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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nature. Author manuscript; available in PMC May 31, 2011.
Published in final edited form as:
PMCID: PMC3104398
NIHMSID: NIHMS291902

Preplay of future place cell sequences by hippocampal cellular assemblies

Abstract

During spatial exploration, hippocampal neurons show a sequential firing pattern in which individual neurons fire specifically at particular locations along the animal’s trajectory (place cells1,2). According to the dominant model of hippocampal cell assembly activity, place cell firing order is established for the first time during exploration, to encode the spatial experience, and is subsequently replayed during rest36 or slow-wave sleep710 for consolidation of the encoded experience11,12. Here we report that temporal sequences of firing of place cells expressed during a novel spatial experience occurred on a significant number of occasions during the resting or sleeping period preceding the experience. This phenomenon, which is called preplay, occurred in disjunction with sequences of replay of a familiar experience. These results suggest that internal neuronal dynamics during resting or sleep organize hippocampal cellular assemblies1315 into temporal sequences that contribute to the encoding of a related novel experience occurring in the future.

We recorded neuronal firing sequences from the CA1 area of the mouse hippocampus (Supplementary Fig. 1) during periods of awake rest (Fam-Rest) alternating with periods of running (Fam-Run) on a familiar track (Fam session; Supplementary Fig. 2a) that preceded the exploration of a novel linear arm in contiguity with the familiar track (Contig-Run on L-shaped track; Fig. 1, Supplementary Fig. 2a and Methods). All the place cells active on the novel arm during Contig-Run, whether previously silent16 (19% in both directions and 31% in at least one direction; Methods and Supplementary Tables 1–3) or active during Fam-Run (subpanels a in Fig. 1), fired during Fam-Rest at the ends of the familiar track (range, 0.17–11.7 Hz; Supplementary Fig. 3) as part of a number of ‘spiking events’. The spiking events were defined as epochs composed of multiple individual spikes from at least four different place cells active on the novel arm or familiar track, separated by less than 50 ms and flanked by at least 50 ms of silence3,4. More significantly, the temporal sequence in which the cells active on the novel arm fired during Fam-Rest (subpanels b in Fig. 1) was significantly correlated with the spatial sequence in which they fired later as place cells on the novel arm during Contig-Run (subpanels c in Fig. 1), despite being uncorrelated with their spatial sequence as place cells on the familiar track during Fam-Run. This is illustrated as place cell sequences during Contig-Run (subpanels c in Fig. 1) and Fam-Run (subpanels a in Fig. 1) compared with the firing sequences of these cells within individual spiking events observed during Fam-Rest (subpanels b in Fig. 1). We refer to this process as ‘preplay’ of place cell sequences because the temporal sequence of firing during Fam-Rest had occurred before the actual exploration of the novel arm in the subsequent Contig-Run and was not a replay of the place cell sequences from the previous Fam-Run.

Figure 1
Preplay of novel place cell sequences

To quantify the significance of preplay and to compare it with replay, we created place cell sequence templates according to the spatial order of the peak firing of place cells3,4,10 on the novel arm during Contig-Run (novel arm templates; subpanels c in Fig. 1 and Methods) and on the familiar track during Fam-Run (familiar track templates) for each run direction. The spikes of all the place cells used to construct the two types of template that were emitted during Fam-Rest were sorted by time, and spiking events were determined as explained above (subpanels b in Fig. 1). For each spiking event, we calculated a rank-order correlation between the novel armtemplates and the temporal sequence of firing of the corresponding cells in the spiking events during Fam-Rest. The event correlation was considered significant if it exceeded the 97.5th percentile of a distribution of correlations resulting from randomly shuffling the order of place cells in the novel arm templates 200 times (P < 0.025). Forward4 and reverse3,4 preplay refers to the cases in which the sequence of place cells during Contig-Run and the firing order of the corresponding cells in Fam-Rest were in the same and opposite directions, respectively. In 91% of the preplay cases, the spiking events were correlated with the novel armtemplate in one direction only. The distribution of event correlation values obtained using the original novel arm templates was significantly shifted towards higher positive or negative values in comparison with the distribution of correlation values obtained using shuffled templates (Fig. 2a and Supplementary Fig. 4). Figure 2a also shows the distribution of significant preplay events (in red). Of all the spiking events detected as above and in which at least four novel arm place cells were active, 14.2% were significant preplay events for the place cell sequence on the novel arm(P < 10−32, binomial probability test4) in the forward or reverse order (Fig. 2b).

