One retrieval trial induces reconsolidation in an appetitive learning paradigm in honeybees (Apis mellifera)

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Abstract

Combining memory retrieval with the application of a protein synthesis-inhibitor leads to an amnestic effect that is referred to as the reconsolidation phenomenon. Several behavioural studies demonstrate that only a few or weak retrieval trials (that do not result in significant extinction) lead to this phenomenon. In contrast, many trials (that result in significant extinction) combined with a protein synthesis inhibitor result in an inhibition of the extinction memory. Based on these findings it was suggested that extinction is the boundary condition for reconsolidation: when extinction is induced the consolidation of the extinction memory is the dominant process. Recently we were not able to confirm this hypothesis in the honeybee (Apis mellifera): We did not find the reconsolidation phenomenon after one retrieval trial, but demonstrated reconsolidation after five retrieval trials that led to extinction. To exclude that this observation resembles a special case in insects we here wanted to know if one retrieval trial induces reconsolidation as it has been demonstrated before in many other species. To do so we used experimental parameters that had been used before to demonstrate consolidation in the honeybee with the exception that this time the protein synthesis-inhibitor was applied 1 h after one memory retrieval instead after acquisition. We thereby demonstrate the reconsolidation phenomenon after one retrieval trial but only when using the doubled dose of protein synthesis-inhibitor that has been used to inhibit consolidation.

Introduction

Retrieving an associative memory repeatedly leads to a decrease in the conditioned response (CR). This behavioral phenomenon is called extinction (Pavlov, 1927). Extinction arises from extinction learning, when the animal learns that the unconditioned stimulus (US) is no longer predicted by the conditioned stimulus (CS) (CS–noUS association) (Bouton & Moody, 2004). The resulting extinction memory undergoes a consolidation process, as indicated by the finding that extinction memory is blocked by protein synthesis-inhibitors (Berman and Dudai, 2001, Power et al., 2006, Santini et al., 2004, Pedreira and Maldonado, 2003, Vianna et al., 2003, Vianna et al., 2001). In some cases the consolidation of the extinction memory is not inhibited. Instead, the application of protein synthesis-inhibitors around the time point of retrieval results in a decrease of the CR. Accordingly a consolidation process following retrieval is inhibited. Initially it was proposed that retrieval makes the consolidated CS–US memory labile and that a second round of consolidation is required to stabilize the retrieved memory. Therefore the process in question was called reconsolidation (Nader, 2003, Sara, 2000, Spear, 1973).

Several studies demonstrate that only a few or weak retrieval trials, that do not result in significant extinction, lead to reconsolidation (Eisenberg et al., 2003, Pedreira and Maldonado, 2003, Power et al., 2006, Suzuki et al., 2004). It was therefore suggested that extinction is a boundary condition for reconsolidation: reconsolidation occurs only when extinction is not induced (Debiec et al., 2002, Eisenberg and Dudai, 2004, Nader, 2003).

We previously examined reconsolidation in an appetitive learning paradigm in the honeybee (Apis mellifera) (Stollhoff, Menzel, & Eisenhardt, 2005). In this paradigm the proboscis extension response (PER) that is elicited by touching the antennae with sucrose solution (US) is conditioned such that it is elicited by a previously neutral odour (CS). Using this paradigm we were unable to demonstrate reconsolidation after one retrieval trial, but found reconsolidation after five retrieval trials, although five retrieval trials led to extinction (Stollhoff et al., 2005). To exclude that this finding resembles a special case in insects we here ask if one retrieval trial induces reconsolidation as it has been demonstrated in many other species and paradigms, but not in the honeybee (Stollhoff et al., 2005). In contrast to our previous study we used the experimental setup that had been used before to demonstrate consolidation in the honeybee with the exception that this time the protein synthesis-inhibitor was applied 1 h after one memory retrieval instead after acquisition (Wüstenberg, Gerber, & Menzel, 1998). We here demonstrate reconsolidation but only when doubling the amount of protein synthesis-inhibitor that is neccessary to inhibit consolidation in the honeybee.

Section snippets

Results

Wüstenberg et al. (1998) demonstrated that 10 mM anisomycin applied 1 h after three CS–US pairings inhibit memory consolidation. To demonstrate reconsolidation after one retrieval trial we used this experimental setup with the exception that 10 mM anisomycin was applied 1 h after a first memory retrieval (FR) instead after acquisition. The FR took place 24 h after acquisition, because protein synthesis-inhibitors applied at this time point after acquisition are not interfering with consolidation

Discussion

In a previous study in the honeybee we were not able to demonstrate the reconsolidation phenomenon after one retrieval trial. This is in contrast to studies in species other then the honeybee Apis mellifera. In this study we excluded that this finding is a special feature of honeybee learning behaviour, because we here demonstrate the reconsolidation phenomenon after one retrieval trial.

The reconsolidation phenomenon demonstrated here does only appear at the 2nd or 3rd retention test after

Materials and methods

Experiments were carried out in Berlin, Germany from 2002–2005. General procedures and injections were described previously (Stollhoff et al., 2005).

Anisomycin (Sigma Aldrich, Deisenhof, Germany) and emetine (Fluka Chemie, Switzerland) were dissolved in PBS (137 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4, adjusted to pH 7.2). Honeybees were trained with three pairings of clove odor (CS) with sucrose (US) with an inter trial-interval of 2 min on day 1. On day 2 they were divided into two

Acknowledgments

This study was supported by the Volkswagenstiftung (I/77378) and a position for D.E. from the Deutsche Forschungsgemeinschaft (EI 512/1-2). We thank Martina Kemmer for technical support, Uwe Greggers for kindly providing us with a computer program, Mary Wurm for correcting the English and Rodrigo De Marco for fruitful discussions.

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