Skip to main content
NCBI home page
The Journal of Physiology logoLink to The Journal of Physiology
. 1977 Dec;273(2):443–457. doi: 10.1113/jphysiol.1977.sp012103

Action of brown widow spider venom and botulinum toxin on the frog neuromuscular junction examined with the freeze-fracture technique.

D W Pumplin, T S Reese
PMCID: PMC1353714  PMID: 202700

Abstract

1. Structural changes which normally accompany transmitter release at frog neuromuscular junctions are visualized with the freeze-fracture technique. The effects of brown widow spider venom and botulinum toxin were evaluated in terms of their ability to block or produce these structural changes. Changes produced by these neuropoisons were correlated with their known effects on neurotransmitter release. 3. Fusion of synaptic vesicles with the presynaptic plasmalemma, normally evoked by electrical stimulation, was abolished at neuromuscular junctions from frogs treated with botulinum toxin. 3. The concentration of large intramembranous particles in the presynaptic plasmalemma, an indication of the excess of synaptic vesicle fusion over recovery of synaptic vesicle membrane, was increased by treatment with brown widow spider venom, even in the presence of botulinum toxin. 4. When external calcium was present, sites of vesicle fusion induced by brown widow spider venom, as well as by electrical stimulation, were located mainly in the active zone. In the absence of external calcium, many plasmalemmal deformations, also though to be sites of vesicle fusion, were more evenly dispersed over the presynaptic surface of nerve terminals. 5. Botulinum toxin decreased the number of vesicle fusion sites in the active zone induced by spider venom in the presence of external calcium but had little effect on the number of fusion sites induced by spider venom in the absence of external calcium. 6. Nerve terminals soaked in a sodium-free Ringer solution were partially depleted of vesicles. Addition of spider venom to this Ringer did not cause additional depletion of vesicles. 7. Formation of cation-permeable channels in the presynaptic membrane could account for these effects of spider venom on the frog neuromuscular junction. Botulinum toxin blocks vesicle fusion by some means which is not yet understood.

Full text

PDF
443

Images in this article

456-1Fig. 1
on p.456-1
456-1Fig. 2
on p.456-1
456-2Fig. 3
on p.456-2
456-2Fig. 4
on p.456-2
456-3Fig. 5
on p.456-3
456-3Fig. 6
on p.456-3

