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. 1992 May 2;117(4):849–861. doi: 10.1083/jcb.117.4.849

Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons

PMCID: PMC2289460  PMID: 1577861

Abstract

In mature neurons synaptic vesicles (SVs) undergo cycles of exo- endocytosis at synapses. It is currently unknown whether SV exocytosis and recycling occurs also in developing axons prior to synapse formation. To address this question, we have developed an immunocytochemical assay to reveal SV exo-endocytosis in hippocampal neurons developing in culture. In this assay antibodies directed against the lumenal domain of synaptotagmin I (Syt I), an intrinsic membrane protein of SVs, are used to reveal exposure of SV membranes at the cell surface. Addition of antibodies to the culture medium of living neurons for 1 hr at 37 degrees C resulted in their rapid and specific internalization by all neuronal processes and, particularly, by axons. Double immunofluorescence and electron microscopy immunocytochemistry indicated that the antibodies were retained within SVs in cell processes and underwent cycles of exo-endocytosis in parallel with SV membranes. In contrast, another endocytotic marker, wheat germ agglutinin, was rapidly cleared from the processes and transported to the cell body. Antibody-labeled SVs were still present in axons several days after antibody loading and became clustered at presynaptic sites in parallel with synaptogenesis. These results demonstrate that SVs undergo multiple cycles of exo-endocytosis in developing neuronal processes irrespective of the presence of synaptic contacts.

