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PubMed2004

Technique to quantify local clustering of synaptic vesicles using single section data.

Synaptic vesicles are organelles that specialize in the storage of a neurotransmitter that continuously undergo an exo-endocytotic cycle. During this cycle vesicles change their positions within a presynaptic terminal and their numbers as well as spatial arrangement can provide insight into a neurotransmitter turnover. This article introduces a technique based on the nearest-neighbor formalism to quantify the proximity of vesicles to active zones and vesicle clustering in different regions of a terminal. The technique, implemented in a software package, uses the two-dimensional coordinates of features identified in digitized electron micrographs as an input. It has been validated in the analysis of asymmetric synapses of the rat hippocampal CA1 stratum radiatum affected by transient cerebral ischemia. It was shown that a 15-minute-long ischemic episode influenced the spatial arrangement of vesicles that were more distant from active zones and had larger intervesicle spacings with respect to the control. The latter effect was apparently stronger within 200 nm distance of active zones.

Animals↗

PubMed1986

Changes in the size and shape of the synaptic vesicles in the sensorimotor cortex of the rat brain in the initial phases of kindling.

The sensorimotor area of rat cerebral cortex was subjected to repeated electrical stimulation at 10-min intervals, with resultant formation and progressive lengthening of self-sustained after-discharges (SSAD). One and 60 min after the third SSAD ended, we carried out an electron microscopy morphometric analysis of the agranular synaptic vesicles in type I synapses (after Gray) in the second cortical layer of the homotopic area of the unstimulated hemisphere. One minute after the seizure ended, 5.8% enlargement of the synaptic vesicles compared with the control was demonstrated in zone II of the synapse (0.1-0.2 micron from the active zone of the synapse). Neither the size nor the shape of the synaptic vesicles in the other parts of the synaptic apparatus altered. Sixty min after the seizure ended, a 5.5% enlargement of the synaptic vesicles in zone I (0.0-0.1 micron) and a 5.4% enlargement of those in zone II was found. The synaptic vesicles in zone I in the experimental animals were more oval than in the controls. Our findings support the vesicular theory and testify that hyperfunction, up to temporary exhaustion of the synaptic apparatuses, produces a change in the transmitter content of the synaptic vesicles. A raised amount of transmitter in the synaptic vesicles near the active zone could be one of the factors responsible for continued hyperexcitability of the tissue one hour after the seizure had ended. The results likewise support the concept of two mechanisms of synaptic vesicle formation, and hence of the existence of two different vesicle populations.

Animals↗

PubMed1991

Association of Rab3A with synaptic vesicles at late stages of the secretory pathway.

Rab3A is a small GTP-binding protein highly concentrated on synaptic vesicles. Like other small GTP-binding proteins it is thought to cycle between a soluble and a membrane-associated state. To determine at which stage of the life cycle of synaptic vesicles rab3A is associated with their membranes, the localization of the protein in neurons and neuroendocrine cells at different developmental and functional stages was investigated. In all cases, rab3A was colocalized with synaptic vesicle markers at the cell periphery, but was absent from the Golgi area, suggesting that rab3A associates with vesicles distally to the Golgi complex and dissociates from vesicle membranes before they recycle to this region. Immunofluorescence experiments carried out on frog motor end plates demonstrated that massive exocytosis of synaptic vesicles is accompanied by a translocation of rab3A to the cell surface. The selective localization of rab3A on synaptic vesicles at stages preceding their fusion with the plasmalemma suggests that the protein is part of a regulatory machinery that is assembled onto the vesicles in preparation for exocytosis.

Animals↗

PubMed1999

Empty synaptic vesicles recycle and undergo exocytosis at vesamicol-treated motor nerve terminals.