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Boroff D. A., del Castillo J., Evoy W. H., Steinhardt R. A. Observations on the action of type A botulinum toxin on frog neuromuscular junctions. J Physiol. 1974 Jul;240(2):227–253. doi: 10.1113/jphysiol.1974.sp010608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Castillo J. D., Pumplin D. W. Discrete and discontinuous action of brown widow spider venom on the presynaptic nerve terminals of frog muscle. J Physiol. 1975 Nov;252(2):491–508. doi: 10.1113/jphysiol.1975.sp011154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ceccarelli B., Hurlbut W. P., Mauro A. Depletion of vesicles from frog neuromuscular junctions by prolonged tetanic stimulation. J Cell Biol. 1972 Jul;54(1):30–38. doi: 10.1083/jcb.54.1.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clark A. W., Mauro A., Longenecker H. E., Jr, Hurlbut W. P. Effects of black widow spider venom on the frog neuromuscular junction. Effects on the fine structure of the frog neuromuscular junction. Nature. 1970 Feb 21;225(5234):703–705. doi: 10.1038/225703a0. [DOI] [PubMed] [Google Scholar]
  5. Cull-Candy S. G., Lundh H., Thesleff S. Effects of botulinum toxin on neuromuscular transmission in the rat. J Physiol. 1976 Aug;260(1):177–203. doi: 10.1113/jphysiol.1976.sp011510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DEL CASTILLO J., ENGBAEK L. The nature of the neuromuscular block produced by magnesium. J Physiol. 1954 May 28;124(2):370–384. doi: 10.1113/jphysiol.1954.sp005114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DEL CASTILLO J., KATZ B. Changes in end-plate activity produced by presynaptic polarization. J Physiol. 1954 Jun 28;124(3):586–604. doi: 10.1113/jphysiol.1954.sp005131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dreyer F., Peper K., Akert K., Sandri C., Moor H. Ultrastructure of the "active zone" in the frog neuromuscular junction. Brain Res. 1973 Nov 23;62(2):373–380. doi: 10.1016/0006-8993(73)90699-9. [DOI] [PubMed] [Google Scholar]
  9. FINKELSTEIN A., Rubin L. L., Tzeng M. C. Black widow spider venom: effect of purified toxin on lipid bilayer membranes. Science. 1976 Sep 10;193(4257):1009–1011. doi: 10.1126/science.948756. [DOI] [PubMed] [Google Scholar]
  10. Frontali N., Ceccarelli B., Gorio A., Mauro A., Siekevitz P., Tzeng M. C., Hurlbut W. P. Purification from black widow spider venom of a protein factor causing the depletion of synaptic vesicles at neuromuscular junctions. J Cell Biol. 1976 Mar;68(3):462–479. doi: 10.1083/jcb.68.3.462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grasso A. Preparation and properties of a neurotoxin purified from the venom of black widow spider (Latrodectus mactans tredecimguttatus). Biochim Biophys Acta. 1976 Aug 9;439(2):406–412. doi: 10.1016/0005-2795(76)90077-5. [DOI] [PubMed] [Google Scholar]
  12. Harris A. J., Miledi R. The effect of type D botulinum toxin on frog neuromuscular junctions. J Physiol. 1971 Sep;217(2):497–515. doi: 10.1113/jphysiol.1971.sp009582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Heuser J. E., Reese T. S., Landis D. M. Functional changes in frog neuromuscular junctions studied with freeze-fracture. J Neurocytol. 1974 Mar;3(1):109–131. doi: 10.1007/BF01111936. [DOI] [PubMed] [Google Scholar]
  15. Kao I., Drachman D. B., Price D. L. Botulinum toxin: mechanism of presynaptic blockade. Science. 1976 Sep 24;193(4259):1256–1258. doi: 10.1126/science.785600. [DOI] [PubMed] [Google Scholar]
  16. Kendrick N. C., Blaustein M. P., Fried R. C., Ratzlaff R. W. ATP-dependent calcium storage in presynaptic nerve terminals. Nature. 1977 Jan 20;265(5591):246–248. doi: 10.1038/265246a0. [DOI] [PubMed] [Google Scholar]
  17. Lloyd S., Pickford M. The effect of oxytocin and adrenaline on blood flow in the hind limb of the dog following chronic lumbar sympathectomy. J Physiol. 1967 Sep;192(1):43–52. doi: 10.1113/jphysiol.1967.sp008286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Longenecker H. E., Jr, Hurlbut W. P., Mauro A., Clark A. W. Effects of black widow spider venom on the frog neuromuscular junction. Effects on end-plate potential, miniature end-plate potential and nerve terminal spike. Nature. 1970 Feb 21;225(5234):701–703. doi: 10.1038/225701a0. [DOI] [PubMed] [Google Scholar]
  19. Lowe D. A., Richardson N. P., Taylor P., Donatsch P. Increasing intracellular sodium triggers calcium release from bound pools. Nature. 1976 Mar 25;260(5549):337–338. doi: 10.1038/260337a0. [DOI] [PubMed] [Google Scholar]
  20. McCrone J. D., Netzloff M. L. An immunological and electrophoretical comparison of the venoms of the North American Latrodectus spiders. Toxicon. 1965 Nov;3(2):107–110. doi: 10.1016/0041-0101(65)90004-8. [DOI] [PubMed] [Google Scholar]
  21. Ornberg R. L., Smyth T., Jr, Benton A. W. Isolation of a neurotoxin with A presynaptic action from the venom of the black widow spider (Latrodectus mactans, Fabr.). Toxicon. 1976;14(4):329–333. doi: 10.1016/0041-0101(76)90030-1. [DOI] [PubMed] [Google Scholar]
  22. Pfenninger K. H., Rovainen C. M. Stimulation- and calcium-dependence of vesicle attachment sites in the presynaptic membrane: a freeze-cleave study on the lamprey spinal cord. Brain Res. 1974 May 31;72(1):1–23. doi: 10.1016/0006-8993(74)90646-5. [DOI] [PubMed] [Google Scholar]
  23. Pfenninger K., Akert K., Moor H., Sandri C. The fine structure of freeze-fractured presynaptic membranes. J Neurocytol. 1972 Sep;1(2):129–149. doi: 10.1007/BF01099180. [DOI] [PubMed] [Google Scholar]
  24. Pumplin D. W., del Castillo J. Release of packets of acetylcholine and synaptic vesicle elicited by brown widow spider venom in frog motor nerve endings poisoned by botulinum toxin. Life Sci. 1975 Jul 1;17(1):137–141. doi: 10.1016/0024-3205(75)90249-0. [DOI] [PubMed] [Google Scholar]
  25. Rose B., Loewenstein W. R. Calcium ion distribution in cytoplasm visualised by aequorin: diffusion in cytosol restricted by energized sequestering. Science. 1975 Dec 19;190(4220):1204–1206. doi: 10.1126/science.1198106. [DOI] [PubMed] [Google Scholar]
  26. Spitzer N. Miniature end-plate potentials at mammalian neuromuscular junctions poisoned by botulinum toxin. Nat New Biol. 1972 May 3;237(70):26–27. doi: 10.1038/newbio237026a0. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES