Summary
The sequence of structural changes occurring in the presynaptic membrane during transmitter release was studied at the mouse neuromuscular junction using the combined quick-freezing and cryosubstitution techniques. The mouse levator auris longus (LAL) muscle was stimulated by two means: either, chemically, by soaking 5 min before freezing in a physiological solution containing 25mm potassium chloride or, electrically, by applying, 10 ms before freezing, a single supramaximal stimulus to the nerve-muscle preparation treated with 50 μM 3,4-diaminopyridine (3,4-DAP) and 100 μM (+)tubocurarine. In both cases, the preparations were maintained at approximately 5 °C, 5 min prior to freezing, in order to prolong nerve membrane changes. In most experiments, tannic acid (0.1%) was added to the substitution medium for better preservation of membranes. The different steps of warming in the substitution medium were strictly controlled from −90 °C to 4 °C. When fixed under chemical stimulation, the presynaptic membrane appeared very sinuous and synaptic vesicles were seen apposed to specialized sites facing subjunctional folds. When submitted to a single electrical stimulus, after treatment with 3,4-diaminopyridine, features of synaptic vesicle fusion were observed at these specialized sites which appear similar by their morphology, their macromolecular organization (already described) and their functional changes to active zones of the frog neuromuscular junction. Other images suggested that with 3,4-diaminopyridine which causes a pronounced and long-lasting release of transmitter, some vesicles collapse after exocytosis instead of being locally reformed by endocytosis.
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Angaut-Petit, D., Molgo, J., Connold, A. L. &Faille, L. (1987) Thelevator auris longus muscle of the mouse: a convenient preparation for studies of short- and long-term presynaptic effects of drugs or toxins.Neuroscience Letters 82, 83–8.
Ceccarelli, B., Hurlbut, W. P. &Mauro, A. (1972) Depletion of vesicles from frog neuromuscular junctions by prolonged tetanic stimulation.Journal of Cell Biology 54, 30–8.
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., Grohovaz, F. &Hurlbut, W. P. (1979) Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter.Journal of Cell Biology 81, 178–92.
Ceccarelli, B. &Hurlbut, W. P. (1980) Vesicle hypothesis of the release of quanta of acetylcholine.Physiological Reviews 60, 396–441.
Chandler, D. E. &Heuser, J. E. (1980) Arrest of membrane fusion events in mast cells by quick-freezing.Journal of Cell Biology 86, 666–74.
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.
De Robertis, E., Rodrigues De Lores Arnaiz, G., Salganicoff, L., De Iraldi, A. &Zieher, L. M. (1963) Isolation of synaptic vesicles and structural organisation of the acetylcholine system within the brain nerve endings.Journal of Neurochemistry 10, 225–35.
Del Castillo, J. &Katz, B. (1954) Quantal components of the end-plate potential.Journal of Physiology 124, 560–73.
Ebersold, H. R., Lüthy, P., Cordier, J. L. &Müller, M. (1981) A freeze-substitution and freeze-fracture study of bacterial spore structures.Journal of Ultrastructure Research 76, 71–81.
Ellisman, M. H., Rash, J. E., Staehelin, A. &Porter, K. R. (1976) Studies of excitable membranes. II. A comparison of specializations at neuromuscular junctions and nonjunctional sarcolemmas of mammalian fast and slow twitch muscle fibers.Journal of Cell Biology 68, 752–74.
Escaig, J. (1982) New instruments which facilitate rapid freezing at 83K and 6K.Journal of Microscopy 126, 221–9.
Gonzalez-Aguilar, F., Rodriguez, J. A., Anzola, R. H. &Lupidio, M. C. (1988) Synaptic vesicle relationships with the presynaptic membrane as shown by a new method of fast chemical fixation.Neuroscience 24, 9–17.
Grohovaz, F., Hurlbut, W. P. &Ceccarelli, B. (1980) Exo-endocytosis in relation to the release of quanta of acetylcholine at frog neuromuscular junctions. InLa transmission neuromusculaire, les médiateurs et le milieu intérieur (edited byColloque Claude Bernard) pp. 2–37. Paris: Masson.
Heuser, J. E. (1977) Synaptic vesicle exocytosis revealed in quick-frozen frog neuromuscular junctions treated with 4-amino-pyridine and given a single electrical shock. InNeuroscience Symposia (edited byCowan, W. M. &Ferrendelli, J. A.) pp. 215–39. Society for Neurosciences: Bethesda.
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, 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.
Hirokawa, N. &Heuser, J. E. (1981) Structural evidence that botulinum toxin blocks neuromuscular transmission by impairing the calcium influx that normally accompanies nerve depolarization.Journal of Cell Biology 88, 160–71.
