Synaptic structure and diffusion dynamics of synaptic receptors

Biology of the Cell 95 (2003) 465–476 www.elsevier.com/locate/bicell

Review article

Synaptic structure and diffusion dynamics of synaptic receptors Antoine Triller a,*, Daniel Choquet b a b

Biologie Cellulaire de la Synapse N&P, INSERM U497, Ecole Normale Supérieure, Paris, France Physiologie Cellulaire de la Synapse, UMR 5091 CNRS/Université de Bordeaux 2, Bordeaux, France Received 2 July 2003; accepted 4 July 2003

Abstract Neurotransmitter receptors are concentrated at synaptic sites through interactions with specific scaffolding proteins and cytoskeletal elements, but can also be found in intracellular compartments or dispersed in the membrane. The notion has emerged in recent years that this distribution results from a dynamic equilibrium between the different pools of receptors. This equilibrium is regulated by neuronal activity and interactions with scaffolding proteins. A change in its set point can result in rapid variations in the number of functional receptors at synapses such as seen during plasticity. Trafficking of receptors in and out of synapses has up to now mainly been viewed within the framework of endo/exocytotic processes. After a brief presentation of the strucure of excitatory and inhibitory synapses, we review data indicating that the lateral diffusion of receptors in the plane of the membrane is also a key step to regulate receptor stabilization and accumulation at synapses. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Synapse; Scaffold; Postsynaptic density; Diffusion; Receptor trafficking

1. Introduction

2. Structure of the postsynaptic neuronal membrane

The receptive somatodendritic membrane contains many types of receptors. They are accumulated in front of presynaptic terminal boutons therefore forming functional microdomains which overlap with the postsynaptic densities (PSDs) (Peters et al., 1976). The electron-dense PSDs correspond to the subsynaptic scaffold constituted of a high density of proteins. There is usually a perfect match between the secreted neurotransmitter and the apposed postsynaptic receptors (Craig and Boudin, 2001; Nusser, 2000). The scaffolding proteins stabilize excitatory and inhibitory receptors, and therefore the neuronal membrane resembles a patchwork forming a ’harlequin’s coat’. In the recent years it has been demonstrated that the composition of the postsynaptic membrane fluctuates rapidly, accounting for construction and plasticity of the synapse. Endo/exocytosis and diffusion within the plane of the plasma membrane are thought to be the general mechanisms accounting for the local changes in the number and/or type of receptors at synapses. The traffic of molecules forming postsynaptic densities is also involved at steady state through basal turnover of proteins.

Fluorescence and electron microscopy have for a long time revealed that the neuronal membrane is composed of patches of membrane enriched with given receptors. Extrasynaptic receptors are at a lower density. For more than ten years, it has been assumed that receptors were immobilized at synapses (Fig. 1) but recent data (see below) challenge this static view.

* Corresponding author for publication. E-mail address: [email protected] (A. Triller). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/j.biolcel.2003.07.001

3. Excitatory synapses In the central nervous system (CNS), excitation is mostly mediated by glutamate where it activates ionotropic (Dingledine et al., 1999) or G-protein coupled (Pin and Duvoisin, 1995) receptors. The distribution pattern of the ionotropic receptor within the PSDs, depends upon receptor types and the type of synapse studied. The AMPA receptor distribution is either uniform (Nusser et al. 1994) or skewed with higher concentrations at the edge of synaptic complexes (Matsubara et al. 1996). The NMDA receptors are either evenly distributed at the PSDs (Clarke and Bolam, 1998) or concentrated at its center (Somogyi et al., 1998). The postsynaptic type I metabotropic receptors are concentrated in an annulus surrounding the postsynaptic differentiation (Matsubara et al.,

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Excitatory Synapse

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Fig. 1. Schematic representation of the structural organization of generic glutamatergic excitatory (left) and inhibitory (right) synapses. A, transverse view of synaptic contacts with emphasis on the postsynaptic scaffold network organization. A1: Organization of PSD at excitatory synapses. Transmembrane proteins include adhesive proteins such as cadherin-catenin complexes (1) at the periphery of the synapse, neuroligin (1′), as well as NMDA (2), AMPA (2′) or metabotropic (2′′) glutamate receptors, and the AMPA receptor interacting protein stargazin (3). In a first layer, the postsynaptic scaffold includes PSD-95 (3′) that interacts with NMDAR, neuroligin and stargazin, GRIP (3′′) that interacts with AMPAR, and Homer (3′′′) which binds to mGluR. In a second layer, PSD-95 binds to GKAP (4), which in turn connects to Shank (4′). The cytoskeleton (5) interacts at various levels with the postsynaptic synaptic molecular network. A2: Organization of PSD at inhibitory synapses. Transmembrane proteins include adhesive proteins such as cadherins (1) at the periphery of the synapse. Glycine (2) and GABA (2′) receptors bind directly and indirectly, respectively, through a nonidentified intermediate molecule (3′) to gephyrin (3). B: top view of the synapse. The cadherins are concentrated on an annulus around both excitatory and inhibitory synapses. Neuroligins are only detected at excitatory synapses. Transmembrane proteins are shown with the same color code as in A.

