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PubMed1994

Differential expression of synaptic vesicle protein 2 (SV2) isoforms.

The synaptic vesicle proteins SV2A and SV2B (SV2 = synaptic vesicle protein 2) are two highly related proteins belonging to a family of transporters. As a first step toward identifying the function of the SV2 proteins, we examined the expression of SV2A and SV2B in the rat brain by in situ hybridization, immunohistochemistry, and immunoprecipitation with isoform-specific antibodies. These analyses revealed that one isoform, SV2A, is expressed ubiquitously throughout the brain at varying levels. The other isoform, SV2B, has a more limited distribution with varying degrees of coexpression with SV2A. Immunoprecipitation of brain synaptic vesicles with isoform-specific antibodies followed by Western analyses suggests that both isoforms can be present on the same synaptic vesicle. The expression of the SV2 proteins did not correlate either with neurotransmitter phenotype or with the expression of other synaptic vesicle protein isoforms. SV2B expression was observed to change during development; it is more widely expressed in the immature brain and is found in cells that have yet to establish synaptic contacts. The ubiquitous and overlapping expression of the SV2s suggests that they perform a function common to all synaptic vesicles. Variable and changing coexpression of the SV2 isoforms may indicate that SV2 function is regulated by the isoform composition of synaptic vesicles. The observation that the synaptic vesicle proteins, all occurring in multiple isoforms, are differentially expressed with respect to each other indicates that up to 90 different vesicle types are possible.

Animals↗

PubMed2001

Drosophila stoned proteins regulate the rate and fidelity of synaptic vesicle internalization.

At an initial step during synaptic vesicle recycling, dynamin and adaptor proteins mediate the endocytosis of synaptic vesicle components from the plasma membrane. StonedA and stonedB, novel synaptic proteins encoded by a single Drosophila gene, have predicted structural similarities to adaptors and other proteins implicated in endocytosis. Here, we test possible roles of the stoned proteins in synaptic vesicle internalization via analyses of third instar larval neuromuscular synapses in two Drosophila stoned (stn) mutants, stn(ts) and stn(8P1). Both mutations reduce presynaptic levels of stonedA and stonedB, although stn(ts) has relatively weak effects. The mutations cause retention of synaptic vesicle proteins on the presynaptic plasma membrane but do not alter the levels or distribution of endocytosis proteins, dynamin, alpha-adaptin, and clathrin. In addition, stn(8P1) mutants exhibit depletion and enlargement of synaptic vesicles. To determine whether these defects arise from altered synaptic vesicle endocytosis or from defects in synaptic vesicle biogenesis, we implemented new methods to assess directly the efficiency of synaptic vesicle recycling and membrane internalization at Drosophila nerve terminals. Behavioral and electrophysiological analyses indicate that stn(ts), an allele with normal evoked release and synaptic vesicle number, enhances defects in synaptic vesicle recycling shown by Drosophila shi(ts) mutants. A dye uptake assay demonstrates that slow synaptic vesicle recycling in stn(ts) is accompanied by a reduced rate of synaptic vesicle internalization after exocytosis. These observations are consistent with a model in which stonedA and stonedB act to facilitate the internalization of synaptic vesicle components from the plasma membrane.

Animals↗

PubMed2006

GTP uptake into rat brain synaptic vesicles.

Uptake of neurotransmitters into synaptic vesicles is driven by an electrochemical gradient generated by a vacuolar-type proton pump ATPase. This uptake implies a key role for synaptic vesicles in the regulation of neurotransmitter systems. Guanine nucleoside and nucleotides are involved in the inhibition of glutamate-induced cellular responses via an extracellular action and diverse trophic, proliferative, and modulatory effects of guanine nucleotides on neural cells have been shown. Here, we characterized the uptake of GTP into synaptic vesicles isolated from whole rat brain, by using a tritiated poorly-hydrolyzable GTP analog, 5'-guanylylimidodiphosphate ([3H]GppNHp). Uptake of GTP into synaptic vesicles is saturable, time- and temperature-dependent, and relies on a proton-eletrochemical gradient. However, [3H]GMP and [3H]GDP radioactive labeling in synaptic vesicles is not dependent on temperature and vesicular ATPase activity, which indicates that these nucleotides only bind to and are not taken up into synaptic vesicles. GTP is taken up by the same eletrochemical gradient-dependent transport system, as are neurotransmitters storage, which indicates that this guanine nucleotide may also function as a neurotransmitter.

Adenosine Triphosphate↗

PubMed2003

Technique to study three-dimensional spatial arrangement of synaptic vesicles using data from single sections.

