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PubMed1989

Glutamate uptake into synaptic vesicles: competitive inhibition by bromocriptine.

The ATP-dependent uptake of L-glutamate into synaptic vesicles has been well characterized, implicating a key role for synaptic vesicles in glutamatergic neurotransmission. In the present study, we provide evidence that vesicular glutamate uptake is selectively inhibited by the peptide-containing halogenated ergot bromocriptine. It is the most potent inhibitor of the agents tested: the IC50 was determined to be 22 microM. The uptake was also inhibited by other ergopeptines such as ergotamine and ergocristine, but with less potency. Ergots devoid of the peptide moiety, however, such as ergonovine, lergotrile, and methysergide, had little or no effect. Although bromocriptine is known to elicit dopaminergic and serotonergic effects, its inhibitory effect on vesicular glutamate uptake was not mimicked by agents known to interact with dopamine and serotonin receptors. Kinetic data suggest that bromocriptine competes with glutamate for the glutamate binding site on the glutamate translocator. It is proposed that this inhibitor could be useful as a prototype probe in identifying and characterizing the vesicular glutamate translocator, as well as in developing a more specific inhibitor of the transport system.

Adenosine Triphosphate↗
PubMed1998

Defect in synaptic vesicle precursor transport and neuronal cell death in KIF1A motor protein-deficient mice.

The nerve axon is a good model system for studying the molecular mechanism of organelle transport in cells. Recently, the new kinesin superfamily proteins (KIFs) have been identified as candidate motor proteins involved in organelle transport. Among them KIF1A, a murine homologue of unc-104 gene of Caenorhabditis elegans, is a unique monomeric neuron- specific microtubule plus end-directed motor and has been proposed as a transporter of synaptic vesicle precursors (Okada, Y., H. Yamazaki, Y. Sekine-Aizawa, and N. Hirokawa. 1995. Cell. 81:769-780). To elucidate the function of KIF1A in vivo, we disrupted the KIF1A gene in mice. KIF1A mutants died mostly within a day after birth showing motor and sensory disturbances. In the nervous systems of these mutants, the transport of synaptic vesicle precursors showed a specific and significant decrease. Consequently, synaptic vesicle density decreased dramatically, and clusters of clear small vesicles accumulated in the cell bodies. Furthermore, marked neuronal degeneration and death occurred both in KIF1A mutant mice and in cultures of mutant neurons. The neuronal death in cultures was blocked by coculture with wild-type neurons or exposure to a low concentration of glutamate. These results in cultures suggested that the mutant neurons might not sufficiently receive afferent stimulation, such as neuronal contacts or neurotransmission, resulting in cell death. Thus, our results demonstrate that KIF1A transports a synaptic vesicle precursor and that KIF1A-mediated axonal transport plays a critical role in viability, maintenance, and function of neurons, particularly mature neurons.

Animals↗
PubMed1983

Effect of lanthanum ions on the amplitude distributions of miniature endplate potentials and on synaptic vesicles in frog neuromuscular junctions.

Miniature endplate potential (MEPP) amplitude distributions, MEPP frequencies and percentages of small MEPPs were determined as well as synaptic vesicle diameters and numbers in the frog neuromuscular junction during La3+ treatment. MEPP frequencies initially increased by two orders of magnitude and then fell to very low values. La3+ treatment had an initial postsynaptic effect making the MEPPs larger. Prolonged treatment had a variable effect on MEPP amplitudes. There were considerable variations in MEPP frequencies in adjacent junctions so single junctions on edge muscle fibers were recorded for the duration of many experiments and later identified in the electron microscope. Therefore, the physiological and morphological conditions of a given identified junction could be compared. There was a loss of synaptic vesicles and no change in mean diameter during depletion. During high MEPP frequencies infoldings occurred on the axolemma and these disappeared when MEPP frequencies decreased towards the end of the La3+ treatment. After 3-4 h of La3+ treatment, the overall frequency of MEPPs dropped and many were composed of a small class of MEPPs. It is suggested that the morphological correlate of small MEPPs, as well as the classical bell-MEPPs is likely to be synaptic vesicles.

Animals↗
PubMed1992

The phosphorylation of kinesin regulates its binding to synaptic vesicles.

