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PubMed1991

Cytoplasmic architecture of the axon terminal: filamentous strands specifically associated with synaptic vesicles.

Cytoplasmic architecture of axon terminals in rat central nervous tissue was examined by quick-freeze deep-etch method to determine how synaptic vesicles and their associated cytoplasmic environment are organized in the terminal and to know how these structures participate in the mechanism for neurotransmitter release. The axoplasm is divisible into two domains: one occupied by mitochondria in the middle of the terminal, called the mitochondrial domain, the other situated in the periphery and exclusively filled with spherical synaptic vesicles, 50-60 nm in diameter, the synaptic vesicle domain. The most characteristic feature of the mitochondrial domain was the appearance of many microtubules connected with mitochondria by filamentous strands. Large vesicles, 80-100 nm in diameter, were preferentially associated with the mitochondrial domain, and linked with microtubules wherever they appeared. The cytoplasmic matrix of the synaptic vesicle domain showed a more fibrillar texture than that of the mitochondrial domain because of the distribution of filamentous strands associated with synaptic vesicles. These strands were significantly thicker and longer (mean 11.7 nm thick and 42.7 nm long) than those linking membrane-bound organelles to microtubules (mean 8.3 nm thick and 23.0 nm long), and connected vesicles to one another or to the plasma membrane, making a complicated network around the vesicles. Further, both strands were significantly different in dimension from actin filaments (mean 9.9 nm thick and 73.5 nm long) showing 5-nm axial periodicity. These strands, especially synaptic vesicle-associated ones including their network, were readily broken down in the most part by detergent treatment or chemical fixation, indicating that they are very delicate in nature. Granular materials, which are spherical and vary in size (6-20 nm in diameter), are also more conspicuous in the synaptic vesicle domain than in the mitochondrial domain. More fibrillar and granular cytoplasmic structure of the synaptic vesicle domain may be crucial for synaptic vesicles to perform an essential role in releasing the transmitter.

Animals↗
PubMed2006

The synapsin domain E accelerates the exoendocytotic cycle of synaptic vesicles in cerebellar Purkinje cells.

Synapsins are synaptic-vesicle-associated phosphoproteins implicated in the regulation of neurotransmitter release and excitability of neuronal networks. Mutation of synapsin genes in mouse and human causes epilepsy. To understand the role of the highly conserved synapsin domain E in the dynamics of release from mammalian inhibitory neurons, we generated mice that selectively overexpress the most conserved part of this domain in cerebellar Purkinje cells. At Purkinje-cell-nuclear-neuron synapses, transgenic mice were more resistant to depression induced by short or prolonged high-frequency stimulations. The increased synaptic performance was accompanied by accelerated release kinetics and shorter synaptic delay. Despite a marked decrease in the total number of synaptic vesicles, vesicles at the active zone were preserved or slightly increased. The data indicate that synapsin domain E increases synaptic efficiency by accelerating both the kinetics of exocytosis and the rate of synaptic vesicle cycling and decreasing depression at the inhibitory Purkinje-cell-nuclear-neuron synapse. These effects may increase the sensitivity of postsynaptic neurons to inhibition and thereby contribute to the inhibitory control of network activity.

Amino Acid Sequence↗
PubMed1994

Synaptic targeting of rabphilin-3A, a synaptic vesicle Ca2+/phospholipid-binding protein, depends on rab3A/3C.

rab3A, a low molecular weight GTP-binding protein of synaptic vesicles with a putative function in synaptic vesicle docking, interacts in a GTP-dependent manner with rabphilin-3A, a peripheral membrane protein that binds Ca2+ and phospholipids. We now show that rabphilin-3A is an evolutionarily conserved synaptic vesicle protein that is attached to synaptic vesicle membranes via its N terminus and exhibits a heterogeneous distribution among synapses. In rab3A-deficient mice, rabphilin-3A is decreased in synapses belonging to neurons that primarily express rab3A and accumulates in the perikarya of these neurons. In contrast, neurons expressing significant levels of rab3C still contain normal levels of rabphilin-3A in a synaptic pattern, and rabphilin-3A binds rab3C in vitro. These results suggest that analogous to the membrane recruitment of raf by ras, rab3A and rab3C may function in recruiting rabphilin-3A to the synaptic vesicle membrane in a GTP-dependent manner.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Botulinum neurotoxin A blocks synaptic vesicle exocytosis but not endocytosis at the nerve terminal.

The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K(+) depolarization, in the presence of Ca(2+), triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A-blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca(2+) is required for synaptic vesicle membrane retrieval.

Animals↗
PubMed1993

Expression of synaptotagmin in Drosophila reveals transport and localization of synaptic vesicles to the synapse.

Synaptotagmin is a synaptic vesicle-specific integral membrane protein that has been suggested to play a key role in synaptic vesicle docking and fusion. By monitoring Synaptotagmin's cellular and subcellular distribution during development, it is possible to study synaptic vesicle localization and transport, and synapse formation. We have initiated the study of Synaptotagmin's expression during Drosophila neurogenesis in order to follow synaptic vesicle movement prior to and during synapse formation, as well as to localize synaptic sites in Drosophila. In situ hybridizations to whole-mount embryos show that synaptotagmin (syt) message is present in the cell bodies of all peripheral nervous system neurons and many, if not all, central nervous system neurons during neurite outgrowth and synapse formation, and in mature neurons. Immunocytochemical staining with antisera specific to Synaptotagmin indicates that the protein is present at all stages of the Drosophila life cycle following germ band retraction. In embryos, Synaptotagmin is only transiently localized to the cell body of neurons and is transported rapidly along axons during axonogenesis. After synapse formation, Synaptotagmin accumulates in a punctate pattern at all identifiable synaptic contact sites, suggesting a general role for Synaptotagmin in synapse function. In embryos and larvae, the most intense staining is found along two broad longitudinal tracts on the dorsal side of the ventral nerve cord and the brain, and at neuromuscular junctions in the periphery. In the adult head, Synaptotagmin localizes the discrete regions of the neurophil where synapses are predicted to occur. These data indicate that synaptic vesicles are present in axons before synapse formation, and become restricted to synaptic contact sites after synapses are formed. Since a similar expression pattern of Synaptotagmin has been reported in mammals, we propose that the function of Synaptotagmin and the mechanisms governing localization of the synaptic vesicle before and after synapse formation are conserved in invertebrate and vertebrate species. The ability to mark synapses in Drosophila should facilitate the study of synapse formation and function, providing a new tool to dissect the molecular mechanisms underlying these processes.

Animals↗
PubMed2006

Fusion of synaptic vesicles and plasma membrane in the presence of synaptosomal soluble proteins.

Fusion between synaptic vesicles and plasma membranes isolated from rat brain synaptosomes is regarded as a model of neurosecretion. The main aim of current study is to investigate whether the synaptosomal soluble proteins are essential members of Ca(2+)-triggered fusion examined in this system. Fusion experiments were performed using fluorescent dye octadecylrhodamine B, which was incorporated into synaptic vesicle membranes at self-quenching concentration. The fusion of synaptic vesicles, containing marker octadecylrhodamine B, with plasma membranes was detected by dequenching of the probe fluorescence. Membrane fusion was not found in Ca(2+)-supplemented buffer solution, but was initiated by the addition of the synaptosomal soluble proteins. When soluble proteins were treated with trypsin, they lost completely the fusion activity. These experiments confirmed that soluble proteins of synaptosomes are sensitive to Ca(2+) signal and essential for membrane fusion. The experiments, in which members of fusion process were treated with monoclonal antibodies raised against synaptotagmin and synaptobrevin, have shown that antibodies only partially inhibited fusion of synaptic vesicles and plasma membranes in vitro. These results indicate that other additional component(s), which may or may not be related to synaptobrevin or synaptotagmin, mediate this process. It can be assumed that fusion of synaptic vesicles with plasma membranes in vitro depends upon the complex interaction of a large number of protein factors.

Animals↗
PubMed1981

Lipids of synaptic vesicles: relevance to the mechanism of membrane fusion.

