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.     

Molecules involved in the formation of synaptic connections in muscle and brain.

Synapses are highly specialized structures designed to guarantee precise and efficient communication between neurons and their target cells. Molecules of the extracellular matrix have an instructive role in the formation of the neuromuscular junction, the best-characterized synapse. In this review, the molecular mechanisms underlying these instructive signals will be discussed with particular emphasis on the receptors involved. Additionally, recent evidence for the involvement of specific adhesion complexes in the formation and modulation of synapses in the central nervous system will be reviewed. Synapses are specialized junctions between neurons and their target cells where information is transferred from the pre- to the postsynaptic cell. At most vertebrate synapses, this transfer is accomplished by the release of a specific neurotransmitter from the presynaptic nerve terminal. The release of neurotransmitter is initiated by the action potential and the subsequent influx of Ca(2+) into the presynaptic nerve terminal. This results in the rapid fusion of vesicles with the nerve membrane and the release of the neurotransmitter into the synaptic cleft. The neurotransmitter then diffuses across the cleft and binds to specific postsynaptic receptors, resulting in a change in the membrane potential of the postsynaptic cell. This can result in the generation of an action potential. The high precision of synaptic transmission requires that pre- and postsynaptic structures are both highly organized and in juxtaposition to each other. In addition, alterations in synaptic transmission are the basis of learning and memory and are likely to be accompanied by the remodeling of synaptic structures (Toni et al., 1999). Thus, the study of how synapses are formed during development is also of relevance for the understanding of the cellular and molecular processes involved in learning and memory. This review focuses on the molecular mechanisms involved in the formation and the function of synapses.     

Preferential localization of glutamate receptors opposite sites of high presynaptic release

BACKGROUND: The localization of glutamate receptors is essential for the formation and plasticity of excitatory synapses. These receptors cluster opposite neurotransmitter release sites of glutamatergic neurons, but these release sites have heterogeneous structural and functional properties. At the Drosophila neuromuscular junction, receptors expressed in a single postsynaptic cell are confronted with an array of hundreds of apposed active zones. Hence, this is an ideal preparation for the investigation of whether receptor clustering is sensitive to the morphological and physiological properties of the apposed active zones. RESULTS: To investigate the relationship between the localization of glutamate receptors and the properties of the apposed active zones, we investigated receptor localization in mutants in which receptors are limited. We find that receptors are not uniformly distributed opposite the full array of active zones but that some active zones have a disproportionately large share of receptors as assayed by receptor levels and response to transmitter. The active zones at which receptors preferentially cluster are larger and have a higher neurotransmitter release probability than the average active zone. We find a similar relationship between glutamate receptor clusters and active-zone size at wild-type synapses. CONCLUSIONS: When confronted with an array of active zones, glutamate receptors preferentially cluster opposite the largest and most physiologically active sites. These results suggest an activity-dependent matching of pre- and postsynaptic function at the level of a single active zone.     

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.     

CPG2: a brain- and synapse-specific protein that regulates the endocytosis of glutamate receptors

Long-term maintenance and modification of synaptic strength involve the turnover of neurotransmitter receptors. Glutamate receptors are constitutively and acutely internalized, presumptively through clathrin-mediated receptor endocytosis. Here, we show that cpg2 is a brain-specific splice variant of the syne-1 gene that encodes a protein specifically localized to a postsynaptic endocytotic zone of excitatory synapses. RNAi-mediated CPG2 knockdown increases the number of postsynaptic clathrin-coated vesicles, some of which traffic NMDA receptors, disrupts the constitutive internalization of glutamate receptors, and inhibits the activity-induced internalization of synaptic AMPA receptors. Manipulating CPG2 levels also affects dendritic spine size, further supporting a function in regulating membrane transport. Our results suggest that CPG2 is a key component of a specialized postsynaptic endocytic mechanism devoted to the internalization of synaptic proteins, including glutamate receptors. The activity dependence and distribution of cpg2 expression further suggest that it contributes to the capacity for postsynaptic plasticity inherent to excitatory synapses.     

Analytical description of the activation of multi-state receptors by continuous neurotransmitter signals at brain synapses.

Chemical synaptic transmission is a fundamental component of interneuronal communications in the central nervous system (CNS). Discharge of a presynaptic vesicle containing a few thousand molecules (a quantum) of neurotransmitter into the synaptic cleft generates a transmitter concentration signal that drives postsynaptic ion-channel receptors. These receptors exhibit multiple states, with state transition kinetics dependent on neurotransmitter concentration. Here, a novel and simple analytical approach for describing gating of multi-state receptors by signals with complex continuous time courses is used to describe the generation of glutamate-mediated quantal postsynaptic responses at brain synapses. The neurotransmitter signal, experienced by multi-state N-methyl-D-aspartate (NMDA)- and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors at specific points in a synaptic cleft, is approximated by a series of step functions of different intensity and duration and used to drive a Markovian, multi-state kinetic scheme that describes receptor gating. Occupancy vectors at any point in time can be computed interatively from the occupancy vectors at the times of steps in transmitter concentration. Multi-state kinetic schemes for both the low-affinity AMPA subtype of glutamate receptor and for the high-affinity NMDA subtype are considered, and expected NMDA and AMPA components of synaptic currents are calculated. The amplitude of quantal responses mediated by postsynaptic receptor clusters having specific spatial distributions relative to foci of quantal neurotransmitter release is then calculated and related to the displacement between the center of the postsynaptic receptor cluster and the focus of synaptic vesicle discharge. Using this approach we show that the spatial relation between the focus of release and the center of the postsynaptic receptor cluster affects synaptic efficacy. We also show how variation in this relation contributes to variation in synaptic current amplitudes.     

Modulation of synaptic signalling complexes by Homer proteins

The number of neurotransmitter receptors in the postsynaptic membrane and their functional coupling to intracellular signalling cascades are important determinants of synaptic strength--and hence potential targets for plasticity related modulation. In this context, Homer/Vesl proteins have gained particular interest for three main reasons: (i) they constitute part of the molecular scaffold at postsynaptic densities of excitatory synapses in the mammalian brain; (ii) they physically link type-I metabotropic glutamate receptors to the postsynaptic density and to inositol 1,4,5-triphosphate receptors in the subsynaptic endoplasmic reticulum; and (iii) Homer-1a, which has been categorized as an immediate early gene isoform, exerts dominant-negative activity, suggesting that it is involved in activity dependent rearrangements at synaptic junctions. Although these fundamental aspects have been reviewed previously by Xiao et al., this review will address primarily more recent studies on the regulation of Homer 1a expression and on the role of Homer/Vesl proteins in spine morphogenesis and receptor targeting and signalling.     

Physiology of synapse: from molecular modules to retrograde modulation

Synapses are highly organized, specific structures assuring rapid and highly selective interactions between cells. Synaptic transmission involves the release of neurotransmitter from presynaptic neurons and its detection by specific ligand-gated ion channels at the surface membrane of postsynaptic neurons. The protenomic analysis shows that for self-formation and functioning of synapses nearly 2000 proteins are involved in mammalian brain. The core complex in excitatory synapses includes glutamate receptors, potassium channels, CaMKII, scaffolding protein and actin. These proteins exist as part of a highly organized protein complex known as the postsynaptic density (PSD). The coordinated functioning of the different PSD components determines the strength of signalling between the pre- and postsynaptic neurons. Synaptic plasticity is regulated by changes in the amount of receptors on the postsynaptic membrane, changes in the shape and size of dendritic spines, posttranslational modification of PSD components, modulation kinetics of synthesis and degradation of proteins. Integration of these processes leads to long-lasting changes in synaptic function and neuronal networks underlying learning-related plasticity, memory and information treatment in nervous system of multicellular organisms.     

Neuregulin and ErbB receptor signaling pathways in the nervous system

The neuregulins are a complex family of factors that perform many functions during neural development. Recent experiments have shown that neuregulins promote neuronal migration and differentiation, and regulate the selective expression of neurotransmitter receptors in neurons and at the neuromuscular junction. They also regulate glial commitment, proliferation, survival and differentiation. At interneuronal synapses, neuregulin ErbB receptors associate with PDZ-domain proteins at postsynaptic densities where they can modulate synaptic plasticity. How this combinatorial network - comprising many neuregulin ligands that signal through distinct combinations of dimeric ErbB receptors - elicits its multitude of biological effects is beginning to be resolved.     

Structural and Functional Maturation of Active Zones in Large Synapses

Virtually all functions of the nervous system rely upon synapses, the sites of communication between neurons and between neurons and other cells. Synapses are complex structures, each one comprising hundreds of different types of molecules working in concert. They are organized by adhesive and scaffolding molecules that align presynaptic vesicular release sites, namely, active zones, with postsynaptic neurotransmitter receptors, thereby allowing rapid and reliable intercellular communication. Most synapses are relatively small, and acting alone exerts little effect on their postsynaptic partners. Some, however, are much larger and stronger, reliably driving the postsynaptic cell to its action potential threshold, acting essentially as electrical relays of excitation. These large synapses are among the best understood, and two of these are the subject of this review, namely, the vertebrate neuromuscular junction and the calyx of Held synapse in the mammalian auditory pathway of the brain stem. Both synapses undergo through a complex and well-coordinated maturation process, during which time the molecular elements and the biophysical properties of the secretory machinery are continuously adjusted to the synapse size and to the functional requirements. We here review the morphological and functional changes occurring during postnatal maturation, noting particular similarities and differences between these two large synapses.     