1996; Baude et al., 1993; Lujan et al., 1997). However, the amount of metabotropic glutamate receptor (mGluR) is still high at a distance from the PSD and 75% of the total surface immunoreactivity is on nonsynaptic membrane (Lujan et al., 1997). The glutamate receptors interact directly or indirectly with a number of intracellular proteins (Walikonis et al., 2000) accumulated at PSDs. The network of interacting molecules between the cytoskeleton and the transmembrane receptors (Sheng and Sala, 2001; Garner et al., 2000) define the subsynaptic scaffold (schematized in Fig. 1A). Scaffolding proteins contain sequences such as PDZ, SH3 or proline-rich domains that are implicated in protein-protein interactions. Scaffold proteins can be divided into two groups: depending upon their interactions with receptors. They include molecules such as PSD 95/SAP90, which interact directly with the NMDA receptor (Kornau et al., 1995), GRIP with AMPA receptor (Dong et al., 1997; Scannevin and Huganir, 2000) and Homer with mGluR (Brakeman et al., 1997). These proteins are coupled to “secondary” scaffolding molecules such as, GKAP, that interacts with PSD-95 (Naisbitt et al., 1997). GKAP binds to Shank (Naisbitt et al., 1999) which in turn couples the NMDA/PSD-95/GKAP and the mGluR/Homer complexes (Tu et al., 1999). In the PSD, the

scaffolding molecules also interact with signaling molecules that play important roles, but the NMDA receptor can itself interact directly with signaling molecules such as CAMKII (Wyszynski et al., 1997). Among the postsynaptic proteins within and around the PSDs, stargazin plays an important role (Chen et al. 2000a). It interacts directly with PSD-9 (Schnell et al. 2002) and AMPARs and controls the number of synaptic AMPA receptors. Another possible classification of scaffolding proteins could rely on the stabilization versus trafficking properties. However, given the molecular interaction network, the distinction between these two functions is often hazy. For example, the shift of AMPAR binding preference from GRIP to PICK during LTD-associated endocytosis (Xia et al., 1999; Kim et al., 2001) indicates that it may be difficult to distinguish between these two properties. One may wonder also whether PSD-95 is a genuine stabilizer of NMDARs since the NMDAR distribution is not disorganized in mice knockout for PSD-95 (Migaud et al., 1998). In these mice, downstream signaling and long term potentiation (LTP) were enhanced. It is therefore likely that PSD-95 is rather a traffic regulator or an AMPA-stabilizing protein through stargazin. Although interaction models have been proposed, they do not fully explain the fine structural organization of the postsynaptic membrane. The ultimate point is that of the perisynaptic annulus of metabotropic receptors whose localization cannot be explained only by the known interaction properties of the subsynaptic scaffold proteins. For example, mGluR are at the periphery of the synapse while Homer proteins are evenly distributed at the PSD (Xiao et al., 1998). 4. Inhibitory synapses Glycine and GABA are the two main transmitters mediating fast chloride-dependent inhibition in the CNS (for their structures, see Moss et al. 2001). Glycine (GlyR) and many types of GABA (GABAR) receptors are accumulated at the PSD where they are stabilized by a subsynaptic scaffold (Schematized on Fig. 1B). Gephyrin is the core protein of the scaffold at inhibitory synapses. The GlyR was the first receptor in the CNS which was demonstrated, with immunogold electron microscopy, to form microdomains (Triller et al., 1985). Anti-sense experiments (Kirsch et al., 1993) and knockout mice (Feng et al., 1998) have been used to establish that gephyrin is involved in the synaptic localization of GlyR. The interaction of gephyrin with the GlyR is mediated by a 20-amino-acid sequence present on its b subunit (Meyer et al., 1995). On its cytoplasmic side, gephyrin also interacts with the actin- and microtubule-based cytoskeleton (Kirsch and Betz, 1995). The distributions of GABA receptors at the neuronal surface depend upon their subunit composition; some are accumulated at synapses while others spread throughout the somatodendritic membrane independently of synapses (Nusser et al., 1995; Nusser et al. 1998). At synapses, the GABA receptors are co-localized with gephyrin (Sassoe-Pognetto et

