Long-Term Cultures of Embryonic and Mature Insect Nervous and Neuroendocrine Systems

  • R. Levi-Montalcini
  • K. R. Seshan
Part of the Current Topics in Neurobiology book series (CTNB)


More than a half century of extensive work on the vertebrate nervous system cultured in vitro under different experimental conditions has brought to light the merits and the limitations of these techniques. While a considerable amount of information has been gathered on growth and differentiation of nerve cells, on axonal growth, on the relationship between glial and nerve cells, and, recently, also on bioelectrical properties of neuronal circuits in vitro (Crain and Peterson, 1964, 1967; Crain et al., 1970), little has been learned concerning the organization of nerve cells at the supracellular level and no attempts have been made to explore, with the aid of these techniques, the problem of neuronal specificity and the building of wiring circuits between nerve cell populations and between nerve cells and their end organs. The reasons which suggested restriction of the study to only a few neurobiological problems are numerous. To mention only some of the limiting factors, we remind the reader that this system in vertebrates is from its very inception a highly organized system and cannot operate when submitted to dissociation into small fragments, with each one cultured alone or in proximity to other parts of the same system or of nonnervous structures. Disruption of the continuity of the neuraxis and destruction of the blood capillary network, which permeates the entire system and provides the nutrition and blood supply of individual nerve units, are most damaging factors which cannot possibly be mitigated by any technical skill or ingenious device. In order to permit survival if not proper function of the nerve cell populations, which must rely on diffusion rather than on blood vascular channels, the explants must be reduced to what has been defined as “the critical cubic millimeter” (Lumsden, 1968). The fragments of the nervous system undergo, as a rule, flattening and thinning in long-term cultures. This condition favors exchanges with the medium, and nerve cells located at the periphery of the explants survive reasonably well, but cell-to-cell interconnections through nerve circuits are grossly altered and nothing can be learned about their normal function, leaving aside the more complex problem of the operation of neuronal circuits between distant nuclei and between these and their end organs.


