Annual changes of Neohelice granulata cognitive abilities indicate opposition between short- and long-term memory retention

Neohelice is a long-standing model for memory studies for its strong retention of a reduced escape response when trained to iterative presentations of a visual danger stimulus (VDS). Here we present year-round changes that are related to the memory acquisition, storage, and expression. First, we evaluated exploratory activity and response to the VDS, as necessary for memory acquisition and expression. Both parameters change year-round. Second, short-term memory (STM) and two types of long-term memory (LTM) were assessed throughout the year. STM and long-term context-dependent signal memory (CSM) change between periods of the year, whereas signal memory (SM) does not, indicating that the cognitive abilities of the crab display circannual rhythms. Third, during the reproductive period, STM retention is higher than both CSM and SM, indicating a trade-off between STM and LTM. This is the first report of memory retention abilities changing seasonally as a trade-off between short- and long-term memories.Fil: Anahi, Rosso. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Biología Celular e Histología; ArgentinaFil: Freudenthal, Ramiro A. M.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Biología Celular e Histología; Argentin

Characterization of the beta amyloid precursor protein-like gene in the central nervous system of the crab Chasmagnathus. Expression during memory consolidation

Background: Human β-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the β-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information.Results: Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl), showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions. We observed a wide distribution of cappl mRNA in the nervous system as well as in muscle and gills. The protein localized in all tissues analyzed with the exception of muscle. Immunofluorescence revealed localization of cAPPL in associative and sensory brain areas. We studied gene and protein expression during long-term memory consolidation using a well characterized memory model: the context-signal associative memory in this crab species. mRNA levels varied at different time points during long-term memory consolidation and correlated with cAPPL protein levels. Conclusions: cAPPL mRNA and protein is widely distributed in the central nervous system of the crab and the time course of expression suggests a role of cAPPL during long-term memory formation.Fil: Fustiñana, María Sol. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Ariel, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Federman, Maria Noel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Freudenthal, Ramiro A. M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Romano, Arturo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Characterization of the cardiac ganglion in the crab Neohelice granulata and immunohistochemical evidence of GABA-like extrinsic regulation

The aim of the present work is to provide an anatomical description of the cardiac system in the crab Neohelice granulata and evidence of the presence of GABA by means of immunohistochemistry. The ganglionic trunk was found lying on the inner surface of the heart's dorsal wall. After dissection, this structure appeared as a Y-shaped figure with its major axis perpendicular to the major axis of the heart. Inside the cardiac ganglion, we identified four large neurons of 63.7 μm ± 3.7 in maximum diameter, which were similar to the motor neurons described in other decapods. All the GABA-like immunoreactivity (GABAi) was observed as processes entering mainly the ganglionic trunk and branching in slender varicose fibers, forming a network around the large neurons suggesting that GABAi processes contact them. Our findings strengthen previous results suggesting that the GABAergic system mediates the cardio-inhibitory response upon sensory stimulation.Fil: Yang, Margarita.Fil: Carbo, Martin.Fil: Freudenthal, Ramiro A. M..Fil: Hermitte, Gabriela

Reconsolidation or Extinction: Transcription Factor Switch in the Determination of Memory Course after Retrieval

In fear conditioning, aversive stimuli are readily associated with contextual features. A brief reexposure to the training context causes fear memory reconsolidation, whereas a prolonged reexposure induces memory extinction. The regulation of hippocampal gene expression plays a key role in contextual memory consolidation and reconsolidation. However, the mechanisms that determine whether memory will reconsolidate or extinguish are not known. Here, we demonstrate opposing roles for two evolutionarily related transcription factors in the mouse hippocampus. We found that nuclear factor-κB (NF-κB) is required for fear memory reconsolidation. Conversely, calcineurin phosphatase inhibited NF-κB and induced nuclear factor of activated T-cells (NFAT) nuclear translocation in the transition between reconsolidation and extinction. Accordingly, the hippocampal inhibition of both calcineurin and NFAT independently impaired memory extinction, whereas inhibition of NF-κB enhanced memory extinction. These findings represent the first insight into the molecular mechanisms that determine memory reprocessing after retrieval, supporting a transcriptional switch that directs memory toward reconsolidation or extinction. The precise molecular characterization of postretrieval processes has potential importance to the development of therapeutic strategies for fear memory disorders.Fil: de la Fuente, Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Freudenthal, Ramiro A. M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Romano, Arturo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Protein degradation by ubiquitin-proteasome system in formation and labilization of contextual conditioning memory

