synaptic tagging - Semantic Scholar

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SYNAPTIC TAGGING — WHO’S IT? Kelsey C. Martin* and Kenneth S. Kosik‡ A synaptic tag transiently marks a synapse after activation in a way that allows the local recognition of transcriptional products to effect an enduring change in transmission efficiency. The idea of the synaptic tag as a single molecule might be misguided; instead, a process such as activation of local translation or cytoskeletal reorganization could mark a synapse. A change of this nature might be modulated directly or indirectly by transcriptional products that, for example, modulate translational activity, mRNA stability or protein degradation, or are ensconced in a cytoskeletal configuration, and thereby lead to long-term changes in synaptic strength.

*Department of Psychiatry and Biobehavioral Sciences and Department of Biological Chemistry, Brain Research Institute, University of California, Los Angeles, 695 Charles Young Drive South, Los Angeles, California 90095-1761, USA. ‡ Department of Neurology, Brigham and Women’s Hospital and Harvard Medical School, Harvard Institute of Medicine, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. e-mails: [email protected]; [email protected] doi:10.1038/nrn942

Our current view of the integrated cellular and molecular units that seamlessly operate as a neural information-storage system has emerged from experiments in a diverse range of organisms. A significant challenge for the neuroscience community is to fit the stock of ‘learning-and-memory’ molecules that have been identified so far with the cellular and physiological observations that are associated with enduring synaptic changes. Single synapses or sets of synapses can undergo highly selective modifications when stimulated and, although any given neuron receives thousands of synaptic contacts, each of them can potentially be modified in an independent manner for long periods of time. Because this type of synaptic modification requires both transcription and translation, the problem of targeting gene products from the nucleus to the few activated synapses in a vast dendritic tree has been solved by the neuron in ways that we do not yet fully understand. The synaptic-tagging hypothesis has been put forward as a way to address this problem. This hypothesis proposes that the products of gene expression are delivered throughout the cell, but that they function to increase synaptic strength only at synapses that have been ‘tagged’ by previous synaptic activity. Despite strong evidence for some type of synaptic tag, which we discuss in this review, its identification and the mechanisms by which the products of gene expression are ‘captured’ by tagged synapses have remained elusive.

NATURE REVIEWS | NEUROSCIENCE

Evidence for tagging

Key experiments in two systems — Aplysia and rat — have confirmed the existence of a synaptic tag or synaptic ‘mark’. In a culture system, a single bifurcated Aplysia sensory neuron can form synaptic contacts with two spatially separated motor neurons (FIG. 1). Delivery of five puffs of serotonin (5-hydroxytryptamine or 5-HT) to one contact selectively enhances synaptic efficacy at that synapse without altering the efficacy of the other contact (a phenomenon that has been termed branch-specific facilitation)1. This increase in synaptic potency persists for more than 24 hours and depends on transcription, as it can be blocked by the transcriptional inhibitor actinomycin D. Transcriptional activation seems to be mediated by the cyclic-AMP-responsive element (CRE)binding protein (CREB), because microinjection of anti-CREB antibodies into the sensory neuron blocks the branch-specific facilitation. So, a single cell can change the strength of its connections in a manner that depends on transcription in the nucleus, but is spatially restricted to a single subset of synapses (FIG. 1). Synaptic tagging can be shown in this experimental system when a single puff of serotonin is delivered to one contact and five puffs are applied to the other connection. A single puff of serotonin produces only transient facilitation, but if five puffs are applied to the other contact, the facilitation produced by a single puff is long-lasting 1 (FIG. 1). Importantly, to produce long-term facilitation (LTF), the single pulse of serotonin must be VOLUME 3 | OCTOBER 2002 | 8 1 3