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al., 2000). The GABAR gamma2 subunit is influential in the synaptic localization since mice lacking this subunit had a significant reduction in the number of GABA receptor clusters at the synapse (Essrich et al., 1998). This effect is mediated through gephyrin as demonstrated by antisense experiments (Essrich et al., 1998), or by examining gephyrin knockout mice (Kneussel et al., 1999). However, a direct link between GABARs and gephyrin could never be demonstrated. In the spinal cord, as well as in the brain stem, inhibitory synapses are either GABAergic, glycinergic or mixed, using the two neurotransmitters (Maxwell et al., 1995). Indeed, the corresponding receptors accumulate to form microdomains enriched with GABAR, GlyR, or a mixture of both, and the composition of the postsynaptic membrane is determined by the identity of the presynaptic element (Levi et al., 1999). This raises the question of how differences in the composition of the postsynaptic scaffold lead to the postsynaptic accumulation of given inhibitory receptors at the PSDs. Indeed multiple spliced variants of gephyrin exist (Ramming et al., 2000) with distinct functions with respect to binding to the GlyR b subunits (Meier et al., 2000). Yeast two hybrid and immunoprecipitation experiments have identified various molecules, that directly interact with gephyrin or inhibitory receptors, such as GABARAP, Raft1 (rapamycin FKB12 target protein) and Collibistin, Plic1 or dynein light chain 1 and 2 (Dlc1 and 2). Although these proteins are not structural components of the subsynaptic scaffold, they may participate in the regulation of receptor traffic and subsequently their number at synapses. GABARAP (GABA receptor-associated protein) binds to GABAR c2 subunit (Wang et al. 1999) and to gephyrin (Kneussel et al., 2000), but it is only occasionally present at the synapse (Kittler et al., 2001; Sabatini et al., 1999). GABARAP binds directly to N-ethylmaleimide-sensitive factor (NSF) and shares homologies with GATE16. It is likely that GABARAP is involved in the intracellular trafficking of the receptor rather than in its stabilization at synapses. Raft1 also binds to gephyrin (Sabatini et al., 1999) and interestingly is implicated in the control of mRNA translation. It could therefore be involved in the synaptic regulation of GlyR synthesis in dendrites, since high concentrations of GlyR a subunit mRNA are present in the vicinity of PSDs (Racca et al. 1997; Gardiol et al., 1999). Collibistin is a GTP/GDP exchange factor (GEF), that enhances the Cdc42 rac/rho like GTPase (Kins et al., 2000). Between two splice variants (I and II), collibistin II may be involved in the translocation of the GlyR b subunit with gephyrin to the plasma membrane (Kins et al., 2000). As a Cdc42-specific GEF, collibistin could serve many other functions including the control of the actin based subsynaptic cytoskeleton, since Cdc42 is implicated in the regulation of actin polymerization (Nobes and Hall., 1995). It has also been reported that profilin, another regulator of the actin-based cytoskeleton, can bind to gephyrin (Mammoto et al., 1998). Plic-1, a ubiquitinlike protein that interacts directly with the GABAaR, is

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enriched at inhibitory synapses and is detected in association with subsynaptic membranes (Bedford et al. 2001). Plic 1 augments the number of surface GABA receptors without increasing its internalization. It rather increases the number of receptors available for insertion in the plasma membrane by increasing the stability of intracellular GABAR as a consequence of the inhibition of its poly-ubiquination, therefore reducing its targeting to the proteasome, as proposed for other ubiquitinin-like proteins (Jentsch et al., 2000). When gephyrin was used as bait for yeast two hybrid experiments, Dlc 1 and 2 were also identified (Fuhrmann et al., 2002). Dlc proteins are enriched in hippocampal synapses; Dlc was localized at the edge of some PSDs as well as at the periphery of Golgi stacks. Interestingly, Dlc1 and 2 are also enriched in dendritic spines, where they bind directly to GKAP (Naisbitt et al. 1999). Dlc 1 and 2 are components of the cytoplasmic dynein and myosin V complexes (Espindola et al., 2000). The association of gephyrin with Dlc1 and 2 suggests that dynein could be involved in dynamically regulating the number of gephyrin molecules at synapses. 5. Adhesion proteins at synapses Other transmembrane molecules such as adhesion proteins that bridge pre- and postsynaptic membranes are present at synaspes. They include molecules such as the neurexin-neuroligin molecules with heterophilic interactions, and others such as cadherins and new Ig-like molecules involved in homophilic interactions. These bridging elements have a micro-structured topological organization defining sub-domains a few tens of nanometers wide at the synapse. Cadherins have been detected at excitatory synapses were they are associated with catenins (review in Fannon and Colman 1996), which interact with the actin-based cytoskeleton. Cadherins and b−catenins are present pre- and postsynaptically at the periphery of synaptic complexes (Husi et al., 2000; Uchida et al., 1996) where they mediate adhesion (Bruses 2000). This system could play an important role in recruiting specific receptors at synaptic sites (Coussen et al., 2002). In reconstituted systems, cadherins directly recruit kainate receptors at cadherin-mediated adhesion sites, and this interaction is negatively regulated by PSD-95. Cadherins are also regulators of synapse shape and function (Togashi et al., 2002). Therefore regulation of adhesion at synapses is likely to be a mechanism for plasticity of neuronal transmission, and symmetrically, synaptic activity locally modifies cadherin-cadherin interactions and consequently synaptic adhesion (Tanaka et al., 2000). Depolarization induces a translocation of b−catenin from the dendritic shaft to the spine where its interaction with cadherins modifies the spine shape and the synaptic strength (Murase et al., 2002). Interestingly, disruption by various means of cadherin-mediated adhesion leads to a calciumdependent reduction in long-term potentiation induction (Tang et al., 1998). Cadherins and b−catenins are also present at inhibitory synapses (Uchida et al., 1996; Togashi et al.,