We investigated whether recycled cholinergic synaptic vesicles, which were not refilled with ACh, would join other synaptic vesicles in the readily releasable store near active zones, dock, and continue to undergo exocytosis during prolonged stimulation. Snake nerve-muscle preparations were treated with 5 microM vesamicol to inhibit the vesicular ACh transporter and then were exposed to an elevated potassium solution, 35 mM potassium propionate (35 KP), to release all preformed quanta of ACh. At vesamicol-treated endplates, miniature endplate current (MEPC) frequency increased initially from 0.4 to >300 s-1 in 35 KP but then declined to <1 s-1 by 90 min. The decrease in frequency was not accompanied by a decrease in MEPC average amplitude. Nerve terminals accumulated the activity-dependent dye FM1-43 when exposed to the dye for the final 6 min of a 120-min exposure to 35 KP. Thus synaptic membrane endocytosis continued at a high rate, although MEPCs occurred infrequently. After a 120-min exposure in 35 KP, nerve terminals accumulated FM1-43 and then destained, confirming that exocytosis also still occurred at a high rate. These results demonstrate that recycled cholinergic synaptic vesicles that were not refilled with ACh continued to dock and undergo exocytosis after membrane retrieval. Thus transport of ACh into recycled cholinergic vesicles is not a requirement for repeated cycles of exocytosis and retrieval of synaptic vesicle membrane during prolonged stimulation of motor nerve terminals.

Acetylcholine↗

PubMed2005

Tyrosine phosphorylation of synaptophysin in synaptic vesicle recycling.

The integral SV (synaptic vesicle) protein synaptophysin was one of the first nerve terminal proteins identified. However its role, if any, in the SV life cycle remains undetermined. One of the most prominent features of synaptophysin is that its cytoplasmic C-terminus largely consists of pentapeptide repeats initiated by a tyrosine residue. Synaptophysin is heavily phosphorylated by tyrosine kinases in the nerve terminal, suggesting that this phosphorylation is central to its function. This review will cover the evidence for tyrosine phosphorylation of synaptophysin and how this phosphorylation may control its function in the SV life cycle.

Amino Acid Sequence↗

PubMed2002

Phosphorylation of 46-kDa protein of synaptic vesicle membranes is stimulated by GTP and Ca2+/calmodulin.

The release of neurotransmitter is regulated in the processes of membrane docking and membrane fusion between synaptic vesicles and presynaptic plasma membranes. Synaptic vesicles contain a diverse set of proteins that participate in these processes. Small GTP-binding proteins exist in the synaptic vesicles and are suggested to play roles for the regulation of neurotransmitter release. We have examined a possible role of GTP-binding proteins in the regulation of protein phosphorylation in the synaptic vesicles. GTPgammaS stimulated the phosphorylation of 46 kDa protein (p46) with pI value of 5.0-5.2, but GDPbetaS did not. The p46 was identified as protein interacting with C-kinase 1 (PICK-1) by MALDI-TOF mass spectroscopy analysis, and anti-PICK-1 antibody recognized the p46 spot on 2-dimensional gel electrophoresis. Rab guanine nucleotide dissociation inhibitor (RabGDI), which dissociates Rab proteins from SVs, did not affect phosphorylation of p46. Ca(2+)/calmodulin (CaM), which causes the small GTP-binding proteins like Rab3A and RalA to dissociate from the membranes and stimulates CaM-dependent protein kinase(s) and phosphatase, strongly stimulate the phosphorylation of p46 in the presence of cyclosporin A and cyclophylin. However, RhoGDI, which dissociates Rho proteins from membranes, reduced the phosphorylation of p46 to the extent of about 50%. These results support that p46 was PICK-1, and its phosphorylation was stimulated by GTP and Ca(2+)/CaM directly or indirectly through GTP-binding protein(s) and Ca(2+)/CaM effector protein(s). The phosphorylation of p46 (PICK-1) by GTP and Ca(2+)/CaM may be important for the regulation of transporters and neurosecretion.

Animals↗

PubMed2004

Differential regulation of synaptic vesicle proteins by antidepressant drugs.

