Aversive memory conditioning induces fluoxetine-dependent anxiety-like states in the crab Neohelice granulata.

The interactions between memory processes and emotions are complex. Our previous investigations in the crab Neohelice led to an adaptation of the affective extension of sometimes opponent processes (AESOP) model. The model proposes that emotions generate separate emotive memory traces, and that the unfolding of emotional responses is a crucial component of the behavioral expression of reactivated memories. Here, we show that an aversive conditioning, that used changes in an innate escape response to an aversive visual stimulus, induced an emotional behavior that endured beyond the stimuli: the aversive memory training built an anxiety-like state evaluated in a dark/light plus-maze. We found that, after the training session, crabs displayed aversion to maze light areas, and an increased time immobilized in the dark zones of the maze, an anxiety-like behavior induced by stressors or physiological conditions in other crustaceans. The training-dependent anxiety-like behavior was blocked by pretraining administration of fluoxetine, suggesting an underlying serotonin-dependent phenomenon. We hypothesize that this training-induced anxiety-like state generates a separate emotive memory trace that is reinstated and crucial for the modulation of memory expression once the memory is reactivated.

Contextual memory reactivation modulates Ca2+-activity network state in a mushroom body-like center of the crab N. granulata.

High-order brain centers play key roles in sensory integration and cognition. In arthropods, much is known about the insect high-order centers that support associative memory processes, the mushroom bodies. The hypothesis that crustaceans possess structures equivalent to the mushroom bodies -traditionally called hemiellipsoid body- has been receiving neuroanatomical endorsement. The recent functional support is limited to the short term: in a structure of the true crab Neohelice granulata that has many insect-like mushroom bodies traits, the plastic learning changes express the context attribute of an associative memory trace. Here, we used in vivo calcium imaging to test whether neuronal activity in this structure is associated with memory reactivation in the long-term (i.e., 24 h after training). Long-term training effects were tested by presenting the training-context alone, a reminder known to trigger memory reconsolidation. We found similar spontaneous activity between trained and naïve animals. However, after training-context presentation, trained animals showed increased calcium events rate, suggesting that memory reactivation induced a change in the underlying physiological state of this center. Reflecting the change in the escape response observed in the paradigm, animals trained with a visual danger stimulus showed significantly lower calcium-evoked transients in the insect-like mushroom body. Protein synthesis inhibitor cycloheximide administered during consolidation prevented calcium mediated changes. Moreover, we found the presence of distinct calcium activity spatial patterns. Results suggest that intrinsic neurons of this crustacean mushroom body-like center are involved in contextual associative long-term memory processes.

Embodiment of an Emotional State Concurs with a Stress-Induced Reconsolidation Impairment Effect on an Auditory Verbal Word-List Memory.

Stress alters memory. Understanding how and when acute stress improves or impairs memory is a challenge. Stressors can affect memory depending on a combination of factors. Typically, mild stressors and stress hormones might promote consolidation of memory processing and impair memory retrieval. However, studies have shown that during reconsolidation, stressors may either enhance or impair recalled memory. We propose that a function of reconsolidation is to induce changes in the behavioral expression of memory. Here, we adapted the Rey Auditory Verbal Learning Test (RAVLT) to evaluate the effect of cold pressor stress (CPS) during the reconsolidation of this declarative memory. A decay in memory performance attributable to forgetting was found at the time of memory reactivation 5 d after training (day 6). Contrary to our initial predictions, the administration of CPS after memory reactivation impaired long-term memory expression (day 7), an effect dependent on the presence of a mismatch during Reactivation Session. No differences in recognition tests were found. To assess putative sources of the negative memory modulation effects induced during reconsolidation, current emotional state was evaluated immediately after Testing Session (day 7). An increase in arousal was revealed only when CPS was administered concurrently with memory reactivation-labilization. The possibility of integration during reconsolidation of independent associations of these emotive components in the trace is a critical factor in modulating neutral memories during reconsolidation by stressors.