The biochemical anatomy of cortical inhibitory synapses

Classical electron microscopic studies of the mammalian brain revealed two major classes of synapses, distinguished by the presence of a large postsynaptic density (PSD) exclusively at type 1, excitatory synapses. Biochemical studies of the PSD have established the paradigm of the synapse as a complex signal-processing machine that controls synaptic plasticity. We report here the results of a proteomic analysis of type 2, inhibitory synaptic complexes isolated by affinity purification from the cerebral cortex. We show that these synaptic complexes contain a variety of neurotransmitter receptors, neural cell-scaffolding and adhesion molecules, but that they are entirely lacking in cell signaling proteins. This fundamental distinction between the functions of type 1 and type 2 synapses in the nervous system has far reaching implications for models of synaptic plasticity, rapid adaptations in neural circuits, and homeostatic mechanisms controlling the balance of excitation and inhibition in the mature brain.     

Clathrin-mediated endocytosis at synapses

Neurons are communication specialists that convert electrical into chemical signals at specialized cell-cell junctions termed synapses. Arrival of an action potential triggers calcium-regulated exocytosis of neurotransmitter (NT) from small synaptic vesicles (SVs), which then diffuses across the synaptic cleft and binds to postsynaptic receptors to elicit specific changes within the postsynaptic cell. Endocytosis of pre- and postsynaptic membrane proteins including SV components and postsynaptic NT receptors is essential for the proper functioning of the synapse. During the past several years, we have witnessed enormous progress in our understanding of the mechanics of clathrin-mediated endocytosis (CME) and its role in regulating exo-endocytic vesicle cycling at synapses. Here we summarize the molecular machinery used for recognition of synaptic membrane protein cargo and its clathrin-dependent internalization, and describe the inventory of tools that can be used to monitor vesicle cycling at synapses or to inhibit CME in a stage-specific manner.     

Presynaptic kainate receptors are localized close to release sites in rat hippocampal synapses

The subsynaptic distribution of kainate receptors is still a matter of much debate given its importance to understand the way they influence neuronal communication. Here, we show that, in synapses of the rat hippocampus, presynaptic kainate receptors are localized within the presynaptic active zone close to neurotransmitter release sites. The activation of these receptors with low concentrations of agonists induces the release of [(3)H]glutamate in the absence of a depolarizing stimulus. Furthermore, this modulation of [(3)H]glutamate release by kainate is more efficient when compared with a KCl-evoked depolarization that causes a more than two-fold increase in the intra-terminal calcium concentration but no apparent release of [(3)H]glutamate, suggesting a direct receptor-mediated process. Using a selective synaptic fractionation technique that allows for a highly efficient separation of presynaptic, postsynaptic and non-synaptic proteins we confirmed that, presynaptically, kainate receptors are mainly localized within the active zone of hippocampal synapses where they are expected to be in a privileged position to modulate synaptic phenomena.     

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.     

Heterogeneity of receptor immunoreactivity at synapses of glycine-utilizing neurons.

Neurons often contain, and probably release, more than one neuroactive substance that may have diverse or opposite actions on the postsynaptic cell. It remains unexplained how these neurons utilize their multiple neuroactive substances while maintaining appropriate resolution of neurotransmitter functions. Here, we have examined the ultrastructural localization of glycine receptors by using a monoclonal antibody directed to the intracellular domain of the strychnine-sensitive glycine receptor. We have found that glycine receptors are only localized to 56% of the synapses made by presumed 'glycinergic' (more accurately, glycine-utilizing) amacrine cells in the turtle retina. The remaining synapses made by these same boutons show no evidence of glycine receptors. As there is no evidence to suggest the presence of a second type of glycine receptor, these data indicate that only a portion of the postsynaptic sites contacted by the glycine-utilizing neurons can respond to glycine. They also suggest that a neuron containing multiple neuroactive substances can selectively affect postsynaptic elements by means of heterogeneous receptor localization.     

Postsynaptic actin and neuronal plasticity.

In the adult brain, actin is concentrated in dendritic spines where it can produce rapid changes in their shape. Through various synaptic junction proteins, this postsynaptic actin is linked to neurotransmitter receptors, influencing their function and, in turn, being influenced by them. Thus, the actin cytoskeleton is emerging as a key mediator between signal transmission and anatomical plasticity at excitatory synapses.     

