Memory built in conjunction with a stressor is privileged: Reconsolidation-resistant memories in the crab Neohelice

Journal Pre-proof Memory built in conjunction with a stressor is privileged: reconsolidation-resistant memories in the crab Neohelice Heidi Gonzalez (Data curation) (Formal analysis) (Investigation) (Validation) (Visualization)Writing – original draft)Writing – review and editing), Leonardo Bloise (Data curation) (Formal analysis) (Investigation) (Validation) (Visualization)Writing – original draft)Writing – review and editing), Francisco J. Maza (Conceptualization) (Data curation) (Investigation) (Visualization)Writing – review and editing), V´ıctor A. Molina (Conceptualization)Writing – review and editing), Alejandro Delorenzi (Conceptualization) (Funding acquisition) (Project administration) (Resources) (Supervision) (Data curation) (Formal analysis) (Visualization) (Investigation) (Validation)Writing – original draft)Writing – review and editing)

PII:

S0361-9230(19)30839-1

DOI:

https://doi.org/10.1016/j.brainresbull.2020.01.014

Reference:

BRB 9847

To appear in:

Brain Research Bulletin

Received Date:

28 October 2019

Revised Date:

14 January 2020

Accepted Date:

21 January 2020

Please cite this article as: Gonzalez H, Bloise L, Maza FJ, Molina VA, Delorenzi A, Memory built in conjunction with a stressor is privileged: reconsolidation-resistant memories in the crab Neohelice, Brain Research Bulletin (2020), doi: https://doi.org/10.1016/j.brainresbull.2020.01.014

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Title Page: Memory built in conjunction with a stressor is privileged: reconsolidationresistant memories in the crab Neohelice.

Heidi Gonzalez1†, Leonardo Bloise1†, Francisco J. Maza 1, Víctor A. Molina 2,

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Alejandro Delorenzi 1. († Both authors contributed equally to this manuscript)

Institutions:

FCEyN, Universidad de Buenos Aires, Ciudad Universitaria

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CONICET,

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(1) Departamento de Fisiologíay Biología Molecular y Celular, IFIByNE-

C1428EHA, Argentina.

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(2) Departamento de Farmacología, Facultad de Ciencias Químicas, IFEC-

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Argentina.

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CONICET-Universidad Nacional de Córdoba, Ciudad Universitaria, Córdoba,

Corresponding autor: Alejandro Delorenzi: [email protected]; Heidi

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Gonzalez:

[email protected];

Leonardo

Bloise:

[email protected]; Francisco J.Maza: [email protected]; Víctor A. Molina: [email protected];

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Highlights Some

conditions,

including

strong

training,

reconsolidation. 

limit

memory

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

In crabs, memory does undergo reconsolidation even after strong

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training. However, strong training plus stressors form reconsolidationresistant memory.

The induction of resistant memories may allow the preservation of

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Abstract

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privileged information.

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

The dynamics of memory processes are conserved throughout evolution, a

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feature based on the hypothesis of a common origin of the high-order memory centers in bilateral animals. Reconsolidation is just one example. The possibility to interfere with long-term memory expression during reconsolidation has been proposed as potentially useful in clinical application to treat traumatic memories. However, several pieces of evidence in rodents show that either robust fear memories or stressful events applied before acquisition promote 2

reconsolidation-resistant memories, i.e., memories that are resistant to the interfering effect of drugs on memory reconsolidation. Conceivably, the generation of these reconsolidation-resistant fear memories also occurs in humans. Is the induction of reconsolidation-resistant memories part of the dynamics of memory processes conserved throughout evolution? In the semiterrestrial crab Neohelice granulata, memory reconsolidation is triggered by a short reminder without reinforcement. Here, we show that an increase in the

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salience of the aversive stimulus augmented the memory strength; nonetheless,

the protein synthesis inhibitor cycloheximide still disrupted the reconsolidation

However, crabs stressed by a water-deprivation episode before a

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process.

strong training session built up a memory that was now reconsolidation-

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resistant. We tested whether these reconsolidation-resistant effects can be

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challenged by changing parametric conditions of memory-reminder sessions; multiple memory reactivations without reinforcement were not able to trigger

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the labilization-reconsolidation of this resistant memory. Overall, the present findings suggest that generation of reconsolidation-resistant memories can be

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another part of the dynamics of memory processes conserved throughout

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evolution that protects privileged information from change.

Keywords

memory, evolution, stress, reconsolidation, resistant memories, memory expression.

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Abbreviations VDS: visual danger stimulus; TR: trained group; UN: untrained group; TCx: training context; NCx: novel context; Veh: vehicle; CHX: cycloheximide; WD: water deprivation.

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1. Introduction

Although learning skills vary across species depending upon specific

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adaptations (Menzel, 2007), animals share much of the essential mechanisms involved in memory processes. Indeed, memory’s dynamics are highly

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conserved throughout evolution (Glanzman, 2010; Menzel, 1999), a feature

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based on the hypothesis of a common origin of the high-order memory centers in bilateral animals (Francisco Javier Maza et al., 2016; Tomer et al., 2010; Wolff

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and Strausfeld, 2016). Reconsolidation is yet another example of shared dynamics. Evolutionary distant species show vulnerability to pharmacological

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interventions during reconsolidation, from protein synthesis inhibitors to

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neuromodulators' agonists or antagonists, and to behavioral interventions (Dodd and Lukowiak, 2015; Lewis, 1979; Pedreira and Romano, 2013). Indeed, the possibility of disrupting memory during the reconsolidation phase has been proposed as potentially useful in clinical application to treat traumatic memories (Ecker et al., 2015; Faliagkas et al., 2018). However, not all reminder sessions lead to memory reconsolidation, and not all memories can enter 4

reconsolidation. Some conditions appear to limit the occurrence of memory destabilization/reconsolidation after retrieval. Like learning, the initiation of the reconsolidation process is crucially dependent on errors in outcome prediction during retrieval sessions (Barto et al., 2013; Fernández et al., 2016; Rescorla, 1972). Other boundary conditions are memory age (Suzuki et al., 2004), extinction ((Merlo et al., 2014, 2019) but see (Almeida-Corrêa and Amaral, 2014), and, here significant to highlight, the memory strength. Over time,

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studies in rodents have shown that stronger memories can become resistant to

interference following reactivation. For instance, increasing the number of

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training trials results in a memory trace that is more resistant to interference after reactivation (Rodriguez-Ortiz et al., 2008; Suzuki et al., 2004; Wang et al.,

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2009). Also, stressful experiences before fear learning prevent the onset of

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reconsolidation (Bustos et al., 2010; Wang et al., 2009). Likewise, chronic stress also can induce memories resistant to reconsolidation disruption (Hoffman et

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al., 2015).

We hypothesize the generation of reconsolidation-resistant memories is an

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integral part of the dynamic of memory processes conserved throughout evolution that protects privileged information from change. Consequently, this

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resistance can be disclosed in evolutionarily distant species. The first working hypothesis tested here, not confirmed by the results, was that more intense training produces reconsolidation-resistant memories in Neohelice. Then, we hypothesized that, in this semiterrestrial crab, a stronger memory plus a stressful experience –water deprivation- may produce a long-term memory

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trace that is resistant to interference after reactivation. The associative memory paradigm used in Neohelice (formerly Chasmagnathus) is based on the crab’s escape response elicited by a visual danger stimulus (VDS) resembling an aerial predator (Tomsic and Romano, 2013). The memory understudy arises as a consequence of an association between the context and the VDS. During training, the escape response decreases in intensity and is progressively replaced by a freezing response; in trained animals, this preference for freezing

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is present in testing sessions (Pereyra et al., 2000). Throughout a 15-trial training, robust freezing is also acquired throughout. Memory is dependent on

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the context in which memory was tested; noticeable retention is found when

tested in the same training-context with the VDS presentation in the long-term

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(1–4 days). This decrease in activity in testing sessions is due to an increase in

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the number of animals displaying the freezing response (Frenkel et al., 2005a; Fustiñana et al., 2013; Pedreira et al., 2002, 1998, 1998; Pereyra et al., 2000;

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Tomsic et al., 1998; Tomsic and Romano, 2013). Studies at the behavioral, anatomical and cellular levels have provided an integrated description of

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different memory phases of a contextual-associative memory in Neohelice granulata (Tomsic and Romano, 2013). Many aspects of acquisition,

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consolidation, extinction, retrieval and memory expression, and reconsolidation phases have been characterized (Barreiro et al., 2013; Caffaro et al., 2012; Delorenzi et al., 2014; Frenkel et al., 2005a, 2005b; Fustiñana et al., 2013; Merlo et al., 2019; Pedreira et al., 2004; Pedreira and Romano, 2013; Romano et al., 2006, 1996). One advantage of addressing the generation of reconsolidation-resistant

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memories in crabs is that the reconsolidation processes and the parametric structure to induce labilization in the reminder sessions have been intensely studied. For instance, the notion that the reminder presentation per se does not trigger memory destabilization/reconsolidation, given that the interfering or enchaining effects depend on the reminder fulfills mismatch conditions was initially originated in this paradigm (Frenkel et al., 2005a; Fustiñana et al., 2013; Pedreira et al., 2004). The present findings support the view that the generation

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of reconsolidation-resistant memories may be part of the dynamics of memory

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processes conserved throughout evolution.

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2. Experimental procedures

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2.1. Animals

Neohelice granulata is a burrowing semiterrestrial crab that inhabits the

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intertidal zone of estuaries, salt marshes, and mangroves of the South-western Atlantic Ocean (Spivak, 2010). Intermolt adult male crabs measuring between

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2.7 and 3.0 cm across the carapace were collected from the narrow coastal inlets of San Clemente del Tuyú, Buenos Aires Province, Argentina. In the laboratory,

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crabs were kept on a 12:12 h light-dark cycle, in collective plastic tanks (20 animals each) filled up to 2 cm depth with 12% seawater prepared from Coral Pro Salt, Red Sea, Israel, pH 7.4-7.6. The holding and experimental rooms were kept at 22–24 °C and 80 ± 10% relative humidity. Experiments were carried out at daytime within the first two weeks after the arrival of animals. Each crab was

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used in one experiment only. All efforts were made to minimize the number of animals. The research was conducted following the Ethical Reference Frame for Biomedical Investigations of CONICET, equivalent to the standard procedures for animal care and use of the NIH of the U.S.A.

2.2. The experimental device

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The experimental device, the actometer (Tomsic and Romano, 2013), referred to as the training context, consisted of a bowl-shaped opaque container, illuminated by a bottom light (5W bulb), with a steep concave wall 12 cm high

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(23 cm top diameter and 9 cm floor diameter) covered to a depth of 0.5 cm with brackish water, where the crab was lodged before each experimental session. In

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the training protocol used here, termed Contextual Pavlovian Conditioning,

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eighteen seconds before each trial, the bottom light went off, and a top light (5Wbulb) was turned on. Then, a 9 s trial was given, in which an opaque

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rectangular screen (25–7.5 cm), termed visual danger stimulus (VDS) and located horizontally 12 or 6 cm over the actometer, was moved twice over the

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animal at a constant speed, cyclically from left to right and vice versa. During this 9s trial, the top light was kept on (Fustiñana et al., 2013). The VDS

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provoked an escape response in crabs and consequent container vibrations, which were converted into electrical signals through a piezoelectric transducer placed on the external wall of the container. These signals were amplified, integrated during each 9s trial and translated into numerical units ranging from 0 to 23.999, before being processed by a computer. The intensity of container

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vibrations during VDS presentation (a trial) depends on the magnitude of the defensive responses that each crab displays when faced with an impending threat. Throughout this study, data were recorded during the trial time, i.e., during the 9 s VDS presentation. No escape response is displayed either during the adaptation time or during intertrial intervals (Tomsic et al., 1991). The experimental room had 40 devices separated from each other by partitions. Between Training, Reminder, and Testing Sessions crabs stayed in resting

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containers (cylindrical plastic containers, 15 cm in diameter and 15 cm in height) covered to a depth of 0.5 cm with brackish water and kept inside dimly

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lit drawers.

2.3. Experimental procedure and design

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Reconsolidation experiments included three sessions: Training Session (Day 1), Reminder Session (Day 2) 24 h later, and Testing Session (Day 3) 48 h after the

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Training Session. The experimental protocols involve pairs of crab groups, where each pair consists of a trained group (TR) and an untrained group (UN).

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UN animals remained in the actometer during the entire Training Session under

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the same conditions that TR, but received only one VDS presentation that was simultaneous to the first training trial of the trained group. This trial was used to control homogeneity in the escape response of all experimental groups at the beginning of the experiments. For the untrained groups, the response was recorded in intervals of 9s coincident with VDS presentation in the respective trained group. During the Reminder Session and Testing Session, untrained 9

groups were treated in the same way as the trained groups. Experimental groups started with 30 or 40 crabs. Animals that did not respond in the first VDS trial, presented damaged appendages or died were excluded from the analysis. Before training, each crab was turned on its back and only animals that without delay returned to the normal position were used. Crabs that present slow reactions show low responsiveness to a broad diversity of stimuli, and at a later time, they usually present unhealthy symptoms. Memory expression is

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operatively disclosed at the testing sessions when the TR group shows a level of responsiveness to the VDS presentation that is statistically lower than that of

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the UN group (Caffaro et al., 2012; Fustiñana et al., 2013; Maldonado, 2002;

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al., 1991; Tomsic and Romano, 2013).

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Francisco Javier Maza et al., 2016; Merlo et al., 2019; Tano et al., 2013; Tomsic et

2.4.1. Day 1, Training session

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TR group: This group underwent a training protocol. First, each crab was placed in an individual training context for 10 min, without being stimulated

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with VDS. After this adaptation period, animals received 5 or 15 training trials

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(Section 2.3.), separated by intertrial intervals of 3 min. Finally, animals were immediately placed in individual resting containers until the following day. UN group: Each crab was placed in the training context (Section 2.2.) and remained there for the same time and conditions as the TR group but receiving only the first presentation of the VDS (2.3.). Then, animals were immediately placed in individual resting containers until the following day. In experiments that 10

involved water deprivation, crabs were water-deprived for 2hs before training. Except where the training protocol consisted of only 3 training trials, one trial was presented before the training to measure the responsiveness of each animal.

2.4.2. Day 2 – Reminder session

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Three situations may arise in this session according to the design of the experiment in question (Barreiro et al., 2013; Caffaro et al., 2012; Fustiñana et al.,

2013; Merlo et al., 2019; Tano et al., 2013): (a) Crabs were re-exposed to the

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training context for 5 min, in which the bottom light went off, and a top light

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came on for the last 27 s without the VDS presentation. Then, animals were returned to their respective resting containers until the following day. This

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procedure reactivates and turns memory into the destabilization process, initiating the reconsolidation process. Note here that the presentation of the

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VDS is not necessary for memory reactivation (Frenkel et al., 2005b; Fustiñana et al., 2013; Pedreira et al., 2004), (b) Crabs were re-exposed to the training

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context for 5 min, in which the bottom light went off, and a top light came on

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for the last 27 s, with the VDS being presented for the last 9 s. This procedure prevents memory labilization, and reconsolidation is not initiated. (c) Crabs were exposed to a cylindrical and striped context (different context)(a cylindrical 15 cm in diameter and 15 cm in height plastic container with black and white striped walls, covered to a depth of 0.5 cm with brackish water) and the VDS was not presented. This context is perceived as a novel context, and 11

thus, it does not trigger memory labilization and reconsolidation. After the presentation of each reminder, crabs were returned to their respective resting containers until the following day.

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2.4.3. Testing sessions

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Crabs were re-exposed to the training context for 10 min, and then memory

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expression was tested with one VDS trial as in Section 2.3.

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2.5. Drug administration

Crustacean physiological saline solution (Hoeger and Florey, 1989) was used as

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the vehicle. 50 µl of vehicle or drug dissolved in vehicle solution were given through the right side of the dorsal cephalothoraxic-abdominal membrane,

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using a syringe fitted with a sleeve to control the depth of penetration to 4 mm, thus ensuring that the injected solution was released in the pericardial sac. The

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protein synthesis inhibitor Cycloheximide (CHX) was administered at a final dose of 2.35 µg/g. Injections were carried out one hour after the reminder session; in previous studies in Neohelice, reconsolidation has shown a time window of 4–6-h sensitivity to CHX (Caffaro et al., 2012; Pedreira et al., 2004,

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2002) All drugs were purchased from Sigma Chemical Co. Saint Louis, Missouri, USA.

2.6. Memory retention criterion and data analysis In the experiments in which there were two TR groups, the training session was analyzed using a one-way ANOVA for the first VDS trial of each training

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session, trials of the TR groups were analyzed with an ANOVA for repeated measures. Long-term memory expression was assessed on Day 2 or 3 by focusing data analysis on test trial scores, i.e., by estimating the difference

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between the response level of the trained group (TR) and that of the respective

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untrained group (UN). Rescorla convincingly argued in favor of using this sort of analysis instead of a paired training–testing comparison, stressing the need

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to clearly distinguish between the time of input (Training Session) and the time of assessment (Testing Session) (Rescorla, 1988). This approach is justified in the

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present case since it has been demonstrated that memory expression in crabs is independent of the escape response level at training (Tomsic and Romano,

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2013). Data were analyzed using analysis of variance (ANOVA) and a priori

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planned comparisons. The variable under measure (container vibrations as a result of complex motor activities) reflects qualitatively different responses (for example, freezing and escape), and therefore the variance of this parameter is different when different responses prevail. As the variance of activity scores increases with the mean, thus violating the homogeneity of variance assumption of ANOVA, data were log2 transformed. All experiments described 13

in this paper used one or two untrained–trained pairs (UN–TR). Three types of contrast per experiment were used for two pairs: the first, between the two untrained groups of each pair; the second, between UN and TR of one pair; and the third, between UN and TR of the other pair. A TR group was said to show memory when its mean response level at the test trial was statistically lower than the respective UN group. The values, resulting from the 9 s vibration integration measured by four microphones, are expressed in arbitrary units. We

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use the transformed values (log2 of response), which have a normal distribution

to show the activity of groups of animals as mean and standard errors. When

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only one pair of groups was compared, a t-test was used. We analyzed data

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using STATISTICA (StatSoft 8.0).

