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PubMed1990

Recycled synaptic vesicles contain vesicle but not plasma membrane marker, newly synthesized acetylcholine, and a sample of extracellular medium.

To monitor the fate of the synaptic vesicle membrane compartment, synaptic vesicles were isolated under varying experimental conditions from blocks of perfused Torpedo electric organ. In accordance with previous results, after low-frequency stimulation (0.1 Hz, 1,800 pulses) of perfused blocks of electric organ, a population of vesicles (VP2 type) can be separated by density gradient centrifugation and chromatography on porous glass beads that is denser and smaller than resting vesicles (VP1 type). By simultaneous application of fluorescein isothiocyanate-dextran as extracellular volume marker and [3H]acetate as precursor of vesicular acetylcholine, and by identifying the vesicular membrane compartment with an antibody against the synaptic vesicle transmembrane glycoprotein SV2, we can show that the membrane compartment of part of the synaptic vesicles becomes recycled during the stimulation period. It then contains both newly synthesized acetylcholine and a sample of extracellular medium. Recycled vesicles have not incorporated the presynaptic plasma membrane marker acetylcholinesterase. Cisternae or vacuoles are presumably not involved in vesicle recycling. After a subsequent period of recovery (18 h), all vesicular membrane compartments behave like VP1 vesicles on subcellular fractionation and still retain both volume markers. Our results imply that on low-frequency stimulation, synaptic vesicles are directly recycled, equilibrating their luminal contents with the extracellular medium and retaining their membrane identity and capability to accumulate acetylcholine.

Acetates↗

PubMed1998

Synaptic vesicles retain their identity through the endocytic cycle.

After fusion of synaptic vesicles with presynaptic membrane and secretion of the contents of the vesicles into the synaptic cleft (a process known as exocytosis), the vesicular membrane is retrieved by endocytosis (internalization) for re-use. Several issues regarding endocytosis at central synapses are unresolved, including the location of membrane retrieval (relative to the active zone, where exocytosis occurs), the time course of various endocytic steps, and the recycling path taken by newly endocytosed membranes. The classical model of synaptic-vesicle recycling, proposed by analogy to other cellular endocytic pathways, involves retrieval of the membrane, fusion of the membrane with endosome-like compartments and, finally, budding of new synaptic vesicles from endosomes, although the endosomal station may not be obligatory. Here we test the classical model by using the fluorescent membrane dye FM1-43 with quantitative fluorescence microscopy. We find that the amount of dye per vesicle taken up by endocytosis equals the amount of dye a vesicle releases on exocytosis; therefore, we conclude that the internalized vesicles do not, as the classical picture suggests, communicate with intermediate endosome-like compartments during the recycling process.

Action Potentials↗

PubMed2002

Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton.

Actin is an abundant component of nerve terminals that has been implicated at multiple steps of the synaptic vesicle cycle, including reversible anchoring, exocytosis, and recycling of synaptic vesicles. In the present study we used the lamprey reticulospinal synapse to examine the role of actin at the site of synaptic vesicle recycling, the endocytic zone. Compounds interfering with actin function, including phalloidin, the catalytic subunit of Clostridium botulinum C2 toxin, and N-ethylmaleimide-treated myosin S1 fragments were microinjected into the axon. In unstimulated, phalloidin-injected axons actin filaments formed a thin cytomatrix adjacent to the plasma membrane around the synaptic vesicle cluster. The filaments proliferated after stimulation and extended toward the vesicle cluster. Synaptic vesicles were tethered along the filaments. Injection of N-ethylmaleimide-treated myosin S1 fragments caused accumulation of aggregates of synaptic vesicles between the endocytic zone and the vesicle cluster, suggesting that vesicle transport was inhibited. Phalloidin, as well as C2 toxin, also caused changes in the structure of clathrin-coated pits in stimulated synapses. Our data provide evidence for a critical role of actin in recycling of synaptic vesicles, which seems to involve functions both in endocytosis and in the transport of recycled vesicles to the synaptic vesicle cluster.

Actin Cytoskeleton↗

PubMed1996

Synaptotagmin, a synaptic vesicle protein, is present in human cerebrospinal fluid: a new biochemical marker for synaptic pathology in Alzheimer disease?