Interaction of the N-terminal domain of the AMPA receptor GluR4 subunit with the neuronal pentraxin NP1 mediates GluR4 synaptic recruitment

Synaptogenesis requires recruitment of neurotransmitter receptors to developing postsynaptic specializations. We developed a coculture system reconstituting artificial synapses between neurons and nonneuronal cells to investigate the molecular components required for AMPA-receptor recruitment to synapses. With this system, we find that excitatory axons specifically express factors that recruit the AMPA receptor GluR4 subunit to sites of contact between axons and GluR4-transfected nonneuronal cells. Furthermore, the N-terminal domain (NTD) of GluR4 is necessary and sufficient for its recruitment to these artificial synapses and also for GluR4 recruitment to native synapses. Moreover, we show that axonally derived neuronal pentraxins NP1 and NPR are required for GluR4 recruitment to artificial and native synapses. RNAi knockdown and knockout of the neuronal pentraxins significantly decreases GluR4 targeting to synapses. Our results indicate that NP1 and NPR secreted from presynaptic neurons bind to the GluR4 NTD and are critical trans-synaptic factors for GluR4 recruitment to synapses.     

Asymmetry of glia near central synapses favors presynaptically directed glutamate escape

Recent findings demonstrate that synaptically released excitatory neurotransmitter glutamate activates receptors outside the immediate synaptic cleft and that the extent of such extrasynaptic actions is regulated by the high affinity glutamate uptake. The bulk of glutamate transporter systems are evenly distributed in the synaptic neuropil, and it is generally assumed that glutamate escaping the cleft affects pre- and postsynaptic receptors to a similar degree. To test whether this is indeed the case, we use quantitative electron microscopy and establish the stochastic pattern of glial occurrence in the three-dimensional (3D) vicinity of two common types of excitatory central synapses, stratum radiatum synapses in hippocampus and parallel fiber synapses in cerebellum. We find that the occurrence of glia postsynaptically is strikingly higher (3-4-fold) than presynaptically, in both types of synapses. To address the functional consequences of this asymmetry, we simulate diffusion and transport of synaptically released glutamate in these two brain areas using a detailed 3D compartmental model of the extracellular space with glutamate transporters arranged unevenly, in accordance with the obtained experimental data. The results predict that glutamate escaping the synaptic cleft is 2-4 times more likely to activate presynaptic compared to postsynaptic receptors. Simulations also show that postsynaptic neuronal transporters (EAAT4 type) at dendritic spines of cerebellar Purkinje cells exaggerate this asymmetry further. Our data suggest that the perisynaptic environment of these common central synapses favors fast presynaptic feedback in the information flow while preserving the specificity of the postsynaptic input.     

Dynamics of postsynaptic glutamate receptor targeting

Targeting of glutamate receptors to synapses is an important event in both developing and mature neurons. Glutamate receptors are delivered to nascent synapses during synaptogenesis and to existing synapses during activity-dependent synaptic strengthening. Increasing evidence suggests that glutamate receptors are inserted into the plasma membrane before they accumulate at the synapse. Lateral diffusion of receptors occurs at both synaptic and non-synaptic membranes, and glutamate receptors can exchange rapidly between synaptic and extrasynaptic sites. In addition, recent studies show that postsynaptic scaffold molecules can be highly mobile. The dynamic nature of the synapse suggests that many mechanisms might be involved in regulating synapse formation and synaptic plasticity.     

Ligand-Induced Cell Adhesion as a New Mechanism to Promote Synapse Formation

The formation of neuronal synapses is a finely organized process that involves the presynaptic assembly of the machinery responsible for neurotransmitter release and the postsynaptic recruitment of neurotransmitter receptors and scaffold proteins to the postsynaptic density (PSD). The molecular cues guiding the establishment of synaptic connections are now beginning to be identified. Recent evidences indicate that cell adhesion molecules (CAMs) participate prominently in the key steps of synapse formation, inducing trans-synaptic adhesion and promoting a precise alignment of pre- and postsynaptic terminals. This addendum describes a new mechanism of cell-cell interaction that combines features of both diffusible and membrane-bound synaptogenic factors. It particularly points out the key role played by GDNF triggering trans-homophilic binding between GFRα1 molecules and cell adhesion between GFRα1-expressing cells. In this model GFRα1 functions as a ligand-induced cell adhesion molecule (LICAM) to establish precise synaptic contacts and promote the assembly of presynaptic terminals. In this overview, I summarize the current concepts of synapse formation in the limelight of this new mechanism of ligand-induced cell adhesion.