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PubMed1990

Artificially imposed electrical potentials drive L-glutamate uptake into synaptic vesicles of bovine cerebral cortex.

L-Glutamate is a major excitatory neurotransmitter in the central nervous system. MgATP-dependent glutamate uptake and H(+)-pumping ATPase activity were reported in highly purified synaptic vesicles [Naito & Ueda (1983) J. Biol. Chem. 258, 696-699; Shioi, Naito & Ueda (1989) Biochem. J. 258, 499-504], and it is hypothesized that an electrochemical H+ gradient across the vesicle membrane, the so-called protonmotive force, elicits the neurotransmitter uptake. An inside-positive diffusion potential across the vesicle membrane was established with valinomycin plus Rb+. This artificial electrical potential promoted the uptake of glutamate, but not aspartate, in the synaptic vesicles prepared from bovine cerebral cortex. The uptake was inhibited by the protonmotive-force dissipators carbonyl cyanide p-trifluoro-methoxyphenylhydrazone or nigericin, and was enhanced by concomitant imposition of a pH jump (alkalinization) in the external medium. Subcellular and subvesicular distributions showed the uptake system to be predominantly associated with small synaptic vesicles. The results support the hypothesis that glutamate uptake into synaptic vesicles is coupled with a H+ efflux down the electrochemical potential gradient, which is generated by H(+)-pumping ATPase.

Adenosine Triphosphate↗

PubMed1986

Demonstration of a receptor in Torpedo synaptic vesicles for the acetylcholine storage blocker L-trans-2-(4-phenyl[3,4-3H]-piperidino) cyclohexanol.

Transport and storage of acetylcholine by purified Torpedo electric organ synaptic vesicles is blocked by the drug L-trans-2-(4-phenylpiperidino)cyclohexanol (AH-5183). This study sought evidence of a specific receptor for the drug. Highly tritiated L-trans-2-(4-phenyl [3,4-3H] piperidino)-cyclohexanol (L-[3H] AH5183) was synthesized. An excess of nonradioactive L-isomer competed with L-[3H]AH5183 for binding to purified Torpedo synaptic vesicles whereas nonradioactive D-isomer did so poorly. Dissociation of bound L-[3H]AH5183 was first order with a rate constant at 23 degrees C of 0.23 +/- 0.03 min-1, and association was too rapid to study. At equilibrium the amount of L-[3H]AH5183 bound at saturation varied in different vesicle preparations, but in one typical preparation specific binding of 181 +/- 15 pmol L-[3H]AH5183 per mg of synaptic vesicle protein was observed with a dissociation constant of 34 +/- 6 nM. Neither acetylcholine nor choline compete effectively with L-[3H]AH5183 for binding. The evidence suggests that about 3.7 +/- 0.3 enantioselective receptors for L-[3H]AH5183 are typically present in each cholinergic synaptic vesicle, and the L-AH5183 binding site does not recognize acetylcholine.

Acetylcholine↗

PubMed1986

Selectivity and regulation in the phospholipase A2-mediated attack on cholinergic synaptic vesicles by beta-bungarotoxin.

The total fatty acid composition of purified Torpedo californica electric organ synaptic vesicles was determined by GLC analysis of methyl esters. Limit amounts of fatty acids released by high concentrations of either beta-bungarotoxin (beta-BuTx) or Naja naja venom phospholipase A2 (PLA2) acting in deoxycholate are reported. The time and enzyme concentration dependence for beta-BuTx- and PLA2-induced release of fatty acids from intact synaptic vesicles indicate that PLA2 is 100- to 1,000-fold more active. The Ca2+ dependence for beta-BuTx-induced release of fatty acids also was determined. ATP inhibits beta-BuTx- but not PLA2-induced release of fatty acids from vesicles in a manner that can not be ascribed only to chelation of the required Ca2+. ATP, other nucleotides, and adenosine have complex effects on beta-BuTx-induced release of fatty acids from egg yolk phosphatidylcholine dispersed in deoxycholate. The results suggest that beta-BuTx-mediated hydrolysis of the cholinergic synaptic vesicle membrane is approximately 10- to 100-fold more effective at causing uncoupling of vesicles than is PLA2 and that the enzymatic activity of beta-BuTx is subject to regulation by nucleotide-like factors.

