Limited replicability of drug-induced amnesia after contextual fear memory retrieval in rats

https://doi.org/10.1016/j.nlm.2019.107105Get rights and content

Highlights

  • We aimed to induce post-retrieval amnesia for contextual fear memories in rats.

  • Midazolam, propranolol, or cycloheximide were injected systemically after retrieval.

  • Only 2 of our replication attempts provided evidence for amnesia.

  • Current literature is likely biased.

  • Dependence on subtle, unknown moderators hampers clinical translation.

Abstract

With the ultimate goal of investigating boundary conditions for post-reactivation amnesia, we set out to replicate studies in which systemic, post-reactivation administration of midazolam, propranolol, or cycloheximide resulted in amnesia for contextual fear memories. Our experiments involved conceptual as well as exact replications of previously published studies. In most of our experiments, we adopted a procedure that conformed to the standard 3-day protocol typically used in the literature, with contextual fear conditioning on day 1, unreinforced re-exposure to the conditioning context followed by systemic injection of the amnestic drug on day 2, and a memory retention test on day 3. Given the plethora of successful studies with large effects sizes and the absence of any failed replications in the literature, we were surprised to find that we were generally unable to replicate those findings. Our results suggest that post-reactivation amnesia by systemic drug administration in rats is more difficult to obtain than what would be expected based on published empirical reports. At present, it remains unclear which conditions determine the success of this procedure.

Introduction

In a ground-breaking paper, Misanin, Miller, and Lewis (1968) showed that fear memory retention in rats was impaired when an electroconvulsive shock was given after a brief, unreinforced presentation of a previously conditioned cue. Interest in the phenomenon of reactivation-dependent amnesia was renewed around the turn of the century, when several publications reported impaired memory expression after post-reactivation administration of pharmacological agents such as anisomycin, MK-801 or propranolol (PROP) (Nader et al., 2000, Przybyslawski and Sara, 1997, Przybyslawski et al., 1999, Sara, 2000). Based on those results, it was hypothesized that adequate memory retention after reactivation required – at least partial – recapitulation of cellular events that occur during initial consolidation (such as protein synthesis and NMDA-dependent long-term potentiation) (Przybyslawski & Sara, 1997). Based on this analogy, the term ‘reconsolidation’ was proposed to describe the cascade of molecular events required for adequate retention of destabilized memories, and post-reactivation pharmacologically-induced amnesia was attributed to (partial) blockage of these molecular events.

The view of post-reactivation amnesia as resulting from reconsolidation interference has been challenged by observations of recovery from amnesia (e.g., after passage of time, reinstatement, or after a change in the internal context by re-administration of the amnestic agent) (DeVietti and Larson, 1971, Eisenberg and Dudai, 2004, Gisquet-Verrier and Riccio, 2018, Gisquet-Verrier et al., 2015, Lattal and Abel, 2004, Power, 2006, Trent et al., 2015). Similar memory preservation despite initial amnesia has been observed when amnestic treatments were administered after initial learning (Ryan, Roy, Pignatelli, Arons, & Tonegawa, 2015). The fact that recovery from amnesia is sometimes observed after post-reactivation pharmacological interventions, suggests that different or multiple mechanisms might be at play when observing (temporary) post-reactivation amnesia. Elsey, Van Ast, and Kindt (2018) have provided a range of control conditions that should (minimally) be met in order to infer the occurrence of reconsolidation. In the remainder of the paper we use a terminology that refers to the expected behavioral outcome (i.e., ‘post-reactivation amnesia’) without committing to a specific underlying mechanism.

In the past 20 years, post-reactivation amnesia has been demonstrated for various types of memory and in a variety of species, indicating its ubiquitous nature. After initial observations of post-reactivation amnesia induction for Pavlovian fear memories, this procedure was successfully applied to other types of aversive memories (e.g., inhibitory avoidance and conditioned taste aversion) (Gisquet-Verrier et al., 2015; but see Muravieva & Alberini, 2010), as well as appetitive memories (Milton, Lee, Butler, Gardner, & Everitt, 2008). Apart from demonstrations in rodents, post-reactivation amnesia has been established in slugs, chicks, crabs, fish and humans (Kindt et al., 2009, Sara, 2000; but see Bos et al., 2014, Hardwicke et al., 2016, Schroyens et al., 2017, Thome et al., 2016). Studies in clinical populations have provided mixed results, emphasizing the necessity of a deeper understanding regarding the underlying mechanisms and conditions required for post-reactivation amnesia induction (Beckers and Kindt, 2017, Soeter and Kindt, 2015, Wood et al., 2015). To gain a better insight into these conditions on a neurobiological and behavioral level, we set out to establish post-reactivation amnesia in rodent contextual and cued fear conditioning.