Synaptic vesicles are membrane-bound organelles storing neurotransmitters in presynaptic terminals and releasing them into the synaptic cleft. Coordinated movements of synaptic vesicles relate to synaptic function and their spatial arrangement can provide useful information about the activity of a synapse. This article presents a technique to extract quantitative information about three-dimensional (3D) spatial arrangement of synaptic vesicles from measurements performed on single ultrathin random sections of a presynaptic terminal. The technique presumes quantification of a 2D density as well as 2D spatial pattern formed by vesicle profiles using a minimum spanning tree (MST) algorithm, in digitized micrographs of a presynaptic terminal. Further, original software was used to simulate a 3D spatial arrangement of synaptic vesicles and their random sectioning. A 3D density and pattern of synaptic vesicles were used as basic input parameters of the model, while a 2D density and MST quantities for vesicle profiles served as output, model-derived parameters allowing one to compare and fit simulated distributions to experimental ones. Pilot simulations performed to check the validity of the technique have shown that a 2D density and MST quantities of vesicle profiles closely relate to a 3D density and spatial pattern of vesicles. The technique was demonstrated in the analysis of spatial distribution of synaptic vesicles in axonal terminals forming asymmetric synaptic densities in the stratum radiatum of the CA1 subfield of the murine hippocampus.

Animals↗

PubMed1984

Extensive cross-linking of calf brain plain synaptic vesicle proteins.

Calf brain plain synaptic vesicle proteins have been cross-linked with bis[2-(succinimidooxycarbonyloxy)ethyl] sulfone, a homobifunctional, cleavable reagent, as well as with N-hydroxysuccinimidyl 4-azidobenzoate, a photosensitive, heterobifunctional reagent. These results demonstrate the generality of a recent report that synaptic vesicle proteins can be cross-linked, in contrast to a prior report that no cross-linking could be observed. The reagents gave some differences in the proteins that were preferentially cross-linked. A protein at Mr = 173 000, which comigrates with clathrin, is present in the plain synaptic vesicle fraction and appears to be involved in cross-linking. A high degree of association and structural organization of synaptic vesicle proteins is suspected, since extensive cross-linking of most synaptic vesicle proteins with high-molecular-mass proteins, which are probably structural in nature, is observed. A protein with an Mr of 249 000 is specifically cross-linked to a protein of Mr 42 000, probably actin, suggesting that the 249 000-Mr protein may be a spectrin-like molecule. The present results suggest that synaptic vesicles may be organized by a spectrin-like matrix similar to that observed in erythrocytes and other cells.

Animals↗

PubMed1990

Partial purification and characterization of the vacuolar H(+)-ATPase of mammalian synaptic vesicles.

Several major proteins of synaptic vesicles from rat or cow brain sediment as a large complex on sucrose density gradients when solubilized in nonionic detergents. A vacuolar H(+)-ATPase identified by sensitivity to bafilomycin A1 appears to be associated with this oligomeric protein complex. Two subunits of this complex, synaptic vesicle proteins S and U, correspond to the 57-kDa (B) and 39-kDa accessory (Ac39) subunits, respectively, of bovine chromaffin granule vacuolar H(+)-ATPase as shown by Western immunoblot analysis. The five subunits of the oligomeric complex constitute approximately 20% of the total protein of rat brain synaptic vesicles. Taken together, these results strongly suggest that the abundant, multisubunit complex partially purified from brain synaptic vesicles by density gradient centrifugation is a vacuolar H(+)-ATPase. Bafilomycin A1 completely blocks proton pumping in rat brain synaptic vesicles as measured by [14C]methylamine uptake and also blocks catecholamine accumulation measured by [3H]dopamine uptake. Moreover, ATPase activity, [14C]methylamine uptake, and [3H]dopamine uptake are inhibited by bafilomycin A1 at similar I50 values of approximately 1.7 nmol/mg of protein. These findings indicate that the vacuolar H(+)-ATPase is essential for proton pumping as well as catecholamine uptake by mammalian synaptic vesicles.

Adenosine Triphosphatases↗

PubMed1985

Saturable [D-Ala2, D-Leu5]-enkephalin transport into cholinergic synaptic vesicles.

Incubation of cholinergic synaptic vesicles purified from the electric organ of Torpedo ocellata with radiolabeled [D-Ala2, D-Leu5]-enkephalin results in its accumulation in the vesicles. Kinetic analysis of the initial rates of uptake reveals temperature-dependent saturation kinetics which are best fitted by a single transport system (KT = 12 +/- 2 microM and Vmax = 0.85 +/- 0.24 pmol/mg protein per min). The specific rates and extents of [3H]-[D-Ala2, D-Leu5]-enkephalin uptake, like those of [3H]acetylcholine uptake, are highest in the purified synaptic vesicles fraction. These findings suggest that Torpedo cholinergic synaptic vesicles contain an opioid peptide transporter. The physiological significance of this transporter is discussed in view of the recent observation that Torpedo nerve terminals contain an endogenous enkephalin-like peptide and presynaptic opiate receptors.

