Long-term Facilitation In Aplysia Research Articles

Mathematical modeling and bistability analysis for synaptic tagging and synapse-specific long-term facilitation in Aplysia

Mathematical modeling and bistability analysis for synaptic tagging and synapse-specific long-term facilitation in Aplysia

A decrease in eukaryotic elongation factor 2 phosphorylation is required for local translation of sensorin and long-term facilitation in Aplysia.

Mechanistic target of rapamycin complex 1 (mTORC1)-dependent protein synthesis is required for many forms of synaptic plasticity and memory, but the downstream pathways important for synaptic plasticity are poorly understood. Long-term facilitation (LTF) in Aplysia is a form of synaptic plasticity that is closely linked to behavioral memory and an attractive model system for examining the important downstream targets for mTORC1 in regulating synaptic plasticity. Although mTORC1-regulated protein synthesis has been strongly linked to translation initiation, translation elongation is also regulated by mTORC1 and LTF leads to an mTORC1-dependent decrease in eukaryotic elongation factor 2 (eEF2) phosphorylation. The purpose of this study is to test the hypothesis that the decrease in eEF2 phosphorylation is required for mTORC1-dependent translation and plasticity. We show that the LTF-induced decrease in eEF2 phosphorylation is blocked by expression of an eEF2 kinase (eEF2K) modified to be resistant to mTORC1 regulation. We found that expression of this modified kinase blocked LTF. LTF requires local protein synthesis of the neuropeptide sensorin and importantly, local sensorin synthesis can be measured using a dendra fluorescent protein containing the 5' and 3' untranslated regions (UTRs) of sensorin. Using this construct, we show that blocking eEF2 dephosphorylation also blocks the increase in local sensorin synthesis. These results identify decreases in eEF2 phosphorylation as a critical downstream effector of mTOR required for long-term plasticity and identify an important translational target regulated by decreases in eEF2 phosphorylation.

Enhanced memory persistence is blocked by a DNA methyltransferase inhibitor in the snail Lymnaea stagnalis.

Lymnaea stagnalis provides an excellent model system for studying memory because these snails have a well-described set of neurons, a single one of which controls expression of long-term memory of operantly conditioned respiratory behavior. We have shown that several different manipulations, including pre-training exposure to serotonin (5-HT) or methamphetamine, submersion of snails after training to prevent memory interference, and exposure to effluent from predatory crayfish (CE), enhance memory persistence. Changes in DNA methylation underlie formation of strong memories in mammals and 5-HT-enhanced long-term facilitation in Aplysia. Here we determined the impact of the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-AZA; 87 μmol l(-1)), on enhanced memory persistence by all four manipulations. We found that 5-HT (100 μmol l(-1)) enhanced memory persistence, which was blocked by 5-AZA pretreatment. Snails pre-exposed to 3.3 μmol l(-1) Meth 4 h prior to training demonstrated memory 72 h later, which was not present in controls. This memory-enhancing effect was blocked by pre-treatment with 87 μmol l(-1) 5-AZA. Similarly, submersion to prevent interference learning as well as training in CE produced memory that was not present in controls, and these effects were blocked by pre-treatment with 87 μmol l(-1) 5-AZA. In contrast, 5-AZA injection did not alter expression of normal (non-enhanced) memory, suggesting that these four stimuli enhance memory persistence by increasing DNA methyltransferase activity, which, in turn, increases expression of memory-enhancing genes and/or inhibits memory suppressor genes. These studies lay important groundwork for delineating gene methylation changes that are common to persistent memory produced by different stimuli.

Spontaneous transmitter release is critical for the induction of long-term and intermediate-term facilitation inAplysia

Long-term plasticity can differ from short-term in recruiting the growth of new synaptic connections, a process that requires the participation of both the presynaptic and postsynaptic components of the synapse. How does information about synaptic plasticity spread from its site of origin to recruit the other component? The answer to this question is not known in most systems. We have investigated the possible role of spontaneous transmitter release as such a transsynaptic signal. Until recently, relatively little has been known about the functions of spontaneous release. In this paper, we report that spontaneous release is critical for the induction of a learning-related form of synaptic plasticity, long-term facilitation in Aplysia. In addition, we have found that this signaling is engaged quite early, during an intermediate-term stage that is the first stage to involve postsynaptic as well as presynaptic molecular mechanisms. In a companion paper, we show that spontaneous release from the presynaptic neuron acts as an orthograde signal to recruit the postsynaptic mechanisms of intermediate-term facilitation and initiates a cascade that can culminate in synaptic growth with additional stimulation during long-term facilitation. Spontaneous release could make a similar contribution to learning-related synaptic plasticity in mammals.