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3. Results

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3.1. Stronger memories enter in reconsolidation. Briefly, the memory paradigm used in Neohelice is based on the crab’s escape

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response elicited by a visual danger stimulus (VDS, an opaque rectangular screen 25 x 7.5 cm located 12 cm above the container) resembling an aerial

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predator. Upon repeated presentations (15 spaced trials) of the VDS, the escape response declines over trials turning into a freezing behavior. This change in behavior persists in the long term and is mediated by an association between training context and VDS (Tomsic et al., 1998; Tomsic and Romano, 2013). Memory expression is revealed at the testing session as a significant decrease in

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the escape response upon stimulation with the VDS. Here we used the version of the training protocol that enhances the associative strength between VDS and context where the actometer was illuminated from above during the training trials and from below during inter-trial intervals, termed Contextual Pavlovian Conditioning in Neohelice (Fustiñana et al., 2013). A standard training protocol to generate long-term memory consists of 15 VDS trials (located 12 cm above the container, VDS-12cm) separated by 3 minutes intervals. After a weak

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training protocol, consisting of 6 or less VDS trials, crabs do not show memory expression in the long term (Caffaro et al., 2012; Delorenzi et al., 1995; Fustiñana

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et al., 2013; Smal et al., 2011). The associative strength of a memory depends on stimulus salience (Rescorla, 1988, 1972); consequently, it is expected that a 5

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trials training protocol using greater salience of the VDS would build up a

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memory that is expressed in the long term. To estimate a putative increase in the salience by bringing the aversive stimulus closer, we first compared the

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escape response of groups of animals for one trial stimulated with the VDS located 6 cm (VDS-6cm) vs. 12 cm (VDS-12cm) above the training context

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(Figure 1A). As expected, the group of animals stimulated VDS-6cm showed a higher escape response (log2-transformed) (11.75 ± 0.23) than the VDS-12cm

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group (10.95 ± 0.28) (t(65) = 2.26, p = 0.027). Hereafter, in all experimental series, the VDS was located 6 cm instead of 12 cm above the container. In the next experiment, we evaluated whether a training protocol of just 5 trials but with the greater salience of the VDS-6cm generates a memory that is expressed in the long term (Figure 1B).

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No difference was found for the initial trial between trained (TR, n = 29) and untrained (UN, n = 30) groups (t(57) = 0.78, p = 0.43) and a significant trial effect was disclosed for the TR group (F trial (4, 116) = 8.57; p < 0.001). During the memory retention test, one day later, both groups received one trial with VDS-6cm presentation. Although trained with just 5 trials, crabs exhibited longterm memory revealed as a significant reduction of the escape response (TR vs. UN groups, t(57) = 2.67, p = 0.0098) (Figure 1B-Day 2). Moreover, a training

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protocol consisting of only 3 training trials (TR group vs. 0 trials, UN group) with the VDS-6cm was enough to build up a memory that is expressed in the

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long term (Figure 1C). Training session, Day 1: a significant trial effect was

disclosed for TR group for trial 1 to 3 (F trial (2, 54) = 10.6; p < 0.001). Testing

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Session, Day 2: UN vs. TR, t(54) = 2.63, p < 0.05). Unlike 5 VDS-12cm training

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trial, where crabs do not show memory expression in the long term (Fustiñana et al., 2013; Tano et al., 2013), 5 trials training protocol with an increased

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salience (VDS-6cm) builds up a memory that is expressed in the long term. Moreover, only 3 trials appear to be enough to do it.

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In the Neohelice paradigm, it has been shown that the memory generated by training with the VDS-12 entered into reconsolidation when crabs were re-

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exposed to the training context for 5 min, in which the bottom light went off, and a top light came on for the last 27 s without the VDS presentation (Caffaro et al., 2012; Fustiñana et al., 2013; Pedreira et al., 2004). Since the objective of the study is to assess whether an increase in the strength of memory causes it to be resistant to reconsolidation, in the next experiment, we assessed whether the

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memory reconsolidation process took place when five VDS-6cm training trials generated the memory. We evaluated whether the administration of cycloheximide following the Reminder Session that triggers reconsolidation disrupts memory (Figure 1D). Two pairs of UN–TR groups of crabs underwent the Training Session. No difference was found for the first trial between TR and UN groups (F (3, 128) = 0.36; p = 0.78) and a significant trial effect was disclosed for the trained groups (Ftrial(4, 256) = 13.99; p < 0.001). On Day 2, a pair of UN–

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TR groups was re-exposed to the original training context (TCx) for 5 min

without presentation of the VDS. The nonappearance of the VDS during the

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reminder session triggers reconsolidation since there is a mismatch with the

predictions generated by the re-activated memory (Pedreira et al., 2004); then,

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crabs were returned to their resting containers (UN–TCx and TR–TCx groups).

