Abstract
It is widely accepted that fear memories are consolidated through protein synthesis-dependent changes in the basolateral amygdala complex (BLA). However, recent studies show that protein synthesis is not required to consolidate the memory of a new dangerous experience when it is similar to a prior experience. Here, we examined whether the protein synthesis requirement for consolidating the new experience varies with its spatial and temporal distance from the prior experience. Female and male rats were conditioned to fear a stimulus (S1, e.g., light) paired with shock in stage 1 and a second stimulus (S2, e.g., tone) that preceded additional S1-shock pairings (S2-S1-shock) in stage 2. The latter stage was followed by a BLA infusion of a protein synthesis inhibitor, cycloheximide, or vehicle. Subsequent testing with S2 revealed that protein synthesis in the BLA was not required to consolidate fear to S2 when the training stages occurred 48 h apart in the same context; was required when they were separated by 14 d or occurred in different contexts; but was again not required if S1 was re-presented after the delay or in the different context. Similarly, protein synthesis in the BLA was not required to reconsolidate fear to S2 when the training stages occurred 48 h apart but was required when they occurred 14 d apart. Thus, the protein synthesis requirement for consolidating/reconsolidating fear memories in the BLA is determined by similarity between present and past experiences, the time and place in which they occur, and reminders of the past experiences.
Significance Statement
Protein synthesis in the basolateral amygdala complex (BLA) is not required to consolidate the memory of a new dangerous experience when it is similar to a prior experience. This study is significant in showing that (1) when the new experience occurs after a delay or in a different context, the protein synthesis requirement is reinstated; (2) the effects of the delay and context shift are reversed by reminding animals of their prior experience; and (3) the delay affects reconsolidation of a new experience in the same way that it affects the initial consolidation. Thus, the neural mechanisms underlying memory are regulated by similarity/dissimilarity between present and past experiences, the time/place in which those experiences occur, and reminders of past experience.
Introduction
People readily learn about stimuli that signal danger. In the laboratory, this learning is studied using pavlovian threat or fear conditioning in rodents. In a standard protocol, rats are placed in a distinctive chamber where they receive pairings of an initially neutral conditioned stimulus (CS), such as a tone or a light, and an innately aversive unconditioned stimulus (US), such as footshock. Rats display defensive responses such as freezing when subsequently presented with the CS alone. These responses are taken to mean that the pairings produce an enduring memory of the association between the CS and US, which is retrieved by the CS and expressed in defensive responses indicative of fear in people.
The consolidation of a CS–US association in long-term memory depends on an array of cellular processes, including synthesis of new proteins in the basolateral amygdala complex (BLA). This is supported by findings that infusion of a protein synthesis inhibitor into the BLA immediately before or after a conditioning session spares the acquisition of fear responses to the CS but impairs their expression when the CS is tested days later (Schafe and LeDoux, 2000; Maren et al., 2003; Desgranges et al., 2008). Such findings have been taken to mean that new proteins are essential for stabilizing learning-induced changes in BLA neurons. However, these findings have emerged from studies involving a single fearful experience (one conditioning session), and recent evidence shows that the molecular mechanisms that support a second fearful experience may differ from those that support an initial fearful experience as a function of their similarity.
This evidence comes from studies that have used a two-stage training protocol (Leidl et al., 2018; Williams-Spooner et al., 2019). In stage 1, rats were exposed to pairings of a novel stimulus (S1; e.g., tone) and shock, which resulted in formation of an initial S1-shock fear memory. In stage 2, rats were exposed to a second novel stimulus (S2; e.g., light) in sequence with S1 and shock (S2-S1-shock sequence), which resulted in formation of a second fear memory involving the S2. Intra-BLA infusions of the protein synthesis inhibitor, cycloheximide, before or after stage 2 had no effect on the new S2 fear memory. In contrast, a BLA cycloheximide infusion disrupted fear to S2 among rats exposed to S2-S1-shock sequences in the absence of any prior training; and among rats exposed to S2-[trace]-shock pairings in stage 2 after having been exposed to S1-shock pairings in stage 1. Thus, the protein synthesis requirement for consolidating fear to S2 appears to be determined by the similarity between present and past experiences. When the experiences are dissimilar, consolidation of fear to S2 requires protein synthesis in the BLA; but when the experiences are similar (S1 is conditioned in stage 1 and present in stage 2), consolidation of fear to S2 does not require protein synthesis in the BLA.
The present study used the protocol described above to examine whether context and time regulate the protein synthesis requirement for consolidating fear to S2. It focused on context and time as both factors influence memory encoding and recall (Goodwin et al., 1969; Godden and Baddeley, 1975; Kahana, 1996; Yonelinas et al., 2019), but it is unknown whether they influence the molecular mechanisms of memory consolidation in the BLA. The initial experiments confirmed that, when a session of S2-S1-shock sequences occurs 48 h after a session of S1-shock pairings and in the same context, consolidation of fear to S2 does not require protein synthesis in the BLA. Subsequent experiments then examined whether the protein synthesis requirement is reinstated when the two stages of training are separated by 14 d or administered in different physical contexts; and whether any reinstatement can be reversed by reminding rats of the initial training after the 14 d delay or in the different context. We also examined the effect of a delay on the protein synthesis requirement for reconsolidating fear to the S2.
Materials and Methods
Subjects
Subjects were 307 experimentally naive adult (8–11 weeks old at the start of the experiment), male and female Long–Evans rats, obtained from the breeding facility at the University of New South Wales. Male and female rats were housed separately in plastic tubs (22 cm high × 67 cm long × 40 cm wide) with four males and up to eight females per tub. Rats were housed in a temperature-controlled colony room (22°C) on a 12 h light/dark cycle (lights on at 0700) with continuous access to food and water. Rats were handled once a day for at least 3 d prior to beginning behavioral procedures.
Apparatus
Behavioral procedures took place in two distinct contexts referred to as contexts A and B. Context A consisted of four identical chambers (30 cm high × 26 cm long × 30 cm wide) with a grid floor composed of rods that were 7 mm in diameter, spaced 18 mm apart. Context B was located in a different room and consisted of four identical chambers (30 cm high × 26 cm long × 30 cm wide) with a grid floor composed of rods that were 2 mm in diameter, spaced 15 mm apart. The front and back walls of both sets of chambers were made of Plexiglas and their side walls of aluminum. A tray containing corn cob bedding was located below the floor of each chamber. Each chamber was located in its own wooden sound- and light-attenuating compartment. A light source and speaker were mounted on the back wall of each compartment. The chambers were cleaned with water upon removal of a rat, and those in context B were scented with peppermint essence (four drops onto a plastic weigh boat located in the sound-attenuating cubicle). Each chamber was illuminated by an infrared light, and sessions were recorded via a camera mounted on the rear wall of the cubicle and connected to a computer located in another room in the laboratory.
