Hippocampal awake replay in fear memory retrieval

Abstract

Hippocampal place cells are key to episodic memories. How these cells participate in memory retrieval remains unclear. After rats acquired a fear memory by receiving mild footshocks in a shock zone on a track, we analyzed place cells when the animals were placed on the track again and displayed an apparent memory retrieval behavior: avoidance of the shock zone. We found that place cells representing the shock zone were reactivated, despite the fact that the animals did not enter the shock zone. This reactivation occurred in ripple-associated awake replay of place cell sequences encoding the paths from the animal's current positions to the shock zone but not in place cell sequences within individual cycles of theta oscillation. The result reveals a specific place-cell pattern underlying inhibitory avoidance behavior and provides strong evidence for the involvement of awake replay in fear memory retrieval.

Figure 1

Behavior in the linear IA task. (a) Experimental procedure. On Day1, rats were allowed to freely move on a two-segmented (light, dark) linear track in two sessions (Run1, Run2). On Day2, rats freely moved in the same track before (Pre) and after (Post) receiving mild foot-shocks at a shock zone (SZ). Afterward, rats were placed at the SZ to make them travel through the entire track (Re-exposure). The sessions were separated by resting in an enclosed box. The duration of every session and rest was 10 – 15 minutes. (b) A rat’s trajectories in Pre and Post. Shaded area: positions in the dark segment. Dashed line: boundary of SZ. ◀: SZ-avoiding turns, ▶: LE-avoiding turns. (c) Average percentage of time (mean ± s.e.) spent at each location (occupancy) of the track across all animals before (Pre/Day1) and after (Post) the shocks. (d) Occupancy of each rat (o) within the SZ in Pre/Day1 and Post. *P = 0.01, t₃ = 5.8, paired t-test (N = 4 rats).

Figure 2

Sequential firing of place cells occurred prior to the first SZ-avoiding turn in every rat. Activities of track-active place cells are plotted for each of the 4 animals (Rat1 – Rat4). Left: firing rate curves of place cells in Pre, each showing firing rate (normalized to its peak rate) of a cell along the track. Cells are ordered by peak locations. Red: place fields overlapping with the SZ. Dashed line: SZ boundary. Middle: spike raster of the same place cells as ordered on the left and the rat’s trajectory during the first SZ-avoiding turn in Post. Each row shows spikes of a cell plotted at its peak firing location on the track (y-axis). Red: spikes of those cells with place fields overlapping with the SZ. Dashed line: SZ boundary. Right: expanded view of the spike raster within a time window in the middle (arrows) and the filtered LFP in the ripple band within the same window (bottom). Note the sequential firing initiated by cells with place fields close to current locations, but terminated by those with place fields in the SZ (red). Also note the simultaneous increase in ripple activity.

Figure 3

SZ-avoiding turns in Post were preceded by replay of place cell activities leading to the SZ. (a) Counts of PBEs and replay events within each 2 s bin of pausing, as well as animals’ average speed (blue line, mean ± s.e.), around LE-avoiding turns in Pre/Day1 and around SZ-avoiding turns in Post. The counts were normalized by number of turns. ▲: turning time. (b) Replay trajectories during pausing (red), as well as animals’ actual trajectories (blue), around LE-avoiding turns in Pre/Day1 (N = 65, from all 4 rats) and around SZ-avoiding turns in Post (N = 37). The trajectories are aligned at the animal’s position and time of turning (▼). Red arrowheads: end positions of replay trajectories; upward arrows: direction to the LE or SZ. (c) Same replay trajectories in (b), but plotted against animals’ current positions (y-axis) on the track. Red arrowheads: end positions of replay trajectories; black dashed line: LE/SZ boundary; gray dashed line: equal animal and decoded positions. (d) Fraction of those replay trajectories during pausing that ended near the LE before LE-avoiding turns in Pre/Day1 (LE-avoiding, N = 15), fraction of those that ended near the SZ before SZ-avoiding turns in Post (SZ-avoiding, N = 44), and fraction of those that ended near the SZ during the rest of the time periods in Post (Post-other, N = 194). The fractions are plotted for each rat (o) and for all animals combined (bar). ***P = 4 × 10⁻⁴ between LE-avoiding turns and SZ-avoiding turns, 1 × 10⁻⁵ between SZ-avoiding turns and Post-other, binomial test (Chi-Square test among all 3 types: P = 2 × 10⁻⁵). (e) Activation probability and mean spike count of LE cells within PBEs during pausing periods before LE-avoiding turns in Pre/Day1, and for same measures of SZ cells within PBEs before SZ-avoiding turns in Post. Number of cells: N = 30 (LE), 26 (SZ); *P = 0.0057, **P = 0.0040, ranksum test. (f) Same as e, but within replay events. **P = 0.0039, ***P = 8 × 10⁻⁴.