Figure 2
Quantification of the preplay phenomenon and comparison with replay

The occurrence of significant preplay events was correlated with the occurrence of high-frequency ripple oscillations in CA1 (Fig. 2c). The majority of the significant preplay events (81.1%; Fig. 2d, total, blue) took place at the junction between the familiar and novel arms, and the remaining 18.9% took place at the free end of the familiar track (Fig. 2d, total, purple). The proportion of significant preplay events among the total events at each of the two track ends was higher at the junctional end (15.2%, P < 10−26) than at the free end (8.5%, P < 10−4) of the familiar track (P < 0.035, Z-test; Fig. 2d, normalized).

We found a relatively high correlation between the place field maps (Fig. 1A, B and Supplementary Fig. 5) of the familiar track before and after the novel experience (median r = 0.66; Fig. 2e, familiar track, blue); it was significantly higher than the correlations obtained when the cell identities were shuffled (median r = 0.23, P < 10−4; Fig. 2e, familiar track, black).A similar correlation analysis showed a relatively high stability of the newly formed place fields on the novel arm from the beginning to the end of Contig-Run (median r = 0.62 (newly formed) versus median r = 0.21 (shuffled), P < 10−3; Fig. 2e, novel arm, blue versus grey). These results suggest that preplay of the novel arm does not occur over an entirely new (that is, remapped) representation of the whole L-shaped track but rather benefits from the relative stability of the familiar track representation across sessions and perhaps facilitates the rapid, stable encoding of the novel arm experience.

Using the familiar track templates and spiking events during Fam-Rest, constructed as above, we determined that 16.2% (P < 10−91; data not shown) were significant replay events36,17 among the spiking events in which a minimum of four familiar track place cells were active. All significant preplay events occurring during Fam-Rest (n = 75) were tested for possible replay of the familiar track spatial sequence: these spiking events were more correlated with the novel arm template (Fig. 2f, red) than the familiar track template (Fig. 2f, blue). Seventy-two percent (n = 54) of the significant events previously considered to be preplay had no significant correlation with the familiar track template. An additional 16% (n = 12) of those events were better correlated with the novel arm templates (mean absolute r = 0.92) than with the familiar track template (mean absolute r = 0.67, P < 10−3). Together, these findings reject the hypothesis that the preplay events simply represent a replay of the familiar track activity (see additional controls in Supplementary Information). Moreover, we found that the proportion of events exclusively composed of silent cells that perfectly matched the novel arm spatial templates was 0.67 (16 of 24 triplets), which is significantly greater (P < 0.025) than the proportion of by-chance perfect matches (0.33).

To illustrate the distribution and relative proportions of preplay and replay events among all significant spiking events during Fam-Rest, we calculated a ‘template specificity index’ (Fig. 2g and Methods) for each event. Pure preplay events (Fig. 2g, red) and pure replay events (Fig. 2g, blue) were segregated, and only a minority of events were significant for both preplay and replay (Fig. 2g, yellow). Consistent with this segregation of preplay and replay events, the novel arm and the ‘corresponding familiar track’ templates were not significantly correlated (Fig. 2h and Methods). The ratio between the number of pure replay events (n = 171) and the number of pure preplay events (n = 54) during Fam-Rest was about 3.1 (Fig. 2g, inset; see Supplementary Information for proportions of events). Preplay and replay events were distributed in time across Fam-Rest (Supplementary Fig. 6a–c) and their occurrences were generally uncorrelated (Supplementary Fig. 6d). The majority (79.9%) of the spiking events during Fam-Rest did not significantly correlate with either of the two templates (data not shown).

