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PubMed1978

Electrogenic behavior of synaptic vesicles from Torpedo californica.

Electrical potential changes in pure synaptic vesicles from Torpedo californica were monitored with the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide. Vesicles resuspended in variable external sodium ion in the presence of gramicidin established sodium ion membrane diffusion potentials. Vesicles resuspended in choline or acetylcholine chloride became hyperpolarized upon addition of gramicidin. Hyperpolarization was subsequently partially reversed spontaneously by choline or acetylcholine influx, which was confirmed by gel filtration, to yield a new, less negative, stable membrane potential. Thus, acetylcholine and choline are taken up electrogenically by synaptic vesicles.

Acetylcholine↗

PubMed1994

Rabphilin-3A is associated with synaptic vesicles through a vesicle protein in a manner independent of Rab3A.

Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein, which is implicated in regulated secretion, particularly in neurotransmitter release. Rabphilin-3A is associated with synaptic vesicles, although it has no transmembrane segment. Here we have studied how rabphilin-3A is associated with synaptic vesicles. Treatment of the synaptic vesicles isolated from rat brain with 1 M NaCl completely solubilized rabphilin-3A from the vesicles. These vesicles deprived of rabphilin-3A still contained Rab3A and synaptophysin. Exogenous rabphilin-3A bound to the vesicles deprived of endogenous rabphilin-3A in dose-dependent and saturable manners. The concentration of exogenous rabphilin-3A giving a half-maximal binding was about 50 nM and maximally 5 +/- 1 molecules of exogenous rabphilin-3A bound to one vesicle. Addition of exogenous Rab3A bound to one vesicle. Addition of exogenous Rab3A or removal of endogenous Rab3A by the action of Rab GDI did not affect the binding of exogenous rabphilin-3A to the vesicles. However, treatment of the vesicles with trypsin completely abolished the binding of exogenous rabphilin-3A. These results suggest that rabphilin-3A is associated with synaptic vesicles at least through a vesicle protein in a manner independent of Rab3A.

Adaptor Proteins, Signal Transducing↗

PubMed1992

Characterization of the P-type and V-type ATPases of cholinergic synaptic vesicles and coupling of nucleotide hydrolysis to acetylcholine transport.

Both phosphointermediate- and vacuolar-type (P- and V-type, respectively) ATPase activities found in cholinergic synaptic vesicles isolated from electric organ are immunoprecipitated by a monoclonal antibody to the SV2 epitope characteristic of synaptic vesicles. The two activities can be distinguished by assay in the absence and presence of vanadate, an inhibitor of the P-type ATPase. Each ATPase has two overlapping activity maxima between pH 5.5 and 9.5 and is inhibited by fluoride and fluorescein isothiocyanate. The P-type ATPase hydrolyzes ATP and dATP best among common nucleotides, and activity is supported well by Mg2+, Mn2+, or Co2+ but not by Ca2+, Cd2+, or Zn2+. It is stimulated by hyposmotic lysis, detergent solubilization, and some mitochondrial uncouplers. Kinetic analysis revealed two Michaelis constants for MgATP of 28 microM and 3.1 mM, and the native enzyme is proposed to be a dimer of 110-kDa subunits. The V-type ATPase hydrolyzes all common nucleoside triphosphates, and Mg2+, Ca2+, Cd2+, Mn2+, and Zn2+ all support activity effectively. Active transport of acetylcholine (ACh) also is supported by various nucleoside triphosphates in the presence of Ca2+ or Mg2+, and the Km for MgATP is 170 microM. The V-type ATPase is stimulated by mitochondrial uncouplers, but only at concentrations significantly above those required to inhibit ACh active uptake. Kinetic analysis of the V-type ATPase revealed two Michaelis constants for MgATP of approximately 26 microM and 2.0 mM. The V-type ATPase and ACh active transport were inhibited by 84 and 160 pmol of bafilomycin A1/mg of vesicle protein, respectively, from which it is estimated that only one or two V-type ATPase proton pumps are present per synaptic vesicle. The presence of presumably contaminating Na+,K(+)-ATPase in the synaptic vesicle preparation is demonstrated.

Acetylcholine↗

PubMed1989

Microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of presynaptic nerve terminals.

