Abstract
We have utilized immunofluorescence techniques to look for synaptic vesicle antigens on the plasma membrane of resting and active nerve terminals. Rabbit antiserum was raised against purified cholinergic synaptic vesicles from the electric organ of Narcine brasiliensis, a marine electric ray. Antibodies to synaptic vesicles were shown to bind selectively to nerve terminals in cryostat sections of frog nerve-muscle preparations. Binding was demonstrated indirectly by using fluorescein-labeled goat anti-rabbit antibodies. Structures in cross sections that bound antiserum were identified as nerve terminals because of their size, shape, and position and because they coincided with sites that bound rhodamine-conjugated alpha-bungarotoxin and had acetylcholine esterase activity. Presumably, sectioning gave antibodies access to binding sites within the nerve terminal. However, when antibodies to synaptic vesicles were added to the bathing medium of intact neuromuscular preparations prior to sectioning, antibody binding was marginal or undetectable, suggesting that few vesicle antigens were normally accessible on the outer surface of resting nerve terminals. When intact preparations were stimulated to release their vesicular acetylcholine by the addition of 1 mM LaCl3, antibody binding to the intact nerve terminals became striking. These findings suggest that the synaptic vesicle membrane and the synaptic terminal plasma membrane differ in composition. They also provide further support for the exocytotic hypothesis of neurotransmitter release, which predicts that vesicle markers should be exposed on the outside of nerve terminals when vesicles fuse with the plasma membrane during stimulation.
Full text
PDF
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Carlson S. S., Kelly R. B. An antiserum specific for cholinergic synaptic vesicles from electric organ. J Cell Biol. 1980 Oct;87(1):98–103. doi: 10.1083/jcb.87.1.98. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carlson S. S., Wagner J. A., Kelly R. B. Purification of synaptic vesicles from elasmobranch electric organ and the use of biophysical criteria to demonstrate purity. Biochemistry. 1978 Apr 4;17(7):1188–1199. doi: 10.1021/bi00600a009. [DOI] [PubMed] [Google Scholar]
- DEL CASTILLO J., KATZ B. Quantal components of the end-plate potential. J Physiol. 1954 Jun 28;124(3):560–573. doi: 10.1113/jphysiol.1954.sp005129. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DeBassio W. A., Schnitzler R. M., Parsons R. L. Influence of lanthanum on transmitter release at the neuromuscular junction. J Neurobiol. 1971;2(3):263–278. doi: 10.1002/neu.480020307. [DOI] [PubMed] [Google Scholar]
- Heuser J. E., Reese T. S., Dennis M. J., Jan Y., Jan L., Evans L. Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J Cell Biol. 1979 May;81(2):275–300. doi: 10.1083/jcb.81.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Heuser J., Miledi R. Effects of lanthanum ions on function and structure of frog neuromuscular junctions. Proc R Soc Lond B Biol Sci. 1971 Dec 14;179(1056):247–260. doi: 10.1098/rspb.1971.0096. [DOI] [PubMed] [Google Scholar]
- Hooper J. E., Carlson S. S., Kelly R. B. Antibodies to synaptic vesicles purified from Narcine electric organ bind a subclass of mammalian nerve terminals. J Cell Biol. 1980 Oct;87(1):104–113. doi: 10.1083/jcb.87.1.104. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Israel M., Dunant Y., Manaranche R. The present status of the vesicular hypothesis. Prog Neurobiol. 1979;13(3):237–275. doi: 10.1016/0301-0082(79)90017-0. [DOI] [PubMed] [Google Scholar]
- KARNOVSKY M. J., ROOTS L. A "DIRECT-COLORING" THIOCHOLINE METHOD FOR CHOLINESTERASES. J Histochem Cytochem. 1964 Mar;12:219–221. doi: 10.1177/12.3.219. [DOI] [PubMed] [Google Scholar]
- Kajimoto N., Kirpekar S. M. Effect of manganese and lanthanum on spontaneous release of acetylcholine at frog motor nerve terminals. Nat New Biol. 1972 Jan 5;235(53):29–30. doi: 10.1038/newbio235029a0. [DOI] [PubMed] [Google Scholar]
- Ravdin P., Axelrod D. Fluorescent tetramethyl rhodamine derivatives of alpha-bungarotoxin: preparation, separation, and characterization. Anal Biochem. 1977 Jun;80(2):585–592. doi: 10.1016/0003-2697(77)90682-0. [DOI] [PubMed] [Google Scholar]
- Sanes J. R., Carlson S. S., Von Wedel R. J., Kelly R. B. Antiserum specific for motor nerve terminals in skeletal muscle. Nature. 1979 Aug 2;280(5721):403–404. doi: 10.1038/280403a0. [DOI] [PubMed] [Google Scholar]
- Stadler H., Tashiro T. Isolation of synaptosomal plasma membranes from cholinergic nerve terminals and a comparison of their proteins with those of synaptic vesicles. Eur J Biochem. 1979 Nov 1;101(1):171–178. doi: 10.1111/j.1432-1033.1979.tb04229.x. [DOI] [PubMed] [Google Scholar]
- Suszkiw J. B., Whittaker V. P. Role of vesicle recycling in vesicular storage and release of acetylcholine in Torpedo electroplaque synapses. Prog Brain Res. 1979;49:153–162. doi: 10.1016/S0079-6123(08)64629-4. [DOI] [PubMed] [Google Scholar]