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The experimental design included another pair where the interfering effect should not be present since memory was not re-activated because crabs were

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exposed to a novel context (NCx) for 5 min; UN–NCx and TR–NCx groups. One hour after the Reminder Session, all crabs were administered with

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cycloheximide 2.35 µg/ g. On Day 3, all animals underwent a testing session. As expected, planned comparisons on escape response (ANOVA F(3,128) =

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3.15, p = 0.027) disclosed significant differences in escape response between the groups exposed to a novel context at Day 2 (NCx groups), revealing memory expression (p = 0.008) despite the administration of the amnesic agent. However, no differences between the groups exposed to the training context at Day 2 (UN-TCx vs. TR-TCx groups, p = 0.28) (Figure 1D-Day 3). In addition, no

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differences between untrained groups, UN-TCx vs. UN-NCx (p = 0.96), were disclosed. Therefore, results show that like a training protocol of 15 trials with VDS-12cm, five VDS-6cm training trials also generate a memory that is expressed in the long-term and enters reconsolidation when the reminder that reactivates and initiates reconsolidation was presented. Some studies have described that stronger memories are more resistant to

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reactivation-induced interference (Suzuki et al., 2004; Wang et al., 2009). A standard training protocol consisted of 15 VDS-12cm trials. In the next experiment, we assessed whether the reconsolidation process takes place when

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memory is generated by a strong training protocol of 15 trials but now with the

greater salience of the VDS-6cm (Figure 2). On Day 1 two pairs of UN–TR

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groups of crabs underwent the training session of 15 trials with VDS-6 cm; no

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difference was found for the first trial between TR and UN groups (F(3, 99) = 1.13; p = 0.340) and a significant trial effect was disclosed for the trained groups

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(Ftrial 4, 200) = 15.43; p < 0.001). At Day 2, one TR-UN pair was presented with the VDS (TCx+VDS) (this procedure reactivates but not initiates memory

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reconsolidation); trained crabs exhibited memory revealed as a significant reduction of the escape response in comparison to the UN groups (t(52) = 2.52,

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p = 0.015)(Figure 2-Day 2). The other pair was exposed 5 min to the training context (TCx) and crabs were not stimulated with the VDS presentation (this procedure reactivates and initiates memory destabilization-reconsolidation). One hour after re-exposition, all groups were injected with cycloheximide (CHX). On Day 3, all animals underwent a testing session. Planned

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comparisons on activity scores on Day 3 [ANOVA F(3,99) = 3.86, p = 0.0116] disclosed significant differences in escape response between TCx+VDS groups, revealing memory expression (p = 0.003) but no differences between the TCx groups (p = 0.60) (Figure 2-Day 3). No differences between untrained groups, UN- TCx+VDS vs. UN- TCx (p = 0.88), were disclosed. Therefore, the strong training protocol of 15 training trials with the greater salience (VDS-6cm) also generates a memory that was not resistant to the interference by cycloheximide

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during reconsolidation.

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3.2. The concurrence of water deprivation plus strong training generates

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reconsolidation-resistant memories

Environmental demanding experiences previous to training prevents the onset

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of memory reconsolidation in rodents (Bustos et al., 2010; Espejo et al., 2016; Giachero et al., 2013). In Neohelice, water deprivation both increases heat shock

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protein HSP70 expression in the brain (Frenkel et al., 2011, 2008) and improves memory consolidation, retrieval, and reconsolidation through the activation of

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angiotensin II-like receptors (Frenkel et al., 2010a, 2005b, 2005a, 2002). The

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following experiment was carried out to evaluate whether water deprivation before training prevents the onset of memory reconsolidation after memory reactivation (Figure 3). On Day 1 a pair of UN–TR groups of crabs underwent 2 hours of water deprivation (WD pair) episode before the strong training protocol (15 training trials with the greater salience of the VDS-6cm) while another pair continued to be in brackish water (W), not exposed to the water 19

deprivation episode. No difference was found for the first trial between TR and UN groups (F(3, 110) = 2.52; p = 0.061) and a significant trial effect was disclosed for the trained groups (Ftrial (14, 770) = 27.36; p < 0.00001)(Figure 3Day 1). In agreement with previous studies (Frenkel et al., 2002), note the decreased escape response in water-deprived animals during training (trial 15 TR-W vs. TR-WD: p = 0.0358).

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On day 2, both UN-TR pairs were placed for 5min in the training context without VDS presentation, the Reminder Session that initiates memory

reconsolidation. One hour after re-exposition, all groups were injected with

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CHX. On Day 3, all animals were placed in the training context and tested with one VDS-6cm trial [ANOVA, a priori planned comparisons, (F(3, 110) = 7.55, p

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= 0.00012)] (Figure 3-Day 3). Crabs without water deprivation before training

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did not show memory on Day 3; there were no differences in escape response (TR-W vs. UN-W: p = 0.76). Conversely, memory expression was disclosed for

na

the water deprivation group (TR-WD vs. UN-WD: p = 0.0079). No difference was found between untrained groups (UN-W vs. UN-WD: p = 0.11). This result

ur

strongly suggests that the episode of water deprivation plus a strong training

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protocol can generate reconsolidation-resistant memories. As the memory generated by 15 trials with the greater salience of the VDS-6cm plus the water deprivation episode before the training session appears to be reconsolidation-resistant, in the following experiment the protocol was the same as previously but the Reminder session of Day 2 was modified in order to reiterate the mismatch component (Figure 4). Several studies suggested that 20

multiple short reactivations can modulate the reactivated memory because multiple rounds of reconsolidation are possible (Almeida-Corrêa and Amaral, 2014; Forcato et al., 2011; Inda et al., 2011). The working hypothesis here was that multiple reactivations would surpass the induced resilience to reconsolidation. All animals were water-deprived for 2 hours before training (15 trials, VDS-

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6cm). No difference was found for the first trial between TR and UN groups (F(3, 114) = 0.712; p = 0.546)) and a significant trial effect was disclosed for the trained groups (Ftrial (14, 812) = 25.28; p < 0.001)(Figure 4-Day 1).

