Lessons From a Crab: Molecular Mechanisms in Different Memory Phases of Chasmagnathus
http://www.100md.com
《生物学报告》
Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. IFIByNE, CONICET. Ciudad Universitaria, Pab. II, 2do piso (1428EHA). Buenos Aires, Argentina
Abstract
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-B) transcription factor, and the role of synaptic proteins such as amyloid ? 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.
Abbreviations: APP, amyloid precursor protein ? CSM, context-signal memory ? ERK, extracellular-signal-regulated kinase ? ITI, intertrial interval ? JNK, c-Jun N-terminal kinase ? LTM, long-term memory ? MAPK, mitogen-activated protein kinase ? NF-B, nuclear factor kappa B ? NF, natural fibril ? PKA, protein kinase A ? TF, transcription factor
Context-signal memory in the crab Chasmagnathus, an invertebrate model for learning and memory studies
The development of memory models in different species, and particularly in invertebrates such as molluscs and insects, has led to considerable progress in the understanding of the molecular mechanisms underlying memory formation and processing (e.g.,Kandel, 2001; Menzel, 2001; Dubnau et al., 2003, Roberts and Glanzman, 2003; Crow, 2004). In the last 20 years, a research effort has been focused on the study of learning and memory in the grapsid crab Chasmagnathus. In this model, the repeated presentation of a danger stimulus (an opaque screen passing over the animal) provokes the decline of the escape response that was initially elicited (Lozada et al., 1990). Long-term memory (LTM) is defined at testing as a significantly lower response to the danger stimulus by a trained group of animals than by an untrained control group. LTM, lasting at least for a week, is generated by strong training (15 or more trials) with the spaced presentation of the stimulus (e.g., 171 s of intertrial interval, ITI). Conversely, massed training (e.g., 300 trials of stimulus presentations, with 4-s ITI) induces retention that lasts no more than 3 days and entails a different mechanism than that underlying LTM (Pedreira et al., 1998; Hermitte et al., 1999). The effect of both massed and spaced training has been analyzed at the behavioral level. Massed training induces a nonassociative habituation learning that does not show up in the first testing trial but only in subsequent trials, the so-called retraining phase of the testing session. An extreme case of massed training, the continuous-stimulation training protocol (ITI 0 s), induces a strong, short-duration decrement of the escape response but causes no long-term retention despite the enormous amount of training received (e.g., 600 trials) (Freudenthal et al., 1998) (Fig. 1). This protocol is used in active control groups to disclose the effects of visual stimulation, motor activity, stress, novelty, etc., that could induce molecular changes in a way not directly associated with memory formation. In turn, spaced training induces a context-dependent memory that is associative in nature (context-signal associative learning), showing characteristics of conditioning, such as latent inhibition and extinction (Tomsic et al., 1998). This association between the iterated stimulus and contextual features is expressed mainly in the first testing trial but also in the retraining phase (Pedreira et al., 1998).
A universal feature of long-lasting forms of memory is their dependence on macromolecular synthesis (Squire and Davis, 1981). Accordingly, LTM in Chasmagnathus, so-called context-signal memory (CSM), proved to be sensitive to the protein synthesis inhibitor cycloheximide during the first hours after training, and was also sensitive to the mRNA synthesis inhibitor actinomycin D (Pedreira et al., 1995; Pedreira et al., 1996).
Extinction of the context-signal association is obtained after a prolonged reexposure of the animals to the training context (typically for 2 h) one day after training. This process is sensitive to cycloheximide when administered after reexposure but not 6 h later (Pedreira and Maldonado, 2003). However, a brief reexposure (5 min) to the training context reactivates the memory, but extinction does not occur. This treatment induces a reconsolidation process that opens a new period of lability in which protein synthesis is again required (Pedreira et al., 2002).
Studies at the cellular level, using intracellular recording in vivo, described movement-detector neurons from the lobula (the third optic ganglion) that respond to the danger stimulus and modify their response during learning. These modifications strikingly correlate with the acquisition rate and with the duration of retention, indicating that information during learning is in part processed within optic neuropiles (Tomsic et al., 2003).
Several studies with a pharmacological approach demonstrated the participation of neurotransmitters such as glutamate (Troncoso and Maldonado, 2002; Pedreira et al., 2002), acetylcholine (Beron de Astrada and Maldonado, 1999), and the neuropeptide angiotensin II (Delorenzi et al., 1996; Frenkel et al., 2002) in this memory model.
Role of protein kinase A in memory consolidation
Using the crab model, our group has focused the research on signal transduction mechanisms that are activated by learning and participate in memory storage. Initially we studied the participation of the cAMP-dependent protein kinase (PKA). A body of evidence has implicated the cAMP pathway in neural plasticity related to memory formation (e.g., Castelluci et al., 1982; Frey et al., 1993). With the crab model, we obtained the first data showing that the manipulation of PKA activity by means of cAMP analogs affects memory formation.
In fact, the administration of a PKA activator, CPT-cAMP, together with a phosphodiesterase inhibitor, IBMX, before or after a weak training, facilitated long-term memory in a dose-dependent manner (Romano et al., 1996a). Although this result supports the view that cAMP augmentation and PKA activation are important steps in memory consolidation, an activation of the cGMP-PKG pathway could not be ruled out, since CPT-cAMP also activates cGMP-dependent protein kinase and both IBMX and CPT-cAMP increase cGMP levels. Therefore, we used two membrane-permeable cAMP analogs, Sp-5,6-DCl-cBIMPS and Rp-8-Cl-cAMPS, that are highly specific for PKA. Sp-5,6-DCl-cBIMPS, a PKA activator, facilitated memory, whereas Rp-8-Cl-cAMPS, a PKA inhibitor, induced amnesia (Romano et al., 1996b).
Further analysis using the PKA inhibitor revealed two critical periods during which PKA activity was necessary for CSM consolidation: one during training and other between 4 and 8 h after training (Locatelli et al., 2002) (Fig. 2a).
Thus far, the involvement of PKA in CSM formation had been based only on pharmacological results. To evaluate whether PKA activation is in fact part of the cellular mechanisms triggered by training, we next measured PKA activity in the central brain (supraesophageal ganglion) during the critical periods for CSM formation. In agreement with our previous findings, PKA was activated in the brain immediately and 6 h after training. In contrast, PKA activity increased immediately but not 6 h after exposure to a novel context (Locatelli and Romano, 2005) (Fig. 2b).
Two major families of PKA isoforms (PKA I and PKA II) that differ in their respective R subunits (R I and R II) have been described in different animals and tissues. PKA isoforms may show different subcellular distributions, so different activation kinetics and cAMP sensitivity (Cadd et al., 1990) are considered to provide specificity to PKA action (Sk?lhegg and Tasken, 2000). We demonstrated that PKA I and PKA II are present in Chasmagnathus neural tissues, showing characteristics similar to those described in other groups. Indeed, Chasmagnathus PKA I is 10 times more sensitive than PKA II to cAMP (Locatelli et al., 2001). Studying the activity profile of both PKA isoforms after training, we found different activation patterns. We obtained evidence for PKA II activation immediately after exposure to the training context without stimulus presentation. For animals trained in the CSM paradigm, we found a significant increase in the total level of PKA I 6 h after training. Considering the higher sensitivity of PKA I to cAMP, its increase can account for the PKA activation found 6 h after training (Fig. 2b) and is proposed as a novel mechanism providing the prolonged PKA activation during memory consolidation.
Mitogen-activated protein kinases in context-signal memory
The participation of other protein kinases that are included in the family of mitogen-activated protein kinases (MAPKs), namely extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), was studied in this model. This is a highly conserved family of protein kinases, present in species as different as yeast, worms, molluscs, insects, and mammals (Noselli, 1998; Villanueva et al., 2001). These Ser/Thr protein kinases are found in a variety of tissues, regulating development, mitogenesis, and the stress response. Substantial evidence has involved ERK in synaptic plasticity since the initial work of English and Sweatt (1996) in long-term potentiation and Martin et al. (1997) in long-term facilitation in Aplysia neurons. Subsequent studies showed the involvement of this kinase in memory processes in rodents (e.g., Atkins et al., 1998; Alonso et al., 2002), Hermissenda (Crow et al., 1998), and Aplysia (Purcell et al., 2003, Sharma et al., 2003). Although MAPKs are highly conserved, no evidence was found for their participation in memory models in animal groups other than rodents and molluscs.
