Extended Figure 3. Neurons active during Aversive encoding are selectively reactivated offline and during Aversive recall.

A) Representative maximum intensity projection of the field-of-view of one example session (left). Spatial footprints of all recorded cells during the session, randomly color-coded (right).

B) Schematic of a single aversive experience. Mice had an Aversive experience followed by a 1hr offline session in the homecage. The next day, mice were tested in the Aversive context, followed by a test in a Novel context one day later. Calcium imaging in hippocampal CA1 was performed during all sessions.

C) Mice acquired within-session freezing during Aversive encoding (left); main effect of time (F_8,56 = 12.59, p = 3.87e-10, N = 8 mice). And mice responded robustly to all three foot shocks, though their locomotion generally decreased across shocks, driven by increased freezing (right); main effect of shock number (F_2,14 = 7.45, p = 0.0154, N = 8 mice) and main effect of PreShock vs Shock (F_1,7 = 581, p = 5.38e-8, N = 8 mice), and no interaction.

D) Mice displayed a modest decrease in locomotion across the 1hr offline period (R² = 0.064, p = 1.9e-8, N = 8 mice).

E) Mice froze significantly more in the Aversive context than in a Novel context during recall (t₇ = 165, p = 4e-6, N = 8 mice).

F) Cells that were active during Aversive encoding and reactivated offline were significantly more likely to be reactivated during Aversive recall than cells active during Aversive encoding and not reactivated offline (t₇ = 19.41, p = 2e-7, N = 8 mice).

G) A larger fraction of cells active during Aversive recall than during Novel recall were previously active during Aversive encoding (t₇ = 6.897, p = 0.0002, N = 8 mice).

H) During the offline period, ~40% of the population was made up of cells previously active during Aversive encoding (top). This Aversive ensemble was much more highly active than the rest of the population during the offline period (bottom; A.U.) (t₇ = 8.538, p = 0.00006, N = 8 mice).

I) Each cell’s activity was compared during locomotion vs during quiet rest (left; A.U.). A regression line was fit to the cells in the Aversive ensemble and in the Remaining ensemble separately, for each mouse. The Remaining ensemble showed greater activity during locomotion than during quiet rest (i.e., a less positive slope). The Aversive ensemble showed relatively greater activity during quiet rest than locomotion (i.e., a more positive slope) across mice (right) (t₇ = 5.76, p = 0.047, N = 8 mice).

J) Cells that had high levels of activity (A.U.) during Aversive encoding continued to have high levels of activity during the offline period (example mouse; left). There was a linear relationship between how active a cell was during Aversive encoding and how likely it was to be reactivated during the offline period (all mice; right) (R² = 0.726, p = 1.25e-23, N = 8 mice).

K) During the offline period, cells that would go on to become active during recall were more highly active than the Remaining ensemble during the offline period. The top represents the proportion of each ensemble (legend to its right). The cells that would become active during both Aversive and Novel recall were most highly active (A.U.). There was no difference in activity in the cells that would go on to be active in Aversive or Novel. Main effect of Ensemble (F_3,21 = 27.81, p = 1.65e-7, N = 8 mice). Post-hoc tests: for Aversive vs Novel (t₇ = 1.33, p = 0.22), for Remaining vs Aversive ∩ Novel (t₇ = 11.95, p = 0.000007), for Remaining vs Aversive (t₇ = 3.97, p = 0.005), for Remaining vs Novel (t₇ = 7.47, p = 0.0001).