Alejandro Delorenzi

κ-B like DNA-binding activity is enhanced after spaced training that induces long-term memory in the crab Chasmagnathus

Regulation of gene expression has been involved in long-term memory consolidation. Present results support the role of Rel/ NFkappa-B like activation in this process. In the crab Chasmagnathus, the spaced presentation of 15 or more danger stimuli induces long-term habituation (LTH), while no LTH is observed after a massed training of 600 trials. When a group trained with 30 spaced trials was compared with a passive control group and massed trained groups, a higher level of specific Rel/kappa-B like DNA-binding activity was found in brain nuclear extracts. These results strongly suggest that the enhancement of Rel/kappa-B like DNA-binding activity in the brain is specifically related to LTH formation.

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.

The enhancement of reconsolidation with a naturalistic mild stressor improves the expression of a declarative memory in humans.

The reconsolidation hypothesis proposes that a previously consolidated memory recalled by a reminder enters an unstable state (memory labilization) during which it is transiently sensitive to disruption. Although this process has been shown in very diverse species and types of memories, including human declarative memory, elucidating the role of this process is still an open challenge. The hypothesis that reconsolidation allows the incorporation of new information has recently been demonstrated in humans. However, the findings show that, during the reconsolidation phase, memory retention can be increased by pharmacological modulation or real life events in animals have not been found in humans yet. In order to evaluate this, we used a paradigm of human declarative memory whose reminder structure allows us to differentiate between a retrieved labile memory state and a retrieved but non-labile state. Volunteers learned an association between five cue-syllables and their respective response-syllables. 6 days later, the paired-associate memory was reactivated by exposing the subjects to the reminder, and then they received a mild stressor, cold pressor stress (CPS). Poor memory performance was found at both the time of memory reactivation (day 6 after training) and at testing of all groups that were designed as controls (day 7). Conversely, robust memory performance was shown at testing when the CPS administration was concurrent with the retrieved-labile memory state. Results from the present study reveal that a naturalistic mild stressor can enhance reconsolidation, improving the long-term expression of this declarative memory. This finding might have significant implications for the comprehension of memory persistence and memory expression, and add new evidence in order to understand the adaptive meaning of the reconsolidation process.

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.

Angiotensin II enhances long-term memory in the crab Chasmagnathus

An opaque screen moving overhead provokes an escape response in the crab Chasmagnathus granulatus that habituates after a few presentations of the eliciting stimulus. Fifteen trials with a 180-s intertrial interval or 30 trials with a 90-s interval (strong training protocol) ensures long-term habituation (LTH) of the response for 24 h, whereas 10 trials (weak training protocol) fail to induce it. However, robust LTH is obtained when crabs are injected with human angiotensin (All; 50 pmol) immediately after a weak training protocol. This memory-enhancing effect of All is dose-dependent, reversible by saralasin (5 pmol), and vanishes either when the weak training protocol is reduced to only five trials, or when the peptide is given before training or 1 h after. LTH is impaired by saralasin (5 pmol) administered before or after the strong training protocol, but no amnestic effect is disclosed when the antagonist is given 1 h after. On the other hand, both All-like immunoreactivity and angiotensin-converting enzyme-like activity are described in diverse tissues of Chasmagnathus, namely, in gills and in both thoracic and supraesophageal ganglia. Results support the view that some components of the renin-angiotensin system and their influence on memory might have emerged early in evolution.

Effects of activation and inhibition of cAMP-dependent protein kinase on long-term habituation in the crab Chasmagnathus.

On sudden presentation of a danger stimulus, the crab Chasmagnathus elicits an escape response that habituates promptly and for a long period. We have previously reported that administration of a cAMP-permeable analog (CPT-cAMP) along with a phosphodiesterase inhibitor (IBMX) improves long-term habituation (LTH). In present experiments we studied the effect of systemic administration of the protein kinase A (PKA) activator Sp-5,6-DCl-cBIMPS and that of the PKA inhibitor Rp-8-Cl-cAMPS on LTH tested 24 h after a weak training protocol (5 trials of danger stimulus presentation) or a strong training protocol (15-30 trials), respectively. A 50 microliters pre-training injection of 75 microM Sp-5,6-DCl-cBIMPS, and to a lesser degree of 25 microM, improved retention of the habituated response but not affect short-term habituation (STH). Like pre-training injection, post-training administration of Sp-5,6-DCl-cBIMPS proved to exert a facilitatory action on retention though with 75 microM dose only. Conversely, both pre- and post-training injection of 25 microM Rp-8-Cl-cAMPS impaired LTH without affecting STH. Thus, the PKA activator Sp-5,6-DCl-cBIMPS enables a weak training to produce LTH while the PKA inhibitor Rp-8-Cl-cAMPS impairs LTH when a strong training is given. Activation of crab PKA by Sp-5,6-DCl-cBIMPS and its inhibition by Rp-8-Cl-cAMPS were assessed using an in vitro PKA activity assay. These results provide independent evidences supporting the view that PKA plays a key role in long-term memory storage in this learning paradigm.

