Activity‐Dependent Regulation of Transcription During Development of Synapses

ACTIVITY-DEPENDENT REGULATION OF TRANSCRIPTION DURING DEVELOPMENT OF SYNAPSES

Subhabrata Sanyal* and Mani Ramaswami*,y,z *Department of Molecular and Cellular Biology, Life Sciences South, University of Arizona Tucson, Arizona 85721, USA y Smurfit Institute of Genetics, University of Dublin, Trinity College, Dublin-2, Ireland z Trinity College Institute for Neuroscience, University of Dublin, Trinity College Dublin-2, Ireland

I. Introduction II. Mechanisms of Transcriptional Activation During Long-Term Plasticity III. Experimental Paradigms of Protein Synthesis-Dependent Long-Term Plasticity in Drosophila IV. NMJ as a Model Synapse to Study Transcriptional Regulation of Developmental Plasticity A. Transcriptional Control of MN Cell Fate B. Transcriptional Regulation of Synaptic Development C. Transcriptional Events in Postsynaptic Muscle That Regulate Plasticity V. Open Questions and Areas of Convergence References

The ability of neurons to alter activity in response to previous experience forms the principle substrate of learning and memory. Intracellular signaling cascades operate during these alterations to transduce patterned neural activity into structural and functional modifications. Thus, during the establishment of long-term changes, new proteins are synthesized in a highly context-dependent fashion. This is brought about both by the translation of preexisting mRNAs as well as by the activation of specific transcription factors. The Drosophila larval neuromuscular junction (NMJ) has provided a unique paradigm in which the role of transcription in neural development and plasticity has been assessed with remarkable resolution. At this motor synapse, the eVect of modulating signaling and transcription factor activation on synaptic strength and size can be determined easily. The availability of classical and modern genetic and genomic methods further enhances the utility of this system. Current results indicate the presence of conserved activitydependent signaling networks that trigger particular transcription factors during long-term synaptic plasticity. Further, as the NMJ grows through development, communication between the pre- and postsynaptic compartments provides signals and constraints to maintain parity. Taken together, the regulation of transcription and protein synthesis during developmental plasticity of the NMJ oVers valuable insights into conserved mechanisms of learning and memory across species. INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 75 DOI: 10.1016/S0074-7742(06)75013-9

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Copyright 2006, Elsevier Inc. All rights reserved. 0074-7742/06 $35.00

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I. Introduction

Perhaps one of the most intriguing physiological processes in biology is the ability of the brain to acquire, process, store, and retrieve information. Encountering new experiences, making associations, and recalling them at will underscore the utility of learning and memory. Most animals, however primitive, have a semblance of a ‘‘neural circuitry’’ that subserves this function. Instances in which this function of the nervous system is compromised oVer one of the best (and the most unfortunate) opportunities for their study. Nowhere is the ability to form new memories more obvious than in cases in which it is lacking, for example, anterograde amnesia (Sacks, 1998). EVorts to understand memory and describe its underpinnings have been remarkably productive. As in most of experimental biology, a top-down approach has been complemented continually by bottomup, reductionist ones. Thus, while an incredible amount of knowledge exists now on information processing by various vertebrate brains, equally substantial progress has been made in understanding the cellular and molecular correlates that operate in neurons. When an organism learns, there are discrete changes in relevant neural circuits. These changes occur simultaneously at several levels and fundamentally alter the way in which neurons connect to one another. Consequently, both the physical connectivity and firing properties of these neurons change (Fregnac, 1996). These modifications, a necessity for long-term plasticity, are brought about by cellular processes that direct synthesis of new proteins. In several animal models, blocking protein synthesis precludes long-term plasticity (Castellucci et al., 1986; Frey et al., 1988; Stanton and Sarvey, 1984). Based on comparable experiments designed to discriminate between discrete steps of plasticity, the following largely conserved sequential steps can be identified (Fig. 1). On initial

FIG. 1. Conceptual outline of sequential stages of learning and memory formation. This diagram also shows that these stages can be queried at various levels of analysis, organism, cell, and molecules. A comprehensive understanding would involve collating knowledge from all these approaches.

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patterned neural activity in a given circuit (such as in response to encountering novel stimuli), short-term changes are quickly set in motion. These changes typically do not require protein synthesis and instead utilize modifications of preexisting proteins in the relevant neurons (Abraham et al., 1991; Bailey et al., 2000). Membrane properties are altered as a consequence of protein modifications and signaling mechanisms are activated. However, these changes are transient and, in turn, are further consolidated by continued neural activity and long-lasting activation of intracellular signaling (such as those that might be triggered by repetitive trials; Mauelshagen et al., 1996, 1998). This late phase, invariably involves the synthesis of new proteins, resulting in structural modifications and changes in membrane and synaptic properties that are more permanent. This later phase is known as late long-term plasticity (L-LTP) of synapses. This sequence during the establishment of long-term changes has been consistently observed in several systems, both in vivo and in ‘‘reduced’’ preparations (Sharma et al., 2003; Waddell and Quinn, 2001). Thus, formation of longterm memory requires protein synthesis either through translation of preexisting RNA (Klann and Dever, 2004; Martin, 2004; Richter, 2001; Steward and Schuman, 2001; Sutton and Schuman, 2005) or through transcription driven primarily by transcription factors that are activated following instructive neural activity and intracellular signaling (Hevroni et al., 1998; Lonze and Ginty, 2002). In this chapter, we focus on the role of signaling and transcription during long-term neural change. At the outset, it is important to bear in mind that although protein synthesis is a must for L-LTP, it is usually not the identity of the proteins but rather the ‘‘place’’ where this happens that encodes the ‘‘type’’ of memory. Protein synthesis thus forms the necessary permissive step that underlies synaptic modifications. It does not dictate what kind of memory will be formed and therefore all long-term changes are expected to share a large majority of new proteins being made.

