AMPA and NMDA glutamate receptor trafficking: multiple roads for reaching and leaving the synapse

Glutamate receptor trafficking in and out of synapses is one of the core mechanisms for rapid changes in the number of functional receptors during synaptic plasticity. Recent data have shown that the fast gain and loss of receptors from synaptic sites are accounted for by endocytic/exocytic processes and by their lateral diffusion in the plane of the membrane. These events are interdependent and regulated by neuronal activity and interactions with scaffolding proteins. We review here the main cellular steps for AMPA and NMDA receptor synthesis, traffic within intracellular organelles, membrane exocytosis/endocytosis and surface trafficking. We focus on new findings that shed light on the regulation of receptor cycling events and surface trafficking and the way that this might reshape our thinking about the specific regulation of receptor accumulation at synapses.     

New insights in endosomal dynamics and AMPA receptor trafficking

The trafficking mechanisms that control the density of synaptic AMPA-type glutamate receptors have received significant attention because of their importance for regulating excitatory synaptic transmission and synaptic plasticity in the hippocampus. AMPA receptors are synthesized in the neuronal cell body and reach their postsynaptic targets after a complex journey involving multiple transport steps along different cytoskeleton structures and through various stages of the endocytic pathway. Dendritic spines are important sites for AMPA receptor trafficking and contain the basic components of endosomal recycling. On induction of synaptic plasticity, internalized AMPA receptors undergo endosomal sorting and cycle through early endosomes and recycling endosomes back to the plasma membrane (model for long-term potentiation) or target for degradation to the lysosomes (model for long-term depression). Exciting new studies now provide insight in actin-mediated processes that controls endosomal tubule formation and receptor sorting. This review describes the path of AMPA receptor internalization up to sites of recycling and summarizes recent studies on actin-mediated endosomal receptor sorting.     

Learning from NMDA receptor trafficking: clues to the development and maturation of glutamatergic synapses

Activity-dependent changes in excitatory transmission allow the brain to develop, mature, learn and retain memories, and underlie many pathological states of the central nervous system. A principal mechanism by which neurons regulate excitatory transmission is by altering the number and composition of glutamate receptors at the postsynaptic plasma membrane. The dynamic trafficking of glutamate receptors to and from synaptic sites involves a complex series of events including receptor assembly, trafficking through secretory compartments, membrane insertion and endocytic cycling. While these events have become widely appreciated as critical processes regulating AMPA-type glutamate receptors during synaptic plasticity, the mechanisms that control the trafficking of NMDA-type glutamate receptors (NMDARs) are only now beginning to be understood. Until recently, NMDARs were considered immobile receptors, tightly anchored to the postsynaptic membrane. Here, we review recent evidence that challenges this view, focusing on the role that activity plays in altering NMDAR trafficking and how such dynamic regulation of NMDARs may impact on the plasticity of neural circuits.     

Differential Regulation of Kainate Receptor Trafficking by Phosphorylation of Distinct Sites on GluR6

Kainate receptors are widely expressed in the brain, and are present at pre- and postsynaptic sites where they play a prominent role in synaptic plasticity and the regulation of network activity. Within individual neurons, kainate receptors of different subunit compositions are targeted to various locations where they serve distinct functional roles. Despite this complex targeting, relatively little is known about the molecular mechanisms regulating kainate receptor subunit trafficking. Here we investigate the role of phosphorylation in the trafficking of the GluR6 kainate receptor subunit. We identify two specific residues on the GluR6 C terminus, Ser⁸⁴⁶ and Ser⁸⁶⁸, which are phosphorylated by protein kinase C (PKC) and dramatically regulate GluR6 surface expression. By using GluR6 containing phosphomimetic and nonphosphorylatable mutations for these sites expressed in heterologous cells or in neurons lacking endogenous GluR6, we show that phosphorylation of Ser⁸⁴⁶ or Ser⁸⁶⁸ regulates receptor trafficking through the biosynthetic pathway. Additionally, Ser⁸⁴⁶ phosphorylation dynamically regulates endocytosis of GluR6 at the plasma membrane. Our findings thus demonstrate that phosphorylation of PKC sites on GluR6 regulates surface expression of GluR6 at distinct intracellular trafficking pathways, providing potential molecular mechanisms for the PKC-dependent regulation of synaptic kainate receptor function observed during various forms of synaptic plasticity.     

Dual role of the exocyst in AMPA receptor targeting and insertion into the postsynaptic membrane

Intracellular membrane trafficking of glutamate receptors at excitatory synapses is critical for synaptic function. However, little is known about the specialized trafficking events occurring at the postsynaptic membrane. We have found that two components of the exocyst complex, Sec8 and Exo70, separately control synaptic targeting and insertion of AMPA-type glutamate receptors. Sec8 controls the directional movement of receptors towards synapses through PDZ-dependent interactions. In contrast, Exo70 mediates receptor insertion at the postsynaptic membrane, but it does not participate in receptor targeting. Thus, interference with Exo70 function accumulates AMPA receptors inside the spine, forming a complex physically associated, but not yet fused with the postsynaptic membrane. Electron microscopic analysis of these complexes indicates that Exo70 mediates AMPA receptor insertion directly within the postsynaptic density, rather than at extrasynaptic membranes. Therefore, we propose a molecular and anatomical model that dissects AMPA receptor sorting and synaptic delivery within the spine, and uncovers new functions of the exocyst at the postsynaptic membrane.     

Modeling synaptic dynamics driven by receptor lateral diffusion

The synaptic weight between a pre- and a postsynaptic neuron depends in part on the number of postsynaptic receptors. On the surface of neurons, receptors traffic by random motion in and out from a microstructure called the postsynaptic density (PSD). In the PSD, receptors can be stabilized at the membrane when they bind to scaffolding proteins. We propose a mathematical model to compute the postsynaptic counterpart of the synaptic weight based on receptor trafficking. We take into account the receptor fluxes at the PSD, which can be regulated by neuronal activity, and the interactions of receptors with the scaffolding molecules. Using a Markovian approach, we estimate the mean and the fluctuations of the number of bound receptors. When the number of receptors is large, a deterministic system is also derived. Moreover, these equations can be used, for example, to fit fluorescence-recovery-after-photobleaching experiments to determine, in living neurons, the chemical binding constants for the receptors/scaffolding molecules interaction at synapses.     

Initiation of synapse formation by Wnt-induced MuSK endocytosis

In zebrafish, the MuSK receptor initiates neuromuscular synapse formation by restricting presynaptic growth cones and postsynaptic acetylcholine receptors (AChRs) to the center of skeletal muscle cells. Increasing evidence suggests a role for Wnts in this process, yet how muscle cells respond to Wnt signals is unclear. Here, we show that in vivo, wnt11r and wnt4a initiate MuSK translocation from muscle membranes to recycling endosomes and that this transition is crucial for AChR accumulation at future synaptic sites. Moreover, we demonstrate that components of the planar cell polarity pathway colocalize to recycling endosomes and that this localization is MuSK dependent. Knockdown of several core components disrupts MuSK translocation to endosomes, AChR localization and axonal guidance. We propose that Wnt-induced trafficking of the MuSK receptor to endosomes initiates a signaling cascade to align pre- with postsynaptic elements. Collectively, these findings suggest a general mechanism by which Wnt signals shape synaptic connectivity through localized receptor endocytosis.     

Photoinactivation of native AMPA receptors reveals their real-time trafficking

AMPA receptors mediate the majority of the fast excitatory transmission in the central nervous system. Much evidence suggests that the fast trafficking of AMPA receptors into and out of the postsynaptic membrane underlies changes in synaptic strength thought to be necessary for higher cognitive functions such as learning and memory. Despite the abundance of research conducted in this area, a direct, real-time functional assay that measures the trafficking of native AMPA receptors has been lacking. Toward this aim, we use a photoreactive, irreversible antagonist of AMPA receptors, ANQX, to rapidly silence surface AMPA receptors and investigate directly the trafficking of native AMPA receptors in real time. We find that the most dynamic movement of AMPA receptors occurs by lateral movement across the surface of neurons. Fast cycling of surface AMPA receptors with receptors from internal stores does occur but exclusively at extrasynaptic somatic sites. The cycling of synaptic AMPA receptors only occurs on a much longer timescale with complete exchange requiring at least 16 hr. This cycling is not dependent on protein synthesis or action potential driven network activity. These data suggest a revised model of AMPA receptor trafficking wherein a large internal store of AMPA receptors exchanges rapidly with extrasynaptic somatic AMPA receptors, and these newly inserted AMPA receptors then travel laterally along dendrites to reside stably at synapses.     

Modeling the role of lateral membrane diffusion in AMPA receptor trafficking along a spiny dendrite

AMPA receptor trafficking in dendritic spines is emerging as a major postsynaptic mechanism for the expression of plasticity at glutamatergic synapses. AMPA receptors within a spine are in a continuous state of flux, being exchanged with local intracellular pools via exo/endocytosis and with the surrounding dendrite via lateral membrane diffusion. This suggests that one cannot treat a single spine in isolation. Here we present a model of AMPA receptor trafficking between multiple dendritic spines distributed along the surface of a dendrite. Receptors undergo lateral diffusion within the dendritic membrane, with each spine acting as a spatially localized trap where receptors can bind to scaffolding proteins or be internalized through endocytosis. Exocytosis of receptors occurs either at the soma or at sites local to dendritic spines via constitutive recycling from intracellular pools. We derive a reaction–diffusion equation for receptor trafficking that takes into account these various processes. Solutions of this equation allow us to calculate the distribution of synaptic receptor numbers across the population of spines, and hence determine how lateral diffusion contributes to the strength of a synapse. A number of specific results follow from our modeling and analysis. (1) Lateral membrane diffusion alone is insufficient as a mechanism for delivering AMPA receptors from the soma to distal dendrites. (2) A source of surface receptors at the soma tends to generate an exponential-like distribution of receptors along the dendrite, which has implications for synaptic democracy. (3) Diffusion mediates a heterosynaptic interaction between spines so that local changes in the constitutive recycling of AMPA receptors induce nonlocal changes in synaptic strength. On the other hand, structural changes in a spine following long term potentiation or depression have a purely local effect on synaptic strength. (4) A global change in the rates of AMPA receptor exo/endocytosis is unlikely to be the sole mechanism for homeostatic synaptic scaling. (5) The dynamics of AMPA receptor trafficking occurs on multiple timescales and varies according to spatial location along the dendrite. Understanding such dynamics is important when interpreting data from inactivation experiments that are used to infer the rate of relaxation to steady-state.     

The dynamics of synaptic scaffolds

Complex functions of the central nervous system such as learning and memory are believed to result from the modulation of the synaptic transmission between neurons. The sequence of events leading to the fusion of synaptic vesicles at the presynaptic active zone and the detection of this signal at the postsynaptic density involve the activity of ion channels and neurotransmitter receptors. Their accumulation and dynamic exchange at synapses are dependent on their interaction with synaptic scaffolds. These are synaptic structures composed of adaptor proteins that provide binding sites for receptors and channels as well as other synaptic proteins. While in its entirety the synaptic scaffold is a relatively stable structure, individual adaptor proteins exchange at a fast time scale. These properties of scaffolds help to ensure the stability of synaptic transmission while permitting the modulation of synaptic strength. Here, we review the dynamics of the synaptic scaffold and of adaptor proteins in relation to their roles in the organisation of the synapse as well as in the clustering and trafficking of receptor proteins.     

AMPA receptor trafficking at excitatory synapses

Excitatory synapses in the CNS release glutamate, which acts primarily on two sides of ionotropic receptors: AMPA receptors and NMDA receptors. AMPA receptors mediate the postsynaptic depolarization that initiates neuronal firing, whereas NMDA receptors initiate synaptic plasticity. Recent studies have emphasized that distinct mechanisms control synaptic expression of these two receptor classes. Whereas NMDA receptor proteins are relatively fixed, AMPA receptors cycle synaptic membranes on and off. A large family of interacting proteins regulates AMPA receptor turnover at synapses and thereby influences synaptic strength. Furthermore, neuronal activity controls synaptic AMPA receptor trafficking, and this dynamic process plays a key role in the synaptic plasticity that is thought to underlie aspects of learning and memory.     

Clustering of nicotinic acetylcholine receptors: From the neuromuscular junction to interneuronal synapses

Fast and accurate synaptic transmission requires high-density accumulation of neurotransmitter receptors in the postsynaptic membrane. During development of the neuromuscular junction, clustering of acetylcholine receptors (AChR) is one of the first signs of postsynaptic specialization and is induced by nerve-released agrin. Recent studies have revealed that different mechanisms regulate assembly vs stabilization of AChR clusters and of the postsynaptic apparatus. MuSK, a receptor tyrosine kinase and component of the agrin receptor, and rapsyn, an AChR-associated anchoring protein, play crucial roles in the postsynaptic assembly. Once formed, AChR clusters and the postsynaptic membrane are stabilized by components of the dystrophin/utrophin glycoprotein complex, some of which also direct aspects of synaptic maturation such as formation of postjunctional folds. Nicotinic receptors are also expressed across the peripheral and central nervous system (PNS/CNS). These receptors are localized not only at the pre- but also at the postsynaptic sites where they carry out major synaptic transmission. In neurons, they are found as clusters at synaptic or extrasynaptic sites, suggesting that different mechanisms might underlie this specific localization of nicotinic receptors. This review summarizes the current knowledge about formation and stabilization of the postsynaptic apparatus at the neuromuscular junction and extends this to explore the synaptic structures of interneuronal cholinergic synapses.     

Local control of AMPA receptor trafficking at the postsynaptic terminal by a small GTPase of the Rab family

The delivery of neurotransmitter receptors into the synaptic membrane is essential for synaptic function and plasticity. However, the molecular mechanisms of these specialized trafficking events and their integration with the intracellular membrane transport machinery are virtually unknown. Here, we have investigated the role of the Rab family of membrane sorting proteins in the late stages of receptor trafficking into the postsynaptic membrane. We have identified Rab8, a vesicular transport protein associated with trans-Golgi network membranes, as a critical component of the cellular machinery that delivers AMPA-type glutamatergic receptors (AMPARs) into synapses. Using electron microscopic techniques, we have found that Rab8 is localized in close proximity to the synaptic membrane, including the postsynaptic density. Electrophysiological studies indicated that Rab8 is necessary for the synaptic delivery of AMPARs during plasticity (long-term potentiation) and during constitutive receptor cycling. In addition, Rab8 is required for AMPAR delivery into the spine surface, but not for receptor transport from the dendritic shaft into the spine compartment or for delivery into the dendritic surface. Therefore, Rab8 specifically drives the local delivery of AMPARs into synapses. These results demonstrate a new role for the cellular secretory machinery in the control of synaptic function and plasticity directly at the postsynaptic membrane.     

Glutamate receptor dynamics in dendritic microdomains

Among diverse factors regulating excitatory synaptic transmission, the abundance of postsynaptic glutamate receptors figures prominently in molecular memory and learning-related synaptic plasticity. To allow for both long-term maintenance of synaptic transmission and acute changes in synaptic strength, the relative rates of glutamate receptor insertion and removal must be tightly regulated. Interactions with scaffolding proteins control the targeting and signaling properties of glutamate receptors within the postsynaptic membrane. In addition, extrasynaptic receptor populations control the equilibrium of receptor exchange at synapses and activate distinct signaling pathways involved in plasticity. Here, we review recent findings that have shaped our current understanding of receptor mobility between synaptic and extrasynaptic compartments at glutamatergic synapses, focusing on AMPA and NMDA receptors. We also examine the cooperative relationship between intracellular trafficking and surface diffusion of glutamate receptors that underlies the expression of learning-related synaptic plasticity.     

Measurement and characteristics of neurotransmitter receptor surface trafficking (Review)

Neurotransmitter receptor trafficking in and out synapses has emerged as a key process to regulate synaptic transmission during synaptic development and plasticity both at excitatory and inhibitory synapses. Lateral diffusion of surface neurotransmitter receptors has recently emerged as a key pathway to regulate receptor trafficking to and from synapses. Receptors enter and exit synapses mainly by lateral diffusion within the plane of the membrane while their retrieval and addition from and to the plasma membrane by endo and exocytotic processes occur largely at extrasynaptic sites. As a consequence, regulation of receptor surface trafficking is likely to be a major process to regulate receptor numbers at synapses. Measurement of receptor surface diffusion has required the development of new experimental approaches to specifically label and track surface receptor with appropriate time- and space-resolutions. In this review, we first discuss the approaches that have been used to measure receptor surface diffusion, such as the ensemble approach that measure average diffusion of a defined surface receptor population and the single molecule/particle approaches that measure the surface diffusion of isolated receptors. To date, surface diffusion has been described for a variety of neurotransmitter receptors that exhibit common as well as specific features. These points are discussed in a comparative manner and emerging rules of surface trafficking as well as potential interplay between receptor classes are further commented. Because our knowledge on neurotransmitter receptor surface diffusion is fairly recent, open questions and experimental challenges facing the field are highlighted throughout the review.     

Measurement and characteristics of neurotransmitter receptor surface trafficking (Review)

Neurotransmitter receptor trafficking in and out synapses has emerged as a key process to regulate synaptic transmission during synaptic development and plasticity both at excitatory and inhibitory synapses. Lateral diffusion of surface neurotransmitter receptors has recently emerged as a key pathway to regulate receptor trafficking to and from synapses. Receptors enter and exit synapses mainly by lateral diffusion within the plane of the membrane while their retrieval and addition from and to the plasma membrane by endo and exocytotic processes occur largely at extrasynaptic sites. As a consequence, regulation of receptor surface trafficking is likely to be a major process to regulate receptor numbers at synapses. Measurement of receptor surface diffusion has required the development of new experimental approaches to specifically label and track surface receptor with appropriate time- and space-resolutions. In this review, we first discuss the approaches that have been used to measure receptor surface diffusion, such as the ensemble approach that measure average diffusion of a defined surface receptor population and the single molecule/particle approaches that measure the surface diffusion of isolated receptors. To date, surface diffusion has been described for a variety of neurotransmitter receptors that exhibit common as well as specific features. These points are discussed in a comparative manner and emerging rules of surface trafficking as well as potential interplay between receptor classes are further commented. Because our knowledge on neurotransmitter receptor surface diffusion is fairly recent, open questions and experimental challenges facing the field are highlighted throughout the review.     

Endocytosis of neurotransmitter receptors: location matters

Endocytosis of excitatory glutamate receptors from the postsynaptic plasma membrane plays a fundamental role in synaptic function and plasticity. In a recent study published in Neuron, Lu et al. (2007) describe protein interactions that link zones of receptor endocytosis directly to the postsynaptic scaffold and propose that local trafficking of receptors facilitated by these endocytic zones is required to maintain synaptic responsiveness.     

Synaptic strength regulated by palmitate cycling on PSD-95

Dynamic regulation of AMPA-type glutamate receptors represents a primary mechanism for controlling synaptic strength, though mechanisms for this process are poorly understood. The palmitoylated postsynaptic density protein, PSD-95, regulates synaptic plasticity and associates with the AMPA receptor trafficking protein, stargazin. Here, we identify palmitate cycling on PSD-95 at the synapse and find that palmitate turnover on PSD-95 is regulated by glutamate receptor activity. Acutely blocking palmitoylation disperses synaptic clusters of PSD-95 and causes a selective loss of synaptic AMPA receptors. We also find that rapid glutamate-mediated AMPA receptor internalization requires depalmitoylation of PSD-95. In a nonneuronal model system, clustering of PSD-95, stargazin, and AMPA receptors is also regulated by ongoing palmitoylation of PSD-95 at the plasma membrane. These studies suggest that palmitate cycling on PSD-95 can regulate synaptic strength and regulates aspects of activity-dependent plasticity.     

Caldendrin-Jacob: a protein liaison that couples NMDA receptor signalling to the nucleus

NMDA (N-methyl-D-aspartate) receptors and calcium can exert multiple and very divergent effects within neuronal cells, thereby impacting opposing occurrences such as synaptic plasticity and neuronal degeneration. The neuronal Ca2+ sensor Caldendrin is a postsynaptic density component with high similarity to calmodulin. Jacob, a recently identified Caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Strictly depending upon activation of NMDA-type glutamate receptors, Jacob is recruited to neuronal nuclei, resulting in a rapid stripping of synaptic contacts and in a drastically altered morphology of the dendritic tree. Jacob's nuclear trafficking from distal dendrites crucially requires the classical Importin pathway. Caldendrin binds to Jacob's nuclear localization signal in a Ca2+-dependent manner, thereby controlling Jacob's extranuclear localization by competing with the binding of Importin-alpha to Jacob's nuclear localization signal. This competition requires sustained synapto-dendritic Ca2+ levels, which presumably cannot be achieved by activation of extrasynaptic NMDA receptors, but are confined to Ca2+ microdomains such as postsynaptic spines. Extrasynaptic NMDA receptors, as opposed to their synaptic counterparts, trigger the cAMP response element-binding protein (CREB) shut-off pathway, and cell death. We found that nuclear knockdown of Jacob prevents CREB shut-off after extrasynaptic NMDA receptor activation, whereas its nuclear overexpression induces CREB shut-off without NMDA receptor stimulation. Importantly, nuclear knockdown of Jacob attenuates NMDA-induced loss of synaptic contacts, and neuronal degeneration. This defines a novel mechanism of synapse-to-nucleus communication via a synaptic Ca2+-sensor protein, which links the activity of NMDA receptors to nuclear signalling events involved in modelling synapto-dendritic input and NMDA receptor-induced cellular degeneration.