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Morphological studies of neurotransmitter release and membrane recycling in sympathetic nerve terminals in culture

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Summary

The morphological correlates of transmitter release from synapses and varicosities were examined in mature cultures of sympathetic neurons dissociated from neonatal rat superior cervical ganglia. The number of synaptic vesicles decreased in synapses and varicosities depolarized with 53 mM K+. The decrease in vesicle number was accompanied by striking changes in the appearance of the synaptic terminals and an increase in their mean circumference. Coated pits and membrane-bound cisternae were observed more frequently in synapses and varicosities of depolarized neurons than in terminals of resting neurons. These morphological changes were not seen when the neurons were depolarized in the presence of Co2+, consistent with the Ca2+-dependence of transmitter release from these neurons. In freeze-fracture replicas of depolarized neurons, numerous dimples were observed in the cytoplasmic leaflet of synapses and varicosities, adjacent to large 12–14 nm particles. After a period of recovery in 5mm K+ medium, the number of synaptic vesicles and the shape of synaptic terminals returned to normal. When horseradish peroxidase (HRP) was included in the medium as an extracellular tracer during depolarization and recovery, a significant proportion of small, synaptic vesicles contained reaction product. Label was also present in coated vesicles and cisternae. Neurons which were depolarized in medium containing Co2+ or were exposed to HRP without depolarization contained few labelled synaptic vesicles. The proportion of labelled vesicles was not significantly different in synapses and varicosities, nor did it vary consistently with the transmitter identity of the neurons. These observations are consistent with the hypothesis that transmitter release occurs from varicosities as well as from synapses of postganglionic sympathetic neurons by exocytosis of the small synaptic vesicles, and that at least some new vesicles are formed from the nerve terminal membrane.

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References

  1. Akert, K., Pfenninger, K., Sandri, C. &Moor, H. (1972) Freeze etching and cytochemistry of vesicles and membrane complexes in synapses of the central nervous system. InStructure and Function of Synapses (edited byPappas, G. D. andPurpura, D. P.), pp. 67–86. New York: Raven Press.

  2. Atwood, H. L., Lang, F. &Morin, W. A. (1972) Synaptic vesicles: Selective depletion in crayfish excitatory and inhibitory axons.Science 176, 1353–55.

  3. Basbaum, C. &Heuser, J. (1979) Morphological studies of stimulated adrenergic axon varicosities in the mouse vas deferens.Journal of Cell Biology 80, 310–25.

  4. Bennett, M. V. L., Model, P. G. &Highstein, S. M. (1976) Stimulation induced depletion of vesicles, fatigue of transmission, and recovery processes at a vertebrate central synapse.Cold Spring Harbor Symposium on Quantitative Biology 40, 25–35.

  5. Bisby, M. A. &Fillenz, M. (1972) The storage of endogenous NA in sympathetic nerve terminals.Journal of Physiology 215, 163–79.

  6. Bisby, M. A., Fillenz, M. &Smith, A. D. (1973) Evidence for presence of DβH in both populations of NA storage vesicles in sympathetic nerve terminals of the rat vas deferens.Journal of Neurochemistry 20, 245–8.

  7. Bloom, F. E. (1973) Ultrastructural identification of catecholamine-containing central synaptic terminals.Journal of Histochemistry and Cytochemistry 21, 333–48.

  8. Buckley, J. M. &Landis, S. C. (1983) Morphological studies of synapse and varicosities in dissociated cell cultures of sympathetic neurons.Journal of Neurocytology 12, 67–92.

  9. Burton, H. &Bunge, R. (1975) A comparison of uptake and release of [3H]-norepinephrine in rat autonomic and sensory ganglia in tissue culture.Brain Research 97, 157–62.

  10. Ceccarelli, B., Grohovaz, F. &Hurlbut, W. P. (1979) 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. II. Effects of electrical stimulation and high potassium.Journal of Cell Biology 81, 163–92.

  11. 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.

  12. 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.

