Summary
Frog, snake and rat neuromuscular junctions were prepared for electron microscopy by the quick-freeze, deep-etch, rotary replication procedure. The postsynaptic membrane was exposed by treating muscles with 1 mg/ml collagenase to remove the basal lamina. Present on the apices of the postsynaptic folds are regular arrays of 8–9 nm protrusions. These are not seen in the depths of the folds nor elsewhere on the muscle surface, thus they presumably represent the heads of cholinergic receptor molecules. These protrusions tend to be arranged in parallel rows two-abreast. Their high concentration (10 000/μm2) and their orderly arrangement is basically similar to the receptors seen inTorpedo postsynaptic membrane. Their distribution did not appear to change after denervation. Efforts were made to expose possible anchoring structures of these receptors, by treating muscles with 0.1% Saponin immediately before and/or during fixation in 1% formaldehyde, or by homogenizing muscles after brief formaldehyde fixation. This washed most soluble protein out of the cytoplasm and exposed a submembraneous meshwork just beneath the postsynaptic membrane. This meshwork appears to connect the membrane to underlying bundles of intermediate filaments which course through the postsynaptic processes that border each fold. This meshwork is presumably equivalent to the postsynaptic ‘density’ seen in thin sections. Its three-dimensional structure suggests that it could anchor receptor molecules to underlying cytoskeletal elements and thus immobilize receptors in the plane of the postsynaptic membrane.
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References
Albuquerque, E. X., Barnard, E. A., Porter, C. W. &Warnick, J. E. (1974) The density of acetylcholine receptors and their sensitivity in the postsynaptic membrane of muscle endplates.Proceedings of the National Academy of Science USA 71, 2818–22.
Allen, T., Baerwald, R. &Potter, L. T. (1977) Postsynaptic membranes in the electric tissue of Narcine. II. A freeze-fracture study of nicotinic receptor molecules.Tissue and Cell 9, 595–608.
Anderson, M. J. &Cohen, M. W. (1974) Fluorescent staining of acetylcholine receptors in vertebrate skeletal muscle.Journal of Physiology 237, 385–400.
Anderson, C. R. &Stevens, C. F. (1973) Voltage clamp analysis of acetylcholine produced end-plate current fluctuations at frog neuromuscular junction.Journal of Physiology 235, 655–92.
Axelrod, D., Ravdin, P., Koppel, D. E., Schlessinger, J., Webb, W. W., Elson, E. L. &Podleski, T. R. (1976) Lateral motion of fluorescently labelled acetylcholine receptors in membranes of developing muscle fibres.Proceedings of the National Academy of Science USA 73, 4594–8.
Betz, W. &Sakmann, B. (1973) Effects of proteolytic enzymes on function and structure of frog neuromuscular junctions.Journal of Physiology 230, 673–88.
Birks, R., Huxley, H. E. &Katz, B. (1960a) The fine structure of the neuromuscular junction of the frog.Journal of Physiology 150, 134–44.
Birks, R., Katz, B. &Miledi, R. (1960b) Physiological and structural changes at the amphibian myoneural junction, in the course of nerve degeneration.Journal of Physiology 150, 145–68.
Brockes, J. P., Berg, D. K. &Hall, Z. W. (1975) The biochemical properties and regulation of acetylcholine receptors in normal and denervated muscle.Cold Spring Harbor Symposium on Quantitative Biology 40, 253–62.
Burden, S. J., Sargent, P. B. &McMahan, U. J. (1979) Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of the nerve.Journal of Cell Biology 82, 412–25.
Burrage, T. G. &Lentz, T. L. (1979) Surface specialization associated with high density accumulations of acetylcholine receptors in embryonic chick muscle.Abstracts of the Society for Neurosdence USA 5, 477.
Cartaud, J., Benedetti, E. L., Cohen, J. B., Meunier, J. C. &Chanceux, J. -P. (1973) Presence of a lattice structure in membrane fragments rich in nicotinic receptor protein from the electric organ ofTorpedo marmorata.FEBS Letters 33, 109–13.
