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. 1979 Jul 1;82(1):195–211. doi: 10.1083/jcb.82.1.195

Localization of acetylcholine receptors by means of horseradish peroxidase-alpha-bungarotoxin during formation and development of the neuromuscular junction in the chick embryo

PMCID: PMC2110414  PMID: 479297

Abstract

The localization of acetylcholine receptors (AChR) in the surface of developing myogenic cells of the chick embryo anterior and posterior latissimus dorsi muscles in relation to the process of innervation has been studied at the ultrastructural level utilizing a horseradish peroxidase-alpha-bungarotoxin conjugate. Localized concentrations of AChR were found in small regions 0.1-0.4 micron in width on the surface of myogenic cells of 10- to 14-d-old muscles. Surface specializations consisting of an external coating of extraneous material and an internal accumulation of dense material are associated with the plasma membrane in the regions of AChR concentration. As the muscle fibers are innervated, reactive surface patches are found at the region of contact of the growing nerve fiber and the surface of myotubes or their fusing myoblasts. After the establishment of contact, the patches of reaction product become more numerous and coextensive within the region of the neuromuscular junction and its immediate surroundings forming a dense continuous deposit on the postsynaptic sarcolemma. Activity becomes increasingly restricted to the site of the neuromuscular junction as the embryos approach hatching. At all stages, specializations external and internal to the plasmalemma are found at regions of high density of AChR, suggesting that they play a role in the maintenance of a higher concentration of receptors at these sites. These specializations also occur at the region of initial synaptic contact, indicating that they might be recognized by the nerve and represent preferred sites of innervation. Innervation appears to exert a stabilizing influence on the area of high AChR concentration in contact with the nerve and to induce a further increase in the AChR density of this site while the number of AChR in the remaining portions of the muscle surface declines.