A crabs' high-order brain center resolved as a mushroom body-like structure.

The hypothesis of a common origin for high-order memory centers in bilateral animals presents the question of how different brain structures, such as the vertebrate hippocampus and the arthropod mushroom bodies, are both structurally and functionally comparable. Obtaining evidence to support the hypothesis that crustaceans possess structures equivalent to the mushroom bodies that play a role in associative memories has proved challenging. Structural evidence supports that the hemiellipsoid bodies of hermit crabs, crayfish and lobsters, spiny lobsters, and shrimps are homologous to insect mushroom bodies. Although a preliminary description and functional evidence supporting such homology in true crabs (Brachyura) has recently been shown, other authors consider the identification of a possible mushroom body homolog in Brachyura as problematic. Here we present morphological and immunohistochemical data in Neohelice granulata supporting that crabs possess well-developed hemiellipsoid bodies that are resolved as mushroom bodies-like structures. Neohelice exhibits a peduncle-like tract, from which processes project into proximal and distal domains with different neuronal specializations. The proximal domains exhibit spines and en passant-like processes and are proposed here as regions mainly receiving inputs. The distal domains exhibit a "trauben"-like compartmentalized structure with bulky terminal specializations and are proposed here as output regions. In addition, we found microglomeruli-like complexes, adult neurogenesis, aminergic innervation, and elevated expression of proteins necessary for memory processes. Finally, in vivo calcium imaging suggests that, as in insect mushroom bodies, the output regions exhibit stimulus-specific activity. Our results support the shared organization of memory centers across crustaceans and insects.

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

The dynamics of memory processes are conserved throughout evolution, a 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 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 salience of the aversive stimulus augmented the memory strength; nonetheless, the protein synthesis inhibitor cycloheximide still disrupted the reconsolidation process. However, crabs stressed by a water-deprivation episode before a strong training session built up a memory that was now reconsolidation-resistant. We tested whether these reconsolidation-resistant effects can be challenged by changing parametric conditions of memory-reminder sessions; multiple memory reactivations without reinforcement were not able to trigger the labilization-reconsolidation of this resistant memory. Overall, the present findings suggest that generation of reconsolidation-resistant memories can be another part of the dynamics of memory processes conserved throughout evolution that protects privileged information from change.

Retrieval under stress decreases the long-term expression of a human declarative memory via reconsolidation.

Acute stress impairs memory retrieval of several types of memories. An increase in glucocorticoids, several minutes after stressful events, is described as essential to the impairing retrieval-effects of stressors. Moreover, memory retrieval under stress can have long-term consequences. Through what process does the reactivated memory under stress, despite the disrupting retrieval effects, modify long-term memories? The reconsolidation hypothesis proposes that a previously consolidated memory reactivated by a reminder enters a vulnerability phase (labilization) during which it is transiently sensitive to modulation, followed by a re-stabilization phase. However, previous studies show that the expression of memories during reminder sessions is not a condition to trigger the reconsolidation process since unexpressed memories can be reactivated and labilized. Here we evaluate whether it is possible to reactivate-labilize a memory under the impairing-effects of a mild stressor. We used a paradigm of human declarative memory whose reminder structure allows us to differentiate between a reactivated-labile memory state and a reactivated but non-labile state. Subjects memorized a list of five cue-syllables associated with their respective response-syllables. Seventy-two hours later, results showed that the retrieval of the paired-associate memory was impaired when tested 20min after a mild stressor (cold pressor stress (CPS)) administration, coincident with cortisol levels increase. Then, we investigated the long-term effects of CPS administration prior to the reminder session. Under conditions where the reminder initiates the reconsolidation process, CPS impaired the long-term memory expression tested 24h later. In contrast, CPS did not show effects when administered before a reminder session that does not trigger reconsolidation. Results showed that memory reactivation-labilization occurs even when retrieval was impaired. Memory reactivation under stress could hinder -via reconsolidation- the probability of the traces to be expressed in the long term.

