Pattern detection in the TGFß cascade controls the induction of long-term synaptic plasticity.

Transforming growth factor ß (TGFß) is required for long-term memory (LTM) for sensitization in Aplysia. When LTM is induced using a two-trial training protocol, TGFß inhibition only blocks LTM when administrated at the second, not the first trial. Here, we show that TGFß acts as a "repetition detector" during the induction of two-trial LTM. Secretion of the biologically inert TGFß proligand must coincide with its proteolytic activation by the Bone morphogenetic protein-1 (BMP-1/Tolloid) metalloprotease, which occurs specifically during trial two of our two-trial training paradigm. This paradigm establishes long-term synaptic facilitation (LTF), the cellular correlate of LTM. BMP-1 application paired with a single serotonin (5HT) pulse induced LTF, whereas neither a single 5HT pulse nor BMP-1 alone effectively did so. On the other hand, inhibition of endogenous BMP-1 activity blocked the induction of two-trial LTF. These results suggest a unique role for TGFß in the interaction of repeated trials: during learning, repeated stimuli engage separate steps of the TGFß cascade that together are necessary for the induction of long-lasting memories.

Precise timing of ERK phosphorylation/dephosphorylation determines the outcome of trial repetition during long-term memory formation.

Two-trial learning in Aplysia reveals nonlinear interactions between training trials: A single trial has no effect, but two precisely spaced trials induce long-term memory. Extracellularly regulated kinase (ERK) activity is essential for intertrial interactions, but the mechanism remains unresolved. A combination of immunochemical and optogenetic tools reveals unexpected complexity of ERK signaling during the induction of long-term synaptic facilitation by two spaced pulses of serotonin (5-hydroxytryptamine, 5HT). Specifically, dual ERK phosphorylation at its activating TxY motif is accompanied by dephosphorylation at the pT position, leading to a buildup of inactive, singly phosphorylated pY-ERK. Phosphorylation and dephosphorylation occur concurrently but scale differently with varying 5HT concentrations, predicting that mixed two-trial protocols involving both "strong" and "weak" 5HT pulses should be sensitive to the precise order and timing of trials. Indeed, long-term synaptic facilitation is induced only when weak pulses precede strong, not vice versa. This may represent a physiological mechanism to prioritize memory of escalating threats.

The Journal of Neuroscience's 40th Anniversary: Looking Back, Looking Forward.

Some of us fortunate enough to have published a paper in The Journal of Neuroscience in its inaugural year (1981) have been asked to write a Progressions article addressing our views on the significance of the original work and how ideas about the topic of that work have evolved over the last 40 years. These questions cannot be effectively considered without placing them in the context of the incredible growth of the overall field of neuroscience over these last four decades. For openers, in 1981, the Nobel Prize was awarded to three neuroscience superstars: Roger Sperry, David Hubel, and Torsten Wiesel. Not a bad year to launch the Journal With this as a backdrop, I divide this Progressions article into two parts. First, I discuss our original (1981) paper describing classical conditioning in Aplysia californica, and place our results in the context of the state of the field at the time. Second, I fast forward to the present and consider some of remarkable progress in the broad field of learning and memory that has occurred in the last 40 years. Along the way, I also reflect briefly on some of the amazing advances, both technical and conceptual, that we in neuroscience have witnessed.

ELAV Proteins Bind and Stabilize C/EBP mRNA in the Induction of Long-Term Memory in Aplysia.

