A preregistered, direct replication attempt of the retrieval-extinction effect in cued fear conditioning in rats

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Highlights

  • This is a preregistered, exact replication study of Monfils, Cowansage, Klann, and LeDoux (2009), Science.

  • They found less return of fear when adding a retrieval trial before extinction.

  • We found no evidence for attenuated fear recovery using retrieval-extinction.

  • This questions the validity of moderators derived from reviews and meta-analyses.

  • Experimental evaluation of purported moderators may be the way forward.

Abstract

In 2009, Monfils and colleagues proposed a behavioral procedure that was said to result in a permanent attenuation of a previously established fear memory, thereby precluding a possible return of fear after extinction (Monfils, Cowansage, Klann, & LeDoux, 2009). By presenting a single retrieval trial one hour before standard extinction training, they found an enduring reduction of fear. The retrieval-extinction procedure holds great clinical potential, particularly for anxiety patients, but the findings are not undisputed, and several conceptual replications have failed to reproduce the effect. These failures have largely been attributed to small procedural differences. This preregistered study is the first endeavor to exactly replicate three key experiments of the original report by Monfils et al. (2009), thereby gauging the robustness of their seminal findings.

Despite adhering to the original procedures as closely as possible, we did not find any evidence for reduced return of fear with the retrieval-extinction procedure relative to regular extinction training, as assessed through spontaneous recovery, reinstatement and renewal. Behavior of animals in the control condition (extinction only) was comparable to that in the original studies and provided an adequate baseline to reveal differences with the retrieval-extinction condition. Our null findings indicate that the effect sizes in the original paper may have been inflated and question the legitimacy of previously proposed moderators of the retrieval-extinction effect. We argue that direct experimental evaluation of purported moderators of the retrieval-extinction effect will be key to shed more light on its nature and prerequisites.

Introduction

Anxiety disorders and posttraumatic stress disorder are among the most prevalent psychiatric disorders (Kessler et al., 2005), and are hallmarked by substantial disability and poor quality of life (DSM-5). An effective behavioral treatment strategy for these disorders is exposure (Hofmann and Smits, 2008, Ougrin, 2011), which entails in vivo or imaginal contact with the feared object or situation. An important downside of exposure therapy is the relatively frequent relapse of fear after seemingly successful treatment (Craske and Mystkowski, 2006, Loerinc et al., 2015, Yonkers et al., 2003). Extinction learning of conditioned fear memories in the laboratory bears some resemblance to exposure therapy (Myers and Davis, 2002, Scheveneels et al., 2016, Vervliet et al., 2013). Likewise, under several circumstances, researchers observe a return of fear after successful extinction learning (Bouton, 2002, Bouton, 2004). This has been interpreted as evidence that extinction does not produce an erasure of the fear memory, but rather involves new learning, resulting in an extinction memory that coexists and competes with the original fear memory. Which memory will be retrieved upon confrontation with the conditioned and subsequently extinguished stimulus depends on the circumstances. The fear memory is, for example, more likely to surface again after the passing of time (spontaneous recovery), after experiencing an aversive event (reinstatement) or outside of the extinction context (renewal). Similarly, even after successful exposure therapy, the original fear memory is not erased, thereby forming an enduring risk for relapse.

In a highly influential paper, Monfils and colleagues proposed a modified extinction procedure that was said to result in a more permanent attenuation of the fear memory, thereby precluding a possible return of fear (Monfils, Cowansage, Klann, & LeDoux, 2009). Using a cued fear conditioning procedure in rats, a tone fear memory was formed and then extinguished again one day later. Various recovery assays, including spontaneous recovery after one month, reinstatement, renewal, and reacquisition tests, provided evidence that the fear memory was retained through extinction. However, in a group of animals that received a single isolated tone presentation one hour before extinction training, there was a significant attenuation of the return of fear. These findings were soon confirmed in human subjects (Schiller et al., 2010) and mice (Clem & Huganir, 2010). The recovery-reducing effect of the retrieval-extinction procedure was attributed to the extinction training interfering with reconsolidation of the fear memory after retrieval. It has been argued that consolidated memories can enter a labile state upon retrieval. During this window of instability (<6 h), a fear memory storing the association between a conditioned stimulus (CS) and an aversive unconditioned stimulus (US; e.g., shock) may be impaired using certain pharmacological manipulations (Beckers and Kindt, 2017, Debiec and Ledoux, 2004, Nader et al., 2000) that are assumed to disrupt the reconsolidation of the fear memory into its stable form. Monfils and colleagues proposed that, whereas the administration of amnestic drugs upon memory retrieval can prevent the reconsolidation of a fear memory, extinction training applied within the reconsolidation window can induce updating of the reactivated memory trace, by incorporating non-threatening information about the CS. Their data indeed supported that the retrieval-extinction procedure resulted in a more persistent fear reduction as compared with standard extinction training.