  13. Clark, A. W., Hurlbut, W. P. &Mauro, A. (1972) Changes in the fine structure of the neuromuscular junction of the frog caused by black widow spider venom.Journal of Cell Biology 52, 1–14.

  14. Cohen, S. A. &Pumplin, D. W. (1979) Clusters of intramembrane particles associated with binding sites for α-bungarotoxin in cultured chick myotubes.Journal of Cell Biology 82, 494–516.

  15. Coté, M. G., Palaic, D. &Parrisset, J. C. (1970) Changes in the number of vesicles and the size of sympathetic nerve terminals following nerve stimulation.Revue Canadienne de Biologie 29, 111–4.

  16. Couteaux, R. &Pécot-Dechavassine, M. (1970) Vésicules synaptiques et poches au niveau des ‘zones actives’ de la junction neuromusculaire.Comptes Rendus Hebdomadaires des Seances de L'Academie des Sciences, Series D 271, 2346–9.

  17. Couteaux, R. &Pécot-Dechavassine, M. (1974) Les zones specialisees des membranes pre-synaptique.Comptes Rendus Hebdomadaires des Seances de L'Academie des Sciences, Series D 280, 299–301.

  18. Dreyer, F., Peper, K., Akert, K., Sandri, C. &Moor, H. (1973) Ultrastructure of the ‘active zone’ in the frog neuromuscular junction.Brain Research 62, 373–80.

  19. Fillenz, M. (1971) Fine structure of noradrenaline storage vesicles in nerve terminals of the rat vas deferens.Philosophical Transactions of the Royal Society Series B 261, 319–23.

  20. Fillenz, M. &Howe, P. R. C. (1975) Depletion of noradrenaline stores in sympathetic nerve terminals.Journal of Neurochemistry 24, 683–88.

  21. Fried, G., Lagercrantz, H. &Hokfelt, T. (1978) Improved isolation of small noradrenergic vesicles from rat seminal ducts following castration. A density gradient centrifugation and morphological study.Neuroscience 3, 1271–91.

  22. Geffen, L. B. &Livett, B. G. (1971) Synaptic vesicles in sympathetic neurons.Physiological Reviews 51, 98–157.

  23. Gulley, R. L. (1978) Changes in the presynaptic membrane of the synapses of the cochlear nucleus with different levels of acoustic stimulation.Brain Research 146, 373–9.

  24. Gulley, R. L., Landis, D. M. D. &Reese, T. S. (1978) Internal organization of membranes at end bulbs of Held in the anteroventral cochlear nucleus.Journal of Comparative Neurology 180, 707–42.

  25. Hamilton, R. C. &Robinson, P. M. (1973) Disappearance of small vesicles from adrenergic nerve endings in the rat vas deferens caused by red back spider venom.Journal of Neurocytology 2, 465–9.

  26. Heuser, J. E. &Reese, T. S. (1973) Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction.Journal of Cell Biology 57, 315–44.

  27. Heuser, J. E. &Reese, T. S. (1977) The structure of the synapse. InHandbook of Physiology, Section 1: The Nervous System, Vol. 1 (edited byKandel, E.), pp. 261–94. Bethesda, MD: American Physiological Society.

  28. Heuser, J. E. &Reese, T. S. (1979a) Synaptic vesicle exocytosis captured by quick freezing. InThe Neurosciences: 4th Study Program (edited bySchmitt, F. O. andWorden, F. G.), pp. 573–600. Cambridge, MA: MIT Press.

  29. Heuser, J. E. &Reese, T. S. (1979b) Changes in the structure of presynaptic membranes during transmitter secretion. InNeurobiology of Chemical transmission (edited byOtsuka, M. andHall, Z.), pp. 3–11. Chichester, New York: John Wiley and Sons.

  30. Heuser, J. E. &Reese, T. S. (1981) Structural changes after transmitter release at the frog neuromuscular junction.Journal of Cell Biology 88, 564–80.

  31. Heuser, J. E., Reese, T. S., Dennis, M. J., Jan, J. L. &Evans, L. (1979) Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release.Journal of Cell Biology 81, 275–300.