Long-term memory (LTM) formation is a critical survival process by which an animal retains information about prior experiences to guide future behavior. In the experimentally advantageous marine mollusk Aplysia, LTM for sensitization can be induced by the presentation of two aversive shocks to the animal's tail. Each of these training trials recruits distinct growth factor signaling systems that promote LTM formation. Specifically, whereas intact TrkB signaling during Trial 1 promotes an initial and transient increase of the immediate early gene apc/ebp mRNA, a prolonged increase in apc/ebp gene expression required for LTM formation requires the addition of TGFß signaling during Trial 2. Here we explored the molecular mechanisms by which Trial 2 achieves the essential prolonged gene expression of apc/ebp We find that this prolonged gene expression is not dependent on de novo transcription, but that apc/ebp mRNA synthesized by Trial 1 is post-transcriptionally stabilized by interacting with the RNA-binding protein ApELAV. This interaction is promoted by p38 MAPK activation initiated by TGFß. We further demonstrate that blocking the interaction of ApELAV with its target mRNA during Trial 2 blocks both the prolonged increase in apc/ebp gene expression and the behavioral induction of LTM. Collectively, our findings elucidate both when and how ELAV proteins are recruited for the stabilization of mRNA in LTM formation. Stabilization of a transiently expressed immediate early gene mRNA by a repeated training trial may therefore serve as a "filter" for learning, permitting only specific events to cause lasting transcriptional changes and behavioral LTM.SIGNIFICANCE STATEMENT: In the present paper, we significantly extend the general field of molecular processing in long-term memory (LTM) by describing a novel form of pretranslational processing required for LTM, which relies on the stabilization of a newly synthesized mRNA by a class of RNA binding proteins (ELAVs). There are now compelling data showing that important processing can occur after transcription of a gene, but before translation of the message into protein. Although the potential importance of ELAV proteins in LTM formation has previously been reported, the specific actions of ELAV proteins during LTM formation remained to be understood. Our new findings thus complement and extend this literature by demonstrating when and how this post-transcriptional gene regulation is mediated in the induction of LTM.

The Neurohumanities: An Emerging Partnership for Exploring the Human Experience.

Neuroscience has an extraordinary opportunity to investigate issues historically addressed by the arts, humanities, and social sciences. As a guide, we suggest three features of meaningful progress in the collaborative field, the neurohumanities, which we illustrate through a discussion of "neural schemas."

Postsynaptic effects of Aplysia cysteine-rich neurotrophic factor in the induction of activity-dependent long-term facilitation in Aplysia californica.

The spatial and temporal coordination of growth factor signaling is critical for both presynaptic and postsynaptic plasticity underlying long-term memory formation. We investigated the spatiotemporal dynamics of Aplysia cysteine-rich neurotrophic factor (ApCRNF) signaling during the induction of activity-dependent long-term facilitation (AD-LTF) at sensory-to-motor neuron synapses that mediate defensive reflexes in Aplysia We found that ApCRNF signaling is required for the induction of AD-LTF, and for training-induced early protein kinase activation and late forms of gene expression, exclusively in postsynaptic neurons. These results support the view that ApCRNF is critically involved in AD-LTF at least in part through postsynaptic mechanisms.

The ELAV family of RNA-binding proteins in synaptic plasticity and long-term memory.

The mechanisms of de novo gene expression and translation of specific gene transcripts have long been known to support long-lasting changes in synaptic plasticity and behavioral long-term memory. In recent years, it has become increasingly apparent that gene expression is heavily regulated not only on the level of transcription, but also through post-transcriptional gene regulation, which governs the subcellular localization, stability, and likelihood of translation of mRNAs. Specific families of RNA-binding proteins (RBPs) bind transcripts which contain AU-rich elements (AREs) within their 3' UTR and thereby govern their downstream fate. These post-transcriptional gene regulatory mechanisms are coordinated through the same cell signaling pathways that play critical roles in long-term memory formation. In this review, we discuss recent results that demonstrate the roles that these ARE-binding proteins play in LTM formation.

The Contribution of Spatial and Temporal Molecular Networks in the Induction of Long-term Memory and Its Underlying Synaptic Plasticity.

The ability to form long-lasting memories is critical to survival and thus is highly conserved across the animal kingdom. By virtue of its complexity, this same ability is vulnerable to disruption by a wide variety of neuronal traumas and pathologies. To identify effective therapies with which to treat memory disorders, it is critical to have a clear understanding of the cellular and molecular mechanisms which subserve normal learning and memory. A significant challenge to achieving this level of understanding is posed by the wide range of distinct temporal and spatial profiles of molecular signaling induced by learning-related stimuli. In this review we propose that a useful framework within which to address this challenge is to view the molecular foundation of long-lasting plasticity as composed of unique spatial and temporal molecular networks that mediate signaling both within neurons (such as via kinase signaling) as well as between neurons (such as via growth factor signaling). We propose that evaluating how cells integrate and interpret these concurrent and interacting molecular networks has the potential to significantly advance our understanding of the mechanisms underlying learning and memory formation.

Transforming growth factor ß recruits persistent MAPK signaling to regulate long-term memory consolidation in Aplysia californica.

