Synaptic Connectivity series
Surface trafficking of receptors between synaptic and extrasynaptic membranes: and yet they do move!

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Concentration of neurotransmitter receptors at synapses is thought to result from stable binding to subsynaptic scaffold proteins. Recent data on synaptic plasticity have shown that changes in synaptic strength derive partly from modification of postsynaptic receptor numbers. This has led to the notion of receptor trafficking into and out of synapses. The proposed underlying mechanisms have under-evaluated the role of extrasynaptic receptors. Recent technological advances have allowed imaging of receptor movements at the single-molecule level, and these experiments demonstrate that receptors switch at unexpected rates between extrasynaptic and synaptic localizations by lateral diffusion. Variation in receptor numbers at postsynaptic sites is therefore likely to depend on regulation of diffusion by modification of the structure of the membrane and/or by transient interactions with scaffolding proteins. This review is part of the TINS Synaptic Connectivity series.

Introduction

New imaging methods now allow visualization of receptor movements at the single-molecule level inside and outside synapses. They have revealed that receptors permanently exchange between synaptic and extrasynaptic locations. This has changed our view of the organization of the neuronal membrane, which now includes lateral diffusion of receptors as a key parameter for regulation of synapse function and plasticity. As a consequence, extrasynaptic pools of receptors are likely to have a more important role than previously suspected. The aim of this review is to highlight this point.

Until recently, ultrastructural immunocytochemistry has provided the backbone of our knowledge on the distribution of receptors in neurons. The first receptor to be seen using electron microscopy concentrated at synaptic sites was that for glycine [1], and it was followed by many others 2, 3. This morphological approach has established that most receptor types are concentrated at synapses [4]. The ratio between the numbers of synaptic versus extrasynaptic receptors was generally found to be higher in morphological experiments than in physiological experiments, which generally detect more extrasynaptic receptors (e.g. see Refs 5, 6). This discrepancy resulted from the failure of immunocytochemistry to detect receptors at low density, such as in the extrasynaptic membrane (Figure 1). The consequences of these discrepancies have been overlooked. Indeed, the total number of surface extrasynaptic receptors is likely to be larger than that of synaptic ones, owing to the large surface area of extrasynaptic versus synaptic membrane [7].

The preferential and specific localization of receptors at synapses has long been postulated to result from their interactions with submembranous scaffolding proteins. The first of these to be characterized at central synapses was gephyrin [8], which is localized below receptor microdomains at inhibitory synapses 1, 9. Comparison with the neuromuscular junction encouraged the postulate that gephyrin is involved in the so-called ‘stabilization’ and ‘concentration’ of the receptors [1]. These two concepts, often unduly mixed, were extended to most central synapses and believed to be the heart of synapse-specific receptor localization. This was reinforced by the discovery and characterization of numerous scaffolding molecules interacting with receptors 10, 11. These structural and biochemical observations have perpetuated the false notion that receptors are fixed at synapses and that this accounts for their concentration. Although electrophysiology has long-since provided evidence for the existence of extrasynaptic receptors [12], they were often thought to pertain to a pool of receptors distinct from synaptic ones. More importantly, their physiological roles have been limited to activation by spillover of neurotransmitter outside the synaptic cleft during massive release 13, 14, 15 or during glutamate release by neighboring glia [16]. The notion that extrasynaptic and synaptic receptors are separate entities was reinforced by the fact that some receptor isoforms have specific subcellular distributions. For example, at excitatory synapses, the NR2b subunit of the NMDA receptor is mainly extrasynaptic in the adult, whereas NR2a is detected both inside and outside synapses [6]. At inhibitory synapses, GABA receptors with various subunit compositions have preferential extrasynaptic and synaptic distributions [17].

This classical static view of receptor distribution was challenged a few years ago by evidence that receptor numbers at synapses are tuned during regulation of synaptic strength (reviewed in Refs 18, 19, 20). This is now considered one of the molecular bases of synaptic plasticity and has led to the important notion of receptor flux into and out of synapses, both at rest and during plasticity. It has prompted the development of dynamic real-time imaging approaches in living neurons, such as videomicroscopy of green fluorescent protein (GFP)-tagged receptors, to go beyond the fixed snapshots given by immunocytochemistry. However, these multimolecular approaches have limits and, for example, cannot detect receptor fluxes in basal conditions when synaptic receptor numbers remain globally constant. The advent of single-molecule imaging techniques now enables measurement of individual receptor movements in identified submembranous compartments, and reveals the heterogeneities and new physical parameters important for the understanding of receptor trafficking.

Section snippets

Evidence for rapid traffic of synaptic receptors

Rapid activity-dependent appearance or disappearance of receptors at synapses has been established for GABA and glutamate receptors. The mechanisms underlying these rapid changes involve exocytosis and endocytosis of receptors (reviewed in Refs 18, 19, 20, 21). Until recently, changes in receptor numbers at synapses were mainly considered as cycling between surface and intracellular compartments. Thus, attention has been drawn to intracellular receptors, and large intracellular pools of

Direct visualization of receptor movements and regulation by scaffold proteins

Recent results indicate that glycine receptors 45, 46, GABA receptors (S. Lévi, personal communication), and AMPA [31] and NMDA [47] glutamate receptors are all mobile in synapses and can be exchanged between synapses through lateral diffusion in the plane of the extrasynaptic plasma membrane.

The initial concept, according to which receptors can enter and leave synaptic sites through lateral diffusion, was established at the neuromuscular junction [48]. A role for diffusion of receptors in the

Observing single receptors yet thinking of the synapse as a whole

Real-time imaging of extrasynaptic receptor movements led us to postulate that receptors could escape synapses through lateral diffusion. However, the size of the latex beads precluded direct visualization of synaptic receptor dynamics. In the search for approaches allowing detection of receptor movements in synapses, single-molecule detection techniques have been developed (Box 1), using either organic dyes or the recently developed inorganic fluorescent semiconductor particles known as

Revisiting receptor stabilization in the light of receptor movements

There is an apparent contradiction between the continuous receptor movements and the apparent stability of synapses [19]. This arises from the difference between the concept of stabilization and that of local concentration of receptors. Images obtained using classical immunocytochemistry reveal a patchy distribution for many receptors, with accumulation at synapses. Actually, this approach generates a snapshot of the statistical distribution of receptors at a given time, indicating that

Concluding remarks

Because receptors diffuse in the plasma membrane, in the same way that particles do in a two-dimensional field, they could be transiently trapped at specific loci corresponding to postsynaptic densities. New receptors could pop into or disappear from the plasma membrane by exocytosis and endocytosis, respectively. Another characteristic of the neuronal membrane is that the processes of diffusion, trapping and variation in receptor numbers could be regulated by ‘biological processes’ and could

References (68)

  • K.R. Tovar et al.

    Mobile NMDA receptors at hippocampal synapses

    Neuron

    (2002)
  • A. Luthi

    Hippocampal LTD expression involves a pool of AMPARs regulated by the NSF–GluR2 interaction

    Neuron

    (1999)
  • Y.T. Wang et al.

    Expression of cerebellar long-term depression requires postsynaptic clathrin-mediated endocytosis

    Neuron

    (2000)
  • J. Meier

    Functional heterogeneity of gephyrins

    Mol. Cell. Neurosci.

    (2000)
  • A. Kusumi

    Mobility and cytoskeletal interactions of cell adhesion receptors

    Curr. Opin. Cell Biol.

    (1999)
  • S. Murase et al.

    The role of cell adhesion molecules in synaptic plasticity and memory

    Curr. Opin. Cell Biol.

    (1999)
  • A. Triller

    Distribution of glycine receptors at central synapses: an immunoelectron microscopy study

    J. Cell Biol.

    (1985)
  • Y. Takumi

    The arrangement of glutamate receptors in excitatory synapses

    Ann. N. Y. Acad. Sci.

    (1999)
  • J.R. Cottrell

    Distribution, density, and clustering of functional glutamate receptors before and after synaptogenesis in hippocampal neurons

    J. Neurophysiol.

    (2000)
  • K.R. Tovar et al.

    The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro

    J. Neurosci.

    (1999)
  • J. Kirsch

    Gephyrin antisense oligonucleotides prevent glycine receptor clustering in spinal neurons

    Nature

    (1993)
  • R.H. Scannevin et al.

    Postsynaptic organization and regulation of excitatory synapses

    Nat. Rev. Neurosci.

    (2000)
  • D.S. Faber

    Evidence that receptors mediating central synaptic potentials extend beyond the postsynaptic density

    Proc. Natl. Acad. Sci. U. S. A.

    (1985)
  • B.A. Clark et al.

    Activity-dependent recruitment of extrasynaptic NMDA receptor activation at an AMPA receptor-only synapse

    J. Neurosci.

    (2002)
  • A. Momiyama

    The density of AMPA receptors activated by a transmitter quantum at the climbing fibre–Purkinje cell synapse in immature rats

    J. Physiol.

    (2003)
  • Z. Nusser

    Segregation of different GABAA receptors to synaptic and extrasynaptic membranes of cerebellar granule cells

    J. Neurosci.

    (1998)
  • R. Malinow et al.

    AMPA receptor trafficking and synaptic plasticity

    Annu. Rev. Neurosci.

    (2002)
  • M. Sheng et al.

    Postsynaptic signaling and plasticity mechanisms

    Science

    (2002)
  • R.C. Carroll

    Role of AMPA receptor endocytosis in synaptic plasticity

    Nat. Rev. Neurosci.

    (2001)
  • R.S. Petralia et al.

    Light and electron immunocytochemical localization of AMPA-selective glutamate receptors in the rat brain

    J. Comp. Neurol.

    (1992)
  • W. Ju

    Activity-dependent regulation of dendritic synthesis and trafficking of AMPA receptors

    Nat. Neurosci.

    (2004)
  • S.H. Shi

    Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation

    Science

    (1999)
  • M. Park

    Recycling endosomes supply AMPA receptors for LTP

    Science

    (2004)
  • R.S. Petralia

    Internalization at glutamatergic synapses during development

    Eur. J. Neurosci.

    (2003)
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