  32. 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.

  33. Hökfelt, T. (1968)In vitro studies on central and peripheral monoamine neurons at the ultra-structural level.Zeitschrift fur Zellforschung und mikroskopische Anatomie 91, 1–74.

  34. Holtzman, E. (1977) The origin and fate of secretory packages, especially synaptic vesicles.Neuroscience 2, 327–55.

  35. Lagercrantz, H. (1976) On the composition and function of large dense-cored vesicles in sympathetic nerves.Neuroscience 1, 81–92.

  36. Landis, S. C. (1976) Rat sympathetic neurons and cardiac myocytes developing in microcultures: Correlation of fine structure of endings with neurotransmitter function in single neurons.Proceedings of the National Academy of Sciences USA 73, 4220–4.

  37. Landis, S. C. (1980) Developmental changes in the neurotransmitter properties of dissociated sympathetic neurons: A cytochemical study of the effects of medium.Developmental Biology 77, 349–61.

  38. Llinás, R. R. &Heuser, J. E. (1977) Depolarization-release coupling systems in neurons.Neurosciences Research Program Bulletin. Cambridge, MA: MIT Press.

  39. Lynch, K. (1980) Stimulation-induced reduction of large dense-core vesicle numbers in cholinergic motor nerve endings.Brain Research 194, 249–54.

  40. Lynch, K. (1982) The effects of chronic stimulation on the morphology of the frog neuromuscular junction.Journal of Neurocytology 11, 81–107.

  41. Mains, R. E. &Patterson, P. H. (1973) Primary cultures of dissociated sympathetic neurons. I. Establishment of long-term growth in culture and studies of differentiated properties.Journal of Cell Biology 59, 329–45.

  42. Model, P. G., Highstein, S. M. &Bennett, M. V. L. (1975) Depletion of vesicles and fatigue of transmission at a vertebrate central synapse.Brain Research 98, 209–28.

  43. Nelson, D. L. &Molinoff, P. B. (1976) Distribution and properties of adrenergic storage vesicles in nerve terminals.Journal of Pharmacology and Experimental Therapeutics 196, 346–59.

  44. Orci, L., Carpentier, J.-L., Perrelet, A., Anderson, R., Goldstein, J. &Brown, M. (1978) Occurrence of low density lipoprotein receptors within large pits on the surface of human fibroblasts as demonstrated by freeze-etching.Experimental Cell Research 113, 1–13.

  45. Patterson, P. H. &Chun, L. L. Y. (1974) The influence of nonneuronal cells on acetcholamine and acetylcholine synthesis and accumulation in cultures of dissociated sympathetic neurons.Proceedings of the National Academy of Sciences USA 71, 3607–10.

  46. Patterson, P. H. &Chun, L. L. Y. (1977a) Induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons. I. Effects of conditioned medium.Developmental Biology 56, 263–80.

  47. Patterson, P. H. &Chun, L. L. Y. (1977b) The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons. II. Developmental aspects.Developmental Biology 60, 473–81.

  48. Patterson, P. H., Reichardt, L. F. &Chun, L. L. Y. (1975) Biochemical studies on the development of primary sympathetic neurons in cell culture.Cold Spring Harbor Symposium on Quantitative Biology 46, 380–97.

  49. Pauli, B. U., Weinstein, R. S., Soble, L. W. &Alroy, J. (1977) Freeze-fracture of monolayer cultures.Journal of Cell Biology 72, 763–9.

  50. 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, 437–53.

  51. Pfenninger, K. H. &Rovainen, C. M. (1974) Stimulation and calcium-dependence of vesicle attachment sites in the presynaptic membrane. A freeze-cleave study on the lamprey spinal cord.Brain Research 72, 1–23.

  52. Pumplin, D. W. &Reese, T. S. (1977) Action of spider venom and botulinum toxin on the frog neuromuscular junction examined with the freeze fracture technique.Journal of Physiology 273, 443–56.

