Stoned B mediates sorting of integral synaptic vesicle proteins

A continuous supply of fusion-competent synaptic vesicles is essential for sustainable neurotransmission. Drosophila mutations of the dicistronic stoned locus disrupt normal vesicle cycling and cause functional deficits in synaptic transmission. Although both Stoned A and B proteins putatively participate in reconstituting synaptic vesicles, their precise function is still unclear. Here we investigate the effects of progressive depletion of Stoned B protein (STNB) on the release properties of neuromuscular synapses using a novel set of synthetic stnB hypomorphic alleles. Decreasing neuronal STNB expression to < or =35% of wild-type level causes a strong reduction in excitatory junctional current amplitude at low stimulation frequencies and a marked slowing in synaptic depression during high-frequency stimulation, suggesting vesicle depletion is attenuated by decreased release probability. Recovery from synaptic depression after prolonged stimulation is also decelerated in mutants, indicating a delayed recovery of fusion-ready vesicles. These phenotypes appear not to be due to a diminished vesicle population, since the docked vesicle pool is ultrastructurally unaffected, and the total number of vesicles is only slightly reduced in these hypomorphs, unlike lethal stoned mutants. Therefore, we conclude that STNB not only functions as an essential component of the endocytic complex for vesicle reconstitution, as previously proposed, but also regulates the competence of recycled vesicles to undergo fusion. In support of such role of STNB, synaptic levels of the vesicular glutamate transporter (vGLUT) and synaptotagmin-1 are strongly reduced with diminishing STNB function, while other synaptic proteins are largely unaffected. We conclude that STNB organizes the endocytic sorting of a subset of integral synaptic vesicle proteins thereby regulating the fusion-competence of the recycled vesicle.     

Effect of carbidine on content and deposition of adrenergic neurotransmitter in synaptic vesicles

The effect of the original Soviet psychotropic agent carbidine on the content and deposition of adrenergic mediator in the synaptic vesicles of sympathetic nerve fibers of the rat vas deferens was studied by electron-microscopic cytochemistry. The results showed that carbidine can reduce the reserves of monoamines deposited in synaptic vesicles but does not affect the uptake and accumulation of exogenous noradrenalin by synaptic vesicles.Group for Electron Microscopy, Laboratory of Pharmacology of the Nervous System, Institute of Pharmacology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Zakusov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 80, No. 10, pp. 69–72, October, 1975.     

Effect of NO-generating compound NaNO<Subscript>2</Subscript> on ultrastructure of synaptic vesicles of glutamatergic synapses

Ultrastructure of synaptic vesicles in axon terminals of granule cells from isolated cerebellum of Rana temporaria frogs under the influence of NO-generating compound NaNO₂ in various concentrations and electrical stimulation was evaluated by the method of electron microscopy. NO-generating compound in low concentration induced translocation of synaptic vesicles and formation of small clusters. The size and structure of synaptic vesicles remained unchanged under these conditions. Increasing the concentration of NaNO₂ led to swelling of synaptic vesicles, formation of arranged heaps from individual vesicles or fusion of their content. Electrical stimulation of the cerebellum in the presence of NaNO₂ increased damage to synaptic vesicles. These experimental data model some stages observed in stroke. The formation of clusters from synaptic vesicles is a compensatory and adaptive response maintaining the structure of synaptic vesicles and protecting neurons from high concentrations of glutamate. Glutamate produces a toxic effect on nerve cells and glial cells of the cerebellum under pathological conditions, which is accompanied by impairment of signal transduction from presynaptic to postsynaptic neurons. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 146, No. 7, pp. 13–17, July, 2008     

Spatial organization and dynamic properties of neurotransmitter release sites in the enteric nervous system

