Extinction-reconsolidation boundaries: key to persistent attenuation of fear memories

Abstract

Dysregulation of the fear system is at the core of many psychiatric disorders. Much progress has been made in uncovering the neural basis of fear learning through studies in which associative emotional memories are formed by pairing an initially neutral stimulus (conditioned stimulus, CS; e.g., a tone) to an unconditioned stimulus (US; e.g., a shock). Despite recent advances, the question of how to persistently weaken aversive CS-US associations, or dampen traumatic memories in pathological cases, remains a major dilemma. Two paradigms (blockade of reconsolidation and extinction) have been used in the laboratory to reduce acquired fear. Unfortunately, their clinical efficacy is limited: Reconsolidation blockade typically requires potentially toxic drugs, and extinction is not permanent. Here, we describe a behavioral design in which a fear memory in rats is destabilized and reinterpreted as safe by presenting an isolated retrieval trial before an extinction session. This procedure permanently attenuates the fear memory without the use of drugs.

Figure 1. Finite lability window to prevent return of fear via post retrieval extinction

(A) Rats were fear-conditioned (Fear Cond) with three tone-shock pairings. Twenty-four hours later they were exposed either to an isolated cue retrieval trial (Ret) or context only (No Ret) followed by extinction training (Ext). The time interval between the retrieval trial (or context exposure, n=12) and the extinction was either within (10 min, n=8; 1 hr, n=8), or outside (6 hrs, n=8; 24 hrs, n=8) the reconsolidation window. Twenty-four hours after extinction, all groups were tested for long-term memory (LTM), and one month later for spontaneous recovery. The grey shading represents context A. (B) All groups were equivalent for the last 4 trials of extinction and at the 24 hr LTM test. One month after later, the Ret groups with an interval outside the reconsolidation window (grey), as well as the No Ret group (black), showed increased freezing (spontaneous recovery) relative to the 24 hr LTM test (no retrieval- P<.0001; 6 hr ITI-P=.001; 24 hr ITI- P=.031), however, the groups with an interval within the lability window (red) did not (10 min- P=.888; 1 hr- P=.156) (see SOM for further details). All data points show mean ± SEM.

Figure 2. Attenuation of fear memory by presenting a single isolated retrieval trial followed by an extinction session prevents renewal

(A) Rats were fear-conditioned (Fear Cond) in context A. Twenty-four hours later they were exposed either to an isolated cue retrieval trial (Ret, n=8) or context only (No Ret, n=8) in context B, followed, one hour later, by extinction training (Ext) in context B. Twenty-four hours after extinction, they were tested for long-term memory (LTM) in context B. The grey shading represents context A, and the blue shading represents context B (see SOM for details). (B) Rats from both experimental groups froze equivalently during the LTM test (all ANOVAs, P>.1). When they were placed back in the acquisition context, the No Ret group (black) showed fear renewal (P=.012), but the Ret group (red) did not (p>.1), relative to their respective LTM tests. All data points show mean ± SEM.

Figure 3. Presenting a single isolated retrieval trial prior to an extinction session prevents reinstatement

(A) Rats were fear-conditioned (Fear Cond). The next day, they were exposed either to an isolated cue retrieval trial (Ret, n=8) or context only (No Ret, n=8) followed, one hour later, by extinction training (Ext). Twenty-four hours after extinction, they received 5 unsignaled footshocks, and the next day were tested for reinstatement. The grey shading represents context A. (B) The No Ret and Ret groups froze equivalently to the last 4 CSs of extinction; however, 24 hours after the unsignaled footshocks the No Ret group (black) showed increased freezing (reinstatement) (p<.05), but the Ret group (red) did not (p>.05). All data points show mean ± SEM.

Figure 4. De-phosphorylation of GluR1s845 underlies destabilization, and allows behavioral updating during reconsolidation

(A) Rats were fear conditioned, then 24 hrs later received (i) context exposure only (No CS) and euthanized 3 minutes later (n=6); (ii) a single CS retrieval and euthanized 3 min later (n=4); (iii) a single CS and euthanized 1 hr later (n=6); (iv) 2 CSs with a 3 min interval and euthanized 3 min later (n=6); or (v) 2 CSs with a 1 hr interval and euthanized 3 min later (n=6). (B) Quantification showing an increase in GluR1 phosphorylation at S845 both 3 min and 1 hr after CS presentation (grey). A second CS presented one hour after initial retrieval leads to de-phosphorylation of the GluR1 receptors (red), while the presentation of 2 CSs with a 3 min interval (grey) does not result in de-phosphorylation. * signifies significantly different from the no CS group (black). Four Western blots were run, and all the data are included in the quantification graph. A representative western blot is shown in S1. All data points show mean ± SEM.

Figure 5. Presenting a single isolated retrieval trial prior to an extinction session leads to less fear memory savings than extinction alone

(A) On day 1, rats were fear conditioned (Fear Cond). The next day, they received either No Ret+Ext (n=10), or Ret+Ext (n=10), with a 1 hour interval between the retrieval and extinction phases. Then, on the third experimental day, rats were reconditioned using a single CS-US pairing. The fourth day, we tested the groups and compared them for savings. (B) The No Ret group (black) froze significantly more than the Ret group (red) during the long-term memory (LTM-savings) test presented 24 hours after the single CS-US training session, F(1,18)=11.679, p=.003. The No Ret and Ret groups did not differ during extinction (p>.1), or during the single CS-US pairing session (p>.1), and no significant pre CS freezing was observed. All data points show mean ± SEM.

Figure 6. An isolated retrieval trial followed by an extinction leads to a re-valuation of the stimulus as safe, and retards subsequent acquisition of fear conditioning

(A) On day 1, rats were fear conditioned (Fear Cond). On day 2, they received either a retrieval (Ret+Ext, n=9) or not (No Ret, n=14), followed one hour later by an extinction session (18 CSs for the Ret group, 19 CSs for the No Ret group). On day 3, we reconditioned these groups, as well as conditioned a naïve group of rats (control, n=7), using 5 CS-US pairings, to look at the effect of our treatment on re-acquisition. (B) The isolated retrieval presented before extinction (Ret+Ext, red) retards re-acquisition, relative to a naïve group (white) or the No Ret+ Ext group (black). Repeated measures ANOVA revealed a Main effect of Group, F(1,27)=85.85, p<.0001, and a Group by Trial interaction, F(2,27)=55.687, p=.016. Simple main effect follow-up showed that the Ret+Ext group was significantly lower than the Control (.019) and No Ret (.009) groups. The Control and No Ret groups were not significantly different from one another. All data points show mean ± SEM.