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PubMed1995

Synaptic vesicles and release of transmitters: new insights at the molecular level.

Neurotransmitter release from transmitter storage vesicles is a regulated signalling event that has properties in common with other secretory systems. Biochemical characterization of mammalian synaptic vesicle proteins has recently converged with studies of protein traffic in non-neuronal cells and the genetic dissection of the yeast secretory pathway to give us a considerable amount of new data. Many new synaptic vesicle proteins have been characterized together with plasma membrane proteins with which they interact, and it appears that many of the participating components may be part of a general machinery for secretion. The new results significantly improve our understanding of the molecular mechanisms governing transmitter release. This review discusses the recent progress in terms of synaptic vesicle components and the proposed mechanisms for exocytosis.

Animals↗

PubMed2005

Teaching resources. Calcium-triggered exocytosis and clathrin-mediated endocytosis of synaptic vesicles.

The animation illustrates the recruitment, calcium-triggered exocytosis, and clathrin-mediated endocytosis of synaptic vesicles. The animation shows not only the proteins involved in synaptic vesicle fusion and recycling, but also changes in the lipid composition that are important for targeting of proteins to sites of membrane internalization. The animation would be useful in both neurobiology courses, but also any cell biology course that explores clathrin-mediated endocytic and calcium-regulated secretion events.

Audiovisual Aids↗

PubMed2006

Antagonistic regulation of synaptic vesicle priming by Tomosyn and UNC-13.

Priming of synaptic vesicles (SVs) is essential for synaptic transmission. UNC-13 proteins are required for priming. Current models propose that UNC-13 stabilizes the open conformation of Syntaxin, in which the SNARE helix is available for interactions with Synaptobrevin and SNAP-25. Here we show that Tomosyn inhibits SV priming. Tomosyn contains a SNARE motif, which forms an inhibitory SNARE complex with Syntaxin and SNAP-25. Mutants lacking Tomosyn have increased synaptic transmission, an increased pool of primed vesicles, and increased abundance of UNC-13 at synapses. Behavioral, imaging, and electrophysiological studies suggest that SV priming was reconstituted in unc-13 mutants by expressing a constitutively open mutant Syntaxin, or by mutations eliminating Tomosyn. Thus, priming is modulated by the balance between Tomosyn and UNC-13, perhaps by regulating the availability of open-Syntaxin. Even when priming was restored, synaptic transmission remained defective in unc-13 mutants, suggesting that UNC-13 is also required for other aspects of secretion.

Animals↗

PubMed2006

Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release.

Central synapses exhibit spontaneous neurotransmitter release that is selectively regulated by cAMP-dependent protein kinase A (PKA). We now show that synaptic vesicles contain synaptotagmin-12, a synaptotagmin isoform that differs from classical synaptotagmins in that it does not bind Ca(2+). In synaptic vesicles, synaptotagmin-12 forms a complex with synaptotagmin-1 that prevents synaptotagmin-1 from interacting with SNARE complexes. We demonstrate that synaptotagmin-12 is phosphorylated by cAMP-dependent PKA on serine(97), and show that expression of synaptotagmin-12 in neurons increases spontaneous neurotransmitter release by approximately threefold, but has no effect on evoked release. Replacing serine(97) by alanine abolishes synaptotagmin-12 phosphorylation and blocks its effect on spontaneous release. Our data suggest that spontaneous synaptic-vesicle exocytosis is selectively modulated by a Ca(2+)-independent synaptotagmin isoform, synaptotagmin-12, which is controlled by cAMP-dependent phosphorylation.

Amino Acid Sequence↗

PubMed1986

Translocation of cytosolic acetylcholine into synaptic vesicles and demonstration of vesicular release.

The rate of translocation of newly synthesized acetylcholine (ACh) from the presynaptic cytosol of Torpedo electric organ nerve terminals into synaptic vesicles and the extent to which ACh release from these neurons is mediated by a vesicular mechanism were investigated. For this purpose the compound 2(4-phenylpiperidino)cyclohexanol (AH5183), which inhibits the active transport of ACh into isolated cholinergic synaptic vesicles, was employed. Preincubation of purified Torpedo nerve terminals (synaptosomes) with AH5183 does not affect the intraterminal synthesis of [3H]ACh but results in a marked inhibition (85%) of its Ca2+-dependent K+-evoked release. By contrast, the evoked release of the endogenous nonlabeled ACh is not affected by this compound. When AH5183 is added during radiolabeling, it causes a progressively smaller inhibition of [3H]ACh release which is completely abolished if the drug is added after the preparation has been labeled. These findings suggest that most of the newly synthesized synaptosomal [3H]ACh (85%) is released by a vesicular mechanism and that some [3H]ACh (15%) may be released by a different process. The translocation of cytosolic [3H]ACh into the synaptic vesicles was monitored by determining the time course of the loss of susceptibility of [3H]ACh release to AH5183. It was found not to be coupled kinetically to [3H]ACh synthesis and to lag behind it. The nature of the intraterminal processes underlying this lag is discussed.

Acetylcholine↗

PubMed2000

Association of L-glutamic acid decarboxylase to the 70-kDa heat shock protein as a potential anchoring mechanism to synaptic vesicles.

Recently we have reported that the membrane-associated form of the gamma-aminobutyric acid-synthesizing enzyme, l-glutamate decarboxylase (MGAD), is regulated by the vesicular proton gradient (Hsu, C. C., Thomas, C., Chen, W., Davis, K. M., Foos, T., Chen, J. L., Wu, E., Floor, E., Schloss, J. V., and Wu, J. Y. (1999) J. Biol. Chem. 274, 24366-24371). In this report, several lines of evidence are presented to indicate that l-glutamate decarboxylase (GAD) can become membrane-associated to synaptic vesicles first through complex formation with the heat shock protein 70 family, specifically heat shock cognate 70 (HSC70), followed by interaction with cysteine string protein (CSP), an integral protein of the synaptic vesicle. The first line of evidence comes from purification of MGAD in which HSC70, as identified from amino acid sequencing, co-purified with GAD. Second, in reconstitution studies, HSC70 was found to form complex with GAD(65) as shown by gel mobility shift in non-denaturing gradient gel electrophoresis. Third, in immunoprecipitation studies, again, HSC70 was co-immunoprecipitated with GAD by a GAD(65)-specific monoclonal antibody. Fourth, HSC70 and CSP were co-purified with GAD by specific anti-GAD immunoaffinity columns. Furthermore, studies here suggest that both GAD(65) and GAD(67) are associated with synaptic vesicles along with HSC70 and CSP. Based on these findings, a model is proposed to link anchorage of MGAD to synaptic vesicles in relation to its role in gamma-aminobutyric acid neurotransmission.

Animals↗

PubMed1997

Sodium-dependent increase in quantal secretion induced by brevetoxin-3 in Ca2+-free medium is associated with depletion of synaptic vesicles and swelling of motor nerve terminals in situ.

Brevetoxin-3 at nanomolar concentrations markedly enhanced spontaneous quantal transmitter release from neuromuscular junctions equilibrated in a Ca2+-free EGTA medium. After about 3 h, the sustained increase in miniature endplate potential frequency led to an exhaustion of transmitter release. This increase still occurred after loading the nerve terminals with the Ca2+ chelator bis-(aminophenoxy)ethanetetra-acetate or after pretreatment with various pharmacological agents known to prevent Ca2+ release from intracellular pools, but was completely prevented by the Na+ channel blocker tetrodotoxin. Brevetoxin-3 also increased miniature endplate potential frequency from junctions treated with botulinum type-A toxin, but to a smaller extent than at normal junctions. At normal junctions, brevetoxin-3 exposure for 2 h increased the three-dimensional projected area of living motor nerve terminals in situ by about 74% while at botulinum type-A poisoned junctions a similar toxin exposure caused only a 29% increase. Tetrodotoxin prevented such effects, indicating that they are related to both Na+ entry into the terminals and increased quantal transmitter release. Ultrastructural examination of nerve terminals from junctions exposed for 3 h to brevetoxin-3 revealed profound depletions of clear and large dense core synaptic vesicles and an increase in coated vesicles and axolemma infoldings. These results indicate that brevetoxin-3 impairs the recycling of clear synaptic vesicles and are consistent with our immunofluorescent observations showing that synaptophysin epitopes can be revealed without nerve terminal permeabilization. In contrast, no such changes were detected in nerve terminals poisoned with botulinum type-A toxin which, after 3 h exposure to brevetoxin-3, retained their synaptic vesicles and had a normal appearance. We conclude that tetrodotoxin-sensitive Na+ entry into motor nerve terminals induced by brevetoxin-3 triggers external Ca2+-independent asynchronous quantal transmitter release, blocks synaptic vesicle recycling and induces swelling of the terminals. We suggest that an excess of cytoplasmic Na+ per se can activate the asynchronous neurotransmitter release process.

Animals↗

PubMed1976

Interrelationships between Golgi, GERL and synaptic vesicles in the nerve cells of insect and gastropod ganglia.