Nerve endings of the posterior pituitary are densely populated by dense-core neurosecretory granules which are the storage sites for peptide neurohormones. In addition, they contain numerous clear microvesicles which are the same size as small synaptic vesicles of typical presynaptic nerve terminals. Several of the major proteins of small synaptic vesicles of presynaptic nerve terminals are present at high concentration in the posterior pituitary. We have now investigated the subcellular localization of such proteins. By immunogold electron microscopy carried out on bovine neurohypophysis we have found that three of these proteins, synapsin I, Protein III, and synaptophysin (protein p38) were concentrated on microvesicles but were not detectable in the membranes of neurosecretory granules. In addition, we have studied the distribution of the same proteins and of the synaptic vesicle protein p65 in subcellular fractions of bovine posterior pituitaries obtained by sucrose density centrifugation. We have found that the intrinsic membrane proteins synaptophysin and p65 had an identical distribution and were restricted to low density fractions of the gradient which contained numerous clear microvesicles with a size range the same as that of small synaptic vesicles. The peripheral membrane proteins synapsin I and Protein III exhibited a broader distribution extending into the denser part of the gradient. However, the amount of these proteins clearly declined in the fractions preceding the peak of neurosecretory granules. Our results suggest that microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of all other nerve terminals and argue against the hypothesis that such vesicles represent an endocytic byproduct of exocytosis of neurosecretory granules.

Animals↗

PubMed1988

ATP-dependent glutamate uptake into synaptic vesicles from cerebellar mutant mice.

The ATP-dependent glutamate uptake system in synaptic vesicles prepared from mouse cerebellum was characterized, and the levels of glutamate uptake were investigated in the cerebellar mutant mice, staggerer and weaver, whose main defect is the loss of cerebellar granule cells, and the nervous mutant, whose main defect is the loss of Purkinje cells. The ATP-dependent glutamate uptake is stimulated by low concentrations of chloride, is insensitive to aspartate, and is inhibited by agents known to dissipate the electrochemical proton gradient. These properties are similar to those of the glutamate uptake system observed in the highly purified synaptic vesicles prepared from bovine cortex. The ATP-dependent glutamate uptake system is reduced by 68% in the staggerer and 57-67% in the weaver mutant; these reductions parallel the substantial loss of granule cells in those mutants. In contrast, the cerebellar levels of glutamate uptake are not altered significantly in the nervous mutant, which has lost Purkinje cells, but not granule cells. In view of evidence that granule cells are glutamatergic neurons and Purkinje cells are GABAergic neurons, these observations support the notion that the ATP-dependent glutamate uptake system is present in synaptic vesicles of glutamatergic neurons.

Adenosine Triphosphate↗

PubMed1989

Topogenesis and sorting of synaptophysin: synthesis of a synaptic vesicle protein from a gene transfected into nonneuroendocrine cells.

Diverse nonneuroendocrine (non-NE) cells were forced to express synaptophysin (SY), the major and typical transmembrane glycoprotein of small (30-80 nm) neurotransmitter vesicles of NE cells, using microinjection of RNA synthesized in vitro from cDNA or transient and stable transfections with cDNA brought under SV40 promoter control. The glycoprotein synthesized in non-NE cells is indistinguishable from SY of NE cells and is integrated with similar, if not identical, orientation in the membranes of a specific, novel type of small cytoplasmic vesicle that structurally resembles synaptic vesicles and in which SY is the only major protein detected. A non-N-glycosylated form of SY generated by site-directed mutagenesis showed the same behavior and specific distribution in small vesicles. The results show that the information contained in this protein alone is sufficient to secure its sorting into a special type of vesicle in a heterotypic context, i.e., in the absence of other NE-specific components.

Animals↗

PubMed1988

Redistribution of synaptic vesicle antigens is correlated with the disappearance of a transient synaptic zone in the developing cerebral cortex.

To examine the distribution of synaptic vesicle antigens during development of the cerebral cortex, antibodies against synapsin I and p65 were used on sections of cat cerebral cortex between E40 and adulthood. In the adult, the layers of the cerebral cortex are immunoreactive for each of these antigens, while the white matter is free of staining. In contrast, the fetal and neonatal pattern of immunostaining is reversed: the cortical plate (future cortical layers) is devoid of immunoreactivity, while the marginal (future layer 1) and the intermediate zones (future white matter) are stained. Electron microscopic immunohistochemistry shows that immunolabeling is associated with presynaptic nerve terminals in the adult and during development. These observations suggest that during development the white matter is a transient synaptic neuropil and that a global redistribution of synapses takes place as the mature pattern of connections within the cerebral cortex emerges.