Using a novel approach, including affinity chromatography, reversed-phase chromatography, and chemiluminescence immunoblotting, we have for the first time been able to demonstrate one of the small synaptic vesicle proteins, synaptotagmin I, in cerebrospinal fluid (CSF). Two other small synaptic vesicle proteins, rab3a and synaptophysin, were not detectable. The approximate molecular weight of CSF-synaptotagmin was 65 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Further characterization of CSF synaptotagmin by high-performance capillary electrophoresis (HPCE) showed a single peak. These findings support that the whole synaptotagmin molecule is present in CSF, without significant proteolytic degradation. After high-speed centrifugation of CSF, synaptotagmin was exclusively found in the supernatant, suggesting that synaptotagmin is present in CSF as a free protein, and not as a constituent of synaptic vesicles. In a preliminary study, we found a marked reduction of CSF synaptotagmin in patients with early onset Alzheimer disease (EAD) as compared with age-matched healthy individuals. To elucidate the biological relevance of this finding, we also quantified synaptotagmin in brain tissue. A marked reduction in synaptotagmin was found both in the hippocampus and frontal cortex of EAD, suggesting that a decrease in synaptotagmin in the brain is followed by a concomitant decrease in the CSF. Analysis of CSF synaptotagmin might provide a tool to study synaptic function and pathology in the human brain.

Aged↗

PubMed1986

Acetylcholine, ATP, and proteoglycan are common to synaptic vesicles isolated from the electric organs of electric eel and electric catfish as well as from rat diaphragm.

Cholinergic synaptic vesicles were isolated from the electric organs of the electric eel (Electrophorus electricus) and the electric catfish (Malapterurus electricus) as well as from the diaphragm of the rat by density gradient centrifugation followed by column chromatography on Sephacryl-1000. This was verified by both biochemical and electron microscopic criteria. Differences in size between synaptic vesicles from the various tissue sources were reflected by their elution pattern from the Sephacryl column. Specific activities of acetylcholine (ACh; in nmol/mg of protein) of chromatography-purified vesicle fractions were 36 (electric eel), 2 (electric catfish), and 1 (rat diaphragm). Synaptic vesicles from all three sources contained ATP in addition to ACh (molar ratios of ACh/ATP, 9-12) as well as binding activity for an antibody raised against Torpedo cholinergic synaptic vesicle proteoglycan. Synaptic vesicles from rat diaphragm contained binding activity for the monoclonal antibody asv 48 raised against a rat brain 65-kilodalton synaptic vesicle protein. Antibody asv 48 binding was absent from electric eel and electric catfish synaptic vesicles. These antibody binding results, which were obtained by a dot blot assay on isolated vesicles, directly correspond to the immunocytochemical results demonstrating fluorescein isothiocyanate staining in the respective nerve terminals. Our results imply that ACh, ATP, and proteoglycan are common molecular constituents of motor nerve terminal-derived synaptic vesicles from Torpedo to rat. In addition to ACh, both ATP and proteoglycan may play a specific role in the process of cholinergic signal transmission.

Acetylcholine↗

PubMed1993

Relative properties and localizations of synaptic vesicle protein isoforms: the case of the synaptophysins.

Synaptophysins are abundant synaptic vesicle proteins present in two forms: synaptophysin, also referred to as synaptophysin I (abbreviated syp I), and synaptoporin, also referred to as synaptophysin II (abbreviated syp II). In the present study, the properties and localizations of syp I and syp II were investigated to shed light on their relative functions. Our results reveal that syp II, similar to syp I, is an abundant, N-glycosylated membrane protein that is part of a heteromultimeric complex in synaptic vesicle membranes. Cross-linking studies indicate that syp II is linked to a low-molecular-weight protein in this complex as has been observed before for syp I. Furthermore, after transfection into CHO cells, syp II, similar to syp I, is targeted to the receptor-mediated endocytosis pathway. Immunocytochemistry of rat brain sections reveals that syp II expression is highly heterogeneous, with high concentrations of syp II only in selected neuronal populations, whereas syp I is more homogeneously expressed in most nerve terminals. In general, nerve terminals expressing syp II also express syp I. In addition to high levels of syp II observed in selected neurons, a rostrocaudal gradient of syp II expression was observed in the cerebellar cortex. Immunoelectron microscopy confirmed that syp II is localized to synaptic vesicles. Immunoprecipitations of synaptic vesicles from rat brain with antibodies to syp I demonstrated that syp II is colocalized with syp I on the same vesicles. However, after detergent solubilization, no coimmunoprecipitations of the two proteins were observed, suggesting that they are not complexed with each other although they are on the same vesicles. Together our results demonstrate that syp I and syp II have similar properties and are present on the same synaptic vesicles but do not coassemble. The presence of the two proteins in the same nerve terminal suggests that they have similar but nonidentical functions and that the relative abundance of the two proteins may contribute to the functional heterogeneity of nerve terminals.