Context-dependent memory traces in the crab's mushroom bodies: Functional support for a common origin of high-order memory centers.

The hypothesis of a common origin for the high-order memory centers in bilateral animals is based on the evidence that several key features, including gene expression and neuronal network patterns, are shared across several phyla. Central to this hypothesis is the assumption that the arthropods' higher order neuropils of the forebrain [the mushroom bodies (MBs) of insects and the hemiellipsoid bodies (HBs) of crustaceans] are homologous structures. However, even though involvement in memory processes has been repeatedly demonstrated for the MBs, direct proof of such a role in HBs is lacking. Here, through neuroanatomical and immunohistochemical analysis, we identified, in the crab Neohelice granulata, HBs that resemble the calyxless MBs found in several insects. Using in vivo calcium imaging, we revealed training-dependent changes in neuronal responses of vertical and medial lobes of the HBs. These changes were stimulus-specific, and, like in the hippocampus and MBs, the changes reflected the context attribute of the memory trace, which has been envisioned as an essential feature for the HBs. The present study constitutes functional evidence in favor of a role for the HBs in memory processes, and provides key physiological evidence supporting a common origin of the arthropods' high-order memory centers.

Memory expression is independent of memory labilization/reconsolidation.

There is growing evidence that certain reactivation conditions restrict the onset of both the destabilization phase and the restabilization process or reconsolidation. However, it is not yet clear how changes in memory expression during the retrieval experience can influence the emergence of the labilization/reconsolidation process. To address this issue, we used the context-signal memory model of Chasmagnathus. In this paradigm a short reminder that does not include reinforcement allows us to evaluate memory labilization and reconsolidation, whereas a short but reinforced reminder restricts the onset of such a process. The current study investigated the effects of the glutamate antagonists, APV (0.6 or 1.5 µg/g) and CNQX (1 µg/g), prior to the reminder session on both behavioral expression and the reconsolidation process. Under conditions where the reminder does not initiate the labilization/reconsolidation process, APV prevented memory expression without affecting long-term memory retention. In contrast, APV induced amnesic effects in the long-term when administered before a reminder session that triggers reconsolidation. Under the present parametric conditions, the administration of CNQX prior to the reminder that allows memory to enter reconsolidation impairs this process without disrupting memory expression. Overall, the present findings suggest that memory reactivation--but not memory expression--is necessary for labilization and reconsolidation. Retrieval and memory expression therefore appear not to be interchangeable concepts.

Behaviorally related neural plasticity in the arthropod optic lobes.

BACKGROUND: Due to the complexity and variability of natural environments, the ability to adaptively modify behavior is of fundamental biological importance. Motion vision provides essential cues for guiding critical behaviors such as prey, predator, or mate detection. However, when confronted with the repeated sight of a moving object that turns out to be irrelevant, most animals will learn to ignore it. The neural mechanisms by which moving objects can be ignored are unknown. Although many arthropods exhibit behavioral adaptation to repetitive moving objects, the underlying neural mechanisms have been difficult to study, due to the difficulty of recording activity from the small columnar neurons in peripheral motion detection circuits. RESULTS: We developed an experimental approach in an arthropod to record the calcium responses of visual neurons in vivo. We show that peripheral columnar neurons that convey visual information into the second optic neuropil persist in responding to the repeated presentation of an innocuous moving object. However, activity in the columnar neurons that convey the visual information from the second to the third optic neuropil is suppressed during high-frequency stimulus repetitions. In accordance with the animal's behavioral changes, the suppression of neural activity is fast but short lasting and restricted to the retina's trained area. CONCLUSIONS: Columnar neurons from the second optic neuropil are likely the main plastic locus responsible for the modifications in animal behavior when confronted with rapidly repeated object motion. Our results demonstrate that visually guided behaviors can be determined by neural plasticity that occurs surprisingly early in the visual pathway.

Dissociation between memory reactivation and its behavioral expression: scopolamine interferes with memory expression without disrupting long-term storage.