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Figure 1 | Synaptic tagging in cultures of sensory and motor Aplysia neurons. a | Photomicrograph of a single, bifurcated sensory neuron making synaptic contact with two spatially separated motor neurons. A perfusion pipette is used to deliver puffs of serotonin (5-hydroxytryptamine or 5-HT) locally to the connection made onto one of the motor neurons. b | Five puffs, but not a single puff, of serotonin produce a long-lasting (24 hour) increase in the amplitude of the excitatory postsynaptic synaptic potential (EPSP), providing a case of branch-specific long-term facilitation (LTF). This branch-specific facilitation is blocked by the bath application of actinomycin D and by the microinjection of anti-CREB (cyclic-AMP-responsive element (CRE)-binding protein) antibodies into the sensory neuron, indicating that LTF requires CREB-mediated transcription in the sensory neuron. c | The LTF produced by five puffs of serotonin can be ‘captured’ by the opposite branch if a single pulse of serotonin is given within a discrete time window with respect to the five puffs. Applying one puff of serotonin to the other branch, either simultaneously (left) or within 1 hour of the five puffs (middle), results in LTF in both branches. This is not the case if the single pulse is given after 4 hours (right). d | Schematic of these phenomena. Five puffs of serotonin activate a retrograde signal that induces CREB-mediated transcription in the nucleus (blue arrow). The products of gene expression are delivered throughout the cell (red arrows), but only function to increase synaptic strength at sites that have been ‘tagged’ by synaptic activity. Five puffs of serotonin produce a synaptic tag and also turn on gene expression; a single puff of serotonin produces a synaptic tag and captures the products of gene expression that are induced by the five puffs to the other branch.

SCHAFFER COLLATERALS

Axons of the CA3 pyramidal cells of the hippocampus that form synapses with the apical dendrites of CA1 neurons.

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given within a discrete time window — either 1–2 hours before, or 1–4 hours after the five pulses of serotonin are applied to the other connection. These observations indicate that long-term synaptic changes at one synapse can trigger a cell-wide process that is captured by another synapse that has experienced a level of activation that would otherwise produce only short-term changes. Furthermore, they show that this capture phenomenon has a transient lifetime. The phenomenon of synaptic tagging has also been seen in the rodent hippocampus (FIG. 2). Weak activation of the SCHAFFER COLLATERAL projections to area CA1 produces short-lasting increases in synaptic efficacy — a phenomenon that is termed early long-term potentiation (E-LTP). By contrast, strong stimulation produces a long-lasting form of LTP — late LTP (L-LTP) — that depends on transcription and translation. Synaptic tagging occurs when weak stimulation that activates a

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small number of synapses is followed by strong stimulation of another set of synapses that terminate on the same postsynaptic neuron. This causes the synapses that were activated by the weak stimulation to express L-LTP2, but only if the weak stimulation is delivered 1–2 hours before or less than 2.5–3 hours after the strong stimulation2,3 (FIG. 2). Again, these results indicate that the weak stimulus created a synaptic tag that could ‘hijack’ the products of gene expression, resulting in persistent synaptic strengthening of synapses that would otherwise express only E-LTP. These experiments show that the products of transcription are delivered throughout the cell, and that they persist for a specific period, during which they can be captured by subthreshold synaptic activity at another site in the cell. Together, the studies in Aplysia and rat illustrate two important temporal features of the synaptic tag. First, once a neuron has been stimulated to undergo long-term,

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Figure 2 | Synaptic tagging in rodent hippocampal neurons. a | In a hippocampal slice, two stimulating electrodes are used to stimulate two independent pathways — S1 and S2 — that project to the same neuronal population in area CA1. b | A single train of high-frequency stimulation to one of the pathways produces long-term potentiation (LTP) that decays after 1.5 hours (control). By contrast, three trains produce LTP that persists for at least 8 hours and is sensitive to inhibitors of gene transcription. If a single train is given to S2 either before (right panel) or after (left panel) three tetanic stimuli are applied to S1, persistent LTP occurs in both pathways. This indicates that the single tetanus produces a synaptic tag that can ‘capture’ the products of gene expression that are induced by the three trains. c | This synaptic tag does not depend on local protein synthesis. Blockade of protein synthesis by anisomycin (red bars) during the three trains of stimulation inhibits persistent LTP in both pathways (left panel). However, once the three trains have been given to S1, subsequent blockade of protein synthesis does not inhibit LTP in S2, indicating that a single tetanus produces a protein-synthesis-independent tag that can capture persistent LTP.