The ubiquitin-proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this case, the inhibition of the UPS during consolidation impairs memory. Similar results were reported for memory reconsolidation. However, in other cases, the inhibition of UPS had no effect on memory consolidation and reconsolidation but impedes the amnesic action of protein synthesis inhibition after retrieval. The last finding suggests a specific action of the UPS inhibitor on memory labilization. However, another interpretation is possible in terms of the synthesis/degradation balance of positive and negative elements in neural plasticity, as was found in the case of long-term potentiation. To evaluate these alternative interpretations, other reconsolidation-interfering drugs than translation inhibitors should be tested. Here we analyzed initially the UPS inhibitor effect in contextual conditioning in crabs. We found that UPS inhibition during consolidation impaired long-term memory. In contrast, UPS inhibition did not affect memory reconsolidation after contextual retrieval but, in fact, impeded memory labilization, blocking the action of drugs that does not affect directly the protein synthesis. To extend these finding to vertebrates, we performed similar experiments in contextual fear memory in mice. We found that the UPS inhibitor in hippocampus affected memory consolidation and blocked memory labilization after retrieval. These findings exclude alternative interpretations to the requirement of UPS in memory labilization and give evidence of this mechanism in both vertebrates and invertebrates.Fil: Fustiñana, María Sol. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: de la Fuente, Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Federman, Maria Noel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Freudenthal, Ramiro A. M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Romano, Arturo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Participación de factores de transcripción de la familia Rel/NF-kappa B en procesos de plasticidad y memoria

