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At least 865 records · Page 48Linked to original sources

PubMed1995

Cysteine-string proteins as templates for membrane fusion: models of synaptic vesicle exocytosis.

Cysteine-string proteins are relatively small, cysteine-rich components of synaptic vesicle membranes. Recent investigations demonstrated that at least 11 of the 13 cysteine residues of the Torpedo cysteine-string protein are fatty acylated. This exceptional level of fatty acylation occurs along a short stretch (less than 25 residues) of amino acids which are flanked on either side by very polar amino and carboxy termini. This amphipathic structure may have unique capabilities to catalyze events at membrane interfaces. We propose two distinct pathways to explain how these capabilities might subserve membrane fusion and exocytosis.

Animals↗

PubMed1975

The smooth endoplasmic reticulum: structure and role in the renewal of axonal membrane and synaptic vesicles by fast axonal transport.

The spatial arrangement of the smooth endoplasmic reticulum (SER) was studied in 0.5-2 mum thick sections of rat spinal and chick ciliary ganglia previously impregnated with heavy metal salts. Electron microscopy at low (10-5 V) or high (10-6 V) voltage showed the impregnated SER as a continuous system extending probably from the perikaryon to the axon terminal. Tubules of the SER, which were running in a parallel direction with the axon, were occasionally seen in close apposition with the axonal membrane. Moreover in the preterminal region, anastomosed tubules of the SER formed a subsurface 'primary network' and gave rise to a deeper 'secondary network' made of thinner tubules; synaptic vesicles bulging at the tip of thin tubules of the SER were frequently observed. To specify the role played by the SER in the fast axonal transport, chicken ciliary ganglia were slighty compressed and radioautographed 3 h after the intracerebral injection of [3-H]lysine. Quantitative analysis of the silver grain distribution indicated that labeled proteins, rapidly conveyed down the axon, piled up in regions containing an accumulation of SER profiles. On the basis of these results, it is concluded that: (1) the SER appears as a continuous intraaxonal pathway bridging the perikaryon and the axon terminal; (2) the SER conveys macromolecular components with the fast axonal transport; (3) the conveyed macromolecules, which are delivered to the axonal membrane and to the synaptic vesicles, are probably transferred by means of connections with the SER.

Animals↗

PubMed2005

Phenylarsine oxide is able to dissipate synaptic vesicle acidic pool.

Phenylarsine oxide (PAO) has a number of targets in the neurons, one of them is exocytotic process. In this study, we have focused on the mechanisms of phenylarsine oxide action on Ca(2+)-dependent and Ca(2+)-independent neurotransmitter release from rat brain synaptosomes. We investigated the influence of phenylarsine oxide on: (i) l-[(14)C]glutamate and [(3)H]GABA release and uptake; (ii) plasma membrane potential using a potential-sensitive fluorescent probe rhodamine 6G; (iii) exo/endocytotic process using a pH-sensitive fluorescent probe acridine orange (AO). It has been found that phenylarsine oxide induced deacidification of synaptic vesicles. This effect was completely abolished by preliminary treatment of synaptosomes with a protonophore FCCP indicating that both reagents injured a proton electrochemical gradient. Dissipation of the proton gradient by low concentrations of phenylarsine oxide (not exceed 1 microM) did not prevent KCl-triggered exocytotic response, but essentially modified endocytotic one. At higher concentrations of phenylarsine oxide (up to 10 microM), the proton gradient dissipation was intensified and the exocytotic response was fully abolished. The reagent did not change plasma membrane potential, but depolarized mitochondria. It also caused potent inhibition of the Ca(2+)-stimulated l-[(14)C]glutamate and [(3)H]GABA release and increase the Ca(2+)-independent release of l-[(14)C]glutamate, but not of [(3)H]GABA. Disulfide-reducing reagents (dithiothreitol and beta-mercaptoethanol) completely prevented phenylarsine oxide-evoked injuries. They could also restore the initial levels of the mitochondrial potential, the exocytotic response to KCl and the release and uptake of neurotransmitters. Our data provide the evidence that phenylarsine oxide causes dissipation of synaptic vesicle acidic pool resulting in the reduction of vesicle filling and as consequence in attenuation of Ca(2+)-stimulated neurotransmitter release.

Animals↗

PubMed1984

Characterization of a Mg2+-ATPase and a proton pump in cholinergic synaptic vesicles from the electric organ of Torpedo marmorata.