A protein of the postsynaptic density that can interact with metabotropic glutamate receptors, regulating their membrane insertion and their interaction with other postsynaptic proteins.

transcription-dependent synaptic plasticity, the cell can use stimuli that normally produce transient changes in synaptic strength to induce more persistent changes. This capability persists for a limited duration (1–4 hours). Second, stimuli that produce transient synaptic strengthening produce an alteration at the synapse — the synaptic tag — that persists within a specific time window (1–2 hours), during which the tag can capture products of gene expression that are induced by strong activity elsewhere in the neuron.

UBIQUITIN

Candidate synaptic tags

A molecule that is attached to lysine residues of other proteins, often as a tag for their rapid cellular degradation by the proteasome.

The validity of the synaptic-tagging hypothesis rests on the identification of the tag. Any candidate should fulfil the following criteria. First, it should be spatially restricted (that is, it must be local). Second, it should be

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time-limited and reversible. Third, it should be able to interact with cell-wide molecular events that occur after strong stimulation (most commonly thought to involve changes in gene expression) to produce long-term, synapse-specific strengthening. So, from a broad perspective, anything that provides a spatially restricted trace of activity is a candidate for the synaptic tag. The stimuli that can produce a synaptic tag are not necessarily sufficient to activate transcription, and are therefore frequently considered to be subthreshold for long-term plasticity. Indeed, this characteristic was used to confirm the existence of a tag in Aplysia neurons and in rodent hippocampal neurons, as described above. Such stimuli induce a host of changes at the synapse, and any number of these changes could potentially serve as a tag. Many of these changes are related to the activity and strength of the synapse, such as the rapid addition of AMPA (α-amino-3-hydroxy-5-methyl4-isoxazole propionic acid) receptors to ionotropic glutamate receptor clusters4, the lateral mobility of NMDA (N-methyl-D-aspartate) receptors between synaptic and extrasynaptic sites5, HOMER-mediated insertion of the metabotropic glutamate receptor mGluR5 into the membrane6, and palmitate cycling on postsynaptic density protein 95 (PSD95; REF. 7). Such events could serve as localized traces of previous synaptic activity that are able to produce synaptic strengthening on their own within a limited time period. However, to function as synaptic tags, they would need to be able to interact with cell-wide events to produce local and persistent increases in synaptic efficacy. As such, they would function to integrate activity over temporal and spatial domains. Kinases. Persistently active kinases meet several of the criteria for a tag, as they allow a synapse to ‘remember’ previous activity in a spatially restricted and reversible manner. Calcium/calmodulin-dependent protein kinase II (CaMKII), which becomes autonomously and persistently active by autophosphorylation, has been shown to be activated by, and necessary for, long-term plasticity and long-term memory8. The atypical protein kinase C known as protein kinase Mζ (PKM-ζ), the persistent activity of which requires protein synthesis9, has been shown to be both necessary and sufficient for the maintenance of L-LTP in the hippocampus10. PKM-ζ is not only necessary for, but can also enhance, longterm memory for olfactory learning in Drosophila melanogaster11. Local, persistent changes in the activity of these kinases that are induced by synaptic stimulation could serve as synaptic tags that combine with products of gene expression to produce enduring but local changes in synaptic efficacy. In Aplysia, the ability of a single puff of serotonin to capture LTF depends on the cAMP-dependent protein kinase (PKA)12. Previous studies have shown that UBIQUITIN-mediated proteolysis of the regulatory subunit of PKA results in a persistently active kinase13, and that this degradation involves the transcriptional induction of a ubiquitin carboxy-terminal hydrolase14,15. Together, these findings raise the possibility that local activation of

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REVIEWS PKA and local regulation of the ubiquitin–PROTEASOME pathway can serve as synaptic tags that combine with transcriptional events (for example, the induction of the ubiquitin carboxy-terminal hydrolase) to produce persistent and local synaptic strengthening.

PROTEASOME

A protein complex responsible for degrading intracellular proteins that have been tagged for destruction by the addition of ubiquitin. HYPOMORPH

A mutant that expresses less than the normal amount of a given gene product. ADHERENS JUNCTION

A cell–cell junction also known as zonula adherens, which is characterized by the intracellular insertion of microfilaments. If intermediate filaments are inserted in lieu of microfilaments, the resulting junction is referred to as a desmosome. PHOTOCONDUCTIVE STIMULATION

A method whereby light can be used to stimulate the activity of subpopulations of synapses in vitro. Cells are cultured on a silicon chip and light is applied to single neurons. As the conductivity of silicon changes in response to light, it is possible to pair local illumination of single neurons with subthreshold electrical stimulation of the whole culture. So, the stimulus intensity will reach threshold for activation solely in the illuminated cell. INTERNAL RIBOSOME ENTRY SITE

A sequence that is inserted between the coding regions of two proteins and allows efficient assembly of the ribosome complex in the middle of a transcript, leading to translation of the second protein.

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Adhesion molecules. Changes in adhesion are likely to underlie the morphological changes that are associated with synaptic strengthening16. Studies of activitydependent development of the fly neuromuscular junction have highlighted a potential role for adhesion molecules as synaptic tags. Goodman and colleagues found that downregulation of the adhesion molecule fasciclin II produced increases in the number of boutons at the neuromuscular junction without any alteration in synaptic strength17. Overexpression of CREB in the motor neuron also failed to produce alterations in synaptic strength at the neuromuscular junction. However, if CREB was overexpressed in fasciclin II HYPOMORPHS, an increase in synaptic strength was observed at this synapse18. So, the downregulation of fasciclin II sets the stage for CREB-induced transcription to alter synaptic strength, indicating that changes in adhesion molecules that are induced by activity at the synapse could serve as a synaptic tag. Although the downregulation of fasciclin II in Drosophila seems to occur in a cell-wide manner, localized activity might lead to local downregulation. Consistent with the idea that adhesion-molecule dynamics could serve as synaptic tags, alterations in cell-adhesion molecules at the synapse have been found to occur during many forms of synaptic plasticity. For example, the Aplysia homologue of fasciclin II, apCAM, has been shown to be internalized in response to serotonin19,20, and the concentration of the neural celladhesion molecule (NCAM) has been shown to control synapse number in cultured neurons21. Also, the cadherins have been shown to dimerize and alter their conformation during depolarization22, and the number of cadherin-positive synapses has been shown to increase in a protein-synthesis-dependent manner during LTP23. Moreover, neural activity alters the localization of β-catenin, which couples the cadherins to the actin cytoskeleton; depolarization causes the translocation of β-catenin into the postsynaptic spine24. A neural-specific component of the ADHERENS JUNCTION25 — δ-catenin — is also linked to proteins in the postsynaptic compartment26 and affects spine morphology. These effects might be dependent on synaptic activity (K.S.K., unpublished observations). Although these studies indicate that activity can regulate adhesion molecules at the synapse, it remains to be shown that such changes can occur in a spatially restricted manner and can interact with the products of transcription to produce enduring changes in synaptic strength. Actin network. Another potential candidate for a synaptic tag that has recently received significant attention is the actin microfilament network at the synapse. The actin network in neurons is extremely dynamic, and these dynamics have been shown to change with activity27,28. Changes in the actin cytoskeleton probably


accompany changes in cell-adhesion molecules, as most adhesion molecules are linked to the cytoskeleton. In addition, changes in the actin microfilament network are likely to underlie the growth of new synaptic structures that has been observed after repetitive stimulation of hippocampal synapses29–31 and after serotonergic stimulation of Aplysia sensorimotor connections32. Using non-invasive PHOTOCONDUCTIVE STIMULATION to restrict synaptic stimulation spatially, Colicos et al.33 have shown that repetitive tetanic stimulation can lead to remodelling of the actin network at the synapse, both pre- and postsynaptically. These changes are persistent, lasting for at least 18 hours after stimulation. Such local changes in the cytoskeleton could combine with the products of gene expression induced by strong synaptic stimulation to produce enduring, yet spatially restricted alterations in synaptic strength. Again, a requirement for gene expression or other cell-wide events in stabilizing the local changes in synaptic actin remains to be shown. Ion channels. A synaptic tag that allows a neuron to integrate synaptic stimulation at a single synaptic connection over time has recently been described in studies of LTF of the crayfish neuromuscular junction34. Beaumont et al. 34,35 found that presynaptic I h potassium channels become activated during tetanic stimulation, and that this activation generates a synaptic tag, which allows the motor neuron to increase transmitter output by subsequently responding to further stimuli that would otherwise be ineffective. If the activation of the I h channels occurs concomitantly with elevations in presynaptic calcium, the tagged synapses can undergo proteinsynthesis-dependent LTF in response to subthreshold stimuli that are applied at least 1 hour after the tag was set. These experiments indicate that synaptic tags provide a history of activity at the synapse, and can combine with subthreshold stimuli over a 1-hour period to produce long-lasting changes in synaptic efficacy. Interestingly, this particular synaptic tag was found to be dependent on an intact actin cytoskeleton, which, the authors propose, reflects a requirement for stabilizing the localization of the channels at the synapse. Although this was defined as a synaptic tag, there is an important difference between this type of tag and the tag that was defined for LTF in Aplysia and for LTP in the rodent hippocampus. LTF at the crayfish neuromuscular junction does not depend on transcription, but rather on local protein synthesis35. So, this tag does not need to interact with the cell-wide products of transcription but, instead, with locally synthesized proteins to produce persistent synaptic strengthening. Local translation and protein degradation. The activation of local translation meets many of the requirements for a tag that can integrate activity in both temporal and spatial domains. Protein translation at a synapse can alter the composition of that synapse over an extended period of time, and thereby leave a relatively persistent trace of previous activity. In addition, activating translation locally provides a spatial tag, because messenger RNAs that are delivered from the cell soma will be



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Figure 3 | A model of synaptic tagging in which activity-dependent effects on translation serve as the tag. a | Afferent arrows (blue) represent a depolarization-induced signal that is capable of travelling to the nucleus and activating transcription. Efferent arrows (red) represent induced transcriptional products that can be ‘captured’ by the tag and, in this case, effect local translation of messenger RNAs that reside in RNA granules. b | A depolarizing impulse induces the release of mRNAs from translationally silent RNA granules. Although many mRNAs and ribosomes can be released from each granule, only a few are shown. Once released, the mRNAs undergo local translation. A co-translational event may serve as a tag, which, by definition, is recognized by the transcriptional products. It is important to emphasize that the tag persists beyond the time course of the impulse.


preferentially translated at sites at which translation is active. Notably, the percentage of spines that contain polyribosomes increases markedly in CA1 neurons after LTP induction36. Furthermore, after the induction of LTP, the area of the postsynaptic density (PSD) is significantly larger in the spines that contain polyribosomes than in spines that lack them, indicating that the presence of polyribosomes produces structural changes, which, in turn, produce changes in synaptic strength. How might synaptic stimulation produce increases in local translation? In one scenario, depolarization leads to the release of local (dendritic) mRNAs into the translatable polysome pool (FIG. 3). The mRNAs are present in RNA granules, in which they are maintained in proximity to synapses in a translationally silent state37. Depolarization induces mRNA release into the translationally competent polysome pool. Granules oscillate around a cluster of synapses38, and the release of mRNAs from granules could deliver translational products to several synapses within a discrete locale. Alternatively, synaptic activity might activate components of the translational machinery; for example, by phosphorylating specific translation factors39–41 or perhaps by recruiting 42 INTERNAL RIBOSOME ENTRY SITE (IRES)-driven translation . All of these mechanisms would lead to an increase in the translation of a subset of mRNAs, rather than to a global increase in protein synthesis. Although a product of local translation might serve as the tag, it seems more likely that the activation of translation itself or the reorganization of the RNA in the region of activity can serve as a tag that can be recognized by transcription-dependent products. One of the downstream genes that is turned on by CREB activation is brain-derived neurotrophic factor (BDNF), which can modulate synaptic transmission within seconds43–46. BDNF levels increase after learning-related events, presumably as a result of CREB binding to the CRE sequence in its promoter47–49. Not only is BDNF transcription increased in response to stimulation, but its mRNA, along with the mRNA that encodes its receptor TrkB (tyrosine kinase receptor B) is also targeted to dendrites50. Local translation of this dendritically localized BDNF mRNA could result in localized increases in BDNF concentration at stimulated synapses. The dendritic targeting of BDNF mRNA indicates that local synaptic stimulation can not only alter translation and mRNA dynamics locally, but also seems to be able to alter the trafficking of newly synthesized mRNAs from the cell soma. In a beautiful example of this potential type of synaptic tag, the mRNA that encodes the immediate early gene Arc (activity-regulated cytoskeletalassociated protein) has been shown not only to translocate into dendrites, but also to accumulate specifically at the stimulated synaptic sites51. These experiments indicate that the tag provides an anchor for mRNAs, a mechanism that is reminiscent of mRNA targeting during development in Drosophila embryos52. Alternatively, the Arc mRNA might be delivered throughout the dendrite, but selectively stabilized at stimulated synapses or selectively degraded at unstimulated sites.



