Figure 1. Strong aversive experience drives retrospective memory-linking to a neutral context learned days ago.

A) Schematic of prospective vs retrospective memory-linking behavior experiment. Mice either received a Neutral experience followed by an Aversive experience two days later (Retrospective) or the Aversive experience followed by Neutral (Prospective). One day after the second experience, mice were tested in the Aversive context they were shocked in. The following day, mice were tested in either the previously experienced Neutral context or a Novel context.

B) Freezing during Aversive recall in Prospective vs Retrospective groups. There was no difference in Aversive recall freezing between Prospective & Retrospective conditions (t₃₄ = 0.36, p = 0.72) (Retrospective, N = 16 mice; Prospective, N = 20 mice).

C) Freezing during Neutral vs Novel recall in Prospective vs Retrospective groups. There was a significant interaction between freezing in Neutral vs Novel recall in the Retrospective vs Prospective groups, suggesting the Aversive experience retrospectively linked to the Neutral memory, but not prospectively. Significant interaction between Direction (Prospective vs Retrospective) and Context (Neutral vs Novel), (F_1,32 = 4.90, p = 0.034) (Retrospective Neutral, N = 8 mice; Retrospective Novel, N = 8 mice; Prospective Neutral, N = 12 mice, Prospective Novel, N = 8 mice). Post-hoc, Retrospective (t₃₂ = 2.586, p = 0.029), Prospective (t₃₂ = 0.452, p = 0.6546).

D) Schematic of Low Shock vs High Shock retrospective memory-linking experiment. Mice received a Neutral experience followed by a 1hr offline session in their homecage. Two days later, they received either 3 low shocks (0.25mA) or 3 high shocks (1.5mA, same amplitude as in Figure 1A) in an Aversive context, followed by another 1hr offline session in their homecage. The following day they were tested in the Aversive context, and for the following two days they were tested in the Neutral and Novel contexts, counterbalanced. Calcium imaging was performed during all the sessions.

E) Freezing during Aversive recall in Low vs High Shock mice. Mice froze more in the Aversive context after receiving a high shock vs low shock (t_18.8 = 5.877, p = 0.000012) (Low Shock, N = 10 mice; High Shock, N = 12 mice).

F) Freezing during Neutral vs Novel recall in Low vs High Shock mice. Mice only displayed enhanced freezing in Neutral vs Novel (i.e., retrospective memory-linking) after High Shock and not Low Shock. Significant effect of Context (Neutral vs Novel) (F_1,20 = 17.32, p = 0.000048) and significant interaction between Context and Amplitude (F_1,20 = 4.99, p = 0.037) (Low Shock, N = 10 mice; High Shock, N = 12 mice). High Shock mice froze more in the Neutral vs Novel contexts (t₁₁ = 4.37, p = 0.002) while Low Shock mice froze no differently in the two contexts (t₉ = 1.23, p = 0.249).

G) Correlation between Aversive recall freezing and memory-linking strength. The strength of the aversive memory was correlated with the degree of retrospective memory-linking in High Shock mice (R₂ = 0.45, p = 0.016), but not in Low Shock mice (R² = 0.0003, p = 0.963) (Low Shock, N = 10 mice; High Shock, N = 12 mice).