II. Mechanisms of Transcriptional Activation During Long-Term Plasticity

As mentioned previously, the requirement for protein synthesis is common during plasticity in all systems studied. It is generally believed that transcription factors come in two broad flavors, constitutive and inducible (Herdegen and Leah, 1998). As the name implies, inducible transcription factors are those that are typically found in the cells at low levels and on activation show rapid induction. Several inducible transcription factors are also immediate-early (IE) genes. IE genes are classically defined as those which do not require de novo protein synthesis (transcriptional upregulation) for their induction. For example, the activator protein-1 (AP1) class of transcription factors are canonical IE genes

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whereas cAMP response element binding protein (CREB), another key transcription factor required during plasticity, is a constitutively present transcription factor. Thus, it is possible to envisage at least two major ways of increasing the output of transcription factors. One is to make more of the factor itself, a mechanism likely to be operative for inducible transcription factors like AP1. The other is to alter protein activity by posttranslational means such as phosphorylation. Phosphorylation of CREB driven by upstream activation of kinases, such as extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and Ca2þ/Calmodulin-dependent protein kinase (CaMK), are well-studied examples in which signaling cascades, typical mediators between neural activity and protein synthesis, impinge on target transcription factors (Fig. 2).

FIG. 2. A simplified scheme of neural activity-driven transcriptional activation. In general, inducible transcription factors are recruited as terminal targets of intracellular signaling cascades. These coordinate with basal tissue-specific transcription factors and core transcription machinery to activate transcription of specific genes. Histone modifications play a significant role in making these genes transcription competent.

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Both inducible and constitutive transcription factors work in concert with components of the core transcription machinery in cells such as the TATAbinding proteins (TBPs) and histone-modifying enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). In fact, it has been shown in several cases that acetylation of critically conserved lysine residues in histone N-terminal tails is an important regulatory element of gene transcription during long-term plasticity (Crosio et al., 2003). Two examples of chromatin modification both centered on CREB function are noteworthy. In the first case, Guan et al. (2002) showed that an activator form of CREB in Aplysia, CREB1, works together with HATs to induce transcription in a 5-hydroxytryptamine (5-HT; serotonin) stimulated sensory neuron. In the same report they also showed that CREB2, a transcriptional repressor, operates with specific HDACs to deacetylate histones and repress transcription during FMRFamide-induced long-term depression. Further, in two related studies it was discovered that CREB-binding protein (CBP), a transcriptional cofactor, was necessary for HAT activity (Alarcon et al., 2004; Korzus et al., 2004). Not only was learning and memory impaired in mice with CBP lacking HAT but this was also found to be the basis for cognitive defects in Rubinstein-Taybi syndrome. Changes in chromatin structure are therefore key steps during neural activity–driven gene transcription (Fig. 2).

III. Experimental Paradigms of Protein Synthesis-Dependent Long-Term Plasticity in Drosophila

Several experimental systems have been utilized to study neural plasticity. These diverse preparations oVer unique advantages and perspectives. Before we describe in detail the advances made in this field using the Drosophila NMJ, it is therefore instructive to summarize a few key vertebrate studies as shown in Table I. The contribution of research done using the fruit fly has been as enriching as expected from this vital model organism. There are several factors that render Drosophila a system that is useful for analyzing plasticity at diVerent levels. Flies display robust learning, both associative and nonassociative. Several circuits or at least nervous system regions that mediate these behaviors are well described, it is easy to access neurons that form part of these circuits and, finally, highly specialized genetic tools permit sophisticated analysis of cellular signaling. Before we illustrate how the glutamatergic neuromuscular junction (NMJ) has been a key model synapse, keeping in mind the structure in Fig. 1, we will briefly describe three selected preparations in which studies on the role of transcription factors in neural plasticity have provided fundamental insights. At the behavioral level, flies display robust associative learning and long-term memory. In assays that measure the ability of a population of flies to learn and

\TABLE I TRANSCRIPTION FACTORS IMPLICATED

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Transcription factor

Organism

Drosophila homologue

Fos

Rodents

Kayak

Jun

Rodents

Jra

Zenk/zif268/ NGF1-A

Zebrafinch/ Rodents

?

CREB

Rodents/(Aplysiab)

DCREB2

c/EBP

Rodents/(Aplysia)

Slbo

a b

IN

VERTEBRATE MODELS

OF

PLASTICITY a

Experimental assay Electroconvulsive seizures, sleep deprivation, drug addiction

Neuronal survival, drug addiction Song perception/LTP, enriched environment, sleep deprivation Spatial learning, alcohol addiction, neuronal survival/ (long-term facilitation) Memory storage/ (long-term facilitation)

A summary of vertebrate studies on the role of transcription in long-term plasticity. Aplysia, a marine sea-slug, is also included in this table as a key non-fly invertebrate model organism.

References Bibb et al., 2001; Chen et al., 2000; Cirelli and Tononi, 2000; Cirelli et al., 2005; Dash et al., 1990; Hiroi et al., 1998; Nestler et al., 2001; Pompeiano et al., 1994; Woolf and Costigan, 1999 Zhou et al., 2004 Jones et al., 2001; Mello and Clayton, 1994; Mello and Ribeiro, 1998; Mello et al., 2004; Pinaud, 2004; Wallace et al., 1995 Bartsch et al., 1995, 1998; Chen et al., 2003; Dash et al., 1990; Livingstone et al., 1984 Alberini et al., 1994; Chen et al., 2000

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remember the association between an odor and a noxious stimulus (electric shock), flies perform remarkably well (Davis, 2005; Heisenberg et al., 1985; Tully and Quinn, 1985). More significantly, mutations in several genes that have been shown both in flies and in other systems to constitute molecular determinants of learning and memory profoundly aVect performance in these assays. For example, both dunce (cAMP phosphodiesterase) and rutabaga (adenylyl cyclase) mutants perform poorly in associative learning assays, implicating the cAMP cascade in this process (Byers et al., 1981; Livingstone et al., 1984). At the level of organ systems, the olfactory system in Drosophila has proved to be a highly tractable model not only for studying neural connectivity but also for asking questions as to how this connectivity develops and changes with experience ( JeVeris et al., 2001; Komiyama et al., 2003; Marin et al., 2005). In this seemingly hardwired circuit, olfactory sensory neurons relay information from the periphery to higher centers of olfactory perception in the mushroom body and protocerebrum by synapsing onto projection neurons ( JeVeris et al., 2002). These synapses are located in the antennal lobes, where a glomerular organization is also subject to modulation by inhibitory and excitatory interneurons. This organization, which is remarkably similar to that in vertebrates, is also plastic. Altering the function of critical proteins in a cell-autonomous fashion results in changes in the arborization of these neurons (Komiyama et al., 2003). It is only a matter of time before correlations are made between changes in olfactory behavior and structural and functional alterations in circuit components (Devaud et al., 2001). Although the earlier examples are highly informative, the most valuable model to unravel cellular correlates of plasticity in Drosophila is undeniably the larval NMJ. We describe this preparation in greater detail later, particularly in the context of synaptic plasticity and its regulation by transcriptional induction.