Acetylcholine↗

PubMed1997

Synaptic vesicle endocytosis.

Exocytosis of synaptic vesicles is followed rapidly by reinternalization and recycling of their membranes. Recent studies have confirmed the key role of clathrin-mediated endocytosis in synaptic vesicle reformation and have identified new proteins that participate in this process. In addition, growing evidence suggests that lipids, primarily phosphoinositides, play an important role in synaptic vesicle recycling.

Animals↗

PubMed1996

Ultrastructural distribution of synaptophysin and synaptic vesicle recycling at the frog neuromuscular junction.

Synaptic vesicle recycling after intense acetylcholine (ACh) release was studied at the frog neuromuscular junction (NMJ) using the synaptic vesicle transmembrane protein synaptophysin as immunocytochemical marker of the synaptic vesicle membrane during the process of exo-endocytosis. ACh release in cutaneous pectoris nerve-muscle preparations was stimulated by three different means: K+, Cd2+ in Ca(2+)-free medium, and electrical stimulation in the presence of 4-aminopyridine (4-AP). Cd2+ stimulation produced synaptic vesicle depletion and nerve terminal swelling. Electrical stimulation in the presence of 4-AP produced a reduction in the number of synaptic vesicles, deep axolemmal infoldings, coated pits, and coated vesicles. K+ stimulation did not produce any observable ultrastructural changes. Synaptophysin was labeled using silver-intensified immunogold in dissociated muscle fibers. Unstimulated and K(+)-stimulated preparations showed synaptophysin immunolabeling associated only with synaptic vesicles. In contrast, in Cd(2+)-stimulated preparations, synaptophysin appeared along the axolemma, mainly at the active zones, and after electrical stimulation it appeared in both axolemmal infoldings and the remaining synaptic vesicles. The results show that when synaptic vesicle recycling is inhibited by Cd2+ in Ca(2+)-free medium, or when 4-AP is present during electrical stimulation, synaptic vesicle fusion is accompanied by translocation and incorporation of synaptic vesicle membrane proteins into the axolemma. However, during the latter condition, synaptic vesicles are recycled through coated vesicles arising from the axolemmal infoldings. Conversely, during physiological-like stimulation of ACh release by K+ the synaptic vesicles are rapidly recycled at the active zones, by a double and rapid process of exo-endocytosis, without collapse into the axolemma.

Acetylcholine↗

PubMed1976

Microtubule--synaptic vesicle associations in cultured rat spinal cord neurons.

This paper describes new ultrastructural features of neural processes and of synapses in cultured CNS tissue treated with albumin before fixation using a modification of the technique recently introduced by Gray (1975). Nerve fibre bundles in explants of foetal spinal cord grown in vitro for 15-18 days were transected microsurgically. After transection the cultures were exposed to 20% albumin in distilled water and then fixed in unbuffered osmium tetroxide followed by unbuffered glutaraldehyde. In this material, but not in controls (injured but not exposed to albumin; exposed to albumin without injury) microtubules were found within many axonal varicosities, often situated close to presynaptic membrane specializations. These microtubules were closely associated with vesicles resembling synaptic vesicles, which were occasionally aligned in rows along the microtubules. Similar vesicle-microtubule associations were also found in non-terminal axons. Microtubules were also observed very close to some postsynaptic densities. The possiblity that the microtubule-vesicle associations are involved in vesicle movements (along axons and/or within axon terminals) is discussed. A more direct involvement of microtubules in terminals in the mechanism of transmitter release is also considered.

Animals↗

PubMed2004

Synaptic vesicle pools and plasticity of synaptic transmission at the Drosophila synapse.