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On Day 2 all groups underwent three Reminder Sessions without VDS trying to

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enhance the mismatch component. After 1 hour, a UN-TR pair was administered with cycloheximide CHX 2.35 µg/g (TR- CHX and UN- CHX

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groups), and the other pair of UN–TR groups was administered with physiological saline solution (TR-Veh and UN- Veh). On Day 3 (ANOVA, a

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priori planned comparisons: F(3, 114) = 3.66, p = 0.0145), activity scores showed differences between TR-Veh and UN-Veh (p = 0.034), and between TR- CHX

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and UN- CHX (p = 0.013) indicating memory retention in both pair of groups

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(Figure. 4-Day 3). There was no difference between UN-Veh and UN-CHX (p = 0.77). That is, CHX administered after this putative enhanced reminder procedure did not show impairing memory effects. Results suggest that this type of 3x-reminder also did not trigger the destabilization-reconsolidation process when animals were water-deprived before the strong training session.

21

4. Discussion The key finding of this study is that the resistance to pharmacological interference during reconsolidation described in mammals can also be evidenced in crabs. In accordance with classical models of associative memories (Rescorla, 1972), memory seems to be strengthened via an increase in the salience of the visual-danger stimulus. Five instead of fifteen trials were enough

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to generate a memory that can be expressed in the long term. However, fifteen trials with the increased salience of the visual-danger stimulus generate a strong memory that enters reconsolidation when a short reminder without

-p

reinforcement reactivates it. In Neohelice, an event that induces a memory

resistant to reconsolidation was accomplished by adding a challenging

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environmental episode: when animals were water-deprived before the strong

lP

training protocol, neither a short reminder without reinforcement nor three reminder presentations were able to initiate the destabilization/reconsolidation

na

process.

In Neohelice, the formation of a memory that is expressed in the long-term

ur

requires 15 or more training trials (Fustiñana et al., 2013; Tomsic and Romano,

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2013). Training sessions consisting of five or six training trials -called weak training sessions- were initially suggested as unable to build up long-term memory (Delorenzi et al., 1995; Romano et al., 1996; Smal et al., 2011). However, this initial interpretation was not accurate. Memory reconsolidation occurs even if memories are behaviorally unexpressed (Delorenzi et al., 2014). Weak training protocols generate consolidated memories that can be reactivated even though 22

they are not behaviorally expressed (Barreiro et al., 2013; Caffaro et al., 2012; Delorenzi et al., 2014; Frenkel et al., 2010b, 2005a; Kindt et al., 2014; Klappenbach et al., 2017; F. J. Maza et al., 2016). As expected, via an increment in the salience of the aversive stimulus five, and even three, training trials were sufficient to generate an associative memory that was expressed in the longterm (Figure 1). Our initial hypothesis foresees that stronger training (animals trained with 15 trials with the increased salience of the aversive stimulus, VDS 6

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cm) yield to stronger memory (Rescorla, 1972) and, consequently, resistant to

initiate the reconsolidation process. However, the present findings showed that

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this memory entered reconsolidation when the reminder that triggered destabilization was presented since the cycloheximide administration induced

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memory disruption as revealed in the testing session (Figure 2)(Caffaro et al.,

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2012; Fustiñana et al., 2013). Such interference was reconsolidation-specific since memory was not disrupted by cycloheximide when the brief reminder

na

with reinforcement was used, a procedure that reactivates memory but does not initiate the reconsolidation process (Figure 2) (Pedreira et al., 2004) In rodents,

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increasing the training trials induced memories that are resistant to reconsolidation interference (Suzuki et al., 2004; Wang et al., 2009). Although

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we increased the training protocol in crabs by an augmentation in the salience of the aversive stimulus (Figure 1), the short reminder without reinforcement was still able to reactivate and be susceptible to cycloheximide interference during reconsolidation (Figure 2).

23

It is well established that stress has profound effects on memory processes (Akirav and Maroun, 2013; de Quervain et al., 2017; Dodd and Lukowiak, 2015). The recognized effects of intrinsic stressors (i.e., related to the cognitive task) are to enhance memory consolidation; meanwhile the documented effects of extrinsic stressors (i.e., unrelated to the task) are more heterogeneous (Sandi and Pinelo-Nava, 2007).

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Exposure to acute stressors may have enhancing effect when the event occurs near to acquisition (Cadle and Zoladz, 2015; Lukowiak et al., 2014; Sandi and Pinelo-Nava, 2007) and, in rodents, also prevents the onset of reconsolidation

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following memory reactivation (Bustos et al., 2010). A stressor, applied before

learning, induce the formation of persistent fear memories that are resistant to

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interferences during the reconsolidation process. These studies show that

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robust memories plus intrinsic stressors that surround the learning process are one of the conditions that may limit the occurrence of the reconsolidation

na

process.

Neohelice is a euryhaline and semi-terrestrial crab that inhabits brackish water

ur

and can be confronted with water-deprivation episodes. In crustaceans, heat

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shock and osmotic and environmental stressors significantly increase heat shock protein expression in several tissues, including the central nervous system (Chang, 2005; Spees et al., 2002). A significant increase in HSP70 expression in brain areas of the crab Neohelice is induced by different environmental challenges, including an episode of 2h water deprivation (Frenkel et al., 2011, 2008). Initial studies in Neohelice suggest that endogenous 24

angiotensins appeared early in evolution as a functional link between water shortage and behavioral adaptations, including the modulation of memory processes (De Mello, 2014; Delorenzi et al., 2000, 1995; Frenkel et al., 2002; Salzet et al., 2001). In crabs, this environmental stressor improves, via angiotensin II, memory consolidation, retrieval, and reconsolidation (Caffaro et al., 2012; Frenkel et al., 2005a, 2005b, 2002; Klappenbach et al., 2017). Indeed, in rats, both physiological and psychological stressors elicit the activation of the

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angiotensinergic system (Krause et al., 2011). The studies in Neohelice are in agreement with the view that, when animals cope with water shortages, the

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angiotensinergic system triggers coordinated actions, from osmoregulation to behavior, which enables the animal to survive under this ecological challenge

re

(Albrecht, 2010; De Mello, 2014; Maren et al., 1994; Sierra et al., 2013).