Cholinergic synaptic vesicles from the electric organ of Torpedo marmorata are associated with a Mg2+-ATPase insensitive to ouabain and oligomycin. Treatment of vesicle membranes with dichloromethane releases a Mg2+-ATPase with apparent molecular mass of around 250 kDa as determined by gel filtration. The vesicular ATPase resembles the mitochondrial F1-ATPase in these properties. Gel electrophoresis of the solubilized ATPase shows however that only a single 50-kDa band is present as compared to the alpha-subunit (52 kDa) and beta-subunit (50 kDa) of electric organ mitochondrial F1-ATPase present in this range of molecular mass range. In agreement, covalent photoaffinity labelling of isolated vesicles with azido-ATP shows a 50-kDa band. Vesicle ghosts were found to accumulate [14C]methylamine in an ATP-dependent manner indicating the presence of an inwardly directed proton pump. We conclude that cholinergic vesicles contain a proton pump probably driven by the Mg2+-ATPase here described, which generates an electrochemical gradient across the vesicle membrane and is necessary for uptake and storage of acetylcholine within the vesicles.

Adenosine Triphosphatases↗

PubMed2001

Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion.

Exo-endocytotic turnover of synaptic vesicles (SVs) at synapses between hippocampal neurons in culture was examined by electron microscopy (EM). We carried out photoconversion (PC) of the fluorescent endocytotic marker FM 1-43 by using 3,3'-diaminobenzidine to convert the dye signal into an electron-dense product. Electron-dense products were located almost exclusively in SVs, whose densities were bimodally distributed in two sharply demarcated populations, PC-positive (PC+) and PC-negative (PC-). The median densities of these populations did not vary with the proportion of vesicles stained within a presynaptic terminal (bouton). The proportion of PC+ SVs remained constant across consecutive thin sections of single boutons, but varied greatly from one bouton to another, indicating marked heterogeneity in exo-endocytotic activity. Our experiments indicated that only a minority of SVs were stained in most boutons after stimuli known to cause complete turnover of the functional vesicular pool. A direct spatial correlation was found between FM 1-43 fluorescent spots seen with light microscopy and PC+ boutons by EM. The correlation was clearer in isolated boutons than in clusters of boutons. Photoconversion in combination with FM dyes allows clarification of important aspects of vesicular traffic in central nervous system nerve terminals.

Animals↗

PubMed1989

Progressive restriction of synaptic vesicle protein to the nerve terminal during development of the neuromuscular junction.

Antibodies to synaptic vesicle (SV) proteins and to neurofilament (NF) proteins were used to investigate presynaptic differentiation and its relation to the formation of acetylcholine receptor (AChR) clusters at developing mouse neuromuscular junctions. At all times during development, SV proteins and NF proteins were segregated into neighboring, but separate regions of the presynaptic neurite. At embryonic day (ED) 14 SV proteins were present throughout preterminal neurites but at later ages became progressively restricted to the distal parts of the neurites. NF proteins occupied a more proximal region that extended distally during development until NF proteins occupied the entire axon up to the terminal. The restriction of SV proteins exclusively to the terminal did not occur until the second postnatal week. At the time of their first appearance (ED 14), up to 50% of AChR clusters were not associated with neurites; precise colocalization required 12-36 hr to develop. These findings demonstrate a progressive restriction of both pre- and postsynaptic components to the synapse during development.

Aging↗

PubMed1996

Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles.

A cholinergic locus has recently been identified consisting of a unique mammalian genomic arrangement containing the genes for choline acetyltransferase (ChAT) and a putative vesicular acetylcholine transporter (VAChT). Although transcripts for ChAT and VAChT protein have been localized in cholinergic neurons, little is known about the encoded VAChT protein. Here we describe production of highly specific rabbit polyclonal antibodies, generated using a VAChT C-terminus/glutathione-S-transferase fusion protein, and immunological characterization of the native VAChT protein. These antibodies specifically recognized full-length recombinant VAChT expressed in transfected HeLa cells by Western blotting, with the prominent immunoreactive band at 55 kDa. In rat brain homogenates, a single VAChT-immunoreactive band of approximately 70 kDa was predominant in known areas of cholinergic innervation, including striatum, cortex, hippocampus,and amygdala. Light microscopic immunocytochemistry revealed reaction product in cholinergic cell groups but not in noncholinergic areas. More significantly, immunoreactivity was also concentrated in axonal fibers in many regions known to receive prominent cholinergic innervation, such as cerebral cortex, hippocampus, amygdala, striatum, several thalamic nuclei, and brainstem regions. Electron microscopy using immunoperoxidase revealed that VAChT was localized in axon terminals, and using more precise immunogold techniques, to synaptic vesicles. In VAChT-positive perikarya, the immunogold particles were localized to the cytoplasmic face of the Golgi complex. These findings confirm that VAChT protein is expressed uniquely in cholinergic neurons, concentrated in synaptic vesicles, and at least for the C terminus, topologically oriented as predicted by models.

