Ramiro Freudenthal

Participation of Rel/NF-κB transcription factors in long-term memory in the crab Chasmagnathus

The induction of gene expression has been correlated with long-lasting neuronal plasticity and long-term memory (LTM) formation. The fast activation of constitutive transcription factors by signaling mechanisms is thought to be the link between synaptic events and gene expression. However, only one constitutive transcription factor, CREB, has been shown to play a key role in several memory paradigms, both in vertebrates and invertebrates. Here, we report evidences for Rel/NFkappa-B constitutive transcription factors participation in memory. Using the LTM paradigm in the crab Chasmagnathus, an enhancement of NFkappa-B DNA-binding activity was found after spaced training, which induces LTM, but not after massed training which yields an intermediate-term memory (ITM). Such finding is correlated with the requirement of protein synthesis for LTM consolidation but not for ITM. Furthermore, NFkappa-B activation was observed after 15 or 30 training trials, which are sufficient to induce LTM, but not after 5 or 10 trials, a number of trials insufficient to induce LTM. The kinetics of activation was studied and two waves of DNA-binding activity were found, similar to the time course described in other systems. NFkappa-B activation after training was also found in synaptosomal extracts. The latter result supports the hypothesis of a novel synapse-to-nucleus signaling system, in which the transcription factor is locally activated by synaptic events and then transported to the nucleus.

The IκB kinase inhibitor sulfasalazine impairs long-term memory in the crab Chasmagnathus

Evidence for the participation of Rel/NF-kappaB transcription factors in long-term memory has recently been reported in the context-signal learning paradigm of the crab Chasmagnathus, in which a high correlation between long-term memory formation and NF-kappaB activation was observed. Two components of the NF-kappaB pathway in the crab brain have now been identified by cross-immunoreactivity using mammalian antibodies for IkappaB-alpha and IkappaB kinase alpha. Furthermore, IkappaB kinase-like phosphotransferase activity, which was inhibited by the IkappaB kinase inhibitor sulfasalazine, was detected in brain extracts. We have evaluated the effect of sulfasalazine administration on long-term memory tested at 48 h. Amnesia was found when sulfasalazine was administered pre-training and 5 h after training but not at 0 or 24 h after training. Thus, two periods for sulfasalazine-induced amnesia were found in coincidence with the two phases of NF-kappaB activation previously described (immediately and 6 h after training). The cyclooxygenase inhibitor indomethacin did not induce amnesia when administered pre-training. Thus, the possibility that sulfasalazine induces amnesia by means of cyclooxygenase inhibition is unlikely to be tenable. In vivo sulfasalazine inhibition of basal NF-kappaB activity was found between 30 and 45 min after injection, as assessed by electrophoretic mobility shift assay. On the other hand, in vivo sulfasalazine administration 6 h after training inhibited the second phase of training-induced NF-kappaB activation, providing evidence that the sulfasalazine effect on memory is due to a direct effect of the drug on the NF-kappaB pathway. These results provide the first evidence that IkappaB kinase and NF-kappaB activation are necessary for memory formation.

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.

NF-κB transcription factor is required for inhibitory avoidance long-term memory in mice

Although it is generally accepted that memory consolidation requires regulation of gene expression, only a few transcription factors (TFs) have been clearly demonstrated to be specifically involved in this process. Increasing research data point to the participation of the Rel/nuclear factor‐κB (NF‐κB) family of TFs in memory and neural plasticity. Here we found that two independent inhibitors of NF‐κB induced memory impairment in the one‐trial step‐through inhibitory avoidance paradigm in mice: post‐training administration of the drug sulfasalazine and 2 h pretraining administration of a double‐stranded DNA oligonucleotide containing the NF‐κB consensus sequence (κB decoy). Conversely, one base mutation of the κB decoy (mut‐κB decoy) injection did not affect long‐term memory. Accordingly, the κB decoy inhibited NF‐κB in hippocampus 2 h after injection but no inhibition was found with mut‐κB decoy administration. A temporal course of hippocampal NF‐κB activity after training was determined. Unexpectedly, an inhibition of NF‐κB was found 15 min after training in shocked and unshocked groups when compared with the naïve group. Hippocampal NF‐κB was activated 45 min after training in both shocked and unshocked groups, decreasing 1 h after training and returning to basal levels 2 and 4 h after training. On the basis of the latter results, we propose that activation of NF‐κB in hippocampus is part of the molecular mechanism involved in the storage of contextual features that constitute the conditioned stimulus representation. The results presented here provide the first evidence to support NF‐κB activity being regulated in hippocampus during consolidation, stressing the role of this TF as a conserved molecular mechanism for memory storage.