The reconsolidation hypothesis has challenged the traditional view of fixed memories after consolidation. Reconsolidation studies have disclosed that the mechanisms mediating memory retrieval and the mechanisms that underlie the behavioral expression of memory can be dissociated, offering a new prospect for understanding the nature of experimental amnesia. The muscarinic antagonist scopolamine has been used for decades to induce experimental amnesias The goal of the present study is to determine whether the amnesic effects of scopolamine are due to storage (or retrieval) deficits or, alternatively, to a decrease in the long-term memory expression of a consolidated long-term memory. In the crab Chasmagnathus memory model, we found that scopolamine-induced amnesia can be reverted by facilitation after reminder presentation. This recovery of memory expression was reconsolidation specific since a reminder that does not triggers reconsolidation process did not allow the recovery. A higher dose (5 µg/g) of scopolamine induced an amnesic effect that could not be reverted through reconsolidation, and thus it can be explained as an interference with memory storage and/or retrieval mechanisms. These results, showing that an effective amnesic dose of scopolamine (100 ng/g) negatively modulates long-term memory expression but not memory storage in the crab Chasmagnathus, are consistent with the concept that dissociable processes underlie the mechanisms mediating memory reactivation and the behavioral expression of memory.

Enhancement of long-term memory expression by a single trial during consolidation.

Before the memory trace is stored long term, it must undergo a phase of consolidation during which it remains susceptible to modifications. It has previously been proposed that during consolidation, memories are kept from being stored long term, and can therefore be modified with additional information resulting from ongoing behavior. The Chasmagnathus associative memory model is used here to test whether it is possible during consolidation to modify the long-term expression of a memory generated by a weak training procedure. In this memory model, long-term memory expression is achieved after strong training protocols, a 15-spaced trial procedure. After a weak training protocol (WTP, six spaced trials), crabs do not show memory retention when tested in the long term. Nevertheless, the WTP builds a long-term memory that it is indeed consolidated, but remains unexpressed. Here we show that memory can be modified by experience during this short period after learning: memory expression can be enhanced by a Single Trial Session, on the condition that this session takes place contingent upon the consolidation period. We also found that during this time, the memory built by the WTP is behaviorally expressed, in contrast with what occurs at long term. Our results support the idea that during consolidation memories can be evaluated in the background of concurrent experiences. In particular, we propose that during the consolidation period it is possible for crabs to assess which experiences, among those stored long term, will be expressed long term.

Angiotensin modulates long-term memory expression but not long-term memory storage in the crab Chasmagnathus.

Memory reconsolidation is a dynamic process in which a previously consolidated memory becomes labile following reactivation by a reminder. In a previous study in the crab Chasmagnathus memory model, we showed that a water-shortage episode, via angiotensin modulation during reconsolidation, could reveal a memory that otherwise remains unexpressed: weakly trained animals cannot reveal long-term memory (LTM) except when an episode of noticeable ethological meaning, water deprivation, is contingent upon reconsolidation. However, these results are at variance with two of our previous interpretations: weak training protocols do not build LTM and angiotensin II modulates the strength of the information storing process. A parsimonious hypothesis is that in Chasmagnathus angiotensins regulate LTM expression, but not LTM storage. Here, we tested three predictions of this hypothesis. First, the well-known retrograde amnesic effect of the angiotensin II antagonist saralasin is not due to interference on memory storage, but to modulation of memory expression. Second, the recovery of the LTM memory expression of the apparently amnesic retrograde effect produced by saralasin, through the water-shortage episode contingent upon reconsolidation, must be reconsolidation specific. Consequently, summation-like effects and retrieval deficits cannot explain these results because of the parametric conditions of reconsolidation. Third, weak training protocols build an unexpressed LTM that requires mRNA transcription and translation, a diagnostic characteristic of LTM. Results show that angiotensin modulates LTM expression but not LTM memory storage in the crab Chasmagnathus. The results lead us to suggest that, in Chasmagnathus, LTM expression - the process of gaining appreciable control over behavior of the reactivated trace in the retrieval session - may be considered a distinct attribute of its long-term storage. This strategy, a positive modulation during reconsolidation, is proposed to distinguish between memories that can be reactivated, labilized and are not expressed, and memories that are not stored long term, obliterated or altered in other retrieval mechanisms.