Figure 4

Replay trajectories ending near the SZ were followed by pausing and turning away from the SZ in Post. (a) Replay trajectories and the animal’s moving trajectories within a 10 s window following replay, for the first 7 replay events that ended near the SZ in Pre (Pre-near) and Post (Post-near) and for the first 7 replay events that did not end near the SZ in Post (Post-other) in an example rat. Dashed line: SZ boundary. Black circle: animal’s position at the end of the window. (b) Movement vectors following each replay event, plotted at the end position of its replay trajectory (Replay end), for all replay events across all rats in Pre/Day1 and in Post. The movement vectors following those replays ending near the SZ are plotted in blue (Post-near) or light blue (Pre/Day1-near). Otherwise they are in purple (Post-other) or light purple. Dashed line: SZ boundary. (c) Movement vectors following 3 types of replays (Pre/Day1-near, Post-near, and Post-other). *P = 0.0057, t₂₁₈ = −2.8; ***P = 1 x10⁻⁶, t₂₃₆ = 5.0; t-test. ANOVA comparing all 3 types: P = 1 × 10⁻⁴, F_2,342 = 9.5. Number of replay events: N = 107 (Pre/Day1-near), 113 (Post-near), 125 (Post-other). (d) Proportion of pausing among all movement vectors following the 3 types of replays, plotted for each rat (o, N = 4) and for all animals combined (bars). ***P = 2 × 10⁻⁸, **P = 0.0037, binomial test; Chi-Square test comparing all 3 types: P = 1 × 10⁻⁷. (e) Similar to (d), but for proportion of moving away from the SZ among non-pausing movement vectors. ***P = 8 × 10⁻⁴ (between Pre/Day1-near and Post-near), 9 × 10⁻⁶ (between Post-near and Post-other), binomial test; Chi-Square test among all 3 types: P = 4 × 10⁻⁵. Number of non-pausing vectors: N = 70 (Pre/Day1-near), 31 (Post-near), 57 (Post-other). (f) Overlaps between replay trajectories and animals’ future or past trajectories for all replay events in Pre/Day1 and for those in Post. ***P = 1 × 10⁻¹⁰ (between Future and Past in Pre/Day1), 1 × 10⁻²⁶ (between Pre/Day1 and Post for future overlap), ranksum test. Two-way ANOVA among all 4 overlaps: P = 1 × 10⁻¹³ (Future vs. Past), 6 × 10⁻³⁹ (Pre/Day1 vs. Post); F_1,1,1163 = 56.3, 183.6, respectively. Number of replays: N = 345 (Pre/Day1), 238 (Post). (g) Proportions of replay trajectories that did not overlap with either animals’ future or past trajectories out of those replay trajectories with non-pausing past/future movement in Pre/Day1 and in Post, plotted for each rat (o) and for all animals combined (bars). ***P = 3 × 10⁻¹⁹, binomial test. Number of replays with non-pausing past/future movement: N = 323 (Pre/Day1), 180 (Post).