Stimuli
The conditioned stimuli (CSs) were auditory and visual stimuli, counterbalanced in their roles as S1 and S2. The auditory stimulus was a 620 Hz tone of 70 dB intensity, measured at the center of the chamber (Dick Smith audiometer), and the visual stimulus was a light that flashed at 3.5 Hz with an intensity of 8 lux, measured at the center of the chamber (Lux meter). The unconditioned stimulus (US) was a 0.5 s duration, 0.8 mA intensity shock delivered through the grid floor via a custom-built, constant current generator. An ammeter was used to verify the shock intensity prior to the conditioning sessions.
Experimental design and statistical analyses
Scoring
Freezing was assessed by experimenters who were blind to the experimental group. It was defined as the absence of movement except that required for breathing (Fanselow, 1980) and was measured using a time sampling procedure whereby each rat was scored every 2 s as either freezing or not freezing. The number of samples scored as freezing was divided by the total number of samples to generate a percentage freezing.
Statistical analysis
Freezing data across training was analyzed using orthogonal contrasts with a between-subjects factor of group and a within-subject factor of trial. Freezing during the S2 and S1 test sessions was collapsed across trials, and the data were analyzed using orthogonal contrasts with a between-subjects factor of group. In each case, the criterion for rejection of the null hypothesis was set at α = 0.05. During the test sessions, baseline freezing in the context prior to stimulus presentations was low (<20%) and did not differ significantly between groups.
Surgical procedures
Rats were surgically implanted with bilateral cannulas targeting the BLA. They were anaesthetized with 2–5% inhalant isoflurane delivered in oxygen at a flow rate of 0.5 L/min. Rats received a preoperative subcutaneous injection of the nonsteroidal anti-inflammatory carprofen (5 mg/ml, Cenvet Australia). Following the onset of stable anesthesia, rats were mounted onto a stereotaxic apparatus, and 26-gauge guide cannulas were implanted into the BLA in both hemispheres (AP, −2.7 mm; ML, ±4.9 mm; DV, −8.4 mm from bregma). The guide cannulas were secured with four jeweler's screws and dental cement. Immediately after surgery, rats received a prophylactic, subcutaneous injection of penicillin (0.05 ml, 33 mg/kg) and were placed on a heating pad until recovered from the anesthetic. Rats were then returned to their home tub and allowed to recover for at least 7 d prior to behavioral procedures. During recovery, rats were handled and weighed daily.
Drug infusion
Cycloheximide (Sigma Aldrich) was dissolved in 70% ethanol to yield a 200 µg/µl stock solution which was diluted 1:4 with artificial cerebral spinal fluid (ACSF, Sigma Aldrich) to a final concentration of 40 µg/µl. The vehicle solution was prepared by diluting 70% ethanol 1:4 in ACSF. Infusions were carried out using injectors connected via plastic tubing to a 25 µl Hamilton syringe driven by an infusion pump. Cycloheximide or vehicle was administered at a volume of 0.5 µl bilaterally over a 2 min period (0.25 µl/min). The injectors were left in place for a further 2 min to allow for diffusion of the drug.
Histology
Following behavioral testing, rats received a lethal dose of sodium pentobarbital. Brains were removed and stored at −20°C prior to coronal sectioning (40 µm) on a cryostat. Sections containing the BLA were mounted onto glass slides and stained with cresyl violet. Cannula placements were determined with a light microscope using the BLA boundaries defined in the atlas of Paxinos and Watson (2007). Rats with cannula placements outside of these boundaries were excluded from statistical analysis. Figure 1 shows the cannula placements for all rats that were included in the study.
Cannula placements as verified by cresyl violet-stained sections. The black dots represent the most ventral point of the cannula track for each rat as determined using the atlas of Paxinos and Watson (2007).
Behavioral procedures
Experiment 1
The behavioral procedures in Experiment 1 were used in all subsequent experiments, unless otherwise indicated.
Context pre-exposure
On days 1–2, rats received twice daily exposures to the experimental chambers, one in the morning and the other ∼3 h later, in the afternoon. Each session lasted 20 min and was intended to familiarize rats with the chambers, thereby eliminating any neophobic reactions that could interfere with the detection of conditioned freezing.
Stage 1: S1-shock pairings (first-order conditioning)
On day 3, rats received four S1-shock pairings spaced 5 min apart. Each presentation of the 10 s S1 (light or tone) coterminated with a 0.5 s × 0.8 mA shock. The first S1-shock pairing occurred 5 min after placement in the chamber, and rats were removed from the chamber 1 min after the last shock.
Context extinction
Twenty-four hours prior to serial-order conditioning, rats received two, 20 min exposures to the experimental chambers (context) in the absence of any scheduled events, one in the morning and one in the afternoon. The purpose of these sessions was to extinguish any context-elicited freezing that would otherwise mask freezing to the discrete stimuli during the subsequent stage of conditioning.
Stage 2: S2-S1-shock sequences (serial-order conditioning)
Rats in Groups P2 and U2 received the second conditioning session 2 d after S1-shock pairings (day 5). Rats in Groups P14 and U14 received the second conditioning session 14 d after S1-shock pairings (day 18). Rats in the paired groups (P2 and P14) received four S2-S1-shock sequences spaced 5 min apart. Offset of the 30 s S2 (tone or light) co-occurred with onset of the 10 s S1 (tone or light, respectively), which coterminated in the 0.5 s × 0.8 mA shock. The first S2-S1-shock sequence occurred 5 min after placement in the chamber, and rats were removed from the chamber 1 min after the last shock. Rats in the unpaired groups (U2 and U14) received equivalent exposures to the S2 and S1-shock but in an explicitly unpaired arrangement. Specifically, rats received four S2-alone presentation with a 2.5 min intertrial interval (ITI), followed by four S1-shock pairings, with a 2.5 min intertrial interval.
Context extinction
Twenty-four hours after serial-order conditioning, rats received two, 20 min exposures to the context alone, one in the morning and the other 3 h later in the afternoon. They also received an additional 10 min context-alone exposure the following morning. These exposures were intended to reduce any context-elicited freezing that would obscure the test levels of freezing elicited by S2 and S1.
Testing
Twenty-four hours after context extinction, rats were tested for freezing to the S2. They received S1 testing on the following day. Each test consisted of eight stimulus-alone presentations, spaced 3 min apart. The duration of the stimuli was identical to that used in initial conditioning: 10 s for S1 and 30 s for S2.
Experiment 2
The behavioral procedures were identical to those described for the paired groups in Experiment 1. Briefly, rats received 2 d of context exposure, followed by S1-shock pairings. Twenty-four hours prior to stage 2, rats received context extinction. Stage 2 took place either 2 d (Groups 2 d) or 14 d (Groups 14 d) after initial S1-shock pairings. In stage 2, rats were exposed to S2-S1-shock sequences, followed immediately by intra-BLA infusion of either vehicle or the protein synthesis inhibitor, cycloheximide (CHX). Twenty-four hours later, they received context extinction, followed by testing as previously described in Experiment 1.
Experiment 3
As in Experiment 2, rats received S1-shock pairings (stage 1) followed 2 d or 14 d later by exposure to S2-S1-shock sequences and a BLA infusion of CHX or VEH (stage 2). The difference between the two experiments was that rats received a reminder (the S1 alone) either 2 d after initial conditioning in stage 1 (Early reminder) or 2 d prior to S2-S1-shock sequences in stage 2 (Late reminder). Briefly, rats received 2 d of context exposure, followed by S1-shock pairings. Either 2 (Early reminder) or 12 (Late reminder) days later, rats were returned to the conditioning chambers where they received two presentations of the S1 alone with a 2 min ITI. The first presentation occurred 5 min after placement in the chamber, and rats were removed 2 min after the second presentation. The following day they received context extinction, followed 24 h later by S2-S1-shock sequences and infusion of either CHX or VEH. Rats then received context extinction and testing as previously described in Experiment 1.
Experiment 4
The next experiment examined the requirement for protein synthesis in the reconsolidation of fear to the S2. The design was similar to Experiment 2, except that 48 h after the session containing the S2-S1-shock sequences in stage 2, rats received an S2 reminder session, followed immediately by an intra-BLA infusion of CHX or VEH. Briefly, rats received 2 d of context exposure, followed by S1-shock pairings. Twenty-four hours prior to stage 2, rats received context extinction. Stage 2 took place either 2 d (Groups 2 d) or 14 d (Groups 14 d) after S1-shock conditioning in stage 1 and consisted of four S2-S1-shock sequences. Rats then received another day of context extinction, followed 24 h later by S2 reactivation. S2 reactivation consisted of two presentations of the S2 alone separated by a 2 min ITI. The first presentation occurred 5 min after placement in the chamber and rats were removed 2 min after the second presentation. Immediately after the reactivation session, rats received an intra-BLA infusion of either CHX or VEH. Rats then received context extinction and testing as previously described in Experiment 1.
Experiment 5
The next set of experiments examined the effect of shifting the context between the two stages of training. In Experiment 5, rats received S1-shock training in either the same chambers used in the previous experiments (context A) or a different set of chambers (context B). Briefly, rats received 2 d of context exposure to both sets of chambers (days 1–2). The order of context exposure was counterbalanced, with half of the rats being exposed to context A and then to context B and vice versa for the remainder. On day 3, rats received four S1-shock presentations in either context A (Group Same) or context B (Group Different). On day 4, rats underwent two, 20 min sessions of context extinction, one in context A and one in context B. This was intended to reduce freezing elicited by the S1 conditioning context and, critically, to enable detection of discrimination between the two contexts. On day 5, all rats received stage 2 conditioning in context A. The identity of the stage 2 conditioning context was not counterbalanced so as to maintain continuity in the chambers in which serial-order conditioning took place across all experiments. Rats in group Same Paired and Different Paired received four S2-S1-shock sequences with a 5 min ITI. The remaining rats (Groups Same Unpaired and Different Unpaired) received equivalent exposures to S2 and S1-shock but in an explicitly unpaired arrangement. Specifically, rats received four S2-alone presentation with a 2.5 min ITI, followed by four S1-shock pairings, with a 2.5 min ITI. All groups remained in the chambers for 1 min following the last shock. On day 6, rats received context extinction in context A, followed by S2 testing (day 7) and S1 testing (day 8) in context A.
Experiment 6
Training was similar to the Paired groups in Experiment 5. Briefly, rats received 2 d of context exposure to both sets of chambers (days 1–2). On day 3, rats received four S1-shock pairings in either context A (Groups Same) or context B (Groups Different). On day 4, rats underwent two, 20 min sessions of context extinction, one in context A and one in context B. On day 5, rats received four S2-S1-shock sequences in context A, followed immediately by an intra-BLA infusion of CHX or VEH. On day 6, rats received context extinction in context A, followed by S2 and S1 testing in context A on days 7 and 8, respectively.
Experiment 7
Experiment 7 was similar to Experiment 6, except that rats received a reminder (two presentations of the S1) in either context A or context B prior to stage 2. Briefly, rats received 2 d of context exposure to both sets of chambers (days 1–2) followed by S1-shock pairings in context B (day 3) and context extinction in both sets of chambers (day 4). On day 5, rats were returned to either context A (Groups A reminder) or context B (Groups B reminder) where they received two presentations of the S1 alone with a 2 min ITI. The first presentation occurred 5 min after placement in the chamber, and rats were removed 2 min after the second presentation. They then received S2-S1-shock sequences in context A (day 6). Immediately after serial-order conditioning on day 6, rats received an intra-BLA infusion of either CHX or VEH. On day 7, rats received context extinction in context A, followed by S2 and S1 testing in context A on days 8 and 9, respectively.
Results
Experiment 1
Research indicates that fear conditioning can result in a temporary increase in neural excitability (Zhou et al., 2009; Rashid et al., 2016; Josselyn and Frankland, 2018), which may alter the way that neurons store subsequent information. Accordingly, the first set of experiments examined whether the interval of time between the S1-shock pairings in stage 1 (which are likely to excite amygdala neurons) and S2-S1-shock sequences in stage 2 influences the protein synthesis requirement for consolidating the S2 fear memory. We hypothesized that extending the interval between the training sessions in stages 1 and 2 from 2 d (used previously) to 14 d would reinstate the effect of a BLA cycloheximide infusion in disrupting consolidation of fear to S2. As a first step toward testing this hypothesis, Experiment 1 examined whether acquisition of fear to the S2 in these two conditions (serial-order conditioning of S2 occurring 2 d or 14 d after conditioning of S1) is associatively mediated (i.e., due to S2 immediately preceding the additional S1-shock pairings). Four groups of rats were exposed to S1-shock pairings in stage 1. Two groups received stage 2 training 2 d later (2 d groups), while the other two groups commenced stage 2 training 14 d later (14 d groups). During this stage, one group in each pair was exposed to four S2-S1-shock sequences (Paired; Groups P2 and P14), while the other group was exposed to further S1-shock pairings and temporally separated presentations of S2 alone (Unpaired; Groups U2 and U14). Rats were then tested for fear to S2 and S1. The question of interest was whether fear to the S2 was dependent on its presentation in sequence with the S1 and shock at the two time-points, which would be evident as more freezing to S2 in the paired groups relative to the unpaired groups.
There was a significant increase in freezing across S1-shock pairings (Fig. 2B; F(1,28) = 771.89; Fc = 4.20; p < 0.001). There were no significant between-group differences or group × trend interactions, indicating that acquisition of fear to the S1 was equivalent between the 2 d and the 14 d groups and did not differ between paired and unpaired groups (Fs < 2.0; Fc = 4.20; p > 0.05).
Acquisition of fear to the S2 is associatively mediated when it is conditioned 2 or 14 d after the prior conditioning of S1. A, Experimental timeline of conditioning and testing. Rats received either paired or unpaired presentations of S2 and S1-shock either 2 d or 14 d after S1-shock pairings. B, Mean (±SEM) levels of freezing to presentations of the S1 during S1-shock pairings. C, Mean (±SEM) levels of freezing to presentations of the S2 and S1 during S2-S1-shock sequences. D, E, Mean (+SEM) freezing to test presentations of the S2 and S1 alone. n = 8 for all groups.
