Directly reactivated, but not indirectly reactivated, memories undergo reconsolidation in the amygdala

Abstract

Memory consolidation refers to a process by which newly learned information is made resistant to disruption. Traditionally, consolidation has been viewed as an event that occurs once in the life of a memory. However, considerable evidence now indicates that consolidated memories, when reactivated through retrieval, become labile (susceptible to disruption) again and undergo reconsolidation. Because memories are often interrelated in complex associative networks rather than stored in isolation, a key question is whether reactivation of one memory makes associated memories labile in a way that requires reconsolidation. We tested this in rats by creating interlinked associative memories using a second-order fear-conditioning task. We found that directly reactivated memories become labile, but indirectly reactivated (i.e., associated) memories do not. This suggests that memory reactivation produces content-limited rather than wholesale changes in a memory and its associations and explains why each time a memory is retrieved and updated, the entire associative structure of the memory is not grossly altered.

Fig. 1.

First-order fear conditioning and SOFC each depend on associative learning. Rats received associative (paired, P) or nonassociative (unpaired, U) presentations of the CS and US during first- and second-order conditioning. Fear (freezing) responses were tested after completion of second-order conditioning. Results were analyzed by a two-factor ANOVA, with test CS (CS1, CS2) and training type (PP, PU, UP) as the factors (test CS was a repeated measure). The analysis revealed significant main effects of training type [F (2, 15) = 60.7; P < 0.000001] and test CS [F (1, 15) = 74.5; P < 0.000001] and a significant test CS × training type interaction: F (2, 15) = 34.0; P < 0.00001. Posthoc mean comparisons (Tukey’s honest significant difference text) showed that animals that received pairing of CS1 and the US in first-order conditioning (groups PP and PU) showed equivalent levels of freezing to CS1 (P = 0.99), but animals given unpaired presentations of CS1 and US during first-order conditioning (UP) did not freeze to CS1, indicated by a significant difference in freezing to CS1 between group UP and groups PP and PU (P < 0.001). Similarly, only group PP, which received paired presentations of CS1 and the US during first-order conditioning and paired presentations of CS2 and CS1 during second-order conditioning, showed significant freezing to CS2. Responses to CS2 were significantly greater in PP than in the other two groups (P < 0.001). These results indicate that both the first- and second-order paradigms depend on associative learning: first-order conditioning depends on the CS1–US association, and second-order conditioning depends on the CS2–CS1 association.

Fig. 2.

SOFC results in the formation of an associative network in which responding to CS2 depends on the first-order (CS1–US) association. After completion of first- and second-order conditioning, rats were exposed to CS1 or CS2 30 times on each of 2 consecutive days. Freezing to the CSs was examined during extinction training (a) and in a retention test (b) performed on the day after completion of extinction training. (a) Freezing during extinction training. Average freezing responses elicited by CS1 during the last four trials of CS1 extinction training (trials 57–60) were lower than responses elicited during the first four trials (trials 1–4). Similarly, average freezing responses elicited by CS2 during the last four trials of CS2 extinction training (trials 57–60) were lower than responses elicited during the first four trials (trials 1–4). The data were analyzed by a two-factor ANOVA, with extinction CS (CS1, CS2) and extinction training phase (first four trials, last four trials) as the factors (extinction training phase was a repeated measure). The ANOVA revealed a main effect of extinction training phase, due to the decrease in freezing between the beginning and end of extinction: F (1, 10) = 158.8; P < 0.000001. There was no main effect of stimulus type (P = 0.13), nor was there an interaction between stimulus type and extinction training phase (P = 0.37). Thus, extinction training with CS1 led to extinction of responses elicited by CS1, and extinction training with CS2 led to extinction of responses elicited by CS2 during the extinction session. (b) Freezing during the retention test. One day after the completion of extinction training, the rats received a LTM retention test in which they were exposed to CS2 and then CS1. The data were analyzed with a two-factor ANOVA, with extinction CS (CS1, CS2) and test CS (CS1, CS2) as the factors (test CS was a repeated measure). CS1 extinction training led to low levels of freezing to both CS2 and CS1, whereas CS2 extinction led to low levels of freezing to CS2 but not CS1. ANOVA revealed significant main effects of extinction CS [F (1, 10) = 114.3; P < 0.0001] and test CS [F (1, 10) = 213.8; P < 0.00001], and an extinction CS × test CS interaction [F (1, 10) = 202; P < 0.000001]. Post-hoc Tukey’s honest significant difference tests revealed that rats given extinction training with CS1 showed similarly low levels of freezing when tested with CS2 or CS1 (P = 0.99) (b), and that rats given extinction training with CS2 also had low levels of freezing to CS2 that did not differ from the low level of responses to CS1 (P = 0.52) and CS2 (P = 0.72) after extinction training with CS1. In contrast, freezing levels to CS1 were significantly higher than freezing to CS2 after CS2 extinction (P < 0.001) and significantly higher than both CS1 (P < 0.001) and CS2 (P < 0.001) after CS1 extinction (b). These results demonstrate that, in our protocol, extinction of freezing responses to first-order stimulus (CS1) leads to concurrent impairment of responding to CS2, but extinction of the second-order stimulus (CS2) impairs only freezing to CS2. Freezing to CS2 thus depends on the first-order association. This suggests that our SOFC protocol results in the formation of an associative network involving CS2 → CS1 → US. In this network, the first-order association (CS1–US) is activated directly by presenting CS1 and indirectly by presenting CS2.