Translocation of ERK to the nucleus and indirect activation of transcription factor CREB was classically proposed as the pathway by which this MAPK acts in memory consolidation. In the crab, we found high immunoreactivity with antibodies that recognize specifically the phosphorylated form of ERK and JNK in the central brain. Using these antibodies, we found that ERK, but not JNK, showed memory-specific activation in the brain 1 h after training. This activation, however, was detected in cytosolic but not in nuclear extracts (Fig. 3a). This is a striking finding considering the importance of nuclear targets of these MAPKs in different processes including learning, and their relevance in mechanisms of gene expression regulation in other memory models. Coincident with the temporal course of activation, an inhibitor of the ERK pathway, PD098059, showed amnestic effect when administered 45 min after training, but not immediately pre-training or post-training (Fig. 3b). Additionally, the same drug did not affect short-term memory (Feld et al., 2005).
All these findings suggest that an extranuclear ERK pool is necessary for long-term memory consolidation in this arthropod model. Further research is required to elucidate its specific subcellular localization and potential molecular effectors.
Rel/NF-B, a key transcription factor in consolidation and reconsolidation
Experimental data increasingly supports the participation of the nuclear transcription factor B (NF-B) in memory The first evidence indicating that learning activates NF-B was found in the crab Chasmagnathus (Freudenthal et al., 1998; Freudenthal and Romano, 2000). We detected specific B DNA-binding activity using a double-strand DNA oligonucleotide containing the NF-B consensus sequence in a gel shift assay. With this technique we detected one specific complex in the crab central brain. One of the complex components is a 61-kD protein that could be detected with an antibody against p65/Rel A, a member of the NF-B family in mammals. The gel shift assay allows an estimation of NF-B activity, and a high correlation between memory formation and NF-B activation was demonstrated. Spaced training yields CSM, which correlates with NF-B activation in the cell nucleus of crab central brain. In contrast, NF-B is not activated after massed training. Crabs that received spaced training showed two phases of Rel/NF-B activation. The first one was seen immediately after 10 or more trials, coinciding with the number of trials required for memory formation. Such activation decayed to basal level 3 h after training. The second phase occurred 6 h after training, waning to basal level in 12 and 24 h (Freudenthal and Romano, 2000) (Fig. 4a). Moreover, activated NF-B was found in synaptic terminals after long-term memory induction. The fact that activation of a transcription factor (TF) was found so far from the nucleus gave physiological support for the hypothesis that NF-B has a dual role in synapse-to-nucleus signaling, initially as a synaptic activity detector and then as transcriptional regulator after its retrograde transport to the nucleus (Meberg et al., 1996; Freudenthal and Romano, 2000; Wellmann et al., 2001; Meffert et al., 2003).
We next evaluated the effect of the inhibition of this TF pathway by means of the drug sulfasalazine. This drug is a specific inhibitor of IB kinase (IKK), the protein kinase that activates NF-B by phosphorylation of the inhibitor B (IB) that, after this phosphorylation, is degraded by proteasome, allowing the nuclear translocation of the TF. Sulfasalazine administration induced amnesia during the two periods in which NF-B was active, but not before or after the activation periods (Fig. 4b) (Merlo et al., 2002). This activity profile is similar to that found for PKA activity after training and suggests that these two pathways might be functionally related.
To study the effect of NF-B inhibition in another step of its activation pathway, we used a cell-permeable 26S proteasome inhibitor, MG132. This drug, among other effects, impedes NF-B activation by reducing the degradation of its inhibitor IB. In fact, MG132 impaired training-induced NF-B activation in crab brain when administered in vivo, while the same dose impaired long-term memory (Merlo and Romano, unpubl. obs.).
The requirement of this TF in CSM formation led us to question which neurotransmitters or neuromodulators are involved in NF-B activation during learning. The neuropeptide angiotensin II plays a key role in the crab long-term memory, and we found that nuclear NF-B in the crab brain is activated by angiotansin II administration at doses that facilitated CSM. The activation of the TF was reversed by saralasin, an antagonist of angiotensin II receptors. Accordingly, NF-B activation was also found after water shortage, a treatment that increased angiotensin II levels in the brain and induced memory facilitation (Frenkel et al., 2002).
In the classical postulation, memory is initially labile and becomes resistant to different disruption treatments after a consolidation period. However, some experimental data (e.g., Misanin et al., 1968; Sara, 2000; Nader et al., 2000; Pedreira et al., 2002) challenged this assumption, postulating that a consolidated memory may become labile once again when retrieved and reactivated by a reminder, opening a new consolidation period called reconsolidation. Whether reconsolidation is mechanistically similar to the initial consolidation is a matter of debate (Debiec et al., 2002; Myers and Davis, 2002; Lee et al., 2004; Salinska et al., 2004). We tested whether NF-B is required for reconsolidation and found that NF-B was specifically reactivated in animals reexposed 24 h after training to the same training context but not to a different context (Fig. 5a). Furthermore, NF-B was not activated in animals reexposed to the context after a weak training protocol insufficient to induce long-term memory. Additionally, sulfasalazine impaired reconsolidation when administered 20 min before reexposure to the training context, but was not effective when a different context was used (Merlo et al., 2005) (Fig. 5b). These findings are in keeping with the view that basic molecular mechanisms of consolidation are necessary to restore reactivated memory. At the same time, these findings reveal for the first time that NF-B is activated specifically by retrieval and that this activation is required for memory reconsolidation, supporting the view that this molecular mechanism plays a key role in both consolidation and reconsolidation.
Chasmagnathusas a model for testing naturally secreted amyloid fibrils and amyliod-? peptides in memory
Amyloid ? (A?) peptides are produced by cleavage of a membrane protein, amyloid precursor protein (APP). In humans, these peptides are found in neuritic plaques of patients with Alzheimer’s disease, and they are proposed as the cause of the memory deficit and neurodegeneration observed in the disease. An important research effort has been devoted to the study of neurotoxicity and neurodegeneration caused by these peptides. However, the memory deficit, an early symptom of Alzheimer’s that occurs before neural degeneration, has also been attributed to increased levels of A? peptides in different stages of aggregation, particularly as soluble oligomers (Walsh et al., 2002). These studies normally employed synthetic A? peptides. However, the effect of endogenous neuronal amyloid fibrils on memory processes is unknown and was evaluated only in a model of neural plasticity in mammals (Walsh et al., 2002). Beyond its pathological role, A? has been proposed to have physiological functions as a synaptic transmission depressor (Kamenetz et al., 2003). That possibility prompted us to evaluate the role of A? as a negative memory modulator in the crab, to analyze the effect of the exogenous administration of these mammalian peptides in an invertebrate memory model that lacks endogenous A? (see next section). First, we tested the effect on long-term memory of natural fibrils (NFs) obtained from constitutive secretion of rat cerebellar granule cells. Pre-training administration of very low quantities of NFs (10 to 50 ng/animal) induced amnesia in a dose-dependent manner when tested 24 h after training. In contrast, synthetic fibrillated A? 1–40, one of the forms of A? peptides, at doses two orders of magnitude higher (5 μg/animal) had no effect on memory retention (Romano et al., 2003). To test whether the amnestic effect was due to an irreversible toxicity of fibrils on the crab’s nervous system, NF was administered 24 h before training and had no effect on LTM. NFs impaired CSM when injected immediately after training, but they had no effects when administered 6 h after training. Thus, only the peri-training administration of NFs affected LTM, suggesting that the peptides act during memory consolidation by interfering with normal signaling and that their action is not due to unspecific and irreversible toxic effects. To further characterize the active components included in NFs, we evaluated the action of several synthetic peptides that are normally present in human brain, A? 1–40, A? 1–42, A? 3–42, and A? 11–42. These peptides were used after 3 days of incubation at 37°C, a protocol that allows for in vitro fibrillation. A? 1–42 and A? 3–42 induced remarkable amnestic effect at very low doses; A? 11–42 did not induce amnesia; and A? 1–40 did not affect retention, even at a higher dose than A? 1–42, indicating that the difference in two amino acids determines their effect on memory consolidation. The fibrillation state of the peptides seems to be a requirement for their action on memory, considering that non-fibrillated A? 1–42 did not show an amnestic effect (Feld et al., unpubl. obs.). These results are at variance with the postulation that the early aggregation stages, particularly oligomers, are the active element altering memory processes (Walsh et al., 2002).