IV. NMJ as a Model Synapse to Study Transcriptional Regulation of Developmental Plasticity

Studies using the Drosophila larval NMJ as a model synapse have been rendered facile due to the elegant simplicity of this preparation. As is obvious from other chapters in this book, larval motor synapses are easy to access, they allow independent analysis of synapse size and strength, and have been remarkably well characterized with respect to their subsynaptic constituents (Broadie, 1995). Additionally, several GAL4 drivers have made it possible to perturb protein function in both pre- and postsynaptic compartments in a controlled manner (Brand and Perrimon, 1993; DuVy, 2002). Research indicates that transcription factors play critical roles in determining the identity of motoneurons (MNs),

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in directing axon growth toward their normal muscle targets, and in generating homeostatic responses to changes in both presynaptic (interneurons and sensory neurons) and postsynaptic (muscle) partners. We will examine each of these in greater detail.

A. TRANSCRIPTIONAL CONTROL

OF

MN CELL FATE

Through pioneering work from several laboratories, it became clear that the axons from embryonic MNs rely on initial transcription factor activity to grow along correct nerve tracts and innervate their target muscle. It was shown that a transcription factor code determines whether MNs would innervate dorsally placed body wall muscles or ventral ones (Skeath and Thor, 2003; Thor and Thomas, 2002). Thus, for example, while the LIM homeodomain transcription factors are expressed in ‘‘ventrally innervating’’ MNs, the transcription factor Even-skipped (Eve) is responsible for properly directing dorsally innervating MNs (Broihier and Skeath, 2002; Fujioka et al., 2003; Landgraf et al., 1999; McDonald et al., 2003; Thor et al., 1999). Consistent with this model, in MNs that have lateral muscle targets, a combination of Eve and Islet operates to make this decision. What might be the targets of these transcription factors? In other words, what are the final eVectors that regulate proper axon growth and targeting? Some initial hints came from studies that find that Eve activity controls the expression of one of the two Netrin receptors (Unc-5) and whereas the LIM-HD code regulates the expression of an immunoglobulin-containing cell adhesion molecule encoded by beaten path (Certel and Thor, 2004; Labrador et al., 2005). By controlling the expression of these surface proteins, transcriptional events implement a code that ensures appropriate matching between a neuron and its target muscle. Chapter 2 by Landgraf and Thor discusses these issues in greater detail.

B. TRANSCRIPTIONAL REGULATION

OF

SYNAPTIC DEVELOPMENT

Once accurate targeting and synapse formation has taken place by late embryogenesis, the larval motor synapses continue to enlarge to keep up with the demands of a growing animal. Drosophila larvae undergo three larval molts during which the muscle pattern is left unchanged but muscles increase in size by a factor of about 40. This imposes greater loads on the neurons that depolarize them. Larger muscle volumes necessitate larger currents to depolarize them during crawling. These larger depolarizing currents are brought about, in part, from the expansion of synapses through these larval molts (Schuster et al., 1996). This process forms the basis of a system that has served as a valuable model for activity-dependent long-term plasticity. Studying developmental plasticity of the

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motor synapse oVers several parallels with studies of synaptic plasticity in vertebrate central nervous systems. MNs in Drosophila are predominantly glutamatergic and the growth of this synapse is dependent on neural activity. There is extensive cross talk and homeostatic compensation between pre- and postsynaptic partners. Transcription factors known to operate in learning regulate synapse growth at the NMJ, and the experimental accessibility alluded to before is an undeniable advantage. One of the best illustrations of the utility of the NMJ as an eVective tool to address questions of neural plasticity is the elucidation of the role of the transcription factor CREB in learning. Using the olfactory conditioning assay developed by Tully and Quinn, Yin et al. (1994) showed that activation of DCREB-dependent transcription was required during the acquisition of longterm memory. More strikingly, expression of an ‘‘activator’’ isoform of DCREB was suYcient to convert short-term into long-term memory. In this experiment, stimuli that normally elicited short-term memory now resulted in a long-lasting change (Yin et al., 1995). Parallel experiments performed to ascertain the role of DCREB during developmental plasticity of the NMJ gave rise to consistent results and substantially enhanced our understanding of the phenomenon. Davis et al. (1996) showed that during the plasticity, the NMJ inhibiting DCREB function resulted in a decrease in synaptic strength while keeping the structure or ‘‘size’’ of the synapse essentially wild type. Additionally, in a suitable background where synaptic proliferation was driven by lowering levels of the fly cell adhesion molecule neural cell adhesion molecule (NCAM) homologue, FasciclinII (FasII), DCREB activation was suYcient to produce increases in synapse strength. These experiments for the first time suggested the possibility that synapse size and strength, although probably controlled simultaneously, involve dedicated molecular pathways in their regulation. Significantly, at least in this system, DCREB-dependent transcription was unable to drive changes in all aspects of plasticity. Subsequently, a cofactor of CREB-dependent transcription, DCREB-binding protein, or DCBP was also shown to influence the growth and maturation of the NMJ synapse (Marek et al., 2000). The primary eVect of altering DCBP function was to reduce presynaptic transmitter release. This was seen under conditions in which either DCBP function was increased or decreased. Further, the primary site of action of DCBP appeared to be in postsynaptic muscle. How CBP activity correlates with and aVects that of DCREB remain ambiguous at this point. Once again, starting with the olfactory learning paradigm, Drosophila nalyot mutants [containing mutations in the Myoeloblastosis (Myb)-related transcription factor, alcohol dehydrogenase transcription factor 1(Adf1)] were shown to result in specific deficits in memory (DeZazzo et al., 2000). Adf1 is a widely expressed transcription factor and when tested at the NMJ, mutations of Adf1 showed smaller synapses while keeping synaptic strength intact. Expectedly, neural