Ribosomal Protein S6 Kinase Is a Critical Downstream Effector of the Target of Rapamycin Complex 1 for Long-term Facilitation in Aplysia

Long-term facilitation (LTF) in Aplysia is a leading cellular model for elucidating the biochemical mechanisms of synaptic plasticity underlying learning. In Aplysia, LTF requires translational control downstream of the target of rapamycin (TOR) complex 1 (TORC1). The major known downstream targets of TORC1 are 4E binding protein (4E-BP) and S6 kinase (S6K). By removing the site within these regulators required for their interaction with TORC1, we have generated dominant negative proteins that disrupt specific pathways downstream of TORC1. Expression of dominant negative S6K, but not dominant negative 4E-BP, in Aplysia sensory neurons (SNs) blocked 24-h LTF. TORC1 is directly activated by the small GTP-binding protein, Ras homologue enriched in brain (Rheb). To determine the effects of TORC1 activation on translation in Aplysia neurons, we have examined the effects of expressing a constitutively active form of the Aplysia orthologue of Rheb, ApRheb (ApRheb(Q63L)). Expression of ApRheb(Q63L) increased 4E-BP phosphorylation and the level of general, cap-dependent translation within the SN cell soma in a rapamycin-sensitive manner. This increase in cap-dependent translation was blocked neither by dominant negative 4E-BP nor dominant negative S6K. Thus, we demonstrate that S6K is an important downstream target of TORC1 in Aplysia and that it is necessary for 24-h LTF, but not for TORC1-mediated increases in somatic cap-dependent translation.

Sustained CPEB-Dependent Local Protein Synthesis Is Required to Stabilize Synaptic Growth for Persistence of Long-Term Facilitation in Aplysia

The time course of the requirement for local protein synthesis in the stabilization of learning-related synaptic growth and the persistence of long-term memory was examined using Aplysia bifurcated sensory neuron-motor neuron cultures. We find that, following repeated pulses of serotonin (5-HT), the local perfusion of emetine, an inhibitor of protein synthesis, or a TAT-AS oligonucleotide directed against ApCPEB blocks long-term facilitation (LTF) at either 24 or 48 hr and leads to a selective retraction of newly formed sensory neuron varicosities induced by 5-HT. By contrast, later inhibition of local protein synthesis, at 72 hr after 5-HT, has no effect on either synaptic growth or LTF. These results define a specific stabilization phase for the storage of long-term memory during which newly formed varicosities are labile and require sustained CPEB-dependent local protein synthesis to acquire the more stable properties of mature varicosities required for the persistence of LTF.

Postsynaptic Regulation of Long-Term Facilitation in Aplysia

Repeated exposure to serotonin (5-HT), an endogenous neurotransmitter that mediates behavioral sensitization in Aplysia[1-3], induces long-term facilitation (LTF) of the Aplysia sensorimotor synapse [4]. LTF, a prominent form of invertebrate synaptic plasticity, is believed to play a major role in long-term learning in Aplysia[5]. Until now, LTF has been thought to be due predominantly to cellular processes activated by 5-HT within the presynaptic sensory neuron [6]. Recent work indicates that LTF depends on the increased expression and release of a sensory neuron-specific neuropeptide, sensorin [7]. Sensorin released during LTF appears to bind to autoreceptors on the sensory neuron, thereby activating critical presynaptic signals, including mitogen-activated protein kinase (MAPK) [8, 9]. Here, we show that LTF depends on elevated postsynaptic Ca2+ and postsynaptic protein synthesis. Furthermore, we find that the increased expression of presynaptic sensorin resulting from 5-HT stimulation requires elevation of postsynaptic intracellular Ca2+. Our results represent perhaps the strongest evidence to date that the increased expression of a specific presynaptic neuropeptide during LTF is regulated by retrograde signals.

Ancient rules of memory

The first kiss, the smell of mother's cooking or the vision of a beautiful landscape; memories such as these are etched firmly into our brains for recall in vivid detail, even decades later. Memories and experience influence our decisions throughout life, both consciously and subconsciously—in a sense, our memories make us what we are. Yet, understanding how information is encoded in our brains and how memories are maintained, both at the conceptual and molecular levels, remains one of the greatest challenges in the life sciences. Through decades of research, biologists have answered many fundamental questions about the brain, and more recent work has revealed the identity of several crucial molecules involved in the storage and retrieval of information. This has at last begun to shed light on the major scientific puzzle: how do we form and maintain long‐term memories? ![][1] The field of neurobiology had made relatively little progress toward understanding memory since the Canadian psychologist Donald Hebb (1904–1985) introduced the idea of synaptic plasticity nearly 60 years ago (Hebb, 1949). Hebb explained the establishment of memory by changes in the number and strength of synaptic connections between neurons. According to his theory, synapses respond to stimulation by neurotransmitters in a cumulative manner, and repeated stimulation—for example, by learning—leads to ever‐stronger connections between specific neurons until they become permanent. Over time, neuroscientists accepted Hebb's idea of synaptic plasticity, despite a lack of direct molecular evidence. In fact, it was not until recently that scientists—in large part Eric Kandel at Columbia University in New York, NY, USA—discovered several biochemical mechanisms that now support Hebb's idea. Kandel himself won the Nobel Prize for medicine in 2000 for his discovery that the syntheses of different proteins relate to short‐term and long‐term memory in the sea slug Aplysia . The foundations for this work and … [1]: /embed/graphic-1.gif

Rookie Snail Protein LAPS Veteran C/EBP: Net Transcriptional Proceeds for Long-Term Facilitation

Long-term changes in synaptic plasticity require new protein synthesis. This preview discusses data from Kim et al. (this issues of Neuron) that demonstrate the requirement of a novel, nucleolar protein, LLP (LAPS18-like protein), for the formation of long-term facilitation in Aplysia. LLP binds to and transcriptionally activates C/EBP, thereby promoting the formation of long-term facilitation and behavioral sensitization.

CREB, Synapses and Memory Disorders: Past Progress and Future Challenges

In neurons, appropriate long-term adaptive responses to changes in the environment require the conversion of extracellular stimuli into discrete intracellular signals. Many of these signals involve the regulation of gene expression. The cAMP responsive element binding protein (CREB) is a nuclear transcription factor that modulates transcription of genes containing cAMP responsive elements (CRE sites) in their promoters. CREB is a key part of many intracellular signaling events that critically regulate many neural functions. Numerous studies on invertebrates and vertebrates demonstrate that CREB is critical for long-term memory. Here, we review the key features of CREB-dependent transcription and critically evaluate the data examining the roles of CREB in different forms of plasticity, including long-term memory in mammals. Because learning and memory have been linked to specific types of synaptic plasticity in several species, we also review studies on the role of CREB in long-term facilitation in Aplysia and in hippocampal long-term potentiation (LTP). Several human cognitive disorders have been linked to alterations of CREB-regulated gene expression. Therefore, we explore the possibility of targeting CREB function in developing novel treatment strategies. Finally, we highlight areas of research on CREB that are ripe for further advancement.

ApTrkl, a Trk-like Receptor, Mediates Serotonin- Dependent ERK Activation and Long-Term Facilitation in Aplysia Sensory Neurons

The Trk family of receptor tyrosine kinases plays a role in synaptic plasticity and in behavioral memory in mammals. Here, we report the discovery of a Trk-like receptor, ApTrkl, in Aplysia. We show that it is expressed in the sensory neurons, the locus for synaptic facilitation, which is a cellular model for memory formation. Serotonin, the facilitatory neurotransmitter, activates ApTrkl, which, in turn, leads to activation of ERK. Finally, inhibiting the activation of ApTrkl with the Trk inhibitor K252a or using dsRNA to inhibit ApTrkl blocks the serotonin-mediated activation of ERK in the cell body, as well as the cell-wide long-term facilitation induced by 5-HT application to the cell body. Thus, transactivation of the receptor tyrosine kinase ApTrkl by serotonin is an essential step in the biochemical events leading to long-term facilitation in Aplysia.

