before learning. The probability of firing at goal locations was computed based on the pre-exploration epoch (Online Methods). The correlation between the two measures was not significant for any of the three groups of place cells (Pearson’s test; r = -0.05, P = 0.41, n = 283 for Control; r = -0.06, P = 0.57, n = 81 for Delayed and r = -0.06, P = 0.42, n = 167 for Silenced place cells). This result suggests that a place cell with a place field near the goal is not more likely to remap after learning then a place cell with a place field elsewhere in the maze.

Supplementary Figure 12 SPW-R silenced place cell ensembles show nonstabilized spatial representation as compared to simultaneously recorded Control ensembles after controlling for ensemble size.

(a, b) Comparison of pairs of simultaneously recorded ensembles was conducted after randomly downsampling (up to 100 times) the larger ensemble of the pair to match the number of cells in the smaller ensemble of the pair (see Online Methods). (a) Ensembles of Delayed place cells are similar to their matching Control ensembles with equated ensemble sizes (score = 0.71 ± 0.03 and 0.71 ± 0.02 for Control and Delayed ensembles, respectively; Wilcoxon's signed rank test, P = 0.84, n = 6 pairs). (b) In contrast, ensembles of Silenced place cells show lower stability scores compared to their matching Control ensembles from the same session with equated ensemble sizes (score = 0.65 ± 0.03 and 0.54 ± 0.04 for Control and Silenced ensembles, respectively; *P = 0.02, n = 15 pairs). (c) Within-session differences between the stability scores of optogenetically manipulated ensembles and their matching Control ensembles (0.005 ± 0.02 for Delayed-Control pairs and -0.11 ± 0.04 for Silenced-Control pairs). Mann-Whitney U test: *P = 0.047.

Supplementary Figure 13 Data from the cued version of cheeseboard maze task.

(a) Behavioral performance across trials in the cued (red) and non-cued (black) version of the task. A new set of three baited wells was randomly selected every day but stayed fixed within a given day. In the cued version, the locations of the rewards were indicated to the mouse by prominent objects (cylinders) placed on the maze. Lines with shaded areas show mean ± s.e.m. for n = 29 sessions in 5 mice (non-cued) and n = 10 sessions in 4 mice (cued). Black dots above the graph indicate significant differences between the two conditions (P < 0.05; Mann and Whitney U tests with Bonferroni correction for each trial). (b) Cumulative distributions of the r values obtained for individual place cells in the five groups: Control place cells in the non-cued version of the task (‘learningC’, black, n = 283), Silenced place cells in the non-cued version of the task (‘learningS’, green, n = 167), Delayed place cells in the non-cued version of the task (‘learningD’, blue, n = 81), Control place cells in the cued version of the task (‘cuedC’, red, n = 121), Silenced place cells in the cued version of the task (‘cuedS’, pink, n = 72). Details of statistical tests are shown in the figure (Kruskall Wallis test, ***P < 0.001, followed by Tukey’s post-hoc tests).

Supplementary Figure 14 Changes in activity during slow-wave sleep SPW-Rs are comparable across experimental groups and do not predict the degree of place cell remapping.

For each rest epochs (pre- and post- learning), we computed the rate (total number of spikes during SPW-Rs divided by total SPW-Rs duration), the participation rate (number of events with a least one spike/ total number of events), the mean spike counts (mean number of spikes per event) and the gain (rate during SPW-Rs divided by baseline rate outside SPW-Rs) of each place cell during slow wave sleep SPW-Rs. Changes in the rates, participation, spike count and gain are computed between the pre-learning and the post-learning sleep SPW-Rs (Change = (Post-Pre)/(Post+Pre)). (a) Changes for the four SPW-R activity measures does not differ across groups of Control, Silenced and Delayed place cells (Kruskall Wallis tests, P = 0.73 for rate change, P = 0.66 for participation change, P = 0.79 for spike count change, P = 0.66 for gain change; n = 257, 79, 161 Control, Delayed and Silenced place cells with sleep recordings). (b) Changes in sleep SPW-R rates do not correlate with map stability (correlation coefficient between pre- and post- rate maps) (Pearson’s test; r = -0.04, P = 0.36, n = 497 place cells from the 3 groups). (c) Changes in sleep SPW-R participation rates do not correlate with map stability (r = -0.04, P = 0.35, n = 497 place cells). (d) Changes in sleep SPW-R mean spike counts do not correlate with map stability (r = -0.04, P = 0.39, n = 497 place cells). (e) Changes in sleep SPW-R gains do not correlate with map stability (r = -0.04, P = 0.32, n = 497 place cells).

Supplementary Figure 15 Goal location representation before and after learning.

(a) The probability of place cells to spike in goal areas (15 cm diameter region centered on reward locations used during the learning task, Online Methods) is similar in the pre- and post-learning exploration epochs across all three groups of place cells (Kruskall Wallis test on the 6 conditions: P = 0.02, followed by post-hoc Wilcoxon’s paired signed rank tests with Bonferroni correction: P = 1, n = 283 for Control; P = 1, n = 81 for Delayed and P = 0.45, n = 167 for Silenced place cells). (b) The distance of place field(s) to goal locations is comparable between the pre- and post-learning exploration epochs across the three groups of place cells, whether place field edge (i)(P = 0.09, Kruskall Wallis test on the 6 conditions), peak (ii)(P = 0.07) or centroid (iii)(P = 0.08) is considered. Edge: 11.4 ± 0.6 and 12.5 ± 0.7 cm (Control Pre and Post, respectively), 10.1 ± 1.1 and 13.0 ± 1.2 cm (Delayed Pre and Post, respectively), 9.4 ± 0.7 and 12.7 ± 1.1 cm (Silenced Pre and Post, respectively). Peak: 20.5 ± 0.6 and 21.2 ± 0.7 cm (Control Pre and Post, respectively), 19.4 ± 1.1 and 22.0 ± 1.3 cm (Delayed Pre and Post, respectively), 17.9 ± 0.8 and 21.4 ± 1.1 cm (Silenced Pre and Post, respectively). Centroid: 18.9 ± 0.6 and 19.8 ± 0.7 cm (Control Pre and Post, respectively), 18.3 ± 1.1 and 20.8 ± 1.2 cm (Delayed Pre and Post, respectively), 16.8 ± 0.7 and 20.0 ± 1.1 cm (Silenced Pre and Post, respectively). n = 272 Control, n = 80 Delayed and n = 153 for Silenced place cells with place fields in both pre- and post-exploration epochs.

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Roux, L., Hu, B., Eichler, R. et al. Sharp wave ripples during learning stabilize the hippocampal spatial map. Nat Neurosci 20, 845–853 (2017). https://doi.org/10.1038/nn.4543

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