Activation of Hippocampal Nuclear Factor-κB by Retrieval Is Required for Memory Reconsolidation

Initially, memory is labile and requires consolidation to become stable. However, several studies support that consolidated memories can undergo a new period of lability after retrieval. The mechanistic differences of this process, termed reconsolidation, with the consolidation process are under debate, including the participation of hippocampus. Up to this point, few reports describe molecular changes and, in particular, transcription factor (TF) involvement in memory restabilization. Increasing evidence supports the participation of the TF nuclear factor-κB (NF-κB) in memory consolidation. Here, we demonstrate that the inhibition of NF-κB after memory reactivation impairs retention of a hippocampal-dependent inhibitory avoidance task in mice. We used two independent disruptive strategies to reach this conclusion. First, we administered intracerebroventricular or intrahippocampal sulfasalazine, an inhibitor of IKK (IκB kinase), the kinase that activates NF-κB. Second, we infused intracerebroventricular or intrahippocampal κB decoy, a direct inhibitor of NF-κB consisting of a double-stranded DNA oligonucleotide that contains the κB consensus sequence. When injected immediately after memory retrieval, sulfasalazine or κB decoy (Decoy) impaired long-term retention. In contrast, a one base mutated κB decoy (mDecoy) had no effect. Furthermore, we also found NF-κB activation in the hippocampus, with a peak 15 min after memory retrieval. This activation was earlier than that found during consolidation. Together, these results indicate that NF-κB is an important transcriptional regulator in memory consolidation and reconsolidation in hippocampus, although the temporal kinetics of activation differs between the two processes.

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.

Evolutionarily-conserved role of the NF-κB transcription factor in neural plasticity and memory

NF‐κB is an evolutionarily conserved family of transcription factors (TFs) critically involved in basic cellular mechanisms of the immune response, inflammation, development and apoptosis. In spite of the fact that it is expressed in the central nervous system, particularly in areas involved in memory processing, and is activated by signals such as glutamate and Ca2+, its role in neural plasticity and memory has only recently become apparent. A surprising feature of this molecule is its presence within the synapse. An increasing number of reports have called attention to the role of this TF in processes that require long‐term regulation of the synaptic function underlying memory and neural plasticity. Here we review the evidence regarding a dual role for NF‐κB, as both a signalling molecule after its activation at the synapse and a transcriptional regulator upon reaching the nucleus. The specific role of this signal, as well as the general transcriptional mechanism, in the process of memory formation is discussed. Converging lines of evidence summarized here point to a pivotal role for the NF‐κB transcription factor as a direct signalling mechanism in the regulation of gene expression involved in long‐term memory.

Lessons From a Crab: Molecular Mechanisms in Different Memory Phases of Chasmagnathus

Consolidation of long-term memory requires the activation of several transduction pathways that lead to post-translational modifications of synaptic proteins and to regulation of gene expression, both of which promote stabilization of specific changes in the activated circuits. In search of the molecular mechanisms involved in such processes, we used the context-signal associative learning paradigm of the crab Chasmagnathus. In this model, we studied the role of some molecular mechanisms, namely cAMP-dependent protein kinase (PKA), extracellular-signal-regulated kinase (ERK), the nuclear factor kappa B (NF-κB) transcription factor, and the role of synaptic proteins such as amyloid β precursor protein, with the object of describing key mechanisms involved in memory processing. In this article we review the most salient results obtained over a decade of research in this memory model.

Participation of transcription factors from the Rel/NF-κB family in the circadian system in hamsters

We have studied the presence and activity of components of the nuclear factor-kappaB (NF-kappaB) transcription factor in the hamster circadian system analyzing wheel-running activity, protein expression and DNA binding activity by electrophoresis mobility shift assays (EMSA). Non-rhythmic specific immunoreactive bands corresponding to a NF-kappaB subunit (p65) were found in hamster suprachiasmatic nuclei (SCN) homogenates. The active form of NF-kappaB evidenced by EMSA was clear and specific in SCN nuclear extracts. The administration of the NF-kappaB inhibitor pyrrolidine-dithiocharbamate (PDTC) blocked the light-induced phase advance at circadian time 18 (vehicle+light pulse: 2.08+/-0.46 h, PDTC+light: 0.36+/-0.35 h). These results demonstrate the presence and activity of Rel/NF-kappaB family proteins in the hamster SCN and suggest that these proteins may be related to the entrainment and regulation of circadian rhythms.

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.