REVIEWS Protein degradation provides another means of altering the macromolecular composition of synapses. A plausible scenario is that, without the arrival of transcription-dependent products, translational products from locally released mRNAs, or the mRNAs themselves, will undergo degradation. As described above, a requirement for the protein-degradation machinery has been described during the LTF of Aplysia sensorimotor synapses14, as well as during synaptic growth and transmission in Drosophila53. Both local protein synthesis and protein degradation have been shown to be necessary for axon guidance in Xenopus retinal neurons54,55. In all three studies, ubiquitin-mediated proteolysis has been shown to be required. It is therefore possible that local, ubiquitin-mediated protein degradation of specific molecules in response to synaptic stimulation creates a synaptic tag by altering the composition of the synapse, and that this synaptic tag combines with the products of transcription to produce a persistent type of synaptic strengthening. Local translation and synaptic strengthening

MULTIPHOTON MICROSCOPY

A form of microscopy in which a fluorochrome that would normally be excited by a single photon is stimulated quasisimultaneously by several photons of lower energy. Under these conditions, fluorescence increases as a function of the square of the light intensity, and decreases approximately as the square of the distance from the focus. Because of this behaviour, only fluorochrome molecules near the plane of focus are excited, greatly reducing light scattering and photodamage of the sample.

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Together, protein translation and degradation encompass sufficient diversity to explain changes in transmission efficiency at a wide variety of synapses. The presence of polyribosomes, translation factors and a population of mRNAs in the dendrites of vertebrate neurons and in the neurites of invertebrate neurons56 indicates that the machinery that is necessary for translation is available at the synapse. Furthermore, dendrites are competent to translate proteins. This conclusion was supported by an experiment in which tetanic stimulation of the Schaffer collateral pathway led to an increase in the concentration of α-CaMKII protein in the dendrites of postsynaptic neurons57. This increase occurred as early as 5 minutes after the tetanus at a distance of 100–200 µm, a distance too long to be accounted for by invoking the known dendritic transport mechanisms. Protein synthesis was also observed in dendrites using a protein-synthesis reporter in which the coding sequence of the green fluorescent protein was flanked by the 5′ and 3′ untranslated regions of α-CaMKII, conferring both dendritic mRNA localization and translational regulation58. BDNF, a growth factor that is involved in synaptic plasticity, stimulated protein synthesis of the reporter in intact, mechanically or ‘optically’ isolated dendrites. In an elegant study, MULTIPHOTON MICROSCOPY of green fluorescent protein synthesized in living isolated dendrites was used to measure the increase in fluorescence after stimulation with a glutamate receptor agonist. In contrast to the linear increase in the cell-body signal, dendrites showed an exponential increase within spatially stable hotspots59. Translation of membrane proteins has been shown directly in isolated neurites of Lymnaea 60. In Aplysia, in which distal neurites isolated from their cell bodies can survive for extended periods, translation has been directly measured by metabolic labelling and scintillation counting, and is also regulated by serotonergic stimulation1. These experiments show that dendritic translation occurs, but they do not address the question of whether