Membrane organella are transported bidirectionally in cells, and the axonal transport system has provided an ideal model system for studying this bidirectional transport. Kinesin and cytoplasmic dynein were identified as candidates for the motor molecules of fast axonal transport, which transport organella along microtubules anterogradely and retrogradely. However, the mechanism that controls this bidirectional transport is unknown. Our previous work revealed that kinesin in axons was associated abundantly with anterogradely transported membranous organella, most of which are believed to be precursors of synaptic vesicles and axonal plasma membranes, while the fractions bound to retrogradely transported ones were very small (Hirokawa, N., Sato-Yoshitake, R., Kobayashi, N., Pfister, K. K., Bloom, G. S., and Brady, S. T. (1991) J. Cell Biol. 114, 295-302). Here we demonstrated in vitro that the binding of kinesin to synaptic vesicles was concentration-dependent and saturable and could be released by high salt concentration. When kinesin was phosphorylated by cAMP-dependent protein kinase, its binding to symaptic vesicles was significantly reduced. By motility assay and by statistical analysis using electron microscopy, we further revealed that synaptic vesicles preincubated with phosphorylated kinesin associated less frequently with microtubules than synaptic vesicles preincubated with unphosphorylated kinesin. The phosphorylation of kinesin should therefore play an essential role in regulating the direction of fast axonal transport by inhibiting its binding to membrane organella, thus releasing it from membrane organella at nerve terminals.

Animals↗
PubMed1994

Distribution of synaptic markers in the retina: implications for synaptic vesicle traffic in ribbon synapses.

Synapses in the retina are present in two layers that can easily be distinguished morphologically: the thin outer plexiform layer (OPL) containing the photoreceptor ribbon synapses, and the more complex inner plexiform layer (IPL) containing mostly conventional synapses. In the current study we have used the exquisite spatial separation between ribbon and conventional synapses in the retina to investigate by immunocytochemistry the protein components of these two types of synapses. Our results show that all of the synaptic vesicle proteins tested are present in the ribbon synapses of photoreceptors except for the synapsins which were previously described to be absent from ribbon synapses. Thus, synaptic vesicles of ribbon and conventional synapses are likely to be very similar molecularly in spite of their different modes of trafficking. Furthermore, proteins with a likely function in synaptic vesicle docking and fusion (SNAP-25, munc-18) and endocytosis (dynamin) were also highly enriched in ribbon synapses, indicating that the basic machineries for synaptic membrane traffic are similar in ribbon and conventional synapses. Interestingly, photoreceptor cells contained only low concentrations of syntaxin I (which functions in the synaptic vesicle fusion complex) and GDI (which regulates the membrane association of rab-proteins as a function of GTP/GDP binding). These results raise the possibility that photoreceptors express other isoforms of these proteins, or that the functions of these proteins are not required for the tonical release mode of their synapses.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Ontogeny of glutamate accumulating activity in rat brain synaptic vesicles.

The relationship between the ontogeny of the vesicular glutamate uptake system and synaptogenesis in rats was investigated. For this purpose we have developed a simplified procedure for the preparation of crude synaptic vesicles which are sufficiently pure to demonstrate a highly ATP-dependent glutamate uptake. ATP-dependent glutamate uptake into synaptic vesicles was found to increase dramatically starting on postnatal day 10 and reaching a maximum on day 30 (76 +/- 40 and 657 +/- 40 pmol/mg protein/10 min, respectively), correlating well with the active period of synaptogenesis. Stimulation of uptake by chloride also developed in parallel with the vesicular glutamate uptake. In contrast, combined non-ATP-dependent uptake and non-specific binding remained constant (21 +/- 6 pmol/mg protein/10 min). This development of vesicular glutamate uptake during the period of synaptogenesis supports the notion that synaptic vesicles play an important role in glutamate synaptic transmission.

Aging↗
PubMed2003

Cdk5 and the mystery of synaptic vesicle endocytosis.

Regulation of endocytosis by protein phosphorylation and dephosphorylation is critical to synaptic vesicle recycling. Two groups have now identified the neuronal kinase Cdk5 (cyclin-dependent kinase 5) as an important regulator of this process. Robinson and coworkers recently demonstrated that Cdk5 is necessary for synaptic vesicle endocytosis (SVE) (Tan et al., 2003), whereas a new report in this issue claims that Cdk5 negatively regulates SVE (Tomizawa et al., 2003). Careful examination of the data reveals a model that helps resolve the apparently contradictory nature of these reports.