We used a Bayesian reconstruction algorithm2,5,6,18,19 (Methods) to decode the animals’ position from the spiking activity during Fam-Run (Fig. 3a) or Fam-Rest (Fig. 3b, c). For all original and shuffled6 probability distributions, a line was fitted to the data using a line-finding algorithm6 to represent the decoded virtual trajectory (Methods and Supplementary Information). In 16.36% of cases representing trajectories, the reconstructed trajectory during spiking events in Fam-Rest was contained within the novel arm (Fig. 3c, top), a place the animal had not yet visited (that is, trajectory preplay). Moreover, in 79.8% of the trajectory preplay cases the shuffling procedures resulted in lines that were significantly less or not at all contained within the novel arm (that is, not preplay; Supplementary Information). The remaining trajectories decoded during Fam-Rest represented replay of the familiar track (64.15%; Fig. 3c, middle) or spanned the joint familiar track/novel arm space (19.49%; Fig. 3c, bottom). Means of absolute rank-order correlations between spiking activity and novel arm templates (Fig. 2a) restricted during epochs of trajectory preplay were significantly larger than those between spiking activity and familiar track templates calculated during the same epochs (0.75 versus 0.59, P < 10−4). Overall, these results support the existence of the preplay phenomenon.

Figure 3
Bayesian reconstruction of the animal’s trajectory in the familiar track (replay) and novel arm (preplay)

To investigate the possibility that preplay of novel arm place cell sequences during Fam-Rest depends on the prior run experience on the familiar track, mice with no prior experience on any linear track were placed in a high-walled sleep box and recorded while resting/sleeping. The animals were then transferred to a novel isolated linear track that was in the same room but could not be seen from inside the box, and the recording continued during de novo formation of place cells (Supplementary Fig. 2b, de novo session). We found that in a relatively large proportion (16.1%) of spiking events identified during sleep/rest in the sleep box, the neuronal firing sequences were significantly correlated with the place cell sequences observed during the first run session on the novel track (Fig. 4A, B and Methods); this was the case for all four individual mice (Supplementary Fig. 7). Preplay events were associated with the ripple occurrence (Fig. 4C). The place cells established on the novel track in the de novo session were more dynamic (median r = 0.42; Fig. 4D, blue) than in Contig-Run (median r = 0.62, P < 0.016; Fig. 2e, right, blue).

Figure 4
Preplay of novel place cell sequences before any linear track experience

We have demonstrated that a significant number of temporal firing sequences of CA1 cells during resting periods of a familiar track exploration that preceded a novel track exploration in the same general environment were correlated with the place cell sequences of the novel track rather than the familiar track. This phenomenon, preplay, is temporally opposite to the process of replay310,19,20, when activity during rest or sleep periods recapitulates place cell sequences that have already occurred during previous explorations. Preplay differs fundamentally from replay because it occurs before exploration of novel tracks.

Although our recordings were carried out in CA1, we believe that what we observed could be a reflection of the output of the recurrent cellular assemblies from upstream regions (CA3 or entorhinal cortex). During running on a familiar track, some of the cells in the postulated upstream cellular assemblies fire sequentially at spatial locations while others, although connected anatomically to these cells, remain silent. The lack of expression of preplay sequences during Fam-Run may reflect their state-dependent suppression or subthreshold activation during these exploratory behaviours. Owing to increased net excitation during rest periods predominantly during ripples21, some of these silent cells together with some of the familiar track cells are activated above threshold and fire in a certain sequence. Their sequence of activation may be determined in part by their functional connectivity within the hippocampal formation network. Some of these sequences may in turn be activated on a novel track as place cell sequences (Supplementary Fig. 8). The activation of the novel place cell sequences during running may strengthen their pre-existing assembly organization manifested during preplay.