La memoria de largo término y la plasticidad neuronal comparten varias características,incluyendo las fases temporales de mecanismos celulares y moleculares: la inducción requiere de un aumento de calcio intracelular, la persistencia temprana depende de la activación de quinasas y las etapas tardías necesitan de transcripción génica y de la síntesis de proteínas. Desde los primeros experimentos en los cuales se logro amnesia inhibiendo la síntesis proteica, se especula acerca de la función de factores de transcripción (FT) específicos, en la regulación de la expresión génica necesaria para la perdurabilidad tanto de fenómenos mnésicos como de la plasticidad neuronal. Este trabajo se concentro en dos aspectos de la función del FT NF-kappa B en la consolidación de la memoria de largo término y la plasticidad neuronal. El primer aspecto se refiere a la activación del FT luego de inducidos estos fenómenos, y el segundo a la conservación evolutiva de la función de esta vía de transducción desde crustáceos a mamíferos. Para los propósitos de este trabajo se evaluó la activación de este FT en tres modelos: la consolidación de la memoria de largo termina (MLT) en el cangrejo Chasmagnathus granulatus, un paradigma evitación inhibitoria en ratón y la inducción de potenciación de largo término in vivo de la vía perforante del hipocampo de ratón. La presencia de la vía de regulación transcripcional Rel/NF-kappa B, se determinó en el cangrejo por retardo electroforético (EMSA), Western Blot, inmunoprecipitación, unión covalente con irradiación ultravioleta e inmunohistoquímica. En el cangrejo, un protocolo que induce una MLT es seguido por una activación bifásica de unión al ADN por parte del NF-kappa B, mientras que un protocolo que induce una memoria de corto término no activa al FT. NF-kappa B se encuentra presente en las terminales sinápticas, en donde es activado por protocolos de entrenamiento que inducen MLT. El curso temporal de la activación de NF-kappa B nuclear en hipocampo, se estudió por EMSA, después del entrenamiento de un ensayo, en el paradigma de evitación inhibitoria en ratón. Mostrando, cuando se lo comparo con los animales naive, una inhibición del FT luego de 15 min. del entrenamiento seguida por una activación a los 45 min. tanto en los animales que sufrieron un shock eléctrico, como en los que fueron expuestos al contexto sin shock. En esta misma línea argumental, encontramos que la inyección intra cerebro ventricular (icv) de sulfasalazina inmediatamente post-entrenamiento impide la formación de la memoria de largo término. Más aún, una segunda estrategia independiente para inhibir el NF-kappa B en el cerebro, la administración icv de un oligonucleótido doble cadena de ADN conteniendo la secuencia consenso de NF-kappa B (kB Decoy) también resulta amnésica. Cuando se estudian los EMSAs de extractos nucleares de hipocampo de ratón, se observa que animales tetanizados (protocolo inductor de potenciación de largo término) muestran, luego de 15 min. un aumento en la actividad de unión al ADN, cuando son comparados con, animales estimulados a baja frecuencia (EBF) y animales naive. Los hipocampos al ser analizados por inmunohistoquímica con un anticuerpo contra la forma activa del factor, muestran la misma tendencia general pero con patrones celulares de activación más complejos. Estos resultados indican, primero que la actividad de unión al ADN del NF-kappa B es necesaria para la consolidación de memorias de largo término. Y el tratamiento con inhibidores específicos de NF-kappa B, suficiente para generar amnesia en los animales inyectados. A la vez que muestra un patrón temporal claramente delimitado en el cual el factor es necesario para la consolidación. Igualmente significativa es la activación del FT luego de inducida la plasticidad neuronal, mostrando un papel en el proceso de potenciación. El segundo aspecto de este trabajo sugiere que la vía de transducción señales de NF-kappa B se encuentra conservada, en su función fisiológica en el sistema nervioso, entre invertebrados y vertebrados.Long term memory and neural plasticity share several characteristics, including the temporal phases of molecular and cellular mechanisms: the induction requires an increment on intracellular calcium, early persistence depends on the activation of kinases and the late steps need gene expression and protein synthesis. Since the first experiments in which amnesia was produced by protein synthesis inhibition, it has been speculation about the role of specific transcription factors (TF) in the regulation of the gene expression needed for the lasting of mnesic processes such as neural plasticity. This thesis concentrates on two aspects of the function of TF NF-kappa B during long term memory consolidation and neuronal plasticity. The first, concerns it’s activation after the induction of these phenomena, and the second concerns the evolutionary conservation of this signaling pathway from crustaceans to mammals. For the purpose of this thesis, the activation of NF-kappa B was evaluated in three models: long term memory (LTM) consolidation in the crab Chasmagnathus granulatus, inhibitory avoidance in mice and the induction of in vivo long term potentiation, of the hipocampal perforant pathway in mice. The presence of the Rel/NF-kappa B transcriptional regulatory pathway in Chasmagnathus was studied by Electro Mobility Shift Assay (EMSA), Western Blot, Immunoprecipitation, Ultraviolet Crosslinking and Immunohistochemistry. In the crab, a protocol that induced LTM, is followed by a a bifasic activation of the DNA binding activity of the TF, while a protocol that induces short term memory does not activate NF-kappa B. This TF is present at the synaptic terminals, where it is activated by the LTM inducing training protocols. The temporal course of nuclear NF-kappa B activation in mice hippocampus, was studied by EMSA, after the one trial inhibitory avoidance training. Showing, when compared with naive animals, an inhibition 15 min. after training, followed by activation after 45 min., both in the animals that received shock and the ones only exposed to the context. In the same line of evidence, animals injected intra cerebro ventricular (icv) with sulfasalazine (an inhibitor of NF-kappa B activation) immediately after training impede the formation of LTM. What is more, an independent and more specific form of NF-kappa B inhibition, the icv injection of a double stranded DNA containing the TF consensus sequence (kB decoy), results amnesic. When we studied EMSAs of hippocampal nuclear extracts, tetanized mice (a protocol that induces potentiation) shows, 15 min after, an augment in DNA binding activity, in contrast with low frequency stimulated and naive mice. Immunohistochemical analysis with an antibody against the active form of the transcription factor, showed the same tendency but with a more complicated cellular pattern of activation within the hipocampal structure. These results indicate, first that the DNA binding activity of the TF is necessary for LTM and that the treatment with NF-kappa B specific inhibitors is sufficient to generate amnesia in the injected animals. At the same time it shows that a clear temporal pattern of activation is needed for consolidation. Equally significant is the activation of TF after the induction of neural plasticity, showing a role in this process. The second aspect suggested by this work, is that the Rel/NF-kappa B pathway is conserved in its physiological function between invertebrates and vertebrates.Fil:Freudenthal, Ramiro A.M.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Activation of the transcription factor NF-κB by retrieval is required for long-term memory reconsolidation