Cartaud, J., Benedetti, E. L., Sobel, A. &Chanceux, J. -P. (1978) A morphological Study of the cholinergic receptor protein fromTorpedo marmorata in its membrane environment and its detergent-extracted form.Journal of Cell Science 29, 313–37.
Cartaud, J., Sobel, A., Rousselet, A., Devaux, P. F. &Changeux, J. -P. (1981) Consequences of alkaline treatment for the ultrastructure of the acetylcholine receptor-rich membranes fromTorpedo marmorata electric organ.Journal of Cell Biology 90, 418–26.
Changeux, J. -P., Benedetti, E. L., Bourgeois, J. P., Brisson, A., Cartaud, J., Devaux, P., Grunhagen, H., Moreau, M., Popot, J. L., Sobel, A. &Weber, M. (1976) Some structural properties of the cholinergic receptor protein in its membrane environment relevant to its function as a pharmacological receptor.Cold Spring Harbor Symposium on Quantitative Biology 40, 211–30.
Cohen, R. S., Blomberg, F., Berzines, K. &Siekevitz, P. (1977) The Structure of postsynaptic densities isolated from dog cerebral cortex. I. Overall morphology and protein composition.Journal of Cell Biology 74, 181–203.
Colonnier, M. (1968) Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscope study.Brain Research 9, 268–87.
Couteaux, M. R. (1981) Structure of the subsynaptic sarcoplasm in the interfolds of the frog neuromuscular junction.Journal of Neurocytology 10, 947–62.
Couteaux, M. R. &Pecot-Dechavassine, M. (1968) Particularites structurales du sarcoplasme sous-neural.Comptes Rendus de l'Academie des Sciences D 266, 8–10.
Ellisman, M. H., Rash, J. E., Staehelin, L. A. &Porter, K. R. (1976) Studies of excitable membranes. II. A comparison of specializations at neuromuscular junctions and nonfunctional sarcolemmas of mammalian fast and slow twitch muscle fibers.Journal of Cell Biology 68, 752–74.
Fertuck, H. C. &Salpeter, M. M. (1976) Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscopic autoradiography after125I-α-bungarotoxin binding at mouse neuromuscular junctions.Journal of Cell Biology 69, 144–58.
Frank, E., Gantvik, K. &Sommerschild, H. (1975) Persistence of junctional acetylcholine receptors following denervation.Cold Spring Harbor Symposium on Quantitative Biology 40, 275–81.
Hall, Z. W. &Kelly, R. B. (1971) Enzymatic detachment of endplate acetylcholinesterase from muscle.Nature, New Biology 232, 62–3.
Hamilton, S. L., McLaughlln, M. &Karlin, A. (1979) Formation of disulfide-linked dimers of acetylcholine receptor in membrane fromTorpedo electric tissue.Biochemistry 18, 155–63.
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.
Heuser, J. E., Reese, T. S., Dennis, M. J., Jan, L., Jan, Y. &Evans, L. (1979) Synaptic vesicle exocitosis captured by quick-freezing and correlated with quantal transmitter release.Journal of Cell Biology 81, 275–300.
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.
Heuser, J. E. &Salpeter, S. R. (1979) Organization of acetylcholine receptors in quick frozen, deep-etched, and rotary-replicatedTorpedo postsynaptic membrane.Journal of Cell Biology 82, 150–73.
Hirokawa, N. &Heuser, J. E. (1980) Possible anchoring structures for the organized arrays of receptors seen at neuromuscular junctions.Journal of Cell Biology 87, 75a.
Hirokawa, N. &Heuser, J. E. (1981a) Structural evidence that botulinum toxin blocks neuromuscular transmission by impairing the calcium influx that normally accompanies nerve depolarization.Journal of Cell Biology 88, 160–71.
Hirokawa, N. &Heuser, J. E. (1981b) Quick freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of epithelial cells.Journal of Cell Biology 91, 399–409.
Hirokawa, N. &Kirino, T. (1980) An ultrastructural study of nerve and glial cells by freeze-substitution.Journal of Neurocytology 9, 243–54.