It could be argued that during Contig-Run the animals simply considered the novel arm to be an extension of the familiar arm and, thus, what we considered to be preplay events were replays of the previous runs on the familiar track. If this was the case, preplay events would not be expected to be found when the experience of the familiar track run is eliminated. This idea was refuted by the demonstration of frequent preplay events in the sleep box before the mice were transferred onto a novel linear track (de novo condition). Under this condition, the place cell sequences were more dynamic and a higher proportion of all spiking events correlated with the place cell sequences in these runs than in the later runs on novel linear tracks. These results suggest a shift in the relative contribution of internal22,23 and external drives in the formation of place cell sequences on encounter with a novel track. In the early phase, internal drives originating in the dynamic cellular assembly activities, which probably reflect numerous past experiences distinct from the current one and expressed as preplay, may have a greater role, whereas in the late phase, external drives that come from the specific set of stimuli of the current experience may dominate. Thus, place cell sequences on novel tracks seem to be products of a dynamic interplay between the internal and external drives.

Several previous studies did not reveal preplay7,8,10,20. Although it is difficult to pinpoint the apparent discrepancies between these studies and the present one, we suggest that the use of insufficiently sensitive methods (pairwise correlations) by some studies7,8,20 and small sample sizes by others10 might have precluded detection of preplay in previous work (see Supplementary Information for details). Data from the de novo condition (Fig. 4), in which we observed an even higher proportion of preplay events, have not been reported previously.

Our data showed that novel preplay events coexist in disjunction with familiar replay events during the rest periods on the familiar track. This and the finding that these preplay and replay events together make up fewer than one-quarter of all detected spiking events suggest that they are part of a dynamic repertoire of temporal sequences in the hippocampus that are past-experience dependent (replay) or future-experience expectant24 (preplay). Post-experience replay of place cell sequences during resting36 or slow-wave sleep810 has been proposed to have an important role in memory consolidation11,12. The temporal preplay of new place cell sequences during resting or sleep is consistent with a predictive function for the hippocampal formation25 and may contribute to accelerating learning26 when a new experience is introduced in multiple steps of increasing novelty.

METHODS SUMMARY

We recorded place cells from the CA1 area of the hippocampus with six independently movable tetrodes in four mice during sleep/rest sessions in the sleep box before any experience on linear tracks and during the first run session on a novel track. Following familiarization with the linear track, animals were subsequently allowed to explore a continuous (L-shaped) track in which the now familiar track and a new novel arm were made contiguous. To quantify the significance of the preplay and replay processes, spiking events in which at least four cells were active were detected during sleep/rest (speed, <1 cm s−1) periods in the sleep box or awake rest (speed, <2 cm s−1) periods at the ends of the familiar track and novel arm, predominantly during ripple epochs.

We calculated statistical significance at the P < 0.025 level for each event by comparing the rank-order correlation between the event sequence and the place cell sequence (template) with the distribution of correlation values from 200 templates obtained by shuffling the original order of the place cells. Proportions of significant events were calculated as the ratio between the number of significant events and the total number of spiking events. We calculated the overall significance of preplay or replay processes by comparing the distribution of correlation values of all events with the distribution of correlation values of shuffled templates (Kolmogorov–Smirnov test).The significance of the proportion of significant events out of the total number of spiking events was determined as the binomial probability of observing the number of significant events (as successes) from the total number of spiking events (as independent trials), with a probability of success of 0.025 in any given trial. We reconstructed the position of the animal from the spiking activity emitted during resting periods using Bayesian decoding procedures6.

Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.

Supplementary Material

Published

Acknowledgements

We thank M. A. Wilson for assistance with data acquisition, discussions and comments on an earlier version of the manuscript; J. O’Keefe, A. Siapas, F. Kloosterman, D. L. Buhl for comments on earlier versions of the manuscript; and F. Kloosterman for providing assistance with the line detection for the Bayesian decoding. This work was supported by NIH grants R01-MH078821 and P50-MH58880 to S.T., who was an HHMI Investigator in an earlier part of this study.

Footnotes

Supplementary Information is linked to the online version of the paper at www.nature.com/nature.

Author Contributions S.T. and G.D. conceived the project jointly. G.D. designed and performed the experiments and the analyses. G.D. and S.T. wrote the paper.

Reprints and permissions information is available at www.nature.com/reprints.

Readers are welcome to comment on the online version of this article at www.nature.com/nature.

The authors declare no competing financial interests.

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