Acetylcholine↗

PubMed1980

Changes in the number, size, and shape of synaptic vesicles in an experimental, projected cortical epileptic focus in the rat.

A projected epileptic focus was induced in the brains of experimental rats by applying penicillin G sodium to the contralateral hemisphere. Synapses of type I (after Gray) in the second cortical layer of the mirror area were analyzed morphometrically in the electron microscope 30 min after application of the epileptogenic drug. The number of agranular synaptic vesicles was counted per 0.01 micron2 over the active zone of the presynaptic membrane in an area 0.1 micron wide adjacent to the active zone and in an area of the same width 0.1 micron distant from the active zone. A statistically significant increase in the number of synaptic vesicles was found in the experimental animals as compared with the controls. The number of synaptic vesicles per 0.01 micron2 area was evaluated along the inactive presynaptic membrane, both in the area adjacent to the active zone of the presynaptic membrane and in other areas at successively greater distances from the active zone. The counts in some of these latter areas were statistically significantly higher in the experimental animals. The size and shape of synaptic vesicles were evaluated in three zones, each 0.1 micron wide, parallel to the membrane of the synaptic cleft. Zone I was directly adjacent to the membrane of the synaptic cleft; zone II was 0.1 micron distant; and zone III 0.1 micron distant from the membrane. The results show that the synaptic vesicles of both the experimental and control animals are significantly smaller in zone I than in the more distant zones. This trend is more marked in the controls than in the experimental animals. Histograms of vesical areas of zones II and III in the controls point to two populations of vesicles. The volumes of vesicles are approximately 10% greater in zone I of experimental animals than in the controls. The elongation of the vesicles in the three zones does not differ statistically either in controls or in experimental animals. Experimental animals, however, have statistically more rounded vesicles than the controls. Assuming that the synpatic vesicles contain the transmitters, our results might contribute to a better understanding of the increased excitability of the epileptic focus area.

Animals↗

PubMed2002

Synaptic vesicle protein synaptoporin is differently expressed by subpopulations of mouse hippocampal neurons.

In the hippocampus, the synaptic vesicle protein synaptoporin (SPO) has been reported to be exclusively enriched in the granule cell axons, the mossy fibers. In this study, we show that in adult rats and mice SPO immunoreactivity (IR) is also detectable in strata oriens, radiatum, and lacunosum-moleculare of CA1-CA3, as well as perisomatically in the hippocampus proper and fascia dentata. In situ hybridization confirmed that SPO mRNA was present in granule cells and CA3 pyramidal cells but not in CA1 pyramidal cells. Importantly, cells scattered throughout the hippocampal layers resembling the distribution of interneurons were found to synthesize high amounts of SPO mRNA, too. Thus, these findings indicate that SPO expression in the hippocampus was underestimated until now. Moreover, double-labeling immunohistochemistry and confocal microscopy revealed selective colocalization of SPO and glutamate decarboxylase (GAD 65), a marker for gamma-aminobutyric acid (GABA)ergic terminals. To identify SPO expressing interneurons, in situ hybridization was combined with immunocytochemistry against parvalbumin (PV), calbindin (CB), calretinin (CR), cholecystokinin (CCK), and vasoactive intestinal polypeptide (VIP). We found that SPO transcripts were differentially expressed by various interneuron subpopulations in the hippocampus of C57Bl/6 mice (PV 44.2%, CB 46.3%, CR 19.3%, CCK 38.6%, VIP 59.9%). Immunoelectron microscopy for SPO labeled synaptic vesicle profiles in distinct symmetric and asymmetric synapses. In conclusion, our data demonstrate that hippocampal principal cells and interneurons display a variety of synaptic vesicles that are likely to contribute to the functional characteristics of their output synapses.

Animals↗

PubMed1996

Alzheimer amyloid protein precursor is localized in nerve terminal preparations to Rab5-containing vesicular organelles distinct from those implicated in the synaptic vesicle pathway.

