Transsynaptic channelosomes: non-conducting roles of ion channels in synapse formation

Recent findings demonstrate that synaptic channels are directly involved in the formation and maintenance of synapses by interacting with synapse organizers. The synaptic channels on the pre- and postsynaptic membranes possess non-conducting roles in addition to their functional roles as ion-conducting channels required for synaptic transmission. For example, presynaptic voltage-dependent calcium channels link the target-derived synapse organizer laminin β2 to cytomatrix of the active zone and function as scaffolding proteins to organize the presynaptic active zones. Furthermore, postsynaptic δ2-type glutamate receptors organize the synapses by forming transsynaptic protein complexes with presynaptic neurexins through synapse organizer cerebellin 1 precursor proteins. Interestingly, the synaptic clustering of AMPA receptors is regulated by neuronal activity-regulated pentraxins, while postsynaptic differentiation is induced by the interaction of postsynaptic calcium channels and thrombospondins. This review will focus on the non-conducting functions of ion-channels that contribute to the synapse formation in concert with synapse organizers and active-zone-specific proteins.     

Transsynaptic channelosomes

Recent findings demonstrate that synaptic channels are directly involved in the formation and maintenance of synapses by interacting with synapse organizers. The synaptic channels on the pre- and postsynaptic membranes possess non-conducting roles in addition to their functional roles as ion-conducting channels required for synaptic transmission. For example, presynaptic voltage-dependent calcium channels link the target-derived synapse organizer laminin β2 to cytomatrix of the active zone and function as scaffolding proteins to organize the presynaptic active zones. Furthermore, postsynaptic δ2-type glutamate receptors organize the synapses by forming transsynaptic protein complexes with presynaptic neurexins through synapse organizer cerebellin 1 precursor proteins. Interestingly, the synaptic clustering of AMPA receptors is regulated by neuronal activity-regulated pentraxins, while postsynaptic differentiation is induced by the interaction of postsynaptic calcium channels and thrombospondins. This review will focus on the non-conducting functions of ion-channels that contribute to the synapse formation in concert with synapse organizers and active-zone-specific proteins.     

Activity-dependent regulation of synaptic AMPA receptor composition and abundance by beta3 integrins

At synapses, cell adhesion molecules (CAMs) provide the molecular framework for coordinating signaling events across the synaptic cleft. Among synaptic CAMs, the integrins, receptors for extracellular matrix proteins and counterreceptors on adjacent cells, are implicated in synapse maturation and plasticity and memory formation. However, little is known about the molecular mechanisms of integrin action at central synapses. Here, we report that postsynaptic beta3 integrins control synaptic strength by regulating AMPA receptors (AMPARs) in a subunit-specific manner. Pharmacological perturbation targeting beta3 integrins promotes endocytosis of GluR2-containing AMPARs via Rap1 signaling, and expression of beta3 integrins produces robust changes in the abundance and composition of synaptic AMPARs without affecting dendritic spine structure. Importantly, homeostatic synaptic scaling induced by activity deprivation elevates surface expression of beta3 integrins, and in turn, beta3 integrins are required for synaptic scaling. Our findings demonstrate a key role for integrins in the feedback regulation of excitatory synaptic strength.     

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.     

Synapse proteomics: current status and quantitative applications

Chemical synapses are key organelles for neurotransmission. The coordinated actions of protein networks in diverse synaptic subdomains drive the sequential molecular events of transmitter release from the presynaptic bouton, activation of transmitter receptors located in the postsynaptic density and the changes of postsynaptic potential. Plastic change of synaptic efficacy is thought to be caused by the alteration of protein constituents and their interaction in the synapse. As a first step toward the understanding of the organization of synapse, several proteomics studies have been carried out to profile the protein constituents and the post-translational modifications in various rodent excitatory chemical synaptic subdomains, including postsynaptic density, synaptic vesicle and the synaptic phosphoproteome. Quantitative proteomics have been applied to examine the changes of synaptic proteins during brain development, in knockout mice model developed for studies of synapse physiology and in rodent models of brain disorders. These analyses generate testable hypotheses of synapse function and regulation both in health and disease.     

Synapse proteomics: current status and quantitative applications

Chemical synapses are key organelles for neurotransmission. The coordinated actions of protein networks in diverse synaptic subdomains drive the sequential molecular events of transmitter release from the presynaptic bouton, activation of transmitter receptors located in the postsynaptic density and the changes of postsynaptic potential. Plastic change of synaptic efficacy is thought to be caused by the alteration of protein constituents and their interaction in the synapse. As a first step toward the understanding of the organization of synapse, several proteomics studies have been carried out to profile the protein constituents and the post-translational modifications in various rodent excitatory chemical synaptic subdomains, including postsynaptic density, synaptic vesicle and the synaptic phosphoproteome. Quantitative proteomics have been applied to examine the changes of synaptic proteins during brain development, in knockout mice model developed for studies of synapse physiology and in rodent models of brain disorders. These analyses generate testable hypotheses of synapse function and regulation both in health and disease.     