Is a Prion the Memory Switch?

Prions are proteins that promote the conversion of proteins of the same type to an alternate conformational state; typically, there is a soluble conformation and an insoluble, aggregating conformation. Furthermore, the state of the protein is heritable in an epigenetic manner. In humans, a prion protein is responsible for transmissible spongiform encephalopathies; however, in yeast, prions are not pathogenic and represent a mechanism of epigenetic regulation of protein activity. Si et al. noted that the model organism Aplysia , which is used for studying neurobiological questions such as the mechanisms underlying synaptic plasticity, expressed a neuronal form of the translational regulatory protein cytoplasmic polyadenylation element binding protein (CPEB) that had an N-terminal domain enriched in glutamine and asparagine residues, like that of other prion proteins. When tested in several yeast assays for prion activity (the ability to promote heritable conformational changes in the heterologously expressed protein or proteins to which the N-terminal domain was fused), the Aplysia CPEB behaved as though it were a prion. Surprisingly, the aggregated state of CPEB appeared to be the active form of the protein, at least in the yeast assays, which is in contrast to yeast prion proteins that are inactivated when they aggregate. Analysis of the role of Aplysia CPEB in response to prolonged exposure to or a short pulse of serotonin indicated that CPEB protein increases in a posttranscriptional manner. Furthermore, serotonin-stimulated synthesis of CPEB occurred in isolated neurites that had been detached from the cell bodies. Treatment of cells or processes with antisense oligonucleotides to disrupt CPEB expression resulted in no loss of the initial facilitation response, but impairment of the maintenance of the enhanced synaptic efficacy. Pharmacologic analysis indicated that CPEB stimulation required phosphoinositide 3-kinase but did not require protein kinase A or protein kinase C. What remains to be determined is whether CPEB behaves as a prion in Aplysia and whether changes in the protein's conformation occur in response to synaptic activity associated with long-term potentiation (see Darnell for discussion). K. Si, S. Lindquist, E. R. Kandel, A neuronal isoform of the Aplysia CPEB has prion-like properties. Cell 115 , 879-891 (2003). [Online Journal] K. Si, M. Giustetto, A. Etkin, R. Hsu, A. M. Janisiewicz, M. C. Miniaci, J.-H. Kim, H. Zhu, E. R. Kandel, A neuronal isoform of CPEB regulates local protein synthesis and stabilizes synapse-specific long-term facilitation in Aplysia . Cell 115 , 893-904 (2003). [Online Journal] R. B. Darnell, Memory, synaptic transmission, and ... prions? Cell 115 , 767-768 (2003). [Online Journal]

A Neuronal Isoform of CPEB Regulates Local Protein Synthesis and Stabilizes Synapse-Specific Long-Term Facilitation in Aplysia

Synapse-specific facilitation requires rapamycin-dependent local protein synthesis at the activated synapse. In Aplysia, rapamycin-dependent local protein synthesis serves two functions: (1) it provides a component of the mark at the activated synapse and thereby confers synapse specificity and (2) it stabilizes the synaptic growth associated with long-term facilitation. Here we report that a neuron-specific isoform of cytoplasmic polyadenylation element binding protein (CPEB) regulates this synaptic protein synthesis in an activity-dependent manner. Aplysia CPEB protein is upregulated locally at activated synapses, and it is needed not for the initiation but for the stable maintenance of long-term facilitation. We suggest that Aplysia CPEB is one of the stabilizing components of the synaptic mark.

Time-restricted role for dendritic activation of the mTOR-p70S6K pathway in the induction of late-phase long-term potentiation in the CA1.