Our knowledge on the Drosophila neuromuscular synapse is rapidly expanding. Thus, this synapse offers an excellent model for studies of the molecular mechanism of synaptic transmission and synaptic plasticity. Two synaptic vesicle (SV) pools have been identified and characterized using a fluorescent styryl dye, FM1-43, to stain SVs. They are termed the exo/endo cycling pool (ECP), which corresponds to the readily releasable pool (RRP) defined electrophysiologically, and the reserve pool (RP). These two pools were identified first in a temperature-sensitive paralytic mutant, shibire, and subsequently confirmed in wild-type larvae. The ECP participates in synaptic transmission during low frequency firing of presynaptic nerves and locates in the periphery of presynaptic boutons in the vicinity of release sites, while SVs in the RP spread toward the center of boutons and are recruited only during tetanic stimulation. These two pools are separately replenished by endocytosis. Cyclic AMP facilitates recruitment of SVs from the RP to the ECP. Activation of presynaptic metabotropic glutamate receptors recruits SVs from the RP and enhances SV release by elevation of the cAMP level. Memory mutants that have defects in the cAMP/PKA cascade, dunce and rutabaga, exhibit reduced levels of recruitment of synaptic SVs from the RP to the ECP and have limited short-term synaptic plasticity. SV mobilization between the two pools could be a key step for changes in synaptic efficacy. Since a variety of mutants that have distinct defects in synaptic transmission are available for detailed studies of synaptic function, this direction of approach in Drosophila seems promising.

Animals↗

PubMed2002

Direct, Ca2+-dependent interaction between tubulin and synaptotagmin I: a possible mechanism for attaching synaptic vesicles to microtubules.

The synaptic vesicle protein synaptotagmin I probably plays important roles in the synaptic vesicle cycle. However, the mechanisms of its action remain unclear. In this study, we have searched for cytoplasmic proteins that interact with synaptotagmin I. We found that the cytoskeletal protein tubulin directly and stoichiometrically bound to recombinant synaptotagmin I. The binding depended on mm Ca(2+), and 1 mol of tubulin dimer bound 2 mol of synaptotagmin I with half-maximal binding at 6.6 microm tubulin. The Ca(2+) dependence mainly resulted from Ca(2+) binding to the Ca(2+) ligands of synaptotagmin I. The C-terminal region of beta-tubulin and both C2 domains of synaptotagmin I were involved in the binding. The YVK motif in the C2 domains of synaptotagmin I was essential for tubulin binding. Tubulin and synaptotagmin I were co-precipitated from the synaptosome extract with monoclonal antibodies to tubulin and SNAP-25 (synaptosome-associated protein of 25 kDa), indicating the presence of tubulin/synaptotagmin I complex and tubulin binding to synaptotagmin I in SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. Synaptotagmin I promoted tubulin polymerization and bundled microtubules in the presence of Ca(2+). These results suggest that direct interaction between synaptotagmin I and tubulin provides a mechanism for attaching synaptic vesicles to microtubules in high Ca(2+) concentrations.

Animals↗

PubMed1997

ATP-dependent formation of free synaptic vesicles from PC12 membranes in vitro.

Synaptic vesicles are released from membranes during incubation at 37 degrees C in the presence of ATP (adenosine triphosphate). The donor membranes are a rapidly sedimenting fraction derived from the neuroendocrine cell line PC12 (pheochromocytoma 12). These starting membranes contain the synaptic vesicle proteins, synaptophysin and SV2, and the endosomal markers transferrin receptor and cation-independent MPR (mannose 6-phosphate receptor). Incubating the membranes in vitro increased the amount of organelles that migrate as synaptic vesicles in velocity sedimentation gradients. The synaptic vesicle fractions that contain both synaptophysin and SV2 do not contain endosomal markers. A synaptic vesicle increase in vitro is time-, cytosol-, ATP- and temperature-dependent and is inhibited by NEM (N-ethylmaleimide), BFA (brefeldin A) and aluminum fluoride, but not GTP gamma S (guanosine-5'O-C3-thiotriphosphate). The production of synaptic vesicles under these conditions is unlike the de novo generation of vesicles from endosomes (1). Incubation in vitro under the conditions described here may allow the final stages of synaptic vesicle formation, uncoating or undocking, to occur but not the initiation of formation de novo.

Adenosine Triphosphate↗

PubMed1998

Matching kinetics of synaptic vesicle recycling and enhanced neurotransmitter influx by Ca2+ in brain plasma membrane vesicles.

Using native plasma membrane vesicle suspensions from the rat cerebral cortex under conditions designed to alter intravesicular [Ca2+], we found that Ca2+ induced 47 +/- 5% more influx of [3H]GABA, [3H]D-aspartate and [3H]glycine at 37 degrees C with half-times 1.7 +/- 0.5, 1.3 +/- 0.4 and 1.3 +/- 0.4 min, respectively. We labelled GABA transporter sites with the uptake inhibitor, [3H]-(R,S)-N-[4,4-bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid and found that Ca2+ induced a partial dissociation of the bound inhibitor from GABA transporter sites with a similar half-time. By means of rapid kinetic techniques applied to native plasma membrane vesicle suspensions, containing synaptic vesicles stained with the amphipathic fluorescent styryl membrane probe N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyrid inium dibromide, we have measured the progress of the release and reuptake of synaptic vesicles in response to Ca2+ and high-[K+] depolarization in the 0.0004-100 s range of time. Synaptic vesicle exocytosis, strongly influenced by external [Ca2+], appeared with the kinetics accelerated by depolarization. These results are consistent with the potential involvement of Ca2+ in taking low-affinity transporters to the plasma membrane surface via exocytosis.