In addition to demonstrating synaptic vesicles, staining with the zinc-iodide-osmium tetroxide (ZIO) method reveals the presence of positively reacting GERL membranes in association with the Golgi complex and lysosomes in the nerve cell bodies within ganglia from the locust Schistocerca gregaria and the gastropod molluscs, Limnaea stagnalis and Helix aspersa. A positive response to ZIO occurs in certain Golgi vesicles and saccules, in GERL (Golgi-endoplasmic-reticulum-lysosomes), in multivesicular bodies as well as residual bodies and in small vesicles and cisternae of axonal smooth endoplasmic reticllum (ER). The interrelationships between these organelles are considered in view of the similarity of the ZIO localization to phosphatase-rich sites in the neuronal perikarya and with respect to the possibility that components of the synaptic vesicles are formed in the Golgi region of the cell and migrate via the axonal smooth ER to the synaptic regions.

Animals↗

PubMed1982

31P-NMR analysis of synaptic vesicles. Status of ATP and internal pH.

The status of ATP in cholinergic synaptic vesicles from electric organ of Torpedo marmorata has been studied by 31P-NMR. In isolated vesicles, ATP was found to be the only major phosphate-containing constituent. The chemical shifts and the linewidths of the P gamma and P beta resonances are different from those of uncompartmented ATP added to the suspension. The spectrum of intact electric tissue contains two sets of ATP resonances; one of them belongs to vesicular ATP as identified by its linewidths and chemical shifts. Both spectral characteristics of vesicular ATP, linewidths and chemical shifts, could be explained by an exchange mechanism, which also occurs in model solutions containing acetylcholine, ATP and Mg2+ at a pH around 5.5. We therefore conclude that the cholinergic synaptic vesicles store ATP together with acetylcholine and Mg2+ essentially in free solution at an acidic pH. Since the chemical shift of the P gamma resonance of ATP is largely determined by factors other than pH, the concept of its use as an indicator for the internal pH has to be modified in the case of these organelles.

Acetylcholine↗

PubMed1992

Synaptic vesicle increase correlated to potentiation of transmission at the synapse of the cat superior cervical ganglion in vivo.

Changes in the pattern, number and size of synaptic vesicles during transmitter release were examined in the synapses of the cat superior cervical ganglion (SCG) in vivo in relation to alteration in the amplitude of postganglionic compound action potential (PGP). Stimulation of the preganglionic nerve fibers at 10 Hz caused an increase in the mean amplitude of PGP. It became augmented by approximately 30% compared to control 10-30 sec after starting the stimulation, and then gradually declined to reach a plateau after 4-6 min. This level, about 20% higher in value than control, was sustained until the end of 30 min of stimulation. The nerve terminal was divided into two areas to examine topographically the numerical changes in synaptic vesicles (SVs): zone I on the presynaptic membrane encircled with a diameter equivalent to the active zone length, and zone II occupying the remaining area outside zone I. The synaptic vesicle density in zone I (vesicle number/microns 2) was 96.9 +/- 4.8 (mean +/- S.E.M.) in the unstimulated control ganglia and 128.8 +/- 9.4 (mean +/- S.E.M.) in the ganglia stimulated for 10-30 sec, which was 30% higher in value than control. Then, it decreased slightly reaching a plateau, 20% higher in value than control. The diameter distribution of the SVs showed that their diameters in zone I (56.6 nm mean) were larger than those (51.6 nm mean) in zone II, and that prolonged stimulation induced smaller vesicles in both areas. The results showed that the increase in SV number in zone I correlated well with the elevation of PGP.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗

PubMed2001

Contact-dependent aggregation of functional Ca2+ channels, synaptic vesicles and postsynaptic receptors in active zones of a neuromuscular junction.

To examine whether Ca2+ channels aggregate in a contact-dependent manner, we characterized the distribution of synaptic vesicles and postsynaptic receptors, and compared it to the location of Ca2+ entry sites, in a Xenopus laevis nerve-muscle coculture preparation using a localized Ca2+ detection method. The majority (75%) of Ca2+ entry sites at spontaneously formed nerve-muscle contacts were associated with enhanced immunofluorescence to the synaptic vesicle protein, SV2. In contrast, only 11% of recorded sites without Ca2+ transients exhibited significant SV2 immunofluorescence. When comparing the spatial distribution of synaptic markers with that of Ca2+ entry sites, we found that the majority of Ca2+ entry sites (61%) were associated with both enhanced SV2 immunofluorescence and R-BTX fluorescence, thereby identifying putative neurotransmitter release sites where Ca2+ channels, synaptic vesicles and postsynaptic receptors are colocalized. Using polystyrene beads coated with a heparin binding protein known to mediate in vitro postsynaptic receptor clustering, we show that the location of Ca2+ domains was associated with enhanced SV2 immunofluorescence at neurite-to-bead contacts. We conclude that the localization of functional Ca2+ channels to putative active zones follows a contact-dependent signalling mechanism similar to that known to mediate vesicle aggregation and AChR clustering.