chAPPL, the crab ortholog of amyloid precursor protein family
Members of the amyloid precursor protein (APP) family are transmembrane glycoproteins. In humans, one of these members (A?) is the source of the amyloid peptides found in the neuritic plaques of Alzheimer patients and, as discussed in the last section, has been proposed as the cause of memory deficit and neurodegeneration in this pathology. In the research focused on the neurotoxic properties of A?, several models were used to study the effect of the overexpression and accumulation of such peptides in the nervous system. However, the APP family is composed of transmembrane proteins involved in synaptogenesis and synaptic plasticity, both in vertebrates and invertebrates, acting either in full-length configuration or in their proteolytic fragments (Turner et al., 2003). In spite of the importance of these proteins in synaptic physiology and pathology, little information is available on their function. Two homolog members of this protein family have been cloned in invertebrates, in the nematode Caenorhabditis elegans and in the fruit fly Drosophila melanogaster. We recently cloned most of the mRNA sequence of the Chasmagnathus APP-like protein (chAPPL) gene, the third ortholog member of this family in invertebrates. As expected, this gene shows high similarity with Drosophila APPL, and both genes show low similarity with the A? peptide sequence of vertebrates. Preliminary results support the view that the expression of this gene is upregulated during memory consolidation (Ariel et al., unpubl. obs). The study of genes in the APP family in well-characterized memory models in invertebrates will help elucidate their role in memory storage, and could contribute to the understanding of their role in pathology.
Concluding remarks
The development of learning and memory models in different taxa and the use of nonconventional animals in these studies are important contributions to the search for molecular mechanisms involved in memory storage. On the one hand, each preparation presents particular advantages for study of some aspect of the subject. On the other hand, the availability of models in diverse animal groups allows for a comparative analysis of memory processes. Such analysis will make it possible to define basic mechanisms of memory formation and, in turn, to determine specific characteristics of particular systems and neuronal pathways.
The CSM in the crab Chasmagnathus is a memory model well characterized at the behavioral level and in terms of the phases of memory processing. This characterization constitutes an advantage for the study of the molecular mechanisms of memory because appropriate controls are available to evaluate the effect of factors such as stress, sensorial stimulation, and motor activity and to distinguish them from the specific mechanisms of memory storage.
As an example of comparative studies, the finding of NF-B participation in crab memory led us to question whether the activation of this mechanism is characteristic of this model or is common to different groups of animals. To answer this question, we studied this transcription factor in mice, first in a model of neural plasticity—in vivo long-term potentiation (Freudenthal et al., 2004)—and then in inhibitory avoidance learning. We found that NF-B indeed participates in neural plasticity and memory consolidation (Freudenthal et al., 2005) and reconsolidation (Freudenthal et al., unpubl. obs.) in mice. Together with recent results obtained by other groups in rodents (Yeh et al., 2002; Meffert et al., 2003), these findings are evidence that this transcriptional mechanism is conserved in evolution and important in memory processing.
Acknowledgments
Authors are indebted to Hector Maldonado for his support and guidance during all these years, to Angel Vidal for technical assistance, and to Liliana Orelli for language correction.
Footnotes
Received 7 November 2005; accepted 7 March 2006.
Literature Cited
Alonso, M., H. Viola, I. Izquierdo, and J. H. Medina. 2002. Aversive experiences are associated with a rapid and transient activation of ERKs in the rat hippocampus. Neurobiol. Learn. Mem. 77(1):119R–124.
Atkins, C. M., J. C. Selcher, J. J. Petraitis, J. M. Trzaskos, and J. D. Sweatt. 1998. The MAPK cascade is required for mammalian associative learning. Nature Neurosci. 1(7):602–609.
Beron de Astrada, M., and H. Maldonado. 1999. Two related forms of long-term habituation in the crab Chasmagnathus are differentially affected by scopolamine. Pharmacol. Biochem. Behav. 63(1):109–118.
Cadd, G.G., M.D. Uhler, and G. McKnight. 1990. Holoenzymes of cAMP-dependent protein kinase containing the neural form of type I regulatory subunit have an increased sensitivity to cyclic nucleotides. J. Biol. Chem. 265(32):19502–19506.
Castellucci, V. F., A. Nairn, P. Greengard, J.H. Schwartz, and E.R. Kandel. 1982. Inhibitor of adenosine 3':5'-monophosphate-dependent protein kinase blocks presynaptic facilitation in Aplysia. J. Neurosci. 12:1673–1681.
Crow, T. 2004. Pavlovian Conditioning of Hermissenda: current cellular, molecular, and circuit perspectives. Learn. Mem. 11(3):229–238.
Crow, T., J-J. Xue-Bian, V. Siddiqi, Y. Kang,, and J.T. Neary. 1998. Phosphorylation of mitogen-activated protein kinase by one-trial, and multi-trial classical conditioning. J. Neurosci. 18(9):3480–3487.
Debiec, J., J.E. LeDoux, and K. Nader. 2002. Cellular and systems reconsolidation in the hippocampus. Neuron36:527–538.
Delorenzi, A., E.M. Pedreira, A. Romano, S. García, C. Pirola, V. Nahmod, and H. Maldonado. 1996. Angiotensin II enhances long-term memory in the crab Chasmagnathus. Brain Res. Bull. 47(4):211–219.
Dubnau, J., A. S. Chiang, and T. Tully. 2003. Neural substrates of memory: from synapse to system. J. Neurobiol. 54(1):238–253.
English, J. D., and J. D. Sweatt. 1996. Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. J. Biol. Chem. 271 (40):24329–24332.
Feld, M., B. Dimant, A. Delorenzi, O. Coso, and A. Romano. 2005. Extra-nuclear activation of ERK/MAPK is required for long-term memory consolidation in the crab Chasmagnathus. Behav. Brain Res. 158(2):251–261.
Frenkel, L., R. Freudenthal, A. Romano, V. E. Nahmod, H. Maldonado, and A. Delorenzi. 2002. Angiotensin II and the transcription factor Rel/NF-B link environmental water shortage with memory improvement. Neuroscience 115(4):1079–1087.
Freudenthal, R., and A. Romano. 2000. Participation of NF-B transcription factors in long-term memory in the crab Chasmagnathus. Brain Res. 855:274–281.
Freudenthal, R., F. Locatelli, G. Hermitte, H. Maldonado, A. Delorenzi, C. Lafourcade, and A. Romano. 1998. -B like DNA-binding activity is enhanced after a spaced training that induces long-term memory in the crab Chasmagnathus. Neurosci. Lett. 242:143–146.
Freudenthal, R., A. Romano, and A. Routtenberg. 2004. Activation of the transcription factor NF-B after in vivo perforant path LTP in the mouse hippocampus. Hippocampus14:677–683.
Freudenthal, R., M. M. Boccia, G. B. Acosta, M. G. Blake, E. Merlo, C. M. Baratti, and A. Romano. 2005. NF-B transcription factor is required for inhibitory avoidance long-term memory in mice. Eur. J. Neurosci.21:2845–2852.
Frey, U., Y. Y. Huang, and E. R. Kandel. 1993. Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons. Science260:1661–1664.
Hermitte, G., M. E. Pedreira, D. Tomsic, and H. Maldonado. 1999. Context shift and protein synthesis inhibition disrupt long-term habituation after spaced, but not massed, training in the crab Chasmagnathus. Neurobiol. Learn. Mem. 71:34–49.
Kamenetz, F., T. Tomita, H. Hsieh, G. Seabrook, D. Borchelt, T. Iwatsubo, S. Sisodia, and R. Malinow. 2003. APP processing and synaptic function. Neuron37:925–937.
Kandel, E. R. 2001. The molecular biology of memory storage: a dialog between genes and synapses. Science294:1030–1038.
Lee, J. L., B. J. Everitt, and K. L. Thomas. 2004. Independent cellular processes for hippocampal memory consolidation and reconsolidation. Science304:839–483.
Locatelli, F., and A. Romano. 2005. Differential role of cAMP-dependent protein kinase isoforms during long-term memory consolidation in the crab Chasmagnathus. Neurobiol. Learn. Mem. 83:232–242.
Locatelli, F., C. Lafourcade, H. Maldonado, and A. Romano. 2001. Characterisation of cAMP-dependent protein kinase isoforms in the brain of the crab Chasmagnathus. J. Comp. Physiol. B 171:33–40.
Locatelli, F., H. Maldonado, and A. Romano. 2002. Two critical periods for cAMP-dependent protein kinase activity during long-term memory consolidation in the crab Chasmagnathus. Neurobiol. Learn. Mem. 77(2):234–249.
Lozada, M., A. Romano, and H. Maldonado. 1990. Long-term habituation to a danger stimulus in the crab Chasmagnathus. Physiol. Behav. 47:35–41.
Martin, K. C., D. Michael, J. C. Rose, M. Barad, A. Casadio, H. Zhu, and E. R. Kandel. 1997. MAP kinase translocates into the nucleus of the presynaptic cell and is required for long-term facilitation in Aplysia. Neuron 18(6):899–912.
Meberg, P. J., W. R. Kinney, E. G. Valcourt, and A. Routtenberg. 1996. Gene expression of the transcription factor NF-B in hippocampus: regulation by synaptic activity. Mol. Brain, Res.38:179–190.
Meffert, M. K., J. M. Chang, B. J. Wiltgen, M. S. Fanselow, and D. Baltimore. 2003. NF-B function in synaptic signaling and behavior. Nature Neurosci.6:1072–1078.
Menzel, R. 2001. Searching for the memory trace in a mini-brain, the honeybee. Learn Mem. 8(2):53–62.
Merlo, E., R. Freudenthal, and A. Romano. 2002. The IB kinase inhibitor sulfasalazine impairs long-term memory in the crab Chasmagnathus. Neuroscience 112(1):161–172.
Merlo, E., R. Freudenthal, H. Maldonado, and A. Romano. 2005. Activation of the transcription factor NF-B by retrieval is required for long-term memory reconsolidation. Learn. Mem. 12(1):23–29.
Misanin, J. R., R. R. Miller, and D. I. Lewis. 1968. Retrograde amnesia produced by electroconvulsive shock following reactivation of a consolidated memory trace. Science16:554–555.
Myers, K.M., and M. Davis. 2002. Behavioral and neural analysis of extinction. Neuron36:567–584.
Nader, K., G. E. Schafe, and J. E LeDoux. 2000. Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval. Nature406:722–726.
Noselli, S. 1998. JNK signaling and morphogenesis in Drosophila. Trends Genet. 14:33–38.
Pedreira, M. E., and H. Maldonado. 2003. Protein synthesis subserves reconsolidation or extinction depending on reminder duration. Neuron 38(6):863–869.
Pedreira, M. E., B. Dimant, D. Tomsic, L. A. Quesada-Allue, and H. Maldonado. 1995. Cycloheximide inhibits long-term habituation and context memory in the crab Chasmagnathus. Pharmacol. Biochem. Behav. 52:385–395.
Pedreira, M. E., B. Dimant, and H. Maldonado. 1996. Inhibitors of protein and RNA synthesis block context memory and long-term habituation in the crab Chasmagnathus. Pharmacol. Biochem. Behav. 54:611–617.
Pedreira, M. E., A. Romano, D. Tomsic, M. Lozada, and H. Maldonado. 1998. Massed and spaced training build up different components of long-term habituation in the crab Chasmagnathus. Anim. Learn. Behav. 26:34–45.
Pedreira, M. E., L. M. Perez-Cuesta, and H. Maldonado. 2002. Reactivation and reconsolidation of long-term memory in the crab Chasmagnathus: protein synthesis requirement and mediation by NMDA-type glutamatergic receptors. J Neurosci. 22(18):8305–8311.
Purcell, A. L., S. K. Sharma, M. W. Bagnall, M. A. Sutton, and T. J. Carew. 2003. Activation of a tyrosine kinase-MAPK cascade enhances the induction of long-term synaptic facilitation and long-term memory in Aplysia. Neuron 37(3):473–484.
Roberts, A. C., and D. L. Glanzman. 2003. Learning in Aplysia: looking at synaptic plasticity from both sides. Trends Neurosci. 26(12):662–670.
Romano, A., A. Delorenzi, M. E. Pedreira, D. Tomsic, and H. Maldonado. 1996a. Acute administration of a cAMP analog and a phosphodiesterase inhibitor improve long-term habituation of the crab Chasmagnathus. Behav. Brain Res. 75:119–125.
Romano, A., F. Locatelli, M. E. Pedreira, A. Delorenzi. and H. Maldonado. 1996b. Effect of activation and inhibition of cAMP-dependent proteinkinase on long-term habituation of the crab Chasmagnathus. Brain Res. 735:131–140.
Romano, A., A. L. Serafino, E. Krasnowska, M. T. Ciotti, P. Calissano, F. Ruberti, and C. Galli. 2003. Neuronal fibrillogenesis: amyloid fibrils from primary neuronal cultures impair long-term memory in the crab Chasmagnatus. Behav. Brain Res. 147:73–82.
Salinska, E., R. C. Bourne, and S. P. R. Rose. 2004. Reminder effects: the molecular cascade following a reminder in young chicks does not recapitulate that following training on a passive avoidance task. Eur. J. Neurosci.19:3042–3047.
Sara, S. J. 2000. Retrieval and reconsolidation: toward a neurobiology of remembering. Learn. Mem7:73–84.
Sharma, S. K., C. M. Sherff, J. Shobe, M. W. Bagnall, M. A. Sutton, and T. J. Carew. 2003. Differential role of mitogen-activated protein kinase in three distinct phases of memory for sensitization in Aplysia. J. Neurosci. 23 (9):3899–3907.
Sk?lhegg, B. S., and K. Tasken. 2000. Specificity in the cAMP/PKA signalling pathway. Differential expression, regulation, and subcellular localization of subunits of PKA. Front. Biosci.,5:D678–693.
Squire, L. R., and H. P. Davis. 1981. The pharmacology of memory: a neurobiological perspective. Annu. Rev. Pharmacol. Toxicol.21:323–336.
Tomsic, D., E. M. Pedreira, A. Romano, G. Hermitte, and H. Maldonado. 1998. Association to context as a determinant of long-term habituation in the crab Chasmagnathus. Anim. Learn. Behav. 26:196–209.
Tomsic, D., M. Beron de Astrada, and J. Sztarker. 2003. Identification of individual neurons reflecting short- and long-term visual memory in an arthropod. J. Neurosci. 23(24):8539–8546.
Troncoso, J., and H. Maldonado. 2002. Two related forms of memory in the crab Chasmagnathus are differentially affected by NMDA receptor antagonists. Pharmacol. Biochem. Behav. 72(1–2):251–265.
Turner, P. R., K. O. O’Connor, W. P. Tate, and W. C. Abraham. 2003. Roles of amyloid precursor protein and its fragments in regulating neural activity, plasticity and memory. Prog. Neurobiol.70:1–32.
Villanueva, A., J. Lozano, A. Morales, X. Lin, X. Deng, M. O. Hengartner, and R. N. Kolesnick. 2001. JKK-1 and MEK-1 regulate body movement coordination and response to heavy metals through JNK-1 in Caenorhabditis elegans. EMBO J. 20(18):5114–5128.
Walsh, D. M., I. Klyubin, J. V. Fadeeva, W. K. Cullen, R. Anwyl, M. S. Wolfe, M. J. Rowan, and D. J Selkoe. 2002. Naturally secreted oligomers of amyloid ? protein potently inhibit hippocampal lasting potentiation in vivo. Nature 416:535–539.
Wellmann, H., B. Kaltschmidt, and C. Kaltschmidt. 2001. Retrograde transport of transcription factor NF-B in living neurons. J. Biol. Chem. 276(15):11821–11829.