Acetylcholine↗

PubMed1991

Functional reconstitution of the gamma-aminobutyric acid transporter from synaptic vesicles using artificial ion gradients.

The gamma-aminobutyric acid transporter of rat brain synaptic vesicles was reconstituted in proteoliposomes, and its activity was studied in response to artificially created membrane potentials or proton gradients. Changes of the membrane potential were monitored using the dyes oxonol VI and 3,3'-diisopropylthiodicarbocyanine iodide, and changes of the H+ gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K+ gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of [3H]GABA which was saturable. Similarly, [3H]glutamate uptake was stimulated by valinomycin, indicating that both transporters can be driven by the membrane potential. Proton gradients were generated by the incubation of K(+)-loaded proteoliposomes in a buffer free of K+ or Na+ ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H+ ATPase by incubation of K(+)-loaded proteoliposomes in equimolar K+ buffer in the presence of valinomycin. These proton gradients evoked nonspecific, nonsaturable uptake of GABA and beta-alanine but not of glycine in proteoliposomes as well as protein-free liposomes. Therefore, transporter activity was monitored using glycine as an alternative substrate. Proton gradients generated by both methods elicited saturable glycine uptake in proteoliposomes. Together, our data confirm that the vesicular GABA transporter can be energized by both the membrane potential and the pH gradient and show that transport can be achieved by artificial gradients independently of the endogenous proton ATPase.

Animals↗

PubMed2000

Neuromelanin biosynthesis is driven by excess cytosolic catecholamines not accumulated by synaptic vesicles.

Melanin, the pigment in hair, skin, eyes, and feathers, protects external tissue from damage by UV light. In contrast, neuromelanin (NM) is found in deep brain regions, specifically in loci that degenerate in Parkinson's disease. Although this distribution suggests a role for NM in Parkinson's disease neurodegeneration, the biosynthesis and function of NM have eluded characterization because of lack of an experimental system. We induced NM in rat substantia nigra and PC12 cell cultures by exposure to l-dihydroxyphenylalanine, which is rapidly converted to dopamine (DA) in the cytosol. This pigment was identical to human NM as assessed by paramagnetic resonance and was localized in double membrane autophagic vacuoles identical to NM granules of human substantia nigra. NM synthesis was abolished by adenoviral-mediated overexpression of the synaptic vesicle catecholamine transporter VMAT2, which decreases cytosolic DA by increasing vesicular accumulation of neurotransmitter. The NM is in a stable complex with ferric iron, and NM synthesis was inhibited by the iron chelator desferrioxamine, indicating that cytosolic DA and dihydroxyphenylalanine are oxidized by iron-mediated catalysis to membrane-impermeant quinones and semiquinones. NM synthesis thus results from excess cytosolic catecholamines not accumulated into synaptic vesicles. The permanent accumulation of excess catechols, quinones, and catechol adducts into a membrane-impermeant substance trapped in organelles may provide an antioxidant mechanism for catecholamine neurons. However, NM in organelles associated with secretory pathways may interfere with signaling, as it delays stimulated neurite outgrowth in PC12 cells.

Animals↗

PubMed1993

Calcium-induced translocation of synaptic vesicles to the active site.

The effect of increasing [Ca2+]o on the positioning of synaptic vesicles relative to the active site in resting coxal neuromuscular junctions of Drosophila was investigated. In normal saline (1.8 mM Ca2+) only a very small percentage of sites possess a vesicle docked under the dense body plate close to the plasma membrane in a readily releasable position. However, after exposure to elevated Ca2+ salines (3.6, 9, 18 mM), an increase in the number of active sites possessing docked vesicles was observed. Also, an increase in the average number of docked vesicles/site was seen. Intracellular recordings from coxal muscle fibers in normal saline and elevated Ca2+ salines were made, and it was observed that exposure to elevated Ca2+ saline caused an increase in miniature excitatory junction potential (mejp) frequency and in multiquantal and clustered mejps. Thus, when the number of active sites possessing docked vesicles increases, the frequency of spontaneous release also increases. Furthermore, when the number of docked vesicles/site increases, the number of multiquantal mejps increases. The data suggest that Ca2+ may be involved in vesicle translocation to the active site, and that the concentration of Ca2+ in the terminal may regulate the number of active sites that possess readily releasable vesicles. The effects of increasing the number of docked vesicles on spontaneous release characteristics are discussed.