Karlin, H., Weill, C. L., McNamee, M. G. &Valderrama, R. (1976) Facets of the Structures of acetylcholine receptors fromElectrophorus andTorpedo.Cold Spring Harbor Symposium on Quantitative Biology 40, 203–10.
Katz, B. &Miledi, R. (1972) The statistical nature of the acetylcholine potential and its molecularcomponents.Journal of Physiology 224, 665–99.
Kuffler, S. W. &Yoshikami, D. (1975) The distribution of acetylcholine sensitivity at the post-synaptic membrane of vertebrate skeletal twitch muscles: iontophoretic mapping in the micron range.Journal of Physiology 244, 703–30.
LeBeux, Y. J. &Willemot, J. (1975) An ultrastructural study of the microfilaments in rat brain by means of E-PTA staining and heavy meromyosin labeling. II. The synapses.Cell and Tissue Research 160, 37–68.
Loring, R. H. &Salpeter, M. M. (1980) Denervatiori increases turnover rate of junctional acetylcholine receptors.Proceedings of the National Academy of Science USA 77, 2293–7.
Matus, A. I. &Walters, B. B. (1975) Ultrastructure of the synaptic junctional lattice isolated from mammalian brain.Journal of Neurocytology 4, 369–75.
Matus, A. I., Walters, B. B. &Jones, D. H. (1975) Junctional ultrastructure in isolated synaptic membranes.Journal of Neurocytology 4, 357–67.
Nickel, E. &Potter, L. T. (1973) Ultrastructure of isolated membranes ofTorpedo electric tissue.Brain Research 57, 508–17.
Peper, K., Dreyer, F., Sandri, C. &Akert, K. (1974) Structure and ultrastructure of the frog motor endplate. A freeze-etching study.Cell and Tissue Research 149, 437–55.
Peper, K. &McMahan, U. J. (1972) Distribution of acetylcholine receptors in the vicinity of nerve terminals on skeletal muscle of the frog.Proceedings of the Royal Society of London B 181, 431–40.
Porter, C. W. &Barnard, E. A. (1975) The density of cholinergic receptors at the endplate postsynaptic membrane: ultrastructural studies in two mammalian species.Journal of Membrane Biology 20, 31–49.
Prives, J., Daniels, M. P., Neal, F. M., Bauer, H. -C., Penman, S. &Christian, C. N. (1979) Two classes of acetylcholine receptors on cultured muscle cells distinguished by detergent extraction.Abstracts of the Society for Neuroscience USA 5, 487.
Rash, J. E. &Ellisman, M. H. (1974) Studies of excitable membranes. Macromolecular specializations of the neuromuscular junction and the nonjunctional sarcolemma.Journal of Cell Biology 63, 567–86.
Rash, J. E., Hudson, C. S. &Ellisman, M. H. (1978) Ultrastructure of acetylcholine receptors at the mammalian neuromuscular junction. InCell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach, (edited byBolis, L. &Straub, R. W.), pp. 47–68. New York: Raven Press.
Rosenbluth, J. (1974) Substructure of amphibian motor end plate. Evidence for a granular component projecting from the outer surface of the receptive membrane.Journal of Cell Biology 62, 755–66.
Rosenbluth, J. (1975) Synaptic membrane structure inTorpedo electric organ.Journal of Neurocytology 4, 697–712.
Sanes, J. R. &Hall, Z. W. (1979) Antibodies that bind specifically to synaptic sites on muscle fiber basal lamina.Journal of Cell Biology 83, 357–70.
Schiff, P. B., Fant, J. &Horwitz, S. B. (1979) Promotion of microtubule assemblyin vitro by taxol.Nature 277, 665–7.
Schotton, D. M., Heuser, J. E., Reese, B. F. &Reese, T. S. (1979) Postsynaptic membrane folds of the frog neuromuscular junction visualized by scanning electron microscopy.Neuroscience 4, 427–35.
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Hirokawa, N., Heuser, J.E. Internal and external differentiations of the postsynaptic membrane at the neuromuscular junction. J Neurocytol 11, 487–510 (1982). https://doi.org/10.1007/BF01257990
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DOI: https://doi.org/10.1007/BF01257990