Animals↗
PubMed1998

Phospholipid translocation from the outer to the inner leaflet of synaptic vesicle membranes isolated from the electric organ of Japanese electric ray Narke japonica.

The phospholipid translocation from the outer to the inner leaflet of synaptic vesicles isolated from the electric organ of the Japanese electric ray, Narke japonica, was measured using fluorescent phospholipid probes. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylserine (PS) with a fluorescent NBD-labeled short acyl chain at the sn-2 position was mixed with purified synaptic vesicles and the probe in the outer leaflet of the membranes was reduced with dithionite to quench the fluorescence from time to time. The percentage of fluorescence remaining after the dithionite treatment served as an index for the phospholipid translocation. The results obtained indicated that about 30, 13, and 9% of NBD-PE, NBD-PS, and NBD-PC, respectively, were translocated from the outer to the inner leaflet in 3 h. Thus, the translocation activity in synaptic vesicle membranes was much higher for PE than for PS, in contrast to the previous results obtained with plasma membranes, including synaptosomal membranes. The percentages of the phospholipid in the inner leaflet at equilibrium were estimated to be 41, 31, and 14% for PE, PS, and PC, respectively. The translocation was inhibited by pretreatment with an SH reagent, iodoacetamide, indicating the involvement of a proteinaceous translocator. These data may provide a biochemical basis for elucidating the mechanisms of membrane fusion and exocytosis at nerve endings.

4-Chloro-7-nitrobenzofurazan↗
PubMed2004

Synaptophysin: leading actor or walk-on role in synaptic vesicle exocytosis?

Synaptophysin (Syp) was the first synaptic vesicle (SV) protein to be cloned. Since its discovery in 1985, it has been used by us and by many laboratories around the world as an invaluable marker to study the distribution of synapses in the brain and to uncover the basic features of the life cycle of SVs. Although single gene ablation of Syp does not lead to an overt phenotype, a large body of experimental data both in vitro and in vivo indicate that Syp (alone or in association with homologous proteins) is involved in multiple, important aspects of SV exo-endocytosis, including regulation of SNARE assembly into the fusion core complex, formation of the fusion pore initiating neurotransmitter release, activation of SV endocytosis and SV biogenesis. In this article, we summarise the main results of the studies on Syp carried out by our and other laboratories, and explain why we believe that Syp plays a major role in SV trafficking.

Animals↗
PubMed2002

Essential role of endophilin A in synaptic vesicle budding at the Drosophila neuromuscular junction.

We characterized Drosophila endophilin A (D-endoA), and generated and analysed D-endoA mutants. Like its mammalian homologue, D-endoA exhibits lysophosphatidic acid acyl transferase activity and contains a functional SH3 domain. D-endoA is recruited to the sites of endocytosis, as revealed by immunocytochemistry of the neuromuscular junction (NMJ) of mutant L3 larvae carrying the temperature-sensitive allele of dynamin, shibire. D-endoA null mutants show severe defects in motility and die at the early L2 larval stage. Mutants with reduced D-endoA levels exhibit a range of defects of synaptic vesicle endocytosis, as observed at L3 larvae NMJs using FM1-43 uptake and electron microscopy. NMJs with an almost complete loss of synaptic vesicles did not show an accumulation of intermediates of the budding process, whereas NMJs with only slightly reduced levels of synaptic vesicles showed a striking increase in early-stage, but not late-stage, budding intermediates at the plasma membrane. Together with results of previous studies, these observations indicate that endophilin A is essential for synaptic vesicle endocytosis, being required from the onset of budding until fission.

Acyl Coenzyme A↗
PubMed1998

Substructure and responses of cholinergic synaptic vesicles in the atomic force microscope.

The substructure and responses of individual 100-nm synaptic vesicles to osmotic stress have been probed with an atomic force microscope (AFM) operating in tapping mode. Cholinergic synaptic vesicles from the electric organ of Torpedo californica were imaged continuously as the osmolarity of the buffer was decreased. Vesicles in hyposmotic buffer lysed to form flat circular structures on the mica surface with a diameter about two times that of intact vesicles and a thickness of 7.2 +/- 1.7 nm, which can accommodate the lipid bilayer plus the internal proteoglycan. Images of intact vesicles in air reveal creases in the membrane surface. Phase mode AFM images of lysed vesicles in air show the presence of a material not seen on intact vesicles that might be intravesicular proteoglycan released from the membrane at very low osmotic and ionic strength.