Animals↗

PubMed1999

Identification of synaptic vesicle, pre- and postsynaptic proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing.

Synaptic pathology is central in the pathogenesis of several psychiatric disorders, for example in Alzheimer's disease (AD) and schizophrenia. Quantification of specific synaptic proteins has proved to be a useful method to estimate synapitc density in the brain. Using this approach, several synaptic proteins have been demonstrated to be altered in both AD and schizophrenia. Until recently, the analysis of synaptic pathology has been limited to postmortem tissue. In living subjects, these synaptic proteins may be studied through analysis of cerebrospinal fluid (CSF). In an earlier study performed by us, one synaptic vesicle specific protein, synaptotagmin, was detected in CSF for the first time using a procedure based on affinity chromatography, reversed-phase chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and chemiluminescence immunoblotting. However, other synaptic proteins were not detectable with this procedure. Therefore, we have developed a procedure including precipitation of CSF proteins with trichloroacetic acid, followed by liquid-phase isoelectric focusing using the Rotofor Cell, and finally analysis of Rotofor fractions by Western blotting for identification of synaptic proteins in CSF. Five synaptic proteins, rab3a, synaptotagmin, growth-associated protein (GAP-43), synaptosomal-associated protein (SNAP-25) and neurogranin, have been demonstrated in CSF using this method. The major advantage of liquid-phase isoelectric focusing (IEF) using the Rotofor cell is that it provides synaptic proteins from CSF in sufficient quantities for identification. This method may also be suitable for identification of other types of trace amounts of brain-specific proteins in CSF. These results demonstrate that several synaptic proteins can be identified and measured in CSF to study synaptic function and pathology in degenerative disorders.

Calcium-Binding Proteins↗

PubMed1986

Horseradish peroxidase uptake of the synaptic vesicles in the superior cervical ganglion of the cat.

The function-dependent changes in synaptic vesicle number and horseradish peroxidase (HRP) labeling were studied in the synapses of cat superior cervical ganglia. It was found that only a low percentage of vesicles contained the extracellular marker. Supra-maximal stimulation (15 min) and subsequent rest (5 min) did not result in significant changes in vesicle number and the percentage of labeled vesicles also remained at about the same level. The low and activity-independent HRP labeling rate does not exclude the possibility of vesicle recycling but indicates that it might be only indirectly connected to the transmitter release itself.

Animals↗

PubMed1999

Unexpected localization of the Na+/Cl--dependent-like orphan transporter, Rxt1, on synaptic vesicles in the rat central nervous system.

Numerous features of its primary structure demonstrate that the orphan transporter Rxt1 belongs to the Na+/Cl--dependent neurotransmitter plasma membrane transporter superfamily, which includes the dopamine, norepinephrine, serotonin and gamma-aminobutyric acid (GABA) transporters. Initial immunocytochemical investigations with affinity-purified antibodies have established that Rxt1 is localized, almost exclusively, in axon terminals of glutamatergic neurons and subsets of GABAergic neurons in the CNS. Further studies were carried out to determine its subcellular distribution. In a first series of experiments, PC-12 cells were transfected with plasmids encoding either the dopamine transporter or Rxt1. Immunofluorescence experiments showed that the dopamine transporter was expressed in these cells, and, as expected, addressed to their plasma membrane. Surprisingly, this was never the case with Rxt1, which was targeted to the same subcellular compartment as synaptophysin, a vesicular protein. In a second set of experiments, subcellular fractionation of rat striatum showed that Rxt1, but not the dopamine transporter, was relatively abundant in the purified synaptic vesicle fraction. Finally, electron microscopic immunocytochemistry with anti-Rxt1 antibodies showed peroxidase as well as pre- and post-embedding immunogold labelling confined to the intracellular compartment in various brain regions. Moreover, quantitative analysis of post-embedding experiments demonstrated that the immunogold particles corresponding to Rxt1 immunoreactivity were mostly localized to small synaptic vesicles. These data indicate that, in contrast with the other members of the Na+/Cl--dependent neurotransmitter transporter superfamily, which are targeted to the plasma membrane, Rxt1 is distributed as a vesicular protein in the CNS.

Animals↗

PubMed2005

Essential role of the synaptic vesicle protein synapsin II in formalin-induced hyperalgesia and glutamate release in the spinal cord.

The synaptic vesicle protein synapsin II plays an important role in the regulation of neurotransmitter release and synaptic plasticity. Here, we investigated its involvement in the synaptic transmission of nociceptive signals in the spinal cord and the development of pain hypersensitivity. We show that synapsin II is predominantly expressed in terminals and neuronal fibers in superficial laminae of the dorsal horn (laminae I-II). Formalin injection into a mouse hindpaw normally causes an immediate and strong release of glutamate in the dorsal horn. In synapsin II deficient mice this glutamate release is almost completely missing. This is associated with reduced nociceptive behavior in the formalin test and in the zymosan-induced paw inflammation model. In addition, the formalin evoked increase in the number of c-Fos IR neurons is significantly reduced in synapsin II knockout mice. Touch perception and motor coordination, however, are normal indicating that synapsin II deficiency does not generally disrupt sensory and/or motor functions. Antisense-mediated transient knockdown of synapsin II in the spinal cord of adult animals also reduced the nociceptive behavior. As the antisense effect is independent of a potential role of synapsin II during development we suggest that the hypoalgesia in synapsin II deficient mice does involve a direct 'pain-facilitating' effect of synapsin II and is not essentially dependent on potentially occurring developmental alterations. The distinctive role of synapsin II for pain signaling probably results from its specific localization and possibly from a specific control of glutamate release.

Animals↗

PubMed1996

Synaptic vesicles have two distinct recycling pathways.

In this paper, evidence is presented that two distinct synaptic vesicle recycling pathways exist within a single terminal. One pathway emanates from the active zone, has a fast time course, involves no intermediate structures, and is blocked by exposure to high Mg2+/low Ca2+ saline, while the second pathway emanates at sites away from the active zone, has a slower time course, involves an endosomal intermediate, and is not sensitive to high Mg2+/low Ca2+. To visualize these two recycling pathways, the temperature-sensitive Drosophila mutant, shibire, in which vesicle recycling is normal at 19 degrees C but is blocked at 29 degrees C, was used. With exposure to 29 degrees C, complete vesicle depletion occurs as exocytosis proceeds while endocytosis is blocked. When the temperature is lowered to 26 degrees C, vesicle recycling membrane begins to accumulate as invaginations of the plasmalemma, but pinch-off is blocked. Under these experimental conditions, it was possible to distinguish the two separate pathways by electron microscopic analysis. These two pathways were further characterized by observing the normal recycling process at the permissive temperature, 19 degrees C. It is suggested that the function of these two recycling pathways might be to produce two distinct vesicle populations: the active zone and nonactive zone populations. The possibility that these two populations have different release characteristics and functions is discussed.

Animals↗

PubMed1984

Accumulation of 3H-dopamine by synaptic vesicles from rat striatum in an impermeant medium.

The accumulation of 3H-dopamine by synaptic vesicles from rat striatum was significantly stabilized in a membrane impermeant medium. The characteristics of dopamine accumulation by striatal vesicles were quite similar to those reported for dopamine accumulation by a whole brain vesicle preparation in the same medium, and were significantly different from the characteristics previously reported for vesicular accumulation of norepinephrine.

Adenosine Triphosphate↗

PubMed2007

Stick-and-diffuse and caged diffusion: a comparison of two models of synaptic vesicle dynamics.

Two models were recently proposed to enable us to understand the dynamics of synaptic vesicles in hippocampal neurons. In the caged diffusion model, the vesicles diffuse in small circular cages located randomly in the bouton, while in the stick-and-diffuse model the vesicles bind and release from a cellular cytomatrix. In this article, we obtain analytic expressions for the fluorescence correlation spectroscopy (FCS) autocorrelation function for the two models and test their predictions against our earlier FCS measurements of the vesicle dynamics. We find that the stick-and-diffuse model agrees much better with the experiment. We find also that, due to the slow dynamics of the vesicles, the finite experimental integration time has an important effect on the FCS autocorrelation function and demonstrate its effect for the different models. The two models of the dynamics are also relevant to other cellular environments where mobile species undergo slow diffusionlike motion in restricted spaces or bind and release from a stationary substrate.

Computer Simulation↗

PubMed1999

Identification of a novel process limiting the rate of synaptic vesicle cycling at hippocampal synapses.

During intense presynaptic activity, the readily releasable pool (RRP) of synaptic vesicles empties more quickly than it can be refilled, and short-term depression results. Ordinarily, the pool refills within 20 s, but long, high-frequency trains of action potentials often induce a form of short-term depression that persists for a much longer time. Here, we report that replenishment of the RRP is governed by two simple processes: the previously identified mechanism termed refilling, and another process that appears after extensive exocytosis and produces a transient decrease in the capacity of the pool, lasting for several minutes. The data presented here place stringent constraints on the types of kinetic models that can be used to describe synaptic vesicular cycling and are inconsistent with the traditional multipool models of vesicular mobilization.

Action Potentials↗