Skip to main content
SpringerLink
Log in

Differentiation of sympathicoblasts in cultures of chick ganglia

Light and electron microscopic, fluorescence and enzyme histochemical observations

  • Published:
Anatomy and Embryology Aims and scope Submit manuscript

Summary

Immature sympathetic ganglia prepared from 51/2- or 6-day-old chick embryos were cultured up to one month. The in vitro development was followed by phase microscopy, electron microscopy and using histochemistry for catecholamines, monoamine oxidase and cholinesterases.

During the first week of culture extensive plexuses of nerve fibres were formed between and around the clusters of nerve cells. Mature-looking neurons were observed in the cultures by phase microscopy after three weeks, at which age the mean diameter of the perikarya was more than doubled.

Varying catecholamine fluorescence was observed in the perikarya during the entire culture period. The nerve fibres showed usually only weak fluorescence, but, in the older cultures, bright varicosities were regularly found in the fibres. Monoamine oxidase activity was demonstrated already at three days of culture and the reaction was maintained positive. Weak or moderate acetyl-cholinesterase activity was demonstrated in the sympathicoblasts and young sympathetic neurons and their processes. The axolemma showed acetylcholinesterase activity also around the nerve terminals containing small dense cored vesicles. Reactions for the non-specific cholinesterases were negative.

Electron microscopy of the 30-day-old cultures revealed that the clusters of nerve cells consisted of mature sympathetic neurons, which contained large (60–200 nm) and small (35–60 nm) granular catecholamine-storing vesicles. Glial cells were almost totally lacking. Large numbers of nerve terminals containing both large and small granular vesicles were observed in the clusters, often in synaptic contact with the sympathetic neurons.

It is concluded that the primitive sympathicoblasts are, in favourable conditions, capable of differentiation in culture up to mature sympathetic neurons.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Benitez, H. H., Masurovsky, E. B., Murray, M. R.: Interneurons of the sympathetic ganglia, in organotypic culture. A suggestion as to their function, based on three types of study. J. Neurocytol. 3, 363–384 (1974)

    Google Scholar 

  • Benitez, H. H., Murray, M. R., Coté, L. J.: Responses of sympathetic chain-ganglia isolated in organotypic culture to agents affecting adrenergic neurons: Fluorescence histochemistry. Exp. Neurol. 39, 424–448 (1973)

    Google Scholar 

  • Bunge, M. B., Bunge, R. P., Peterson, E. R.: The onset of synapse formation in spinal cord culture as studied by electron microscopy. Brain Res. 6, 728–743 (1967)

    Google Scholar 

  • Bunge, R. P., Rees, R., Wood, P., Burton, H., Ko, C.-P.: Anatomical and physiological observations on synapses formed on isolated autonomic neurons in tissue culture. Brain Res. 66, 401–412 (1974)

    Google Scholar 

  • Burn, J. H.: Release of noradrenaline from sympathetic endings. Nature (Lond.) 231, 237–240 (1971)

    Google Scholar 

  • Burn, J. H., Rand, M. J.: Sympathetic postganglionic mechanism. Nature (Lond.) 184, 163–165 (1959)

    Google Scholar 

  • Chamley, J. H., Mark, G. E., Campbell, G. R., Burnstock, G.: Sympathetic ganglia in culture. I. Neurons. Z. Zellforsch. 135, 287–314 (1972)

    Google Scholar 

  • Cohen, A. M.: Factors directing the expression of sympathetic nerve traits in cells of neural crest origin. J. exp. Zool. 179, 167–182 (1972)

    Google Scholar 

  • Corrodi, H., Jonsson, G.: The formaldehyde fluorescence method for the histochemical demonstration of biogenic monoamines. A review on the methodology. J. Histochem. Cytochem. 15, 65–78 (1967)

    Google Scholar 

  • Coupland, R. E., Weakley, B. S.: Developing chromaffin tissue in the rabbit: an electron microscopic study. J. Anat. (Lond.) 102, 425–455 (1968)

    Google Scholar 

  • Enemar, A., Falck, B., Håkanson, R.: Observations on the appearance of norepinephrine in the sympathetic nervous system of the chick embryo. Develop. Biol. 11, 268–283 (1965)

    Google Scholar 

  • Eränkö, L.: Ultrastructure of the developing sympathetic nerve cell and the storage of catecholamines. Brain Res. 46, 159–175 (1972)