Acetylcholine↗
PubMed2005

Properties of synaptic vesicle pools in mature central nerve terminals.

Readily releasable and reserve pools of synaptic vesicles play different roles in neurotransmission, and it is important to understand their recycling and interchange in mature central synapses. Using adult rat cerebrocortical synaptosomes, we have shown that 100 mosm hypertonic sucrose caused complete exocytosis of only the readily releasable pool (RRP) of synaptic vesicles containing glutamate or gamma-aminobutyric acid. Repetitive hypertonic stimulations revealed that this pool recycled (and reloaded the neurotransmitter from the cytosol) fully in <30 s and did so independently of the reserve pool. Multiple rounds of exocytosis could occur in the constant absence of extracellular Ca(2+). However, although each vesicle cycle includes a Ca(2+)-independent exocytotic step, some other stage(s) critically require an elevation of cytosolic [Ca(2+)], and this is supplied by intracellular stores. Repetitive recycling also requires energy, but not the activity of phosphatidylinositol 4-kinase, which maintains the normal level of phosphoinositides. By varying the length of hypertonic stimulations, we found that approximately 70% of the RRP vesicles fused completely with the plasmalemma during exocytosis and could then enter silent pools, probably outside active zones. The rest of the RRP vesicles underwent very fast local recycling (possibly by kiss-and-run) and did not leave active zones. Forcing the fully fused RRP vesicles into the silent pool enabled us to measure the transfer of reserve vesicles to the RRP and to show that this process requires intact phosphatidylinositol 4-kinase and actin microfilaments. Our findings also demonstrate that respective vesicle pools have similar characteristics and requirements in excitatory and inhibitory nerve terminals.

1-Phosphatidylinositol 4-Kinase↗
PubMed1989

Glutamate uptake into synaptic vesicles of bovine cerebral cortex and electrochemical potential difference of proton across the membrane.

Measurements have been made of the ATP-dependent membrane potential (delta psi) and pH gradient (delta pH) across the membranes of the synaptic vesicles purified from bovine cerebral cortex, using the voltage-sensitive dye bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxanol and the delta pH-sensitive fluorescent dye 9-aminoacridine respectively. A pre-existing small delta pH (inside acidic) was detected in the synaptic vesicles, but no additional significant contribution by MgATP to delta pH was observed. In contrast, delta psi (inside positive) increased substantially upon addition of MgATP. This ATP-dependent delta psi was reduced by thiocyanate anion (SCN-), a delta psi dissipator, or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a protonmotive-force dissipator. Correspondingly, a substantially larger glutamate uptake occurred in the presence of MgATP, which was inhibited by SCN- and FCCP. A nonhydrolysable analogue of ATP, adenosine 5'-[beta gamma-methylene]triphosphate, did not substitute for ATP in either delta psi generation or glutamate uptake. The results support the hypothesis that a H+-pumping ATPase generates a protonmotive force in the synaptic vesicles at the expense of ATP hydrolysis, and the protonmotive force thus formed provides a driving force for the vesicular glutamate uptake. The delta psi generation by ATP hydrolysis was not affected by orthovanadate, ouabain or oligomycin, but was inhibited by N-ethylmaleimide, quercetin, trimethyltin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid. These results indicate that the H+-pumping ATPase in the synaptic vesicle is similar to that in the chromaffin granule, platelet granule and lysosome.

Adenosine Triphosphatases↗
PubMed1993

An anion binding site that regulates the glutamate transporter of synaptic vesicles.

Glutamate, the major excitatory neurotransmitter of the mammalian central nervous system, is stored in synaptic vesicles and released by exocytosis upon depolarization of the presynaptic nerve terminal. Synaptic vesicles possess an active glutamate-specific transporter that is driven by an electrochemical proton gradient across the vesicle membrane and requires chloride for maximal activity. In this study, we have characterized the role of chloride in vesicular glutamate transport using 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a potent inhibitor of anion translocators. DIDS inhibited glutamate uptake with an IC50 of 0.7 microM or less. In contrast, all energy gradient parameters (membrane potential, pH gradient, and ATPase activity) required at least 5-fold higher concentration of DIDS for inhibition. Furthermore, high concentrations of chloride but not of glutamate or other anions prevented DIDS inhibition of glutamate uptake. In contrast to uptake, glutamate efflux from glutamate-loaded vesicles was independent of chloride over a wide concentration range. However, efflux was still susceptible to DIDS inhibition. DIDS inhibition was prevented by excess chloride. We conclude that the vesicular glutamate transporter possesses a DIDS-sensitive chloride binding site on the cytoplasmic side, distinct from the substrate binding site, which regulates transport activity.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Ammonia added in vitro, but not moderate hyperammonemia in vivo, stimulates glutamate uptake and H(+)-ATPase activity in synaptic vesicles of the rat brain.

The uptake of radiolabelled neurotransmitters: glutamate (GLU), GABA, and dopamine (DA) and the activity of the vacuolar type H(+)-pumping ATPase (H(+)-ATPase), were measured in crude synaptic vesicles treated in vitro with a neurotoxic (3 mM) dose of NH4+ (acetate or chloride), or isolated from rats with a moderate increase of brain ammonia (to approximately 0.6 mM) induced by i.p. administration of ammonium acetate (HA rats) or a hepatotoxin-thioacetamide (HE rats). In vitro treatment with ammonium salts increased the sodium-independent, chloride-dependent uptake of GLU but did not stimulate the uptake of GABA or DA. The in vitro treatment also stimulated the H(+)-ATPase activity. Since H(+)-ATPase generates the electrochemical gradient driving synaptic vesicular neurotransmitter transport, its stimulation by ammonia may have facilitated GLU uptake. However the GLU specificity of the effect must be related to other factors differentially affecting GLU uptake and the uptake of other neurotransmitters. Enhanced GLU accumulation in the synaptic vesicles may contribute to the increase of synaptic GLU exocytosis previously reported to accompany acute increases of brain ammonia to toxic levels. However, GLU uptake and H(+)-ATPase activity, but also the uptake of GABA and DA, were unchanged in synaptic vesicles prepared from rats with HA or HE. This indicates that changes in GLU and/or GABA release reported for moderate hyperammonemic conditions must be elicited by factors unrelated to the synaptic vesicular transport of the amino acids.

Ammonia↗
PubMed2006

Donut-like topology of synaptic vesicles with a central cluster of mitochondria wrapped into membrane protrusions: a novel structure-function module of the adult calyx of Held.

Structural and functional properties of synapses are intricately and reciprocally coupled. To cope with the functional requirements in auditory processing, the calyx of Held developed distinct structural specializations such as a large number of active zones, large size, elaborate morphology, and defined distribution of ion channels. These specializations typically appear during postnatal maturation within the first 3 weeks of life and are accompanied by marked changes in the properties of synaptic transmission. We examined the arrangement of synaptic vesicles at different postnatal stages of maturation by genetically labeling vesicles with the fluorescent fusion protein synaptophysin-enhanced green fluorescent protein. Fluorescence and electron microscopy-based analyses revealed a new anatomical specialization in the mature calyx of Held. Within small, membrane-delimited compartments (swellings), synaptic vesicles formed donut-like assemblies around a central cluster of interconnected mitochondria. Adult calyces contained approximately 100 such structural units, each of them consisting of approximately 800 synaptic vesicles, six to nine mitochondria, and five to nine active zones. A donut of synaptic vesicles measured approximately 1 microm in diameter and was placed in a swelling with a volume of approximately 5 fl. Conspicuously, this structural specialization appears with the onset of hearing and may contribute to maturational changes in presynaptic function.

Age Factors↗
PubMed2006

Evidence for early endosome-like fusion of recently endocytosed synaptic vesicles.

Early endosomes are well-established acceptor compartments of endocytic vesicles in many cell types. Little evidence of their existence or function has been obtained in synapses, and it is generally believed that synaptic vesicles recycle without passing through an endosomal intermediate. We show here that the early endosomal SNARE proteins are enriched in synaptic vesicles. To investigate their function in the synapse, we isolated synaptic nerve terminals (synaptosomes), stimulated them in presence of different fluorescent markers to label the recycling vesicles and used these vesicles in in vitro fusion assays. The recently endocytosed vesicles underwent homotypic fusion. They also fused with endosomes from PC12 and BHK cells. The fusion process was dependent upon NSF activity. Moreover, fusion was dependent upon the early endosomal SNAREs but not upon the SNAREs involved in exocytosis. Our results thus show that at least a fraction of the vesicles endocytosed during synaptic activity are capable of fusing with early endosomes and lend support to an involvement of endosomal intermediates during recycling of synaptic vesicles.

Animals↗