Synaptic communication requires an efficient coupling of vesicle fusion to release neurotransmitter and vesicle retrieval to repopulate the synapse. In synapses of the CNS many proteins involved in exocytosis, endocytosis and refilling of vesicles have been identified. However, little is known about the organization and functioning of synaptic contacts in the enteric nervous system (ENS). We used fluorescent antibodies against presynaptic proteins (synaptobrevin, synaptophysin, synaptotagmin and bassoon) to identify synaptic contacts not only in guinea-pig enteric ganglia but also in the interconnecting fiber strands. Staining patterns were not altered by colchicine (100 μM), ruling out a contribution of protein transport at the time of fixation. Active release sites at fiber intersections and around neuronal cell bodies were labeled with FM1-43 (10 μM) by high K⁺ or electric field stimulation (EFS). During a second round of EFS, vesicles were reused, as reflected by dye loss. Destaining rates increased with stimulus frequency (2–30 Hz), reaching a maximum at about 15 Hz, likely caused by synaptic depression at higher frequencies. Tetrodotoxin (TTX, 1 μM) as well as nominally zero external Ca²⁺ (2 mM EGTA) prevented all destaining. The readily releasable pool (RRP, a subset of vesicles docked at the membrane and ready to fuse upon [Ca²⁺]_i increase) can be specifically released by a hypertonic challenge (500 mM sucrose). We measured this pool to be ∼27% of the total recycling pool, remarkably similar to synapses in the CNS. In whole-mount preparations, FM1-43 also reliably labeled active release sites in ganglia, fiber strands and in muscle bundles. The staining pattern indicated that the presynaptic antibodies mainly labeled active sites. The presence of numerous release sites suggests information processing capability within interconnecting fibers. With FM imaging, enteric synaptic function can be monitored independent of any postsynaptic modulation. Although electron microscopy data suggest that ENS synapses may not be as specialized as hippocampal synapses, remarkably similar release properties were measured.     

N type Ca2+ channels and RIM scaffold protein covary at the presynaptic transmitter release face but are components of independent protein complexes

Fast neurotransmitter release at presynaptic terminals occurs at specialized transmitter release sites where docked secretory vesicles are triggered to fuse with the membrane by the influx of Ca²⁺ ions that enter through local N type (CaV2.2) calcium channels. Thus, neurosecretion involves two key processes: the docking of vesicles at the transmitter release site, a process that involves the scaffold protein RIM (Rab3A interacting molecule) and its binding partner Munc-13, and the subsequent gating of vesicle fusion by activation of the Ca²⁺ channels. It is not known, however, whether the vesicle fusion complex with its attached Ca²⁺ channels and the vesicle docking complex are parts of a single multifunctional entity. The Ca²⁺ channel itself and RIM were used as markers for these two elements to address this question. We carried out immunostaining at the giant calyx-type synapse of the chick ciliary ganglion to localize the proteins at a native, undisturbed presynaptic nerve terminal. Quantitative immunostaining (intensity correlation analysis/intensity correlation quotient method) was used to test the relationship between these two proteins at the nerve terminal transmitter release face. The staining intensities for CaV2.2 and RIM covary strongly, consistent with the expectation that they are both components of the transmitter release sites. We then used immunoprecipitation to test if these proteins are also parts of a common molecular complex. However, precipitation of CaV2.2 failed to capture either RIM or Munc-13, a RIM binding partner. These findings indicate that although the vesicle fusion and the vesicle docking mechanisms coexist at the transmitter release face they are not parts of a common stable complex.     

Ethanol tolerance and enhanced calcium/calmodulin-dependent phosphorylation of synaptic membrane proteins

The chronic effects of ethanol on synaptic membrane proteins was studied in ethanol-tolerant rats. Synaptic plasma membranes and postsynaptic densities were prepared from homogenates of forebrain and incubated in vitro with [gamma-32P]adenosine triphosphate in the presence and absence of calcium and calmodulin. In ethanol-tolerant animals, enhanced phosphorylation of synaptic plasma membrane but not postsynaptic density proteins was demonstrated in the presence of calcium and calmodulin. These results suggest that the development of tolerance to ethanol may involve alteration in neuronal sensitivity to calcium.     