Hirokawa, N. &Kirino, T. (1980) An ultrastructural study of nerve and glial cells by freeze-substitution.Journal of Neurocytology 9, 243–54.
Ikeda, H. (1985) An ultrastructural analysis of the inclusion body in the type II pneumocyte processed by rapid freezing followed by freeze-substitution. An autoradiographic study.Journal of Electron Microscopy 34, 398–410.
Israel, M., Dunant, Y. &Manaranche, R. (1979) The present status of the vesicular hypothesis.Progress in Neurobiology 13, 237–75.
Israel, M., Gautron, J. &Lesbats, B. (1968) Isolement des vésicules synaptiques de l'organe électrique de la Torpile et localisation de l'acétylcholine à leur niveau.Comptes Rendus de l'Académie des Sciences 266, 273–5.
Katz, B. &Miledi, R. (1965) The effect of temperature on the synaptic delay at the neuromuscular junction.Journal of Physiology 181, 656–70.
Lawson, D., Fewtrell, C. &Raff, M. (1978) Localized mast cell degranulation induced by concanavalin A-sepharose beads. Implication for the Ca2+ hypothesis of stimulus-secretion coupling.Journal of Cell Biology 79, 394–400.
Lentz, T. L. &Chester, J. (1982) Synaptic vesicle recycling at the neuromuscular junction in the presence of a presynaptic membrane marker.Neuroscience 7, 9–20.
Li, C. L. (1958) Effect of cooling on neuromuscular transmission in the Rat.American Journal of Physiology 194, 200–6.
Mazzone, R. W., Durand, C. M. &West, J. B. (1979) Electron microscopic appearances of rapidly frozen lung.Journal of Microscopy 117, 269–84.
Molgo, J. (1982) Effects of aminopyridines on neuromuscular transmission. InAdvances in the Biosciences, Vol. 35. Aminopyridines and similarly acting drugs. Effects on nerves, muscles and synapses (edited byLechat et al.) pp. 95–116. Oxford New York: Pergamon.
Molgo, J., Lundh, H. &Thesleff, S. (1980) Potency of 3,4-diaminopyridine and 4-aminopyridine on mammalian neuromuscular transmission and the effect of pH changes.European Journal of Pharmacology 61, 26–34.
Okamura, Y. &Tsukita, S. (1986) Morphology of freeze-substituted myelinated axon in mouse peripheral nerves.Brain Research 383, 146–58.
Ornberg, R. L. &Reese, T. S. (1981) Beginning of exocytosis captured by rapid-freezing of Limulus amebocytes.Journal of Cell Biology 90, 40–54.
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.
Peper, K., Dreyer, F., Sandri, C., Akert, K. &Moor, H. (1974) Structure and ultrastructure of the frog motor endplate. A freeze-etching study.Cell and Tissue Research 149, 437–55.
Rees, R. P. &Reese, T. S. (1981) New structural features of freeze-substituted neuritic growth cones.Neuroscience 6, 247–54.
Roubos, E. W. &Van Der Wal-Divendal, R. M. (1980) Ultrastructural analysis of peptide-hormone release by exocytosis.Cell and Tissue Research 207, 267–75.
Tauc, L. C. (1982) Nonvesicular release of neurotransmitter.Physiological Reviews 62, 857–93.
Torri-Tarelli, F., Grohovaz, R., Fesce, R. &Ceccarelli, 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.
Valtorta, F., Jahn, R., Fesce, R., Greengard, P. &Ceccarelli, B. (1988) 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.
Van Der Kloot, W. (1988) Acetylcholine quanta are released from vesicles by exocytosis (and why some think not).Neuroscience 24, 1–7.
Van Harreveld, A. &Crowell, J. (1964) Electron microscopy after rapid freezing on a metal surface and substitution fixation.Anatomical Record 149, 381–6.
Whittaker, V. P., Michaelson, I. A. &Kirkland, R. J. A. (1964) The separation of synaptic vesicles from disrupted nerve ending particles ‘Synaptosomes’.Biochemical Journal 90, 293–303.
Whittaker, V. P., Essman, W. B. &Dowe, G. H. (1972) The isolation of pure cholinergic synaptic vesicles from the electric organs of elasmobranch fish of the family Torpinidae.Biochemical Journal 128, 833–46.
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Velasco, M.E., Pécot-Dechavassine, M. Membrane events related to transmitter release in mouse motor nerve terminals captured by ultrarapid cryofixation. J Neurocytol 22, 913–923 (1993). https://doi.org/10.1007/BF01186361
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DOI: https://doi.org/10.1007/BF01186361