During stage 2 (Fig. 2C), rats in the paired groups displayed significantly higher freezing to the S2 than those in the unpaired groups (F(1,28) = 26.41; Fc = 4.20; p < 0.001). There was no overall difference in freezing levels between the 2 d and the 14 d groups (F(1,28) = 0.07; Fc = 4.20; p = 0.80) and no significant time interval × pairing interaction (F(1,28) = 4.13; Fc = 4.20; p = 0.052). Averaging across groups, there was a significant increase in freezing across conditioning trials (F(1,28) = 52.37; Fc = 4.20; p < 0.001). This effect was driven by the paired groups, which increased freezing across S2-S1-shock sequences, while freezing in the unpaired groups remained low, resulting in a significant paired × trend interaction (F(1,28) = 10.67; Fc = 4.20; p < 0.01). There was no significant time interval × trend interaction (F(1,28) = 1.61; Fc = 4.20; p = 0.215), although there was a significant time × paired × trend interaction (F(1,28) = 4.34; Fc = 4.20; p < 0.05), indicating that the increase in freezing to the S2 across trials was more pronounced for rats in the paired 2 d group (P2) relative to those in the paired 14 d group (P14). Freezing to the S1 across stage 2 decreased across trials (F(1,28) = 5.60; Fc = 4.20; p < 0.05), likely reflecting an increase in fear-elicited escape behaviors. Freezing levels to the S1 did not differ between the groups, and there were no significant interactions involving any between- or within-subjects factors (Fs < 2.2; Fc = 4.20).
At test (Fig. 2D,E), freezing levels to the S2 were higher in the paired than the unpaired groups (F(1,28) = 105.22; Fc = 4.20; p < 0.001). In contrast, the paired and unpaired groups did not significantly differ in levels of freezing to the S1 (F(1,28) = 0.026; Fc = 4.20; p = 0.87). In both cases, there was no significant effect of the time interval between conditioning stages or time × context interaction (Fs < 2.4; Fc = 4.20). Together, this indicates that responding to S2 was not due to generalized freezing from the conditioned S1. Instead, conditioning to S2 occurred when it was presented in sequence with S1 and shock (S2-S1-shock), and this was true when the interval between conditioning to S2 and the prior conditioning of S1 was 2 d or 14 d.
Experiment 2
This experiment examined whether increasing the interval between the two training stages from 2 to 14 d alters the protein synthesis requirement for consolidating the S2 fear memory. Rats received S2-S1-shock training either 2 d or 14 d after initial S1-shock conditioning. Immediately after the session containing S2-S1-shock sequences, rats were infused with either cycloheximide or vehicle into the BLA. They were then tested for fear to the S2 and also to the S1. We hypothesized that the protein synthesis requirement for consolidating fear to S2 would increase with the interval between stages 1 and 2 of training and, hence, that rats in Group 14 d-CHX would freeze less during the final test with S2 than rats in the remaining groups.
Rats acquired fear to S1 in stage 1 and to S2 in stage 2 (Fig. 3B,C). In stage 1, freezing to the S1 increased across its pairing with shock (F(1,49) = 476.61; Fc = 4.04; p < 0.001). There were no significant group differences or group × trend interactions, indicating that the rate at which freezing was acquired to S1 was the same in each of the groups (Fs < 3.10; Fc = 4.04). Similarly, freezing to S2 increased across the four S2-S1-shock sequences in stage 2 (F(1,49) = 445.72; Fc = 4.04; p < 0.001), and there were no significant differences between groups or group × trend interactions (Fs < 2.0; Fc = 4.04). Freezing to the S1 during stage 2 did not differ between groups, and there were no significant differences in the between- or within-subjects factors (Fs < 3.9; Fc = 4.04).
Protein synthesis is required in the BLA for consolidation of fear to the S2 when the two stages occur 14 d apart, but not when they occur 2 d apart. A, Experimental timeline of conditioning and testing for rats that received intra-BLA infusions of CHX or VEH. B, Mean (±SEM) levels of freezing to presentations of the S1 across its pairings with shock. C, Mean (±SEM) levels of freezing to presentations of the S2 and S1 across the S2-S1-shock sequences. D, E, Mean (±SEM) freezing across test presentations of S2 alone and S1 alone. n = 13–14 for all groups.
During S2 test, rats in Group 14 d-CHX showed significantly lower freezing than rats in the remaining groups (F(1,49) = 11.54; Fc = 4.04; p < 0.01; Fig. 3D), indicating that, in the long delay condition, consolidation of the S2 fear memory had been disrupted by the BLA infusion of cycloheximide. There was no significant difference in freezing levels between the 2 d-CHX group and the two VEH groups (F(1,49) = 0.23; Fc = 4.04), replicating previous research showing that consolidation of fear to the S2 does not require de novo protein synthesis when S1-shock and S2-S1-shock training occur 2 d apart (Leidl et al., 2018; Williams-Spooner et al., 2019). Finally, there were no significant differences between the two VEH control groups (F(1,49) = 0.33; Fc = 4.04), indicating that fear to the S2 was equivalent regardless of whether S1-shock training occurred 2 d or 14 d prior to S2-S1-shock training. During S1 test, there were no significant differences between groups (Fs < 0.62; Fc = 4.04; Fig. 3E). Together, these results indicate that the time between stages influences the protein synthesis requirement for consolidating the S2 fear memory in the BLA. Protein synthesis in the BLA was not needed to consolidate fear to S2 when it was established 2 d after the prior conditioning of S1 but was needed to consolidate fear to S2 when it was established 14 d after the prior conditioning of S1.
Experiment 3
Consolidation of the S2 fear memory required protein synthesis in the BLA when the S2-S1 shock sequences occurred 14 d after the prior S1-shock pairings but not when the sequences occurred just 2 d after the S1-shock pairings. Experiment 3 examined whether reminding rats of their prior conditioning experience with S1 would render consolidation of S2 insensitive to the effects of the protein synthesis inhibitor, cycloheximide. Four groups of rats received S1-shock pairings in stage 1 and, 2 weeks later, S2-S1-shock sequences in stage 2. Immediately after the stage 2 session, two groups of rats received an intra-BLA infusion of cycloheximide, while the remaining two groups received an infusion of vehicle. Within these pairs, the groups differed with respect to when they were reminded of their stage 1 experience, which involved two presentations of the S1 alone. One group in each pair received the reminder session early in the interval between the S1-shock pairings in stage 1 and S2-S1-shock sequences in stage 2 (i.e., 2 d after the S1-shock pairings), while the other group received the reminder session later in the interval (2 d prior to the S2-S1-shock sequences). We hypothesized that, when the reminder occurred close to the conditioning of S2 (i.e., later in the interval), it would functionally bridge the gap between the stage 1 and stage 2 experiences and, thus, reverse the effect of time on the protein synthesis requirement for consolidating fear to S2. Hence, we predicted that the BLA infusion of cycloheximide would disrupt consolidation of fear to S2 among rats exposed to the reminder early in the interval (Early CHX), but not among rats exposed to the reminder later in the interval. That is, we predicted that rats in Group Early CHX would freeze less when tested with S2 alone than rats in the remaining groups and that rats in the remaining groups would not differ from each other.