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overexpression of Adf1 also resulted in significantly larger synapses with normal transmitter release. In conjunction with prior findings on DCREB, these results validated a model proposing that synaptic size and synaptic strength are regulated independently of one another during development. The requirement of distinct transcription factors predicts the existence of distinct sets of downstream target genes or eVectors that underlie this regulation. While the constitutive transcription factor DCREB had received widespread attention in the field of plasticity in both vertebrate and invertebrate fields, it seemed reasonable to assume that there would be inducible transcription factors involved during plasticity. It is likely that following stimuli, neurons on the way to long-term change would transduce this signal through rapidly inducible transcription factors or IE genes. A strong candidate for this role was Fos. Fos, mostly in conjunction with its heteromeric partner Jun, which forms the AP1 transcription factor, had been shown to display rapid induction in rodents following electroconvulsive seizures (Daval et al., 1989; Hiroi et al., 1998; Nakajima et al., 1989). Fos levels were reported to be upregulated during long-term plasticity induction and, perhaps most significantly, expression levels of delta-FosB, a member of the Fos family, in the nucleus accumbens correlated tightly with the progression of drug addiction in mammals (Nestler et al., 2001). Therefore, it was conceivable that Drosophila Fos (or AP1) was recruited early on the hierarchy of signaling events during long-term plasticity. Experiments done in our laboratory showed that this was true. Not only did AP1 positively regulate both synaptic size and strength at the NMJ but it also recruited DCREB (possibly through transcriptional induction) to influence transmitter release (Sanyal et al., 2002, 2003). Thus, an initial network of transcription factors emerges (Fig. 3). While AP1 may be one of the first transcription factors to be induced following stimulus, subsequent downstream transcription factors are then called into play to influence the strength or the size of the NMJ during growth. The Drosophila NMJ provides a simple way to distinguish these possibilities and it will also serve as a valuable model to study the dual problems of signaling cross talk during plasticity and the identity and role of downstream targets of these transcription factors. Once these bona fide target genes are studied in the context of plasticity, we will be able to better explain how changes in synaptic size and strength are brought about by the synthesis of new proteins.

C. TRANSCRIPTIONAL EVENTS REGULATE PLASTICITY

IN

POSTSYNAPTIC MUSCLE THAT

The role of the transcriptional cofactor DCBP in the muscle has been alluded to in the previous section. These results bring up a central issue in the study of plasticity, that of homeostatic regulation during the growth and development of

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FIG. 3. A summary of current understanding of key activity-dependent transcription factors in Drosophila. A significant area of future research is likely to be aimed at identifying downstream targets of these transcription factors and understanding cross talk and overlap between these important regulators of long-term plasticity.

the NMJ. It is widely believed that targeting, pruning, and growth of the motor synapse is regulated significantly by its partner muscle (Broadie and Bate, 1993a). This process is an active one and involves the synchronization of signaling between the neuron and its target muscle (Broadie and Bate, 1993b). It is easy to appreciate that communication between these two entities would be a highly regulated phenomenon and might involve secreted signaling molecules from either or both sides. Several lines of evidence now exist to show that this is true at the Drosophila NMJ. Most significantly, the end points of signaling are transcription factors that in all likelihood are important eVectors of new protein synthesis. Two examples, the Wingless- and the bone morphogenic protein (BMP)-signaling pathway, are particularly informative (Marques, 2005; Chapter 12 by Marque´s and Zhang). Both the Wingless- and the BMP-signaling pathways follow a general format. Both signals are triggered by the binding of secreted ligands to their specific membrane-bound receptors. Once the receptor-ligand complex is formed, a cascade of intracellular signaling is set in motion leading ultimately to the phosphorylationdriven activation and nuclear import of the transcription factor SMAD in the case of BMP signaling (Sanyal et al., 2004; Shi and Massague, 2003). For Wingless, the final outcome in the canonical pathway is the dephosphorylation, stabilization, and

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nuclear entry of the transcriptional cofactor Armadillo ( -catenin) (Seto and Bellen, 2004), or the cleavage of the Wingless receptor and the nuclear import of a receptor fragment (Mathew et al., 2005). SMADS and Armadillo (in concert with the transcription factor lymphoid enhancer factor 1, LEF1), and presumably a cleavage product of the Wingless receptor, can then induce transcriptional activation of selected subsets of genes to bring about the required change. What makes this system unique is that the presence of soluble ligands renders it possible for one cell to aVect another in close apposition. At the NMJ, this is of special significance since the presynaptic terminal forms on the muscle and its growth and development is regulated significantly by signals from the muscle. Thus, it was discovered by workers in the Goodman, Davis, and Budnik laboratories that retrograde and anterograde signaling between MNs and body wall muscles influenced the development of this synapse (Marques, 2005). For instance, one of the BMP ligands, glass bottom boat (Gbb) is thought to be secreted by the muscle cells and to bind to receptors on the presynaptic membrane (McCabe et al., 2003). Transgenic manipulations in MNs have also shown that the TGF- -signaling cascade is active and instructive in determining how the NMJ forms and grows. The Wingless ligand, on the other hand, is released from the presynaptic side and binds to cognate DFrizzled2 receptors on the surface of the muscle (Mathew et al., 2005; Packard et al., 2002). Mechanisms exist, thereby, that allow intimate communication between these two compartments enabling a high degree of synchrony in structure and function. Yet another established transcription factor module that participates in NMJ development are the fly homologues of the NFkappaB-ikappaB family, dorsal and cactus (Cantera et al., 1999). Although it is unclear exactly how these transcription factors influence synaptic growth, it seems that they act on either side of the NMJ, putting them in a position to potentially influence cellular communication. How signaling through these transcription factors correlates with the other candidates mentioned earlier remains unresolved.