Effects of high-potassium solutions and caffeine on synaptic vesicle exoendocytosis processes in the frog neuromuscular junction

Studies on frog skin-pectoris muscle preparations using vital fluorescent microscopy showed that stimulation of transmitter secretion using high-potassium solutions with the endocytosis marker FM 1–43 induced bright spots in all motor nerve terminals, these representing accumulations of vesicles undergoing the exoendocytic cycle in the active zones of nerve endings. Stimulation of transmitter secretion with caffeine evoked bright spots only in some nerve terminals and only in some parts of the terminals. In summer, the number of bright spots on stimulation of transmitter secretion by caffeine increased sharply. Extracellular recording of spontaneous synaptic signals showed that high-potassium solutions, like caffeine, produced dose-dependent increases in the frequency of miniature endplate currents. However, while high-potassium solutions always increased the frequency, this occurred with caffeine in only a proportion of experiments. This leads to the conclusion that exoendocytosis processes can occur both because of the influx of Ca²⁺ ions into nerve endings as a result of depolarization (high-potassium solutions) and because of the release of Ca²⁺ ions from the endoplasmic reticulum (caffeine). The possible spatial localization of the endoplasmic reticulum in nerve endings is discussed. The endoplasmic reticulum is suggested to have a role in synapse remodeling processes. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 91, No. 7, pp. 821–831, July, 2005.     

The vesicle cycle in motor nerve endings of the mouse diaphragm

Experiments on the mouse diaphragm muscle using intracellular microelectrode recordings and fluorescence microscopy were performed to study the dynamics of transmitter secretion and synaptic vesicle recycling processes (the exocytosis-endocytosis cycle) in motor nerve endings (NE) during prolonged rhythmic stimulation (20 impulses/sec). During stimulation, there were triphasic changes in the amplitude of endplate potentials (EPP): an initial rapid reduction, followed by prolonged (1–2 min) stabilization of amplitude, i.e., a plateau, and then a further slow decrease. Restoration of EPP amplitude after stimulation for 3 min occurred over a period of several seconds. Loading of synaptic vesicles with the fluorescent endocytic stain FM1-43 showed that rhythmic stimulation led to a gradual (over 5–6 min) decrease in NE fluorescence, demonstrating exocytosis of synaptic vesicles. Quantum analysis of the electrophysiological data and comparison of these data with results from fluorescence studies suggested that mouse NE have a high rate of endocytosis and reutilization of synaptic vesicles (the mean recycling time was about 50 sec), which may support the maintenance of reliable synaptic transmission during prolonged high-frequency activity. The sizes of the release-ready and recycling pools of synaptic vesicles were determined quantitatively. It is suggested that vesicle recycling in mouse NE occurs via a short, rapid pathway with incorporation into the recycling pool. Vesicles of the reserve pool are not used for transmitter secretion in the stimulation conditions used here. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 94, No. 2, pp. 129–141, February, 2008.     

The mechanisms of multi-component paired-pulse facilitation of neurotransmitter release at the frog neuromuscular junction

We have studied the mechanisms of paired-pulse facilitation (PPF) of neurotransmitter release in isolated nerve-muscle preparations of the frog cutaneous pectoris muscle. In normal extracellular Ca²⁺ concentration ([Ca²⁺]_o, 1.8 mM), as the interpulse interval was increased from 5 to 500 ms, PPF decayed as a sum of two exponential components: a larger but shorter first component (F1) and a smaller but more prolonged second component (F2). In low [Ca²⁺]_o (0.5 mM), both F1 and F2 increased, and a third “early” component (Fe) appeared whose amplitude was larger and whose duration was shorter than F1 or F2. In the presence of the “fast” Ca²⁺ buffer BAPTA-AM, Fe disappeared, whereas F1 and F2 decreased in amplitude and duration. In contrast, the “slow” Ca²⁺ buffer EGTA-AM caused a decrease of Fe and reduction or complete blockade of F2, without any changes of F1. In solutions containing Sr²⁺ (1 mM), the magnitude of Fe was decreased, F1 was significantly reduced and shortened, but F2 was unaffected. Application of the calmodulin inhibitor W-7 (10 μM) at normal [Ca²⁺]_o produced a marked decrease of F2, and at low [Ca²⁺]_o, a complete blockade of Fe. These results suggest that PPF at frog motor nerve terminals is mediated by several specific for different PPF components intraterminal Ca²⁺ binding sites, which trigger neurotransmitter release. These sites have a higher affinity for Ca²⁺ ions and are located farther from the release-controlling Ca²⁺ channels than the Ca ²⁺ sensor that mediates phasic release.     