or not it makes a functional contribution to synaptic strengthening. However, several experiments have shown that synaptic strengthening is dependent on protein synthesis. In Aplysia, forms of synaptic strengthening that are dependent on translation but not on transcription have been described61–63, indicating that synaptic stimulation can regulate translation directly, and that this translation produces synaptic facilitation. Studies in Aplysia have further elucidated a role for local translation during long-term synaptic strengthening. In these experiments, synapse-specific facilitation, induced by the local application of five pulses of serotonin at the connection made onto one motor neuron, was found to require local protein synthesis in the distal processes of the sensory neuron1. When Aplysia sensory neuron somata and their remote motor neuron synapses are simultaneously exposed to serotonin at levels that are insufficient to induce LTF at either site alone, the coincident induction of LTF with spatially distinct properties occurs64. Local protein synthesis occurs immediately at the synapse, followed by transcription and delayed protein synthesis at the soma. In the rodent brain, the enhanced synaptic transmission that occurs after the application of BDNF or neurotrophin 3 (REFS 43–45) depends on local translation65. In these experiments, facilitated transmission was induced at the Schaffer collateral–CA1 pyramidal neuron synapse, and the protein-synthesis inhibitor attenuated the synaptic enhancement within minutes of its application. This result could not be explained by the synthesis and delivery of proteins from the cell soma to the distal site of synaptic enhancement. This conclusion was supported by the isolation of the synaptic regions from either CA1 or CA3, which still showed anisomycin-sensitive enhanced synaptic transmission in response to the trophic factors. More recently, local postsynaptic protein synthesis, triggered by synaptic activation of metabotropic glutamate receptors, was found to modify synaptic transmission within minutes, independently of the cell body 66. One prediction that arises from the hypothesis that dendritic translation is crucial for certain synaptic functions is that a failure to target essential mRNAs to the dendrite will impair function. This prediction has been substantiated by Miller et al.67, who generated a mutant mouse in which the mRNA of α-CaMKII was not present in dendrites, but was restricted to the cell body. They found that the PSD of these mice contained a small fraction of the usual amount of α-CaMKII, indicating that local translation contributes significantly to the amount of this protein in the PSD. Can local translation serve as a tag?

Despite the evidence that local translation can serve as a synaptic tag, it is clear that it is not the only synaptic tag that mediates plasticity and memory. So, although local protein synthesis is required for synapse-specific LTF at Aplysia sensorimotor synapses (that is, it is required at the synapse that receives five pulses of serotonin)1, it is not required for synaptic tagging at rodent hippocampal synapses2,3. As further evidence of the complexity of synaptic tagging, in the case of Aplysia, local translation is



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MICRORNAS

Tiny transcripts of about 22 nucleotides. The functions of these RNAs remain obscure, although they probably serve as antisense regulators of the translation of other RNAs.

not required at the tagged synapse for 24-hour facilitation, but is required for synaptic strengthening at 72 hours, which is accompanied by the stable growth of new synaptic connections12. This finding indicates that local translation might be required for stabilizing new synaptic growth to create persistent synaptic connections and memory. It might be that local protein synthesis will be necessary for synaptic tagging in hippocampal neurons when LTP is examined over more extended time periods. Identification of mRNAs that are localized at the synapse and translated in response to synaptic activity promises to provide further insight into the function of local translation during learning and memory. Identity of the ‘captured’ transcriptional products