Fig. 3.

The effects of protein synthesis inhibition in the LBA on postreactivation LTM depend on whether reactivation involved CS1 or CS2. Rats underwent SOFC and then were exposed to either CS1 or CS2, followed immediately by intraLBA infusion of ANISO or aCSF. Three hours later, postreactivation STM was tested. A separate group of rats underwent the same procedure, except that postreactivation LTM was tested. The two sets of data were analyzed with separate three-factor ANOVAs, with reactivation CS (CS1, CS2), drug (aCSF, ANISO), and retention test CS (CS1, CS2) as the factors (the retention test CS was a repeated-measures factor). (a and b) Protein synthesis inhibition in the LBA has no effect on postreactivation STM. Rats that received ANISO or aCSF infusions after exposure to CS1 (a) or CS2 (b) showed comparable levels of freezing to both CS1 and CS2 during the postreactivation STM test. The main effect of neither reactivation CS (P = 0.13) nor drug (P = 0.67) nor the interaction (P ≥ 0.7) was significant. There was a main effect of retention test CS [F (1, 24) = 45.7; P < 0.00001], indicating overall higher freezing to CS1 than CS2, which is commonly observed in studies of second-order conditioning (–29). (c and d) Protein synthesis inhibition in LBA affects postreactivation LTM but in different ways, depending on which stimulus was reactivated. In contrast to the postreactivation STM test (a and b), in the postreactivation LTM test (c and d), there was a significant drug × reactivation CS × retention test CS interaction [F (1, 25) = 16.5; P < 0.001], indicating that the pattern of ANISO effects on fear memory for CS1 and CS2 depended on whether the reactivation stimulus was CS1 (c) or CS2 (d). The first-order interaction between drug and retention test CS was significant for CS1 reactivation [F (1, 13) = 9.0, P < 0.05] and for CS2 reactivation [F (1, 12) = 7.65; P < 0.05]. Post-hoc Tukey’s honest significant difference test mean comparisons revealed that after reactivation with CS1, ANISO had a significant effect on responses in the postreactivation LTM test elicited by CS2 (P < 0.001) and CS1 (P < 0.045), but after reactivation with CS2, ANISO affected responses elicited by CS2 (P < 0.05) and not CS1 (P = 0.71). These findings indicate that for the first-order associative memory (CS1–US) to undergo protein synthesis-dependent reconsolidation, it must be directly reactivated by CS1. Indirect reactivation of the first-order association by CS2 via the CS2 → CS1 → US associative network, fails to induce reconsolidation.

Fig. 4.

Protein synthesis inhibition in LBA does not affect fear memory in the absence of memory reactivation. To confirm that the SOFC memories are consolidated at the time of reactivation, it is necessary to show that protein synthesis inhibition in the absence of memory reactivation has no effect. Rats underwent SOFC and then were given infusions of ANISO or aCSF into LBA. The next day, they received a LTM retention test in which CS2 and then CS1 were presented. Groups given ANISO and aCSF did not differ. Results were analyzed by two-factor ANOVA, with drug (aCSF, ANISO) and retention test CS (CS1, CS1) as the factors (retention test CS was a repeated measure). The ANOVA indicated that in the absence of stimulus exposure, there was neither a main effect of drug (P = 0.78) nor a CS × drug interaction (P = 0.46). There was a main effect of CS [F (1, 10) = 22.8; P < 0.001], indicating freezing was higher to CS1 than to CS2, a common occurrence in second-order conditioning studies (–29). This experiment demonstrates that reactivation of the memory by an exposure to the CS is necessary for protein synthesis blockade to impair the memory.