Summary
Synaptophysin and synaptobrevin, two integral proteins of synaptic vesicles, have been used as immunocytochemical markers of the synaptic vesicle membrane during Cd2+- or K+-induced ACh release at the frog neuromuscular junction. ACh release was stimulated in cutaneous pectoris nerve-muscle preparations by: (1) 1 mM Cd2+ in Ca2+-free medium for a period of 3 h, (2) 25 or 40mM K+ in normal Ringer's solution. Synaptophysin and synaptobrevin were immunolabelled in single fibres teased from fixed muscles using rabbit antisera raised against synaptophysin and synaptobrevin revealed with fluoresceinconjugated IgG. The postsynaptic ACh receptors were simultaneously labelled with rhodaminated α-bungarotoxin. Unstimulated and K+-stimulated preparations showed synaptophysin and synaptobrevin immunolabelling only after membrane permeabilization with 0.1% Triton X-100. In preparations stimulated with Cd2+ in Ca2+-free medium, the immunofluorescence was also observed in non Triton X-100 treated muscle fibres. Confocal laser scanning microscopy analysis revealed that in unstimulated and K+-stimulated preparations, synaptophysin and synaptobrevin immunofluorescence appears as bands regularly spaced along the permeabilized nerve terminals and that their distribution corresponds to clusters of synaptic vesicles. After Cd2+ stimulation in Ca2+-free medium, labelling for both proteins is irregularly distributed, being more intense at the lateral margins of swollen nerve terminals, suggesting a translocation of synaptic vesicle proteins to the axolemma. At the electron microscopic level, Cd2+ stimulation in Ca2+-free medium produces nerve terminal swelling and synaptic vesicle depletion. The results show that when ACh release is stimulated under an impairment of synaptic vesicle recycling, which leads to synaptic vesicle depletion, synaptophysin and synaptobrevin translocation occurs. These findings are in favour of a permanent incorporation of synaptic vesicle membrane into the axolemma. In contrast, after K+ stimulation, the immunofluorescence and the normal synaptic vesicle population observed, suggest that a double process of synaptic vesicle exo-endocytosis rapidly occurs, without incorporation of synaptic vesicle components into the axolemma.
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References
Baumert, M., Maycox, P. R., Navone, F., De Camilli, P. &Jahn, R. (1989) Synaptobrevin: an integral membrane protein of 18 000 daltons present in small synaptic vesicles of rat brain.EMBO Journal 8, 379–84.
Bennet, M. K., Calakos, N. &Scheller, R. H. (1992) Syntaxin: a synaptic vesicle protein implicated in docking of synaptic vesicles at the presynaptic active zones.Science 257, 255–9.
Betz, W. J., Mao, F. &Bewick, G. S. (1992) Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals.Journal of Neuroscience 12, 363–75.
Ceccarelli, B. &Hurbut, W. P. (1980a) Vesicle hypothesis of the release of quanta of acetylcholine.Physiological Reviews 60, 396–441.
Ceccarelli, B. &Hurlbut, W. P. (1980b) Ca2+-dependent recycling of synaptic vesicles at the frog neuromuscular junction.Journal of Cell Biology 87, 297–303.
Ceccarelli, B., Hurlbut, W. P. &Mauro, A. (1973) Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction.Journal of Cell Biology 57, 499–524.
Ceccarelli, B., Grohovac, F. &Hurlbut, W. P. (1979a) Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. I. Effects of black widow spider venom and Ca2+-free solutions on the structure of the active zone.Journal of Cell Biology 81, 163–77.
Ceccarelli, B., Grohovac, F. &Hurlbut, W. P. (1979b) Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. II. Effects of electrical stimulation and high potasium.Journal of Cell Biology 81, 178–92.
Chavez, E., Briones, R., Michel, B., Bravo, C. &Jay, D. (1985) Evidence for the involvement of dithiol groups in mitochondrial calcium transport: studies with cadmium.Archives of Biochemistry and Biophysics 242, 493–7.
Colasante, C., Brouard, M. O. &Pécot-Dechavas-Sine, M. (1993) Synaptophysin immunolabelling at the mouse neuromuscular junction.Neuromuscular Disorders 3, 395–400.
Couteaux, R. &Pécot-Dechavassine, M. (1970) Vésicules synaptiques et poches au niveau des ‘zones actives’ de la jonction neuromusculaire.Comptes rendus des Séances de l'Académie des Sciences 271, 2346–9.