Animals↗

PubMed2003

Synapsin I-associated phosphatidylinositol 3-kinase mediates synaptic vesicle delivery to the readily releasable pool.

Maintaining synaptic transmission requires replenishment of docked synaptic vesicles within the readily releasable pool (RRP) from synaptic vesicle clusters in the synapsin-bound reserve pool. We show that synapsin forms a complex with phosphatidylinositol 3-kinase (PI 3-kinase) in intact nerve terminals and that synapsin-associated kinase activity increases on depolarization. Disruption of either PI 3-kinase activity or its interaction with synapsin inhibited replenishment of the RRP, but did not affect exocytosis from the RRP. Thus we conclude that a synapsin-associated PI 3-kinase activity plays a role in synaptic vesicle delivery to the RRP. This also suggests that PI 3-kinase contributes to the maintenance of synaptic transmission during periods of high activity, indicating a possible role in synaptic plasticity.

Androstadienes↗

PubMed1990

Synapsin II. Mapping of a domain in the NH2-terminal region which binds to small synaptic vesicles.

The synapsins are a family of neuron-specific phosphoproteins that selectively bind to small synaptic vesicles in the presynaptic nerve terminal. Using the cDNA encoding rat synapsin IIb, we employed an Escherichia coli expression system to synthesize a variety of fusion proteins containing a truncated protein A linked to different portions of the NH2-terminal region of synapsin II. The recombinant proteins were purified by IgG-Sepharose chromatography and tested in vitro for their ability to bind to purified synaptic vesicles. These experiments identified a region between amino acids 43 and 121 of the amino-terminal portion of synapsin II which binds to synaptic vesicles. Mild trypsinization of synaptic vesicles reduces binding of recombinant proteins to synaptic vesicles, suggesting that the interaction between synapsin II and the vesicles is in part mediated by a synaptic vesicle protein. The 42 NH2-terminal amino acids of synapsin II are not necessary for binding to synaptic vesicles, although this domain contains the phosphorylation site for cAMP-dependent protein kinase.

Animals↗

PubMed1996

The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin.

Strong evidence implicates clathrin-coated vesicles and endosome-like vacuoles in the reformation of synaptic vesicles after exocytosis, and it is generally assumed that these vacuoles represent a traffic station downstream from clathrin-coated vesicles. To gain insight into the mechanisms of synaptic vesicle budding from endosome-like intermediates, lysed nerve terminals and nerve terminal membrane subfractions were examined by EM after incubations with GTP gamma S. Numerous clathrin-coated budding intermediates that were positive for AP2 and AP180 immunoreactivity and often collared by a dynamin ring were seen. These were present not only on the plasma membrane (Takei, K., P.S. McPherson, S.L.Schmid, and P. De Camilli. 1995. Nature (Lond.). 374:186-190), but also on internal vacuoles. The lumen of these vacuoles retained extracellular tracers and was therefore functionally segregated from the extracellular medium, although narrow connections between their membranes and the plasmalemma were sometimes visible by serial sectioning. Similar observations were made in intact cultured hippocampal neurons exposed to high K+ stimulation. Coated vesicle buds were generally in the same size range of synaptic vesicles and positive for the synaptic vesicle protein synaptotagmin. Based on these results, we suggest that endosome-like intermediates of nerve terminals originate by bulk uptake of the plasma membrane and that clathrin- and dynamin-mediated budding takes place in parallel from the plasmalemma and from these internal membranes. We propose a synaptic vesicle recycling model that involves a single vesicle budding step mediated by clathrin and dynamin.