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.     

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.     

Superresolution imaging of chemical synapses in the brain

Determination of the molecular architecture of synapses requires nanoscopic image resolution and specific molecular recognition, a task that has so far defied many conventional imaging approaches. Here, we present a superresolution fluorescence imaging method to visualize the molecular architecture of synapses in the brain. Using multicolor, three-dimensional stochastic optical reconstruction microscopy, the distributions of synaptic proteins can be measured with nanometer precision. Furthermore, the wide-field, volumetric imaging method enables high-throughput, quantitative analysis of a large number of synapses from different brain regions. To demonstrate the capabilities of this approach, we have determined the organization of?ten protein components of the presynaptic active?zone and the postsynaptic density. Variations?in synapse morphology, neurotransmitter receptor composition, and receptor distribution were observed both among synapses and across different?brain regions. Combination with optogenetics further?allowed molecular events associated with synaptic plasticity to be resolved at the single-synapse level.     

Bilateral processing in chemical synapses with electrical 'ephaptic' feedback: a theoretical model

I have developed a detailed biophysical model of the chemical synapse which hosts voltage-dependent presynaptic ion channels and takes into account the capacitance of synaptic membranes. I find that at synapses with a relatively large cleft resistance (e.g., mossy fiber or giant calyx synapse) the rising postsynaptic current could activate, within the synaptic cleft, electrochemical phenomena that induce rapid widening of the presynaptic action potential (AP). This mechanism could boost fast Ca(2+) entry into the terminal thus increasing the probability of subsequent synaptic releases. The predicted difference in the AP waveforms generated inside and outside the synapse can explain the previously unexplained fast capacitance transient recorded in the postsynaptic cell at the giant calyx synapse. I propose therefore the mechanism of positive ephaptic feedback that acts between the postsynaptic and presynaptic cell contributing to the basal synaptic transmission at large central synapses. This mechanism could also explain the supralinear voltage dependence of EPSCs recorded at hyperpolarizing membrane potentials in low extracellular calcium concentration.     

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.     

Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers

A lack of methods for measuring the protein compositions of individual synapses in?situ has so far hindered the exploration and exploitation of synapse molecular diversity. Here, we describe the use of array tomography, a new high-resolution proteomic imaging method, to determine the composition of glutamate and GABA synapses in somatosensory cortex of Line-H-YFP Thy-1 transgenic mice. We find that virtually all synapses are recognized by antibodies to the presynaptic phosphoprotein synapsin I, while antibodies to 16 other synaptic proteins discriminate among 4 subtypes of glutamatergic synapses and GABAergic synapses. Cell-specific YFP expression in the YFP-H mouse line allows synapses to be assigned to specific presynaptic and postsynaptic partners and reveals that a subpopulation of spines on layer 5 pyramidal cells receives both VGluT1-subtype glutamatergic and GABAergic synaptic inputs. These results establish a means for the high-throughput acquisition of proteomic data from individual cortical synapses in?situ.     

The ultrastructure of giant fibre and serial synapses in crayfish

Summary The ultrastructure of synapses between the cord giant fibres (lateral and medial) and the motor giant fibres in crayfish, Astacus pallipes, third abdominal ganglia have been examined. These electrotonic synapses are asymmetrical, they have synaptic vesicles only in the presynaptic fibre, and they have synaptic cleft widths normally of about 100 Å but narrowed to about 50 Å in restricted areas. Localized increases in density of the synaptic cleft and adjacent membranes also occur within a synapse, and synaptic vesicles are most tightly grouped at the membrane in such areas. Tight or gap junctions with 30 Å or narrower widths have not been found, but the junctions probably function in a similar way to gap junctions.Three small nerves are closely associated with the synapses between the giant fibres. One of these small nerves has round synaptic vesicles and is thought to be excitatory on morphological grounds; one has flattened vesicles and is thought to be inhibitory; and one is postsynaptic to the lateral giant and the two small presynaptic nerves. It is proposed that these small nerves modulate activity in the much larger giant fibre synapse.     

Drosophila endosomal proteins hook and deep orange regulate synapse size but not synaptic vesicle recycling

To study the function of endosomes at synapses we analyzed the localization and function of two Drosophila endosomal proteins, Hook and Deep orange (Dor), at the larval neuromuscular junction. Hook, a negative regulator of endocytic trafficking, and Dor, a positive regulator of endocytic trafficking, are highly enriched at synapses, especially close to postsynaptic membranes. Mutations in hook (hk) and dor do not affect synaptic vesicle recycling, as assessed by electrophysiological analysis of synaptic transmission and behavioral studies of double mutants with shi(ts) mutations that alter vesicle recycling. However, hk and dor mutations alter the number of presynaptic varicosities (synapse size) in opposing ways. Synapse size is increased in hk(11) mutants and is decreased in dor(4) mutants. Double mutants for dor and hk show a dor-like phenotype. These effects on synapse size parallel known functions of Hook and Dor in endocytosis and strongly indicate a role for endocytic trafficking in the regulation of synapse size in vivo. Our observations suggest a model in which Hook and Dor function in later stages of endocytosis is essential for regulating synaptic plasma membrane composition but not synaptic vesicle recycling.     