    Google Scholar 

  • Eränkö, O.: The practical histochemical demonstration of catecholamines by formaldehyde-induced fluorescence. J. roy. micr. Soc. 87, 259–276 (1967)

    Google Scholar 

  • Eränkö, O., Heath, J. W., Eränkö, L.: Effect of hydrocortisone on the ultrastructure of the small, intensely fluorescent, granule-containing cells in cultures of sympathetic ganglia of newborn rats. Z. Zellforsch. 134, 297–310 (1972)

    Google Scholar 

  • Foelix, R. F., Oppenheim, R.: The development of synapses in the cerebellar cortex of the chick embryo. J. Neurocytol. 3, 277–294 (1974)

    Google Scholar 

  • Giacobini, G., Marchisio, P. C., Giacobini, E., Koslow, S. H.: Developmental changes of cholinesterases and monoamine oxidase in chick embryo spinal and sympathetic ganglia. J. Neurochem. 17, 1177–1185 (1970)

    Google Scholar 

  • Glenner, G. G., Burtner, H. J., Brown, G. W.: The histochemical demonstration of monoamine oxidase activity by tetrazolium salts. J. Histochem. Cytochem. 5, 591–600 (1957)

    Google Scholar 

  • Grillo, M. A.: Electron microscopy of sympathetic tissues. Pharmacol. Rev. 18, 387–399 (1966)

    Google Scholar 

  • Hamburger, V., Hamilton, H. L.: A series of normal stages in the development of the chick embryo. J. Morph. 88, 49–92 (1951)

    Google Scholar 

  • Hermetet, J., Treska, J., Mandel, P.: Histochemical study of isolated neurons in culture from chick embryo sympathetic ganglia. Histochemie 22, 177–186 (1970)

    Google Scholar 

  • Hervonen, A.: Development of catecholamine-storing cells in human fetal paraganglia and adrenal medulla. Acta physiol. scand., Suppl. 368 (1971)

  • Hervonen, H.: Formaldehyde-induced fluorescence in the sympathetic ganglia of chick embryo in maturing organotypic culture. Med. Biol. 52, 154–163 (1974)

    Google Scholar 

  • Hervonen, H., Eränkö, O.: Fluorescence histochemical and electron microscopical observations on sympathetic ganglia of the chick embryo cultured with and without hydrocortisone. Cell Tiss. Res. (1974, in press)

  • Hervonen, H., Rechardt, L.: Observations on closed tissue cultures of sympathetic ganglia of chick embryos in media buffered with N-Tris (Hydroxymethyl)Methyl-Glysine or N-2-Hydroxy-Ethylpiperazine-N-2-Ethanesulfonic acid. Acta physiol. scand. 90, 267–277 (1974a)

    Google Scholar 

  • Hervonen, H., Rechardt, L.: Light and electron microscopic demonstration of cholinesterases of the cultured sympathetic ganglia of the chick embryo. Histochemistry 39, 129–142 (1974b)

    Google Scholar 

  • Hökfelt, T.: Ultrastructural localization of intraneuronal monoamines. Some aspects on methodology. Progr. Brain Res. 34, 213–222 (1971)

    Google Scholar 

  • Ignarro, L. J., Shideman, F. E.: Apperance and concentrations of catecholamines and their biosynthesis in the embryonic and developing chick. J. Pharmacol. exp. Ther. 159, 38–48 (1968)

    Google Scholar 

  • Kanerva, L.: Ultrastructure of sympathetic ganglion cells and granule-containing cells in the paracervical (Frankenhäuser) ganglion of the newborn rat. Z. Zellforsch. 126, 25–40 (1972)

    Google Scholar 

  • Kanerva, L., Teräväinen, H.: Electron microscopy of the paracervical (Frankenhäuser) ganglion of the adult rat. Z. Zellforsch. 129, 161–177 (1972)

    Google Scholar 

  • Kim, S. U., Munkacsi, I.: Cytochemical demonstration of catecholamines and acetylcholinesterase in cultures of chick sympathetic ganglia. Experientia (Basel) 28, 824–825 (1972)

    Google Scholar 

  • Kim, S. U., Munkacsi, I.: Morphological and cytochemical characteristics of neurons in cultures of mouse sympathetic ganglia. Exp. Neurol. 45, 94–103 (1974)