The role of extracellular calcium in exo- and endocytosis of synaptic vesicles at the frog motor nerve terminals

In the present study we combined FM 1-43 imaging and electrophysiological recording of miniature end-plate currents (MEPCs) to determine the role of extracellular calcium in synaptic vesicle exo- and endocytosis at the frog motor nerve terminals. We replaced extracellular Ca2+ ions with other bivalent cations (Sr2+, Ba2+, Cd2+, Mg2+) or used a calcium-free solution and monitored fluorescent staining of the nerve terminals in the presence of caffeine, which promotes the release of Ca2+ from intracellular stores. Caffeine has induced FM1-43 internalization only in the presence of bivalent cations in the external solution. The exposure of the neuromuscular junction to caffeine in a calcium-free solution caused a reversible failure of FM 1-43 loading and an increase in the nerve terminal width. This effect of a calcium-free solution was not due to a decrease in exocytosis, because caffeine-induced FM1-43 unloading from the previously loaded nerve terminals, as well as a degree of the MEPCs frequency increase, was unchanged. We conclude that the presence of Ca2+ or other bivalent cations in extracellular space is necessary for endocytosis but not for exocytosis of synaptic vesicles, while transmitter release is promoted by efflux of Ca2+ from intracellular stores. The effect of extracellular Ca2+ on endocytosis might be driven by the non-specific interactions with membrane lipids.     

Ultrastructural localization of active zone and synaptic vesicle proteins in a preassembled multi-vesicle transport aggregate

Although it has been suggested that presynaptic active zone (AZ) may be preassembled, it is still unclear which entities carry the various proteins to the AZ during synaptogenesis. Here, I propose that aggregates of dense core vesicles (DCV) and small clear vesicles in the axons of young rat hippocampal cultures are carriers containing preformed AZ and synaptic vesicle (SV) components on their way to developing synapses. The aggregates were positively labeled with antibodies against Bassoon and Piccolo (two AZ cytomatrix proteins), VAMP, SV2, synaptotagmin (three SV membrane proteins), and synapsin I (a SV-associated protein). Bassoon and Piccolo labeling were localized at dense material both in the aggregates and at the AZ. In addition to the SV at the synapses, the SV membrane proteins labeled the clear vesicles in the aggregate as well as many other SV-like and pleiomorphic vesicular structures in the axons, and synapsin I labeling was associated with the vesicles in the aggregates. In single sections, these axonal vesicle aggregates were approximately 0.22 by 0.13 microm in average dimensions and contain one to two DCV and five to six small clear vesicles. Serial sections confirmed that the aggregates were not synaptic junctions sectioned en face. Labeling intensities of Bassoon and Piccolo measured from serially sectioned transport aggregates and AZ were within range of each other, suggesting that one or a few aggregates, but not individual DCV, can carry sufficient Bassoon and Piccolo to form an AZ. The present findings provide the first ultrastructural evidence localizing various AZ and SV proteins in a preassembled multi-vesicle transport aggregate that has the potential to quickly form a functional active zone.     

Recruitment of sphingosine kinase to presynaptic terminals by a conserved muscarinic signaling pathway promotes neurotransmitter release

Sphingolipids are potent lipid second messengers that regulate cell differentiation, migration, survival, and secretion, and alterations in sphingolipid signaling have been implicated in a variety of diseases. However, how sphingolipid levels are regulated, particularly in the nervous system, remains poorly understood. Here, we show that the generation of sphingosine-1-phosphate by sphingosine kinase (SphK) promotes neurotransmitter release. Electrophysiological, imaging, and behavioral analyses of Caenorhabditis elegans mutants lacking sphingosine kinase sphk-1 indicate that neuronal development is normal, but there is a significant defect in neurotransmitter release from neuromuscular junctions. SPHK-1 localizes to discrete, nonvesicular regions within presynaptic terminals, and this localization is critical for synaptic function. Muscarinic agonists cause a rapid increase in presynaptic SPHK-1 abundance, whereas reduction of endogenous acetylcholine production results in a rapid decrease in presynaptic SPHK-1 abundance. Muscarinic regulation of presynaptic SPHK-1 abundance is mediated by a conserved presynaptic signaling pathway composed of the muscarinic acetylcholine receptor GAR-3, the heterotrimeric G protein Gαq, and its effector, Trio RhoGEF. SPHK-1 activity is required for the effects of muscarinic signaling on synaptic transmission. This study shows that SPHK-1 promotes neurotransmitter release in vivo and identifies a novel muscarinic pathway that regulates SphK abundance at presynaptic terminals.     