V. Open Questions and Areas of Convergence

It is clear from what we know so far that growth, maturation, and plasticity of the NMJ involves widespread changes in protein synthesis driven by transcription. It also seems reasonable to suppose that at least partially exclusive transcription factors participate in the pre- and postsynaptic compartments. Networks and cascades of signals operate in these cells to bring about changes that are in keeping with concomitant changes in the apposing cell. Means of communication across the boundaries of these two compartments are provided

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by the existence of soluble signaling factors such as Wingless and Gbb. However, several potentially productive avenues of investigation remain. First, the details of how these signaling cascades bring about synaptic changes are far from clear. Although several candidate mechanisms can be deduced from other paradigms in which such signaling is active, contextual diVerences must exist. Ras signaling, for instance, may have distinct distal eVectors in the development of the compound eye and in the growth of the motor synapse (Koh et al., 2002; Silver and Rebay, 2005). Similarly, BMP signaling may be transduced through a unique combination of receptors and target-binding elements in the genome in diVerent cellular backgrounds. Second, the final targets of signaling, the genes whose transcription is activated, need to be identified. These generate the terminal readouts of signaling, and knowledge of the function of these gene products is essential to our understanding of how plasticity is brought about. While, as enumerated earlier, several transcription factors have been implicated, the subset of genes that they target is very poorly understood. Further research will undoubtedly focus on discovering these. Several methods are already being employed, and classical techniques, such as genetic screens, are increasingly being complemented with genomic and proteomic approaches (Guan et al., 2005). Once these targets are pinpointed, their validation in appropriate contexts will be essential. The information arising from these experiments will not only outline the cellular workings of plasticity but it will also address intriguing questions such as how CREB and AP1 influence distinct aspects of long-term plasticity. A related issue is that of detecting transcription factor activity during plasticity. Although Drosophila oVers the power of genetic analysis, the lack of specific in vivo reporters for these signaling cascades is currently a limiting factor. A third question that needs to be addressed more thoroughly is how information is exchanged and coordinated between the pre- and postsynaptic compartment. For instance, how is dendritic growth regulated? Dendritic spines in vertebrates are known to be highly plastic, but it is not clear what the homologous structures in flies are or how they respond to plasticity signals. Research in this area, although not new, still suVers from several gaps. When viewed from the perspective of the whole nervous system, or even of the circuits that are involved in learning and memory storage, it is obvious that diVerent elements contained therein need dedicated machinery for this communication. It is a matter of debate as to what the exact cellular correlates of memory are, but it seems clear that appropriate transactions between pre- and postsynaptic neurons and a proper balance of activating and inhibiting ‘‘factors’’ must be operative. Important questions during MN development are how are the opposing eVects of ERK activation and P38 activation regulated (Guan et al., 2003)? What is their final transcriptional readout? Is there information about the amplitude of the signal (quantity of kinase activated in a cell), or is there merely a threshold that needs to

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be crossed for the observed eVects? Even in a single neuron, synaptic events at terminal boutons have profound eVects in the nuclei. How is this information conveyed? Nuclear transport proteins or importins have been implicated in Aplysia and mice (Thompson et al., 2004), opening the way for further research and speculation regarding these mechanisms. Finally, one of the principle goals of a reductionist approach is to be able to assign physiological relevance to experimental findings. In that sense, putting together all the information on signal transduction, transcription factor activation, and the genomic/proteomic response of the cell or organism in the context of learning is imperative. Thus, experiments that investigate the role of various components of signaling during learning complete the circle of enquiry. It may be found, for example, that not all types of learning require the activation of the same set of transcription factors. Hence, although synthesis of new proteins is believed to be permissive for the formation of new memories, there may be broad categories that are diVerentiated not only by the circuits in which they form but also by the identity of the proteins being synthesized. Definitely, the capacity to encode categories can potentially exist at the level of integrating genomic enhancer elements that are being described with increasing frequency (Flores et al., 2000; Guss et al., 2001). In conclusion, the larval NMJ has proved to be a highly tractable model synapse for addressing several questions in synaptic growth and physiology. The ease and elegance of genetic analysis combined with the clear separation of two parameters of neural plasticity, growth and strength, make this a paradigm that oVers a high degree of resolution. As information from various approaches continues to accumulate, the NMJ will continue to be used to elucidate the function of these ‘‘plasticity genes.’’ If past experience is of any guide, these results will typically be highly conserved and bear directly on our understanding of how we learn, and what goes wrong in cases in which we fail to do so. Acknowledgment

We acknowledge generous support for our work on the roles of transcription at the NMJ by grants from NIH/NIDA (DA15495; DA17749) and the Science Foundation of Ireland to Mani Ramaswami, as well as NIH grant T32 CA09213 to Subhabrata Sanyal. Subhabrata Sanyal wishes to acknowledge Drs. Rick Levine and Sujata Bhattacharyya for suggestions and useful discussions.

References

Abraham, W. C., Dragunow, M., and Tate, W. P. (1991). The role of immediate early genes in the stabilization of long-term potentiation. Mol. Neurobiol. 5, 297–314.