Clearly, these findings have exciting implications regarding the possibility of fear memory modification and hold great clinical potential. However, the fear recovery-reducing effect of the retrieval-extinction procedure is not undisputed and many have questioned whether merely changing the spacing of initial extinction training should result in such a marked loss of the fear memory. Alternative accounts for the superior fear reduction observed after retrieval-extinction relative to regular extinction (e.g., increased variability or spacing of the extinction trials (Rowe and Craske, 1998, Urcelay et al., 2009)) suggest that it may reflect enhanced extinction learning, resulting in a stronger or more retrievable extinction memory, rather than the persistent modification of the initial fear memory (Baker et al., 2013, Ponnusamy et al., 2016). The distinction between the reconsolidation-based explanation of Monfils and colleagues and alternative accounts is not trivial, given that the former implies a permanent disruption of the original fear memory, in which case there could never be a return of fear, under any circumstances.

Although Monfils and colleagues have been able to replicate the effect multiple times, independent conceptual replications have met with varying success (see Table 1). To the best of our knowledge, a significant reduction of the return of cued fear in adult rats has not yet been reported outside of the Monfils lab. Already in 2010, an Australian group published a series of experiments that closely followed the original procedures, but failed to replicate the fear recovery-reducing effect, and even found fear augmentation in some cases (Chan, Leung, Westbrook, & McNally, 2010). Note that these authors did deviate from the original procedure on some aspects (e.g., a different rat strain was used, animals were handled for three days before the start of experiments, animals were housed in groups of eight, and no experiment assessing the long-term effect on spontaneous recovery was included). In a recent meta-analysis, Kredlow, Unger, and Otto (2016) calculated an estimate of the effect size of the retrieval-extinction procedure. Although they reported significant benefits of the retrieval-extinction procedure over regular extinction training in rodent appetitive conditioning and human fear conditioning, this was not the case for rodent fear conditioning. They included 10 fear conditioning reports (involving a total of 34 experiments with 553 rodents) (see Table 1), and found a small, non-significant reduction of the return of fear as compared with standard extinction (Hedges’ g = 0.21). This effect was moderated by the number of animals housed together (i.e., significant effects for animals housed alone) and the delay between the retrieval-extinction procedure and the test for return of fear (i.e., significant effects for spontaneous recovery tests after ≥6 days). Although the meta-analysis was a valuable undertaking, the search for moderators in order to explain the inconsistent results in the literature may be premature, given that we presently have no information regarding the robustness of the initial effect in a direct, independent replication, and given the overall non-significant effect in the existing literature.

The goal of the present series of three experiments was to try and replicate some of the key findings of Monfils and colleagues, adhering to their reported procedures as strictly as possible. More specifically, we compared the return of fear observed after a retrieval-extinction procedure to the return of fear after regular extinction training, as assessed with spontaneous recovery, reinstatement and renewal tests.

Section snippets

Preregistration

This study was registered on the Open Science Framework (OSF) (https://osf.io/aydfs) and the study protocols were previewed by the editor of Neurobiology of Learning and Memory before the start of data collection. Additional details regarding the timing of the behavioral protocols and all raw data can be found on OSF.

Subjects

Male Sprague-Dawley rats (7–8 weeks old, weighing 270 ± 12 g (mean ± SD) three days before the start of the behavioral sessions) (Janvier Labs, Le Genest-Saint-Isle, France) were used

Experiment 1: spontaneous recovery

All rats were included in the final analyses (n = 8 per group) (Fig. 1). Note that extra analyses excluding two subjects with less than 5% freezing on the final acquisition trial reached the same conclusions.

Cued fear was acquired in both groups, without significant group differences, indicating that matching based upon freezing during the third training tone was effective. Accordingly, the RM ANOVA showed a main effect of Trial (F(2, 28) = 12.76, p < 0.001, η2p = 0.48), but no main effect of Group (F(1,

Discussion

This study is a direct replication of three key experiments from the influential 2009 Science paper by Monfils and colleagues, in which they introduced a modified extinction procedure that resulted in a more enduring attenuation of the fear memory, by presenting a single tone stimulus one hour before the start of standard extinction training (Monfils et al., 2009). The findings and proposed mechanism are somewhat controversial (Baker et al., 2013, Chan et al., 2010, Ponnusamy et al., 2016), and

Acknowledgments

We would like to thank Kimberly Jones for her assistance with the behavioral experiments and manual freezing scoring. In addition, we are indebted to Marie H. Monfils for providing additional procedural details during the preparation of this work and for constructive feedback on an earlier version of this manuscript.

Funding

This work was supported by ERC Consolidator Grant 648176 (to Tom Beckers).

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