In order to localize amyloid protein precursor (APP) in nerve terminals, we have immunoisolated vesicular organelles from nerve terminal preparations using antibodies to Rab5 and synaptophysin. These immunoisolates were then analyzed by electron microscopy and by immunoblotting. The synaptophysin immunoisolates represented a nearly homogeneous population of small synaptic vesicles, with less than 10% contamination by other organelles, and very little APP. In contrast, Rab5 immunoisolates contained, in addition to small synaptic vesicles, substantial numbers of large uni- and bilamellar vesicles and high levels of APP. Thus, it appears that nerve terminal APP is contained predominantly in large vesicular organelles, distinct from synaptic vesicles and from the synaptic vesicle recycling pathway.

Alzheimer Disease↗

PubMed2003

Endophilin is required for synaptic vesicle endocytosis by localizing synaptojanin.

Endophilin is a membrane-associated protein required for endocytosis of synaptic vesicles. Two models have been proposed for endophilin: that it alters lipid composition in order to shape membranes during endocytosis, or that it binds the polyphosphoinositide phosphatase synaptojanin and recruits this phosphatase to membranes. In this study, we demonstrate that the unc-57 gene encodes the Caenorhabditis elegans ortholog of endophilin A. We demonstrate that endophilin is required in C. elegans for synaptic vesicle recycling. Furthermore, the defects observed in endophilin mutants closely resemble those observed in synaptojanin mutants. The electrophysiological phenotype of endophilin and synaptojanin double mutants are virtually identical to the single mutants, demonstrating that endophilin and synaptojanin function in the same pathway. Finally, endophilin is required to stabilize expression of synaptojanin at the synapse. These data suggest that endophilin is an adaptor protein required to localize and stabilize synaptojanin at membranes during synaptic vesicle recycling.

Acyltransferases↗

PubMed1998

Ba2+ does not support synaptic vesicle retrieval in rat cerebrocortical synaptosomes.

To investigate whether any specific requirement for extracellular Ca2+ exists in the control synaptic vesicle retrieval, we examined the ability of the divalent cation Ba2+ to substitute for Ca2+ in both vesicle exocytosis and endocytosis. Ba2+ stimulated glutamate release from rat cerebrocortical synaptosomes. Ba2+-evoked release was inhibited by bafilomycin A1, indicating release was via exocytosis of synaptic vesicles. However, Ba2+ did not stimulate vesicle retrieval, monitored by a FM2-10-based retrieval assay. Therefore synaptic vesicle retrieval in central nerve terminals has a specific requirement for extracellular Ca2+ and the Ca2+ receptor for retrieval has a different cation specificity to the Ca2+ receptor for exocytosis.

4-Aminopyridine↗

PubMed2001

Synaptic vesicle acidification and exocytosis studied with acridine orange fluorescence in rat brain synaptosomes.

The acidification of synaptic vesicles (SV) in rat brain synaptosomes was studied using acridine orange (AO) as a fluorescent probe. In synaptosomal suspensions the AO fluorescence was partially quenched, indicating the presence of an acidic compartment. In permeabilized synaptosomes, the quenching was augmented by MgATP and was sensitive to concanamycin A, a specific inhibitor of the V-type H(+)-ATPase known to be present in synaptic vesicles. Some ATP-dependent acidification was also observed without permeabilization, suggesting that a fraction of synaptosomes (ca. 15%) was unsealed, irrespective of the method used to prepare the synaptosomes (sucrose or Ficoll density gradient, sedimentation or flotation). Depolarization of synaptosomes with 30 mM KCl resulted in an immediate, albeit small, rise in AO fluorescence that was prevented by the removal of Ca(2+) or by substituting NaCl for KCl. This response is consistent with depolarization-evoked release of the acidic contents of an exocytosis-competent pool of synaptic vesicles, representing ca. 5% of the total. No further AO release subsequent to the immediate phase was observed in depolarized synaptosomes, which indicates an extremely rapid reacidification. The results demonstrate that AO fluorescence is suitable for monitoring SV acidification within synaptosomes, and may be used to derive an independent estimate of the relative size of the immediately releasable SV pool. In addition, the use of AO might be advantageous for the assessment of synaptosomal integrity by comparing the ATP-dependent acidification in intact and permeabilized synaptosomes.

Acids↗

PubMed1993

Synaptotagmin is endogenously phosphorylated by Ca2+/calmodulin protein kinase II in synaptic vesicles.