GABA and neuroligin signaling: linking synaptic activity and adhesion in inhibitory synapse development

GABA-mediated synaptic inhibition is crucial in neural circuit operations. In mammalian brains, the development of inhibitory synapses and innervation patterns is often a prolonged postnatal process, regulated by neural activity. Emerging evidence indicates that gamma-aminobutyric acid (GABA) acts beyond inhibitory transmission and regulates inhibitory synapse development. Indeed, GABA(A) receptors not only function as chloride channels that regulate membrane voltage and conductance but also play structural roles in synapse maturation and stabilization. The link from GABA(A) receptors to postsynaptic and presynaptic adhesion is probably mediated, partly by neuroligin-reurexin interactions, which are potent in promoting GABAergic synapse formation. Therefore, similar to glutamate signaling at excitatory synapse, GABA signaling may coordinate maturation of presynaptic and postsynaptic sites at inhibitory synapses. Defining the many steps from GABA signaling to receptor trafficking/stability and neuroligin function will provide further mechanistic insights into activity-dependent development and possibly plasticity of inhibitory synapses.     

Role of glia in synapse development

Recent studies suggest that glial cells regulate certain aspects of synapse development. Neurons can form synapses without glia, but may require glia-derived cholesterol to form numerous and efficient synapses. During synapse maturation, soluble and contact-dependent factors from glia may influence the composition of the postsynaptic density. Finally, synaptic connections appear to require glia to support their structural stability. Given the new evidence, it may be time now to acknowledge glia as a source for synaptogenesis-promoting signals. Scrutinizing the molecular mechanisms underlying this new function of glia and testing its relevance in vivo may help to understand how synapses develop and why they degenerate under pathological conditions.     

A role for zinc in postsynaptic density asSAMbly and plasticity?

Chemical synapses are asymmetric cell junctions that mediate communication between neurons. Multidomain scaffolding proteins of the Shank family act as major organizing elements of the "postsynaptic density"--that is, the cytoskeletal protein matrix associated with the postsynaptic membrane. A recent study has shown that the C-terminal sterile alpha-motif or "SAM domain" of Shank3 (also known as ProSAP2) can form two-dimensional sheets of helical fibers. Assembly and packaging of these fibers are markedly enhanced by the presence of Zn2+ ions. Zn2+ can be released together with glutamate from synaptic vesicles and can enter the postsynaptic cell through specific ionotropic receptors. Based on these observations, we propose a new model of synaptic plasticity in which Zn2+ influx directly and instantly modulates the structure and function of the postsynaptic density.     

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.     

Diffusional trapping of GluR1 AMPA receptors by input-specific synaptic activity

Synaptic activity regulates the postsynaptic accumulation of AMPA receptors over timescales ranging from minutes to days. Indeed, the regulated trafficking and mobility of GluR1 AMPA receptors underlies many forms of synaptic potentiation at glutamatergic synapses throughout the brain. However, the basis for synapse-specific accumulation of GluR1 is unknown. Here we report that synaptic activity locally immobilizes GluR1 AMPA receptors at individual synapses. Using single-molecule tracking together with the silencing of individual presynaptic boutons, we demonstrate that local synaptic activity reduces diffusional exchange of GluR1 between synaptic and extraynaptic domains, resulting in postsynaptic accumulation of GluR1. At neighboring inactive synapses, GluR1 is highly mobile with individual receptors frequently escaping the synapse. Within the synapse, spontaneous activity confines the diffusional movement of GluR1 to restricted subregions of the postsynaptic membrane. Thus, local activity restricts GluR1 mobility on a submicron scale, defining an input-specific mechanism for regulating AMPA receptor composition and abundance.     

Transmission,Development, and Plasticity of Synapses

Chemical synapses are sites of contact and information transfer between a neuron and its partner cell. Each synapse is a specialized junction, where the presynaptic cell assembles machinery for the release of neurotransmitter, and the postsynaptic cell assembles components to receive and integrate this signal. Synapses also exhibit plasticity, during which synaptic function and/or structure are modified in response to activity. With a robust panel of genetic, imaging, and electrophysiology approaches, and strong evolutionary conservation of molecular components, Drosophila has emerged as an essential model system for investigating the mechanisms underlying synaptic assembly, function, and plasticity. We will discuss techniques for studying synapses in Drosophila, with a focus on the larval neuromuscular junction (NMJ), a well-established model glutamatergic synapse. Vesicle fusion, which underlies synaptic release of neurotransmitters, has been well characterized at this synapse. In addition, studies of synaptic assembly and organization of active zones and postsynaptic densities have revealed pathways that coordinate those events across the synaptic cleft. We will also review modes of synaptic growth and plasticity at the fly NMJ, and discuss how pre- and postsynaptic cells communicate to regulate plasticity in response to activity.