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301

Alarcon, J. M., Malleret, G., Touzani, K., Vronskaya, S., Ishii, S., Kandel, E. R., and Barco, A. (2004). Chromatin acetylation, memory, and LTP are impaired in CBPþ/
mice: A model for the cognitive deficit in Rubinstein-Taybi syndrome and its amelioration. Neuron 42, 947–959. Alberini, C. M., Ghirardi, M., Metz, R., and Kandel, E. R. (1994). C/EBP is an immediate-early gene required for the consolidation of long-term facilitation in Aplysia. Cell 76, 1099–1114. Bailey, C. H., Giustetto, M., Zhu, H., Chen, M., and Kandel, E. R. (2000). A novel function for serotoninmediated short-term facilitation in Aplysia: Conversion of a transient, cell-wide homosynaptic hebbian plasticity into a persistent, protein synthesis-independent synapse-specific enhancement. Proc. Natl. Acad. Sci. USA 97, 11581–11586. Bartsch, D., Ghirardi, M., Skehel, P. A., Karl, K. A., Herder, S. P., Chen, M., Bailey, C. H., and Kandel, E. R. (1995). Aplysia CREB2 represses long-term facilitation: Relief of repression converts transient facilitation into long-term functional and structural change. Cell 83, 979–992. Bartsch, D., Casadio, A., Karl, K. A., Serodio, P., and Kandel, E. R. (1998). CREB1 encodes a nuclear activator, a repressor, and a cytoplasmic modulator that form a regulatory unit critical for long-term facilitation. Cell 95, 211–223. Bibb, J. A., Chen, J., Taylor, J. R., Svenningsson, P., Nishi, A., Snyder, G. L., Yan, Z., Sagawa, Z. K., Ouimet, C. C., Nairn, A. C., Nestler, E. J., and Greengard, P. (2001). EVects of chronic exposure to cocaine are regulated by the neuronal protein Cdk5. Nature 410, 376–380. Brand, A. H., and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415. Broadie, K. S. (1995). Genetic dissection of the molecular mechanisms of transmitter vesicle release during synaptic transmission. J. Physiol. Paris 89, 59–70. Broadie, K. S., and Bate, M. (1993a). Innervation directs receptor synthesis and localization in Drosophila embryo synaptogenesis. Nature 361, 350–353. Broadie, K. S., and Bate, M. (1993b). Activity-dependent development of the neuromuscular synapse during Drosophila embryogenesis. Neuron 11, 607–619. Broihier, H. T., and Skeath, J. B. (2002). Drosophila homeodomain protein dHb9 directs neuronal fate via crossrepressive and cell-nonautonomous mechanisms. Neuron 35, 39–50. Byers, D., Davis, R. L., and Kiger, J. A., Jr. (1981). Defect in cyclic AMP phosphodiesterase due to the dunce mutation of learning in Drosophila melanogaster. Nature 289, 79–81. Cantera, R., Kozlova, T., Barillas-Mury, C., and Kafatos, F. C. (1999). Muscle structure and innervation are aVected by loss of Dorsal in the fruit fly, Drosophila melanogaster. Mol. Cell. Neurosci. 13, 131–141. Castellucci, V. F., Frost, W. N., Goelet, P., Montarolo, P. G., Schacher, S., Morgan, J. A., Blumenfeld, H., and Kandel, E. R. (1986). Cell and molecular analysis of long-term sensitization in Aplysia. J. Physiol. Paris 81, 349–357. Certel, S. J., and Thor, S. (2004). Specification of Drosophila motoneuron identity by the combinatorial action of POU and LIM-HD factors. Development 131, 5429–5439. Chen, A., Muzzio, I. A., Malleret, G., Bartsch, D., Verbitsky, M., Pavlidis, P., Yonan, A. L., Vronskaya, S., Grody, M. B., Cepeda, I., Gilliam, T. C., and Kandel, E. R. (2003). Inducible enhancement of memory storage and synaptic plasticity in transgenic mice expressing an inhibitor of ATF4 (CREB-2) and C/EBP proteins. Neuron 39, 655–669. Chen, J., Zhang, Y., Kelz, M. B., SteVen, C., Ang, E. S., Zeng, L., and Nestler, E. J. (2000). Induction of cyclin-dependent kinase 5 in the hippocampus by chronic electroconvulsive seizures: Role of (Delta)FosB. J. Neurosci. 20, 8965–8971. Cirelli, C., and Tononi, G. (2000). DiVerential expression of plasticity-related genes in waking and sleep and their regulation by the noradrenergic system. J. Neurosci. 20, 9187–9194. Cirelli, C., LaVaute, T. M., and Tononi, G. (2005). Sleep and wakefulness modulate gene expression in Drosophila. J. Neurochem. 94, 1411–1419.

302

SANYAL AND RAMASWAMI

Crosio, C., Heitz, E., Allis, C. D., Borrelli, E., and Sassone-Corsi, P. (2003). Chromatin remodeling and neuronal response: Multiple signaling pathways induce specific histone H3 modifications and early gene expression in hippocampal neurons. J. Cell Sci. 116, 4905–4914. Dash, P. K., Hochner, B., and Kandel, E. R. (1990). Injection of the cAMP-responsive element into the nucleus of Aplysia sensory neurons blocks long-term facilitation. Nature 345, 718–721. Daval, J. L., Nakajima, T., Gleiter, C. H., Post, R. M., and Marangos, P. J. (1989). Mouse brain c-fos mRNA distribution following a single electroconvulsive shock. J. Neurochem. 52, 1954–1997. Davis, R. L. (2005). Olfactory memory formation in Drosophila: From molecular to systems neuroscience. Annu. Rev. Neurosci. 28, 275–302. Davis, G. W., Schuster, C. M., and Goodman, C. S. (1996). Genetic dissection of structural and functional components of synaptic plasticity. III. CREB is necessary for presynaptic functional plasticity. Neuron 17, 669–679. Devaud, J. M., Acebes, A., and Ferrus, A. (2001). Odor exposure causes central adaptation and morphological changes in selected olfactory glomeruli in Drosophila. J. Neurosci. 21, 6274–6282. DeZazzo, J., Sandstrom, D., de Belle, S., Velinzon, K., Smith, P., Grady, L., DelVecchio, M., Ramaswami, M., and Tully, T. (2000). Nalyot, a mutation of the Drosophila myb-related Adf1 transcription factor, disrupts synapse formation and olfactory memory. Neuron 27, 145–158. DuVy, J. B. (2002). GAL4 system in Drosophila: A fly geneticist’s Swiss army knife. Genesis 34, 1–15. Flores, G. V., Duan, H., Yan, H., Nagaraj, R., Fu, W., Zou, Y., Noll, M., and Banerjee, U. (2000). Combinatorial signaling in the specification of unique cell fates. Cell 103, 75–85. Fregnac, Y. (1996). Dynamics of functional connectivity in visual cortical networks: An overview. J. Physiol. Paris 90, 113–139. Frey, U., Krug, M., Reymann, K. G., and Matthies, H. (1988). Anisomycin, an inhibitor of protein synthesis, blocks late phases of LTP phenomena in the hippocampal CA1 region in vitro. Brain Res. 452, 57–65. Fujioka, M., Lear, B. C., Landgraf, M., Yusibova, G. L., Zhou, J., Riley, K. M., Patel, N. H., and Jaynes, J. B. (2003). Even-skipped, acting as a repressor, regulates axonal projections in Drosophila. Development 130, 5385–5400. Guan, Z., Giustetto, M., Lomvardas, S., Kim, J. H., Miniaci, M. C., Schwartz, J. H., Thanos, D., and Kandel, E. R. (2002). Integration of long-term-memory-related synaptic plasticity involves bidirectional regulation of gene expression and chromatin structure. Cell 111, 483–493. Guan, Z., Kim, J. H., Lomvardas, S., Holick, K., Xu, S., Kandel, E. R., and Schwartz, J. H. (2003). Pp 38 MAP kinase mediates both short-term and long-term synaptic depression in Aplysia. J. Neurosci. 23, 7317–7325. Guan, Z., Saraswati, S., Adolfsen, B., and Littleton, J. T. (2005). Genome-wide transcriptional changes associated with enhanced activity in the Drosophila nervous system. Neuron 48, 91–107. Guss, K. A., Nelson, C. E., Hudson, A., Kraus, M. E., and Carroll, S. B. (2001). Control of a genetic regulatory network by a selector gene. Science 292, 1164–1167. Heisenberg, M., Borst, A., Wagner, S., and Byers, D. (1985). Drosophila mushroom body mutants are deficient in olfactory learning. J. Neurogenet. 2, 1–30. Herdegen, T., and Leah, J. D. (1998). Inducible and constitutive transcription factors in the mammalian nervous system: Control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins. Brain Res. Rev. 28, 370–490. Hevroni, D., Rattner, A., Bundman, M., Lederfein, D., Gabarah, A., Mangelus, M., Silverman, M. A., Kedar, H., Naor, C., Kornuc, M., Hanoch, T., Seger, R., et al. (1998). Hippocampal plasticity involves extensive gene induction and multiple cellular mechanisms. J. Mol. Neurosci. 10, 75–98. Hiroi, N., Marek, G. J., Brown, J. R., Ye, H., Saudou, F., Vaidya, V. A., Duman, R. S., Greenberg, M. E., and Nestler, E. J. (1998). Essential role of the fosB gene in molecular, cellular, and behavioral actions of chronic electroconvulsive seizures. J. Neurosci. 18, 6952–6962.