  53. Pysh, J. J. &Wiley, R. G. (1972) Morphological alterations of synapses in electrically stimulated superior cervical ganglia of the cat.Science 176, 191–3.

  54. Pysh, J. J. &Wiley, R. G. (1974) Synaptic vesicle depletion and recovery in cat sympathetic ganglia electrically stimulatedin vivo.Journal of Cell Biology 60, 365–74.

  55. Reese, R. &Bunge, R. P. (1974) Morphological and cytochemical studies of synapses formed in culture between isolated rat superior cervical ganglion neurons.Journal of Comparative Neurology 157, 1–12.

  56. Richardson, K. C. (1966) Electron microscopic identification of autonomic nerve endings.Nature 210, 756.

  57. Ripps, H., Shakib, M. &MacDonald, E. D. (1976) Peroxidase uptake by photoreceptor terminals of the skate retina.Journal of Cell Biology 70, 86–96.

  58. Schaeffer, S. &Raviola, E. (1978) Membrane recycling in the cone cell endings of the turtle retina.Journal of Cell Biology 79, 802–25.

  59. Siegel, S. (1956)Non-parametric Statistics for the Behavioral Sciences. New York: McGraw-Hill.

  60. Smith, A. D. (1972) Subcellular localization of noradrenaline in sympathetic nerves.Pharmacological Review 24, 435–57.

  61. Smith, A. D., de Potter, W. P., Moerman, E. J. &de Shaepdryver, A. F. (1970) Release of dopamine β-hydroxylase and chromagranin A upon stimulation of the splenic nerve.Tissue and Cell 2, 547–68.

  62. Smith, A. D. &Winkler, H. (1972) Fundamental mechanisms in the release of catecholamines. InCatecholamines, Handbuch der experimentallen Pharmakologie Vol. 33 (edited byBlaschko, H. andMuscholl, E.), pp. 583–617. New York: Springer-Verlag.

  63. Streit, P., Akert, K., Sandri, C., Livingston, R. B. &Moor, H. (1972) Dynamic ultrastructure of presynaptic membranes at nerve terminals in the spinal cord of rats. Anesthetized and unanesthetized preparations compared.Brain Research 48, 11–26.

  64. Thureson-Klein, A., Klein, R. L. &Johansson, O. (1979) Catecholamine-rich cells and varicosities in bovine splenic nerve, vesicle contents and evidence for exocytosis.Journal of Neurobiology 10, 309–24.

  65. Thureson-Klein, A., Stjárne, L. &Brundin, J. (1976) Ultrastructure of the nerves in veins from human omentum.Neuroscience 1, 333–7.

  66. Venzin, M., Sandri, C., Akert, K. &Wyss, Y. R. (1977) Membrane associated particles of the presynaptic active zone in rat spinal cord. A morphometric analysis.Brain Research 130, 393–404.

  67. Von Wedel, R. J., Carlson, S. J. &Kelly, R. B. (1981) Transfer of synaptic vesicle antigens to the presynaptic plasma membrane during exocytosis.Proceedings of the National Academy of Sciences USA 78, 1014–8.

  68. Wakade, A. R. (1979) Recycling of noradrenergic storage vesicles of isolated rat vas deferens.Nature 281, 374–6.

  69. Walicke, P. A., Campenot, R. B. &Patterson, P. H. (1977) Determination of transmitter function by neuronal activity.Proceedings of the National Academy of Sciences USA 74, 5767–71.

  70. Yee, A. G., Fischbach, G. D. &Karnovsky, M. J. (1978) Clusters of intramembranous particles on cultured myotubes at sites that are highly sensitive to acetylcholine.Proceedings of the National Academy of Sciences USA 75, 3004–8.

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Buckley, K.M., Landis, S.C. Morphological studies of neurotransmitter release and membrane recycling in sympathetic nerve terminals in culture. J Neurocytol 12, 93–116 (1983). https://doi.org/10.1007/BF01148089

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Keywords

  • Synaptic Vesicle
  • Transmitter Release
  • Sympathetic Neuron
  • Superior Cervical Ganglion
  • Synaptic Terminal