Adaptor Protein Complex 2↗

PubMed1956

Electron microscope observations on synaptic vesicles in synapses of the retinal rods and cones.

The submicroscopic organization of the rod and cone synapses of the albino rabbit has been investigated with the use of the electron microscope. The most common rod synapse consists of an enlarged expansion of the rod fiber (the so called spherule) into which the dendritic postsynaptic fiber of the bipolar cell penetrates and digitates. The membrane surrounding the terminal consists of a double layer, the external of which is interpreted as belonging to the intervening glial cells. The synaptic membrane has a pre- and a postsynaptic layer with a total thickness of 180 to 300 A. The presynaptic layer is frequently denser and is intimately associated with the adjacent synaptic vesicles. The synaptic membrane shows processes constituted by foldings of the presynaptic layer. The entire spherule is filled with synaptic vesicles varying in diameter between 200 and 650 A with a mean of 386 A. In addition, the spherule contains a few large vacuoles near the rod fiber, interpreted as endoplasmic reticulum, and a matrix in which with high resolution a fine filamentous material can be observed. The postsynaptic fiber is homogeneous and usually does not show synaptic vesicles. In animals maintained in complete darkness for 24 hours vesicles appear to accumulate near the synaptic membrane and its processes. After 9 days there is a sharp decrease in size of the synaptic vesicles. A special rod synapse in which the dendritic postsynaptic expansion penetrates directly into the rod cell body has been identified. In line with Cajal's classification this type of synapse could be considered as a somatodendritic one. The cone synapse has a much larger terminal with a more complex relationship with the postsynaptic fiber. However, the same components recognized in the rod synapse can be observed. In animals maintained for 9 days in complete darkness there is also a considerable diminution in size of the synaptic vesicles.

Animals↗

PubMed2002

Sequential changes in synaptic vesicle pools and endosome-like organelles during depolarization near the active zone of central nerve terminals.

During periods of high-frequency stimulation the maintenance of synaptic transmission depends on a continued supply of synaptic vesicles. Local recycling in the terminals ensures synaptic vesicle replenishment, but the intermediate steps are still a matter of debate. We analyzed changes in synaptic vesicle pools and endosome-like organelles near the active zone in central nerve terminals during depolarization at the ultrastructural level by electron microscopy. A short, 100 ms, depolarization-induced recruitment of synaptic vesicles was observed from a reserve pool to a recruited pool, within 150 nm of the active zone, and the docked pool at the active zone was increased as well. Prolonged, 15 s or 3 min, depolarization decreased the total amount of synaptic vesicles, which was accompanied by a parallel increase in size and amount of endosome-like organelles. After a period of rest, the number of endosome-like organelles decreased and the amount of synaptic vesicles was restored to control level. The endocytotic nature of part of the endosome-like organelles after 15 s and 3 min depolarization was indicated by their labeling with extracellularly added horseradish peroxidase (HRP). In addition, a small number of synaptic vesicles entrapped HRP under these conditions. After repolarization, the number of HRP-loaded endosome-like structures decreased. Simultaneously, a strong increase in amount of HRP-loaded small vesicles did occur. These results indicate that during sub-second depolarization, synaptic vesicles were rapidly recruited from the reserve pool to replenish the releasable pool, whereas prolonged depolarization (s-min) induced local endocytosis in at least two ways, i.e. either directly as vesicles or via endosome-like organelles from which synaptic vesicles were reformed.

Action Potentials↗

PubMed2001

Synaptic vesicle alterations in rod photoreceptors of synaptophysin-deficient mice.