TRANSCRIPTIONAL REGULATION OF NEURAL PLASTICITY

303

JeVeris, G. S., Marin, E. C., Stocker, R. F., and Luo, L. (2001). Target neuron prespecification in the olfactory map of Drosophila. Nature 414, 204–208. JeVeris, G. S., Marin, E. C., Watts, R. J., and Luo, L. (2002). Development of neuronal connectivity in Drosophila antennal lobes and mushroom bodies. Curr. Opin. Neurobiol. 12, 80–86. Jones, M. W., Errington, M. L., French, P. J., Fine, A., Bliss, T. V., Garel, S., Charnay, P., Bozon, B., Laroche, S., and Davis, S. (2001). A requirement for the immediate early gene Zif268 in the expression of late LTP and long-term memories. Nat. Neurosci. 4, 289–296. Klann, E., and Dever, T. E. (2004). Biochemical mechanisms for translational regulation in synaptic plasticity. Nat. Rev. Neurosci. 5, 931–942. Koh, Y. H., Ruiz-Can˜ada, C., Gorczyca, M., and Budnik, V. (2002). The Ras1-mitogen-activated protein kinase signal transduction pathway regulates synaptic plasticity through fasciclin IImediated cell adhesion. J. Neurosci. 22, 2496–2504. Komiyama, T., Johnson, W. A., Luo, L., and JeVeris, G. S. (2003). From lineage to wiring specificity. POU domain transcription factors control precise connections of Drosophila olfactory projection neurons. Cell 112, 157–167. Korzus, E., Rosenfeld, M. G., and Mayford, M. (2004). CBP histone acetyltransferase activity is a critical component of memory consolidation. Neuron 42, 961–972. Labrador, J. P., O’keefe, D., Yoshikawa, S., McKinnon, R. D., Thomas, J. B., and Bashaw, G. J. (2005). The homeobox transcription factor even-skipped regulates netrin-receptor expression to control dorsal motor-axon projections in Drosophila. Curr. Biol. 15, 1413–1419. Landgraf, M., Roy, S., Prokop, A., VijayRaghavan, K., and Bate, M. (1999). Even-skipped determines the dorsal growth of motor axons in Drosophila. Neuron 22, 43–52. Livingstone, M. S., Sziber, P. P., and Quinn, W. G. (1984). Loss of calcium/calmodulin responsiveness in adenylate cyclase of rutabaga, a Drosophila learning mutant. Cell 37, 205–215. Lonze, B. E., and Ginty, D. D. (2002). Function and regulation of CREB family transcription factors in the nervous system. Neuron 35, 605–623. Marek, K. W., Ng, N., Fetter, R., Smolik, S., Goodman, C. S., and Davis, G. W. (2000). A genetic analysis of synaptic development: Pre- and postsynaptic dCBP control transmitter release at the Drosophila NMJ. Neuron 25, 537–547. Marin, E. C., Watts, R. J., Tanaka, N. K., Ito, K., and Luo, L. (2005). Developmentally programmed remodeling of the Drosophila olfactory circuit. Development 132, 725–737. Marques, G. (2005). Morphogens and synaptogenesis in Drosophila. J. Neurobiol. 64, 417–434. Martin, K. C. (2004). Local protein synthesis during axon guidance and synaptic plasticity. Curr. Opin. Neurobiol. 14, 305–310. Mathew, D., Ataman, B., Chen, J., Zhang, Y., Cumberledge, S., and Budnik, V. (2005). Wingless signaling at synapses is through cleavage and nuclear import of receptor DFrizzled2. Science 310, 1344–1347. Mauelshagen, J., Parker, G. R., and Carew, T. J. (1996). Dynamics of induction and expression of long-term synaptic facilitation in Aplysia. J. Neurosci. 16, 7099–7108. Mauelshagen, J., SherV, C. M., and Carew, T. J. (1998). DiVerential induction of long-term synaptic facilitation by spaced and massed applications of serotonin at sensory neuron synapses of Aplysia californica. Learn. Mem. 5, 246–256. McCabe, B. D., Marques, G., Haghighi, A. P., Fetter, R. D., Crotty, M. L., Haerry, T. E., Goodman, C. S., and O’Connor, M. B. (2003). The BMP homolog Gbb provides a retrograde signal that regulates synaptic growth at the Drosophila neuromuscular junction. Neuron 39, 241–254. McDonald, J. A., Fujioka, M., Odden, J. P., Jaynes, J. B., and Doe, C. Q. (2003). Specification of motoneuron fate in Drosophila: Integration of positive and negative transcription factor inputs by a minimal eve enhancer. J. Neurobiol. 57, 193–203. Mello, C. V., and Clayton, D. F. (1994). Song-induced ZENK gene expression in auditory pathways of songbird brain and its relation to the song control system. J. Neurosci. 14, 6652–6666.