Amnesia for contextual or cued fear memories in rodents has been observed after post-reactivation (systemic, intra-amygdala, or intra-hippocampal) administration of various pharmacological agents, e.g., protein synthesis inhibitors (e.g., anisomycin, rapamycin, cycloheximide) (Duvarci and Nader, 2004, Haubrich et al., 2015, Hoffman et al., 2015, Nader et al., 2000), NMDA receptor antagonists (e.g., MK-801) (Cassini et al., 2017, Merlo et al., 2018, Przybyslawski and Sara, 1997), propranolol (PROP, a β-adrenergic receptor antagonist) (Dębiec and Ledoux, 2004, Przybyslawski et al., 1999), and midazolam (MDZ, a positive allosteric modulator of the GABA-A receptor) (Bustos et al., 2006, Espejo et al., 2017, Ortiz et al., 2015). Non-pharmacological interventions, such as electroconvulsive therapy (Misanin et al., 1968, Schneider and Sherman, 1968; but see Dawson & McGaugh, 1969), hypothermia (Mactutus et al., 1982, Mactutus et al., 1979), conducting extinction training (Ferrer Monti et al., 2017, Monfils et al., 2009) or presenting appetitive information shortly after or during CS re-exposure (Ferrer Monti et al., 2016, Haubrich et al., 2015, Ortiz et al., 2016), have also been shown to induce amnesia for fear memories. While there have been several reports of failures to successfully replicate the post-reactivation extinction procedure in rodents (e.g., Chan et al., 2010, Ishii et al., 2015, Luyten and Beckers, 2017; for a meta-analysis, see Kredlow, Unger, & Otto, 2016), the existing literature suggests that pharmacologically-mediated post-reactivation amnesia is a more consistent and robust finding.

With the ultimate goal of obtaining a robust protocol that could be used to investigate constraints on and opportunities of the clinical application of post-reactivation amnesia, we performed a series of experiments aiming to conceptually or exactly replicate published studies using systemic drug administration after unreinforced CS re-exposure in rats. The results of our replication attempts involving contextual fear memories are reported in the current paper. Those involving cued fear conditioning are reported elsewhere (Luyten et al., in prep). In order to induce post-reactivation amnesia for contextual fear memories, we used a standard behavioral protocol. At least 24 h after conditioning, rats were briefly re-exposed to the conditioning context, followed by systemic administration of vehicle or amnestic agent(s). Fear memory retention was assessed 24 h later. Given that our original project aimed to focus on the clinical relevance of post-reactivation amnesia, we limited ourselves to systemic administration of commonly-used and non-invasive drugs that can be safely used in humans as well (except for one study, in which we also injected a drug that directly interferes with protein synthesis (i.e., cycloheximide)). Published studies from other labs have reported robust amnestic effects using similar or identical protocols and midazolam (see Table 1) or propranolol (see Table 2) are commonly used as amnestic agents. Although there are several reports of conditions in which post-reactivation amnesia for contextual fear memories does not occur (e.g., using strong training conditions, with stress induction prior to learning, or depending on the length of the reactivation session; Alfei et al., 2015, Bustos et al., 2009, Cassini et al., 2017, Espejo et al., 2016, Lee and Flavell, 2014), there are currently no publications of failures to replicate amnesia when using a standard contextual fear conditioning paradigm and drug injection/infusion after unreinforced re-exposure to the conditioned context in rodents.