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2002; Benson and Tanaka, 1998). At GABAergic synapses, blockade of the cadherin-cadherin interactions induced a redistribution of the presynaptic glutamic acid decarboxylase, the GABA synthetic enzyme, but, unlike what can be seen at excitatory synapses, the clustered organization of GABAR remained unchanged. As these results were obtained in primary neuronal culture, it was then hypothesized that results would have been hidden by the fact that a large number of clusters are not innervated (Christie et al., 2002). However, the inhibition of cadherin has no effect on inhibitory postsynaptic potential (Tang et al., 1998). To come back to an old story, the structures of inhibitory and excitatory synaptic complexes differ, with a narrower synaptic cleft at inhibitory (20 nm) than at excitatory (30 nm) contacts (ref. In Peters et al., 1976). This probably reflects a specific molecular organization of the adhesive molecules. The neurexin-neuroligin that form a hydrophilic adhesive complex has been observed at excitatory but not at inhibitory synapses. When present, this molecular structure is distributed all along the synapse (Song et al., 1999). Some isoforms of neurexins (see ref in Missler and Sudhof 1998) and neuroligin (Song et al., 1999) are pre- and postsynaptic respectively. Neurexin and neuroligin are likely to be involved in the physical organization of receptors themselves. Intracellularly, the presynaptic neurexins bind to CASK (Butz et al., 1998), while the postsynaptic neuroligins bind to PSD-95 and related proteins (Irie et al., 1997). Therefore, neuroligin is coupled to the NMDA receptor via PSD-95. Furthermore, adhesion proteins such as neuroligin (Scheiffele et al., 2000) and other adhesion molecules including SynCAM (Briederer et al., 2002) or Sidekicks (Yamagatae et al., 2002) contribute to the formation and differentiation of synapses, by interacting with organizing scaffolding molecules, and subsequently to the recruitment of receptors. Therefore, synaptic adhesion molecules not only constitute an obstacle to the diffusion of membrane proteins, they also contribute to their corralling via the actin-based cytoskeleton, and they provide receptor-binding sites via their interaction with scaffolding molecules. 6. Cytoskeleton elements Different cytoskeletal elements bind to postsynaptic scaffold components or directly to receptor subunits via a linker protein. These interactions not only stabilize the scaffoldreceptor complex but also regulate the entry and exit from synapses, as well as in some cases the local density of receptors and the shape of the postsynaptic spines. At excitatory synapses, the different receptors are differentially stabilized by the cytoskeleton. AMPARs are sensitive to F-actin disruption while NMDARs are not (Allisson et al., 2000). At inhibitory synapses, GlyR and GABAR behave differently. With GlyR, alterations of the actin-based and microtubulebased cytoskeleton have different consequences (Kirsch and Betz, 1995). Depolymerization of microtubules induces a lateral spread of the GlyR-enriched postsynaptic micro-

domains, with a reduction in gephyrin density. In contrast, depolymerization of actin filaments generated smaller clusters with increased gephyrin concentration. These observations suggest that the local concentration of GlyR is regulated by the cytoskeleton. The gephyrin-cytoskeleton interactions might be direct or indirect, but the molecular determinants of this link have not been established. These gephyrincytoskeleton interactions were not detected at GABAergic inhibitory connections within hippocampal neurons (Allisson et al. 2000), revealing a striking heterogeneity between inhibitory scaffolds. This could be due to scaffolds containing different gephyrin variants with distinct functions (Meier et al., 2000), or specific associations with other partner molecules specific to GlyR or GABAR. 7. Dynamic processes at synapses The subsynaptic scaffold is a complex molecular machine with functions including spatial stabilization, and trafficking of receptors as well as signal transduction. Our current view of the postsynaptic neuronal membrane has long relied on the fixed snapshot, that does not account for the rapid structural modifications during development, steady state and plasticity. Indeed, the composition of synapses varies on the scale of minutes during synaptogenesis and plasticity. 8. Synaptic dynamics during synaptogenesis Changes in local concentration and number of receptors at synapses may result from diffusion of receptors in the plasma membrane. This notion was initially proposed for AChR accumulation at the neuromuscular junction (NMJ) (Axelrod et al., 1976; Kuromi et al., 1985) and then extended to central synapses (Rao et al., 1998). At the NMJ, the notion that progressive accumulation of receptors in the synapse resulted from lateral diffusion of receptors between the extrasynaptic and synaptic membrane was put forward even before that of regulated endo/exocytosis (Dubinsky et al., 1989). It was subliminal in many articles, and not studied as such, that at central synapses, diffusion of receptors in the plasma membrane, may be involved in setting receptor numbers at the synapse. At glutamatergic synapses (ref in Garner et al., 2002), precise sequences of events lead to the formation of the mature postsynaptic membrane. The adhesive molecules accumulate first at loci of interactions between the future preand postsynaptic elements, followed by PSD scaffold proteins and then by the NMDA and the AMPA receptors. Interestingly, the glutamate receptors redistribute at the cell surface during synapse formation (Mammen et al., 1997), suggesting that diffusion processes or differential turnover could be involved. At inhibitory synapses, postsynaptic scaffold elements appear first in front of synaptic boutons, and then receptors for glycine, GABA or both accumulates at PSDs (Kirsch et