Acute administration of a permeant analog of cAMP and a phosphodiesterase inhibitor improve long-term habituation in the crab Chasmagnathus

A shadow passing overhead acts as a danger stimulus and elicits an escape response in the crab Chasmagnathus that habituates promptly and for a long period. Robust retention is shown at 24 h after 15 trials of shadow presentation or at 120 h after 30 trials, but no significant retention is disclosed at 24 h after 5 trials or at 72 h after 15. A cocktail of the cAMP membrane permeable analog 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), plus the phosphodiesterase inhibitor isobutyl methylxanthine (IBMX), was given by systemic administration. Pretraining injection of the cocktail (25 or 50 microM, 15 min before a 5-trial session) failed to affect short-term habituation, but induced significant retention when tested at 24 h. This facilitatory effect was not shown when a lower dose (5 microM) was used. A post-training injection of 25 microM, immediately after a 5- or 15-trial session, induced retention when tested at 24 or 72 h, respectively. Thus, the administration of CPT-cAMP + IBMX during acquisition of a habituated response or immediately after, improves long-term habituation, a result supporting the view that an increase in the cAMP level is one of the steps in long-term memory consolidation.

Angiotensin II (3–8) induces long-term memory improvement in the crab Chasmagnathus

A shadow moving overhead acts as a danger stimulus and elicits an escape response in the crab Chasmagnathus that habituates after 15 trials and for a long period of time. Previous work showed that angiotensin II enhances this long-term memory. Present results indicate: (1) that the facilitatory effect of angiotensin II is not blocked by either losartan, DUP 753 or the Parke Davis compound PD 123177; (2) that the angiotensin II (3-8) fragment has an enhancing effect on crabs memory stronger than that reported for the integer octopeptide; (3) that the hypermnestic effect of angiotensin II (3-8) is dose-dependent and saralasin reversible. The possibility that the action of angiotensin II on crabs memory were not due to its own action but to that of the degradation fragment angiotensin II (3-8) is discussed.

Angiotensin II and the transcription factor Rel/NF-κB link environmental water shortage with memory improvement

One of the essential requirements even in the most ancient life forms is to be able to preserve body fluid medium. In line with such requirement, animals need to perform different behaviors to cope with water shortages. As angiotensin II (ANGII) is involved on a widespread range of functions in vertebrates, including memory modulation, an integrative role, in response to an environmental water shortage, has been envisioned. Previous work on the semi-terrestrial and brackish-water crab Chasmagnathus granulatus showed that endogenous ANGII enhanced an associative long-term memory and, in addition, that high salinity environment induces both an increase of brain ANGII levels and memory improvement. Here, we show that in the crab Chasmagnathus air exposure transiently increases blood sodium concentration, significantly increases brain ANGII immunoreactivity, and has a facilitatory effect on memory that is abolished by a non-selective ANGII receptor antagonist, saralasin. Furthermore, Rel/NF-kappaB, a transcription factor activated by ANGII in mammals and during memory consolidation in Chasmagnathus brain, is induced in the crabs brain by air exposure. Moreover, nuclear brain NF-kappaB is activated by ANGII, and this effect is reversed by saralasin. Our results constitute the first demonstration in an invertebrate that cognitive functions are modulated by an environmental stimulus through a neuropeptide and give evolutionary support to the role of angiotensins in memory processes. Moreover, these results suggest that angiotensinergic system is preserved across evolution not only in its structure and molecular mechanisms, but also in its capability of coordinating specific adaptative responses.

Memory enhancement by the angiotensinergic system in the crab Chasmagnathus is mediated by endogenous angiotensin II

In previous work with the crab Chasmagnathus, it was reported that either exogenous angiotensin II (ANGII) or angiotensin IV (ANGIV), have an enhancing effect on long-term memory, which involves an association between context and an iterative danger stimulus (context-signal memory, CSM). Present results indicate that Dival, an ANGIV antagonist, reverts the facilitatory effect of ANGIV but not that of ANGII, whereas saralasin but not Dival, disrupts CSM. These findings suggest that ANGII is the endogenous angiotensin that plays a significant role in long-term memory, while the ANGIV receptor would not be encompassed in the cascade of events related to crabs CSM.