Figure 1. Weakening fear memory through deconditioning-update training.

(A) Experimental design: male rats were fear-conditioned with five tone-shock pairings (context A; 5 CS + US, 0.5mA). 48 hr later, the no-footshock and footshock (deconditioning-update) groups were exposed to four daily reactivation sessions (context B). After this, animals underwent test (context B), renewal (context A) and spontaneous recovery (context B) sessions. Black circles represent context A, while white rectangles represent context B. (B) Freezing levels during reactivation sessions. Rats exposed to weak footshocks during reactivation sessions showed a significant reduction in freezing responses, maintained during the test (C), renewal (E) and spontaneous recovery (D) sessions. (F) Experimental design: female rats were fear-conditioned (context A; 5CS+US, 0.5mA). 48 hr later, the no-footshock and footshock groups were exposed to four daily reactivation sessions (context B). After this, all groups underwent test, renewal, and spontaneous recovery sessions. Animals were reconditioned (context A; 3CS+US, 0.5mA) on the next day and retested 24 hr later. (G) Freezing levels during memory reactivation. Rats exposed to weak footshocks showed a significant reduction in freezing responses, maintained during the test (H), renewal (I), spontaneous recovery (J) and retraining test (K) sessions. Bars represent mean ± SEM. Statistical comparisons were performed using two-way repeated-measures ANOVA followed by a Bonferroni post-hoc (reactivation sessions) or one-way ANOVA followed by Tukey post-hoc (test, renewal, spontaneous recovery, and retraining test). *p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001. For full statistics, see Supplementary file 1. For pre-CS freezing values, see Supplementary file 12.

Figure 1—figure supplement 1. Deconditioning-update does not occur with 0.3-mA shocks.

(A) Experimental design: rats were fear-conditioned with five tone-shock pairings (context A; 5 CS + US, 0.5mA). Starting 48 hr later, animals were exposed to four daily reactivation sessions (context B) with or without an intermediate footshock (0.3mA) at the end of tones. Subsequently, all groups underwent test sessions (context B). Black circle represents context A and white rectangles represents context B. (B) Freezing levels during reactivation sessions. Rats exposed to the intermediate footshock (0.3mA) showed less freezing reduction than no-footshock animals across sessions. (C) The no-footshock group expressed lower freezing in the test compared with footshock animals or homecage controls. Statistical comparisons are performed using two-way repeated-measures ANOVA followed by a Bonferroni post-hoc (reactivation sessions) or one-way ANOVA followed by a Tukey post-hoc (test and spontaneous recovery). Bars represent mean ± SEM. *p<0.05; **p<0.005. For full statistics, see Supplementary file 5. For pre-CS freezing, see Supplementary file 15.

Figure 1—figure supplement 2. A single reactivation session does not update fear memory.

(A) Experimental design: rats were fear-conditioned with five tone-shock pairings (context B; 5 CS + US, 0.5mA). 48 hr later, animals were exposed to a single reactivation session (context B) with or without a weak (0.1 mA) footshock. Animals then underwent test (context B), and renewal (context A) sessions. Black circles represent context A and white rectangles represents context B. There were no significant differences in freezing between groups in the reactivation (B), test or renewal sessions (C). Bars represent mean ± SEM. Statistical comparisons are performed using Student’s t test (reactivation sessions) or one-way ANOVA followed by a Tukey post-hoc (test and renewal). For full statistics, see Supplementary file 6. For pre-CS freezing, see Supplementary file 16.

Figure 1—figure supplement 3. Deconditioning-update is not due to US devaluation.

(A) Experimental design for devaluation: animals received five conditioning trial tones (CS) that co-terminated with a 0.5-mA, 1 s footshock (US) in context A. On days 3 to 6, the footshock group received 3 USs (0.1-mA footshock) at the end of the tone in context B, while the devaluation group received the same shocks in a different context (context C) without tone, and control animals remained in their home cages. Black circle represents context A, while white rectangles and hexagon represent contexts B and C, respectively. (B) On day 7, both groups were tested in context B with 3 CSs (tones), and the footshock group showed a decrease in freezing responses compared to the other two groups. (C) Experimental design for reinstatement: rats were fear-conditioned with five tone-shock pairings (context A; 5 CS + US, 0.5mA). Starting 48 hr later, animals were exposed to four daily reactivation sessions (context B) with or without a weak footshock (0.1 mA) at the end of tones. On day 7, both groups were tested. On the following day, animals received two 2-s non-paired footshock (reinstatement) in a different context followed by another test 24 hr later. Freezing levels were similar during reactivation (D) and test sessions, but rats exposed to the weak footshock during reactivation sessions showed less freezing responses after reinstatement (E). Bars represent mean ± SEM. Statistical comparisons are performed using two-way repeated-measures ANOVA followed by a Bonferroni post-hoc (reactivation sessions) or one-way ANOVA followed by a Tukey post-hoc (test and reinstatement sessions). *p<0.05; **p<0.005. ****p<0.0001. For full statistics, see Supplementary file 7. For pre-CS freezing, see Supplementary file 17.

Figure 1—figure supplement 4. Deconditioning-update does not occur with unpaired shocks in the reactivation sessions.

(A) Experimental design: rats were fear-conditioned with five tone-shock pairings (context A; 5 CS + US, 0.5mA). Starting 48 hr later, animals were exposed to four daily reactivation sessions (context B), where weak 0.1 mA footshocks were presented either paired (i.e. at the end of each tone) or unpaired (i.e. in pseudorandom moments during the session) with the CSs. Subsequently, all groups were tested in context B. Black circle represents context A and white rectangles represents context B. (B) Freezing levels during reactivation sessions. Rats exposed to unpaired footshocks showed higher freezing levels across sessions than those exposed to paired ones. (C) The paired CS-US group expressed lower freezing in the test compared with the unpaired group. Statistical comparisons are performed using two-way repeated-measures ANOVA followed by a Bonferroni post-hoc (reactivation sessions) or Student’s t test (test session). Bars represent mean ± SEM. ***p<0.001. For full statistics, see Supplementary file 8. For pre-CS freezing, see Supplementary file 18.