Debello, W. M., Betz, H. &Austine, G. J. (1993) Synaptotagmin and neurotransmitter release.Cell 74, 947–50.
De Camilli, P., Harris, S. M., Huttner, W. &Green-Gard, P. (1983) Synapsin (protein I), a nerve terminalspecific phosphoprotein. II Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose embedded synaptosomes.Journal of Cell Biology 96, 1355–73.
Elferink, L. A., Peterson, M. R. &Scheller, R. H. (1993) A role of synaptotagmin (p65) in regulated exocytosis.Cell 72, 153–9.
Guan, Y. Y., Quastel, D. M. J. &Saint, D. A. (1987) Multiple actions of cadmium on transmitter release at the mouse neuromuscular junction.Canadian Journal of Physiology and Pharmacology 65, 2131–6.
Hagiwara, S. &Byerly, L. (1981) Calcium channel.Annual Review of Neuroscience 4, 69–125.
Heuser, J. E. &Reese, T. S. (1973) Evidence for recycling of synaptic vesicle membrane during transmitter release at frog neuromuscular junction.Journal of Cell Biology 57, 315–44.
Heuser, J. E. &Reese, T. S. (1981) Structural changes after transmitter release at the frog neuromuscular junction.Journal of Cell Biology 88, 564–80.
Heuser, J. E., Reese, T. S. &Landis, D. M. D. (1974) Functional changes in frog neuromuscular junctions studied with freeze-fracture.Journal of Neurocytology 3, 109–31.
Heuser, J. E., Reese, T. S., Dennis, M. J., Jan, Y., Jan, L. &Evans, L. (1979) Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release.Journal of Cell Biology 81, 275–300.
Jahn, R., Schiebler, W., Ouimet, C. &Greengard, P. (1985) A 38 000-dalton membrane protein (p38) present in synaptic vesicles.Proceedings of the National Academy of Science (USA) 82, 4137–41.
Léveque, C. El, Far, O., Martin-Moutot, N., Sato, K., Kato, R., Takahashi, M. &Seagar, M. (1994) Purification of the N-type calcium channel associated with syntaxin and synaptotagmin.Journal of Biological Chemistry 269, 6306–12.
Marxen, P. &Bigalke, H. (1989) Tetanus toxin: inhibitory action in chromaffin cells is initiated by specified types of gangliosides and promoted in low ionic strength solution.Neuroscience Letters 107, 261–6.
Matteoli, M. &De Camilli, P. (1991) Molecular mechanism of neurotransmitter release.Current Opinions in Neurobiology 1, 91–7.
Miller, T. M. &Heuser, J. E. (1984) Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction.Journal of Cell Biology 98, 685–98.
Molgó, J., Pécot-Dechavassine, M. &Thesleff, S. (1989) Effects of cadmium on quantal transmitter release and ultrastructure of frog motor nerve endings.Journal of Neural Transmission 77, 79–91.
Navone, F., Greengard, P. &De Camilli, P. (1984) Synapsin I in nerve terminals: selective association with small synaptic vesicles.Science 226, 1209–11.
Niemann, H., Blasi, J. &Jahn, R. (1994) Clostridial neurotoxins: new tools for dissecting exocytosis.Trends in Cell Biology 4, 179–85.
Palacios-Prü, E., Mendoza, R. V., Palacios, L. &Colasante, C. (1983) Morphological changes in neuromuscular junction during exercise.Journal of Neuroscience Research 9, 371–80.
Pécot-Dechavassine, M. (1982) Synaptic vesicle openings captured by cooling and related to transmitter release at the frog neuromuscular junction.Biology of the Cell 46, 43–50.
Pécot-Dechavassine, M. (1983) Morphological evidence for tracer uptake at the active zones of stimulated frog neuromuscular junction.Experientia 39, 752–3.
Petrenko, A. G., Perin, M. S., Davletov, B. A., Ushkaryov, Y. A., Geppert, M. &Südhof, T. C. (1991) Binding of synaptotagmin to the α-latrotoxin receptor implicates both in synaptic vesicle exocytosis.Nature 353, 65–8.
Poulain, B., Rossetto, O., Deloye, F., Schiavo, G., Tauc, L. &Montecucco, C. (1993) Antibodies against rat brain vesicle-associated membrane protein (synaptobrevin) prevent inhibition of acetylcholine release by tetanus toxin or botulinum neurotoxin type B.Journal of Neurochemistry 61, 1175–8.