Over the past 10–15 years, significant progress has been made in identifying genes that are transcriptionally induced by stimuli that produce long-lasting forms of plasticity and memory. Several approaches have been used, including differential cloning and subtractive hybridization68, microarray analysis69 and genetic screens in model organisms such as Drosophila70. One set of transcriptional products that could be captured by local mRNAs at a tagged synapse are MICRORNAS (miRNAs), a large family of non-coding RNAs71,72. Although a highly speculative possibility at the present time, miRNAs have several properties that make them plausible candidates for recognizing a tag. These mRNAs are not translated into protein, but might modulate translation by binding to mRNAs, thereby regulating their translation. Transcriptional activation might disperse miRNAs widely throughout the dendritic tree, where they recognize mRNAs released locally from RNA granules that are positioned at activated synapses. There is sufficient diversity among miRNAs to implement complex changes in the translational profile of dendritic mRNAs. miRNAs could therefore finely tune the translational response to synaptic activation by affecting the efficiency of translation or the stability of mRNA. Specific insights into the interaction of transcriptionally induced genes with the synaptic tag have recently emerged from studies of mice with restricted and regulated expression of a constitutively active form of the transcriptional activator CREB called VP16CREB73. In these mice, VP16-CREB was expressed selectively in neurons in the hippocampal CA1 field. These neurons, and these neurons alone, showed a reduced threshold for inducing the persistent late phase of LTP, and this induction was independent of transcription. The authors propose that CREB activation turns on

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Martin, K. C. et al. Synapse-specific, long-term facilitation of Aplysia sensory to motor synapses: a function for local protein synthesis in memory storage. Cell 91, 927–938 (1997). Shows that long-lasting, transcription-dependent forms of plasticity can occur in a synapse-specific manner; together with reference 12, this paper provides evidence for synaptic tagging in Aplysia. Frey, U. & Morris, R. G. Synaptic tagging and long-term potentiation. Nature 385, 533–536 (1997). This paper and reference 3 provide evidence for synaptic tagging in rat hippocampal neurons.

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downstream genes, and that the products of these genes are captured by the activated synapses. The interaction of the tag with transcriptional products implements long-lasting LTP. The techniques are now available to identify relevant transcriptional products that are associated with enduring synaptic changes. But it will be more difficult to identify exactly what the transcriptional products recognize among the abundance of changes that occur with synaptic activation. Conclusions

Synaptic tagging allows the synapse, rather than the nucleus, to be the unit of long-lasting plasticity. As the brain contains about 1011 neurons, but 1014 synapses, this greatly increases the information-processing capacity of the brain. Evidence that synaptic tagging can occur during long-lasting forms of plasticity has kindled an intense effort to discover the identity of the synaptic tag. The slate of candidates is wide-ranging, including persistent protein kinases, changes in adhesion molecules at the synapse, alterations in cytoskeletal elements, activation or trafficking of channels, and protein synthesis and translation. Which of these candidates best fits the profile of a synaptic tag? The common characteristic of all of the candidates is that they are able to function as spatially restricted and persistent markers of synaptic activity. At present, the challenge is to understand whether and how each can interact with products of transcription to produce enduring changes in synaptic efficacy. Until this is done, one can only speculate on the advantages of one tag candidate over another. However, one theme that has emerged from studies of synaptic tagging is that, just as multiple molecular mechanisms have been found to underlie LTP induction and memory formation, there seem to be multiple molecular synaptic tags. Each of them might function as a synaptic tag under particular circumstances, and they might be differentially recruited by various stimuli (such as growth factors or neurotransmitters) and mediate plasticity over different time domains (ranging from several hours to several days and beyond). Local translation seems to have a particularly important role in the most persistent forms of plasticity and memory. In fact, from a cell-biological perspective, it would be extremely surprising if a single synaptic molecule or event could integrate synaptic activity over spatial and temporal domains. Instead, it seems more likely that there are several mechanisms by which synaptic activity can leave local traces.

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Acknowledgements We would like to thank M. Barad, M. Mayford, R. Moccia, E. Schuman, W. Sossin and O. Steward for their insightful comments on the manuscript.

Online links DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ actin | AMPA receptors | Arc | BDNF | cadherins | CaMKII | β-catenin | δ-catenin | CREB | mGluR5 | NCAM | neurotrophin 3 | NMDA receptors | PKA | PKM-ζ | TrkB | ubiquitin carboxyterminal hydrolase FURTHER INFORMATION Encyclopedia of Life Sciences: http://www.els.net/ learning and memory | long-term potentiation | protein phosphorylation and long-term synaptic plasticity | protein synthesis and long-term synaptic plasticity Access to this interactive links box is free online.