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Selected References

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  1. Albertini D. F., Anderson E. Microtubule and microfilament rearrangements during capping of concanavalin A receptors on cultured ovarian granulosa cells. J Cell Biol. 1977 Apr;73(1):111–127. doi: 10.1083/jcb.73.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson M. J., Cohen M. W. Nerve-induced and spontaneous redistribution of acetylcholine receptors on cultured muscle cells. J Physiol. 1977 Jul;268(3):757–773. doi: 10.1113/jphysiol.1977.sp011880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson M. J., Cohen M. W., Zorychta E. Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells. J Physiol. 1977 Jul;268(3):731–756. doi: 10.1113/jphysiol.1977.sp011879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Atsumi S. Development of neuromuscular junctions of fast and slow muscles in the chick embryo: a light and electron microscopic study. J Neurocytol. 1977 Dec;6(6):691–709. doi: 10.1007/BF01176380. [DOI] [PubMed] [Google Scholar]
  5. Axelrod D., Ravdin P., Koppel D. E., Schlessinger J., Webb W. W., Elson E. L., Podleski T. R. Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4594–4598. doi: 10.1073/pnas.73.12.4594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bekoff A., Betz W. J. Acetylcholine hot spots: development on myotubes cultured from aneural limb buds. Science. 1976 Sep 3;193(4256):915–917. doi: 10.1126/science.948754. [DOI] [PubMed] [Google Scholar]
  7. Berlin R. D., Oliver J. M., Ukena T. E., Yin H. H. Control of cell surface topography. Nature. 1974 Jan 4;247(5435):45–46. doi: 10.1038/247045a0. [DOI] [PubMed] [Google Scholar]
  8. Betz W., Osborne M. Effects of innervation on acetylcholine sensitivity of developing muscle in vitro. J Physiol. 1977 Aug;270(1):75–88. doi: 10.1113/jphysiol.1977.sp011939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bevan S., Steinbach J. H. The distribution of alpha-bungarotoxin binding sites of mammalian skeletal muscle developing in vivo. J Physiol. 1977 May;267(1):195–213. doi: 10.1113/jphysiol.1977.sp011808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bosmann H. B. Synthesis of glycoproteins in brain: identification, purification and properties of a synaptosomal sialyl transferase utilizing endogenous and exogenous acceptors. J Neurochem. 1973 Apr;20(4):1037–1049. doi: 10.1111/j.1471-4159.1973.tb00075.x. [DOI] [PubMed] [Google Scholar]
  11. Burden S. Development of the neuromuscular junction in the chick embryo: the number, distribution, and stability of acetylcholine receptors. Dev Biol. 1977 Jun;57(2):317–329. doi: 10.1016/0012-1606(77)90218-4. [DOI] [PubMed] [Google Scholar]
  12. Changeux J. P., Benedetti L., Bourgeois J. P., Brisson A., Cartaud J., Devaux P., Grünhagen H., Moreau M., Popot J. L., Sobel A. Some structural properties of the cholinergic receptor protein in its membrane environmental relevant to its function as a pharmacological receptor. Cold Spring Harb Symp Quant Biol. 1976;40:211–230. doi: 10.1101/sqb.1976.040.01.023. [DOI] [PubMed] [Google Scholar]
  13. Changeux J. P., Danchin A. Selective stabilisation of developing synapses as a mechanism for the specification of neuronal networks. Nature. 1976 Dec 23;264(5588):705–712. doi: 10.1038/264705a0. [DOI] [PubMed] [Google Scholar]
  14. Clark D. G., Macmurchie D. D., Elliott E., Wolcott R. G., Landel A. M., Raftery M. A. Elapid neurotoxins. Purification, characterization, and immunochemical studies of -bungarotoxin. Biochemistry. 1972 Apr 25;11(9):1663–1668. doi: 10.1021/bi00759a020. [DOI] [PubMed] [Google Scholar]
  15. Cohen M. W. The development of neuromuscular connexions in the presence of D-tubocurarine. Brain Res. 1972 Jun 22;41(2):457–463. doi: 10.1016/0006-8993(72)90515-x. [DOI] [PubMed] [Google Scholar]
  16. Cohen S. A., Fischbach G. D. Clusters of acetylcholine receptors located at identified nerve-muscle synapses in vitro. Dev Biol. 1977 Aug;59(1):24–38. doi: 10.1016/0012-1606(77)90237-8. [DOI] [PubMed] [Google Scholar]
  17. Edelman G. M. Surface modulation in cell recognition and cell growth. Science. 1976 Apr 16;192(4236):218–226. doi: 10.1126/science.769162. [DOI] [PubMed] [Google Scholar]
  18. Fambrough D., Rash J. E. Development of acetylcholine sensitivity during myogenesis. Dev Biol. 1971 Sep;26(1):55–68. doi: 10.1016/0012-1606(71)90107-2. [DOI] [PubMed] [Google Scholar]
  19. Fertuck H. C., Salpeter M. M. Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after 125I-alpha-bungarotoxin binding at mouse neuromuscular junctions. J Cell Biol. 1976 Apr;69(1):144–158. doi: 10.1083/jcb.69.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fischbach G. D., Cohen S. A. The distribution of acetylcholine sensitivity over uninnervated and innervated muscle fibers grown in cell culture. Dev Biol. 1973 Mar;31(1):147–162. doi: 10.1016/0012-1606(73)90326-6. [DOI] [PubMed] [Google Scholar]
  21. Frank E., Jansen J. K., Lomo T., Westgaard R. H. The interaction between foreign and original motor nerves innervating the soleus muscle of rats. J Physiol. 1975 Jun;247(3):725–743. doi: 10.1113/jphysiol.1975.sp010954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. GINSBORG B. L. Spontaneous activity in muscle fibres of the chick. J Physiol. 1960 Mar;150:707–717. doi: 10.1113/jphysiol.1960.sp006413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gurd J. W. Identification of lectin receptors associated with rat brain postsynaptic densities. Brain Res. 1977 Apr 22;126(1):154–159. doi: 10.1016/0006-8993(77)90222-0. [DOI] [PubMed] [Google Scholar]
  24. Hartzell H. C., Fambrough D. M. Acetycholine receptor production and incorporation into membranes of developing muscle fibers. Dev Biol. 1973 Jan;30(1):153–165. doi: 10.1016/0012-1606(73)90054-7. [DOI] [PubMed] [Google Scholar]