Aging↗

PubMed1996

Inhibition of glutamate uptake and proton pumping in synaptic vesicles by S-nitrosylation.

Nitric oxide (NO; including NO-., NO+, and NO-) was found to inhibit glutamate uptake by isolated synaptic vesicles of rat brain. This was observed when two unrelated NO donors, S-nitrosogluthathione and S-nitroso-N-acetylpenicillamine, were used. The primary target of NO is the H(+)-ATPase found in the synaptic vesicles, which leads to dissipation of the electrochemical proton gradient and inhibition of glutamate uptake. Oxyhemoglobin (12 microM) and, to a much lesser extent, methemoglobin protected the vacuolar H(+)-ATPase from inhibition. Inhibition of H+ pumping by NO was reversed by addition of 0.5 mM dithiothreitol. The results indicate that the vacuolar H(+)-ATPase from synaptic vesicles is inhibited by NO by a mechanism that involves S-nitrosylation of critical sulfhydryl groups in the enzyme. The interaction of NO with synaptic vesicles might be of importance for the understanding of the multiple effects of NO in neuro-transmission.

Animals↗

PubMed2001

Rab3a is involved in transport of synaptic vesicles to the active zone in mouse brain nerve terminals.

The rab family of GTP-binding proteins regulates membrane transport between intracellular compartments. The major rab protein in brain, rab3A, associates with synaptic vesicles. However, rab3A was shown to regulate the fusion probability of synaptic vesicles, rather than their transport and docking. We tested whether rab3A has a transport function by analyzing synaptic vesicle distribution and exocytosis in rab3A null-mutant mice. Rab3A deletion did not affect the number of vesicles and their distribution in resting nerve terminals. The secretion response upon a single depolarization was also unaffected. In normal mice, a depolarization pulse in the presence of Ca(2+) induces an accumulation of vesicles close to and docked at the active zone (recruitment). Rab3A deletion completely abolished this activity-dependent recruitment, without affecting the total number of vesicles. Concomitantly, the secretion response in the rab3A-deficient terminals recovered slowly and incompletely after exhaustive stimulation, and the replenishment of docked vesicles after exhaustive stimulation was also impaired in the absence of rab3A. These data indicate that rab3A has a function upstream of vesicle fusion in the activity-dependent transport of synaptic vesicles to and their docking at the active zone.

Animals↗

PubMed1989

Synapsin I, a neuron-specific phosphoprotein interacting with small synaptic vesicles and F-actin.

Synapsin I is a neuron-specific phosphoprotein which is a substrate for cAMP- and Ca2+/calmodulin-dependent protein kinases. It is specifically localized to the cytoplasmic side of small synaptic vesicles. The interaction of synapsin I with the synaptic vesicle membrane is complex in nature, since it is modulated by phosphorylation and involves binding of different domains of the molecule to phospholipid and protein components of synaptic vesicles. Synapsin I is also able to interact with actin filaments in a phosphorylation-dependent manner. Because of these properties, it has been hypothesized that synapsin I acts as a dynamic link between synaptic vesicles an the actin meshwork of the nerve terminal, thereby modulating the release of neurotransmitter.

Actins↗

PubMed1997

Mg2+/Ca(2+)-ATPase activity is not enriched in synaptic vesicles isolated from rat cerebral cortex.

Neuronal ATPases comprise a wide variety of enzymes which are not uniformly distributed in different membrane preparations. Since purified vesicle fractions have Mg2+/Ca(2+)-ATPase, the purpose of the present study was to know whether such enzyme activities have a preferential concentration in a synaptic vesicle fraction in order to be used as markers for these organelles. Resorting to a procedure developed in this Institute, we fractionated the rat cerebral cortex by differential centrifugation following osmotic shock of a crude mitochondrial fraction and separated a purified synaptic vesicle fraction over discontinuous sucrose gradients. Mg2+/Ca(2+)-ATPase activities and ultrastructural studies of isolated fractions were carried out. It was observed that similar specific activities for Mg2+/Ca(2+)-ATPases were found in all fractions studied which contain synaptic vesicles and/or membranes. Although the present results confirm the presence of Mg2+ and Ca(2+)-ATPase activities in synaptic vesicles preparations, they do not favor the contention that Mg2+/Ca(2+)-ATPase is a good marker for synaptic vesicles.