    Google Scholar 

  • Koelle, G. B.: The elimination of enzymatic diffusion artifacts in the histochemical localization of cholinesterases and a survey of their cellular distribution. J. Pharmacol. exp. Ther. 103, 153–171 (1951)

    Google Scholar 

  • Larrabee, M. G.: Metabolism of adult and embryonic sympathetic ganglia. Fed. Proc. 29, 1919–1928 (1970)

    Google Scholar 

  • Lever, J. D., Presley, M.: Studies on the sympathetic neurone in vitro. Progr. Brain Res. 34, 499–512 (1971)

    Google Scholar 

  • Levi-Montalcini, R., Hamburger, V.: A diffusable agent of mouse sarcoma, producing hyperplasia of sympathetic ganglia and hyperneurotization in the chick embryo. J. exp. Zool. 123, 233–287 (1953)

    Google Scholar 

  • Lewis, P. R., Shute, C. C. D.: The distribution of cholinesterase in cholinergic neurons demonstrated with the electron microscope. J. Cell Sci. 1, 381–390 (1966)

    Google Scholar 

  • Model, P. G., Bornstein, M. B., Crain, S. T., Pappas, G. D.: An electron microscopic study of the development of synapses in cultured fetal mouse cerebrum continuously exposed to xylocaine. J. Cell Biol. 49, 362–371 (1971)

    Google Scholar 

  • Mollenhauer, H. H.: Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111–114 (1964)

    Google Scholar 

  • Murray, M. R.: Nervous tissues isolated in culture. In: Handbook of neurochemistry (ed. A. Lajtha), vol. V, p. 373–438. New York: Plenum Press 1971

    Google Scholar 

  • Murray, M. R., Benitez, H. H.: Deuterium oxide: Direct action on sympathetic ganglia isolated in culture. Science 155, 1021–1024 (1967)

    Google Scholar 

  • Nachmansohn, D., Neuman, E.: Properties and function of proteins in excitable membranes: An integral model of nerve excitability. Ann. N.Y. Acad. Sci. 277, 275–284 (1974)

    Google Scholar 

  • Norr, S. C.: In vitro analysis of sympathetic neuron differentiation from chick neural crest cells. Develop. Biol. 34, 16–38 (1973)

    Google Scholar 

  • O'Lague, P. H., Obata, K., Claude, P., Furshpan, E. J., Potter, D. D.: Evidence for cholinergic synapses between dissociated rat sympathetic neurons in cell culture. Proc. nat. Acad. Sci. (Wash.) 71, 3602–3606 (1974)

    Google Scholar 

  • Olson, M. I., Bunge, R. P.: Anatomical observations on the specificity of synapse formation in tissue culture. Brain Res. 59, 19–33 (1973)

    Google Scholar 

  • Rees, R., Bunge, R. P.: Morphological and cytochemical studies of synapses formed in culture between isolated rat superior cervical ganglion neurons. J. comp. Neurol. 157, 1–12 (1974)

    Google Scholar 

  • Reynolds, E. S.: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208–212 (1963)

    Google Scholar 

  • Romanoff, A. L.: The avian embryo. Structural and functional development. New York: The McMillan Comp. 1960

    Google Scholar 

  • Teichberg, S., Holtzman, E.: Axonal agranular reticulum and synaptic vesicles in cultured embryonic chick sympathetic neurons. J. Cell Biol. 57, 88–108 (1973)

    Google Scholar 

  • Watson, M. L.: Staining of tissue sections for electron microscopy with heavy metals. J. biophys. biochem. Cytol. 4, 475–478 (1958)

    Google Scholar 

  • Wechsler, W., Schmekel, L.: Electronenmikroskopische Untersuchung der Entwicklung der vegetativen (Grenzstrang-) und spinalen Ganglien bei Gallus domesticus. Acta neuroveg. (Wien) 30, 427–444 (1967)

    Google Scholar 

  • Yates, R. D.: A study of cell division in chick embryonic ganglia. J. exp. Zool. 147, 167–181 (1961)

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Anatomy, University of Helsinki, Helsinki, Finland

    Heikki Hervonen

Authors
  1. Heikki Hervonen
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Head: Prof. Olavi Eränkö, M. D., Ph. D.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hervonen, H. Differentiation of sympathicoblasts in cultures of chick ganglia. Anat Embryol 146, 225–243 (1975). https://doi.org/10.1007/BF00302172

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00302172

Key words

Search

Navigation