Figure 5

Shock experience altered place cell activity and coactivity within PBEs. (a) Activation probability of place cells within PBEs in Run1 and Run2 on Day1 (N = 100) and that in Pre and Post on Day2 (N = 147). n.s.: P = 0.47, **P = 0.0018, signrank test. (b) Same as in a, but for mean spike count. n.s.: P = 0.70, **P = 0.0018. (c) Change in activation probability from Pre to Post on Day2 for SZ cells (N = 26) and NSZ cells (N = 85). *P = 0.005, ranksum test. (d) Same as in c, but for change in mean spike count. *P = 0.027. (e) Coactivity of pairs of place cells within PBEs in Run1 and Run2 on Day1 (N = 1591 pairs) and that in Pre and Post on Day2 (N = 2424). n.s.: P = 0.33, *P = 0.017. (f) Changes in coactivity from Pre to Post on Day2 for vicinity pairs with their average peak locations near the SZ (SZ pairs, N = 85) and other vicinity pairs (NSZ pairs, N = 385). ***P = 6 × 10⁻⁷. (g) Change in coactivity within PBEs for every vicinity pair (N = 470) from Pre to Post on Day2, plotted against their average peak firing location. Solid line: linear regression. r, p: correlation coefficient and associated p-value of the regression. Dashed line: SZ boundary.

Figure 6

Theta sequences did not reactivate SZ cells in Post. (a) The plot is arranged similarly to Figure 2b,c, but here to show theta sequences. Left: spike raster of place cells prior to a SZ-avoiding turn in Post. Spikes in identified theta sequences are shown in red. Right: expanded view of two example theta sequences in left (arrows) and associated raw (gray) and filtered LFP trace within the theta band (black). (b) Theta trajectories and replay trajectories in Post, plotted against animals’ positions on the track. All identified theta trajectories pointing toward the SZ from 4 animals are plotted (N = 77). For replay trajectories, we plot a random sample of 77 out of 191 SZ-pointing replay trajectories. Dashed line: SZ boundary. (c) Trajectory lengths of theta (N = 133) and replay (N = 238) trajectories in Post. ***P = 4 × 10⁻¹¹, ranksum test. (d) Percentages of theta/replay trajectories that ended or started near the SZ, among all theta/replay trajectories in Post, plotted for each rat (o, N = 4) and for all animals combined (bars). ***P = 4 × 10⁻¹⁵, binomial test. (e) Activation probability and mean spike count of SZ cells (N = 26) within theta sequences and replay events in Post. ***P = 8 × 10⁻⁶ for both activation probability and mean spike count, signrank test.

Figure 7

Shock experience triggered partial remapping of place cells. (a) Firing rate curves of place cells in Run1 and Run2 on Day1 and Pre and Re-exposure on Day2 for an example rat. Firing rates are normalized to its maximum rate among the two sessions on the same day. Cells are ordered by peak firing locations in Run1 or Pre along the track (x-axis). (b) Cumulative distributions of PV correlations between Run1 and Run2, between Pre and Re-exposure, and between Pre and shuffled Pre. P = 6 × 10⁻¹⁸ (Run1/Run2 vs. Pre/Re-exposure), P = 1 × 10⁻¹³³ (Pre/Re-exposure vs. Pre/Shuffle), ranksum test. Number of spatial bins: N = 216 (Run1/Run2), 288 (Pre/Re-exposure), 288000 (Pre/Shuffle). (c) PV correlations between Run1 and Run2 on Day1, and between Pre and Re-exposure on Day2 along the track. Note that PV correlations within the SZ were relatively similar between Day1 and Day2 (P = 0.08, ranksum test). (d) Distributions of mean firing rate change between Run1 and Run2 on Day1 (N = 100 cells) and between Pre and Re-exposure on Day2 (N = 147). n.s.: P = 0.07, Levene’s test for variance comparison. t-test for mean comparison: P = 0.33, t₂₄₅ = 0.97. (e) Same as (d), but for distributions of spatial correlation. ***P = 2 × 10⁻⁵, ranksum test. (f) Peak firing locations in Pre and Re-exposure of those place cells that became silent, became active, or relocated from Pre to Re-exposure on Day2. Each row is a cell. Dashed line: SZ boundary. (g, h) Distributions of spatial information change (g) and peak location shift (h) between Run1 and Run2 on Day1 and between Pre and Re-exposure on Day2 for those cells active in both sessions (N = 80 on Day1, 106 on Day2). ***P = 1 × 10⁻⁴ (g), n.s.: 0.83 (h), Levene’s test for comparing variances. Ranksum test for comparing medians: P = 0.73 (g), 0.97 (h).