  25. Hausman R. E., Moscona A. A. Purification and characterization of the retina-specific cell-aggregating factor. Proc Natl Acad Sci U S A. 1975 Mar;72(3):916–920. doi: 10.1073/pnas.72.3.916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hoffstein S., Soberman R., Goldstein I., Weissmann G. Concanavalin A induces microtubule assembly and specific granule discharge in human polymorphonuclear leukocytes. J Cell Biol. 1976 Mar;68(3):781–787. doi: 10.1083/jcb.68.3.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hourani B. T., Torain B. F., Henkart M. P., Carter R. L., Marchesi V. T., Fischbach G. D. Acetylcholine receptors of cultured muscle cells demonstrated with ferritin-alpha-bungarotoxin conjugates. J Cell Sci. 1974 Nov;16(2):473–479. doi: 10.1242/jcs.16.2.473. [DOI] [PubMed] [Google Scholar]
  28. Jansen J. K., Van Essen D. C. Re-innervation of rat skeleton muscle in the presence of alpha-bungarotoxin. J Physiol. 1975 Sep;250(3):651–667. doi: 10.1113/jphysiol.1975.sp011075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kelly P. T., Cotman C. W. Identification of glycoproteins and proteins at synapses in the central nervous system. J Biol Chem. 1977 Jan 25;252(2):786–793. [PubMed] [Google Scholar]
  30. Land B. R., Podleski T. R., Salpeter E. E., Salpeter M. M. Acetylcholine receptor distribution on myotubes in culture correlated to acetylcholine sensitivity. J Physiol. 1977 Jul;269(1):155–176. doi: 10.1113/jphysiol.1977.sp011897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lentz T. L., Chester J. Localization of acetylcholine receptors in central synapses. J Cell Biol. 1977 Oct;75(1):258–267. doi: 10.1083/jcb.75.1.258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lentz T. L., Mazurkiewicz J. E., Rosenthal J. Cytochemical localization of acetylcholine receptors at the neuromuscular junction by means of horseradish peroxidase-labeled alpha-bungarotoxin. Brain Res. 1977 Sep 2;132(3):423–442. doi: 10.1016/0006-8993(77)90192-5. [DOI] [PubMed] [Google Scholar]
  33. Low F. N. Developing boundary (basement) membranes in the chick embryo. Anat Rec. 1967 Oct;159(2):231–237. doi: 10.1002/ar.1091590212. [DOI] [PubMed] [Google Scholar]
  34. Marshall L. M., Sanes J. R., McMahan U. J. Reinnervation of original synaptic sites on muscle fiber basement membrane after disruption of the muscle cells. Proc Natl Acad Sci U S A. 1977 Jul;74(7):3073–3077. doi: 10.1073/pnas.74.7.3073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nakane P. K., Kawaoi A. Peroxidase-labeled antibody. A new method of conjugation. J Histochem Cytochem. 1974 Dec;22(12):1084–1091. doi: 10.1177/22.12.1084. [DOI] [PubMed] [Google Scholar]
  36. Nicolson G. L. Transmembrane control of the receptors on normal and tumor cells. I. Cytoplasmic influence over surface components. Biochim Biophys Acta. 1976 Apr 13;457(1):57–108. doi: 10.1016/0304-4157(76)90014-9. [DOI] [PubMed] [Google Scholar]
  37. Podleski T. R., Axelrod D., Ravdin P., Greenberg I., Johnson M. M., Salpeter M. M. Nerve extract induces increase and redistribution of acetylcholine receptors on cloned muscle cells. Proc Natl Acad Sci U S A. 1978 Apr;75(4):2035–2039. doi: 10.1073/pnas.75.4.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Prives J., Silman I., Amsterdam A. Appearance and disappearance of acetycholine receptor during differentiation of chick skeletal muscle in vitro. Cell. 1976 Apr;7(4):543–550. doi: 10.1016/0092-8674(76)90204-x. [DOI] [PubMed] [Google Scholar]
  39. Puro D. G., De Mello F. G., Nirenberg M. Synapse turnover: the formation and termination of transient synapses. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4977–4981. doi: 10.1073/pnas.74.11.4977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schlessinger J., Axelrod D., Koppel D. E., Webb W. W., Elson E. L. Lateral transport of a lipid probe and labeled proteins on a cell membrane. Science. 1977 Jan 21;195(4275):307–309. doi: 10.1126/science.556653. [DOI] [PubMed] [Google Scholar]
  41. Shur B. D., Roth S. Cell surface glycosyltransferases. Biochim Biophys Acta. 1975 Dec 29;415(4):473–512. doi: 10.1016/0304-4157(75)90007-6. [DOI] [PubMed] [Google Scholar]
  42. Simpson D. L., Thorne D. R., Loh H. H. Developmentally regulated lectin in neonatal rat brain. Nature. 1977 Mar 24;266(5600):367–369. doi: 10.1038/266367a0. [DOI] [PubMed] [Google Scholar]
  43. Steinbach J. H., Harris A. J., Patrick J., Schubert D., Heinemann S. Nerve-muscle interaction in vitro. Role of acetylcholine. J Gen Physiol. 1973 Sep;62(3):255–270. doi: 10.1085/jgp.62.3.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sytkowski A. J., Vogel Z., Nirenberg M. W. Development of acetylcholine receptor clusters on cultured muscle cells. Proc Natl Acad Sci U S A. 1973 Jan;70(1):270–274. doi: 10.1073/pnas.70.1.270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Teng N. N., Fiszman M. Y. Appearance of acetylcholine receptors in cultured myoblasts prior to fusion. J Supramol Struct. 1976;4(3):381–387. doi: 10.1002/jss.400040309. [DOI] [PubMed] [Google Scholar]
  46. Vogel Z., Daniels M. P. Ultrastructure of acetylcholine receptor clusters on cultured muscle fibers. J Cell Biol. 1976 May;69(2):501–507. doi: 10.1083/jcb.69.2.501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Vogel Z., Maloney G. J., Ling A., Daniels M. P. Identification of synaptic acetylcholine receptor sites in retina with peroxidase-labeled alpha-bungarotoxin. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3268–3272. doi: 10.1073/pnas.74.8.3268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Vogel Z., Sytkowski A. J., Nirenberg M. W. Acetylcholine receptors of muscle grown in vitro. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3180–3184. doi: 10.1073/pnas.69.11.3180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yahara I., Edelman G. M. Electron microscopic analysis of the modulation of lymphocyte receptor mobility. Exp Cell Res. 1975 Mar 1;91(1):125–142. doi: 10.1016/0014-4827(75)90150-0. [DOI] [PubMed] [Google Scholar]
  50. Yamada K. M., Yamada S. S., Pastan I. The major cell surface glycoprotein of chick embryo fibroblasts is an agglutinin. Proc Natl Acad Sci U S A. 1975 Aug;72(8):3158–3162. doi: 10.1073/pnas.72.8.3158. [DOI] [PMC free article] [PubMed] [Google Scholar]

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