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al., 1993; Colin et al., 1998). The formation of the subsynaptic scaffold and the subsequent accumulation of inhibitory receptors are determined by the presynaptic afferent innervation (Levi et al., 1999). The accumulation of a given receptor type at synaptic sites is paralleled by its disappearance from the extrasynaptic membrane, suggesting that it has been captured by the subsynaptic scaffold. During development, the activity of the glycine receptor is necessary for its postsynaptic accumulation (Levi et al., 1998; Kirsch and Betz 1998). It has been hypothesized (Kirsch and Betz, 1998) that this could be related to the depolarizing action of glycine at early stages of development, which would activate voltage-dependent calcium channels leading to a local increase in calcium in front of inhibitory terminals. This calcium increase allowing the initial formation of the gephyrinbased scaffold would capture the receptors floating in the plasma membrane. Although attractive, this scheme cannot account for the specificity of receptor accumulation at given synapses since activations of GABAR or GluR are also depolarizing during development. Instead, the activity of the GlyR is involved in the insertion-internalization equilibrium and the reduction of GlyR number at the cell surface reduces its capture by the postsynaptic scaffold (Rasmussen et al., 2002). 9. Turnover of synaptic constituents An important aspect of synapse dynamics relies on the relations, still not understood, between the lifetime of the synapse (Lendvai et al., 2000; Trachtenberg et al., 2002) and the turnover of its constitutive elements. The structure of synapses is maintained over time periods spanning days, weeks and even years, although the life-time of given receptors is at most on the order of days (Trachtenberg et al., 2002; Grutzendler et al., 2002). This structural stability is achieved despite a constant flux of molecules. At excitatory synapses, AMPA receptors cycle continuously between the PSD and intracellular pools through endo/exocytotic mechanisms (reviewed in Carroll et al., 2001). The internalization of AMPAR depends upon dynamin (Carroll et al., 1999a) and clathrin assembly (Mann et al., 2000). The exocytotic process is less well characterized but is likely to be SNARE-dependent (Lu et al., 2001). Under basal conditions, the life-time of an individual GluR2-containing AMPAR on the cell surface is estimated to be on the order of 10-30 minutes (Ehlers 2000; Lin et al., 2000; Noel et al., 1999). The turnover of the whole cycle process is regulated by neuronal activity and scaffolding or accessory proteins such as GRIP and NSF (Ehlers et al., 2000; Nishimune et al., 1998; Braithwaite 2002), but the precise step in which these proteins are involved is still a matter of debate (Lee et al., 2002). The NMDA receptors are also trafficking by endo/ exocytosis, and this traffic occurs on a time scale of minutes, the redistribution of NMDAR being regulated by neuronal activity and different kinases (Carroll and Zukin 2002). Furthermore, NMDA receptor-mediated excitatory postsynaptic

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currents show anomalous recovery following “irreversible” block by MK-801(Tovar et al., 2002). This indicated, indirectly, that NMDA receptors moved laterally into the synapse. At least 65% of synaptic NMDA receptors were mobile. At inhibitory synapses, both glycine (Rasmussen et al., 2002) and GABA (Barnes 2000) receptors are recycled on a short time scale. At this stage, the turnover of gephyrin could not be demonstrated directly, but its association with Dlc 2 and the localization of this motor at the periphery of the synapse suggests that it could be involved in the recycling of gephyrin (Fuhrmann et al., 2002). At glutamatergic synapses, continuous remodeling of scaffolding elements such as PSD95on the scale of minutes has been directly demonstrated (Okabe et al., 1999). Recycling of receptors is based on a cycle of endo/ exocytosis from the plasma membrane. Surprisingly, endoor exocytotic vesicles have not been visualized directly at postsynaptic differentiation. The closest ones were seen at the periphery of the synapse: clathrin assembly and disassembly occur at hotspots throughout dendrites, laterally to the PSDs (Blanpied et al., 2002). Therefore, it is likely that diffusion of receptors in the plane of the membrane is necessary for their removal from or addition to the PSDs. Reciprocally, it is not known whether there are also hotspots for exocytosis of receptors outside PSDs. Furthermore, the sites of insertion of receptors in the plasma membrane may differ for different types of receptors. Using cleavable extracellular tags, it was established that the glycine receptor is inserted by constitutive secretion in the somatic plasma membrane and that it secondarily diffuses down dendrites (Rosenberg et al., 2001). This allowed one of the first measurements of the lateral diffusion coefficient in the plasma membrane of central neurons. Dendrites were invaded by GlyRs at a speed of about 1 µm per minute. Using a similar approach, it was found that surface insertion of AMPARs occurs along dendrites in a subunit-dependent manner (Passafaro et al., 2001). At early times after exocytosis of new receptors, GluR1 was diffusively distributed along dendrites, while GluR2 formed clusters at synapses. This might result from an extrasynaptic insertion of GluR1 subunits and from an insertion of GluR2 more directly at or near synapses. Alternatively, these differences could also derive from a different mobility of the two subunits in the extrasynaptic membrane. GluR1- and GluR2containing receptors might be exocytosed at similar sites, but the rate of GluR1 trapping at synapses could be slower than that of GluR2 due to a slower diffusion rate. 10. Synaptic plasticity and postsynaptic receptor number modifications Until recently, trafficking of receptors during development or plasticity was mainly viewed within the framework of endocytic and exocytic processes. The underlying mechanisms may be similar to those operating during constitutive recycling. They are complementary to post-translational