Conditioning was successful. There was a significant increase in freezing across S1-shock pairings in stage 1 (Fig. 4B; F(1,48) = 284.71; Fc = 4.04; p < 0.001). There was an unexpected between-group difference (F(1,48) = 4.37; Fc = 4.30; p < 0.05), with rats in the early reminder group showing more freezing than those in the late reminder group. Importantly, this difference was not maintained across S1 reminder presentations, with all groups showing similar levels of freezing to the S1 (Fig. 4C; Fs < 0.8; Fc = 4.30). It was also not evident across the S2-S1-shock sequences in stage 2: there were no significant group differences or group × trial interactions in levels of freezing to the S1 (Fig. 4D; Fs < 0.3; Fc = 4.30). Importantly, freezing to the S2 increased across the sequences and did so equally across the groups. This was confirmed in a main effect of trial (F(1,48) = 195.07; Fc = 4.04; p < 0.001) but no significant between-group effects or group × trial interactions (Fs < 2.0; Fc = 4.30).
Reminding rats of their prior experience reverses the effect of a time delay on the protein synthesis requirement for consolidating fear to the S2, but only if that reminder occurs shortly prior to stage 2. A, Experimental timeline of conditioning, reminder, and testing for rats that received intra-BLA infusions of CHX or VEH. B, Mean (±SEM) levels of freezing to S1 across its pairing with shock, to S1 across reminder presentations (C), and to S2 and S1 across S2-S1-shock sequences (D). E, F, Mean (±SEM) levels of freezing to S2 (E) and S1 (F) across test presentations. Early VEH, n = 13; Early CHX, n = 14; Late VEH, n = 10; Late CHX, n = 15.
At S2 test (Fig. 4E), rats in Group Early CHX froze significantly less than the remaining groups (F(1,48) = 8.32; Fc = 4.04; p < 0.01). There was no significant difference in freezing levels between-group Late CHX and the VEH groups or between the two VEH control groups (Fs < 2.0; Fc = 4.04). During S1 test, all groups showed similar levels of freezing (Fig. 4F). Statistical analysis confirmed that there was no significant group differences or interactions in levels of freezing to the S1 at test (Fs < 2.1; Fc = 4.04). These results indicate that reminding rats of their stage 1 experience (S1-shock) can render consolidation of the S2 fear memory independent of protein synthesis in the BLA and that the effect of a reminder depends on when the reminder is presented. When the reminder is temporally distant from the session of S2-S1-shock sequences, consolidation of fear to S2 requires protein synthesis in the BLA. In contrast, when the reminder is presented close to the session of S2-S1-shock sequences, consolidation of fear to S2 ceases to require protein synthesis in the BLA.
Experiment 4
Memory retrieval can return consolidated memories to a labile state, from which they must be reconsolidated in long-term memory; and this reconsolidation has been shown to depend on de novo protein synthesis in the amygdala (Nader et al., 2000; Duvarci et al., 2005). The goal of the present experiment was to determine whether the protein synthesis requirement for reconsolidation of fear to S2 in the protocol used above is affected by the interval between training stages in the same way that the protein synthesis requirement for consolidation of fear to S2 is affected by this interval. Four groups of rats were exposed to S1-shock pairings in stage 1 and S2-S1-shock sequences in stage 2. For two groups of rats, the interval between the training stages was 2 d; for the remaining two groups, this interval was 14 d. Two days after stage 2 had been completed, all rats were re-exposed to two presentations of the S2 alone in order to reactivate the S2 fear memory. For one group in each pair, this was immediately followed by a BLA infusion of cycloheximide (Groups 2 d-CHX and 14 d-CHX) or vehicle (Groups 2 d-VEH and 14 d-VEH). Finally, all rats were tested with presentations of S2 alone. We hypothesized that the pattern of test results would mirror that seen for initial consolidation: that is, protein synthesis in the BLA would be required for reconsolidation of fear to S2 when the stages of training were separated by 14 d but not when they were separated by 2 d. This would be evident as less freezing to S2 in Group 14 d-CHX compared with the other groups.
The levels of freezing to S1 and S2 during training are presented in Figure 5B,C. Rats showed an increase in freezing to the S1 across its pairings with shock in stage 1 (F(1,32) = 119.39; Fc = 4.15; p < 0.001) and to the S2 across the S2-S1-shock sequences in stage 2 (F(1,32) = 212.54; Fc = 4.15; p < 0.001). Levels of freezing to the S1 did not change across stage 2 (F(1,32) = 0.069; Fc = 4.15; p = 0.79). There were no significant differences between the groups in levels of freezing to either the S1 or S2 across training, and there were no significant group × trial interactions (Fs < 3.5; Fc = 4.15). Further, there were no significant differences between groups in levels of freezing to the S2 during the reactivation session (Fig. 5D; Fs < 0.8; Fc = 4.15).
Inhibition of protein synthesis disrupts reconsolidation of fear to the S2 when the two stages occur 14 d apart but not when they occur 2 d apart. A, Experimental timeline of conditioning, reminder, and testing. Mean (±SEM) levels of freezing to S1 across its pairing with shock (B), to the S1 and S2 across S2-S1-shock sequences (C), to the S2 across the reactivation session (D). Mean (±SEM) levels of freezing to S2 (E) and S1 (F) across test presentations. 2 d VEH, n = 11; 2 d CHX n = 8; 14 d VEH, n = 9; 14 d CHX, n = 8.
At test, rats in Group 14 d-CHX showed significantly less freezing to the S2 than rats in the other three groups (Fig. 5E; F(1,32) = 21.25; Fc = 4.15; p < 0.001). There were no significant differences in freezing to the S2 between Group 2 d-CHX and the VEH control groups (F(1,32) = 0.01; Fc = 4.15; p = 0.93) or between the two vehicle groups (Fs < 1.3; Fc = 4.15). Finally, there were no significant between-group differences in levels of freezing to the S1 at test (Fig. 5F; Fs < 2.6; Fc = 4.15). Together this indicates that the time between training stages regulates the sensitivity of the S2 to postretrieval disruption by cycloheximide. As with initial consolidation, protein synthesis in the BLA is not required for reconsolidation of the S2 fear memory when the two training stages occur 2 d apart but is required when the two stages occur 14 d apart.
Experiment 5
Experiments 2–4 examined the effect of a shift in temporal context on the requirement for BLA protein synthesis in consolidating the S2 fear memory. Experiments 5–7 examine the effects of a shift in physical context between the S1-shock pairings in stage 1 and the S2-S1-shock sequences in stage 2. We began by examining whether acquisition of fear to S2 in the different context was dependent on its presentation in sequence with the S1 and shock. Four groups of rats were exposed to S1-shock pairings in stage 1, either in the standard chambers (context A, Groups Same) or in a second, different set of chambers (context B, Groups Different). All groups then received stage 2 training in the standard chambers (context A). For one group in each of these pairs, stage 2 consisted in four S2-S1-shock sequences (Groups Same Paired and Different Paired). For the remaining group in each pair, stage 2 training consisted in four S1-shock pairings and four, temporally separated presentations of S2 alone (Groups Same Unpaired and Different Unpaired). Rats were then tested with S2 and then with S1 in the stage 2 context. The question of interest was whether acquisition of fear to the S2 was associatively mediated in the same and different context conditions. This would be evident as more freezing to S2 at test in the paired groups relative to the unpaired groups.