In this study, we explore the mechanistic relationship between growth factor signaling and kinase activity that supports the protein synthesis-dependent phase of long-term memory (LTM) consolidation for sensitization ofAplysia Specifically, we examine LTM for tail shock-induced sensitization of the tail-elicited siphon withdrawal (T-SW) reflex, a form of memory that requires both (i) extracellular signal-regulated kinase (ERK1/2; MAPK) activity within identified sensory neurons (SNs) that mediate the T-SW and (ii) the activation of transforming growth factor ß (TGFß) signaling. We now report that repeated tail shocks that induce intermediate-term (ITM) and LTM for sensitization, also induce a sustained post-training phase of MAPK activity in SNs (lasting at least 1 h). We identified two mechanistically distinct phases of post-training MAPK: (i) an immediate phase that does not require ongoing protein synthesis or TGFß signaling, and (ii) a sustained phase that requires both protein synthesis and extracellular TGFß signaling. We find that LTM consolidation requires sustained MAPK, and is disrupted by inhibitors of protein synthesis and TGFß signaling during the consolidation window. These results provide strong evidence that TGFß signaling sustains MAPK activity as an essential mechanistic step for LTM consolidation.

Synaptic generation of an intracellular retrograde signal requires activation of the tyrosine kinase and mitogen-activated protein kinase signaling cascades in Aplysia.

Cellular changes underlying memory formation can be generated in an activity-dependent manner at specific synapses. Thus an important question concerns the mechanisms by which synaptic signals communicate with the cell body to mediate these cellular changes. A monosynaptic circuit that is enhanced by sensitization in Aplysia is well-suited to study this question because three different subcellular compartments: (i) the sensorimotor SN-MN synapses, (ii) the SN projections to MNs via axonal connections, (iii) the SN cell bodies, can all be manipulated and studied independently. Here, we report that activity-dependent (AD) training in either the entire SN-MN circuit or in only the synaptic compartment, activates MAPK in a temporally and spatially specific pattern. Specifically, we find (i) MAPK activation is first transiently generated at SN-MN synapses during training, (ii) immediately after training MAPK is transiently activated in SN-MN axonal connections and persistently activated in SN cell bodies, and finally, (iii) MAPK is activated in SN cell bodies and SN-MN synapses 1h after training. These data suggest that there is an intracellularly transported retrograde signal generated at the synapse which is later responsible for delayed MAPK activation at SN somata. Finally, we find that this retrograde signal requires activation of tyrosine kinase (TK) and MEK signaling cascades at the synapses.

Distinct Growth Factor Families Are Recruited in Unique Spatiotemporal Domains during Long-Term Memory Formation in Aplysia californica.

Several growth factors (GFs) have been implicated in long-term memory (LTM), but no single GF can support all of the plastic changes that occur during memory formation. Because GFs engage highly convergent signaling cascades that often mediate similar functional outcomes, the relative contribution of any particular GF to LTM is difficult to ascertain. To explore this question, we determined the unique contribution of distinct GF families (signaling via TrkB and TGF-ßr-II) to LTM formation in Aplysia. We demonstrate that TrkB and TGF-ßr-II signaling are differentially recruited during two-trial training in both time (by trial 1 or 2, respectively) and space (in distinct subcellular compartments). These GFs independently regulate MAPK activation and synergistically regulate gene expression. We also show that trial 1 TrkB and trial 2 TGF-ßr-II signaling are required for LTM formation. These data support the view that GFs engaged in LTM formation are interactive components of a complex molecular network.

Activity-dependent inhibitory gating in molecular signaling cascades induces a novel form of intermediate-term synaptic facilitation in Aplysia californica.