In a series of 25 conceptual replication attempts, we varied properties of the training and reactivation session and used several amnestic drugs (MDZ, PROP, and/or cycloheximide) and doses. In one experiment, one of the amnestic drugs, MDZ, was administered before the reactivation session. In some experiments, D-cycloserine was administered before the reactivation session in an attempt to boost memory destabilization (Bustos et al., 2010, Lee et al., 2009). In addition, across experiments, there were variations in the rat strain, amount of handling prior to conditioning, use of cage enrichment, time interval between training and reactivation session, laboratory in which the experiment was performed, and researcher who performed the experiment. We also performed 6 exact replication attempts, in which the methodology of prior reports was followed as precisely as possible after detailed consultation with the authors of these studies (Alfei et al., 2015, Ferrer Monti et al., 2017, Stern et al., 2012).

Section snippets

Preregistration

For some of the current experiments, the adopted study protocols and performed statistical analyses were preregistered on aspredicted.org. The preregistration forms, as well as all raw data and results of preregistered analyses, can be found on the Open Science Framework (OSF) (Schroyens, Alfei, Luyten, & Beckers, 2019). For some studies (i.e., JA01-JA05, JA08), a larger sample size of 8 rats per group was preregistered but not reached given the absence of a promising trend in the first batch

Results

Appendix B contains an overview of descriptive statistics (sample size, mean, SD) and results of statistical analyses (t-value, p-value, Cohen’s d, and BF10) for each experiment in which MDZ or PROP was administered after re-exposure to the conditioning context (Table B.1 and Table B.2, respectively). Fig. 1, Fig. 2 provide an overview of freezing during the test session of all experiments in which we aimed to induce amnesia. Detailed graphical representations of all studies are shown in

Discussion

The ultimate goal of our experiments was to establish a protocol that could be used to investigate (and overcome) boundary conditions for post-reactivation amnesia induction. To this aim, we set out to conceptually or directly replicate previous studies in which systemic, post-reactivation administration of midazolam (MDZ, see Table 1), propranolol (PROP, see Table 2), or cycloheximide (CYCLO; Haubrich et al., 2015) resulted in amnesia for contextual fear memories. In most of the experiments

Acknowledgements

We would like to thank Ineke Pillet and Victoria A. Ossorio Salazar for their assistance with the experimental work and all the researchers who commented on the preprint version of the manuscript.

Funding sources

This work was supported by a Consolidator Grant of the European Research Council (ERC) [T. Beckers, grant number 648176] and a Doctoral Fellowship of the Research Foundation – Flanders (FWO) [N. Schroyens, grant number 1114018N].

Declarations of interest

None.

References (88)

  • A.N. Hoffman et al.

    Chronic stress enhanced fear memories are associated with increased amygdala zif268 mRNA expression and are resistant to reconsolidation

    Neurobiology of Learning and Memory

    (2015)
  • D. Ishii et al.

    An isolated retrieval trial before extinction session does not prevent the return of fear

    Behavioural Brain Research

    (2015)
  • T.K. Koo et al.

    A guideline of selecting and reporting intraclass correlation coefficients for reliability research

    Journal of Chiropractic Medicine

    (2016)
  • L. Luyten et al.

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

    Neurobiology of Learning and Memory

    (2017)
  • V. Ortiz et al.

    Effect of a positive reinforcing stimulus on fear memory reconsolidation in ethanol withdrawn rats: Influence of d-cycloserine

    Behavioural Brain Research

    (2016)
  • J. Przybyslawski et al.

    Reconsolidation of memory after its reactivation

    Behavioural Brain Research

    (1997)
  • M.J.F. Robinson et al.

    Reconsolidation of a morphine place preference: Impact of the strength and age of memory on disruption by propranolol and midazolam

    Behavioural Brain Research

    (2010)
  • A.M. Schneider et al.

    Stress-dependent opioid and adrenergic modulation of newly retrieved fear memory

    Neurobiology of Learning and Memory

    (2014)
  • N. Schroyens et al.

    Post-weaning housing conditions influence freezing during contextual fear conditioning in adult rats

    Behavioural Brain Research

    (2019)
  • M. Soeter et al.

    An abrupt transformation of phobic behavior after a post-retrieval amnesic agent

    Biological Psychiatry

    (2015)
  • S.M. Taubenfeld et al.

    Preclinical assessment for selectively disrupting a traumatic memory via postretrieval inhibition of glucocorticoid receptors

    Biological Psychiatry

    (2009)
  • W. Theilmann et al.