All groups successfully acquired fear to S1 in stage 1, with freezing increasing across the four S1-shock pairings (Fig. 6B; F(1,31) = 61.722; p < 0.001). There were no significant between-group differences or group × trend interactions, indicating that acquisition of fear to S1 was equivalent between groups prior to manipulations of context and did not differ between paired and unpaired groups (F < 1.5; Fc = 4.160).
Fear to the S2 is associatively mediated, even when it is conditioned in a different context to that of the prior S1-shock pairings. In stage 1, rats received S1-shock pairings in either context B or context A. In stage 2, rats received either paired or unpaired presentations of S2 and S1-shock. B, Mean (±SEM) levels of freezing to presentations of the S1 during S1-shock pairings. C, Mean (±SEM) levels of freezing to presentations of the S2 and S1 during S2-S1-shock sequences. D, E, Mean (+SEM) freezing to test presentations of the S2 and S1 alone. Group Same Paired, n = 8; Same Unpaired, n = 9; Different Paired, n = 10; Different Unpaired, n = 8.
In stage 2, freezing developed to S2 among rats that received S2-S1-shock sequences, but there was relatively little freezing to S2 in rats that received the S2 explicitly unpaired from the further S1-shock pairings (Fig. 6C; F(1,31) = 60.555; Fc = 4.160; p < 0.001). Averaging across groups, freezing increased across conditioning trials (F(1,31) = 34.888; Fc = 4.160; p < 0.001). This effect was driven by performance in the paired groups, which increased across presentations of S2 while freezing in the unpaired groups remained low, leading to a significant trend × pairing interaction (F(1,31) = 48.497; Fc = 4.160; p < 0.001). There was no significant effect of context or context × pairing interaction (Fs < 3.2; Fc = 4.160), indicating that freezing to S2 was similar regardless of whether the context was the same or different to that in stage 1. This was not due to a failure to discriminate between the two contexts, as the context-alone exposures after stage 1 revealed significantly higher freezing in the context where the S1-shock pairings had occurred compared with the other context (F(1,68) = 17.910; Fc = 3.982; p < 0.001). Unexpectedly, freezing to the S1 across stage 2 was significantly higher in the different groups compared with that in the same groups (F(1,31) = 5.368; Fc = 4.160; p < 0.05). This freezing did, however, remain stable across this stage (F(1,31) = 3.412; Fc = 4.160; p = 0.074), and there were no significant differences between paired and unpaired groups or trend × pairing interactions (Fs < 3.5; Fc = 4.160).
At test (Fig. 6D,E), freezing levels to S2 were higher in the paired than the unpaired groups (F(1,31) = 79.90; Fc = 4.160; p < 0.001). In contrast, there were no significant differences in freezing to S1 (Fs < 2; Fc = 4.161). In both cases, there was no significant effect of context or context × pairing interaction (Fs < 2.5; Fc = 4.161) indicating that shifting the context between stages 1 and 2 did not affect overall performance to S1 or S2. Together, these results indicate that acquisition of fear to the S2 was conditional on its close temporal contiguity with S1-shock pairings and unaffected by whether the S2-S1-shock sequences were administered in the same or different context to that of the prior S1-shock pairings.
Experiment 6
Experiment 6 examined the effect of a context shift between stages 1 and 2 on the protein synthesis requirement for consolidating fear to the S2. Four groups of rats were exposed to S1-shock pairings in stage 1 and, 2 d later, S2-S1-shock sequences in stage 2. For rats in two groups, both stages of training occurred in the same context, A (Groups Same). For rats in the remaining two groups, the two training stages occurred in different contexts: B in stage 1 and A in stage 2 (Groups Different). Immediately following training in stage 2, rats received a BLA infusion of cycloheximide or vehicle. Rats were then tested for fear to the S2 and S1 in context A. We hypothesized that a context shift would function like a delay in reinstating the protein synthesis requirement for consolidating fear to S2 in the BLA. Hence, we predicted that rats in Group Different CHX would freeze less when tested with S2 than rats in the other groups, which would not differ.
All groups successfully acquired freezing to the S1 in stage 1 (Fig. 7B). There was a significant increase in freezing to the S1 across its pairings with shock (F(1,50) = 210.62; Fc = 4.03; p < 0.001) and no between-group differences in overall levels of freezing or group × trial interactions (Fs < 0.7; Fc = 4.03). Across the S2-S1-shock sequences in context A, rats increased freezing to the S2 (Fig. 7C; F(1,50) = 405.50; Fc = 4.03; p < 0.001) while their freezing to the S1 remained stable (F(1,50) = 0.78; Fc = 4.03; p = 0.38). There were no significant between-group differences in overall freezing to either the S2 or S1 and no significant group × trial interactions (Fs < 2.5; Fc = 4.03).
When the two stages of training occur in different physical contexts, the protein synthesis requirement for consolidating fear to the S2 is reinstated. A, Experimental timeline of conditioning, reminder, and testing for rats that received an intra-BLA infusion of CHX or VEH. B, Mean (±SEM) levels of freezing to presentations of the S1 during S1-shock pairings. C, Mean (±SEM) levels of freezing to presentations of the S2 and S1 during S2-S1-shock sequences. D, E, Mean (+SEM) freezing to test presentations of the S2 and S1 alone. Same VEH, n = 13; Different VEH, n = 15; Same CHX, n = 11; Different CHX, n = 14.
The test levels of freezing to the S2 and S1 are shown in Figure 7D,E. During S2 test, rats in group Different CHX displayed significantly lower freezing than rats in the other three groups (F(1,50) = 4.90; Fc = 4.03; p < 0.05). There were no significant differences in levels of freezing to the S2 between group Same CHX and the vehicle groups (F(1,50) = 1.09; Fc = 4.03; p = 0.30) or between the vehicle groups (F(1,50) = 0.30; Fc = 4.03; p = 0.59). Importantly, the difference between the CHX groups was not due to differences in their ability to discriminate between the contexts, as analysis of context freezing after the initial S1-shock training revealed significantly higher freezing in the shocked context compared with the nonshocked context (F(1,50) = 56.24; Fc = 4.03; p < 0.001), and the size of this difference did not differ significantly between the CHX groups (F(1,50) = 2.09; Fc = 4.03; p = 0.15). During S1 test, there were no significant group differences, with all groups showing robust freezing to the S1 (Fs < 0.4; Fc = 4.03). Taken together, these results show that a BLA infusion of cycloheximide disrupts conditioning to the S2 when there is a shift in the physical context between S1-shock pairings in stage 1 and S2-S1-shock sequences in stage 2.
Experiment 7
This experiment examined whether reminding rats of their stage 1 experience in the stage 2 context would alter the protein synthesis requirement (in the BLA) for consolidating fear to the S2. All rats received S1-shock pairings in context B. Forty-eight hours later, they received two nonshocked presentations of the S1 in either context B or context A. Forty-eight hours later, they were exposed to S2-S1-shock sequences in context A, followed immediately by an intra-BLA infusion of either cycloheximide or vehicle. Finally, rats were tested for freezing to S2 and S1 in context A. We predicted that reminding rats of their stage 1 experience in context B prior to the S2-S1-shock sequences in context A would produce the same result (context shift effect) as that observed in the previous experiment: among these rats, those that received cycloheximide would freeze less to S2 at test than those that received vehicle. The question of interest concerned the rats that were reminded of their stage 1 experience in context A prior to the S2-S1-shock sequences in context A. If this treatment renders the A and B contexts functionally equivalent, consolidation of fear to S2 should be unaffected by the BLA cycloheximide infusion. That is, the reminder should reverse or block the context shift effect, which would be evident as equivalent freezing among rats that received cycloheximide or vehicle after stage 2.
All groups acquired freezing to S1 in stage 1 (Fig. 8B). Specifically, there was a significant increase in freezing to the S1 across the initial S1-shock pairings (F(1,39) = 150.98; Fc = 4.09; p < 0.001), and no significant difference between the groups in overall levels of freezing to the S1, and no significant group × trial interactions (Fs < 1.72; Fc = 4.09). Assessment of context freezing after stage 1 revealed significantly higher freezing in the shocked context compared with the nonshocked context (F(1,39) = 76.91; Fc = 4.09; p < 0.001), and this discrimination did not significantly differ between groups (Fs < 2.16; Fc = 4.09). During the reminder session, there was no significant main effect of drug group, reminder group (context B or context A), or drug × reminder interaction in levels of freezing to the S1 (Fs < 3.7; Fc = 4.09).
Re-exposing rats to the S1 in the stage 2 conditioning context (but not the stage 1 context) alleviates the protein synthesis requirement for consolidating fear to the S2. A, Experimental timeline of conditioning, reminder and testing for rats that received intra-BLA infusions of CHX or VEH. B, Mean (±SEM) levels of freezing to presentations of the S1 during S1-shock pairings. C, Mean (±SEM) levels of freezing to presentations of the S1 during the reminder session. D, Mean (±SEM) levels of freezing to presentations of the S2 and S1 during S2-S1-shock sequences. E, F, Mean (+SEM) freezing to test presentations of the S2 and S1 alone. B reminder VEH, n = 10; B reminder CHX, n = 12; A reminder VEH, n = 12; A reminder CHX, n = 9.
All groups acquired freezing to S2 in stage 2 (Fig. 8D). Specifically, there was a significant increase in freezing to the S2 across S2-S1-shock sequences (F(1,39) = 173.84; Fc = 4.09; p < 0.001), and no significant difference between the groups in overall levels of freezing to the S2, and no significant group × trial interactions (Fs < 1.72; Fc = 4.09). It should be noted that, across this stage, there was a significant linear decrease in freezing to the S1 (F(1,39) = 4.42; Fc = 4.09; p < 0.05), which likely reflects an increase in escape behavior across successive shock administration. Unexpectedly, the VEH groups showed significantly higher freezing to the S1 during S2-S1-shock sequences than the CHX groups (F(1,39) = 15.85; Fc = 4.09; p < 0.001). Importantly, there was no significant difference between rats that received the reminder in context A and those that received the reminder in context B (F(1,39) = 0.44; Fc = 4.09; p = 0.51) and no significant context × drug or group × trial interactions in levels of freezing to the S1 (Fs < 1.5; Fc = 4.09). That is, the levels of freezing to S1 were equivalent between the conditions to which our hypotheses apply.
Test levels of freezing to the S2 and S1 are shown in Figure 8E,F. Rats that received the reminder in the stage 2 context (context A) and were infused with cycloheximide showed significantly lower freezing to the S2 than the other three groups (F(1,39) = 12.75; Fc = 4.09; p < 0.01). Further, there was no significant difference in freezing between rats that received the reminder in the stage 1 context (context B) and were infused with cycloheximide and the two vehicle groups (F(1,39) = 0.22; Fc = 4.09; p = 0.65) or between the two vehicle groups (F(1,39) = 0.02; Fc = 4.09; p = 0.90). During S1 test, there was significantly higher freezing in the groups that received the reminder in the stage 2 context (A) compared with those that received the reminder in the stage 1 context (B; F(1,39) = 5.6; Fc = 4.09; p < 0.05). This is consistent with the idea that exposures to S1-alone presentations (the reminder) in context A allowed for better transfer of the fear that had been conditioned to S1 in context B, resulting in the two contexts being treated as functionally equivalent.
Taken together, these results indicate that the presentation of a reminder in the to-be-conditioned context (but not the alternative context) can reverse the effect of a context shift on the requirement for protein synthesis in consolidation of fear to the S2. It is important to acknowledge pre-existing differences in levels of freezing to the S1 during stage 2 of training, where both of the groups that would subsequently receive a BLA infusion of cycloheximide displayed less freezing than their vehicle-infused counterparts. However, the pattern of results in the S2 test cannot be explained by any such differences as (1) acquisition of freezing to S2 was equivalent across all groups and (2) rats in groups that received the BLA infusion of cycloheximide displayed equivalent freezing to S1 and S2 across both stages of training and only differed in their test levels of freezing to the S2.
A note on the role of systems consolidation in the present study
Research indicates that the neural systems supporting memory may change over time, via a process known as systems consolidation (Frankland and Bontempi, 2005; Wang and Morris, 2010). It has been proposed that this reorganization results in (or is accompanied by) a change in the nature of the memory, with memories becoming less specific and more “gist” like as they are incorporated into cortical circuits (Wiltgen and Silva, 2007; Wiltgen et al., 2010; Winocur and Moscovitch, 2011). In relation to our study, it is possible that such mechanisms could account for the differential requirement for protein synthesis in consolidation of S2 at the 2 d and 14 d time points (i.e., when conditioning to S2 occurred 2 d or 14 d after the initial conditioning to S1). However, this does not explain the differential protein synthesis requirement with a shift in physical context (Experiment 6): the two stages of training occurred just 2 d apart and the rats showed intact context discrimination, indicating that the memory had not generalized. As such, our findings cannot be explained by an account in terms of systems consolidation alone. Instead, we suggest an account based on memory allocation mechanisms that have also been described by others (Josselyn and Frankland, 2018; Rao-Ruiz et al., 2019). This is considered further in the Discussion.