Animals↗

PubMed1988

Endogenous ligands for the quisqualate receptor: presence in rat brain cortex synaptic vesicles.

The presence in highly purified rat brain cortex synaptic vesicles of endogenous ligands for rat brain quisqualate receptors was investigated. The vesicles were extracted, and their contents fractionated by high voltage electrophoresis. Endogenous ligands were detected by a radioreceptor assay in which such ligands competed with 50 nM L-[3H]glutamate for binding to quisqualate receptors present in rat brain postsynaptic densities (PSDs). Binding of L-[3H]glutamate to N-methyl-D-aspartate (NMDA) receptors, also present in PSDs, was blocked by 100 microM NMDA. We found that the endogenous ligands present in brain cortex synaptic vesicles for quisqualate receptors, were glutamate and aspartate, in a molar ratio of about two to one. The quisqualate receptor had an affinity 130-fold higher for glutamate (Kd 0.3 microM) than for aspartate, and the latter amino acid also showed a marked negative cooperative for binding (Hill number 0.29, against 0.67 for glutamate). These findings suggest that glutamate is the natural transmitter that activates quisqualate receptors at some central excitatory synapses, and also that aspartate may be a classical transmitter, the receptor for which still remains to be shown.

Animals↗

PubMed2006

A single vesicular glutamate transporter is sufficient to fill a synaptic vesicle.

Quantal size is the postsynaptic response to the release of a single synaptic vesicle and is determined in part by the amount of transmitter within that vesicle. At glutamatergic synapses, the vesicular glutamate transporter (VGLUT) fills vesicles with glutamate. While elevated VGLUT expression increases quantal size, the minimum number of transporters required to fill a vesicle is unknown. In Drosophila DVGLUT mutants, reduced transporter levels lead to a dose-dependent reduction in the frequency of spontaneous quantal release with no change in quantal size. Quantal frequency is not limited by vesicle number or impaired exocytosis. This suggests that a single functional unit of transporter is both necessary and sufficient to fill a vesicle to completion and that vesicles without DVGLUT are empty. Consistent with the presence of empty vesicles, at dvglut mutant synapses synaptic vesicles are smaller, suggesting that vesicle filling and/or transporter level is an important determinant of vesicle size.

Animals↗

PubMed2001

Interaction of stoned and synaptotagmin in synaptic vesicle endocytosis.

The Drosophila dicistronic stoned locus encodes two distinctive presynaptic proteins, Stoned A (STNA) and Stoned B (STNB); STNA is a novel protein without homology to known synaptic proteins, and STNB contains a domain with homology to the endocytotic protein AP50. Both Stoned proteins colocalize precisely with endocytotic proteins including the AP2 complex and Dynamin in the "lattice network" characteristic of endocytotic domains in Drosophila presynaptic terminals. FM1-43 dye uptake studies in stoned mutants demonstrate a striking decrease in the size of the endo-exo-cycling synaptic vesicle pool and loss of spatial regulation of the vesicular recycling intermediates. Mutant synapses display a significant delay in vesicular membrane retrieval after depolarization and neurotransmitter release. These studies suggest that the Stoned proteins play a role in mediating synaptic vesicle endocytosis. We have documented previously a highly specific synaptic mislocalization and degradation of Synaptotagmin I in stoned mutants. Here we show that transgenic overexpression of Synaptotagmin I rescues stoned embryonic lethality and restores endocytotic recycling to normal levels. Furthermore, overexpression of Synaptotagmin I in otherwise wild-type animals results in increased synaptic dye uptake, indicating that Synaptotagmin I directly regulates the endo-exo-cycling synaptic vesicle pool size. In parallel with recent biochemical studies, this genetic analysis strongly suggests that Stoned proteins regulate the AP2-Synaptotagmin I interaction during synaptic vesicle endocytosis. We conclude that Stoned proteins control synaptic transmission strength by mediating the retrieval of Synaptotagmin I from the plasma membrane.

Adaptor Protein Complex 2↗

PubMed1980

Proton NMR detection of acetylcholine status in synaptic vesicles.