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Selected References

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  1. Bartlett W. P., Banker G. A. An electron microscopic study of the development of axons and dendrites by hippocampal neurons in culture. I. Cells which develop without intercellular contacts. J Neurosci. 1984 Aug;4(8):1944–1953. doi: 10.1523/JNEUROSCI.04-08-01944.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bartlett W. P., Banker G. A. An electron microscopic study of the development of axons and dendrites by hippocampal neurons in culture. II. Synaptic relationships. J Neurosci. 1984 Aug;4(8):1954–1965. doi: 10.1523/JNEUROSCI.04-08-01954.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bergmann M., Lahr G., Mayerhofer A., Gratzl M. Expression of synaptophysin during the prenatal development of the rat spinal cord: correlation with basic differentiation processes of neurons. Neuroscience. 1991;42(2):569–582. doi: 10.1016/0306-4522(91)90399-9. [DOI] [PubMed] [Google Scholar]
  4. Booher J., Sensenbrenner M. Growth and cultivation of dissociated neurons and glial cells from embryonic chick, rat and human brain in flask cultures. Neurobiology. 1972;2(3):97–105. [PubMed] [Google Scholar]
  5. Buchanan J., Sun Y. A., Poo M. M. Studies of nerve-muscle interactions in Xenopus cell culture: fine structure of early functional contacts. J Neurosci. 1989 May;9(5):1540–1554. doi: 10.1523/JNEUROSCI.09-05-01540.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buckley K., Kelly R. B. Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells. J Cell Biol. 1985 Apr;100(4):1284–1294. doi: 10.1083/jcb.100.4.1284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cameron P. L., Südhof T. C., Jahn R., De Camilli P. Colocalization of synaptophysin with transferrin receptors: implications for synaptic vesicle biogenesis. J Cell Biol. 1991 Oct;115(1):151–164. doi: 10.1083/jcb.115.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ceccarelli B., Hurlbut W. P., Mauro A. Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. J Cell Biol. 1973 May;57(2):499–524. doi: 10.1083/jcb.57.2.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ceccarelli B., Hurlbut W. P. Vesicle hypothesis of the release of quanta of acetylcholine. Physiol Rev. 1980 Apr;60(2):396–441. doi: 10.1152/physrev.1980.60.2.396. [DOI] [PubMed] [Google Scholar]
  10. Chun J. J., Shatz C. J. Redistribution of synaptic vesicle antigens is correlated with the disappearance of a transient synaptic zone in the developing cerebral cortex. Neuron. 1988 Jun;1(4):297–310. doi: 10.1016/0896-6273(88)90078-5. [DOI] [PubMed] [Google Scholar]
  11. DEL CASTILLO J., KATZ B. Biophysical aspects of neuro-muscular transmission. Prog Biophys Biophys Chem. 1956;6:121–170. [PubMed] [Google Scholar]
  12. De Camilli P., Cameron R., Greengard P. Synapsin I (protein I), a nerve terminal-specific phosphoprotein. I. Its general distribution in synapses of the central and peripheral nervous system demonstrated by immunofluorescence in frozen and plastic sections. J Cell Biol. 1983 May;96(5):1337–1354. doi: 10.1083/jcb.96.5.1337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. De Camilli P. Co-secretion of multiple signal molecules from endocrine cells via distinct exocytotic pathways. Trends Pharmacol Sci. 1991 Dec;12(12):446–448. doi: 10.1016/0165-6147(91)90631-2. [DOI] [PubMed] [Google Scholar]
  14. De Camilli P., Jahn R. Pathways to regulated exocytosis in neurons. Annu Rev Physiol. 1990;52:625–645. doi: 10.1146/annurev.ph.52.030190.003205. [DOI] [PubMed] [Google Scholar]
  15. De Camilli P., Miller P. E., Navone F., Theurkauf W. E., Vallee R. B. Distribution of microtubule-associated protein 2 in the nervous system of the rat studied by immunofluorescence. Neuroscience. 1984 Apr;11(4):817–846. [PubMed] [Google Scholar]
  16. Dotti C. G., Sullivan C. A., Banker G. A. The establishment of polarity by hippocampal neurons in culture. J Neurosci. 1988 Apr;8(4):1454–1468. doi: 10.1523/JNEUROSCI.08-04-01454.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Evers J., Laser M., Sun Y. A., Xie Z. P., Poo M. M. Studies of nerve-muscle interactions in Xenopus cell culture: analysis of early synaptic currents. J Neurosci. 1989 May;9(5):1523–1539. doi: 10.1523/JNEUROSCI.09-05-01523.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. FATT P., KATZ B. Spontaneous subthreshold activity at motor nerve endings. J Physiol. 1952 May;117(1):109–128. [PMC free article] [PubMed] [Google Scholar]
  19. Fischer von Mollard G., Mignery G. A., Baumert M., Perin M. S., Hanson T. J., Burger P. M., Jahn R., Südhof T. C. rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1988–1992. doi: 10.1073/pnas.87.5.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fletcher T. L., Cameron P., De Camilli P., Banker G. The distribution of synapsin I and synaptophysin in hippocampal neurons developing in culture. J Neurosci. 1991 Jun;11(6):1617–1626. doi: 10.1523/JNEUROSCI.11-06-01617.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Geppert M., Archer B. T., 3rd, Südhof T. C. Synaptotagmin II. A novel differentially distributed form of synaptotagmin. J Biol Chem. 1991 Jul 25;266(21):13548–13552. [PubMed] [Google Scholar]
  22. Gonatas N. K., Kim S. U., Stieber A., Avrameas S. Internalization of lectins in neuronal GERL. J Cell Biol. 1977 Apr;73(1):1–13. doi: 10.1083/jcb.73.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Goslin K., Banker G. Experimental observations on the development of polarity by hippocampal neurons in culture. J Cell Biol. 1989 Apr;108(4):1507–1516. doi: 10.1083/jcb.108.4.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Harper C. G., Gonatas J. O., Stieber A., Gonatas N. K. In vivo uptake of wheat germ agglutinin-horseradish peroxidase conjugates into neuronal GERL and lysosomes. Brain Res. 1980 Apr 28;188(2):465–472. doi: 10.1016/0006-8993(80)90045-1. [DOI] [PubMed] [Google Scholar]
  25. Heuser J. E., Reese T. S. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J Cell Biol. 1973 May;57(2):315–344. doi: 10.1083/jcb.57.2.315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hubbard J. I., Jones S. F., Landau E. M. On the mechanism by which calcium and magnesium affect the spontaneous release of transmitter from mammalian motor nerve terminals. J Physiol. 1968 Feb;194(2):355–380. doi: 10.1113/jphysiol.1968.sp008413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hume R. I., Role L. W., Fischbach G. D. Acetylcholine release from growth cones detected with patches of acetylcholine receptor-rich membranes. Nature. 1983 Oct 13;305(5935):632–634. doi: 10.1038/305632a0. [DOI] [PubMed] [Google Scholar]
  28. Huttner W. B., Schiebler W., Greengard P., De Camilli P. Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation. J Cell Biol. 1983 May;96(5):1374–1388. doi: 10.1083/jcb.96.5.1374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Israël M., Morel N., Lesbats B., Birman S., Manaranche R. Purification of a presynaptic membrane protein that mediates a calcium-dependent translocation of acetylcholine. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9226–9230. doi: 10.1073/pnas.83.23.9226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Johnston P. A., Cameron P. L., Stukenbrok H., Jahn R., De Camilli P., Südhof T. C. Synaptophysin is targeted to similar microvesicles in CHO and PC12 cells. EMBO J. 1989 Oct;8(10):2863–2872. doi: 10.1002/j.1460-2075.1989.tb08434.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Johnston P. A., Südhof T. C. The multisubunit structure of synaptophysin. Relationship between disulfide bonding and homo-oligomerization. J Biol Chem. 1990 May 25;265(15):8869–8873. [PubMed] [Google Scholar]
  32. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  33. Lim N. F., Nowycky M. C., Bookman R. J. Direct measurement of exocytosis and calcium currents in single vertebrate nerve terminals. Nature. 1990 Mar 29;344(6265):449–451. doi: 10.1038/344449a0. [DOI] [PubMed] [Google Scholar]
  34. Linstedt A. D., Kelly R. B. Synaptophysin is sorted from endocytotic markers in neuroendocrine PC12 cells but not transfected fibroblasts. Neuron. 1991 Aug;7(2):309–317. doi: 10.1016/0896-6273(91)90269-6. [DOI] [PubMed] [Google Scholar]
  35. Lockerbie R. O., Miller V. E., Pfenninger K. H. Regulated plasmalemmal expansion in nerve growth cones. J Cell Biol. 1991 Mar;112(6):1215–1227. doi: 10.1083/jcb.112.6.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mason C. A. Axon development in mouse cerebellum: embryonic axon forms and expression of synapsin I. Neuroscience. 1986 Dec;19(4):1319–1333. doi: 10.1016/0306-4522(86)90146-6. [DOI] [PubMed] [Google Scholar]
  37. Matteoli M., De Camilli P. Molecular mechanisms in neurotransmitter release. Curr Opin Neurobiol. 1991 Jun;1(1):91–97. doi: 10.1016/0959-4388(91)90015-y. [DOI] [PubMed] [Google Scholar]
  38. Matteoli M., Takei K., Cameron R., Hurlbut P., Johnston P. A., Südhof T. C., Jahn R., De Camilli P. Association of Rab3A with synaptic vesicles at late stages of the secretory pathway. J Cell Biol. 1991 Nov;115(3):625–633. doi: 10.1083/jcb.115.3.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Matthew W. D., Tsavaler L., Reichardt L. F. Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue. J Cell Biol. 1981 Oct;91(1):257–269. doi: 10.1083/jcb.91.1.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mellman I., Plutner H. Internalization and degradation of macrophage Fc receptors bound to polyvalent immune complexes. J Cell Biol. 1984 Apr;98(4):1170–1177. doi: 10.1083/jcb.98.4.1170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mellman I., Plutner H., Ukkonen P. Internalization and rapid recycling of macrophage Fc receptors tagged with monovalent antireceptor antibody: possible role of a prelysosomal compartment. J Cell Biol. 1984 Apr;98(4):1163–1169. doi: 10.1083/jcb.98.4.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Navone F., Jahn R., Di Gioia G., Stukenbrok H., Greengard P., De Camilli P. Protein p38: an integral membrane protein specific for small vesicles of neurons and neuroendocrine cells. J Cell Biol. 1986 Dec;103(6 Pt 1):2511–2527. doi: 10.1083/jcb.103.6.2511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Penner R., Neher E. The patch-clamp technique in the study of secretion. Trends Neurosci. 1989 Apr;12(4):159–163. doi: 10.1016/0166-2236(89)90059-3. [DOI] [PubMed] [Google Scholar]
