Summary
The extracellular matrix plays important roles in the differentiation of synapses. To identify molecules concentrated specifically in the synaptic extracellular matrix, fluorescently-labelled lectins were applied to neuromuscular junctions. A lectin, peanut agglutinin (PNA), stains the neuromuscular region selectively and irreversibly (up to at least 3 weeksin situ), outlining the periphery of the nerve terminal arborization in the frog. Snake neuromuscular junctions also stain intensely with fluorescent PNA, while mouse diaphragm staining is faint. At the electron microscopic level, the reaction products of horseradish peroxidase-conjugated PNA are found primarily in the extracellular matrix flanking Schwann cells in the frog endplate regions. Fluorescently labelled PNA does not affect synaptic potentials and can serve as a simple stain for correlating functional studies of living neuromuscular junctions. Moreover, it can be combined with a presynaptic dye to observe nerve terminals and synaptic extracellular matrix in the same junctionsin situ. This report reveals the existence of synapse-specific carbohydrates associated with Schwann cell extracellular matrix in the frog neuromuscular junction. The specific binding and its physiological compatibility make PNA a useful probe for further investigation of synaptic differentiation, plasticity and maintenance.
Similar content being viewed by others
References
Anderson, M. J. (1986) Nerve-induced remodeling of muscle basal lamina during synaptogenesis.Journal of Cell Biology 102, 863–77.
Anderson, M. J. &Cohen, M. W. (1974) Fluorescent staining of acetylcholine receptors in vertebrate skeletal muscle.Journal of Physiology 237, 385–400.
Anglister, L. &McMahan, U. J. (1985) Basal lamina directs acetylcholinesterase accumulation at synaptic sites in regenerating muscle.Journal of Cell Biology 101, 735–43.
Bausch, J. N. &Poretz, R. D. (1977) Purification and properties of the hemagglutinin fromMaclura pomifera seeds.Biochemistry 16, 5790–4.
Betz, W. &Sakmann, B. (1973) Effects of proteolytic enzymes on function and structure of frog neuromuscular junctions.Journal of Physiology 230, 673–88.
Borges, L. F. &Sidman, L. R. (1982) Axonal transport of lectins in the peripheral nervous system.Journal of Neuroscience 5, 647–53.
Burden, S. J., Sargent, P. B. &McMahan, U. J. (1979) Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of nerve.Journal of Cell Biology 82, 412–25.
Cooper, N. G. F. &Steindler, D. A. (1986) Lectins demarcate the barrel subfield in the somatosensory cortex of the early postnatal mouse.Journal of Comparative Neurology 249, 157–69.
Covault, J. &Sanes, J. R. (1986) Distribution of N-CAM in synaptic and extrasynaptic portions of developing and adult skeletal muscle.Journal of Cell Biology 102, 716–30.
Del Castillo, J. &Katz, B. J. (1954) Quantal components of the endplate potential.Journal of Physiology 124, 560–73.
Edelman, G. M. (1983) Cell adhesion molecules.Science 219, 450–7.
Fallon, J. R., Nitkin, R. M., Reist, N. E., Wallace, B. G. &McMahan, U. J. (1985) Acetylcholine receptor-aggregating factor is similar to molecules concentrated at neuromuscular junctions.Nature 315, 571–4.
Fatt, P. &Katz, B. (1952) Spontaneous subthreshold activity at motor nerve endings.Journal of Physiology 117, 109–28.
Godfrey, E. W., Nitkin, R. M., Wallace, B. G., Rubin, L. L. &McMahan, U. J. (1984) Components ofTorpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells.Journal of Cell Biology 99, 615–27.
Goldstein, I. J. &Hayes, C. E. (1978) The lectins: carbohydrate-binding proteins of plants and animals.Advanced Carbohydrate Chemistry 35, 127–340.
Graham, R. C. &Karnovsky, M. J. (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubule of the mouse kidney: ultrastructural cytochemistry by a new technique.Journal of Histochemistry and Cytochemistry 14, 291–302.
Grinnel, A. D. &Herrera, A. A. (1981) Specificity and plasticity of neuromuscular connections: long term regulation of motoneuron function.Progress in Neurobiology 17, 203–82.
Indue, S. (1986)Video Microscopy. New York: Plenum Press.
Johnson, L. V., Hageman, G. S. &Blanks, J. C. (1985) Interphotoreceptor matrix domains ensheath vertebrate cone photoreceptor cells.Investigative Ophthalmology and Visual Science 27, 129–35.
Karnovsky, M. J. (1964) The localization of cholineterase activity in rat cardiac muscle by electron microscopy.Journal of Cell Biology 23, 217–32.
Kelly, S. S., Anis, N. &Robbins, N. (1985) Fluorescent staining of living mouse neuromuscular junctions.Pflugers Archiv 404, 97–9.