Synaptic vesicles from the electric organ of the marine ray Narcine brasiliensis, purified to at least 90% homogeneity, were analyzed for the lipid and fatty acid content of their membranes. The major lipids (mol %) were phosphatidylcholine (32.3%), phosphatidylethanolamine (20.5%), phosphatidylserine (6.1%), sphingomyelin (3.0%), and cholesterol (33.3%), a composition which did not differ greatly from that of the parent electric organ. While the number of double bonds per fatty acid molecule was similar for both synaptic vesicle and whole electric organ phospholipids, the vesicles were highly enriched in docosahexenoic acid (22:6). Reaction with the amine labeling reagents isethionylacetimidate and trinitrobenzenesulfonic acid indicated that 40% of the phosphatidylserine and 60% of the phosphatidylethanolamine are present on the external (cytoplasmic) surface of the synaptic vesicle. These data on a natural fusing membrane have relevance to models of membrane fusion, which have been based largely on studies of in vitro fusion using synthetic membranes.

Animals↗
PubMed1991

Synapsin I-mediated interaction of brain spectrin with synaptic vesicles.

We have established a new binding assay in which 125I-labeled synaptic vesicles are incubated with brain spectrin covalently immobilized on cellulosic membranes in a microfiltration apparatus. We obtained saturable, high affinity, salt- (optimum at 50-70 mM NaCl) and pH- (optimum at pH 7.5-7.8) dependent binding. Nonlinear regression analysis of the binding isotherm indicated one site binding with a Kd = 59 micrograms/ml and a maximal binding capacity = 1.9 micrograms vesicle protein per microgram spectrin. The fact that the binding of spectrin was via synapsin was demonstrated in three ways. (a) Binding of synaptic vesicles to immobilized spectrin was eliminated by prior extraction with 1 M KCl. When the peripheral membrane proteins in the 1 M KCl extract were separated by SDS-PAGE, transferred to nitrocellulose paper and incubated with 125I-brain spectrin, 96% of the total radioactivity was associated with five polypeptides of 80, 75, 69, 64, and 40 kD. All five polypeptides reacted with an anti-synapsin I polyclonal antibody, and the 80- and 75-kD polypeptides comigrated with authentic synapsin Ia and synapsin Ib. The 69- and 64-kD polypeptides are either proteolytic fragments of synapsin I or represent synapsin IIa and synapsin IIb. (b) Pure synapsin I was capable of competitively inhibiting the binding of radioiodinated synaptic vesicles to immobilized brain spectrin with a Kl = 46 nM. (c) Fab fragments of anti-synapsin I were capable of inhibiting the binding of radioiodinated synaptic vesicles to immobilized brain spectrin. These three observations clearly establish that synapsin I is a primary receptor for brain spectrin on the cytoplasmic surface of the synaptic vesicle membrane.

Animals↗
PubMed2006

Synaptic vesicle recycling adapts to chronic changes in activity.

Synaptic vesicle recycling is essential for maintaining neurotransmission during rhythmic activity. To test whether the demands imposed by ambient activity influences synaptic vesicle trafficking, we compared the kinetics of synaptic depression in hippocampal versus neocortical cultures, which have high and low levels of intrinsic activity, respectively. In response to moderate 10 Hz stimulation, hippocampal synapses depressed less compared with neocortical synapses, although they reused vesicles more slowly. Therefore, during stimulation, hippocampal synapses used more vesicles from the reserve pool, whereas neocortical synapses relied on vesicle reuse. In hippocampal cultures, chronic block of network activity increased synaptic depression by decreasing the rate of vesicle mobilization, with little effect on the rate of vesicle reuse. In contrast, in neocortical cultures, an increase in the normally low network activity reduced synaptic depression by robustly increasing vesicle reuse with no effect on vesicle mobilization. These results suggest that synaptic vesicle trafficking and the resulting synaptic dynamics adapt to meet the changing demands on neurotransmitter release. Furthermore, during these functional modifications, synapses use alternate strategies to adjust to changes in activity.

Adaptation, Physiological↗
PubMed1984

Delta pH-dependent and -independent uptake of [3H]dopamine by synaptic vesicles isolated from rat brain.