Animals↗

PubMed1996

Synaptic vesicle movements monitored by fluorescence recovery after photobleaching in nerve terminals stained with FM1-43.

We used the fluorescence recovery after photobleaching technique to monitor movements of synaptic vesicles in top views of living frog motor nerve terminals that had been prestained with the fluorescent dye FM1-43. In each experiment, a small portion of a single stained vesicle cluster was bleached with a laser and monitored subsequently for signs of recovery as neighboring, unbleached vesicles moved into the bleached region. In resting terminals, little or no recovery from photobleaching occurred. Repetitive nerve stimulation, which caused all fluorescent spots to grow dim as dye was released from exocytosing vesicles, did not promote recovery from photobleaching. Pretreatment with botulinum toxin (type A, C, or D) blocked exocytosis and destaining, but intense nerve stimulation still did not cause significant recovery in bleached regions. These results suggest that lateral movements of synaptic vesicles are restricted severely in both resting and stimulated nerve terminals. We tested for laser-induced photodamage in several ways. Bleached regions could be restained fully with FM1-43, and these restained regions could be destained fully by nerve stimulation. Partially bleached regions could be destained, although the rate of destaining was lower than normal. Brisk recovery from photobleaching occurred after treatment with okadaic acid, which disrupts synaptic vesicle clusters and causes vesicles to spread throughout the nerve terminal. These results suggest that vesicle translocation and recycling machinery was intact in photobleached regions.

Animals↗

PubMed1999

Role of synaptic vesicle proton gradient and protein phosphorylation on ATP-mediated activation of membrane-associated brain glutamate decarboxylase.

Previously, we have shown that the soluble form of brain glutamic acid decarboxylase (GAD) is inhibited by ATP through protein phosphorylation and is activated by calcineurin-mediated protein dephosphorylation (Bao, J., Cheung, W. Y., and Wu, J. Y. (1995) J. Biol. Chem. 270, 6464-6467). Here we report that the membrane-associated form of GAD (MGAD) is greatly activated by ATP, whereas adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP), a non-hydrolyzable ATP analog, has no effect on MGAD activity. ATP activation of MGAD is abolished by conditions that disrupt the proton gradient of synaptic vesicles, e.g. the presence of vesicular proton pump inhibitor, bafilomycin A1, the protonophore carbonyl cyanide m-chorophenylhydrazone or the ionophore gramicidin, indicating that the synaptic vesicle proton gradient is essential in ATP activation of MGAD. Furthermore, direct incorporation of (32)P from [gamma-(32)P]ATP into MGAD has been demonstrated. In addition, MGAD (presumably GAD65, since it is recognized by specific monoclonal antibody, GAD6, as well as specific anti-GAD65) has been reported to be associated with synaptic vesicles. Based on these results, a model linking gamma-aminobutyric acid (GABA) synthesis by MGAD to GABA packaging into synaptic vesicles by proton gradient-mediated GABA transport is presented. Activation of MGAD by phosphorylation appears to be mediated by a vesicular protein kinase that is controlled by the vesicular proton gradient.

Adenosine Triphosphate↗

PubMed1986

Uncoupling of cholinergic synaptic vesicles by the presynaptic toxin beta-bungarotoxin.

The effect of the presynaptic neurotoxin beta-bungarotoxin (beta-BuTx) on the acetylcholine (ACh) storage system of synaptic vesicles isolated from the electric organ of Torpedo californica was studied. The toxin can totally inhibit active transport of [3H]ACh by the vesicles in a Ca2+-, time-, and concentration-dependent manner. Correlated with these effects is a 50-60% stimulation of the vesicle proton-pumping ATPase activity. The beta-BuTx-mediated transport inhibition and ATPase stimulation are antagonized by delipidated bovine serum albumin, not reversed by excess EGTA, and not mimicked by other cationic proteins or soybean or pancreatic trypsin inhibitors. The behavior is consistent with phospholipase A2 (PLA2)-dependent damage to the vesicle membrane caused by beta-BuTx, which results in uncoupling of the ATPase and ACh transporter systems. The nonneurotoxic Naja naja venom PLA2 causes similar effects, except that it is slightly more potent on a molar basis. About 100-fold more beta-BuTx is required to effect lysis of synaptic vesicles than to uncouple them. ATP is a strong inhibitor of beta-BuTx- but not of N. naja PLA2-mediated uncoupling. The observations suggest that a component of beta-BuTx toxicity in the cholinergic terminal might involve attack on synaptic vesicles or vesicle-like structures and that a nucleotide-like factor might modulate the toxicity.