Discussion
Protein synthesis in the BLA is not required to consolidate the memory of a new dangerous experience when it is similar to a past experience (Leidl et al., 2018; Williams-Spooner et al., 2019). The present study examined whether the timing and location of the new dangerous experience (relative to the past experience) affects the protein synthesis requirement for its consolidation and reconsolidation in the BLA. The first set of experiments showed that the protein synthesis requirement for consolidating fear to S2 varies with the interval between the two training stages. Specifically, consolidation of fear to S2 did not require protein synthesis in the BLA when the two training stages occurred 48 h apart, as previously reported (Leidl et al., 2018; Williams-Spooner et al., 2019) but did require this synthesis when the interval was increased to 14 d (Experiment 2). However, under the latter circumstances, the protein synthesis requirement was additionally influenced by reminders of the stage 1 training prior to stage 2: the BLA cycloheximide infusion disrupted fear to S2 among rats that were re-exposed to S1 12 d prior to stage 2 but had no effect among rats that were re-exposed to S1 just 48 h prior to stage 2 (Experiment 3). Thus, within the BLA, the protein synthesis requirement for consolidating fear to S2 is reinstated with time since the prior conditioning of S1; but this reinstatement can be blocked or reversed by a reminder of the prior S1 conditioning shortly before conditioning to S2. When this occurs, consolidation of fear to S2 remains (or, is again) independent of BLA protein synthesis.
The final experiment in the first set showed that the protein synthesis requirement for reconsolidating fear to S2 also varies with the interval between the two training stages. That is, after fear to S2 had been established, reconsolidation of that fear did not require protein synthesis in the BLA when the two training stages occurred 48 h apart but did require this synthesis when the interval was increased to 14 d (Experiment 4). This pattern of results mirrors the effect of time on the protein synthesis requirement for consolidating fear to S2, indicating that the two processes (consolidation and reconsolidation) share the same molecular requirements. It is consistent with previous research showing that context and time can influence the susceptibility of a fear memory to disruption by protein synthesis inhibitors (Wang et al., 2009; Winters et al., 2009; Jarome et al., 2015; Zinn et al., 2020). However, to the best of our knowledge, they are the first to show that the effect of protein synthesis inhibitors on consolidation and reconsolidation of a fear memory is determined by time since a similar past experience.
The second set of experiments showed that the protein synthesis requirement for consolidating fear to S2 changes when stage 2 is conducted in a different physical context. Here, rats were exposed to the initial S1-shock pairings in one context and, 48 h later, to S2-S1-shock sequences in a second, distinct context. The results showed that fear to S2 was disrupted by a BLA infusion of cycloheximide when the two training stages occurred in different contexts, but not when they occurred in the same context (Experiment 6). However, the effect of cycloheximide observed after shifting contexts was not seen when, 48 h prior to stage 2, rats were reminded of their prior S1 experience in the different context (Experiment 7). Critically, the effect of this reminder was specific to the context in which the reminder occurred; when the reminder was presented in the stage 1 conditioning context, it had no effect on the requirement for protein synthesis in consolidation of S2. Thus, the impact of a context shift on the protein synthesis requirement for consolidating fear to S2 depends on the history or “meaning” of the context in which S2 is conditioned. When the contexts differ with respect to their association with the dangerous cues, the context shift results in protein synthesis-dependent consolidation of fear to S2; but when both contexts are associated with dangerous cues, consolidation of fear to S2 does not require protein synthesis in the BLA.
The finding that a delay and context shift influence consolidation of fear to S2 in a similar way suggests that their effects are mediated by a common mechanism. One way of thinking about this is that (1) time is a gradually changing temporal context (Bouton, 2002) and (2) context plays a hierarchical or occasion-setting role in signaling whether a new situation is the same or different to a prior experience (Bouton and Swartzentruber, 1986; Fraser and Holland, 2019). However, it is important to note that the form of occasion-setting implied by the present findings differs from that which has been previously described, whereby the context (or another CS) regulates the retrieval of specific CS–US relations. That is, the novel implication of the present findings is that context not only influences the processes by which memories are retrieved and expressed in behavior: it also influences the substrates by which new information is consolidated and reconsolidated in the brain, at least in the BLA.
How does the brain compare situations and detect similarity/dissimilarity and what are the consequences of this comparison? A large literature implicates the hippocampus in context processing and computing the overlap between past and present experience based on convergent cortical inputs (Marr, 1971; Kim and Fanselow, 1992; Phillips and LeDoux, 1992; Eichenbaum, 2015). The hippocampus has reciprocal connections with the amygdala and has been shown to regulate context-dependent learning and synaptic changes in the amygdala (Pitkänen et al., 2000; Orsini et al., 2011; Ferrara et al., 2019). Accordingly, the hippocampus may use features of the environment to identify the context as the same or different and transfer this information to the BLA. When both experiences occur in the same physical and temporal context, the hippocampus identifies the current situation as the same as that experienced previously and, consequently, fear that develops to S2 is linked to the established fear of S1. However, when the two experiences occur in different physical/temporal contexts, the mismatch is identified by the hippocampus and fear to S2 is stored independently of fear to S1.
One question that remains to be addressed is how fear to S2 is consolidated independently of protein synthesis in the BLA. We propose that, when the novel S2 is presented in sequence with the already-conditioned S1 and shock, fear to S2 is acquired through activation of the same BLA neurons that encoded/retrieved the prior S1-shock memory: hence, it does not require the full suite of molecular changes for its consolidation. However, with a change in physical or temporal context, the second experience is allocated to a separate population of BLA neurons and protein synthesis is again required for consolidation of fear to S2. This proposal is supported by research demonstrating that dangerous events that occur close in time, or within the same physical context, are more likely to share overlapping neural signatures compared with events that are separated in space and time (Guzowski et al., 1999; Cai et al., 2016; Rashid et al., 2016; Lau et al., 2020). Applied to the present findings, initial conditioning of S1 may lead to a temporary increase in neuronal excitability and plasticity-related products in a population of BLA neurons (Josselyn and Frankland, 2018). When rats are exposed to S2-S1-shock sequences 48 h later, fear to S2 is consolidated via molecular changes in BLA neurons that encoded the prior S1-shock memory. When rats are exposed to S2-S1-shock sequences 14 d later or in a different context, fear to S2 is consolidated via de novo changes in a nonspecific population of BLA neurons. However, re-exposure to S1 prior to stage 2 re-excites the BLA neurons that encoded the prior S1-shock memory: hence, fear to S2 is again consolidated via molecular changes in these neurons (for evidence that a retrieval cue can return engram cells to an excited state, see Pignatelli et al. 2019).
In summary, the present study has shown that the protein synthesis requirement for consolidating and reconsolidating a new dangerous experience in the BLA is regulated by its similarity to a past experience. Specifically, (1) protein synthesis in the BLA is not required for consolidating or reconsolidating fear to S2 when S2-S1-shock sequences occur in the same physical and temporal context as a prior session of S1-shock pairings; (2) the protein synthesis requirement for consolidating fear to S2 is reinstated when the two training stages are separated by a delay or occur in different physical contexts; and (3) the effects of the delay and context shift can be blocked by reminding animals of the prior S1-shock pairings. Thus, the substrates of learning and memory in the mammalian brain are not fixed and immutable. Instead, the way we learn about and remember events reflects the degree to which they match the details of our past experience, which includes information about context and time, and is influenced by recent reminders of our past experience.
Footnotes
This work was supported by Australian Research Council Discovery Project Grants to N.M.H. and R.F.W. (DP200102969) and R.F.W. (DP220103650).
The authors declare no competing financial interests.
- Correspondence should be addressed to Nathan M. Holmes at n.holmes{at}unsw.edu.au.