The abundance of the integral membrane protein synaptophysin in synaptic vesicles and its multiple possible functional contributions to transmitter exocytosis and synaptic vesicle formation stand in sharp contrast to the observed lack of defects in synaptophysin knockout mice. Assuming that deficiencies are compensated by the often coexpressed synaptophysin isoform synaptoporin, we now show that retinal rod photoreceptors, which do not synthesize synaptoporin either in wild-type or in knockout mice, are affected by the loss of synaptophysin. Multiple pale-appearing photoreceptors, as seen by electron microscopy, possess reduced cytoplasmic electron density, swollen mitochondria, an enlarged cell surface area, and, most importantly, a significantly reduced number of synaptic vesicles with an unusually bright interior. Quantification of the number of synaptic vesicles per unit area, not only in these, but also in all other rod terminals of knockout animals, reveals a considerable reduction in vesicles that is even more pronounced during the dark period, i.e., at times of highest synaptic activity. Moreover, activity-dependent reduction in synaptic vesicle diameter, typically occurring in wild-type mice, is not detected in knockout animals. The large number of clathrin-coated pits and vesicles in dark-adapted synaptophysin knockout mice is taken as an indication of compensatory usage of synaptophysin-independent pathway(s), and, conversely, in view of the overall reduction in the number of synaptic vesicles, as an indication for the presence of another synaptophysin-dependent synaptic vesicle recycling pathway. Our results provide in vivo evidence for the importance of the integral membrane protein synaptophysin for synaptic vesicle recycling and formation.

Action Potentials↗

PubMed1989

Most synaptic vesicles isolated from rat brain carry three membrane proteins, SV2, synaptophysin, and p65.

We have prepared highly purified synaptic vesicles from rat brain by subjecting vesicles purified by our previous method to a further fractionation step, i.e., equilibrium centrifugation on a Ficoll gradient. Monoclonal antibodies to three membrane proteins enriched in synaptic vesicles--SV2, synaptophysin, and p65--each were able to immunoprecipitate specifically approximately 90% of the total membrane protein from Ficoll-purified synaptic vesicle preparations. Anti-SV2 precipitated 96% of protein, anti-synaptophysin 92%, and anti-p65 83%. These results demonstrate two points: (1) Ficoll-purified synaptic vesicles appear to be greater than 90% pure, i.e., less than 10% of membranes in the preparation do not carry synaptic vesicle-associated proteins. These very pure synaptic vesicles may be useful for direct biochemical analyses of mammalian synaptic vesicle composition and function. (2) SV2, synaptophysin, and p65 coexist on most rat brain synaptic vesicles. This result suggests that the functions of these proteins are common to most brain synaptic vesicles. However, if SV2, synaptophysin, or p65 is involved in synaptic vesicle dynamics, e.g., in vesicle trafficking or exocytosis, separate cellular systems are very likely required to modulate the activity of such proteins in a temporally or spatially specific manner.

Animals↗

PubMed1994

SV2 and o-rab3 remain associated with recycling synaptic vesicles.

o-rab3 is an electric ray homologue of low molecular weight of GTP-binding proteins thought to be involved in targeting of secretory vesicles to sites of exocytosis. The stimulation-dependent association of o-rab3 with synaptic vesicles was compared with that of the membrane-integral synaptic vesicle protein 2 (SV2). On application of immunoelectron microscopy and the colloidal gold technique, antibodies against either protein labeled the synaptic vesicle membrane compartment. Synaptic vesicles recycled under conditions of low frequency stimulation (0.1 Hz) retained their complement of both SV2 and o-rab3. Isolation of synaptic vesicles by density-gradient centrifugation and subsequent column chromatography yielded no indication of a stimulation-dependent release of o-rab3 from synaptic vesicles. In contrast, multivesicular bodies and vacuoles occasionally observed in the nerve terminals contained SV2 but little if any o-rab3. It is concluded that o-rab3 remains associated with the synaptic vesicle membrane compartment during stimulation-induced cycles of repeated exo- and endocytosis. o-rab3 may be lost once the vesicle enters the prelysosomal pathway.

Animals↗

PubMed1995

Defective recycling of synaptic vesicles in synaptotagmin mutants of Caenorhabditis elegans.

Synaptotagmin, an integral membrane protein of the synaptic vesicle, binds calcium and interacts with proteins of the plasma membrane. These observations suggest several possible functions for synaptotagmin in synaptic vesicle dynamics: it could facilitate exocytosis by promoting calcium-dependent fusion, inhibit exocytosis by preventing fusion, or facilitate endocytosis of synaptic vesicles from the plasma membrane by acting as a receptor for the endocytotic proteins of the clathrin AP2 complex. Here we show that synaptic vesicles are depleted at synaptic terminals in synaptotagmin mutants of the nematode Caenorhabditis elegans. This depletion is not caused by a defect in transport or by increased synaptic vesicle release, but rather by a defect in retrieval or synaptic vesicles from the plasma membrane. Thus we propose that, as well as being involved in exocytosis, synaptotagmin functions in vesicular recycling.