Animals↗

PubMed1982

Ca2+ and calmodulin-dependent phosphorylation of endogenous synaptic vesicle tubulin by a vesicle-bound calmodulin kinase system.

Endogenous synaptic vesicle alpha- and beta-tubulin were shown to be the major substrates for a Ca2+-calmodulin-regulated protein kinase system in enriched synaptic vesicle preparations from rat cortex as determined by two-dimensional gel electrophoresis and peptide mapping. The activation of this endogenous tubulin kinase system was dependent on Ca2+ and the Ca2+ binding protein, calmodulin. Under maximally stimulated conditions, approximately 40% of the tubulin present in enriched synaptic vesicles was phosphorylated within less than 50 s by the vesicle Ca2+-calmodulin kinase. Evidence is presented indicating that the Ca2+-calmodulin tubulin kinase is an enzyme system distinct from previously described cyclic AMP protein kinases. alpha-Tubulin and beta-tubulin were identified as major components of previously designated vesicle phosphorylation bands DPH-L and DPH-M. The Ca2+-calmodulin tubulin kinase is very labile and specialized isolation procedures were necessary to retain activity. Ca2+-activated synaptic vesicle tubulin phosphorylation correlated with vesicle neurotransmitter release. Depolarization-dependent Ca2+ uptake in intact synaptosomes simultaneously stimulated the release of neurotransmitters and the phosphorylation of synaptic vesicle alpha- and beta-tubulin. The results indicate that regulation of the synaptic vesicle tubulin kinase by Ca2+ and calmodulin may play a role in the functional utilization of synaptic vesicle tubulin and may mediate some of the effects of Ca2+ on vesicle function and neurosecretion.

Animals↗

PubMed2003

Ectopic release of synaptic vesicles.

Exocytosis of synaptic vesicles is generally assumed to occur only at ultrastructurally defined presynaptic active zones. If release is restricted to these sites, receptors not located within the synaptic cleft must be activated by transmitter that diffuses out of the cleft or not be activated at all. Here we report that AMPA receptor-mediated quantal events resulting from climbing fiber release are observed in Bergmann glial cells in the cerebellar cortex. These quantal events are not coincident with quanta recorded in neighboring Purkinje cells which receive input from the same climbing fiber. As Bergmann glial membranes are excluded from the synaptic cleft, we propose that exocytosis can occur from climbing fiber release sites located directly across from Bergmann glial membranes. Such ectopic release may account for the majority of the Bergmann glial AMPA response evoked by climbing fiber stimulation.

Animals↗

PubMed1993

Identification of a 34 kDa protein specific to synaptic vesicles.

In this study, we used synaptic vesicles purified from the electric organ of marine electric rays to search for novel molecules which have important functions in synaptic transmission. Proteins that copurified with synaptic vesicles were used to immunize rats, and the resulting antisera were then used to further characterize the vesicle proteins. One of the antisera recognizes a protein of 34 kDa, p34, that has several characteristics which suggest it is a synaptic vesicle specific protein: (1) it copurifies exclusively with the synaptic vesicle peak during permeation chromatography on a controlled pore glass beads column, (2) it can be immunoprecipitated with intact synaptic vesicles and (3) it is specifically localized to the nervous system. The results suggest that p34 is a synaptic vesicle specific protein with a widespread distribution in the nervous system.

Amino Acid Sequence↗

PubMed1992

Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones.

Synaptic vesicles store neurotransmitters that are released during calcium-regulated exocytosis. The specificity of neurotransmitter release requires the localization of both synaptic vesicles and calcium channels to the presynaptic active zone. Two 35-kilodalton proteins (p35 or syntaxins) were identified that interact with the synaptic vesicle protein p65 (synaptotagmin). The p35 proteins are expressed only in the nervous system, are 84 percent identical, include carboxyl-terminal membrane anchors, and are concentrated on the plasma membrane at synaptic sites. An antibody to p35 immunoprecipitated solubilized N-type calcium channels. The p35 proteins may function in docking synaptic vesicles near calcium channels at presynaptic active zones.

Amino Acid Sequence↗