The cytoplasmic domain of synaptotagmin (a synaptic vesicle-specific protein) has a high degree of homology with the Ca(2+)-phospholipid binding domain of protein kinase C. The Ca(2+)-phospholipid binding activity of synaptotagmin has been implicated in the docking and fusion of synaptic vesicles with the presynaptic membrane during Ca(2+)-induced exocytosis. The protein sequence contains potential phosphorylation sites for various protein kinases which could modulate its binding activity. At present there is no clear evidence that the protein is endogenously phosphorylated in intact vesicles. Here it is reported that phospho-synaptotagmin was immunoprecipitated from endogenously phosphorylated synaptic vesicles. The conditions used indicate that synaptotagmin, as synapsin I, is phosphorylated by Ca2+/calmodulin-dependent protein kinase II.

Animals↗

PubMed1981

Morphology of synaptic vesicles in axo-dendritic and axo-somatic collateral terminals of two feline spinocervical tract cells stained intracellularly with horseradish peroxidase.

Synaptic vesicles in initial collateral terminals of two feline spinocervical tract cells have been investigated after intracellular staining with horseradish peroxidase. A total of 5325 vesicles in 52 axodendritic and 26 axo-somatic terminals were analysed after aldehyde-osmium fixation. The greatest length and longest perpendicular width of each vesicle were measured, and the ratio and geometric mean of the diameters (gm-diameters) were calculated. The vesicles were divided into classes with round, elliptical and flat organelles. The variations in vesicle length, gm-diameter, and diameter ratio were statistically analysed by means of one- and two-way analyses of variance and t-tests. The diameter ratio, the length of round vesicles, and the gm-diameters of round and elliptical vesicles differed significantly between the cells. The length and gm-diameter of the elliptical vesicles differed significantly between the groups of axo-dendritic and axo-somatic terminals of each cell. Round vesicles were significantly longer in the axo-somatic than in the axo-dendritic terminals of each cell, and the gm-diameter showed this relation for both round and elliptical vesicles. It is assumed that a one-size vesicle could not account for all the measured profiles. The variability of synaptic vesicles within and between the functionally similar cells emphasize the difficulties in using the morphology of synaptic vesicles for a discrimination of axon terminals of different origin.

Animals↗

PubMed1999

Two distinct domains in hsc70 are essential for the interaction with the synaptic vesicle cysteine string protein.

The cysteine string protein (csp) is a synaptic vesicle protein found to be essential for normal neurotransmitter release. The precise function of csp in the synaptic vesicle cycle is still enigmatic. By interacting with the heat-shock cognate hsc70, a cochaperone-chaperone complex with an unknown function is formed. We report here that the formation of this complex is mediated by two distinct domains in hsc70. The ATPase domain and the substrate-binding domain must cooperate to create a binding site for csp. The C-terminal domain of hsc70 seems to function as a regulator for the formation of the cochaperone-chaperone complex. We also show that the interaction of csp with heat-shock proteins is confined to hsc70 and hsp70. Other heat-shock proteins, like hsp60 and hsp90, do not interact with csp.

Animals↗

PubMed2000

Accumulation of zinc in degenerating hippocampal neurons of ZnT3-null mice after seizures: evidence against synaptic vesicle origin.

In several brain injury models, zinc accumulates in degenerating neuronal somata. Suggesting that such zinc accumulation may play a causal role in neurodegeneration, zinc chelation attenuates neuronal death. Because histochemically reactive zinc is present in and released from synaptic vesicles of glutamatergic neurons in the forebrain, it was proposed that zinc translocation from presynaptic terminals to postsynaptic neurons may be the mechanism of toxic zinc accumulation. To test this hypothesis, kainate seizure-induced neuronal death was examined in zinc transporter 3 gene (ZnT3)-null mice, a strain that completely lacks histochemically reactive zinc in synaptic vesicles. Intraperitoneal injection of kainate induced seizures to a similar degree in wild type and ZnT3-null mice. Staining of hippocampal sections with a zinc-specific fluorescent dye, N-(6-methoxy-8-quinolyl)-p-carboxybenzoylsulfonamide, revealed that zinc accumulated in degenerating CA1 and CA3 neurons in both groups, indicating that zinc originated from sources other than synaptic vesicles. Injection of CaEDTA into the cerebral ventricle almost completely blocked zinc accumulation in ZnT3-null mice, suggesting that increases in extracellular zinc concentrations may be a critical event for zinc accumulation. Arguing against the possibility that zinc accumulation results from nonspecific breakdown of zinc-containing proteins, injection of kainate into the cerebellum did not induce zinc accumulation in degenerating granule neurons. Taken together, these results support the existing idea that zinc is released into extracellular space and then enters neurons to exert a cytotoxic effect. However, the origin of zinc is not likely to be synaptic vesicles, because zinc accumulation robustly occurs in ZnT3-null mice lacking synaptic vesicle zinc.

Animals↗

PubMed1989

Cholinergic synaptic vesicles contain a V-type and a P-type ATPase.

Fifty to eighty-five percent of the ATPase activity in different preparations of cholinergic synaptic vesicles isolated from Torpedo electric organ was half-inhibited by 7 microM vanadate. This activity is due to a recently purified phosphointermediate, or P-type, ATPase, Acetylcholine (ACh) active transport by the vesicles was stimulated about 35% by vanadate, demonstrating that the P-type enzyme is not the proton pump responsible for ACh active transport. Nearly all of the vesicle ATPase activity was inhibited by N-ethylmaleimide. The P-type ATPase could be protected from N-ethylmaleimide inactivation by vanadate, and subsequently reactivated by complexation of vanadate with deferoxamine. The inactivation-protection pattern suggests the presence of a vanadate-insensitive, N-ethylmaleimide-sensitive ATPase consistent with a vacuolar, or V-type, activity expected to drive ACh active transport. ACh active transport was half-inhibited by 5 microM N-ethylmaleimide, even in the presence of vanadate. The presence of a V-type ATPase was confirmed by Western blots using antisera raised against three separate subunits of chromaffin granule vacuolar ATPase I. Both ATPase activities, the P-type polypeptides, and the 38-kilodalton polypeptide of the V-type ATPase precisely copurify with the synaptic vesicles. Solubilization of synaptic vesicles in octaethyleneglycol dodecyl ether detergent results in several-fold stimulation of the P-type activity and inactivation of the V-type activity, thus explaining why the V-type activity was not detected previously during purification of the P-type ATPase. It is concluded that cholinergic vesicles contain a P-type ATPase of unknown function and a V-type ATPase which is the proton pump.

Adenosine Triphosphatases↗

PubMedDate not supplied

Quantification of synaptic vesicles in hippocampus of aging rats and initial studies of possible relations to neurophysiology.

Synaptic vesicle populations were quantified in Schaffer-commissural synapses which terminate on CA1 pyramidal cell apical dendrites, in aging and young-mature rats. Vesicles were found to be reduced with age, and this effect was most pronounced in the oldest animals (e.g., 28 months) within the aged group. Numerical density of synaptic vesicles in aged rat hippocampus was reduced whether expressed as vesicles per terminal, vesicles per mu 2 of terminal, or vesicles per mu2 of terminal corrected for shrinkage or swelling as assessed by mitochondrial cross-sectional diameters. Counted synaptic terminal areas were not significantly different in the aged animals, although a trend toward reduced terminal size with aging was seen. The latter observation apparently rules out increased terminal swelling in aged rat synapses as a basis for reduced vesicle density. In some rats, neurophysiological studies were concomitantly performed. A significant correlation was found between the amplitude of the monosynaptic population spike after 20 min of 4 Hz stimulation of the Schaffer-commissural fibers and synaptic vesicle populations in terminals of these stimulated pathways. However, because of a low n this result must be viewed as preliminary.

Aging↗

PubMed1985

Mg-ATPase and Torpedo cholinergic synaptic vesicles.