Mechanistically distinct forms of long-lasting plasticity and memory can be induced by a variety of different training patterns. Although several studies have identified distinct molecular pathways that are engaged during these different training patterns, relatively little work has explored potential interactions between pathways when they are simultaneously engaged in the same neurons and circuits during memory formation. Aplysia californica exhibits two forms of intermediate-term synaptic facilitation (ITF) in response to two different training patterns: (1) repeated trial (RT) ITF (induced by repeated tail nerve shocks [TNSs] or repeated serotonin [5HT] application) and (2) activity-dependent (AD) ITF (induced by sensory neuron activation paired with a single TNS or 5HT pulse). RT-ITF requires PKA activation and de novo protein synthesis, while AD-ITF requires PKC activation and has no requirement for protein synthesis. Here, we explored how these distinct molecular pathways underlying ITF interact when both training patterns occur in temporal register (an "Interactive" training pattern). We found that (1) RT, AD, and Interactive training all induce ITF; (2) Interactive ITF requires PKC activity but not de novo protein synthesis; and (3), surprisingly, Interactive training blocks persistent PKA activity 1 h after training, and this block is PKC-independent. These data support the hypothesis that sensory neuron activity coincident with the last RT training trial is sufficient to convert the molecular signaling already established by RT training into an AD-like molecular phenotype.

A novel cysteine-rich neurotrophic factor in Aplysia facilitates growth, MAPK activation, and long-term synaptic facilitation.

Neurotrophins are critically involved in developmental processes such as neuronal cell survival, growth, and differentiation, as well as in adult synaptic plasticity contributing to learning and memory. Our previous studies examining neurotrophins and memory formation in Aplysia showed that a TrkB ligand is required for MAPK activation, long-term synaptic facilitation (LTF), and long-term memory (LTM) for sensitization. These studies indicate that neurotrophin-like molecules in Aplysia can act as key elements in a functionally conserved TrkB signaling pathway. Here we report that we have cloned and characterized a novel neurotrophic factor, Aplysia cysteine-rich neurotrophic factor (apCRNF), which shares classical structural and functional characteristics with mammalian neurotrophins. We show that apCRNF (1) is highly enriched in the CNS, (2) enhances neurite elongation and branching, (3) interacts with mammalian TrkB and p75(NTR), (4) is released from Aplysia CNS in an activity-dependent fashion, (5) facilitates MAPK activation in a tyrosine kinase dependent manner in response to sensitizing stimuli, and (6) facilitates the induction of LTF. These results show that apCRNF is a native neurotrophic factor in Aplysia that can engage the molecular and synaptic mechanisms underlying memory formation.

Growth factor signaling and memory formation: temporal and spatial integration of a molecular network.

Growth factor (GF) signaling is critically important for developmental plasticity. It also plays a crucial role in adult plasticity, such as that required for memory formation. Although different GFs interact with receptors containing distinct types of kinase domains, they typically signal through converging intracellular cascades (e.g., Ras-MEK-MAPK) to mediate overlapping functional endpoints. Several GFs have been implicated in memory formation, but due to a high level of convergent signaling, the unique contributions of individual GFs as well as the interactions between GF signaling cascades during the induction of memory is not well known. In this review, we highlight the unique roles of specific GFs in dendritic plasticity, and discuss the spatial and temporal profiles of different GFs during memory formation. Collectively, the data suggest that the roles of GF signaling in long-lasting behavioral and structural plasticity may be best viewed as interactive components in a complex molecular network.

Pattern and predictability in memory formation: from molecular mechanisms to clinical relevance.

Most long-term memories are formed as a consequence of multiple experiences. The temporal spacing of these experiences is of considerable importance: experiences distributed over time (spaced training) are more easily encoded and remembered than either closely spaced experiences, or a single prolonged experience (massed training). In this article, we first review findings from studies in animal model systems that examine the cellular and molecular properties of the neurons and circuits in the brain that underlie training pattern sensitivity during long-term memory (LTM) formation. We next focus on recent findings which have begun to elucidate the mechanisms that support inter-trial interactions during the induction of LTM. Finally, we consider the implications of these findings for developing therapeutic strategies to address questions of direct clinical relevance.

MAPK establishes a molecular context that defines effective training patterns for long-term memory formation.

Although the importance of spaced training trials in the formation of long-term memory (LTM) is widely appreciated, surprisingly little is known about the molecular mechanisms that support interactions between individual trials. The intertrial dynamics of ERK/MAPK activation have recently been correlated with effective training patterns for LTM. However, whether and how MAPK is required to mediate intertrial interactions remains unknown. Using a novel two-trial training pattern which induces LTM in Aplysia, we show that the first of two training trials recruits delayed protein synthesis-dependent nuclear MAPK activity that establishes a unique molecular context involving the recruitment of CREB kinase and ApC/EBP and is an essential intertrial signaling mechanism for LTM induction. These findings provide the first demonstration of a requirement for MAPK in the intertrial interactions during memory formation and suggest that the kinetics of MAPK activation following individual experiences determines effective training intervals for LTM formation.