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When water-deprived crabs were trained with the strong training protocol plus the augmentation in the salience of the aversive stimulus, the short reminder

na

without reinforcement was not able to induce a memory susceptible to interference during reconsolidation (Figure 3). However, the conditions for

ur

triggering reconsolidation are long recognized as complex (Ecker et al., 2015; Fernández et al., 2016; Kindt, 2018), and the search of the specific parametric

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conditions in memories that are resilient to be labile after reactivations is a challenge. The reconsolidation-resistant memory presented here is just another example of how the complex interactions between both extrinsic and intrinsic stressors, learning history, and reactivations can modify memory properties (Martijena and Molina, 2012; Sandi and Pinelo-Nava, 2007). Our attempt to

25

abolish resistance by increasing the number of memory reactivations sessions was not successful since cycloheximide did not induce a disruptive effect on memory reconsolidation (Figure 4). Like other reconsolidation-resilient memories such as those induced by robust training, old memories, or those affected by extrinsic stressors, it is predictable that different parametric conditions during the reminder session will restore the vulnerability to reconsolidation interference, extending the periods of context-presentation for

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instance (Pedreira and Maldonado, 2003; Suzuki et al., 2004). Additionally, it is possible that the stressor, applied before and during learning with the danger

-p

stimulus more intense, led to a stronger memory that is not only

reconsolidation resistant but, as predictable with the addition of the stressor,

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perhaps also insensitive to the standard extinction training protocol (Fustiñana

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et al., 2013).

Results lead to the question about whether there are differences between a

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strengthened memory and a memory accompanied by stressful events previous and during the consolidation -and reconsolidation- processes. Canonical view

ur

highlights that in the core of the mechanisms of the emotional influences on memory are the release of endogenous hormones and neuromodulators

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triggered by either the learning experience itself or the extrinsic stressors that surrounded the event modulate memory consolidation; influencing the activity and plasticity in brain regions involved in the memory processing (LaLumiere et al., 2017; Lukowiak et al., 2014; McGaugh, 2000; Sandi and Pinelo-Nava, 2007). Therefore, this view supports the notion that despite differential

26

neuromodulatory mechanisms can be involved in each particular interaction between the stressful events with the memory trace, the final products of a strengthened memory as a result of extensive training and memory enhanced by stressful events might be similar. However, the neuromodulatory effects on encoding and consolidation should not necessarily be seen as additive but interactive; the signals triggered by neuromodulators during the encoding might determine different actions of the neuromodulatory effects during

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consolidation (Lupien et al., 2007; Roozendaal and Hermans, 2017). Consequently, the generation of different traces generated by a strengthened

-p

memory because intensive training or a memory boosted by surrounding

stressful events is also probable. As mentioned before, the relationship between

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the role of the endogenous neuromodulators triggered by stressors and

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memories are indeed complex with multifaceted interactions (Sandi and PineloNava, 2007). For instance, our previous results in crabs and humans support the that

during

consolidation

and

reconsolidation,

endogenous

na

view

neuromodulators can determine the ability of the memory to guide behavior by

ur

decreasing or increasing its behavioral expression, without disturbing both its persistence and its capacity to be reactivated (Caffaro et al., 2012; Coccoz et al.,

Jo

2013; Delorenzi et al., 2014; Larrosa et al., 2017). In Neohelice, endogenous angiotensin II modulates long term memory expression but does not determine memory persistence (Frenkel et al., 2010b). Angiotensin II participates in the consolidation, retrieval and reconsolidation processes in Neohelice. Angiotensin II induces memory enhancements after

27

weak trainings. Also, the administration of an angiotensin II antagonist, before or after 15 trials (VDS-12 cm) training session, disrupts memory expression but without affecting the potential of persistent memories to be activated and become labile (Delorenzi and Maldonado, 1999; Frenkel et al., 2010b, 2002). Like the actions postulated in mammals (Saavedra and Benicky, 2007), also in crabs, the brain angiotensinergic system might play an important role as a stress

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hormone. It is also possible to argue that the reminder session was not able to initiate

reconsolidation because water deprivation induces a state-dependency resulting

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from the change in experimental conditions between training (with water

deprivation) and testing (without water deprivation). Similar to the

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phenomenon called encoding specificity by Tulving and Thomson (Tulving and

lP

Thomson, 1973), the neurohumoral state -as the triggered by water-deprivationupon different memory phases is crucial for the state-dependency examination

na

where the same internal state becomes necessary to warrant an adequate retrieval (Gisquet-Verrier et al., 2015; Izquierdo and Dias, 1983). In an original

ur

study in rats, Sierra et al. (Sierra et al., 2013) showed that a fear memory reactivated in a specific endogenous state might become state-dependent.

Jo

Consequently, posterior retrieval depends on the reinstallment of this internalstate. Moreover, this state-dependency effect developed during reconsolidation depends on endogenous angiotensins II. As proposed by Gisquet-Verrier et al. (Gisquet-Verrier et al., 2015), this case is another example of how interfering effects induced during both consolidation and reconsolidation can be explained

28

by state dependency. However, this argument does not fit with results showing that on Day 3 (Figures 3 and 4), in both VEH or CHX injected crabs there are not retrieval deficits as the result from the change in water deprivation condition between training and testing sessions. This situation was expected in Neohelice given that cognitive capabilities vary across species depending upon specific adaptations (Menzel, 2007); in this semiterrestrial and intertidal crab, there are not memory-expression deficits if an episode of 2-h water deprivation takes

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place only during consolidation, reconsolidation or retrieval phases (Caffaro et al., 2012; Frenkel et al., 2005a, 2005b, 2002; Klappenbach et al., 2017).