    Behavioral differences of male Wistar rats from different vendors in vulnerability and resilience to chronic mild stress are reflected in epigenetic regulation and expression of p11

    Brain Research

    (2016)
  • S.-H. Wang

    Novelty enhances memory persistence and remediates propranolol-induced deficit via reconsolidation

    Neuropharmacology

    (2018)
  • N.E. Wood et al.

    Pharmacological blockade of memory reconsolidation in posttraumatic stress disorder: Three negative psychophysiological studies

    Psychiatry Research

    (2015)
  • P.R. Abdullahi et al.

    Oxytocin receptor antagonist atosiban impairs consolidation, but not reconsolidation of contextual fear memory in rats

    Brain Research

    (2018)
  • J.M. Alfei et al.

    Prediction error and trace dominance determine the fate of fear memories after post-training manipulations

    Learning & Memory

    (2015)
  • T. Beckers et al.

    Memory reconsolidation interference as an emerging treatment for emotional disorders: Strengths, limitations, challenges, and opportunities

    Annual Review of Clinical Psychology

    (2017)
  • M.G.N. Bos et al.

    Noradrenergic blockade of memory reconsolidation: A failure to reduce conditioned fear responding

    Frontiers in Behavioral Neuroscience

    (2014)
  • J.F. Briggs et al.

    Reexposure to the amnestic agent alleviates cycloheximide-induced retrograde amnesia for reactivated and extinction memories

    Learning & Memory

    (2013)
  • S.G. Bustos et al.

    Previous stress attenuates the susceptibility to midazolam’s disruptive effect on fear memory reconsolidation: Influence of pre-reactivation D-cycloserine administration

    Neuropsychopharmacology

    (2010)
  • S.G. Bustos et al.

    Disruptive effect of midazolam on fear memory reconsolidation: Decisive influence of reactivation time span and memory age

    Neuropsychopharmacology

    (2009)
  • L.F. Cassini et al.

    On the transition from reconsolidation to extinction of contextual fear memories

    Learning & Memory

    (2017)
  • A. Chalkia et al.

    Acute but not permanent effects of propranolol on fear memory expression in humans

    Frontiers in Human Neuroscience

    (2019)
  • W.Y.M. Chan et al.

    Effects of recent exposure to a conditioned stimulus on extinction of Pavlovian fear conditioning

    Learning & Memory

    (2010)
  • R.G. Dawson et al.

    Electroconvulsive shock effects on a reactivated memory trace: Further examination

    Science

    (1969)
  • T.L. DeVietti et al.

    ECS effects: Evidence supporting state-dependent learning in rats

    Journal of Comparative and Physiological Psychology

    (1971)
  • S. Duvarci et al.

    Characterization of fear memory reconsolidation

    The Journal of Neuroscience

    (2004)
  • M. Eisenberg et al.

    Reconsolidation of fresh, remote, and extinguished fear memory in medaka: Old fears don’t die

    European Journal of Neuroscience

    (2004)
  • J.W.B. Elsey et al.

    Human memory reconsolidation: A guiding framework and critical review of the evidence

    Psychological Bulletin

    (2018)
  • F. Faul et al.

    G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences

    Behavior Research Methods

    (2007)
  • R.I. Ferrer Monti et al.

    A comparison of behavioral and pharmacological interventions to attenuate reactivated fear memories

    Learning & Memory

    (2017)
  • R.I. Ferrer Monti et al.

    An appetitive experience after fear memory destabilization attenuates fear retention: Involvement GluN2B-NMDA receptors in the Basolateral Amygdala Complex

    Learning & Memory

    (2016)
  • R.W. Flint et al.

    Cycloheximide impairs reconsolidation of a contextually reactivated memory in a conditioned taste aversion paradigm

    Behavioral Neuroscience

    (2007)
  • L. Gazarini et al.

    PTSD-like memory generated through enhanced noradrenergic activity is mitigated by a dual step pharmacological intervention targeting its reconsolidation

    International Journal of Neuropsychopharmacology

    (2015)
  • Cited by (0)

    1

    Both authors contributed equally to this work.

    2

    Present address.

    View full text