extracellular matrix molecules and synaptic plasticity

REVIEWS

EXTRACELLULAR MATRIX MOLECULES AND SYNAPTIC PLASTICITY Alexander Dityatev and Melitta Schachner Interactions between cells and the extracellular matrix (ECM) have long been accepted to have pivotal roles in neural development and regeneration. Recent data also support the involvement of several ECM molecules in synaptic plasticity. Here, we review the present knowledge of the underlying mechanisms. These include interactions with cell surface receptors for ECM molecules coupled to the cytoskeleton and tyrosine kinase activities, and interactions with ion channels or neurotransmitter receptors. We hypothesize that ECM molecules derived from neurons and glia might also shape synaptic plasticity through regulation of organelle trafficking, and by imposing diffusion constraints for neurotransmitters and trophic factors. PROTEOGLYCANS

The proteoglycans have a much higher ratio of polysaccharide to protein than do collagens, fibronectin, and glycoproteins in the extracellular matrix. The polysaccharide chains in proteoglycans are long repeating linear polymers of specific disaccharides called glycosaminoglycans. Usually one sugar is a uronic acid and the other is either N-acetylglucosamine or N-acetylgalactosamine.

Zentrum für Molekulare Neurobiologie, University of Hamburg, Martinistr. 52, 20246 Hamburg, Germany. Correspondence to A.D. or M.S. e-mails: dityatev@zmnh. uni-hamburg.de; melitta.schachner@zmnh. uni-hamburg.de doi:10.1038/nrn1115

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The architecture of a tissue is determined by recognition mechanisms that involve not only cell–cell interactions, but also interactions between cells and the extracellular matrix (ECM). In animals, organized groups of cells are surrounded by an ECM of collagens, PROTEOGLYCANS and glycoproteins. Molecules in the matrix do not only interact with each other — they also activate signal transduction pathways through diverse cell-surface receptors. These pathways orchestrate the inputs from equally diverse ECM molecules to coordinate cell functions such as proliferation, migration, and morphological and biochemical differentiation. In the nervous system, they also coordinate synaptogenesis and synaptic activity. A large proportion of the brain volume is thought to consist of extracellular space1,2 that has few distinguishing features at the ultrastructural level, but the extrasynaptic space contains distinct aggregations of ECM molecules, the functions of which have remained largely unknown. Conspicuous structures that are enriched in ECM molecules in the central nervous system (CNS) are the so-called perineuronal nets3–5, which surround cell bodies and proximal dendrites in a mesh-like structure that interdigitates with synaptic contacts (FIG. 1). Perineuronal nets are heterogeneous in structure and composition, and nets that are associated with different sets of neurons are characterized by unique molecular

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compositions6,7. Furthermore, the synaptic cleft and extrasynaptic space can be regarded as an elaborate form of ECM specialization, the structural and molecular diversity of which remains to be elucidated. Over the last few decades, substantial knowledge has accumulated about the morphogenetic effects of ECM molecules during development and regeneration in the nervous system5,8–13. In addition, the ECM is involved in physiological processes in the adult brain, such as synaptic plasticity, and in pathological conditions such as trauma. In this review, we focus on the role of ECM molecules in regulating synaptic plasticity in the adult nervous system. But before embarking on this discussion, it is worthwhile reviewing some of the basic terminology that is used in the field of synaptic plasticity. What is synaptic plasticity?

The efficacy of synaptic transmission is not fixed, but can vary in an activity-dependent manner. This phenomenon, known as synaptic plasticity, exists in many forms. For example, trains of presynaptic action potentials produce post-tetanic potentiation — an increase in transmitter release that usually lasts for several minutes. An increase in calcium concentration in the presynaptic terminals underlies these changes in release. As this process is often superimposed on other mechanisms,

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a

b

c

d Hyaluronan Lectican

Tenascin-R

Figure 1 | Extracellular matrix (ECM) in the brain. a | Drawing of perineuronal nets by Ramón y Cajal. b,c | Confocal images of perineuronal net ensheathing interneurons in the CA1 region of the hippocampi of wild-type (b) and tenascin-R-deficient (c) mice. In mutants, the perineuronal nets have an abnormal appearance and do not expand into dendrites as in wild-type mice. The nets were labelled with the biotinylated Wisteria floribunda agglutinin and visualized using streptavidin bound to a fluorochrome. Scale bar, 20 µm. Images courtesy of G. Brückner and J. Grosche, Paul Flechsig Institute for Brain Research, University of Leipzig. d | Hypothetical model of the brain ECM. Molecules of the lectican family of chondroitin sulphate proteoglycans (CSPGs), such as aggrecan, versican, brevican and neurocan (red), bind hyaluronan (green) and tenascin-R (blue) and form a ternary complex. To simplify the model, this figure depicts a situation in which all tenascin-R molecules are present as trimers. Other configurations, without tenascin-R and including interactions of ECM molecules with cell surface receptors, are also conceivable. Modified, with permission, from REF. 5  Birkhäuser Publishing (2000).

SPINES

Specialized regions of the dendrite that receive synaptic inputs from other neurons. ACTIVE ZONE

A portion of the presynaptic membrane that faces the postsynaptic density across the synaptic cleft. It constitutes the site of synaptic vesicle clustering, docking and transmitter release. METABOTROPIC

A term that describes a receptor that is associated with G proteins and exerts its effects through enzyme activation.

the operational term of short-term potentiation is used to refer to a transient increase in synaptic efficacy14. In many synapses, intense synaptic activity induces not only short-term synaptic plasticity but also alterations in synaptic efficacy that last for hours, days or even months. The persistent enhancement or reduction of synaptic strength of stimulated synapses is known as long-term potentiation (LTP) or long-term depression (LTD), respectively15,16. Long-term synaptic plasticity involves signalling cascades. The initial triggering events (induction) result in pre- and postsynaptic modifications, which underlie changes in the efficacy of synaptic transmission (expression) that are subsequently stabilized by additional mechanisms (maintenance). In mechanistic terms, induction events for both LTP and LTD might involve presynaptic and/or postsynaptic mechanisms, most commonly the depolarization of neurons and the activation of NMDA (N-methyl-D-aspartate) receptors and/or voltage-dependent Ca2+ channels, which triggers Ca2+ influx into the cells. Ca2+ levels bi-directionally control synaptic efficacy by influencing the balance between the activity of protein kinases and phosphatases, which in turn control the activities of transmitter receptors and other proteins through phosphorylation. These activities are synapseand induction-protocol-specific. For example, Ca2+/ calmodulin-dependent kinase II (CaMKII), a serine/ threonine protein kinase, is a key component of the molecular machinery that underlies LTP in the widely studied CA1 region of the hippocampus17. In addition to protein kinases A and C, and other serine/threonine protein kinases, tyrosine kinase activities have also been implicated in the regulation of synaptic plasticity18. Usually, potentiation in brain slices is recorded within 1 hour after induction of LTP. This early LTP does not involve de novo protein synthesis, in contrast to the late LTP (> 3 hours), for which protein synthesis is an

NATURE REVIEWS | NEUROSCIENCE

important ingredient (reviewed in REF. 19). So, changes in transcription and translation events add another level of complexity to the regulation of synaptic function. How are signalling events translated into an increase or reduction in synaptic responses? Expression of plasticity involves postsynaptic changes in phosphorylation and synaptic delivery of AMPA (α-amino-3-hydroxy-5methyl-4-isoxazole propionic acid) receptors to the postsynaptic membrane20. Potentiation is also accompanied by an increase in the proportion of presynaptically active terminals21. Some forms of LTP involve rapid coordinated changes in the distribution of proteins in the presynaptic and postsynaptic neurons22. A visible manifestation of synaptic plasticity occurs at the structural level — impressive changes in the shapes of dendritic SPINES and axon terminals, profiles of ACTIVE ZONES and postsynaptic densities and, in some instances, of synapse number, can be observed, often within tens of minutes after the stimulus23–25. These changes underscore the flexibility of the adult nervous system to activate morphogenetic programs of differentiation. It is increasingly recognized that neuronal — and probably also glial — activity can result in changes in the predisposition of synapses to undergo subsequent synaptic plasticity, which often appear as changes in the threshold values of synaptic activity and postsynaptic depolarization that are necessary to induce LTP or LTD. The term metaplasticity is used to refer to this ‘higherorder plasticity of synaptic plasticity’26. Metaplasticity might involve little, if any, apparent change in excitatory synaptic transmission, but it impinges significantly on subsequent plasticity. Induction mechanisms that underlie metaplasticity include modulation of NMDAreceptor-mediated and GABA (γ-aminobutyric acid)mediated synaptic transmission, and METABOTROPIC glutamate receptor-mediated second messenger pathways27. One of the manifestations of metaplasticity is the observation that postsynaptic responses are more easily

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REVIEWS reduced by low-frequency stimulation after induction of LTP than in naive synapses. This reversal of LTP is known as depotentiation28. Although these different forms of plasticity have been found throughout the mammalian brain, they have been studied most extensively in the hippocampal formation, particularly at the synapses between the CA3 and CA1 pyramidal neurons — the SCHAFFER COLLATERALS. Most of the following discussion is centred on findings made at this synaptic connection. ECM: from development to synaptic plasticity

Initially, ECM molecules were recognized as important constituents of the neuromuscular junction, owing to their ability to induce accumulation of postsynaptic acetylcholine receptors and presynaptic proteins. Genetic analysis of synaptogenesis with loss- and gain-offunction mutants, combined with in vitro reconstitution of postsynaptic differentiation of the neuromuscular junction, highlighted a crucial role for the nerve-derived heparan sulphate proteoglycan (HSPG) agrin (reviewed in REF. 29). These studies provided a framework for studying synaptogenesis in other types of synapses. Interest in ECM recognition molecules as important contributors to synaptic function increased in the late 1980s, when subsets of synapses in the hippocampus and cerebellum of adult mice were identified to be

immunoreactive for the HSPG-binding neural cell adhesion molecule NCAM180 (REF. 30). Seminal studies by Hockfield and colleagues31–32 in the lateral geniculate nucleus, visual cortex and spinal cord emphasized the activity-dependent expression of CHONDROITIN SULPHATE PROTEOGLYCANS (CSPGs). Soon afterwards, integrins, which are cell surface receptors for several different ECM ligands, were found to be involved in LTP in the hippocampus33. These studies in the mouse were followed by an extensive account of the role of the Aplysia homologue of NCAM, apCAM, in plasticity of sensory–motor synapses34. It took several years to realize that ECM molecules might act together with their cell surface receptors to modulate synaptic efficacy. Investigations into the functional role of the ECM were impressively spurred on in recent years, when mouse mutants became available and could be investigated in parallel with perturbation studies using antibodies, ECM molecule fragments and ECM-degrading enzymes34–40. Early studies on the contributions of ECM molecules to synaptic plasticity might be viewed with suspicion, owing to the large number of molecules that seemed to be involved in LTP41. Because of the complexity and diversity of synaptic plasticity mechanisms, it is not difficult to imagine that more than one ECM molecule would affect synapse formation and synaptic modifications in the adult. However, some problems might have arisen

Box 1 | Criteria to show that extracellular matrix molecules are required for synaptic plasticity 1. Synaptic plasticity is modified by injections of the extracellular matrix (ECM) molecule itself, fragments derived from it, antibodies raised against it, or enzymes that block its functions. 2. Mice that are deficient in an ECM molecule, or transgenic mice with increased expression of the molecule, show abnormal levels of synaptic plasticity in the absence of overt developmental abnormalities. As the ECM has an important role during development, data obtained with conditional mutants, which only become deficient in a molecule after the main developmental events are complete, should be considered to be the most reliable. 3. Abnormal synaptic plasticity in mice that are deficient for an ECM molecule can be rescued by acute application or inducible expression of the molecule. 4. Genetic or pharmacological blockade of receptor(s) to the ECM molecule should provide similar (if the molecule activates a receptor) or opposite (if the molecule inhibits a receptor) phenotypes to the loss of the ECM molecule itself. 5. The ECM molecule or its receptor(s) are directly linked to established mechanisms that are involved in the induction and expression of specific forms of synaptic plasticity, but do not influence forms of plasticity that depend on other molecular mechanisms. These core criteria can be supported by supplementary criteria, which either outline the specificity of action of the ECM molecule, or highlight its importance at the structural or whole-organism level: 6. Application or inducible expression of the ECM molecule in mice that are deficient in its receptors do not rescue the deficit in synaptic plasticity in the mutant. SCHAFFER COLLATERALS

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

Important components of the extracellular matrix and connective tissue. These proteins contain hydrophilic, negatively charged polymers of glucuronic acid and sulphated N-acetyl glucosamine residues.

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7. Electrophysiological recordings of plasticity are complemented by other methods that directly show that the ECM molecule induces biochemical or morphological changes that reflect synaptic strength, such as the number of synapses, rate of vesicle recycling or accumulation of glutamate receptors. 8. Experimental manipulations of the ECM molecule that affect synaptic plasticity lead to modulation of some forms of learning and/or memory. 9. The ECM molecule and its receptor(s) are present at synaptic and extrasynaptic sites — for example, at the interface with glia that surround the synapse. It should be noted that other patterns of expression — for example, postsynaptically at the soma or at the axonal hillock — could also affect the firing patterns of neurons and thereby modulate synaptic plasticity. 10. The ECM molecule is expressed in the adult in an activity-dependent manner to provide a new microenvironment during or after induction of LTP.




REVIEWS through poorly controlled factors, such as the genetic background, and the stress response before, and transient anoxia during, sample preparation. Furthermore, unknown downstream effectors that mediate indirect actions on synaptic plasticity — for instance, through regulation of energy metabolism or early developmental events — might have confounded the issue. Because of these caveats, we have suggested some criteria that need to be fulfilled to show that an ECM molecule is required for synaptic plasticity (BOX 1). We do not expect that all of these criteria will be met in all cases, but for each one that is fulfilled, the likelihood increases that a particular ECM molecule has a specific role in activity-dependent synaptic plasticity. Our analysis (TABLE 1) shows that no single ECM molecule has yet been shown to satisfy all of these criteria. Nevertheless, we would like to summarize the available data from this newly-emerging field, and outline a few speculative mechanisms for the involvement of the ECM in synaptic plasticity. These mechanisms involve cell surface receptors for ECM glycoproteins (such as the integrins) as mediators of cellular responses, acting directly through the cytoskeleton or more indirectly through intracellular signalling cascades. We will also mention the heparin-binding growth-associated molecule (HB-GAM — also known as pleiotrophin), which binds to syndecans — cell surface receptors that probably

act through the cytoskeleton or kinase signalling pathways. Less conventional mechanisms involve the ECM glycoprotein Narp and the tenascins, which influence neurotransmitter receptors and ion channels. Finally, CSPGs have come into focus, not only because of their barrier functions during development and regeneration, but also because of their involvement in synaptic plasticity through as yet unknown mechanisms. Laminins and their integrin cell surface receptors. Although the presence of laminins in the adult CNS remained controversial for some time, they are now widely accepted to induce both pre- and postsynaptic differentiation of the neuromuscular junction and to influence its activity-dependent modulation42–46. Recent data show that mRNAs for all α-, β-, and γ-chains, which form at least 10 different laminins, can be detected in the adult hippocampus47. Laminin is an important substrate for the protease plasmin, which can inhibit the stabilization of LTP37. Preincubation of hippocampal slices with laminin-1 antibodies prevented both the degradation of laminin and the impairment of LTP by plasmin, indicating that laminin-mediated interactions between cells and the ECM might be necessary for the maintenance of LTP. However, plasmin might affect other proteins — for example, the cell-adhesion molecule L1 (REF. 48) — and

Table 1 | Extracellular matrix (ECM) molecules involved in synaptic plasticity ECM molecule

Glycosylation ECM partners

Laminin*

Agrin

Reelin*

Cell surface receptors

Intracellular signalling

Regulation of LTP/LTD in the CA1 area

Criteria to be essentially involved in synaptic plasticity (BOX 1) 1

2

3

4

5

6

7 8

9

10

Integrins*

Rapsin, Src*, Fyn*

Promotes LTP

+

?

?

+

?

?

?

?

+

?

ApoER2*, VLDLR*, α3β1, CNR

Dab1, Src*, Fyn*, JIP

Promotes STP and LTP

+

+? ?

+

?

+

+

+

+

+

HB-GAM*

Agrin, phosphacan

N-syndecan*, RPTPβ

Cortactin*, Src*, Fyn*

Inhibits LTP

+

+? +

+? ?

+

?

+

+

+

Narp

?

GluR1-3 receptors*

?

?

?

?

?

?

+

?

+

?

+

+

Tenascin-R* HNK-1*

Brevican*, phosphacan, neurocan*, aggrecan, fibronectin

GABAB receptors*, K+ outflux integrins*, through GIRK contactin*, neurofascin, MAG

Promotes LTP

+

+? ?

+

?

?

+

+

+

+

Tenascin-C* HNK-1*

Phosphacan, neurocan*, perlecan, fibronectin

Integrins*, contactin*, TAG-1, syndecan*, glypican

Ca2+ influx through L-type VGCC*

Promotes VGCCdependent LTP and LTD

+

+? ?

+

+

?

?

+

+

+

Brevican*

Chondroitin sulphate*

Hyaluronan, tenascin-R*

Sulphatides, sulphoglucuronyl glycolipids

?

Promotes LTP

+

+? ?

?

?

?

?

–+ +

?

Neurocan*

Chondroitin sulphate*

Hyaluronan, tenascin-R* and -C*

NCAM*, L1*, TAG-1, sulphatides

?

Promotes late LTP

?

+? ?

?

?

?

?

?

?

+

ApoER2, apolipoprotein E receptor 2; CNR, cadherin-related neuronal receptor; GluR, glutamate receptor of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid) subtype; GluR1–3, subunits of AMPA glutamate receptors; LTD, long-term depression; LTP, long-term potentiation; MAG, myelin-associated glycoprotein; Narp, neuronal activity-regulated pentraxin; STP, short-term potentiation; RPTP, receptor protein tyrosine phosphatase; VDCC, voltage-dependent Ca2+ channel; VLDLR, very-low-density lipoprotein receptor. ?, not determined; +, satisfied criterion; –+, data are not conclusive; +?, data were obtained in constitutive knockout mice and have to be viewed with a possiblity that developmental abnormalities underlie the phenomena. * Molecules shown to be involved in LTP and/or LTD.


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a Reelin

3,461 aa

1 A

2 B

A

3 B

A

4 B

A

5 B

A

Reelin repeat (350–390 aa)

CR50 epitope F-spondin-like

b

6 B

A

7 B

A

EGF

8 B

A

B

Carboxy terminus is required for secretion

VLDLR/ ApoER2 α3

Reelin β1 Ca2+

Ca2+ N M D A

N P x Y

Y

mDAB1

P P

Ca2+

Binding

JIP Src

N M D A Ca2+

P

Fyn

Axonal transport

Microtubule reorganization

Increased LTP

Figure 2 | Reelin and regulation of long-term potentiation. a | Domain structure of reelin. Reelin contains a cleavable signal peptide at the amino terminus, followed by a region of similarity to F-spondin, a secreted protein produced by floor plate cells that controls cell migration and neurite outgrowth. The most striking feature of reelin is the presence of a series of eight internal repeats comprising 350–390 amino acids (aa). These so-called reelin repeats contain two related subdomains, A and B, separated by a stretch of 30 amino acids harbouring an epidermal growth factor (EGF)-like motif. A region rich in arginine residues at the carboxy terminus is required for secretion. Adapted, with permission, from Nature REF. 134  Macmillan Magazines Ltd. b | Hypothetical model of the actions of reelin in LTP induction through apolipoprotein E receptor 2 (ApoER2) and the very-low-density lipoportein receptors (VLDLRs), triggering adaptor protein disabled 1 (Dab1) tyrosine phosphorylation and binding of ApoER2 to members of the jun N-terminal kinase interacting protein (JIP) family of scaffolding proteins. Integrin (α3b1) might serve as a co-receptor for reelin signalling. Blue ovals designate alternatively spliced ligand binding repeats in ApoER2. NMDA, N-methyl-D-aspartate receptors; P, phosphorylation. Reproduced, with permission, from REF. 59  (2002) The American Society for Biochemistry and Molecular Biology.

CONTEXTUAL FEAR CONDITIONING

anti-laminin antibodies could sterically inhibit the access of plasmin to other laminin-associated proteins. Additional support for a role for laminin in LTP came from data showing that integrins, which are the main laminin receptors, might be involved in LTP. Many β1integrin-subunit-containing integrins are expressed in the adult hippocampus49,50, and application of peptides that block the interactions between integrins and their ECM ligands was found to affect the stabilization of LTP33,51,52. Also, injection of snake toxins (disintegrins), which preferentially inhibit the binding of ligands to β1- or β3-containing integrins, was efficient at blocking the stabilization of LTP53. Injection of blocking antibody to α3 integrins facilitated depotentiation54. It is therefore reasonable to assume that interactions between α3β1 integrin receptors and laminins are important for the stabilization of LTP.

Hippocampus-dependent learning task in which animals associate a specific location and its surroundings with an electric shock.

Reelin and apolipoprotein E receptors. Integrins have also been recognized as receptors for an ECM glycoprotein called reelin (Reln). This molecule was named after the

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spontaneously-occurring reeler mutant mouse, which shows abnormal migration of neurons during embryonic development55. These deficits result in an outside–in instead of an inside–out positioning of migrating neuronal cell bodies in the cerebral cortex. The Reln molecule was identified as the product of the reeler gene in a screen where a fos transgene was introduced randomly into the mouse genome56. One of the founder lines showed the striking motor abnormalities that are characteristic of the reeler mutant, and the fos-containing gene was subsequently identified as the reeler locus. A flurry of studies that characterized Reln as an ECM recognition molecule soon followed, and two cell surface receptors were implicated as recognition molecules: the very-low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Mice that are deficient in these receptors show the reeler phenotype. Also, a mutant that is deficient in the cytoplasmic adaptor protein disabled 1 (Dab1), which mediates signalling through VLDLR and ApoER2, show the same phenotype, linking Reln to Dab1 signalling pathways (reviewed in REF. 57). Reeler mice show normal LTP in some — but abnormal LTP in other — layers of the hippocampus58. Application of Reln to wild-type hippocampal slices produced a significant increase in LTP59. A modest decrease in short-term potentiation was observed in VLDLR-deficient mice, but LTP was nearly identical to that in wild-type mice. By contrast, ApoER2 mutants were strongly impaired in LTP. Application of receptorassociated protein (RAP), a specific and broad-spectrum inhibitor of ligand binding to all LDL receptor familymembers, almost completely blocked LTP59,60. Importantly, in slices from VLDLR-deficient or ApoER2-deficient mice, the LTP-enhancing effect of Reln was abolished. When they were tested in the CONTEXTUAL FEAR CONDITIONING learning model, which depends on the hippocampus, both the VLDLR- and ApoER2-deficient mice showed significant deficits. So, each LDL receptor seems to have a role in hippocampal synaptic plasticity and associative memory formation, by signalling downstream of Reln (FIG. 2). The underlying mechanisms, however, are not clear. An interesting possibility is that the binding of Reln to ApoER2 or VLDLR stimulates tyrosine kinase activity and induces Dab1 tyrosine phosphorylation. This could lead to the activation of non-receptor tyrosine kinases of the Src family, which might stimulate NMDA receptor activity, thereby increasing Ca2+ influx and LTP18. ApoER2 also binds with its cytoplasmic tail to members of the jun N-terminal kinase interacting protein (JIP) family of scaffolding proteins, thereby interacting indirectly with the microtubule-associated molecular motor kinesin and influencing intracellular transport mechanisms61. Beside these influences, one cannot exclude the possibility that impairments in synaptic plasticity in mutants that are deficient in components of Reln signalling are related to developmental abnormalities. For example, Reln is important for the positioning of hippocampal interneurons58, so it might modulate LTP through regulation of GABA-mediated inhibition.



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ELEVATED PLUS-MAZE

In this experiment, animals are placed in the centre of an elevated four arm plus-like maze, in which two arms are closed and two arms are open. So, the animal can fall down from the open arms. Avoidance of being in open arms is taken as a measure of fearful behaviour. IMMEDIATE-EARLY GENES

Genes that are induced rapidly and transiently. Many immediate-early genes, such as fos, control the transcription of other genes, and thereby regulate expression of sets of proteins. DOMINANT-NEGATIVE

A mutant molecule that binds to an interaction partner of the normal molecule and thereby blocks the functional complex.

HB-GAM and N-syndecan. Another ECM-associated molecule that provides a link between development and synaptic plasticity is HB-GAM. This has been implicated in the regulation of neurite outgrowth, axon guidance and synaptogenesis in vitro (for a review, see REF. 62). In the adult hippocampus, application of HB-GAM inhibited NMDA receptor-dependent LTP, but did not affect L-type voltage-dependent calcium channel (VDCC)-dependent LTP that was induced by application of the K+ channel blocker tetraethylammonium. This indicates that the action of HB-GAM is limited to NMDA-receptor-dependent LTP35. In HB-GAMdeficient mice, a lower threshold for induction of LTP was observed, which was restored to the wild-type level by application of HB-GAM63. Consistent with these findings is the observation that LTP is attenuated in transgenic mice that overexpress HB-GAM. These changes in LTP that are measured in vitro are accompanied by behavioural alterations. For example, mice that overexpressed HB-GAM learned more quickly in the water maze and were less anxious in the ELEVATED PLUS-MAZE than were wildtype mice, whereas HB-GAM-deficient mice learned less well in the water maze and were more anxious in the elevated plus-maze64. HB-GAM binds to the HSPG syndecan 3 (Synd3, also known as N-syndecan), and the role of this molecule in LTP was also investigated. Injection of heparin or removal of heparan sulphate by heparitinase treatment inhibited LTP, showing that LTP depends on endogenous heparan sulphates. Furthermore, Synd3 was identified as one of the main HSPGs that are expressed by pyramidal neurons of the hippocampus, and injection of purified Synd3 was found to inhibit LTP65. Mice lacking Synd3, on the other hand, exhibited enhanced levels of CA1 LTP and were not responsive to HB-GAM. Behavioural testing of the Synd3-deficient mice showed impaired performance in hippocampusdependent learning tasks. These data indicate that Synd3 acts as a receptor for HB-GAM, and thereby influences synaptic plasticity and hippocampusdependent memory66. Interaction of Synd3 with the intracellular cytoskeleton-regulating molecules cortactin and Fyn kinase is important for neurite outgrowth67, and this interaction is increased after induction of LTP65. Furthermore, cortactin is a Shank binding protein68, providing a link between the postsynaptic density and the actin cytoskeleton. Therefore, cortactin is an interesting candidate for mediating signalling downstream of Synd3. Narp and clustering of AMPA receptors. In addition to clustering of neurotransmitter receptors through the intracellular postsynaptic scaffold (for a review, see REF. 69), a mechanism involving aggregation of AMPA receptors through direct interaction with an ECM molecule — the product of the IMMEDIATE EARLY GENE neuronal activity-regulated pentraxin (Narp) — has recently been described70,71. Narp is a secreted protein that is selectively enriched at excitatory synapses on the dendritic shafts of cultured spinal and aspiny hippocampal interneurons, but is not present at excitatory synapses on dendritic


spines in vitro and in vivo. In Narp-transfected cells, the protein interacts with itself, forming large surface clusters that co-aggregate AMPA receptor subunits. Narp molecules cluster and co-immunoprecipitate with AMPA receptor subunits GluR1–3, but not with the AMPA receptor subunit GluR4, the NMDA receptor subunits NR1 and NR2A, or the kainate receptor subunit GluR6. Expression of DOMINANT-NEGATIVE Narp mutant proteins that prevent its accumulation at synapses led to a marked decrease in the ability of transfected cells to induce GluR1 clusters72, whereas exogenous application of Narp to cultured hippocampal neurons induced clusters of AMPA receptors. Surprisingly, this also led to clustering of NMDA receptors on interneurons, but not on pyramidal cells73. The most straightforward interpretation of this observation is that there is an interneuron-specific co-receptor for Narp. Because Narp does not directly aggregate NMDA receptors, it is possible that it promotes clustering of NMDA receptors in hippocampal interneurons indirectly through cytoskeletal scaffolds containing synaptic AMPA receptors (FIG. 3). As expression of Narp is upregulated after induction of LTP71, it is likely that deposition of Narp in the ECM will be crucial for the expression of LTP in excitatory synapses on interneurons. Another interesting link between AMPA receptors and ECM proteoglycans was uncovered when it was shown that heparin increases the open probability of AMPA receptors74,75. So, it is likely that members of the HSPG family interact directly with AMPA receptors. These possibly interconnected findings beg the question of whether Narp could affect the activity of AMPA receptors by direct binding, and whether heparan sulphates could stimulate aggregation of AMPA receptors. Tenascin-C and the L-type Ca2+ channel. The ECM glycoprotein tenascin-C (TN-C) displays dual functional features — as a uniform substrate, it enhances neurite outgrowth, whereas if it is presented as a barrier, it inhibits neuritogenesis8,76. These opposite functions of TN-C are probably due to distinct molecular domains acting differentially in various situations, depending on the previous history of the responding neuron. This functional duality needs to be kept in mind when interpreting observations on synaptic plasticity. The connection between TN-C expression and synaptic plasticity was first recognized when TN-C was found to be upregulated in the hippocampus, both at the mRNA and protein levels, within hours after stimulation of synaptic activity77,78. In TN-C-deficient mutants, stimulation of Schaffer collaterals led to a reduction of CA1 LTP, whereas CA1 LTD was completely abolished39. Nifedipine, an antagonist of L-type VDCCs, did not affect LTP in TN-C deficient mice, but reduced LTP in wild-type mice to the levels seen in the mutant, implying a link between VDCCs and TN-C in the regulation of synaptic plasticity. Furthermore, chemical induction of VDCC-dependent LTP in the CA1 region by application of the K+ channel blocker tetraethylammonium resulted in impaired LTP in TN-C-deficient mice. As NMDAreceptor-mediated responses and three forms of L-type

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a

b CB

PFS

CB

c Glutamatergic terminal Narp NMDAR Dendrite

GluR

STEP-DOWN PASSIVE AVOIDANCE TASK

A behavioural experiment, in which an animal learns to associate stepping down from a raised platform with an aversive stimulus, such as electric shock. The name of the task derives from the fact that the animal learns to passively stay at the platform to avoid the stimulus. WATER-MAZE TASK

A learning task in which an animal is placed in a pool filled with opaque water and has to learn to escape to a hidden platform that is placed at a constant position. The animal must learn to use distal cues, and the spatial relationship between them and the platform. PATCH CLAMP

Technique whereby a small electrode tip is sealed onto a patch of cell membrane, making it possible to record the flow of current through individual ion channels or pores within the patch. Disruption of the patch membrane provides the possibility to record currents from the whole cell.

462

Figure 3 | Narp and postsynaptic differentiation of glutamatergic synapses on interneurons. a | Domain structure of Narp with pentraxin family signature (PFS) and two calcium binding domains (CB) deduced from sequence homology with other members of the pentraxin family. Narp is predicted to contain 15 antiparallel β-strands that form two β-sheets70. b | Three-dimensional structure of C-reactive protein, a pentraxin homologous to Narp, which forms a pentameric ring135. Yellow represents protein backbone and the red mesh is a representation of the molecular surface. Image courtesy of T. J. Greenhough & co-workers from Keele University, UK © Keele University. c | Clustering of glutamate receptors by Narp. Pre- and postsynaptically localized Narp is released into the extracellular space. Narp aggregates induce clustering of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (GluR) through direct interaction and promote accumulations of N-methyl-D-aspartate receptors (NMDAR), presumably through cytoskeletal proteins associated with AMPAR scaffolds.

VDCC-independent LTP seemed to be normal in TN-Cdeficient mice, all the data point to a specific role for TN-C in L-type VDCC-mediated signalling. In a parallel study79, we found that TN-C-deficient mice, as well as wild-type mice that had received an intrahippocampal injection of the fragment comprising TN-C fibronectin-like repeats 6–8, were impaired in memory recall in a STEP-DOWN PASSIVE AVOIDANCE TASK. These findings correlate with electrophysiological experiments, in which a strong reduction of LTP was shown in the CA1 hippocampal area after injection of the fragment. The fragment bound to the somata of pyramidal cells and showed promoting or repellent activity for hippocampal neurite outgrowth in vitro when presented as a


uniform substrate or as a barrier, respectively79,80. As a control, the TN-C fragment comprising fibronectin-like repeats 3–5 did not show any distinct binding pattern in the CA1 area, did not repel neurites and had no effect on LTP and learning and memory. The combined observations not only support the view that TN-C is acutely involved in synaptic plasticity, but also that a particular domain exerts this function. It is noteworthy in this context that the fragment containing fibronectin-like repeats 6–8 binds to fibronectin that is expressed in the hippocampus in the adult37. The fragment could act by competition with endogenous TN-C, disrupting the targeting of TN-C to the ECM scaffold through fibronectin.As fibronectin-like repeats 3–5 are the domains that bind to integrins, it is unlikely that integrins mediate the influence of TN-C on synaptic plasticity. However, as TN-C and the structurally-related tenascin-R (TN-R) affects Na+ channel activity by binding to the channel81–83, and as these channels share structural features with the L-type VDCC, we suggest that TN-C influences LTP and learning and memory through regulation of this Ca2+ channel activity. Tenascin-R and GABA-mediated transmission. TN-R is enriched at the nodes of Ranvier in the CNS and in the perineuronal nets82,84. Interestingly, the distribution of ECM molecules associated with perineuronal nets was altered in TN-R-deficient mice82,84 (FIG. 1b), and these mice also showed impaired CA1 LTP85,38, but it is not clear how this deficiency relates to the abnormal disposition of perineuronal nets. TN-R is one of the main carriers of the unusual carbohydrate HNK-1, which was first discovered on human natural killer cells, hence the name86. This carbohydrate structure contains a 3’ sulphated glucuronic acid, and interestingly, mice deficient in glucuronyltransferase and HNK-1 sulphotransferase, which are the final pathway enzymes in the synthesis of the HNK-1 carbohydrate, showed a similar reduction in CA1 LTP as was found in TN-R-deficient mice87,88. Furthermore, performance in the WATER-MAZE TASK was impaired in both glucuronyltransferase- and HNK-1 sulphotransferase-deficient mutants. Reduced LTP in TN-R- and HNK-1 sulphotransferase-deficient mice was accompanied by increased basal excitatory synaptic transmission in synapses formed on CA1 pyramidal neurons85,88. We hypothesized that this phenomenon might indicate how an ECM molecule can regulate inhibitory transmission, which in turn might modulate synaptic plasticity of excitatory synapses through metaplastic processes. Wholecell PATCH CLAMP recording of the TN-R mutant disclosed a shift in the thresholds for induction of LTP and LTD (O. Bukalo, A.D. and M.S., unpublished observations), which is usually considered to be a reflection of metaplastic processes. TN-R and its associated carbohydrate HNK-1 decorate perisomatic interneurons, and the amplitudes of unitary perisomatic inhibitory postsynaptic currents were smaller in TN-R mutants and HNK-1 antibody-treated wild-type mice36,85. These data correlate with the results of a quantitative electron-microscopic



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Figure 4 | Regulation of perisomatic inhibition in the CA1 region of the hippocampus by glycoprotein tenascin-R and its associated HNK-1 carbohydrate. a | Domain structure of tenascin-R. EGF, epidermal growth factor; R1, alternatively spliced exon. b | Chemical structure of an HNK-1-carbohydrate-carrying glycolipid. c | Hypothetical mechanism by which TN-R and associated HNK-1 regulate perisomatic inhibition. The HNK-1 carried by the extracellular matrix glycoprotein tenascin-R (TN-R) interacts directly with postsynaptic GABABRs (γ-aminobutyric acid metabotropic receptors) on the soma of a pyramidal neuron. This interaction inhibits the activation of GABABRs by the inhibitory neurotransmitter GABA, released by perisomatic interneurons onto pyramidal cells in the CA1 region of the mouse hippocampus. Under these conditions, GABA (triangles) released from presynaptic terminals of these inhibitory interneurons activates postsynaptic GABAARs but not GABABRs. d | Genetic ablation of TN-R or application of monoclonal antibody (Ab) directed against the HNK-1 carbohydrate neutralizes the inhibition of postsynaptic GABABRs by the HNK-1 carbohydrate. As a consequence, tonic GABA release activates postsynaptically both GABAARs and GABABRs. Activation of postsynaptic GABABRs leads to an increase in K+ conductance and accumulation of extracellular K+. This in turn depolarizes the presynaptic terminals (+), elevates the rate of spontaneous asynchronous vesicle release and decreases the evoked GABA release through impairment of the presynaptic action potentials shown next to the presynaptic terminal. GIRK, G-protein-coupled inwardly rectifying K+ channel.

G-PROTEIN-COUPLED INWARDLY RECTIFYING K+ CHANNELS

K+ channels that are regulated by neurotransmitters and hormones through G-proteincoupled receptors. They are called inward rectifiers because current flows through them more easily into than out of cells. PEPTIDOMIMETICS

Peptides mimicking other molecules — for instance carbohydrates — in their ability to bind other molecules.

analysis of TN-R-deficient mice, which showed a strong reduction in the density, and abnormal architecture, of symmetrical perisomatic synapses in the CA1 area of the hippocampus89. Changes in the density and spatial arrangement of synaptic vesicles in the synaptic terminals also provided ultrastructural evidence for reduced inhibitory synaptic activity in TN-R mutants. Analysis of perisomatic inhibition in HNK-1deficient mutants has not yet been performed, however perturbation with the HNK-1 antibody produced a strong reduction in perisomatic inhibitory currents after application of HNK-1 antibody to hippocampal slices36. Antagonists to GABAB receptors and G-PROTEIN-COUPLED INWARDLY RECTIFYING K CHANNELS (GIRKs) abolished the effects of the HNK-1 antibody on perisomatic inhibition, indicating that activation of GIRKs through GABAB receptors is involved in HNK-1 carbohydrate function90. Interestingly, synthetic HNK-1 carbohydrate or its 90 PEPTIDOMIMETIC inhibited GABA receptor-activated B +


GIRK currents, but not GIRKs directly. As the HNK-1 carbohydrate and its peptidomimetic bind to GABAB receptors, the results imply that the HNK-1 antibody relieves a constitutive block of GABAB receptors exerted by endogenous HNK-1 carbohydrate in perisomatic synapses 90 (FIG. 4). Is the action of HNK-1 presynaptic or postsynaptic? Infusion of K+ channel-blocking Cs+ ions into postsynaptic CA1 pyramidal neurons diminished the effects of the HNK-1 antibody on perisomatic inhibitory currents, indicating that the HNK-1 carbohydrate regulates activation of postsynaptic K+ channels 90. However, the consequences of this activation impinge on presynaptic GABA release36. As elevation of extracellular K+ concentration mimicked and occluded the effects of the HNK-1 antibody on inhibitory currents 90, it is plausible that outflow of K+ from postsynaptic cells induces changes in excitability and/or presynaptic machinery (FIG. 4). Measurements of K+ concentrations at perisomatic synapses and further analysis of signalling events downstream of K+ will be necessary to complete the picture of how the HNK-1 carbohydrate affects perisomatic inhibition. The functional link between the HNK-1 carbohydrate and TN-R as its cognate ‘carrier’ was disclosed by experiments in which HNK-1 antibody was applied to hippocampal slices from mice deficient in recognition molecules that carry the HNK-1 carbohydrate. The antagonistic effect of the HNK-1 antibody on inhibitory currents was abolished in TN-R mutant mice, but not in NCAM-deficient mice, indicating that TN-R is the predominant carrier of the HNK-1 carbohydrate that modulates perisomatic inhibition36. The deficit in the number of perisomatic contacts observed in TN-R mutants implies that a higher activity of postsynaptic GABAB receptors could lead to impaired formation or destabilization of inhibitory synapses. Chondroitin sulphate proteoglycans. CSPGs act as molecular ‘barriers‘ in locations that form boundaries for axon growth in vivo. In vitro, CSPGs inhibit neurite outgrowth in a barrier situation, but are conducive to outgrowth on a uniform substrate8,91. These features are reminiscent of those of tenascins, to which CSPGs bind. CSPGs probably also act as barriers in the adult CNS after trauma. For example, if a spinal cord lesion is treated with chondroitinase ABC, which specifically removes chondroitin sulphates from their protein carrier backbones, neuronal regeneration is enhanced92. Chondroitinase ABC treatment was also found to prolong synaptic plasticity in the visual system93. CSPGs are also important components of perineuronal nets, where it has been indicated that they surround perisomatic synapses, thereby stabilizing them or prohibiting new synapse formation. In this respect, CSPGs might be similar to TN-C and TN-R. Infusion of chondroitinase ABC into the brains of adult wild-type animals did not result in an abnormal density of perisomatic synapses84. However, removal of chondroitin sulphates reduced both LTP and LTD in the CA1 region38. Strikingly, LTP was abolished in mice that were deficient

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REVIEWS in the CSPG brevican, and also after injection of antibrevican antibodies94. Interestingly, brevican mutants had normal spontaneous inhibitory postsynaptic currents, normal spatial learning, but possibly altered spatial memory. Although the mechanisms underlying the contribution of brevican to synaptic plasticity are currently not clear, it is conceivable that they involve the interaction of brevican with the HNK-1 carbohydrate95. Interestingly, no deficit in early LTP was observed in mice that were deficient in another CSPG, neurocan, highlighting the specificity of the LTP deficit in brevicandeficient mice. Neurocan-deficient mice, however, showed impairments in late LTP recorded 2–5 hours after multiple tetanization of Schaffer collaterals96. So, different CSPGs seem to be involved in distinct stages of LTP, through mechanisms that have not yet been identified. Perspectives

OCULAR DOMINANCE

In the mature primary visual cortex of mammals, most neurons respond predominantly to visual inputs from one eye or the other. This phenomenon is called ocular dominance. Cells that respond to a given eye are arranged in stripes — the ocular dominance columns — that alternate with stripes of neurons that respond to the other eye.

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As we have seen, several molecules that regulate early development and synaptogenesis are now known to affect LTP and learning and memory in adult animals (TABLE 1). Of the seven molecules studied so far, six promote CA1 LTP, whereas only HB-GAM inhibits it. Interestingly, LTP measured 1 hour after induction was usually only partially inhibited after manipulation of an ECM molecule or its receptor. An exception was found in the case of the brevican-deficient mutant, which showed a complete block of LTP. The partial reduction in LTP might reflect either a predominant inhibition of one mechanism underlying LTP or partial inhibition of several distinct mechanisms. Changes in LTP were not usually associated with changes in basal excitatory transmission, except in TN-R- and HNK-1 sulphotransferase-deficient mice, which displayed enhanced excitatory transmission. Also, short-term potentiation was typically not affected after manipulation of ECM molecules, except for Reln, which increased short-term potentiation after application. Although such distinct functions are reassuring regarding the specificity of function of the molecules, the question arises of how these functions are linked to developmental events. Heparin and HSPGs have been shown predominantly to promote neurite outgrowth, whereas CSPGs and the tenascins can both promote and inhibit neurite outgrowth. Interpreting these functions in the context of synaptic plasticity, both neurite outgrowth promotion and inhibition could come into play in modifying synaptic strength. On the one hand, sprouting of neurites enhanced by a uniformly conducive microenvironment could promote the formation of new synapses. On the other hand, it is equally conceivable that the barrier functions of these molecules are required for stabilization of nascent contacts. The facility to visualize formation and stabilization of synapses by time-lapse cinematography, combined with the available repertoire of biochemical and genetic tools, should make it feasible to determine the morphological and molecular events that underlie the actions of ECM molecules in a short temporal and spatial range of resolution. The following sections highlight the questions that remain to be answered.


What forms of plasticity do ECM molecules regulate? In most ECM molecule-deficient mice, LTP has been tested exclusively in the CA1 region. Recordings in the CA3 region could help further in the search for mechanisms, as different intracellular signalling cascades are involved in CA1 and CA3 LTP. Also, intracellular recordings and pairing protocols — which allow precise control of depolarization of postsynaptic cells during induction of synaptic plasticity and determination of thresholds for induction of LTP and LTD97 — have not yet been applied to the analysis of ECM molecules. These would be instrumental in distinguishing the possible effects of ECM molecules on the induction of synaptic plasticity. For example, if modification of the ECM reduces the degree of depolarization of the postsynaptic neuron during a tetanic stimulus, LTP could be reduced simply because the NMDA receptors open less. The use of intracellular recordings would also allow the analysis of synaptic plasticity in excitatory synapses on postsynaptic interneurons and activity-dependent modulation of inhibitory currents — two aspects that are particularly interesting with regard to Narp, tenascins and CSPGs. Recent studies on the importance of CSPGs and GABA-mediated inhibition for establishing OCULAR DOMINANCE in the visual cortex93,98 highlight this form of experience-dependent developmental plasticity as an attractive in vivo model to elucidate the contributions of ECM molecules to synaptic function. Another potentially important issue is whether the ECM composition could affect spillover of neurotransmitters and neurotrophic factors through regulation of extracellular space, volume and tortuosity2,99. In particular, interfaces at the edges of synaptic cleft determine their leakiness, which might in turn affect the neighbouring synapses that were not directly stimulated during the induction event. Whether cross-talk between neighbouring synapses is increased in ECM-deficient mice, and whether this leads to abnormal levels of synaptic plasticity, remains to be determined. ECM molecules and Ca2+ influx. Now that several ECM molecule–receptor pairs have been implicated in LTP, there are several signalling cascades that should be studied. In particular, it is important to distinguish whether an ECM molecule affects NMDA- or VDCC-dependent components of LTP, and whether tyrosine kinase signalling triggered through ECM receptors modifies Ca2+ influx or affects other downstream mechanisms. NMDA-receptor-mediated responses were measured in CA1 synapses, and were found to be normal under conditions of deficient ECM molecule expression35,38,39,85,94. It needs to be emphasized, however, that weak stimulation protocols were used to evaluate NMDA receptor function in these studies. Normally, protocols based on several episodes of strong stimulation are used to induce LTP, leaving open the possibility that intracellular signalling — possibly activated through triggering of ECM receptors — could modify NMDA receptor-mediated currents and thereby affect the induction of LTP18. Ca2+ imaging during induction of LTP after modification of the ECM could be useful to resolve this issue.



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Figure 5 | Hypothetical mechanisms by which extracellular matrix (ECM) molecules could regulate synaptic plasticity. Cell surface receptors for ECM molecules, exemplified by integrins (α,β) and the neural cell adhesion molecule (NCAM), form signalling complexes with receptor tyrosine kinases (RTK) at the edges of pre- and postsynaptic appositions. ECM molecules serve as crosslinkers of pre- and postsynaptic molecular complexes, which affect the cytoskeleton and receptor and vesicle trafficking (arrows labelled with question marks). The integration of signals triggered by glutamate release and rearrangements in the ECM occur at the level of Ca2+ signalling, exemplified by the putative interaction of tenascin-C with the voltage-dependent Ca2+ channel (VDCC), or by signalling through Ras and Rap. CaMKII, calcium/calmodulin-dependent protein kinase II; GluR, glutamate receptor of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) subtype; NMDAR, N-methyl-D-aspartate receptor.

SYNAPTOSOMES

A preparation of presynaptic terminals and postsynaptic membranes, isolated after subcellular fractionation. These structures can take up, store and release neurotransmitters and could contain postsynaptic densities.

ECM molecules and signalling in endocytic zones. The available data indicate that ECM molecules accumulate largely at the edges of pre- and postsynaptic appositions, close to the interface between neurons and astrocytes, and that they surround perisomatic inhibitory synapses and in some instances also dendritic spines100,101. These edges overlap with endocytic and periactive zones, which surround the active zones and might be important sites for initiation of structural synaptic modifications102. Currently, it is not known how the machinery of endocytic zones is targeted to be outside, but closely associated with, active zones presynaptically, and with postsynaptic densities on the postsynaptic side. Drawing a parallel with Narp-induced clustering of AMPA receptors71, it is plausible that extracellular scaffolds formed by ECM molecules at endocytic zones could regulate the location and function of clusters of cell surface receptors, which would in turn accumulate transmitter receptors and ion channels at the periphery of synapses, under the scaffolding influence of associated cytoskeletal proteins (FIG. 5). Interactions of the HNK-1 carbohydrate groups that are carried by TN-R with GABAB receptors 90, which are predominantly expressed extrasynaptically, raise the interesting possibility of a direct regulation of metabotropic receptors by ECM molecules.


The importance of ECM recognition molecules for the organization of membrane recycling in synapses is underscored by a recent study, which demonstrated that association of trans-Golgi organelles with clusters of NCAM helps to trap these organelles at nascent synapses within minutes after initial contact formation103. A direct link between laminin and the vesicle recycling machineries is implied by its occurrence in SYNAPTOSOMES in complex with SV2, a synaptic vesicle transmembrane proteoglycan. SV2 binds with high affinity to purified laminin 1, indicating that a synaptic vesicle component might act as a laminin receptor on the presynaptic plasma membrane. These data imply that there is a link between laminindependent regulation of adhesion and the number of unbound SV2 molecules that could recycle at synapses104. Apart from providing a basis for structural organization, extracellular scaffolds could also induce oligomerization of receptor tyrosine kinases (RTK), thereby activating intracellular signalling. The association of αvβ3 integrin with receptors for EGF, PDGF, ATP and insulin implicates these complexes as general signalling platforms105. Strikingly, the αvβ3 integrin is necessary for maturation of hippocampal synapses106, and activation of insulin receptors regulates endo- and exocytosis of AMPA, NMDA and GABAA receptors107–110. ECM molecules and small GTPases. A recent study showed that the small GTPases Ras and Rap relay NMDA receptor signalling, thereby driving synaptic delivery and removal of AMPA receptors during LTP and LTD, respectively111. Also, the activity-dependent growth of dendritic arborizations is triggered through activation of NMDA receptors, and requires a decrease in RhoA and an increase in Rac and Cdc42 activities112. Ca2+-dependent exocytosis of post-Golgi organelles might insert new membrane patches into pre- and postsynaptic membranes, and along with inhibition of endocytosis, this would provide conditions for the extension of synaptic structures. So, regulation of small GTPases in a Ca2+ dependent manner could be an important unifying theme in receptor trafficking and structural remodelling of synapses. As signalling through ECM receptors that are activated during developmental events is essentially mediated by small GTPases of the Ras family113–115, an interesting overlap between the mechanisms that underlie synaptic plasticity and early development is conceivable. We propose that Ca2+-dependent secretion of ECM components could modulate and/or initiate formation of new signalling complexes, which guide dendritic filopodial movements in activated synapses116, induce the reorganization of actin filaments surrounding active zones117 and/or redistribute neurotransmitter receptors at postsynaptic sites20. Recent technological advances allow us to visualize directly the effects of ECM molecules on synaptic plasticity in neuronal cultures. For example, to monitor redistribution of the pre- and postsynaptic machineries118, an ECM molecule could be applied alone or together with depolarizing reagents, such as K+ or glutamate, to cells transfected with pre- and postsynaptic proteins tagged with green fluorescent protein.

