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