Mammalian target of rapamycin (mTOR) is a key regulator of translational capacity. The mTOR inhibitor rapamycin can prevent forms of protein synthesis-dependent synaptic plasticity such as long-term facilitation in Aplysia and late-phase long-term potentiation (L-LTP) in the hippocampal CA1 region of rodents. In the latter model, two issues remain to be addressed: defining the L-LTP phase sensitive to rapamycin and identifying the site of rapamycin-sensitive protein synthesis. Here, we show that L-LTP is sensitive to application of rapamycin only during the induction paradigm, whereas rapamycin application after the establishment of L-LTP was ineffective. Second, we observed that Thr-389-phosphorylated p70 S6 kinase (p70S6K), the main active phosphoform of the mTOR effector p70S6K, was induced in an N-methyl-D-aspartate and phosphatidylinositol 3-kinase-dependent manner throughout the dendrites but not in the cell bodies of CA1 neurons in hippocampal slices after L-LTP induction. A similar dendrite-wide activation of p70S6K was induced in primary hippocampal neurons by depolarization with KCL or glutamate. In primary hippocampal neurons, the sites of dendritic activation of p70S6K appeared as discrete compartments along dendritic shafts like the hotspots for fast dendritic translation. Conversely, only a subset of dendritic spines also displayed activated p70S6K. Taken together, the present data suggest that the N-methyl-d-aspartate-, phosphatidylinositol 3-kinase-dependent dendritic activation of the mTOR-p70S6K pathway is necessary for the induction phase of protein synthesis-dependent synaptic plasticity. Newly synthesized proteins in dendritic shafts could be targeted selectively to activity-tagged synapses. Thus, coordinated activation of dendrite-wide translation and synaptic-specific activation is likely to be necessary for long-term synaptic plasticity.

Presynaptic Activation of Silent Synapses and Growth of New Synapses Contribute to Intermediate and Long-Term Facilitation in Aplysia

The time course and functional significance of the structural changes associated with long-term facilitation of Aplysia sensory to motor neuron synaptic connections in culture were examined by time-lapse confocal imaging of individual sensory neuron varicosities labeled with three different fluorescent markers: the whole-cell marker Alexa-594 and two presynaptic marker proteins— synaptophysin-eGFP to monitor changes in synaptic vesicle distribution and synapto-PHluorin to monitor active transmitter release sites. Repeated pulses of serotonin induce two temporally, morphologically, and molecularly distinct presynaptic changes: (1) a rapid activation of silent presynaptic terminals by filling of preexisting empty varicosities with synaptic vesicles, which parallels intermediate-term facilitation, is completed within 3–6 hr and requires translation but not transcription and (2) a slower generation of new functional varicosities which occurs between 12–18 hr and requires transcription and translation. Enrichment of empty varicosities with synaptophysin accounts for 32% of the newly activated synapses at 24 hr, whereas newly formed varicosities account for 68%.

Overexpression and RNA interference of Ap-cyclic AMP-response element binding protein-2, a repressor of long-term facilitation, in Aplysia kurodai sensory-to-motor synapses

cyclic AMP-response element binding protein-2 (CREB2) is a member of the CREB/transcription factor (CREB/ATF4) family. CREB2 is a transcription factor known to be involved in Aplysia long-term facilitation. To further examine the role of ApCREB2 on long-term synaptic facilitation, we isolated ApCREB2 from Aplysia kurodai in full-length cDNA library, and found that the overexpression of ApCREB2 blocked 5-hydroxytryptamine (5-HT)-induced long-term synaptic facilitation in Aplysia sensory-to-motor synapses. Furthermore, a single pulse of 5-HT, which normally induces only short-term facilitation, in the presence of ApCREB2 inhibition by RNA interference, induced long-term facilitation in Aplysia sensory-to-motor synapses. These results suggest that ApCREB2 is a functional repressor of long-term facilitation in Aplysia sensory-to-motor synapses.