Aldicarb↗

PubMed1994

Release of ATP from cholinergic synaptic vesicles during freeze-thaw cycling.

Cholinergic synaptic vesicles were isolated from Torpedo californica and subjected to repeated freeze/thraw (F/T) cycles. Both vesicular (trapped) and released (free) ATP were measured after each cycle. It was found that a constant percentage of vesicular content was released during each F/T cycle. In solutions low in Ca2+ and Mg2+ and high in sucrose, 25% of the ATP is released by each F/T cycle. When synaptic vesicles are resuspended in Tornedo Ringer's (which contains less sucrose, higher Ca2+ and urea), 35-40% of the trapped ATP is released during each F/T cycle. These data contradict the hypothesis that F/T does not disrupt synaptic vesicle membranes (although F/T does disrupt cell membranes and synaptosomes). This nonrupture hypothesis was assumed by C. Solsona, C. Saltó, and A. Ymbern (1991, Biochim. Biophys. Acta 1095, 57-62) to calculate cytosolic ATP as 40% of total ATP. Our results indicate that cytosolic ATP is 10-15%. These results may also explain some of the discrepancies in reported values for cytosolic acetylcholine (ACh). Values of 35-50% were obtained by previous workers using the nonrupture hypothesis, and values of 8-22% in experiments that did not depend on the nonrupture hypothesis. Our results refute the nonrupture hypothesis and thus support a lower value for cytosolic ACh.

Adenosine Triphosphatases↗

PubMed1985

A new method for scanning and transmission electron microscopy of synaptic vesicles isolated from the cerebral cortex.

Spheroid and flattened synaptic vesicles were isolated from the brain homogenate of guinea pigs by a modified purification method. For scanning and transmission electron microscopy, a simple dipping method of preparation was developed and used. The purest and richest fraction of synaptic vesicles was obtained from a 0.1 M sucrose fraction of density gradients. The pellets of synaptic vesicles were easily resuspended without aggregate after ultracentrifugation at 40,000 rpm for 5 min. The isolated synaptic vesicles were dispersed as a monolayer on the surface of a copper grid covered with Formvar membrane. Adequate contrast was obtained by metal impregnation of specimens and gold coating at magnifications as high as 100,000 times using an acceleration voltage of 25 to 40 kV. The specimens were fixed in 0.75% glutaraldehyde (0.1 M phosphate buffer, pH 7.3) and then postfixed in 1% osmium tetroxide. After dipping for 1 to 2 min each in tannic acid, phosphotungstic acid, lead citrate and uranyl acetate, they were dehydrated with graded ethanol and coated with gold by ion sputtering at 400 to 560 volts for 4 min. The preparation method is reported on and technical problems are discussed.

Animals↗

PubMed1997

Glutamate uptake occurs at an early stage of synaptic vesicle recycling.

Rapid membrane recycling in nerve terminals is required to maintain rapid synaptic transmission. Following the fusion of synaptic vesicles with synaptic plasma membranes, recycling can occur via clathrin-coated vesicles (CCVs) [1-3]. The fate of these vesicles is uncertain: they could simply uncoat and acquire other proteins from the cytosol to regenerate synaptic vesicles or they may fuse with endosomal structures from which synaptic vesicles could then bud. We have purified both CCVs and synaptic vesicles from rat brain, and measured the ability of these vesicle fractions to take up the excitatory neurotransmitter glutamic acid. We found that the normalized levels of glutamate uptake by the two types of vesicle were very similar. For each vesicle fraction, uptake required ATP and Cl- and could be fully inhibited by the specific vacuolar proton pump (v-ATPase) inhibitor concanamycin. We suggest that this ability to refill vesicles with neurotransmitter at the earliest intermediate on the recycling pathway - the CCV - may allow uncoated vesicles to immediately enter the releasable pool without sacrificing the quantal nature of neurotransmitter release.

Adenosine Triphosphate↗