Redistribution of secretory granule components precedes that of synaptic vesicle proteins during differentiation of a neuronal cell line in serum-free medium.

Incubation of the rat sensory neuron-derived cell line ND7 in serum-free medium results in the arrest of mitosis and the appearance of numerous neuronal processes. During this differentiation event, secretory granule components such as chromogranins, neuropeptide Y and the C-flanking peptide of pro-neuropeptide Y move to the tips of the majority of the neuronal processes regardless of process length. In contrast, the synaptic vesicle component, synaptophysin, is found only at the tips of the very long processes which appear following prolonged periods of culture in serum-free medium. A similar restriction of synaptophysin to long processes is also observed following differentiation and process formation induced by other treatments such as incubation in reduced serum or treatment with cyclic AMP or phorbol myristate acetate. Hence the regulated secretory pathway associated with the chromogranins and neuropeptides appears to be segregated into the processes at an earlier stage of ND7 differentiation than the synaptophysin-associated synaptic vesicle pathway. ND7 cells therefore provide a model system for studying the processes regulating these pathways and the redistribution of their components during neuronal differentiation.     

免疫突触形成过程中T细胞受体的重定向运动:涡旋驱动模型

目的：建立T细胞受体（TCR）在免疫突触形成过程中作重定向（reorientation）运动的机制模型.方法：基于经典流体力学环境中的双分子反应传能原理,提出了T细胞受体的涡旋驱动模型,利用免疫突触内耦合的受体或配体分子作为涡源驱动T细胞受体分子的募集.结果：模型计算的结果表明,在强度及作用频率同时具备一定范围的涡旋连续驱动下,TCR重定向运动速度可达到实验测定的范围（0.04～0.1 μm/s）.结论：本模型证明突触内受体/配体对耦合时通过将其结合自由能转化为细胞内外流体涡旋运动的机械能可能直接提供了TCR重定向运动的驱动力.     

A quantitative assessment of glutamate uptake into hippocampal synaptic terminals and astrocytes: new insights into a neuronal role for excitatory amino acid transporter 2 (EAAT2)

The relative distribution of the excitatory amino acid transporter 2 (EAAT2) between synaptic terminals and astroglia, and the importance of EAAT2 for the uptake into terminals is still unresolved. Here we have used antibodies to glutaraldehyde-fixed d-aspartate to identify electron microscopically the sites of d-aspartate accumulation in hippocampal slices. About 3/4 of all terminals in the stratum radiatum CA1 accumulated d-aspartate-immunoreactivity by an active dihydrokainate-sensitive mechanism which was absent in EAAT2 glutamate transporter knockout mice. These terminals were responsible for more than half of all d-aspartate uptake of external substrate in the slices. This is unexpected as EAAT2-immunoreactivity observed in intact brain tissue is mainly associated with astroglia. However, when examining synaptosomes and slice preparations where the extracellular space is larger than in perfusion fixed tissue, it was confirmed that most EAAT2 is in astroglia (about 80%). Neither d-aspartate uptake nor EAAT2 protein was detected in dendritic spines. About 6% of the EAAT2-immunoreactivity was detected in the plasma membrane of synaptic terminals (both within and outside of the synaptic cleft). Most of the remaining immunoreactivity (8%) was found in axons where it was distributed in a plasma membrane surface area several times larger than that of astroglia. This explains why the densities of neuronal EAAT2 are low despite high levels of mRNA in CA3 pyramidal cell bodies, but not why EAAT2 in terminals account for more than half of the uptake of exogenous substrate by hippocampal slice preparations. This and the relative amount of terminal versus glial uptake in the intact brain remain to be discovered.     