Neuroanatomical distribution of angiotensin-II-like neuropeptide within the central nervous system of the crab Chasmagnathus; physiological changes triggered by water deprivation.

The angiotensins constitute a neuropeptidergic system that emerged early in evolution. Their classical osmoregulatory and dipsogenic functions and their mnemonic actions have been demonstrated both in vertebrates and in some invertebrates. Previously, we have shown that, in the euryhaline and semiterrestrial crab Chasmagnathus granulatus, water deprivation correlates with an increased level of brain angiotensin-II-like neuropeptide/s (ANGII-like) and improves memory processes through ANGII receptors. We have proposed that the release of brain angiotensins in response to water shortages is an ancient mechanism for coordinating various functions that, together, enable organisms to tolerate this environmental change. Here, we have evaluated the physiological changes in ANGII-like levels in diverse structures of the central nervous system of these animals during water deprivation. The neuroanatomical distribution of ANGII-like is described in the optic lobes and brain of Chasmagnathus granulatus and the physiological changes in ANGII-like distribution in various brain neuropils is evaluated after water deprivation. Our results indicate that ANGII-like is widely distributed, especially in the medial protocerebrum. After 2 h of water deprivation, ANGII-like immunoreactivity increases in the central body and decreases in the olfactory neuropil and, after 6 h of water deprivation, is markedly reduced in several brain areas. Although further experiments are needed to establish that the angiotensinergic system is involved in the balance of body fluids in this crab, our results suggest that ANGII regulates several functions during water shortages.

Updating contextual information during consolidation as result of a new memory trace.

Reconsolidation studies have led to the hypothesis that memory, when labile, would be modified in order to incorporate new information. This view has reinstated original propositions suggesting that short-term memory provides the organism with an opportunity to evaluate and rearrange information before storing it, since it is concurrent with the labile state of consolidation. The Chasmagnathus associative memory model is used here to test whether during consolidation it is possible to change some attribute of recently acquired memories. In addition, it is tested whether these changes in behavioral memory features can be explained as modifications on the consolidating memory trace or as a consequence of a new memory trace. We show that short-term memory is, unlike long-term memory, not context specific. During this short period after learning, behavioral memory can be updated in order to incorporate new contextual information. We found that, during this period, the cycloheximide retrograde amnesic effect can be reverted by a single trial in a new context. Finally, by means of memory sensitivity to cycloheximide during consolidation and reconsolidation, we show that the learning of a new context (CS) during this short-term memory period builds up a new memory trace that sustains the behavioral memory update.

Both heat shock and water deprivation trigger Hsp70 expression in the olfactory lobe of the crab Chasmagnathus granulatus.

Heat-shock proteins (Hsp) are synthesized in the central nervous system in response to traumas but also after physical exercise and psychophysiological stress. Therefore, an increase in Hsp expression is a good marker of changes in metabolic activity. In the crab Chasmagnathus, a powerful memory paradigm has been established. Memory modulation is possible by water shortage. The brain areas activated by either training protocols and/or water-deprivation are still unknown. Hsp expression might be a marker to sensing the increase in metabolic activity in crab Chasmagnathus brain neuropils engaged in the physiological responses triggered by water deprivation and cognitive processing. Here, we observed an increase in brain Hsp of 70kDa (Hsp70) expression after a heat-shock treatment. Additionally, immunohistochemistry analysis revealed that, under basal conditions, some glomeruli of the olfactory lobes showed Hsp70 immunoreactivity in an on-off manner. Both a hot environment and water deprivation increased the number of glomeruli expressing Hsp70. This marker of neuropil's activity might turn out to be a powerful tool to test whether crustacean olfactory lobes not only process olfactory information but also integrate multimodal signals.