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modifications of receptors involved in long-term modifications of synaptic transmissions such as phosphorylation of the AMPA receptors (Barria et al., 1997; Kameyama et al., 1998). The commonly accepted framework of receptor cycling has undervalued the fact that a decrease in postsynaptic current due to a loss of receptors from a postsynaptic site could be due not only to disappearance of receptors from the cell surface but also to dispersal of synaptic receptors to the extrasynaptic membrane. Reciprocally, the gain of receptors in the PSD could result not only from exocytosis at synapses, but also from insertion of receptors at extrasynaptic loci followed by their recruitment by means of diffusion in the plane of the plasma membrane. Numbers of classical paradigms for long-term changes in synaptic efficacy such as NMDA-dependent LTP or LTD or mGluR-dependent LTD involves changes in the trafficking of AMPARs (ref in Scannevin and Huganir, 2000; Carroll et al., 2001; Malinow et al., 2000; Sheng and Kim, 2002; Turrigiano, 2000). Schematically, postsynaptic LTP involves increased exocytosis of AMPARs and LTD increased endocytosis of receptors. Indeed, insertion and synaptic delivery of GluR1-containing AMPARs is induced by activitydependent LTP or the activation of NMDARs, resulting in synaptic potentiation (Lu et al;, 2001; Passafaro et al., 2001; Hayashi et al., 2000; Shi et al., 1999). This process is blocked by intracellular tetanus toxin (TeTx), therefore implying a SNARE-dependent exocytosis (Lu et al., 2001). As for constitutive cycling, this exocytotic event seems to occur first at extrasynaptic sites, followed by lateral translocation into synapses (Passafaro et al., 2001). Reciprocally, endocytosed AMPARs (Luthi et al., 1999; Wang et al., 2000; Carroll et al 1999b) was shown to be involved during LTD or glutamate application. However, glutamate by itself did not increase the efficiency of the endocytotic pathway (Zhou et al., 2001). Further, internalization of AMPARs is induced by depolymerization of the actin cytoskeleton, and a drug which stabilizes actin filaments blocked the internalization. This has led to the suggestion that glutamate could induce a dissociation of AMPARs from their anchors and that this may be followed by entry of AMPARs in a constitutive endocytotic pathway in the extrasynaptic domain of the membrane. If this mechanism is true, diffusion of receptor from synaptic to extrasynaptic sites is a obligatory step. Studies of the function of stargazin which links AMPARs to PSD-95 (Schnell et al. 2002; Chen et al 2000b) is a base for molecular mechanisms involved in the regulation of the accumulation of AMPARs at synaptic sites through lateral diffusion. Indeed, the increase of stargazin raises specifically the level of extrasynaptic AMPARs with no change in AMPA synaptic response. Furtheremore the disruption of stargazinPSD-95 interaction leads to a massive reduction in the amount of synaptic AMPARs and to an increase in extrasynaptic AMPARs. Finally, raising the level of expression of synaptic PSD-95 leads to an increase in synaptic proteins including AMPARs. Altogether, stargazin might regulate AMPAR trafficking between the synaptic and extrasynaptic

membrane through trapping of diffusing AMPARs by binding to synaptic PSD-95. Interestingly, acutely blocking palmitoylation disperses synaptic clusters of PSD-95 and causes a selective loss of synaptic AMPA receptors while rapid glutamate-mediated AMPA receptor internalization requires depalmitoylation of PSD-95 (El-Husseini 2002). 11. Direct visualization of receptor movements Strong experimental data support the notion that neurotransmitter receptors are aggregated and stabilized through interactions with specific scaffolding proteins. However, the distinction between aggregation and stabilization is often unclear and is most often not addressed as such. Coexpression experiments in heterologous systems of given receptors with their corresponding scaffold element(s) usually lead to the formation of small receptor clusters in the plasma membrane, and the disruption of these interactions resulted in receptor spreading (Kornau et al., 1995; Naisbitt et al., 1999). An intriguing observation, is that receptorscaffold complexes form small clusters homogeneously distributed at the cell surface but not a single large cluster of receptors, a situation expected from scaffold-induced crosslinking of receptors. We postulate that understanding how receptor clusters are formed, is directly linked to the mechanism underlying the dynamic organization of the PSD. The movement of individual or small clusters of receptors, with or without interaction with scaffolding proteins, have been followed in real time using single particle tracking in cultured neurons (Meier et al. 2001; Borgdorff 2002; Sergé et al., 2002) (Fig. 2). To this aim 0.5-µm latex particles coated with specific antibodies and bound to various receptors on the cell surface were tracked. The movements of glycine and AMPA subtype glutamate ionotropic receptors, as well as of metabotropic type 5 glutamate receptors, have been studied. In the case of GlyRs and mGluR5, an epitope tag was added at the extracellular N-terminus of the receptors which were transiently expressed in cultured neurons. For AMPARs, anti-GluR2 antibody recognizing native receptors were used. Receptor movements in the plane of the plasma membrane displayed features in common with fast random movements, exploring micrometer-sized domains within minutes. These fast movements were interspersed by periods of reduced mobility during which diffusion was several orders of magnitude slower. The presence of scaffolding proteins, the maturation state of the neurons and neuronal activity modify the relative time spent in each state. Periods of fast diffusion correspond to Brownian movements of the receptors powered solely by thermal agitation. This was evidenced by the characteristic linear relationship between the mean squared displacement of the receptors and time. During these periods, the diffusion coefficient ranged from 0.05 µm2/sec to 0.5 µm2/sec and was similar to that reported for diffusing proteins in other systems or by other techniques such as FRAP and single molecule tracking (Saxton and Jacobson 1997).

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Fig. 2. Receptor diffusion in neurons measured by single particle tracking. A: superimposed image of the trajectory of 500 nm beads bound to GlyR (left panel) and mGluR5 (right panel) with the fluorescent image (green) of GFP-tagged gephyrin (left) and Homer (right). Periods of free diffusion (blue) and confinement (red) in the trajectories are detected using a confinement index (bottom graphs). B: Plots of the average MSD function during periods of free diffusion (left) and confinement (right) for GlyRs. Note the difference in both shape and amplitude of the MSDs. The negatively curved shape of the MSD is characteristic of movement in a confined space. C: Plots of the cumulative distributions of dwell times for GlyR in the confined state in the presence (filled triangles) and absence (open triangles) of gephyrin. Distributions are fitted with the sum of two exponentials whose time constants increase in the presence of gephyrin. D: model for the exchange of receptors between dispersed (freely diffusing) and clustered (confined movement) states. Receptors might also diffuse within the clusters. A, and C: modified from Meier et al., 2001, and B from Sergé et al., 2002.

Periods of slow mobility (with diffusion coefficients below 10–2 µm2/sec) corresponded to stretches of movement during which receptors explored a smaller surface area than expected if they were diffusing freely. During this period receptor diffusion was not Brownian, but was confined (restricted in space) as indicated by the MSD function during these periods that was not linear, but negatively curved. This type of restriction, has usually been attributed to limitation of receptor movements by barriers acting as fences (Jacobson et al., 1995; Kusumi and Sako, 1996), by obstacles in the membrane (Daumas et al., 2003) or by transient association with specialized lipid microdomains (Dietrich et al., 2002; Anderson and Jacobson, 2002). We have demonstrated that they also originate from transient association with receptorscaffold clusters (Meier et al., 2001; Sergé et al., 2002). This was based on the following: 1) during periods of confinement both GlyR and mGluR5 are most often located over clusters of their corresponding scaffolding proteins; and 2) repetitive binding and unbinding of receptors from the periphery of individual clusters. However, even when receptor-scaffold interactions are totally prevented, by removing their scaffold interaction sequences, receptors still spend about 10% of the time in states of slow diffusion. Receptors are not immobilized but still diffuse, albeit slowly while associated with clusters. This could be due either to diffusion of the clusters themselves and/or to diffusion of receptors within the clusters. In a model membrane, a cluster of a 100 receptors should have a diffusion coefficient only two times lower than that of individual receptors. In agreement with this theory, by tracking clusters of GFP-

tagged mGluR5 by epifluorescence, we found some clusters that diffused about as fast as free Brownian diffusing receptors (Sergé et al. 2002). However, most clusters diffused one to three orders of magnitude slower than these fast onesThis was not a surprise since the diffusion of clusters is more likely to be impeded by membrane obstacles and cytoskeletal fences than that of individual receptors. Altogether, this indicates that extrasynaptic clusters of receptors can display a wide range of mobility properties and further support the notion that aggregation and stabilization of receptors are two distinct processes. In any cases, binding of clusters or of individual receptors to rigid elements such as the cytoskeleton or components of the extracellular surroundings is imperative for immobilization. Such is the case for PSD scaffolds that are anchored both to the cytoskeleton and to the presynaptic terminals through adhesion proteins (see above). Exchanges of receptors between clustered and unclustered states are directly related to the molecular scaffold arrangement and to diffusion within a cluster (Fig. 3). Direct exchanges of receptors between confined domains through unconstrained diffusion for both GlyR and mGluR5 have been obesrved. Furthermore, in Homer-mediated clusters of mGluR5, about 50% of the receptors can be replaced within minutes by ones outside the cluster through lateral diffusion as indicated by FRAP experiments. This exchange is only possible if receptors diffuse within clusters, in which case diffusion is expected to be confined due to the presence of the multiple obstacles. Therefore, we postulate that the composition of clusters is continuously regenerated. This results from the reversibility of receptor scaffold interactions and

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A

(a)

(c)

(b)

(e)

B

(e)

(a)

(e)

(b)

(d) Receptors Scaffold elements cytoskeleton

Immobile receptors Fig. 3. Kinetic diagram of dynamic instability and equilibrium of the receptor-scaffold complex. A: Receptor, scaffold and cytoskeleton can have various levels of associations. The receptor can be single or dispersed (a), single and associated with scaffold proteins (b), form microclusters without (c) or with (d) associated scaffold proteins, and, the receptor-scaffold clusters can bind to the cytoskeleton (e) at the PSD (gray square). The (c), (d) and (e) associations have been demonstrated directly for excitatory and inhibitory synapses, (a) and (b) have not yet been visualized directly. We postulate that these associations are at equilibrium, and double-headed arrows indicate the possible transitions. Note that the immobilized receptor-scaffold complex (e) can be at equilibrium with a single receptor (a), a single receptor-scaffold (b) or a microcluster bound to scaffold (d). B: Diffusing receptors, single (a) or associated with scaffold proteins (b), may bind to free slots in PSDs (e) on scaffold or cytoskeleton, respectively.

from the 2D structure of the scaffold below the plasma membrane that leaves free receptor-binding sites and free membrane space in between binding sites. Receptor diffusion and exchanges would then occur – within that space. The residence time of the receptor in the cluster directly sets the rate of postsynaptic receptor renewal. Therefore, this rate depends on the density of obstacles in the cluster and on the affinity of the receptor for the scaffold elements. Measurements of the residence times of single receptor molecules in the confined state, which largely corresponds to the clustered state of the receptor, would give access to “corralling” and affinity parameters. However, at the present time, we cannot assign a time constant to given physical states of the receptor. According to a “corralling-affinity” model, the size of a given cluster depends both on the affinity of the receptors for scaffold molecules and on interactions between scaffolding molecules. If they bind one another sufficiently tightly, they should aggregate to form a single and large domain in the membrane. Conversely, if they bind one another with the same affinity as they bind to other proteins or lipid molecules, then they should remain dispersed in the plasma membrane. The relatively small size of the receptor-scaffold clusters therefore indicates that receptor and scaffold molecules have an affinity for each other, which equilibrates Brownian spreading forces. In other terms, aggregated receptors and scaffolding proteins are in equilibrium with their free forms,

so that receptors and scaffolding proteins are added to and leave the clusters at equal rates. A crucial question is whether a similar equilibrium could account for receptor movements through lateral diffusion between the extrasynaptic and the synaptic plasma membrane. For GluR2-containing AMPA receptors, zones of confinement were often topographically related to synaptic structures, suggesting that around synapses there is a region of lower receptor diffusion. This could correspond to reversible AMPA receptor binding to the external border of the postsynaptic density (Fig. 4). A clear limitation of these experiments however, was that particle size prevents measurement of receptor diffusion inside the synapse. Since receptor trafficking in and out of synapses is under the control of neuronal activity, we found that basal neuronal activity in synaptically connected neurons could also control AMPA receptor movement. Activity-dependent elevation in postsynaptic [Ca2+]i is known to be a central element in the regulation of synaptic transmission efficacy. We found that raising intracellular calcium triggers rapid receptor immobilization and local accumulation on the neuronal surface. This suggests that calcium influx prevents AMPAR from diffusing. The molecular mechanism underlying calciumdependent AMPAR immobilization is unknown, but likely derives from calcium-induced binding of the receptors to

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The work of AT and DC presented in this review has been supported by the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Fondation pour la Recherche Médicale, the Conseil Régional d’Aquitaine, the Association Française contre les Myopathies, the Institut de Recherche sur la Moelle Epinière. We wish to thank Christian Vannier and Laurent Cognet for their precious comments on the manuscript, and the members of AT and DC laboratories involved in this work. Synaptic

References Recycling

Synthesis Degradation

Fig. 4. Receptor exchanges between synaptic, extrasynaptic and intracellular compartments are regulated by confinement. Upper : The extrasynaptic diffusive receptors (blue) are immobilized (red) by the scaffold-cytoskeleton complex (pink) at PSDs. The extrasynaptic receptors are exchanging with the intracellular recycling pool (blue arrows). Increasing or diminishing the number (or affinity) of scaffold molecules at the synapse (+ and – respectively) changes proportionally the confinement capabilities, and subsequently the number of receptors at equilibrium. Note that each synapse behaves as a receptor donor/acceptor module. Lower : Bloc diagram of exchanges between cellular and membranous compartments.

scaffolding proteins. This process has some specificity, as it is not observed for other receptors such as mGluR5.

12. Conclusion The perpetual movement of receptors must be taken into account in the context of the formation and plasticity of post-synaptic microdomains. Although few non clustered receptors may exist at a given time point on the neuronal membrane, any stabilized receptor may enter a diffusive state within a short time period and thus travel long distances. This allows receptors to exchange rapidly between regional specializations such as synapses and enter processes likely to only occur outside the synapse. The stability of the postsynaptic membrane must be considered as a molecular assembly with the turnover of its constitutive elements and the plasticity considered as a change in the set point of the equilibrium between synaptic and extrasynaptic receptor pools (Fig. 4). All these processes being regulated by the interaction with scaffolding molecules. Changes in either the number of stabilizing molecules or their affinity for receptors would regulate these transitions. Such mechanisms can operate in synaptogenesis and synaptic plasticity.

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