Ko, C.-P. &Propst, J. W. (1986) Freeze-fracture of physiologically identified neuromuscular junctions from single frog muscle fibers.Journal of Electron Microscopy Technique 4, 147–56.
Kuffler, D. P. (1986), Thickness of the basal lamina at the frog neuromuscular junction.Journal of Comparative Neurocytology 250, 236–44.
Letinsky, M. S., Fishbeck, K. W. &McMahan, U. J. (1976) Precision of reinnervation of original postsynaptic sites in muscle after a nerve crush.Journal of Neurocytology 5, 691–718.
Lichtman, J. W., Magrassi, L. &Purves, D. (1986) Repeated visualization of neuromuscular junctions in the living mouse.Society for Neuroscience Abstracts 12, 390.
Lichtman, J. W., Magrassi, L. &Purves, D. (1987) Visualization of neuromuscular junctions over a period of several months in living mice.Journal of Neuroscience 7, 1215–22.
Lotan, R., Skutelsky, E., Danon, D. &Sharon, N. (1975) The purification, composition, and specificity of the anti-T lectin from peanut (Arachis hypogaea).Journal of Biological Chemistry 250, 8518–23.
Magrassi, L., Lichtman, J. W. &Purves, D. (1987) Fluorescent probes that stain living nerve terminals.Journal of Neuroscience 7, 1207–14.
McMahan, U. J. &Slater, C. R. (1984) The influence of basal lamina on the accumulation of acetylcholine receptors at synaptic sites in regenerating muscle.Journal of Cell Biology 98, 1453–73.
Nitkin, R. M., Wallace, B. G., Spira, M. E., Godfrey, E. W. &McMahan, U. J. (1983) Molecular components of the synaptic basal lamina that direct differentiation of regenerating neuromuscular junctions.Cold Spring Harbor Symposia on Quantitative Biology 48, 653–65.
Osawa, T., Irimura, T. &Kawaguchi, T. (1978)Bauhinia pupurea agglutinin.Methods in Enzymology 50, 367–72.
Propst, J. W. &KO, C.-P. (1987) Correlations between active zone ultrastructure and synaptic function studied with freeze-fracture of physiologically identified neuromuscular junctions.Journal of Neuroscience (in press).
Purves, D., Hadley, R. D. &Voyvodic, J. T. (1986) Dynamic changes in the dendritic geometry of individual neurons visualized over periods of up to three months in the superior cervical ganglion of living mice.Journal of Neuroscience 6, 1051–60.
Rich, M. M. &Lichtman, J. W. (1986) Remodelling of endplate sites during muscle reinnervation in the living mouse.Society for Neuroscience Abstracts 12, 390.
Ridge, R. M. A. P. (1971) Different types of extra fusal muscle fibres in snake costocutaneous muscles.Journal of Physiology 217, 393–418.
Sanes, J. R. (1982) Laminin, fibronectin, and collagen in synaptic and extrasynaptic portions of muscle fiber basement membrane.Journal of Cell Biology 93, 442–51.
Sanes, J. R. (1983) Roles of extracellular matrix in neural development.Annual Review of Physiology 45, 581–600.
Sanes, J. R. &Cheney, J. M. (1982) Lectin binding reveals a synapse-specific carbohydrate in skeletal muscle.Nature 300, 646–7.
Sanes, J. R. &Chiu, A. Y. (1983) The basal lamina of the neuromuscular junction.Cold Spring Harbor Symposia on Quantitative Biology 48, 667–78.
Sanes, J. R., Marshall, L. M. &McMahan, U. J. (1978) Reinnervation of muscle fiber basal lamina after removal of myofibers. Differentiation of regenerating axons at original synaptic sites.Journal of Cell Biology 78, 176–98.
Sanes, J. R., Schachner, M. &Covault, J. (1986) Expression of several adhesive macromolecules (N-CAM, LI, JI, NILE, Uromorulin, Laminin, Fibronectin, and a Heparan Sulfate Proteoglycan) in embryonic, adult and denervated adult skeletal muscle.Journal of Cell Biology 102, 420–31.
Smith, C. E. &Ko, C.-P. (1986) A plant lectin, peanut agglutinin, binds specifically at the neuromuscular junction in the frog.Society for Neuroscience Abstracts 12, 1500.
Wu, A. M. (1984) Differential binding characteristics and applications of D-galactose-(1→ 3) D-galactosamine specific lectins.Molecular and Cellular Biochemistry 61, 131–41.
Yoshikami, D. &Okun, L. M. (1984) Staining of living presynaptic nerve terminals with selective fluorescent dyes.Nature 310, 53–6.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Ko, CP. A lectin, peanut agglutinin, as a probe for the extracellular matrix in living neuromuscular junctions. J Neurocytol 16, 567–576 (1987). https://doi.org/10.1007/BF01668509
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01668509