Memory strengthening by a real-life episode during reconsolidation: an outcome of water deprivation via brain angiotensin II.

A considerable body of evidence reveals that consolidated memories, recalled by a reminder, enter into a new vulnerability phase during which they are susceptible to disruption again. Consistently, reconsolidation was shown by the amnesic effects induced by administration of consolidation blockers after memory labilization. To shed light on the functional value of reconsolidation, we explored whether an endogenous process activated during a concurrent real-life experience improved this memory phase. Reconsolidation of long-term contextual memory has been well documented in the crab Chasmagnathus. Previously we showed that angiotensin II facilitates memory consolidation. Moreover, water deprivation increases brain angiotensin and improves memory consolidation and retrieval through angiotensin II receptors. Here, we tested whether concurrent water deprivation improves reconsolidation via endogenous angiotensin and therefore strengthens memory. We show that memory reconsolidation, induced by training context re-exposure, is facilitated by a concurrent episode of water deprivation, which induces a raise in endogenous brain angiotensin II. Positive modulation is expressed by full memory retention, despite a weak training, 24 or 72 but not 4 h after memory reactivation. This is the first evidence that memory can be positively modulated during reconsolidation through an identified endogenous process triggered during a real-life episode. We propose that the functional value for reconsolidation would be to make possible a change in memory strength by the influence of a concurrent experience. Reconsolidation improvement would lead to memory re-evaluation, not by altering memory content but by modifying the behaviour as an outcome of changing the hierarchy of the memories that control it.

Retrieval improvement is induced by water shortage through angiotensin II.

Angiotensin II (ANGII) has an evolutionary preserved role in determining adaptative responses to water-shortages. In addition, it has been shown to modulate diverse phases of memory. Still, it is not clear whether ANGII improves or spoils memory. We demonstrated that endogenous angiotensins enhance consolidation of a long-term associative memory in the crab Chasmagnathus and that water shortage improves memory consolidation through brain ANGII actions. Here, we show that weakly trained crabs, when water-deprived, exhibit enhanced retrieval. Subsequently, memory retention is indistinguishable from that of strongly trained crabs. ANGII, but not angiotensin IV, is a necessary and sufficient condition for such enhancing effect. We conclude that ANGII released due to water shortage leads to enhanced memory retrieval. Thus, it seems that ANGII has an evolutionary preserved role as a multifunction coordinator that enables an adaptative response to water-shortage. The facilitation of memory consolidation and retrieval would be among those coordinated functions.

Phosphorylation of extra-nuclear ERK/MAPK is required for long-term memory consolidation in the crab Chasmagnathus.

It was previously demonstrated that mitogen-activated protein kinase (MAPK) signaling plays a pivotal role in neural plasticity and memory processes both in rodents and mollusks. Although the MAPK pathways are highly conserved, no evidence was found for its participation in memory models in other animal groups. Here we found ERK-like and JNK-like cross-immunoreactivity in the crab brain with phospho-specific antibodies and we estimated ERK and JNK activity during long-term memory consolidation in the context-signal learning paradigm of the crab Chasmagnathus. At 0, 1, 3 and 6h after training ERK and JNK activity was measured. ERK-like activation was found 1h after spaced training in cytosolic but not in nuclear fractions of brain homogenates, while JNK activity remained unchanged in both fractions. Passive (context exposure) and active (continuous stimulation) controls showed cytosolic ERK and JNK activation immediately after training, which decayed 1h later. In coincidence with this time course of activity, an ERK1/2 pathway inhibitor, PD098059, induced amnesia only when administered 45 min after training but not when administered immediately pre- or post-training. These data support that: (1) cytoplasmic but not nuclear ERK substrates must be differentially phosphorylated during memory consolidation, and (2) ERK phosphorylation and consequent activation 1h after training is necessary for long-term memory consolidation in this arthropod model.