Yeh, S. H., C. H. Lin, C. F. Lee, and P. W. Gean. 2002. A requirement of nuclear factor-B activation in fear-potentiated startle. J. Biol. Chem. 277(48):46720–9.(Arturo Romano, Fernando L)
Abstract
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-B) transcription factor, and the role of synaptic proteins such as amyloid ? 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.
Abbreviations: APP, amyloid precursor protein ? CSM, context-signal memory ? ERK, extracellular-signal-regulated kinase ? ITI, intertrial interval ? JNK, c-Jun N-terminal kinase ? LTM, long-term memory ? MAPK, mitogen-activated protein kinase ? NF-B, nuclear factor kappa B ? NF, natural fibril ? PKA, protein kinase A ? TF, transcription factor
Context-signal memory in the crab Chasmagnathus, an invertebrate model for learning and memory studies
The development of memory models in different species, and particularly in invertebrates such as molluscs and insects, has led to considerable progress in the understanding of the molecular mechanisms underlying memory formation and processing (e.g.,Kandel, 2001; Menzel, 2001; Dubnau et al., 2003, Roberts and Glanzman, 2003; Crow, 2004). In the last 20 years, a research effort has been focused on the study of learning and memory in the grapsid crab Chasmagnathus. In this model, the repeated presentation of a danger stimulus (an opaque screen passing over the animal) provokes the decline of the escape response that was initially elicited (Lozada et al., 1990). Long-term memory (LTM) is defined at testing as a significantly lower response to the danger stimulus by a trained group of animals than by an untrained control group. LTM, lasting at least for a week, is generated by strong training (15 or more trials) with the spaced presentation of the stimulus (e.g., 171 s of intertrial interval, ITI). Conversely, massed training (e.g., 300 trials of stimulus presentations, with 4-s ITI) induces retention that lasts no more than 3 days and entails a different mechanism than that underlying LTM (Pedreira et al., 1998; Hermitte et al., 1999). The effect of both massed and spaced training has been analyzed at the behavioral level. Massed training induces a nonassociative habituation learning that does not show up in the first testing trial but only in subsequent trials, the so-called retraining phase of the testing session. An extreme case of massed training, the continuous-stimulation training protocol (ITI 0 s), induces a strong, short-duration decrement of the escape response but causes no long-term retention despite the enormous amount of training received (e.g., 600 trials) (Freudenthal et al., 1998) (Fig. 1). This protocol is used in active control groups to disclose the effects of visual stimulation, motor activity, stress, novelty, etc., that could induce molecular changes in a way not directly associated with memory formation. In turn, spaced training induces a context-dependent memory that is associative in nature (context-signal associative learning), showing characteristics of conditioning, such as latent inhibition and extinction (Tomsic et al., 1998). This association between the iterated stimulus and contextual features is expressed mainly in the first testing trial but also in the retraining phase (Pedreira et al., 1998).
A universal feature of long-lasting forms of memory is their dependence on macromolecular synthesis (Squire and Davis, 1981). Accordingly, LTM in Chasmagnathus, so-called context-signal memory (CSM), proved to be sensitive to the protein synthesis inhibitor cycloheximide during the first hours after training, and was also sensitive to the mRNA synthesis inhibitor actinomycin D (Pedreira et al., 1995; Pedreira et al., 1996).
Extinction of the context-signal association is obtained after a prolonged reexposure of the animals to the training context (typically for 2 h) one day after training. This process is sensitive to cycloheximide when administered after reexposure but not 6 h later (Pedreira and Maldonado, 2003). However, a brief reexposure (5 min) to the training context reactivates the memory, but extinction does not occur. This treatment induces a reconsolidation process that opens a new period of lability in which protein synthesis is again required (Pedreira et al., 2002).
Studies at the cellular level, using intracellular recording in vivo, described movement-detector neurons from the lobula (the third optic ganglion) that respond to the danger stimulus and modify their response during learning. These modifications strikingly correlate with the acquisition rate and with the duration of retention, indicating that information during learning is in part processed within optic neuropiles (Tomsic et al., 2003).
Several studies with a pharmacological approach demonstrated the participation of neurotransmitters such as glutamate (Troncoso and Maldonado, 2002; Pedreira et al., 2002), acetylcholine (Beron de Astrada and Maldonado, 1999), and the neuropeptide angiotensin II (Delorenzi et al., 1996; Frenkel et al., 2002) in this memory model.
Role of protein kinase A in memory consolidation
Using the crab model, our group has focused the research on signal transduction mechanisms that are activated by learning and participate in memory storage. Initially we studied the participation of the cAMP-dependent protein kinase (PKA). A body of evidence has implicated the cAMP pathway in neural plasticity related to memory formation (e.g., Castelluci et al., 1982; Frey et al., 1993). With the crab model, we obtained the first data showing that the manipulation of PKA activity by means of cAMP analogs affects memory formation.
In fact, the administration of a PKA activator, CPT-cAMP, together with a phosphodiesterase inhibitor, IBMX, before or after a weak training, facilitated long-term memory in a dose-dependent manner (Romano et al., 1996a). Although this result supports the view that cAMP augmentation and PKA activation are important steps in memory consolidation, an activation of the cGMP-PKG pathway could not be ruled out, since CPT-cAMP also activates cGMP-dependent protein kinase and both IBMX and CPT-cAMP increase cGMP levels. Therefore, we used two membrane-permeable cAMP analogs, Sp-5,6-DCl-cBIMPS and Rp-8-Cl-cAMPS, that are highly specific for PKA. Sp-5,6-DCl-cBIMPS, a PKA activator, facilitated memory, whereas Rp-8-Cl-cAMPS, a PKA inhibitor, induced amnesia (Romano et al., 1996b).
Further analysis using the PKA inhibitor revealed two critical periods during which PKA activity was necessary for CSM consolidation: one during training and other between 4 and 8 h after training (Locatelli et al., 2002) (Fig. 2a).
Thus far, the involvement of PKA in CSM formation had been based only on pharmacological results. To evaluate whether PKA activation is in fact part of the cellular mechanisms triggered by training, we next measured PKA activity in the central brain (supraesophageal ganglion) during the critical periods for CSM formation. In agreement with our previous findings, PKA was activated in the brain immediately and 6 h after training. In contrast, PKA activity increased immediately but not 6 h after exposure to a novel context (Locatelli and Romano, 2005) (Fig. 2b).
Two major families of PKA isoforms (PKA I and PKA II) that differ in their respective R subunits (R I and R II) have been described in different animals and tissues. PKA isoforms may show different subcellular distributions, so different activation kinetics and cAMP sensitivity (Cadd et al., 1990) are considered to provide specificity to PKA action (Sk?lhegg and Tasken, 2000). We demonstrated that PKA I and PKA II are present in Chasmagnathus neural tissues, showing characteristics similar to those described in other groups. Indeed, Chasmagnathus PKA I is 10 times more sensitive than PKA II to cAMP (Locatelli et al., 2001). Studying the activity profile of both PKA isoforms after training, we found different activation patterns. We obtained evidence for PKA II activation immediately after exposure to the training context without stimulus presentation. For animals trained in the CSM paradigm, we found a significant increase in the total level of PKA I 6 h after training. Considering the higher sensitivity of PKA I to cAMP, its increase can account for the PKA activation found 6 h after training (Fig. 2b) and is proposed as a novel mechanism providing the prolonged PKA activation during memory consolidation.
Mitogen-activated protein kinases in context-signal memory
The participation of other protein kinases that are included in the family of mitogen-activated protein kinases (MAPKs), namely extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), was studied in this model. This is a highly conserved family of protein kinases, present in species as different as yeast, worms, molluscs, insects, and mammals (Noselli, 1998; Villanueva et al., 2001). These Ser/Thr protein kinases are found in a variety of tissues, regulating development, mitogenesis, and the stress response. Substantial evidence has involved ERK in synaptic plasticity since the initial work of English and Sweatt (1996) in long-term potentiation and Martin et al. (1997) in long-term facilitation in Aplysia neurons. Subsequent studies showed the involvement of this kinase in memory processes in rodents (e.g., Atkins et al., 1998; Alonso et al., 2002), Hermissenda (Crow et al., 1998), and Aplysia (Purcell et al., 2003, Sharma et al., 2003). Although MAPKs are highly conserved, no evidence was found for their participation in memory models in animal groups other than rodents and molluscs.