Several studies support that stored memories undergo a new period of consolidation after retrieval. It is not known whether this process, termed reconsolidation, requires the same transcriptional mechanisms involved in consolidation. Increasing evidence supports the participation of the transcription factor NF-κB in memory. This was initially demonstrated in the crab Chasmagnathus model of associative contextual memory, in which re-exposure to the training context induces a well characterized reconsolidation process. Here we studied the role of NF-κB in reconsolidation. NF-κB was specifically activated in trained animals re-exposed to the training context but not to a different context. NF-κB was not activated when animals were re-exposed to the context after a weak training protocol insufficient to induce long-term memory. A specific inhibitor of the NF-κB pathway, sulfasalazine, impaired reconsolidation when administered 20 min before re-exposure to the training context but was not effective when a different context was used. These findings indicate for the first time that NF-κB is activated specifically by retrieval and that this activation is required for memory reconsolidation, supporting the view that this molecular mechanism is required in both consolidation and reconsolidation

Learning a non-neutral conditioned stimulus: place preference in the crab Neohelice granulata

In the wild, being able to recognize and remember specific locations related to food sources and the associated attributes of landmarks is a cognitive trait important for survival. In the present work, we show that the crab Neohelice granulata can be trained to associate a specific environment with an appetitive reward in a conditioned place preference task. After a single training trial, when the crabs were presented with a food pellet in the target quadrant of the training arena, they were able to form a long-term memory related to the event. This memory was evident at least 24 h after training and was protein synthesis dependent. Importantly, the target area of the arena proved to be a non-neutral environment, given that animals initially avoided the target quadrant. In the present work, we introduce for the first time an associative one-trial memory paradigm including a conditioned stimulus with a clear valence performed in a crustacean.Fil: Klappenbach, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Medina, Candela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Freudenthal, Ramiro A. M.. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; Argentin

Synaptic NF-kappa B pathway in neuronal plasticity and memory

Several transcription factors are present at the synapse, and among these are the Rel-NF-kappa B pathway components. NF-kappa B is a constitutive transcription factor, with a strong presence in the brain of which a considerable part is located in the neuropiles. This localization of the transcription factor, plus evidence pointing to different functions, is what gave place to two general hypotheses for synaptic NF-kappa B: (a) The transcription factor plays a role in the synapse to nucleus communication, and it is retrogradely transported from polarized localizations to regulate gene expression; (b) The transcription factor modulates the synaptic function locally. Evidence indicates that both mechanisms can operate simultaneously; here we will present different possibilities of these hypotheses that are supported by an increasing amount of data. We pay special attention to the local role of the transcription factor at the synapse, and based in the described evidence from different animal models, we propose several processes in which the transcription factor may change the synaptic strength.Fil: Salles, Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Romano, Arturo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Freudenthal, Ramiro A. M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin