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.     

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.     

受体、突触和记忆

大脑中神经元突触间的信号传递是由许多神经递质受体介导的。在过去,Richard L．Huganir实验室一直致力于神经递质受体功能调节的分子机制。而最近,该实验室又聚焦到大脑中一种最主要的兴奋性受体的研究——谷氨酸受体。谷氨酸受体主要可以分为两大类：AMPA受体和NMDA受体。AMPA受体主要介导了快速的兴奋性突触传递;而NMDA受体则在神经可塑性和发育中起到重要作用。实验发现,AMPA受体和NMDA受体都可以被一系列的蛋白激酶磷酸化,而磷酸化的水平则直接影响了这些受体的功能特性,包括通道电导和受体膜定位等。AMPA受体磷酸化的水平同时还在学习和记忆的细胞模型中发生改变,如长时程增强（LTP）和长时程抑制（LTD）。此外,AMPA受体中GluR1亚单位的磷酸化对于各种形式的可塑性以及空间记忆的维持有重要的作用。实验室主要研究突触部位谷氨酸受体在亚细胞水平的定位和聚集的分子机制。最近,一系列可以直接或间接与AMPA和NMDA受体相互作用的蛋白质得以发现,其中包括一个新发现的蛋白家族GRIPs（glutamate receptor interacting proteins）。GRIPs可以直接和AMPA受体的GluR2／3亚单位的C端结合。GRIPs包含7个PDZ结构域,可以介导蛋白与蛋白直接的相互连接,从而把各个AMPA受体交互连接在一起并与其他蛋白相连。另外,GluR2亚单位的c端还可以和兴奋性突触中的蛋白激酶C结合蛋白（PICK1）的PDZ结构域相互作用。另外,GluR2亚单位的C端也可以与一种参与膜融合的蛋白NSF相互作用。这些与AMPA受体相互作用的蛋白质对于受体在膜上的运输以及定位有至关重要的作用。同时,受体与PICK1和GRIP的结合对于小脑运动学习中的LTD有重要作用。总体上说,该实验室发现了一系列可以调节神经递质受体功能的分子机制,这些工作提示受体功能的调节可能是?     

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.     

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.     

AMPA receptor biogenesis and trafficking

AMPA-type glutamate receptors mediate the majority of fast excitatory transmission in the central nervous system. The trafficking of AMPA receptors to and from synapses alters synaptic strength and has been recognized as a central mechanism underlying various forms of synaptic plasticity. Both secretory and endocytic trafficking events seem to be driven by the subunit composition of AMPA receptor tetramers. Moreover, recent work suggests that synapses employ different tetramer combinations in response to altered synaptic input, suggesting the existence of signalling pathways that mediate remodelling of AMPA receptors. These latest developments and recent progress in elucidating the mechanisms that underlie channel assembly and trafficking are the subject of this review.     

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.     

Subunit-specific surface mobility of differentially labeled AMPA receptor subunits

Lateral mobility of AMPA-type glutamate receptors as well as their trafficking between plasma membrane and intracellular compartments are major mechanisms for the regulation of synaptic plasticity. Here we applied a recently established labeling technique in combination with lentiviral expression in hippocampal neurons to label individual ACP-tagged AMPA receptor subunits specifically at the surface of neurons. We show that this technique allows the differential labeling of two receptor subunits on the same cell. Moreover, these subunits are integrated into heteromeric receptors together with endogenous subunits, and these labeled receptors are targeted to active synapses. Sequential labeling experiments indicate that there is basal surface insertion of GluR1, GluR2 and GluR3, and that this insertion is strongly increased following potassium depolarization. Moreover, we found that ACP-labeled GluR3 shows the highest surface mobility among GluR1, GluR2, and GluR3. In double-infected neurons the diffusion coefficient of labeled GluR2 at the surface of living neurons is significantly higher in GluR2/GluR3-infected neurons compared to GluR1/GluR2-infected neurons suggesting a higher mobility of GluR2/3 receptors compared to GluR1/2 receptors. These results indicate that surface mobility is regulated by different subunit compositions of AMPA receptors.     

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.     

Neurobeachin regulates neurotransmitter receptor trafficking to synapses

The surface density of neurotransmitter receptors at synapses is a key determinant of synaptic efficacy. Synaptic receptor accumulation is regulated by the transport, postsynaptic anchoring, and turnover of receptors, involving multiple trafficking, sorting, motor, and scaffold proteins. We found that neurons lacking the BEACH (beige-Chediak/Higashi) domain protein Neurobeachin (Nbea) had strongly reduced synaptic responses caused by a reduction in surface levels of glutamate and GABA_A receptors. In the absence of Nbea, immature AMPA receptors accumulated early in the biosynthetic pathway, and mature N-methyl-d-aspartate, kainate, and GABA_A receptors did not reach the synapse, whereas maturation and surface expression of other membrane proteins, synapse formation, and presynaptic function were unaffected. These data show that Nbea regulates synaptic transmission under basal conditions by targeting neurotransmitter receptors to synapses.     

Direct imaging of lateral movements of AMPA receptors inside synapses

Trafficking of AMPA receptors in and out of synapses is crucial for synaptic plasticity. Previous studies have focused on the role of endo/exocytosis processes or that of lateral diffusion of extra-synaptic receptors. We have now directly imaged AMPAR movements inside and outside synapses of live neurons using single-molecule fluorescence microscopy. Inside individual synapses, we found immobile and mobile receptors, which display restricted diffusion. Extra-synaptic receptors display free diffusion. Receptors could also exchange between these membrane compartments through lateral diffusion. Glutamate application increased both receptor mobility inside synapses and the fraction of mobile receptors present in a juxtasynaptic region. Block of inhibitory transmission to favor excitatory synaptic activity induced a transient increase in the fraction of mobile receptors and a decrease in the proportion of juxtasynaptic receptors. Altogether, our data show that rapid exchange of receptors between a synaptic and extra-synaptic localization occurs through regulation of receptor diffusion inside synapses.     

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.     

Receptor trafficking and the plasticity of excitatory synapses

Newly discovered features of the trafficking of AMPA receptors to and from the postsynaptic membrane of excitatory synapses are now bringing the mechanisms of synaptic plasticity into focus. Recent advances, including the existence of slots, anchors, transport factors and pathways for activity-dependent control, have elucidated the role of the individual AMPA receptor subunits and their binding partners. The latest views describe how subunit type dictates the assembly of heteromeric receptors, and how these heteromers interact with the receptor trafficking machinery and synaptic anchorage factors. Moreover, phosphorylation may play an important role in receptor transport and synaptic turnover.     

Regulation of AMPA receptor trafficking by N-cadherin

Dendritic spines are dynamically regulated, both morphologically and functionally, by neuronal activity. Morphological changes are mediated by a variety of synaptic proteins, whereas functional changes can be dramatically modulated by the regulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor trafficking. Although these two forms of plasticity appear to be highly coordinated, the connections between them are not fully understood. In this study the synaptic cell adhesion molecule N-cadherin was found to associate with AMPA receptors and regulate AMPA receptor trafficking in neurons. N-cadherin and beta-catenin formed a protein complex with AMPA receptors in vivo, and this association was regulated by extracellular Ca2+. In addition, these proteins co-clustered at synapses in cultured neurons. In heterologous cells and in cultured neurons, overexpression of wild-type N-cadherin specifically increased the surface expression level of the AMPA receptor subunit glutamate receptor 1 (GluR1) and this effect was reversed by a dominant-negative form of N-cadherin. Finally, GluR1 increased the surface expression of N-cadherin in heterologous cells. Importantly, recent studies suggest that N-cadherin and beta-catenin play key roles in structural plasticity in neurons. Therefore, our data suggest that the association of N-cadherin with AMPA receptors may serve as a biochemical link between structural and functional plasticity of synapses.     

Delivering the goods: Activity-induced exocytosis and AMPA receptor insertion within a post-synaptic membrane compartment depends on syntaxin 4

The activity-dependent strengthening of neural transmission at individual synapses has long been postulated to underlie learning and memory in the brain, and current wisdom strongly suggests that molecular modifications within both the pre- and post-synaptic nerve terminals contribute to this strengthening process (i.e. long-term potentiation or LTP). At excitatory, glutamatergic synapses, the dynamic insertion and retrieval of ionotropic glutamate receptors into and from the post-synaptic plasma membrane have been implicated in synaptic plasticity, however, the site(s) for these trafficking events and the molecules involved have not be clearly elucidated. Biochemical studies have identified SNARE proteins as critical mediators of membrane fusion events in many cell types, including neurons, and several bacterial toxins are known to interfere with neurotransmission by disrupting the function of membrane-bound SNARE proteins, such as VAMP and syntaxin. Using high resolution imaging techniques, the authors have characterized the molecular processes underlying activity-driven membrane fusion events within dendritic spines, tiny membrane protrusions only 1-2 microns in size. Their data demonstrate that syntaxin 4 functions as a key SNARE protein for the exocytic insertion of glutamate receptors and the membrane trafficking events contributing to synaptic plasticity.     

Regulation of AMPA receptor trafficking and synaptic plasticity

AMPA receptors (AMPARs) mediate the majority of fast excitatory synaptic transmission in the brain. Dynamic changes in neuronal synaptic efficacy, termed synaptic plasticity, are thought to underlie information coding and storage in learning and memory. One major mechanism that regulates synaptic strength involves the tightly regulated trafficking of AMPARs into and out of synapses. The life cycle of AMPARs from their biosynthesis, membrane trafficking, and synaptic targeting to their degradation are controlled by a series of orchestrated interactions with numerous intracellular regulatory proteins. Here we review recent progress made toward the understanding the regulation of AMPAR trafficking, focusing on the roles of several key intracellular AMPAR interacting proteins.     

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.     

Synaptic trafficking of glutamate receptors by MAGUK scaffolding proteins

Synaptic transmission underlies every aspect of brain function. Excitatory synapses, which release the neurotransmitter glutamate, are the most numerous type of synapse in the brain. The trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors to and from these synapses controls the strength of excitatory synaptic transmission. However, the underlying mechanisms controlling this trafficking have remained elusive. Recent studies, drawing from advances in molecular biology and electrophysiology techniques, have established an essential role for a family of synaptic scaffolding molecules, known as membrane associate guanylate kinases (MAGUKs), in this trafficking process. These studies highlight the remarkable orchestration of AMPA-type glutamate receptor synaptic trafficking by multiple MAGUKs at different synapses within the same neuron and at different developmental stages.     

Ionotropic glutamate receptor trafficking--there and back again

Glutamate is a major excitatory neurotransmitter in brain. It engages mainly ionotropic glutamate receptors of AMPA and NMDA type. Thus, regulation of the number and properties of the receptors is crucial for correct neuronal communication, but also contributes to various forms of synaptic plasticity, namely neuronal development, learning and memory. Glutamate receptors are not static components of synapses. On the contrary, they are continuously delivered and removed from postsynaptic membranes and this process is regulated by synaptic activity, Receptor trafficking to synapses is a multi-step process, involving exit from endoplasmic reticulum, transport along dendrites, incorporation to postsynaptic membrane and finally removing them from synapses. The transport is regulated by numerous proteins, especially those bearing PDZ domains, or by receptors themselves.