Acetylcholine↗

PubMedDate not supplied

Substance P increases the polymorphism of the synaptic vesicles in the temporal isocortex cultured in vitro.

Rat's temporal cortex was cultured in vitro in a feeding medium enriched with substance P. Normal controls were used for correlation. The ultrastructural study of the explant exposed to substance P revealed a marked increase of the polymorphism of the synaptic vesicles in the presynaptic terminals. Large numbers of the synaptic vesicles were large, of a mean diameter of 142.5 A, dark-looking like the catecholaminergic vesicles. Coated vesicles were seen in the presynaptic terminals as well as in the postsynaptic ones. Large elongated vesicles and S-like vesicles were seen in the axonic terminals mainly in axosomatic synapses and in axodendritic synapses with large dendritic profiles. In most of the synapses, a remarkable thickness of the postsynaptic membrane was noticed. In a large number of postsynaptic terminals, the cisternae of the smooth endoplasmic reticulum were dilated and intermixed with dilated vesicles and fragmented microtubules. Some of the presynaptic terminals demonstrated marked thickness of the presynaptic membrane. Glycogen granules were frequently seen in the postsynaptic terminals. In conclusion, substance P administered in vitro, increases the polymorphism of the synaptic vesicles in the presynaptic terminals of neurons derived from the temporal isocortex.

Animals↗

PubMed2002

The synaptic vesicle cycle: exocytosis and endocytosis in Drosophila and C. elegans.

Advances in the study of Drosophila melanogaster and Caenorhabditis elegans have provided key insights into the processes of neurotransmission and neuromodulation. Work in the past year has revealed that Unc-13 and Rab3a-interacting molecule regulate the conformational state of syntaxin to prime synaptic vesicle fusion. Analyses of synaptotagmin support its role as a putative calcium sensor triggering vesicular fusion and highlight the possible role of SNARE complex oligomerization in the fusion mechanism. Characterization of endophilin mutants demonstrates that kiss-and-run endocytosis is a major component of synaptic vesicle recycling. In neuromodulation, dcaps mutants provide the first genetic insight into possible roles of the CAPS protein in mediating dense core vesicle fusion and modulating synaptic vesicle fusion.

Animals↗

PubMed1984

Differences in the osmotic fragility of recycling and reserve synaptic vesicles from the cholinergic electromotor nerve terminals of Torpedo and their possible significance for vesicle recycling.

In this study we demonstrate differences in the osmotic fragility of two metabolically and physically heterogeneous synaptic vesicle populations from stimulated electromotor nerve terminals. When synaptic vesicles isolated on sucrose density gradients are submitted to solutions of decreasing osmolarity 50% of VP2-type vesicles lysed at (mean + S.E. (number of experiments] 332 +/- 14 (4) mosM and 50% of VP1-type vesicles lysed at 573 +/- 8 (3) mosM. These results indicate that recycling vesicles are more resistant to hypo-osmotic lysis and they are consistent with our earlier conclusion that changes in water content on recycling are secondary to changes in the content of the osmotically active small-molecular-mass constituents acetylcholine and ATP.

Acetylcholine↗

PubMed1987

G-proteins in Torpedo marmorata electric organ. Differential distribution in pre- and post-synaptic membranes and synaptic vesicles.

The nature of the G-proteins present in the pre- and post-synaptic plasma membranes and in the synaptic vesicles of cholinergic nerve terminals purified from the Torpedo electric organ was investigated. In pre- and post-synaptic plasma membranes, Bordetella pertussis toxin, known to catalyze the ADP-ribosylation of the alpha-subunit of several G-proteins, labels two substrates at 41 and 39 kDa. The 39 kDa subunit detected by ADP-ribosylation in the synaptic plasma membrane fractions was immunologically similar to the Go alpha-subunit purified from calf brain. In contrast to bovine chromaffin cell granules, no G-protein could be detected in Torpedo synaptic vesicles either by ADP-ribosylation or by immunoblotting.

Adrenal Medulla↗