Schiavo, G., Benfenati, P., Poulain, B., Rosseto, O., Polverino, De Laureto, P., Dasgupta, B. R. &Montecucco, C. (1992) Tetanus toxin and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevm.Nature 359, 832–5.
Schiebler, W., Jahn, R., Doucet, J. P., Rothlein, J. &Greengard, P. (1986) Characterization of Synapsin I binding to small synaptic vesicles.Journal of Biological Chemistry 261, 8383–90.
Simons, T. J. B. &Pocock, G. (1987) Lead enters bovine adrenal medullary cells through calcium channels.Journal of Neurochemistry 48, 383–9.
Söllner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempts, P. &Rothman, J. E. (1993a) SNAP receptor implicated in vesicle targeting and fusion.Nature 362, 318–24.
Söllner, T., Bennet, K. M., Whiteheart, S. W., Scheller, R. H. &Rothman, J. E. (1993b) A protein assembly-disassembly pathwayin vitro that may correspond to sequential steps of synaptic vesicle docking, activation and fusion.Cell 75, 409–18.
Torri-Tarelli, F., Grohovac, F., Fesce, P. &Ceccar-Elli, B. (1985) Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine.Journal of Cell Biology 101, 1386–99.
Torri-Tarelli, F., Villa, A., Valtorta, F., De Camilli, P., Greengard, P. &Ceccarelli, B. (1990) Redistribution of synaptophysin and synapsin I during α-latrotoxin-induced release of neurotransmitter at the neuromuscular junction.Journal of Cell Biology 110, 449–59.
Toury, R., Boissonneau, E., Stelly, N., Dupuis, Y., Berville, A. &Perasso, R. (1985a) Mitochondria alterations in Cd2+-treated rats: general regression of inner membrane cristae and electron transport impairment.Biology of the Cell 55, 71–86.
Toury, R., Stelly, N., Boissonneau, E. &Dupuis, Y. (1985b) Degenerative processes in skeletal muscle of Cd2+-treated rats and Cd2+ inhibition of mitochondrial Ca2+ transport.Toxicology and Applied Pharmacology 77, 19–35.
Trimble, W. S., Cowan T. S. &Scheller, R. H. (1988) VAMP-1: A synaptic vesicle-associated integral membrane protein.Proceedings of the National Academy of Sciences (USA) 85, 4538–2.
Tsien, R. W. (1983) Calcium channels in excitable cell membranes.Annual Review of Physiology 45, 341–58.
Valtorta, P., Villa, A., Jahn, J. E., De Camilli, P., Greengard, P. &Ceccarelli, B. (1988a) Localization of synapsin I at the frog neuromuscular junction.Neuroscience 24, 593–603.
Valtorta, F., Jahn, R., Fesce, R., Greengard, P. &Ceccarelli, B. (1988b) Synaptophysin (p38) at the frog neuromuscular junction: its incorporation into the axolemma and recycling after intense quantal secretion.Journal of Cell Biology 107, 2717–27.
Valtorta, P., Fesce, R., Grohovac, F., Haimann, C., Hurlbut, W. P., Iezzi, N., Torri-Tarelli, F., Villa, A. &Ceccarelli, B. (1990) Neurotransmitter release and synaptic vesicle recycling.Neuroscience 35, 477–89.
Vega, M. T., Villalobos, C., Garrido, B., Gandia, L., Bulbena, O., Garcia-Sancho, J., Garcia, A. G. &Artalejo, A. R. (1994) Permeation by zinc of bovine chromaffin cells calcium channels: relevance to secretion.European Journal of Physiology 429, 231–9.
Velasco, M. E. &Pécot-Dechavassine, M. (1993) Membrane events related to transmitter release in mouse motor nerve terminals captured by ultrarapid cryofixation.Journal of Neurocytology 22, 913–23.
Wiedenmann, B. &Franke, W. W. (1985) Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38 000 characteristic of synaptic vesicles.Cell 41, 1017–28.
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Colasante, C., Pécot-Dechavassine, M. Cd2+- and K+-evoked ACh release induce different synaptophysin and synaptobrevin immunolabelling at the frog neuromuscular junction. J Neurocytol 24, 547–558 (1995). https://doi.org/10.1007/BF01257371
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DOI: https://doi.org/10.1007/BF01257371