Translocation of ERK to the nucleus and indirect activation of transcription factor CREB was classically proposed as the pathway by which this MAPK acts in memory consolidation. In the crab, we found high immunoreactivity with antibodies that recognize specifically the phosphorylated form of ERK and JNK in the central brain. Using these antibodies, we found that ERK, but not JNK, showed memory-specific activation in the brain 1 h after training. This activation, however, was detected in cytosolic but not in nuclear extracts (Fig. 3a). This is a striking finding considering the importance of nuclear targets of these MAPKs in different processes including learning, and their relevance in mechanisms of gene expression regulation in other memory models. Coincident with the temporal course of activation, an inhibitor of the ERK pathway, PD098059, showed amnestic effect when administered 45 min after training, but not immediately pre-training or post-training (Fig. 3b). Additionally, the same drug did not affect short-term memory (Feld et al., 2005).
All these findings suggest that an extranuclear ERK pool is necessary for long-term memory consolidation in this arthropod model. Further research is required to elucidate its specific subcellular localization and potential molecular effectors.
Rel/NF-B, a key transcription factor in consolidation and reconsolidation
Experimental data increasingly supports the participation of the nuclear transcription factor B (NF-B) in memory The first evidence indicating that learning activates NF-B was found in the crab Chasmagnathus (Freudenthal et al., 1998; Freudenthal and Romano, 2000). We detected specific B DNA-binding activity using a double-strand DNA oligonucleotide containing the NF-B consensus sequence in a gel shift assay. With this technique we detected one specific complex in the crab central brain. One of the complex components is a 61-kD protein that could be detected with an antibody against p65/Rel A, a member of the NF-B family in mammals. The gel shift assay allows an estimation of NF-B activity, and a high correlation between memory formation and NF-B activation was demonstrated. Spaced training yields CSM, which correlates with NF-B activation in the cell nucleus of crab central brain. In contrast, NF-B is not activated after massed training. Crabs that received spaced training showed two phases of Rel/NF-B activation. The first one was seen immediately after 10 or more trials, coinciding with the number of trials required for memory formation. Such activation decayed to basal level 3 h after training. The second phase occurred 6 h after training, waning to basal level in 12 and 24 h (Freudenthal and Romano, 2000) (Fig. 4a). Moreover, activated NF-B was found in synaptic terminals after long-term memory induction. The fact that activation of a transcription factor (TF) was found so far from the nucleus gave physiological support for the hypothesis that NF-B has a dual role in synapse-to-nucleus signaling, initially as a synaptic activity detector and then as transcriptional regulator after its retrograde transport to the nucleus (Meberg et al., 1996; Freudenthal and Romano, 2000; Wellmann et al., 2001; Meffert et al., 2003).
We next evaluated the effect of the inhibition of this TF pathway by means of the drug sulfasalazine. This drug is a specific inhibitor of IB kinase (IKK), the protein kinase that activates NF-B by phosphorylation of the inhibitor B (IB) that, after this phosphorylation, is degraded by proteasome, allowing the nuclear translocation of the TF. Sulfasalazine administration induced amnesia during the two periods in which NF-B was active, but not before or after the activation periods (Fig. 4b) (Merlo et al., 2002). This activity profile is similar to that found for PKA activity after training and suggests that these two pathways might be functionally related.
To study the effect of NF-B inhibition in another step of its activation pathway, we used a cell-permeable 26S proteasome inhibitor, MG132. This drug, among other effects, impedes NF-B activation by reducing the degradation of its inhibitor IB. In fact, MG132 impaired training-induced NF-B activation in crab brain when administered in vivo, while the same dose impaired long-term memory (Merlo and Romano, unpubl. obs.).
The requirement of this TF in CSM formation led us to question which neurotransmitters or neuromodulators are involved in NF-B activation during learning. The neuropeptide angiotensin II plays a key role in the crab long-term memory, and we found that nuclear NF-B in the crab brain is activated by angiotansin II administration at doses that facilitated CSM. The activation of the TF was reversed by saralasin, an antagonist of angiotensin II receptors. Accordingly, NF-B activation was also found after water shortage, a treatment that increased angiotensin II levels in the brain and induced memory facilitation (Frenkel et al., 2002).
In the classical postulation, memory is initially labile and becomes resistant to different disruption treatments after a consolidation period. However, some experimental data (e.g., Misanin et al., 1968; Sara, 2000; Nader et al., 2000; Pedreira et al., 2002) challenged this assumption, postulating that a consolidated memory may become labile once again when retrieved and reactivated by a reminder, opening a new consolidation period called reconsolidation. Whether reconsolidation is mechanistically similar to the initial consolidation is a matter of debate (Debiec et al., 2002; Myers and Davis, 2002; Lee et al., 2004; Salinska et al., 2004). We tested whether NF-B is required for reconsolidation and found that NF-B was specifically reactivated in animals reexposed 24 h after training to the same training context but not to a different context (Fig. 5a). Furthermore, NF-B was not activated in animals reexposed to the context after a weak training protocol insufficient to induce long-term memory. Additionally, sulfasalazine impaired reconsolidation when administered 20 min before reexposure to the training context, but was not effective when a different context was used (Merlo et al., 2005) (Fig. 5b). These findings are in keeping with the view that basic molecular mechanisms of consolidation are necessary to restore reactivated memory. At the same time, these findings reveal for the first time that NF-B is activated specifically by retrieval and that this activation is required for memory reconsolidation, supporting the view that this molecular mechanism plays a key role in both consolidation and reconsolidation.
Chasmagnathusas a model for testing naturally secreted amyloid fibrils and amyliod-? peptides in memory
Amyloid ? (A?) peptides are produced by cleavage of a membrane protein, amyloid precursor protein (APP). In humans, these peptides are found in neuritic plaques of patients with Alzheimer’s disease, and they are proposed as the cause of the memory deficit and neurodegeneration observed in the disease. An important research effort has been devoted to the study of neurotoxicity and neurodegeneration caused by these peptides. However, the memory deficit, an early symptom of Alzheimer’s that occurs before neural degeneration, has also been attributed to increased levels of A? peptides in different stages of aggregation, particularly as soluble oligomers (Walsh et al., 2002). These studies normally employed synthetic A? peptides. However, the effect of endogenous neuronal amyloid fibrils on memory processes is unknown and was evaluated only in a model of neural plasticity in mammals (Walsh et al., 2002). Beyond its pathological role, A? has been proposed to have physiological functions as a synaptic transmission depressor (Kamenetz et al., 2003). That possibility prompted us to evaluate the role of A? as a negative memory modulator in the crab, to analyze the effect of the exogenous administration of these mammalian peptides in an invertebrate memory model that lacks endogenous A? (see next section). First, we tested the effect on long-term memory of natural fibrils (NFs) obtained from constitutive secretion of rat cerebellar granule cells. Pre-training administration of very low quantities of NFs (10 to 50 ng/animal) induced amnesia in a dose-dependent manner when tested 24 h after training. In contrast, synthetic fibrillated A? 1–40, one of the forms of A? peptides, at doses two orders of magnitude higher (5 μg/animal) had no effect on memory retention (Romano et al., 2003). To test whether the amnestic effect was due to an irreversible toxicity of fibrils on the crab’s nervous system, NF was administered 24 h before training and had no effect on LTM. NFs impaired CSM when injected immediately after training, but they had no effects when administered 6 h after training. Thus, only the peri-training administration of NFs affected LTM, suggesting that the peptides act during memory consolidation by interfering with normal signaling and that their action is not due to unspecific and irreversible toxic effects. To further characterize the active components included in NFs, we evaluated the action of several synthetic peptides that are normally present in human brain, A? 1–40, A? 1–42, A? 3–42, and A? 11–42. These peptides were used after 3 days of incubation at 37°C, a protocol that allows for in vitro fibrillation. A? 1–42 and A? 3–42 induced remarkable amnestic effect at very low doses; A? 11–42 did not induce amnesia; and A? 1–40 did not affect retention, even at a higher dose than A? 1–42, indicating that the difference in two amino acids determines their effect on memory consolidation. The fibrillation state of the peptides seems to be a requirement for their action on memory, considering that non-fibrillated A? 1–42 did not show an amnestic effect (Feld et al., unpubl. obs.). These results are at variance with the postulation that the early aggregation stages, particularly oligomers, are the active element altering memory processes (Walsh et al., 2002).