Local synaptic integration of mitogen-activated protein kinase and protein kinase A signaling mediates intermediate-term synaptic facilitation in Aplysia.

It is widely appreciated that memory processing engages a wide range of molecular signaling cascades in neurons, but how these cascades are temporally and spatially integrated is not well understood. To explore this important question, we used Aplysia californica as a model system. We simultaneously examined the timing and subcellular location of two signaling molecules, MAPK (ERK1/2) and protein kinase A (PKA), both of which are critical for the formation of enduring memory for sensitization. We also explored their interaction during the formation of enduring synaptic facilitation, a cellular correlate of memory, at tail sensory-to-motor neuron synapses. We find that repeated tail nerve shock (TNS, an analog of sensitizing training) immediately and persistently activates MAPK in both sensory neuron somata and synaptic neuropil. In contrast, we observe immediate PKA activation only in the synaptic neuropil. It is followed by PKA activation in both compartments 1 h after TNS. Interestingly, blocking MAPK activation during, but not after, TNS impairs PKA activation in synaptic neuropil without affecting the delayed PKA activation in sensory neuron somata. Finally, by applying inhibitors restricted to the synaptic compartment, we show that synaptic MAPK activation during TNS is required for the induction of intermediate-term synaptic facilitation, which leads to the persistent synaptic PKA activation required to maintain this facilitation. Collectively, our results elucidate how MAPK and PKA signaling cascades are spatiotemporally integrated in a single neuron to support synaptic plasticity underlying memory formation.

Transsynaptic coordination of presynaptic and postsynaptic modifications underlying enduring synaptic plasticity.

Neurexins and neuroligins are cell adhesion molecules that form transsynaptic interactions. In this issue of Neuron, Choi et al. report that neurexin-neuroligin signaling plays a critical role in functional and structural synaptic plasticity underlying memory formation in Aplysia.

The tail-elicited tail withdrawal reflex of Aplysia is mediated centrally at tail sensory-motor synapses and exhibits sensitization across multiple temporal domains.

The defensive withdrawal reflexes of Aplysia californica have provided powerful behavioral systems for studying the cellular and molecular basis of memory formation. Among these reflexes the tail-elicited tail withdrawal reflex (T-TWR) has been especially useful. In vitro studies examining the monosynaptic circuit for the T-TWR, the tail sensory-motor (SN-MN) synapses, have identified the induction requirements and molecular basis of different temporal phases of synaptic facilitation that underlie sensitization in this system. They have also permitted more recent studies elucidating the role of synaptic and nuclear signaling during synaptic facilitation. Here we report the development of a novel, compartmentalized semi-intact T-TWR preparation that allows examination of the unique contributions of processing in the SN somatic compartment (the pleural ganglion) and the SN-MN synaptic compartment (the pedal ganglion) during the induction of sensitization. Using this preparation we find that the T-TWR is mediated entirely by central connections in the synaptic compartment. Moreover, the reflex is stably expressed for at least 24 h, and can be modified by tail shocks that induce sensitization across multiple temporal domains, as well as direct application of the modulatory neurotransmitter serotonin. This preparation now provides an experimentally powerful system in which to directly examine the unique and combined roles of synaptic and nuclear signaling in different temporal domains of memory formation.

Small G protein signaling in neuronal plasticity and memory formation: the specific role of ras family proteins.

Small G proteins are an extensive family of proteins that bind and hydrolyze GTP. They are ubiquitous inside cells, regulating a wide range of cellular processes. Recently, many studies have examined the role of small G proteins, particularly the Ras family of G proteins, in memory formation. Once thought to be primarily involved in the transduction of a variety of extracellular signals during development, it is now clear that Ras family proteins also play critical roles in molecular processing underlying neuronal and behavioral plasticity. We here review a number of recent studies that explore how the signaling of Ras family proteins contributes to memory formation. Understanding these signaling processes is of fundamental importance both from a basic scientific perspective, with the goal of providing mechanistic insights into a critical aspect of cognitive behavior, and from a clinical perspective, with the goal of providing effective therapies for a range of disorders involving cognitive impairments.