The storage granules of cholinergic nerve terminals, the synaptic vesicles, have a central role in the mechanism of cholinergic transmission, and direct evidence as to whether acetylcholine is stored in them in a free or partially immobilized form is therefore of interest. In some secretory systems it has become evident that low molecular weight secretion products are stored within the granules as complexes with proteins or proteoglycans. The application of high resolution NMR spectroscopy to isolated granules has recently elucidated the internal structure of secretory granules. We report here that proton NMR analysis of isolated cholinergic synaptic vesicles from Torpedo marmorata allows the identification of acetylcholine within the organelle. The spectrum shows that all the acetylcholine is dissolved in an essentially fluid phase within the core. Vesicular and uncompartmentalized acetylcholine can be distinguished from each other and in addition, any hydrolysis to choline and acetate can be monitored. The results open the possibility of studying dynamics of acetylcholine pools and their breakdown in cholinergic tissue by a direct and non-perturbing method.

Acetylcholine↗

PubMed1981

Glycoproteins from adult rat brain synaptic vesicles. Fractionation on four immobilized lectins.

Glycoproteins obtained from large amounts of highly purified synaptic vesicles isolated from adult rat brain were fractionated by sequential affinity chromatography in the presence of SDS on four different immobilized lectins: concanavalin A, Ulex europeus lectin, Ricinus sanguinis lectin and wheat germ agglutinin. 83% of the total protein-bound sugar of synaptic vesicles can be adsorbed on the lectins and separated from the bulk of carbohydrate free proteins. Nine fractions containing only glycoproteins and differing by their terminal sugars were separated by analysed for their carbohydrate composition and electrophoretic profiles. A considerable heterogeneity of the glycoprotein population was observed which cannot be explained solely by the microheterogeneity of the glycans of the synaptic vesicle glycoproteins.

Animals↗

PubMed1981

Saturable acetylcholine transport into purified cholinergic synaptic vesicles.

The uptake of [3H[acetylcholine ([3H]AcCho) into cholinergic synaptic vesicle ghosts purified from Torpedo electric organ was studied at concentration of [3H]AcCho ranging from 0.1 to 10 mM. The accumulated [3H]AcCho can be released either by hypoosmotic buffer or by low levels of the detergent Triton X-100. Kinetic analysis of the initial rate of [3H]AcCho uptake reveals temperature-dependent saturation kinetics which are best fitted by high-affinity (KTh approximately 0.3 mM) and low-affinity (KT) approximately 10 mM) vesicular [3H]AcCho transport systems. Several lines of evidence suggest that [3H]AcCho transport is mediated by vesicle-associated transport systems and not by a contaminant of other subcellular moieties such as the plasma membrane choline transport system. (i) The specific activity of the [3H]AcCho transport systems is higher in the purest vesicular fraction than in the less-pure fractions. (ii) Ghosts prepared from isolated synaptosomes manifest only low levels of low-affinity [3H]AcCho transport and no high-affinity [3H]AcCho transport. (iii) The vesicular AcCho transport systems lack some of the typical characteristics of synaptosomal choline transport, such as Na+ activation. (iv) The ratio of uptakes of [3H]AcCho and [3H]choline (10 microM) is about 5-fold higher in the pure vesicles than in isolated synaptosomal membranes. Addition of Mg2+-ATP decreases the rate of vesicular [3H]AcCho uptake by about 50%. The simultaneous addition of NaHCO3 and Mg2+-ATP results in activation of [3H]AcCho uptake to about 125% (relative to control), which is a 2.5-fold enhancement relative to the rate observed with Mg2+-ATP. The present findings demonstrate the presence of novel vesicle-associated AcCho transport systems. Their physiological role in the life cycle of the cholinergic synaptic vesicle and nerve terminal are discussed.

Acetylcholine↗

PubMed1977

Chlorpromazine induces population increase of synaptic vesicles in the rat hypothalamic ventromedial nucleus.

The population of synaptic vesicles per synaptic area in the hypothalamic ventromedial nucleus of rats treated with chlorpromazine (CP) for 3 days was compared with that of controls. In type I synapse, which contained 400-800 A small spherical vesicles (s-vesicles), together with 800-1200 A large spherical dense-cored vesicles (lc-vesicles), the population of s-vesicles increased 57.8% in the CP rats, In type II synapse, which contained only s-vesicles, the rate of increase among CP animals was 15.4%. Findings suggested that CP blocks the release of the vesicles from the presynaptic site or inhibits the transport function for biogenic amines through the presynaptic membrane, and type I synapse may be aminergic.

Animals↗