-p

Also, pharmacological treatments have been shown useful to restore

vulnerability to interference in memories of stressed animals (Espejo et al.,

re

2016). In rats, the initiation of the molecular cascades triggered by the

GluN2B-NMDA

lP

destabilization-reconsolidation process, such as an increased expression of subunit

and

Zif-268,

for

instance,

is

restricted

in

na

reconsolidation-resistant memories (Espejo et al., 2016; Wang et al., 2009). Whether the molecular cascade triggered in the reconsolidation process is

ur

restricted in the reconsolidation-resistant memory protocol used here needs to be evaluated, the NF-kappaB transcription factor is a prominent candidate

Jo

(Tomsic and Romano, 2013). On the other side, research regarding the mechanisms behind the water deprivation that make memory reconsolidationresistant would open the opportunity to reverse the development of this resilience; studies that include the use of angiotensins II antagonists could shed light on this point. Indeed, in mammals, several reports have shown an

29

essential role for the angiotensinergic system in the stress response (Albrecht, 2010).

5. Conclusions Reconsolidation studies have stressed on the dynamics of memories and proposed a lively process through which memories are built up, updated, and

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maintained. Why consolidated memories become fragile again during

reconsolidation? The hypotheses, proposing that reconsolidation opens the

opportunity to update, strengthen, and modulate consolidated memories

-p

(Alberini et al., 2013; Sara, 2000), have been tested and reshaped during two

re

decades by several experimental approaches (Delorenzi et al., 2014; Dudai et al., 2015; Faliagkas et al., 2018; Lukowiak et al., 2007; Pedreira and Romano, 2013).

lP

Also, the functions of retrieval-induced reconsolidation have been analyzed under evolutionary perspectives (Delorenzi et al., 2014; Lukowiak et al., 2007;

na

Pedreira and Romano, 2013). Here we show that, in Neohelice, strong training plus stressors form reconsolidation-resistant memory. The mechanisms that

ur

restrict some memories to enter reconsolidation perhaps can be conceived as an

Jo

adaptive process, conserved across evolution, which allows the preservation of privileged information.

Conflict of interest The authors declared no conflict of interest. 30

Author statement (CRediT Roles): Heidi Gonzalez Gonzalez: Data curation, Formal Analysis, Investigation, Validation, Visualization, Writing – original draft, Writing – review & editing. Leonardo Bloise Heidi: Data curation, Formal Analysis, Investigation, Validation, Visualization, Writing – original draft, Writing – review & editing.

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Francisco J. Maza: Conceptualization, Data curation, Investigation, Visualization, Writing – review & editing.

Víctor A. Molina: Conceptualization, Writing – review & editing.

-p

Alejandro Delorenzi: Conceptualization, Funding acquisition, Project administration,

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Resources, Supervision, Data Curation, Formal Analysis, Visualization, Investigation,

na

lP

Validation, Writing – original draft, Writing – review & editing.

ur

Acknowledgments—This work was supported by Universidad de Buenos

Jo

Aires (UBA 20020110100071), CONICET (PIP 11220120100170CO), and ANPCYTs (PICT2013-1020 and PICT 2016-1875). We thank Angel Vidal for technical support. The authors declare that there are no competing financial interests.

31

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Figure captions Figure 1. A context-specific long-term memory that enters a reconsolidation phase. A) Different distance of the VDS with different escape response. B) Memory after a 5 trial VDS-6cm training protocol. Day 1, training session, (TR, black circles; UN, grey circles), 5 trials with VDS-6cm. Day 2, escape response during tests. Memory retention was tested with a single VDS

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presentation. Memory expression is evidenced in the TR group (dark bar) as lower escape response to the VDS in comparison to the untrained group (UN, grey bar), UN-TR N = 29–30.

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C) Memory after a 3 trial VDS-6cm training protocol. Day 1, training session,

(TR, black circles; UN, grey circles), 3 trials with VDS-6cm. Day 2, escape

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response during tests. Memory expression is evidenced in the TR group (dark

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bar) as lower escape response to the VDS in comparison to the untrained group (UN, grey bar); UN-TR N = 28–28.

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D) Day 1, training session. Day 2, a UN-TR pair was re-exposed to the training context (TCx) for 5 min (this procedure reactivates and initiates memory

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labilization-reconsolidation) and the other UN-TR pair to a novel context (NCx). After 1 hour, all groups were injected with cycloheximide (CHX). Day 3 (testing

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session). N= 33 for all groups. Only the NCx pair showed memory. ** p < 0.01; * p < 0.05.

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Figure 2. A stronger training protocol by increasing the danger stimulus salience produces a memory that can be interfered during reconsolidation. Day 1, 15 trials training session. Day 2, a UN–TR pair was re-exposed to the training context (TCx) for 5 min with a single VDS-6cm presentation, the reminder session that does not trigger reconsolidation. The other UN-TR pair was re-exposed to the original training context (TCx) for 5 min without presentation of the VDS, the reminder session that triggers reconsolidation.

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After 1 hour, all groups were injected with CHX. Day 3 (testing session),

memory retention was tested with a single VDS-6cm presentation. UN-

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TCx+VDS, N = 27; TR-TCx+VDS, N = 27; UN-TCx, N = 24; TR-TCx N = 25. * = p

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< 0.05; ** p < 0.01.

Figure 3. Water deprivation before strong training prevents memory reconsolidation. 40

On Day 1, 2 hours before the training session, a UN-TR pair was water deprived. Day 2, all crabs were re-exposed to the training context for 5 min, the reminder session that does not trigger reconsolidation, and were injected with CHX after 1 hour. Day 3 (testing session), memory was tested with a single VDS presentation. UN-TCx, N = 27; TR-TCx, N = 27; UN-WD, N = 30; TR-WD, N =

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30. ** p < 0.01.

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ro of -p re lP na ur Jo Figure 4. Water deprivation before strong training prevents memory reconsolidation even after a multiple-reminder session.

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On Day 1, 2 hours before the training session, both pairs UN-TR were water deprived. Day 2, all the crabs were three times re-exposed to the training context and, after 1 hour, were injected with Veh or CHX. On Day 3, memory was tested with a single VDS presentation. UN-Veh, N = 30; TR-Veh, N = 29; UN-CHX, N = 28; TR- CHX, N = 31.

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* = p < 0.05.

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