Synaptic vesicle proteins (SVP) play a critical role in neurotransmitter release and neural plasticity, and have been implicated in the pathophysiology of psychiatric disorders such as depression. Antidepressant drugs not only alter the level of neurotransmitters, but also modulate de novo gene transcription and synthesis of proteins involved in neural plasticity. In order to investigate the effects of antidepressant compounds on SVP-mRNA levels, the expressions of synaptophysin, synaptotagmin, VAMP, and synapsin-I were analysed by in situ hybridization in rats which had been treated with desipramine, fluoxetine, tranylcypromine, or saline. The results demonstrate that chronic treatment with fluoxetine and tranylcypromine leads to an increased expression of synaptophysin, but decreased expression of synaptotagmin and VAMP in the hippocampus and cerebral cortex. Additionally, synapsin I-mRNA levels in the hippocampus and cerebral cortex are significantly reduced in tranylcypromine-treated animals. This identifies SVP genes as target genes of antidepressant treatment.

Animals↗

PubMed1984

Calcium-dependent calmodulin binding to cholinergic synaptic vesicles.

Calmodulin is a major nerve terminal protein and a potential mediator of calcium-dependent nerve terminal functions. Calcium-dependent calmodulin binding has been reported in secretory membrane preparations including chromaffin granules and crude rat brain vesicles. Here we demonstrate a calcium-dependent calmodulin-binding site on cholinergic synaptic vesicles from electric organ. It is saturable with high affinity (KD = 10 nM; Bmax = 80 pmol/mg). The binding is inhibited by trifluoperazine (I50 = 8 microM) and is at least 1000-fold specific for calmodulin over troponin C. Association and dissociation rates (k = 3.1 X 10(6) M-1S-1; k-1 = 1.3 X 10(-2) S-1) are consistent with the dissociation constant measured at equilibrium. Intact synaptic vesicles bind to calmodulin immobilized on polyacrylamide matrix, suggesting that the binding site is cytoplasmically oriented in the vesicle population. Intact synaptic vesicles bind calmodulin up to 80-fold more effectively than do side fractions from the vesicle purification. The quantitative difference is largely due to latency of binding sites since it disappears when the binding is assayed in detergent. Binding of calmodulin to proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that a subset of nerve terminal and electric organ calmodulin-binding proteins are found in synaptic vesicles.

Adenosine Triphosphate↗

PubMed2006

Synaptic vesicle proteins under conditions of rest and activation: analysis by 2-D difference gel electrophoresis.

Synaptic vesicles are organelles of the nerve terminal that secrete neurotransmitters by fusion with the presynaptic plasma membrane. Vesicle fusion is tightly controlled by depolarization of the plasma membrane and a set of proteins that may undergo post-translational modifications such as phosphorylation. In order to identify proteins that undergo modifications as a result of synaptic activation, we induced massive exocytosis and analysed the synaptic vesicle compartment by benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE and difference gel electrophoresis (DIGE) followed by MALDI-TOF-MS. We identified eight proteins that revealed significant changes in abundance following nerve terminal depolarization. Of these, six were increased and two were decreased in abundance. Three of these proteins were phosphorylated as detected by Western blot analysis. In addition, we identified an unknown synaptic vesicle protein whose abundance increased on synaptic activation. Our results demonstrate that depolarization of the presynaptic compartment induces changes in the abundance of synaptic vesicle proteins and post-translational protein modification.

Animals↗

PubMed2004

The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam.

Here, we show that the synaptic vesicle protein SV2A is the brain binding site of levetiracetam (LEV), a new antiepileptic drug with a unique activity profile in animal models of seizure and epilepsy. The LEV-binding site is enriched in synaptic vesicles, and photoaffinity labeling of purified synaptic vesicles confirms that it has an apparent molecular mass of approximately 90 kDa. Brain membranes and purified synaptic vesicles from mice lacking SV2A do not bind a tritiated LEV derivative, indicating that SV2A is necessary for LEV binding. LEV and related compounds bind to SV2A expressed in fibroblasts, indicating that SV2A is sufficient for LEV binding. No binding was observed to the related isoforms SV2B and SV2C. Furthermore, there is a high degree of correlation between binding affinities of a series of LEV derivatives to SV2A in fibroblasts and to the LEV-binding site in brain. Finally, there is a strong correlation between the affinity of a compound for SV2A and its ability to protect against seizures in an audiogenic mouse animal model of epilepsy. These experimental results suggest that SV2A is the binding site of LEV in the brain and that LEV acts by modulating the function of SV2A, supporting previous indications that LEV possesses a mechanism of action distinct from that of other antiepileptic drugs. Further, these results indicate that proteins involved in vesicle exocytosis, and SV2 in particular, are promising targets for the development of new CNS drug therapies.

Animals↗

PubMed1997

Rim is a putative Rab3 effector in regulating synaptic-vesicle fusion.

Rab3 is a neuronal GTP-binding protein that regulates fusion of synaptic vesicles and is essential for long-term potentiation of hippocampal mossy fibre synapses. More than thirty Rab GTP-binding proteins are known to function in diverse membrane transport pathways, although their mechanisms of action are unclear. We have now identified a putative Rab3-effector protein called Rim. Rim is composed of an amino-terminal zinc-finger motif and carboxy-terminal PDZ and C2 domains. It binds only to GTP (but not to GDP)-complexed Rab3, and interacts with no other Rab protein tested. There is enrichment of Rab3 and Rim in neurons, where they have complementary distributions. Rab3 is found only on synaptic vesicles, whereas Rim is localized to presynaptic active zones in conventional synapses, and to presynaptic ribbons in ribbon synapses. Transfection of PC12 cells with the amino-terminal domains of Rim greatly enhances regulated exocytosis in a Rab3-dependent manner. We propose that Rim serves as a Rab3-dependent regulator of synaptic-vesicle fusion by forming a GTP-dependent complex between synaptic plasma membranes and docked synaptic vesicles.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Hydrogen peroxide inhibits the vacuolar H+-ATPase in brain synaptic vesicles at micromolar concentrations.

Hydrogen peroxide (H2O2) is produced from several sources in brain and may be involved in neurodegeneration and second messenger signaling. Little is known about the effects of H2O2 on transmitter storage in brain synaptic vesicles. Neurotransmitter uptake into synaptic vesicles is driven by an electrochemical proton gradient generated by the vacuolar H+-ATPase (V-ATPase) in the vesicle membrane. We report here that the VATPase in bovine brain synaptic vesicles is highly sensitive to inhibition by micromolar concentrations of H2O2. Glutamate uptake by the vesicles is also inhibited, very likely as a secondary consequence of ATPase inactivation. Dithiothreitol or reduced glutathione reverse H2O2-induced inhibition of the V-ATPase, and ATP or GTP partially protect the ATPase from inhibition by H2O2. These and other results suggest that the mechanism of inhibition of the V-ATPase by H2O2 involves oxidation of a reactive cysteine sulfhydryl group in the ATP binding site. Inhibition of V-ATPase activity would decrease the amount of transmitter stored in synaptic vesicles and thus down-regulate transmitter release during episodes of oxidative stress or in response to second messenger signaling.

Adenosine Triphosphate↗

PubMed2003

Differential distribution and developmental expression of synaptic vesicle protein 2 isoforms in the mouse retina.