A Role of the Ubiquitin–Proteasome System in Neuropathic Pain

Neuropathic pain (characterized by hyperalgesia and allodynia to mechanical and thermal stimuli) causes cellular changes in spinal dorsal horn neurons, some of which parallel those in synaptic plasticity associated with learning. Ubiquitin C-terminal hydrolase (UCH) appears to play a key role in long-term facilitation in Aplysia. The cooperation of UCH with the proteolytic enzyme complex known as the proteasome is required for the degradation of a number of signaling molecules within the cell that may remove normal restraints on synaptic plasticity. We have used electrophysiology, in situ hybridization histochemistry, semiquantitative RT-PCR, Western blotting, and in vivo behavioral reflex analysis to investigate the ubiquitin-proteasome system in a model of neuropathic pain. In neuropathic animals, ionophoretic application of selective proteasome inhibitors attenuated dorsal horn neuron firing evoked by normally innocuous brush or cold stimuli and by noxious mustard oil stimuli. In control animals, only mustard oil-evoked responses were inhibited. Intrathecal administration of proteasome inhibitors attenuated hyperalgesia and allodynia in neuropathic rats. Expression of UCH-L1 (a rat homolog of Aplysia neuronal UCH and of the human UCH-L1, also known as PGP 9.5) and its mRNA were selectively increased within the ipsilateral dorsal horn of neuropathic rats, supporting the idea of a role for the ubiquitin-proteasome system in nociceptive processing. Proteasome inhibitors selectively attenuate allodynic and hyperalgesic responses in neuropathic pain, with some reduction in normal nociceptive, but not non-nociceptive responses, and potentially represent a novel therapeutic strategy for neuropathic pain.

Regulation of Neuronal Cell Adhesion Molecule Expression by NF-κB

The neuronal cell adhesion molecule (NCAM) is a key mediator of structural plasticity in the central nervous system, but the mechanisms that control its expression are unknown. Equally, although the transcription factor NF-kappaB is present in the brain, few NF-kappaB-regulated genes relevant for central nervous system function have been identified. We have previously demonstrated that NF-kappaB is activated in neuronal cultures treated with kainic acid or nitric oxide. We show here that kainic acid or nitric oxide also increase the levels of NCAM mRNA and protein in neurons and that this induction of NCAM expression is sensitive to dexamethasone and to antisense, but not missense, oligonucleotides designed to suppress NF-kappaB synthesis. Nitric oxide also stimulates protein binding to an NF-kappaB site in the promoter of the NCAM gene. This indicates that NF-kappaB, which has recently been implicated in synaptic plasticity and also in the etiology of neurodegenerative disease, plays a crucial role in the activity-dependent regulation of NCAM gene expression. In addition, since both NCAM and NF-kappaB are present in the post-synaptic density, this represents a route allowing direct communication between the synapse and the nucleus.

Activation and degradation of the transcription factor C/EBP during long-term facilitation in Aplysia.

Long-term facilitation (LTF) of the sensory-to-motor synapses that mediate defensive reflexes in Aplysia requires induction of the transcription factor Aplysia CCAAT/enhancer binding protein (ApC/EBP) as an early response gene. We examined the time course of ApC/ EBP DNA binding during the induction of LTF: Binding activity was detected within 1 h of the sensitization treatment with serotonin, reached a maximum at 2 h, and decreased after 6 h. How are DNA binding and the turnover of ApC/EBP regulated? We find that phosphorylation of ApC/EBP by mitogen-activated protein (MAP) kinase is essential for binding. MAP kinase appears to be activated through protein kinase C. We also showed that ApC/EBP is degraded through the ubiquitin-proteasome pathway but that phosphorylation by MAP kinase renders it resistant to proteolysis. Thus, phosphorylation by MAP kinase is required for ApC/EBP to act as a transcription activator as well as to assure its stability early in the consolidation phase, when genes essential for the development of LTF begin to be expressed.

Mechanisms for Generating the Autonomous cAMP-Dependent Protein Kinase Required for Long-Term Facilitation in Aplysia

The formation of a persistently active cAMP-dependent protein kinase (PKA) is critical for establishing long-term synaptic facilitation (LTF) in Aplysia. The injection of bovine catalytic (C) subunits into sensory neurons is sufficient to produce protein synthesis–dependent LTF. Early in the LTF induced by serotonin (5-HT), an autonomous PKA is generated through the ubiquitin–proteasome-mediated proteolysis of regulatory (R) subunits. The degradation of R occurs during an early time window and appears to be a key function of proteasomes in LTF. Lactacystin, a specific proteasome inhibitor, blocks the facilitation induced by 5-HT, and this block is rescued by injecting C subunits. R is degraded through an allosteric mechanism requiring an elevation of cAMP coincident with the induction of a ubiquitin carboxy-terminal hydrolase.