chAPPL, the crab ortholog of amyloid precursor protein family
Members of the amyloid precursor protein (APP) family are transmembrane glycoproteins. In humans, one of these members (A?) is the source of the amyloid peptides found in the neuritic plaques of Alzheimer patients and, as discussed in the last section, has been proposed as the cause of memory deficit and neurodegeneration in this pathology. In the research focused on the neurotoxic properties of A?, several models were used to study the effect of the overexpression and accumulation of such peptides in the nervous system. However, the APP family is composed of transmembrane proteins involved in synaptogenesis and synaptic plasticity, both in vertebrates and invertebrates, acting either in full-length configuration or in their proteolytic fragments (Turner et al., 2003). In spite of the importance of these proteins in synaptic physiology and pathology, little information is available on their function. Two homolog members of this protein family have been cloned in invertebrates, in the nematode Caenorhabditis elegans and in the fruit fly Drosophila melanogaster. We recently cloned most of the mRNA sequence of the Chasmagnathus APP-like protein (chAPPL) gene, the third ortholog member of this family in invertebrates. As expected, this gene shows high similarity with Drosophila APPL, and both genes show low similarity with the A? peptide sequence of vertebrates. Preliminary results support the view that the expression of this gene is upregulated during memory consolidation (Ariel et al., unpubl. obs). The study of genes in the APP family in well-characterized memory models in invertebrates will help elucidate their role in memory storage, and could contribute to the understanding of their role in pathology.
Concluding remarks
The development of learning and memory models in different taxa and the use of nonconventional animals in these studies are important contributions to the search for molecular mechanisms involved in memory storage. On the one hand, each preparation presents particular advantages for study of some aspect of the subject. On the other hand, the availability of models in diverse animal groups allows for a comparative analysis of memory processes. Such analysis will make it possible to define basic mechanisms of memory formation and, in turn, to determine specific characteristics of particular systems and neuronal pathways.
The CSM in the crab Chasmagnathus is a memory model well characterized at the behavioral level and in terms of the phases of memory processing. This characterization constitutes an advantage for the study of the molecular mechanisms of memory because appropriate controls are available to evaluate the effect of factors such as stress, sensorial stimulation, and motor activity and to distinguish them from the specific mechanisms of memory storage.
As an example of comparative studies, the finding of NF-B participation in crab memory led us to question whether the activation of this mechanism is characteristic of this model or is common to different groups of animals. To answer this question, we studied this transcription factor in mice, first in a model of neural plasticity—in vivo long-term potentiation (Freudenthal et al., 2004)—and then in inhibitory avoidance learning. We found that NF-B indeed participates in neural plasticity and memory consolidation (Freudenthal et al., 2005) and reconsolidation (Freudenthal et al., unpubl. obs.) in mice. Together with recent results obtained by other groups in rodents (Yeh et al., 2002; Meffert et al., 2003), these findings are evidence that this transcriptional mechanism is conserved in evolution and important in memory processing.
Acknowledgments
Authors are indebted to Hector Maldonado for his support and guidance during all these years, to Angel Vidal for technical assistance, and to Liliana Orelli for language correction.
Footnotes
Received 7 November 2005; accepted 7 March 2006.
Literature Cited
Alonso, M., H. Viola, I. Izquierdo, and J. H. Medina. 2002. Aversive experiences are associated with a rapid and transient activation of ERKs in the rat hippocampus. Neurobiol. Learn. Mem. 77(1):119R–124.
Atkins, C. M., J. C. Selcher, J. J. Petraitis, J. M. Trzaskos, and J. D. Sweatt. 1998. The MAPK cascade is required for mammalian associative learning. Nature Neurosci. 1(7):602–609.
Beron de Astrada, M., and H. Maldonado. 1999. Two related forms of long-term habituation in the crab Chasmagnathus are differentially affected by scopolamine. Pharmacol. Biochem. Behav. 63(1):109–118.
Cadd, G.G., M.D. Uhler, and G. McKnight. 1990. Holoenzymes of cAMP-dependent protein kinase containing the neural form of type I regulatory subunit have an increased sensitivity to cyclic nucleotides. J. Biol. Chem. 265(32):19502–19506.
Castellucci, V. F., A. Nairn, P. Greengard, J.H. Schwartz, and E.R. Kandel. 1982. Inhibitor of adenosine 3':5'-monophosphate-dependent protein kinase blocks presynaptic facilitation in Aplysia. J. Neurosci. 12:1673–1681.
Crow, T. 2004. Pavlovian Conditioning of Hermissenda: current cellular, molecular, and circuit perspectives. Learn. Mem. 11(3):229–238.
Crow, T., J-J. Xue-Bian, V. Siddiqi, Y. Kang,, and J.T. Neary. 1998. Phosphorylation of mitogen-activated protein kinase by one-trial, and multi-trial classical conditioning. J. Neurosci. 18(9):3480–3487.
Debiec, J., J.E. LeDoux, and K. Nader. 2002. Cellular and systems reconsolidation in the hippocampus. Neuron36:527–538.
Delorenzi, A., E.M. Pedreira, A. Romano, S. García, C. Pirola, V. Nahmod, and H. Maldonado. 1996. Angiotensin II enhances long-term memory in the crab Chasmagnathus. Brain Res. Bull. 47(4):211–219.
Dubnau, J., A. S. Chiang, and T. Tully. 2003. Neural substrates of memory: from synapse to system. J. Neurobiol. 54(1):238–253.
English, J. D., and J. D. Sweatt. 1996. Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. J. Biol. Chem. 271 (40):24329–24332.
Feld, M., B. Dimant, A. Delorenzi, O. Coso, and A. Romano. 2005. Extra-nuclear activation of ERK/MAPK is required for long-term memory consolidation in the crab Chasmagnathus. Behav. Brain Res. 158(2):251–261.
Frenkel, L., R. Freudenthal, A. Romano, V. E. Nahmod, H. Maldonado, and A. Delorenzi. 2002. Angiotensin II and the transcription factor Rel/NF-B link environmental water shortage with memory improvement. Neuroscience 115(4):1079–1087.
Freudenthal, R., and A. Romano. 2000. Participation of NF-B transcription factors in long-term memory in the crab Chasmagnathus. Brain Res. 855:274–281.
Freudenthal, R., F. Locatelli, G. Hermitte, H. Maldonado, A. Delorenzi, C. Lafourcade, and A. Romano. 1998. -B like DNA-binding activity is enhanced after a spaced training that induces long-term memory in the crab Chasmagnathus. Neurosci. Lett. 242:143–146.
Freudenthal, R., A. Romano, and A. Routtenberg. 2004. Activation of the transcription factor NF-B after in vivo perforant path LTP in the mouse hippocampus. Hippocampus14:677–683.
Freudenthal, R., M. M. Boccia, G. B. Acosta, M. G. Blake, E. Merlo, C. M. Baratti, and A. Romano. 2005. NF-B transcription factor is required for inhibitory avoidance long-term memory in mice. Eur. J. Neurosci.21:2845–2852.
Frey, U., Y. Y. Huang, and E. R. Kandel. 1993. Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons. Science260:1661–1664.
Hermitte, G., M. E. Pedreira, D. Tomsic, and H. Maldonado. 1999. Context shift and protein synthesis inhibition disrupt long-term habituation after spaced, but not massed, training in the crab Chasmagnathus. Neurobiol. Learn. Mem. 71:34–49.
Kamenetz, F., T. Tomita, H. Hsieh, G. Seabrook, D. Borchelt, T. Iwatsubo, S. Sisodia, and R. Malinow. 2003. APP processing and synaptic function. Neuron37:925–937.
Kandel, E. R. 2001. The molecular biology of memory storage: a dialog between genes and synapses. Science294:1030–1038.
Lee, J. L., B. J. Everitt, and K. L. Thomas. 2004. Independent cellular processes for hippocampal memory consolidation and reconsolidation. Science304:839–483.