Synaptic vesicle protein 2 (SV2), a ubiquitous synaptic vesicle protein, is known to participate in the regulation of Ca(2+)-mediated synaptic transmission, although its precise function has not been established. Three SV2 isoforms (SV2A, SV2B, SV2C) have been identified recently, each of which has a unique distribution in brain, suggesting synapse-specific functions. To determine if SV2A, -B, and -C are differentially distributed among synapses in the retina and the sequence of their development, we examined their distribution and expression patterns immunocytochemically in adult and developing mouse retina. The three SV2 isoforms were differentially distributed in the synapses of the two plexiform layers in the adult retina. SV2A was present in cone, but not rod, terminals in the outer plexiform layer (OPL) and in many synaptic terminals in the inner plexiform layer (IPL). SV2B was present only in the ribbon synapse-containing terminals of rod and cone photoreceptors and bipolar cells. SV2C was present in starburst amacrine cells, other conventional synapses in the IPL of unknown origin, and in presumptive interplexiform cell terminals in the INL and OPL. Each SV2 isoform was expressed in its distinct presynaptic terminals early and throughout postnatal development. In addition, SV2A was transiently expressed by developing horizontal cells. The unique distribution of each isoform suggests potentially distinct functions at different types of synapses, with SV2B having ribbon synapse-specific functions, and SV2C being important for the functions of starburst amacrine cells. Rod and cone terminals contain different complements of SV2 isoforms, indicating that ribbon synapses are not all identical. The early expression of SV2 isoforms prior to initiation of synapse formation suggests that they may have important synapse-specific roles during synaptogenesis.

Amacrine Cells↗

PubMed2006

Inhibition of dynamin completely blocks compensatory synaptic vesicle endocytosis.

The ability of synapses to sustain signal propagation relies on rapid recycling of transmitter-containing presynaptic vesicles. Clathrin- and dynamin-mediated retrieval of vesicular membrane has an undisputed role in synaptic vesicle recycling. There is also evidence for other modes of vesicle retrieval, including bulk retrieval and the so-called kiss-and-run recycling. Whether dynamin in required for these other modes of synaptic vesicle endocytosis remains unclear. Here, we have tested the role of dynamin in synaptic vesicle endocytosis by using a small molecule called dynasore, which rapidly inhibits the GTPase activity of dynamin with high specificity. Endocytosis after sustained or brief stimuli was completely and reversibly blocked by dynasore in cultured hippocampal neurons expressing the fluorescent tracer synaptopHluorin. By contrast, dynasore had no effect on exocytosis. In the presence of dynasore, low-frequency stimulation led to sustained accumulation of synaptopHluorin and other vesicular proteins on the surface membrane at a rate predicted from net exocytosis. These vesicular components remained on surface membranes even after the stimulus was terminated, suggesting that all endocytic events rely on dynamin during low-frequency activity as well as in the period after it. Ultrastructural analysis revealed a reduction in the density of synaptic vesicles and the presence of endocytic structures only at synapses that were stimulated in the presence of dynasore. In sum, our data indicate that dynamin is essential for all forms of compensatory synaptic vesicle endocytosis including any kiss-and-run events.

Animals↗

PubMed2000

Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size.

While the transporters that accumulate classical neurotransmitters in synaptic vesicles have been identified, little is known about how their expression regulates synaptic transmission. We have used adenoviral-mediated transfection to increase expression of the brain vesicular monoamine transporter VMAT2 and presynaptic amperometric recordings to characterize the effects on quantal release. In presynaptic axonal varicosities of ventral midbrain neurons in postnatal culture, VMAT2 overexpression in small synaptic vesicles increased both quantal size and frequency, consistent with the recruitment of synaptic vesicles that do not normally release dopamine. This was confirmed using noncatecholaminergic AtT-20 cells, in which VMAT2 expression induced the quantal release of dopamine. The ability to increase quantal size in vesicles that were already competent for dopamine release was shown in PC12 cells, in which VMAT2 expression increased the quantal size but not the number of release events. These results demonstrate that vesicle transporters limit the rate of transmitter accumulation and can alter synaptic strength through two distinct mechanisms.

Adenoviridae↗

PubMed1990

Immunogold electron microscopic demonstration of glutamate and GABA in normal and deafferented cerebellar cortex: correlation between transmitter content and synaptic vesicle size.