The reported presence of Mg-ATPase activity in cholinergic synaptic vesicles from the electric organ of Torpedo marmorata was reinvestigated in view of possible contamination of vesicles by other subcellular fractions. After dilution in concentrated sucrose, the vesicular fraction isolated on a sedimentation sucrose gradient was purified further on a flotation density gradient. It appears that this treatment allows separation of the vesicles according to their content. The two vesicular content markers, acetylcholine and ATP, are recovered as sharp coincident peaks at a density close to 0.48 M sucrose. Empty vesicles are identified in denser regions by the protein pattern on gel electrophoresis which is identical to the pattern obtained for filled vesicles. Refractionation of vesicles depleted of their acetylcholine content by valinomycin leads to an extreme picture, with a massive shift of the vesicles toward denser regions. We have then shown that a ouabain-insensitive Mg-ATPase is indeed associated with the vesicle membrane, but the activity is fully apparent only when vesicles are permeabilized either as the result of the fractionation procedure or after detergent treatment. The relative insensitivity of the Mg-ATPase associated with the synaptic vesicles to oligomycin, N,N'-dicyclohexylcarbodiimide, and azide indicates that this enzyme differs from the classic F1F0 mitochondrial enzyme. The most striking finding is the sensitivity to vanadate of the vesicular Mg-ATPase, which suggests the involvement of a phosphorylated intermediate. On the basis of both the difference in inhibitor sensitivity between untreated and detergent-treated vesicles and of the pronase experiments, the possibility that the enzyme has an inward orientation is discussed.

Adenosine Triphosphatases↗

PubMed2004

Expression of the iron transporter ferroportin in synaptic vesicles and the blood-brain barrier.

Iron homeostasis in the mammalian brain is an important and poorly understood subject. Transferrin-bound iron enters the endothelial cells of the blood-brain barrier from the systemic circulation, and iron subsequently dissociates from transferrin to enter brain parenchyma by an unknown mechanism. In recent years, several iron transporters, including the iron importer DMT1 (Ireg1, MTP, DCT1) and the iron exporter ferroportin (SLC11A3, Ireg, MTP1) have been cloned and characterized. To better understand brain iron homeostasis, we have characterized the distribution of ferroportin, the presumed intestinal iron exporter, and have evaluated its potential role in regulation of iron homeostasis in the central nervous system. We discovered using in situ hybridization and immunohistochemistry that ferroportin is expressed in the endothelial cells of the blood-brain barrier, in neurons, oligodendrocytes, astrocytes, and the choroid plexus and ependymal cells. In addition, we discovered using techniques of immunoelectron microscopy and biochemical purification of synaptic vesicles that ferroportin is associated with synaptic vesicles. In the blood-brain barrier, it is likely that ferroportin serves as a molecular transporter of iron on the abluminal membrane of polarized endothelial cells. The role of ferroportin in synaptic vesicles is unknown, but its presence at that site may prove to be of great importance in neuronal iron toxicity. The widespread representation of ferroportin at sites such as the blood-brain barrier and synaptic vesicles raises the possibility that trafficking of elemental iron may be instrumental in the distribution of iron in the central nervous system.

Animals↗

PubMed2002

Glycolysis and glutamate accumulation into synaptic vesicles. Role of glyceraldehyde phosphate dehydrogenase and 3-phosphoglycerate kinase.

Glucose is the major source of brain energy and is essential for maintaining normal brain and neuronal function. Hypoglycemia causes impaired synaptic transmission. This occurs even before significant reduction in global cellular ATP concentration, and relationships among glycolysis, ATP supply, and synaptic transmission are not well understood. We demonstrate that the glycolytic enzymes glyceraldehyde phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (3-PGK) are enriched in synaptic vesicles, forming a functional complex, and that synaptic vesicles are capable of accumulating the excitatory neurotransmitter glutamate by harnessing ATP produced by vesicle-bound GAPDH/3-PGK at the expense of their substrates. The GAPDH inhibitor iodoacetate suppressed GAPDH/3-PGK-dependent, but not exogenous ATP-dependent, [(3)H]glutamate uptake into isolated synaptic vesicles. It also decreased vesicular [(3)H]glutamate content in the nerve ending preparation synaptosome; this decrease was reflected in reduction of depolarization-induced [(3)H]glutamate release. In contrast, oligomycin, a mitochondrial ATP synthase inhibitor, had minimal effect on any of these parameters. ADP at concentrations above 0.1 mm inhibited vesicular glutamate and dissipated membrane potential. This suggests that the coupled GAPDH/3-PGK system, which converts ADP to ATP, ensures maximal glutamate accumulation into presynaptic vesicles. Together, these observations provide insight into the essential nature of glycolysis in sustaining normal synaptic transmission.

Adenosine Diphosphate↗