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REVIEWS Trans-synaptic cross-talk through ECM. During potentiation, there is an increase in the number of clusters of postsynaptic glutamate receptors containing the subunit GluR1. This is accompanied by a rapid and long-lasting increase in the number of clusters of the presynaptic protein synaptophysin, and in the number of sites at which synaptophysin and GluR1 are colocalized22, indicating that pre- and postsynaptic changes could go hand-in-hand. As receptors to ECM molecules are present on both presynaptic and postsynaptic membranes, ECM molecules are well placed to coordinate changes in presynaptic and postsynaptic machineries. It is worth emphasizing here that these components could derive either from neurons or from glia, whose contribution to synaptic plasticity remains largely unexplored. In this context, it is noteworthy that αvβ3 integrin is required to convert immature hippocampal synaptic contacts, which express the NR2B subunit of NMDA receptors and have high release probability, into mature synapses, which lack NR2B and have lower release probability106. Trans-synaptic signalling mediated by direct interaction of pre- and postsynaptic recognition molecules, including those located in the synaptic cleft, seems to be important for synaptogenesis and coordinated pre- and postsynaptic changes. Known examples include cadherin–cadherin, neuroligin–neurexin, synaptic cell adhesion molecule (synCAM)–synCAM, and ephrin–Eph receptor interactions119–122. Complementary to such ‘duets’, there could be trans-synaptic signalling at the edges of synapses through ensembles of recognition molecules crosslinked by ECM components (FIG. 5). A putative example of such arrangement is provided by NCAM, which is expressed pre- and postsynaptically, and is known to interact with HSPGs, including agrin123,124. Furthermore, NCAM–heparan sulphate complexes could provide a substrate for dimerization of fibroblast growth factor (FGF) and its receptors, thereby stimulating FGF receptor-mediated signalling125. MOSSY FIBRES

Axons of dentate gyrus granule cells, which constitute the main excitatory input to CA3 pyramidal cells in the hippocampus.

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ECM remodelling as a framework for plasticity. In addition to LTP-induced upregulation in the expression of ECM molecules71,78, synaptic activity might trigger endocytosis and calpain-mediated proteolysis of ECM receptors and promote the release of proteases

Ruoslahti, E. Brain extracellular matrix. Glycobiology 6, 489–492 (1996). Nicholson, C. & Sykova, E. Extracellular space structure revealed by diffusion analysis. Trends Neurosci. 21, 207–215 (1998). Bruckner, G. et al. Perineuronal nets provide a polyanionic, glia-associated form of microenvironment around certain neurons in many parts of the rat brain. Glia 8, 183–200 (1993). Celio, M. R., Spreafico, R., de Biasi, S. & VitellaroZuccarello, L. Perineuronal nets: past and present. Trends Neurosci. 21, 510–515 (1998). This article provides a historical overview on visualization, composition and possible functions of perineuronal nets. Yamaguchi, Y. Lecticans: organizers of the brain extracellular matrix. Cell. Mol. Life Sci. 57, 276–289 (2000). Bruckner, G. et al. Extracellular matrix organization in various regions of rat brain grey matter. J. Neurocytol. 25, 333–346 (1996).

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that digest ECM molecules at neuron–neuron or neuron–astrocyte interfaces40,126. Furthermore, proteases can shed the extracellular domains of transmembrane recognition receptors, such as NCAM, amyloid precursor protein and L1 (REFS 127,128), and these could be incorporated into the ECM to modify its qualities. Secretion of ECM ligands and proteolysis-dependent remodelling of the ECM probably have an instructive role in synaptic plasticity through several distinct mechanisms. ECM ligand–receptor interactions might amplify intracellular signalling events that are involved in induction of synaptic plasticity, or might affect the motility of neuronal and astrocytic processes, providing the basis for changes in cellular morphology. For example, proteolytic degradation of the CSPG phosphacan by the tissue plasminogen activator/plasmin system during epileptic seizures leads to formation of MOSSY FIBRE recurrent collaterals in the dentate gyrus 129. Tissue plasminogen activator/plasmin also controls several forms of LTP, in part through proteolysis of laminin37,130,131. Yet another protease, neuropsin, is involved in LTP and synaptogenesis132,133. Therefore, it will be important to understand when and where different proteases operate during LTP and LTD, and which ECM molecules they affect in remodelling the extracellular space. A modified extracellular environment might also change the conditions for induction of synaptic plasticity during subsequent episodes of synaptic activity — for example, through modulation of inhibitory synaptic transmission or redistribution of ion channels. Activitydependent remodelling of the ECM might significantly contribute to adjustment of the dynamic range of synaptic modifications in response to synaptic activity, thereby forming part of a metaplastic process. Summary

In summary, the available experimental evidence indicates that ECM components affect synaptic plasticity through interactions with cell surface recognition molecules, ionotropic/metabotropic receptors and ion channels. Furthermore, an interplay between ECM molecules and trophic factor receptors or small GTPases of the Ras family could be important for disclosing new links between developmental mechanisms and synaptic plasticity in adults.

Matthews, R. T. et al. Aggrecan glycoforms contribute to the molecular heterogeneity of perineuronal nets. J. Neurosci. 22, 7536–7547 (2002). 8. Faissner, A. & Steindler, D. Boundaries and inhibitory molecules in developing neural tissues. Glia 13, 233–254 (1995). 9. Stichel, C. C. & Muller, H. W. The CNS lesion scar: new vistas on an old regeneration barrier. Cell Tissue Res. 294, 1–9 (1998). 10. Deller, T., Haas, C. A. & Frotscher, M. Reorganization of the rat fascia dentata after a unilateral entorhinal cortex lesion. Role of the extracellular matrix. Ann. NY Acad. Sci. 911, 207–220 (2000). 11. Jones, F. S. & Jones, P. L. The tenascin family of ECM glycoproteins: structure, function, and regulation during embryonic development and tissue remodeling. Dev. Dyn. 218, 235–259 (2000). 12. Bandtlow, C. E. & Zimmermann, D. R. Proteoglycans in the developing brain: new conceptual insights for old proteins. Physiol. Rev. 80, 1267–1290 (2000).


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Acknowledgements We greatly appreciate helpful discussions with H. Beck, V. Bolshakov, G. Bruckner, P. Giese, M. Kneussel, D. Kullmann, A. Luthi and contributions from all members of our laboratory cited in the references. This work was supported by grants from the Deutsche Forschungsgemeinschaft.

Online links DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ agrin | ApoER2 | brevican | fibronectin | GluR1–3 | HB-GAM | integrins | laminins | Narp | neurocan | phosphacan | Reln | Synd3 | TN-C | TN-R | VLDLR Access to this interactive links box is free online.