Locatelli, F., and A. Romano. 2005. Differential role of cAMP-dependent protein kinase isoforms during long-term memory consolidation in the crab Chasmagnathus. Neurobiol. Learn. Mem. 83:232–242.
Locatelli, F., C. Lafourcade, H. Maldonado, and A. Romano. 2001. Characterisation of cAMP-dependent protein kinase isoforms in the brain of the crab Chasmagnathus. J. Comp. Physiol. B 171:33–40.
Locatelli, F., H. Maldonado, and A. Romano. 2002. Two critical periods for cAMP-dependent protein kinase activity during long-term memory consolidation in the crab Chasmagnathus. Neurobiol. Learn. Mem. 77(2):234–249.
Lozada, M., A. Romano, and H. Maldonado. 1990. Long-term habituation to a danger stimulus in the crab Chasmagnathus. Physiol. Behav. 47:35–41.
Martin, K. C., D. Michael, J. C. Rose, M. Barad, A. Casadio, H. Zhu, and E. R. Kandel. 1997. MAP kinase translocates into the nucleus of the presynaptic cell and is required for long-term facilitation in Aplysia. Neuron 18(6):899–912.
Meberg, P. J., W. R. Kinney, E. G. Valcourt, and A. Routtenberg. 1996. Gene expression of the transcription factor NF-B in hippocampus: regulation by synaptic activity. Mol. Brain, Res.38:179–190.
Meffert, M. K., J. M. Chang, B. J. Wiltgen, M. S. Fanselow, and D. Baltimore. 2003. NF-B function in synaptic signaling and behavior. Nature Neurosci.6:1072–1078.
Menzel, R. 2001. Searching for the memory trace in a mini-brain, the honeybee. Learn Mem. 8(2):53–62.
Merlo, E., R. Freudenthal, and A. Romano. 2002. The IB kinase inhibitor sulfasalazine impairs long-term memory in the crab Chasmagnathus. Neuroscience 112(1):161–172.
Merlo, E., R. Freudenthal, H. Maldonado, and A. Romano. 2005. Activation of the transcription factor NF-B by retrieval is required for long-term memory reconsolidation. Learn. Mem. 12(1):23–29.
Misanin, J. R., R. R. Miller, and D. I. Lewis. 1968. Retrograde amnesia produced by electroconvulsive shock following reactivation of a consolidated memory trace. Science16:554–555.
Myers, K.M., and M. Davis. 2002. Behavioral and neural analysis of extinction. Neuron36:567–584.
Nader, K., G. E. Schafe, and J. E LeDoux. 2000. Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval. Nature406:722–726.
Noselli, S. 1998. JNK signaling and morphogenesis in Drosophila. Trends Genet. 14:33–38.
Pedreira, M. E., and H. Maldonado. 2003. Protein synthesis subserves reconsolidation or extinction depending on reminder duration. Neuron 38(6):863–869.
Pedreira, M. E., B. Dimant, D. Tomsic, L. A. Quesada-Allue, and H. Maldonado. 1995. Cycloheximide inhibits long-term habituation and context memory in the crab Chasmagnathus. Pharmacol. Biochem. Behav. 52:385–395.
Pedreira, M. E., B. Dimant, and H. Maldonado. 1996. Inhibitors of protein and RNA synthesis block context memory and long-term habituation in the crab Chasmagnathus. Pharmacol. Biochem. Behav. 54:611–617.
Pedreira, M. E., A. Romano, D. Tomsic, M. Lozada, and H. Maldonado. 1998. Massed and spaced training build up different components of long-term habituation in the crab Chasmagnathus. Anim. Learn. Behav. 26:34–45.
Pedreira, M. E., L. M. Perez-Cuesta, and H. Maldonado. 2002. Reactivation and reconsolidation of long-term memory in the crab Chasmagnathus: protein synthesis requirement and mediation by NMDA-type glutamatergic receptors. J Neurosci. 22(18):8305–8311.
Purcell, A. L., S. K. Sharma, M. W. Bagnall, M. A. Sutton, and T. J. Carew. 2003. Activation of a tyrosine kinase-MAPK cascade enhances the induction of long-term synaptic facilitation and long-term memory in Aplysia. Neuron 37(3):473–484.
Roberts, A. C., and D. L. Glanzman. 2003. Learning in Aplysia: looking at synaptic plasticity from both sides. Trends Neurosci. 26(12):662–670.
Romano, A., A. Delorenzi, M. E. Pedreira, D. Tomsic, and H. Maldonado. 1996a. Acute administration of a cAMP analog and a phosphodiesterase inhibitor improve long-term habituation of the crab Chasmagnathus. Behav. Brain Res. 75:119–125.
Romano, A., F. Locatelli, M. E. Pedreira, A. Delorenzi. and H. Maldonado. 1996b. Effect of activation and inhibition of cAMP-dependent proteinkinase on long-term habituation of the crab Chasmagnathus. Brain Res. 735:131–140.
Romano, A., A. L. Serafino, E. Krasnowska, M. T. Ciotti, P. Calissano, F. Ruberti, and C. Galli. 2003. Neuronal fibrillogenesis: amyloid fibrils from primary neuronal cultures impair long-term memory in the crab Chasmagnatus. Behav. Brain Res. 147:73–82.
Salinska, E., R. C. Bourne, and S. P. R. Rose. 2004. Reminder effects: the molecular cascade following a reminder in young chicks does not recapitulate that following training on a passive avoidance task. Eur. J. Neurosci.19:3042–3047.
Sara, S. J. 2000. Retrieval and reconsolidation: toward a neurobiology of remembering. Learn. Mem7:73–84.
Sharma, S. K., C. M. Sherff, J. Shobe, M. W. Bagnall, M. A. Sutton, and T. J. Carew. 2003. Differential role of mitogen-activated protein kinase in three distinct phases of memory for sensitization in Aplysia. J. Neurosci. 23 (9):3899–3907.
Sk?lhegg, B. S., and K. Tasken. 2000. Specificity in the cAMP/PKA signalling pathway. Differential expression, regulation, and subcellular localization of subunits of PKA. Front. Biosci.,5:D678–693.
Squire, L. R., and H. P. Davis. 1981. The pharmacology of memory: a neurobiological perspective. Annu. Rev. Pharmacol. Toxicol.21:323–336.
Tomsic, D., E. M. Pedreira, A. Romano, G. Hermitte, and H. Maldonado. 1998. Association to context as a determinant of long-term habituation in the crab Chasmagnathus. Anim. Learn. Behav. 26:196–209.
Tomsic, D., M. Beron de Astrada, and J. Sztarker. 2003. Identification of individual neurons reflecting short- and long-term visual memory in an arthropod. J. Neurosci. 23(24):8539–8546.
Troncoso, J., and H. Maldonado. 2002. Two related forms of memory in the crab Chasmagnathus are differentially affected by NMDA receptor antagonists. Pharmacol. Biochem. Behav. 72(1–2):251–265.
Turner, P. R., K. O. O’Connor, W. P. Tate, and W. C. Abraham. 2003. Roles of amyloid precursor protein and its fragments in regulating neural activity, plasticity and memory. Prog. Neurobiol.70:1–32.
Villanueva, A., J. Lozano, A. Morales, X. Lin, X. Deng, M. O. Hengartner, and R. N. Kolesnick. 2001. JKK-1 and MEK-1 regulate body movement coordination and response to heavy metals through JNK-1 in Caenorhabditis elegans. EMBO J. 20(18):5114–5128.
Walsh, D. M., I. Klyubin, J. V. Fadeeva, W. K. Cullen, R. Anwyl, M. S. Wolfe, M. J. Rowan, and D. J Selkoe. 2002. Naturally secreted oligomers of amyloid ? protein potently inhibit hippocampal lasting potentiation in vivo. Nature 416:535–539.
Wellmann, H., B. Kaltschmidt, and C. Kaltschmidt. 2001. Retrograde transport of transcription factor NF-B in living neurons. J. Biol. Chem. 276(15):11821–11829.
Yeh, S. H., C. H. Lin, C. F. Lee, and P. W. Gean. 2002. A requirement of nuclear factor-B activation in fear-potentiated startle. J. Biol. Chem. 277(48):46720–9.(Arturo Romano, Fernando L)