  44. Perin M. S., Brose N., Jahn R., Südhof T. C. Domain structure of synaptotagmin (p65) J Biol Chem. 1991 Jan 5;266(1):623–629. [PubMed] [Google Scholar]
  45. Perin M. S., Fried V. A., Mignery G. A., Jahn R., Südhof T. C. Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature. 1990 May 17;345(6272):260–263. doi: 10.1038/345260a0. [DOI] [PubMed] [Google Scholar]
  46. Perin M. S., Johnston P. A., Ozcelik T., Jahn R., Francke U., Südhof T. C. Structural and functional conservation of synaptotagmin (p65) in Drosophila and humans. J Biol Chem. 1991 Jan 5;266(1):615–622. [PubMed] [Google Scholar]
  47. Petrenko A. G., Perin M. S., Davletov B. A., Ushkaryov Y. A., Geppert M., Südhof T. C. Binding of synaptotagmin to the alpha-latrotoxin receptor implicates both in synaptic vesicle exocytosis. Nature. 1991 Sep 5;353(6339):65–68. doi: 10.1038/353065a0. [DOI] [PubMed] [Google Scholar]
  48. Reetz A., Solimena M., Matteoli M., Folli F., Takei K., De Camilli P. GABA and pancreatic beta-cells: colocalization of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion. EMBO J. 1991 May;10(5):1275–1284. doi: 10.1002/j.1460-2075.1991.tb08069.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Smith S. J., Augustine G. J. Calcium ions, active zones and synaptic transmitter release. Trends Neurosci. 1988 Oct;11(10):458–464. doi: 10.1016/0166-2236(88)90199-3. [DOI] [PubMed] [Google Scholar]
  50. Stoeckel K., Schwab M., Thoenen H. Role of gangliosides in the uptake and retrograde axonal transport of cholera and tetanus toxin as compared to nerve growth factor and wheat germ agglutinin. Brain Res. 1977 Aug 26;132(2):273–285. doi: 10.1016/0006-8993(77)90421-8. [DOI] [PubMed] [Google Scholar]
  51. Sun Y. A., Poo M. M. Evoked release of acetylcholine from the growing embryonic neuron. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2540–2544. doi: 10.1073/pnas.84.8.2540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Südhof T. C., Baumert M., Perin M. S., Jahn R. A synaptic vesicle membrane protein is conserved from mammals to Drosophila. Neuron. 1989 May;2(5):1475–1481. doi: 10.1016/0896-6273(89)90193-1. [DOI] [PubMed] [Google Scholar]
  53. Südhof T. C., Jahn R. Proteins of synaptic vesicles involved in exocytosis and membrane recycling. Neuron. 1991 May;6(5):665–677. doi: 10.1016/0896-6273(91)90165-v. [DOI] [PubMed] [Google Scholar]
  54. Torri-Tarelli F., Grohovaz F., Fesce R., Ceccarelli B. Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine. J Cell Biol. 1985 Oct;101(4):1386–1399. doi: 10.1083/jcb.101.4.1386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Torri-Tarelli F., Villa A., Valtorta F., De Camilli P., Greengard P., Ceccarelli B. Redistribution of synaptophysin and synapsin I during alpha-latrotoxin-induced release of neurotransmitter at the neuromuscular junction. J Cell Biol. 1990 Feb;110(2):449–459. doi: 10.1083/jcb.110.2.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Valtorta F., Jahn R., Fesce R., Greengard P., Ceccarelli B. Synaptophysin (p38) at the frog neuromuscular junction: its incorporation into the axolemma and recycling after intense quantal secretion. J Cell Biol. 1988 Dec;107(6 Pt 2):2717–2727. doi: 10.1083/jcb.107.6.2717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wendland B., Miller K. G., Schilling J., Scheller R. H. Differential expression of the p65 gene family. Neuron. 1991 Jun;6(6):993–1007. doi: 10.1016/0896-6273(91)90239-v. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wiedenmann B., Rehm H., Knierim M., Becker C. M. Fractionation of synaptophysin-containing vesicles from rat brain and cultured PC12 pheochromocytoma cells. FEBS Lett. 1988 Nov 21;240(1-2):71–77. doi: 10.1016/0014-5793(88)80342-9. [DOI] [PubMed] [Google Scholar]
  60. Xie Z. P., Poo M. M. Initial events in the formation of neuromuscular synapse: rapid induction of acetylcholine release from embryonic neuron. Proc Natl Acad Sci U S A. 1986 Sep;83(18):7069–7073. doi: 10.1073/pnas.83.18.7069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Young S. H., Poo M. M. Spontaneous release of transmitter from growth cones of embryonic neurones. Nature. 1983 Oct 13;305(5935):634–637. doi: 10.1038/305634a0. [DOI] [PubMed] [Google Scholar]

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