Cofilin: a redox sensitive mediator of actin dynamics during T-cell activation and migration

Cofilin is an actin-binding protein that depolymerizes and/or severs actin filaments. This dual function of cofilin makes it one of the major regulators of actin dynamics important for T-cell activation and migration. The activity of cofilin is spatio-temporally regulated. Its main control mechanisms comprise a molecular toolbox of phospho-, phospholipid, and redox regulation. Phosphorylated cofilin is inactive and represents the dominant cofilin fraction in the cytoplasm of resting human T cells. A fraction of dephosphorylated cofilin is kept inactive at the plasma membrane by binding to phosphatidylinositol 4,5-bisphosphate. Costimulation via the T-cell receptor/CD3 complex (signal 1) together with accessory receptors (signal 2) or triggering through the chemokine SDF1α (stromal cell-derived factor 1α) induce Ras-dependent dephosphorylation of cofilin, which is important for immune synapse formation, T-cell activation, and T-cell migration. Recently, it became evident that cofilin is also highly sensitive for microenvironmental changes, particularly for alterations in the redox milieu. Cofilin is inactivated by oxidation, provoking T-cell hyporesponsiveness or necrotic-like programmed cell death. In contrast, in a reducing environment, even phosphatidylinositol 4,5-bisphosphate -bound cofilin becomes active, leading to actin dynamics in the vicinity of the plasma membrane. In addition to the well-established three signals for T-cell activation, this microenvironmental control of cofilin delivers a modulating signal for T-cell-dependent immune reactions. This fourth modulating signal highly impacts both initial T-cell activation and the effector phase of T-cell-mediated immune responses.     

Changes in the biochemical and biophysical parameters of cholinergic synaptic vesicles on transmitter release and during a subsequent period of rest

Synaptic vesicles were isolated on sucrose density gradients from perfused blocks of Torpedo electric organ under varying experimental conditions. Newly synthesized acetylcholine was labelled with [³H]acetate in order to distinguish synaptic vesicles which had been recycled during stimulation-induced transmitter release and, in consequence, had become smaller and denser from the larger, lighter vesicles characteristic of unstimulated tissue. After 1800 pulses at 0.1 Hz, the density of these smaller vesicles increased from a pre-stimulation value of 1.056 g.ml^?1 to 1.067 g.ml^?1, whereas their water space (measured as the space occupied by the permeant solute glycerol), decreased by 34% from 65% to 43% of vesicle volume. During a subsequent 12 h rest period, these changes were partially reversed; water space returned to 52% of control and this change was highly correlated with a decrease in vesicle density and an increase in vesicular acetylcholine. The diameter of vesicles in whole tissue sections showed corresponding changes. An additional 12 h rest period did not lead to further significant recovery, suggesting that the preparation had a limited suitability for following long term processes depending on normal energy metabolism.The results can be explained on the assumption that when vesicles reform after releasing transmitter their core has a lower osmotic pressure than that of fully loaded vesicles. Reloading is accompanied by osmotically induced rehydration.     

Heterogeneous Ca2+ influx along the adult calyx of held: a structural and computational study

The calyx of Held is a morphologically complex nerve terminal containing hundreds to thousands of active zones. The calyx must support high rates of transient, sound-evoked vesicular release superimposed on a background of sustained release, due to the high spontaneous rates of some afferent fibers. One means of distributing vesicle release in space and time is to have heterogeneous release probabilities (Pr) at distinct active zones, which has been observed at several CNS synapses including the calyx of Held. Pr may be modulated by vesicle proximity to Ca2+ channels, by Ca2+ buffers, by changes in phosphorylation state of proteins involved in the release process, or by local variations in Ca2+ influx. In this study, we explore the idea that the complex geometry of the calyx also contributes to heterogeneous Pr by impeding equal propagation of action potentials through all calyx compartments. Given the difficulty of probing ion channel distribution and recording from adult calyces, we undertook a structural and modeling approach based on computerized reconstructions of calyces labeled in adult cats. We were thus able to manipulate placement of conductances and test their effects on Ca2+ concentration in all regions of the calyx following an evoked action potential in the calyceal axon. Our results indicate that with a non-uniform distribution of Na+ and K+ channels, action potentials do not propagate uniformly into the calyx, Ca2+ influx varies across different release sites, and latency for these events varies among calyx compartments. We suggest that the electrotonic structure of the calyx of Held, which our modeling efforts indicate is very sensitive to the axial resistivity of cytoplasm, may contribute to variations in release probability within the calyx.