Selective labeling of mossy fiber terminals and parallel fibers was obtained in rat cerebellar cortex by a glutamate antibody produced and characterized by Hepler et al. The high-resolution electron microscopic immunogold demonstration of this amino acid offered the possibility of determining the size and shape of synaptic vesicles in glutamate-positive mossy endings. Mossy terminals that stained with the glutamate antibody formed two distinct populations, one with spherical synaptic vesicles with an average diameter of 34.0 nm (more than 90% of all mossy fiber endings) and one with pleomorphic and smaller synaptic vesicles which had an average diameter of 28.5 nm. We present experimental evidence that the mossy terminals with large round vesicles are of extracerebellar origin, whereas those with small pleomorphic synaptic vesicles are endings of nucleocortical fibers. The presence of two distinct classes of gamma-aminobutyric acid (GABA)-containing axon terminals within cerebellar glomeruli has also been demonstrated; those originating from the cerebellar nuclei contain large (36.2 nm) synaptic vesicles, whereas the majority of GABA-stained axon terminals that are of local (cortical) origin contain small (29.1 nm) synaptic vesicles. It therefore appears that, at least in the case of glutamate and GABA, morphological characterization of the axon terminals based on the size and shape of synaptic vesicles is not a reliable indicator of their functional nature (i.e., whether they are excitatory or inhibitory); convincing evidence for the identity of the transmitter can be obtained only by electron microscopic immunostaining procedures. Our results also suggest the existence of both inhibitory and excitatory feedback from cerebellar nuclei to cerebellar cortex.

Animals↗

PubMed1985

A 38,000-dalton membrane protein (p38) present in synaptic vesicles.

A protein with an apparent molecular mass of 38,000 daltons designated p38 was found in synaptic vesicles from rat brain. The subcellular distribution of p38 and some of its properties were determined with the aid of polyclonal and monoclonal antibodies. The subcellular distribution of p38 was similar to that of synapsin I, a synaptic-vesicle specific phosphoprotein. p38 in the synaptic vesicle fraction purified by controlled-pore glass bead chromatography showed an enrichment of more than 20-fold over the crude homogenate. Immunostaining of sections through various brain regions revealed an intense labeling of most, and possibly all, nerve terminals. Only faint reaction in the region of the Golgi apparatus and no detectable labeling of axons and dendrites was observed. Two-dimensional electrophoresis revealed that p38 has an acidic pI. Solubilization experiments, as well as phase separation experiments using Triton X-114, indicated that p38 is an integral membrane protein. Binding of antibodies to intact synaptic vesicles, as well as controlled proteolytic digestion of intact and detergent-treated vesicles, revealed that p38 has a domain exposed on the cytoplasmic surface.

Animals↗

PubMed1989

The relationship between the number of synaptic vesicles and the amount of transmitter released.

The relationship between the number of synaptic vesicles and the amount of transmitter released from identified synapses was investigated in the dorsal longitudinal flight muscle (DLM) of the temperature-sensitive endocytosis mutant of Drosophila melanogaster, shibirets-1(shi). In the shi fly at 29 degrees C, vesicle recyling is blocked, but transmitter release proceeds normally. Thus, by inducing transmitter release at 29 degrees C, shi synapses gradually become depleted of synaptic vesicles. In this way it was possible to regulate the number of vesicles in a synapse. Intracellular recordings were made from individual fibers of the DLM in shi flies after various periods at 29 degrees C while stimulating at 0.5 Hz. The amplitude of the evoked excitatory junction potential (ejp), gradually decreased with longer exposure and was brought to various levels. The fiber was then rapidly fixed for electron microscopy. The number of vesicles per synapse was compared with the amplitude of the ejp at the time of fixation. It was observed that the smaller the ejp amplitudes became, the fewer vesicles were in the synapses. Also, as the ejp amplitude decreased, an increased number of synapses contained no vesicles. It is concluded that synaptic vesicles are directly involved in the release process.

Action Potentials↗