304

SANYAL AND RAMASWAMI

Mello, C. V., and Ribeiro, S. (1998). ZENK protein regulation by song in the brain of songbirds. J. Comp. Neurol. 393, 426–438. Mello, C. V., Velho, T. A., and Pinaud, R. (2004). Song-induced gene expression: A window on song auditory processing and perception. Ann. NY Acad. Sci. 1016, 263–281. Nakajima, T., Daval, J. L., Gleiter, C. H., Deckert, J., Post, R. M., and Marangos, P. J. (1989). C-fos mRNA expression following electrical-induced seizure and acute nociceptive stress in mouse brain. Epilepsy Res. 4, 156–159. Nestler, E. J., Barrot, M., and Self, D. W. (2001). DeltaFosB: A sustained molecular switch for addiction. Proc. Natl. Acad. Sci. USA 98, 11042–11046. Packard, M., Koo, E. S., Gorczyca, M., Sharpe, J., Cumberledge, S., and Budnik, V. (2002). The Drosophila Wnt, wingless, provides an essential signal for pre- and postsynaptic diVerentiation. Cell 111, 319–330. Pinaud, R. (2004). Experience-dependent immediate early gene expression in the adult central nervous system: Evidence from enriched-environment studies. Int. J. Neurosci. 114, 321–333. Pompeiano, M., Cirelli, C., and Tononi, G. (1994). Immediate-early genes in spontaneous wakefulness and sleep: Expression of c-fos and NGFI-A mRNA and protein. J. Sleep Res. 3, 80–96. Richter, J. D. (2001). Think globally, translate locally: What mitotic spindles and neuronal synapses have in common. Proc. Natl. Acad. Sci. USA 98, 7069–7071. Sacks, O. (1998). ‘‘The Man Who Mistook His Wife for a Hat: And Other Clinical Tales.’’ Touchstone Publishing Company, New York, USA. Sanyal, S., Sandstrom, D. J., HoeVer, C. A., and Ramaswami, M. (2002). AP-1 functions upstream of CREB to control synaptic plasticity in Drosophila. Nature 416, 870–874. Sanyal, S., Narayanan, R., Consoulas, C., and Ramaswami, M. (2003). Evidence for cell autonomous AP1 function in regulation of Drosophila motor-neuron plasticity. BMC Neurosci. 4, 20. Sanyal, S., Kim, S. M., and Ramaswami, M. (2004). Retrograde regulation in the CNS; neuronspecific interpretations of TGF-beta signaling. Neuron 41, 845–848. Schuster, C. M., Davis, G. W., Fetter, R. D., and Goodman, C. S. (1996). Genetic dissection of structural and functional components of synaptic plasticity. I. Fasciclin II controls synaptic stabilization and growth. Neuron 17, 641–654. Seto, E. S., and Bellen, H. J. (2004). The ins and outs of Wingless signaling. Trends Cell Biol. 14, 45–53. Sharma, S. K., SherV, C. M., Shobe, J., Bagnall, M. W., Sutton, M. A., and Carew, T. J. (2003). DiVerential role of mitogen-activated protein kinase in three distinct phases of memory for sensitization in Aplysia. J. Neurosci. 23, 3899–3907. Shi, Y., and Massague, J. (2003). Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113, 685–700. Silver, S. J., and Rebay, I. (2005). Signaling circuitries in development: Insights from the retinal determination gene network. Development 132, 3–13. Skeath, J. B., and Thor, S. (2003). Genetic control of Drosophila nerve cord development. Curr. Opin. Neurobiol. 13, 8–15. Stanton, P. K., and Sarvey, J. M. (1984). Blockade of long-term potentiation in rat hippocampal CA1 region by inhibitors of protein synthesis. J. Neurosci. 4, 3080–3088. Steward, O., and Schuman, E. M. (2001). Protein synthesis at synaptic sites on dendrites. Annu. Rev. Neurosci. 24, 299–325. Sutton, M. A., and Schuman, E. M. (2005). Local translational control in dendrites and its role in long-term synaptic plasticity. J. Neurobiol. 64, 116–131. Thompson, K. R., Otis, K. O., Chen, D. Y., Zhao, Y., O’Dell, T. J., and Martin, K. C. (2004). Synapse to nucleus signaling during long-term synaptic plasticity; a role for the classical active nuclear import pathway. Neuron 44, 997–1009.

TRANSCRIPTIONAL REGULATION OF NEURAL PLASTICITY

305

Thor, S., and Thomas, J. (2002). Motor neuron specification in worms, flies and mice: Conserved and ‘‘lost’’ mechanisms. Curr. Opin. Genet. Dev. 12, 558–564. Thor, S., Andersson, S. G., Tomlinson, A., and Thomas, J. B. (1999). A LIM-homeodomain combinatorial code for motor-neuron pathway selection. Nature 397, 76–80. Tully, T., and Quinn, W. G. (1985). Classical conditioning and retention in normal and mutant Drosophila melanogaster. J. Comp. Physiol. [A] 157, 263–277. Waddell, S., and Quinn, W. G. (2001). Flies, genes, and learning. Annu. Rev. Neurosci. 24, 1283–1309. Wallace, C. S., Withers, G. S., Weiler, I. J., George, J. M., Clayton, D. F., and Greenough, W. T. (1995). Correspondence between sites of NGFI-A induction and sites of morphological plasticity following exposure to environmental complexity. Brain Res. Mol. Brain Res. 32, 211–220. Woolf, C. J., and Costigan, M. (1999). Transcriptional and posttranslational plasticity and the generation of inflammatory pain. Proc. Natl. Acad. Sci. USA 96, 7723–7730. Yin, J. C., Wallach, J. S., Del Vecchio, M., Wilder, E. L., Zhou, H., Quinn, W. G., and Tully, T. (1994). Induction of a dominant negative CREB transgene specifically blocks long-term memory in Drosophila. Cell 79, 49–58. Yin, J. C., Del Vecchio, M., Zhou, H., and Tully, T. (1995). CREB as a memory modulator: Induced expression of a DCREB2 activator isoform enhances long-term memory in Drosophila. Cell 81, 107–115. Zhou, F. Q., Walzer, M. A., and Snider, W. D. (2004). Turning on the machine: Genetic control of axon regeneration by c-Jun. Neuron 43, 1–2.