The dopamine (DA)-translocating mechanism of synaptic vesicles isolated from rat brain has been studied in the presence of an artificially imposed delta pH on the vesicle membranes (acidic inside with respect to the external medium) without the aid of ATP-Mg2+. Under the experimental conditions, [3H]DA uptake by the synaptic vesicles was driven by two different, i.e. delta pH-dependent and -independent processes. Both processes appeared to be carrier-mediated based on the inhibition by NEM (N-ethylmaleimide), an -SH reagent, and by nomifensine, a DA uptake blocker at nerve terminals. The DA carrier of the vesicles was similar to that of the nerve terminal plasma membrane with respect to their susceptibility to nomifensine. The delta pH-dependent uptake was transient and most of the incorporated DA was easily lost from the vesicles. On the other hand, the delta pH-independent uptake increased with time and the amine was retained in the vesicles. The initial rate of the delta pH-independent uptake was lower than that of the delta pH-dependent one but their extents were comparable with each other. These results indicate that rat brain synaptic vesicles have a DA uptake system requiring no ATP hydrolysis. A preparation of synaptic vesicles used here exhibited an inwardly directed proton translocation in the presence of ATP-Mg2+ when monitored by following changes in the fluorescence of ANS (8-anilino-1-naphthalene sulfonate). However, the time course of the delta pH-generation was not influenced by the addition of 1 mM DA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed2004

Normal biogenesis and cycling of empty synaptic vesicles in dopamine neurons of vesicular monoamine transporter 2 knockout mice.

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.

Animals↗
PubMed2003

Role of Munc18-1 in synaptic vesicle and large dense-core vesicle secretion.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex formation between a vesicle and the target membrane is a central aspect of probably all vesicle fusion reactions. The sec1/munc18 (SM) protein family is also involved in membrane trafficking and fusion events. However, in contrast with the consensus on SNARE protein function, analysis of SM proteins in different systems has produced different ideas about their exact role, their site of action and their relationship to SNARE proteins. Deletion of the SM protein involved in secretory vesicle release in mice, Munc18-1, results in a complete block of exocytosis. Manipulation of Munc18-1 protein levels in neurons and adrenal chromaffin cells argues for a positive role of this protein in vesicle secretion, as overexpression results in an increase in vesicle secretion. A decrease in Munc18-1 protein levels, on the other hand, leads to a decrease in vesicle secretion.

Animals↗
PubMed2006

Synaptic vesicle reuse and its implications.

Presynaptic nerve terminals are exquisite vesicle trafficking machines. Neurotransmission is sustained by constant recycling of a handful of vesicles. Therefore, the rate and the pathway of vesicle trafficking can critically determine synaptic efficacy during activity. However, it is yet unclear whether synaptic vesicle recycling becomes rate limiting on a rapid time scale during physiologically relevant forms of activity in the brain. Several forms of synaptic plasticity arise from persistent alterations in the dynamics of vesicle trafficking in presynaptic terminals. What makes presynaptic forms of plasticity particularly interesting is that they not only increase or decrease the amplitude of synaptic responses but also cause frequency-dependent changes in neurotransmission. In this manner, plasticity can alter the information coding in neural circuits beyond simple scaling of synaptic responses. However, studying the synaptic vesicle cycle beyond exocytosis and endocytosis has been difficult. In the past decade, several methods have been developed to infer vesicles' trajectory during their cycle in the synapse. Nevertheless, several questions remain. A better understanding of the role of synaptic vesicle trafficking in neurotransmission will require novel approaches that either combine existing methods or the development of new methods to trace vesicles during their cycle. Recent evidence suggests that various presynaptic proteins involved in the synaptic function and homeostasis are either mutated or altered in their expression in several neurological and psychiatric disorders. Therefore, elucidation of the mechanisms that underlie the synaptic vesicle cycle may reveal novel therapeutic targets for brain disorders.

Animals↗
PubMed1986

Molecular characterization of synaptophysin, a major calcium-binding protein of the synaptic vesicle membrane.

Synaptophysin, a mol. wt 38 000 glycopolypeptide of the synaptic vesicle membrane, was solubilized using Triton X-100 and purified by immunoaffinity or ion-exchange chromatography. From gel permeation and sucrose-density centrifugation in H2O/D2O, a Stokes radius of 7.3 nm, a partial specific volume of 0.830 and a total mol. wt of 119 000 were calculated for the native protein. Cross-linking of synaptic vesicles with glutaraldehyde, dimethylsuberimidate, or Cu2+ -o-phenantroline, resulted in the formation of a mol. wt 76 kd dimer of synaptophysin. Crosslinking of the purified protein in addition produced tri- and tetrameric adducts of the polypeptide. Native synaptophysin thus is a homooligomeric protein. Synaptophysin is N-glycosylated, since cultivation of the rat phaeochromocytoma cell line PC12 in the presence of tunicamycin reduced its mol. wt by about 6 kd. Upon transfer to nitrocellulose and incubation with 45Ca2+, synaptophysin behaved as one of the major calcium-binding proteins of the synaptic vesicle membrane. Pronase treatment of intact synaptic vesicles abolished this 45Ca2+ binding indicating that the Ca2+ binding site of synaptophysin must reside on a cytoplasmic domain of the transmembrane polypeptide. Based on these data, we propose that synaptophysin may play an important role in Ca2+-dependent neurotransmitter release.

Animals↗
PubMed1978

Reversible depletion of synaptic vesicles induced by application of high external potassium to the frog neuromuscular junction.

1. Reversible depletion of synaptic vesicles from frog cutaneous pectoris neuromuscular junctions was studied by application of a Ringer solution containing 115 mM-K propionate.2. During the release of transmitter, the synaptic vesicle membrane is added to the axolemmal membrane. Under the conditions of high K(+)-induced release, the synaptic vesicle membrane accumulates as folds formed in the region of the axolemmal membrane between the active zones. In depleted terminals, large vesicular structures appear and the evidence shows that some of them (possibly all) are formed as axolemmal infoldings. During formation of such infoldings the active zones remain fixed in position with respect to the post-junctional membrane.3. During recovery in normal Ringer solution, which followed 30 min depolarization in high K(+) Ringer solution, spontaneous m.e.p.p.s were detected as early as 9 min after the start of the recovery period and the average time for their reappearance was 17 min.4. At the end of a 20 min recovery period which followed K(+) depolarization, small accumulations of synaptic vesicles were again found within the terminal close to the active zones. At this time coated vesicles and coated pits were seen associated with the prejunctional axolemma and its infoldings. It appears that synaptic vesicles are re-formed directly from these coated vesicles.5. After 60 min recovery from K(+) depolarization, at which time stimulation of the motor nerve induced a muscle twitch, the structure of the terminals closely resembled that of control preparations.6. The entire synaptic vesicle recycling process can take place in the absence of the neurone soma.

Animals↗
PubMed1991

GABA and glycine in synaptic vesicles: storage and transport characteristics.

gamma-Aminobutyric acid (GABA) and glycine are major inhibitory neurotransmitters that are released from nerve terminals by exocytosis via synaptic vesicles. Here we report that synaptic vesicles immunoisolated from rat cerebral cortex contain high amounts of GABA in addition to glutamate. Synaptic vesicles from the rat medulla oblongata also contain glycine and exhibit a higher GABA and a lower glutamate concentration than cortical vesicles. No other amino acids were detected. In addition, the uptake activities of synaptic vesicles for GABA and glycine were compared. Both were very similar with respect to substrate affinity and specificity, bioenergetic properties, and regional distribution. We conclude that GABA, glycine, and glutamate are the only major amino acid neurotransmitters stored in synaptic vesicles and that GABA and glycine are transported by similar, if not identical, transporters.

Animals↗
PubMed1998

Calcium dependence of synaptic vesicle recycling before and after synaptogenesis.

Using an immunocytochemical assay to monitor synaptic vesicle exocytosis/endocytosis independently of neurotransmitter release, we have investigated some aspects of vesicle recycling in hippocampal neurons at different developmental stages. A calcium- and depolarization-dependent exocytotic/endocytotic recycling of synaptic vesicles was found to take place in neurons already before the formation of synaptic contacts. The analysis of synaptic vesicle recycling at different calcium concentrations revealed the presence of two release components: the first one activated by low calcium concentrations and sustaining vesicle recycling before synaptogenesis, and a second one activated by high calcium concentrations, which is specifically turned on after the establishment of synaptic contacts. These data suggest that formation of synapses correlates with the activation of a putative low-affinity calcium sensor, which allows synaptic vesicle exocytosis to be triggered and turned off over extremely short time scales, in response to large increases in the level of intracellular calcium.

Animals↗