Nerve Fiber Nerve Cell Alimentary Canal Neuroendocrine System Thoracic Ganglion 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Aggarwal, S. K., and King, R. C. 1971. An electron microscopic study of the corpus cardiacum of adult Drosophila melanogaster and its afferent nerves. J. Morphol. 134: 437–446.PubMedCrossRefGoogle Scholar
  2. Amaldi, P. 1970. Incorporation of RNA and protein precursors into embryonic nerve cells of Periplaneta americana cultured in vitro. Brain Res. 21:305–308.PubMedCrossRefGoogle Scholar
  3. Attardi, D. G., and Sperry, R. W. 1963. Preferential selection of central pathways by regenerating optic fibers. Exptl. Neurol. 7:46–64.CrossRefGoogle Scholar
  4. Beattie, T. M. 1971. Histology, histochemistry, and ultrastructure of neurosecretory cells in the optic lobe of the cockroach, Periplaneta americana. J. Insect Physiol. 17(10): 1843–1855.CrossRefGoogle Scholar
  5. Bern, H. A. 1966. On the production of hormones by neurons and the role of neurosecretion in neuroendocrine mechanisms. Symp. Soc. Exptl. Biol. 20:325–344.Google Scholar
  6. Bern, H. A., and Hagadorn, I. R. 1965. Neurosecretion, pp. 353–429. In T. H. Bullock and G. A. Horridge (eds.). Structure and Function in the Nervous Systems of Invertebrates. W. H. Freeman & Co., San Francisco and London.Google Scholar
  7. Bowers, B., and Johnson, B. 1966. An electron microscopic study of the corpora cardiaca and secretory neurons in the aphid, Myzus persicae Sulz. Gen. Comp. Endocrinol. 6: 213–230.Google Scholar
  8. Brown, B. E. 1965. Pharmacologically active constituents of the cockroach corpus cardiacum: Resolution and some characteristics. Gen. Comp. Endocrinol. 5:387–401.PubMedCrossRefGoogle Scholar
  9. Bullock, T. H., and Horridge, G. A. 1965. Structure and Function in the Nervous Systems of Invertebrates, Vols. I and II. W. H. Freeman & Co., San Francisco. 1719 pp.Google Scholar
  10. Callec, J. J., Guillet, J. C., Pichon, Y., and Boistel, J. 1971. Further studies on synaptic transmission in insects. II. Relations between sensory information and its synaptic integration at the level of a single giant axon in the cockroach. J. Exptl. Biol. 55:123–149.Google Scholar
  11. Cassier, P., and Fain-Maurel, M. A. 1970. Contribution à l’étude infrastructurale du système neurosécréteur rétrocérébral chez Locusta migratoria migratoriodes (R. et F.) 1. Les corpora cardiaca. Z. Zellforsch. 111:471–482.PubMedCrossRefGoogle Scholar
  12. Cazal, M., Joly, L., and Porte, A. 1971. Étude ultrastructurale des corpora cardiaca et de quelques formations annexés chez Locusta migratoria L. Z. Zellforsch. 114:61–72.PubMedCrossRefGoogle Scholar
  13. Chen, J. S., and Levi-Montalcini, R. 1969. Axonal outgrowth and cell migration in vitro from nervous system of cockroach embryos. Science 166:631–632.PubMedCrossRefGoogle Scholar
  14. Chen, J. S., and Levi-Montalcini, R. 1970. Long term cultures of dissociated nerve cells from the embryonic nervous system of the cockroach Periplaneta americana. Arch. Ital. Biol. 108:503–537.PubMedGoogle Scholar
  15. Cohen, J. J. 1967. Correlations between structure, function and RNA metabolism in central neurons of insects, pp. 65–78. In C. A. G. Wiersma (ed.). Invertebrate Nervous Systems. University of Chicago Press, Chicago.Google Scholar
  16. Cornwell, P. B. 1968. The Cockroach, Vol. 1. Hutchinson, London.Google Scholar
  17. Crain, S. M., and Peterson, E. R. 1964. Complex bioelectric activity in organized tissue cultures of spinal cord (human, rat and chick). J. Cell. Comp. Physiol. 64:1–13.CrossRefGoogle Scholar
  18. Crain, S. M., and Peterson, E. R. 1967. Onset and development of functional interneuronal connections in expiants of rat spinal cord-ganglia during maturation in culture. Brain Res. 6:750–762.PubMedCrossRefGoogle Scholar
  19. Crain, S. M., Peterson, E. R., and Bornstein, M. B. 1968. Formation of functional interneuronal connections between expiants of various mammalian central nervous tissues during development in vitro, pp. 13–31. In G. E. W. Wolstenholme and M. O’Connor (eds.). Ciba Foundation Symposium on Growth of the Nervous System. J. & A. Churchill Ltd., London.Google Scholar
  20. Crain, S. M., Alfei, L., and Peterson, E. R. 1970. Neuromuscular transmission in cultures of adult human and rodent skeletal muscle after innervation in vitro by fetal rodent spinal cord. J. Neurobiol. 1:471–489.PubMedCrossRefGoogle Scholar
  21. Echalier, G., and Ohanessian, A. 1970. In vitro culture of Drosophila melanogaster embryonic cells. In vitro 6:162–172.PubMedCrossRefGoogle Scholar
  22. Farley, R. D., and Milburn, N. S. 1969. Structure and function of the giant fiber system in the cockroach, Periplaneta americana. J. Insect Physiol. 15:457–476.CrossRefGoogle Scholar
  23. Frontali, N., and Mancini, G. 1970. Studies on the neuronal organization of cockroach corpora pedunculata. J. Insect Physiol. 16:2293–2301.CrossRefGoogle Scholar
  24. Gabe, M. 1966. Neurosecretion. Pergamon Press, Oxford, London, New York. 872 pp.Google Scholar
  25. Gersch, M., Richter, K., Böhm, G.-A., and Stürzebecher, J. 1970. Selektive Ausschuttung von Neurohormonen nach elektrischer Reizung der Corpora Cardiaca von Periplaneta americana in vitro. J. Insect Physiol. 16:1991–2013.CrossRefGoogle Scholar
  26. Gilbert, L. I. 1964. Physiology of growth and development: Endocrine aspects, pp. 149–225. In M. Rockstein (ed.). Physiology of Insecta. Academic Press, New York.Google Scholar
  27. Hagadorn, I. R. 1967. Neurosecretory mechanisms, pp. 115–124. In C. A. G. Wiersma (ed.). Invertebrate Nervous Systems: Their Significance for Mammalian Neurophysiology. University of Chicago Press, Chicago.Google Scholar
  28. Highnam, K. C. 1969. Neurosecretion in insects. Progr. Endocrinol., Excerpta Med. Internat. Congr. Ser., No. 184, pp. 351–355.Google Scholar
  29. Highnam, K. C., and Goldsworthy, G. J. 1972. Regenerated corpora cardiaca and hyperglycemic factor in Locusta migratoria. Gen. Comp. Endocrinol. 18:83–88.PubMedCrossRefGoogle Scholar
  30. Hyde, A. C. Ta. 1972. Regeneration, post-embryonic induction and cellular interaction in the eye of Periplaneta americana. J. Embryol. Exptl. Morphol. 27:367–379.Google Scholar
  31. Johnson, B., and Bowers, B. 1963. Transport of neurohormones from the corpora cardiaca in insects. Science 141:264–266.PubMedCrossRefGoogle Scholar
  32. Kater, S. B. 1968. Cardioaccelerator release in Periplaneta americana (L). Science 160: 765–767.PubMedCrossRefGoogle Scholar
  33. Keeley, L. L., and Friedman, S. 1967. Corpus cardiacum as a metabolic regulator in Blaberus discoidalis Serville (Blattidae). Gen. Comp. Endocrinol. 8:129–134.CrossRefGoogle Scholar
  34. Landureau, J. C. 1966. Cultures in vitro de cellules embryonnaires de Blattes. Exptl. Cell Res. 41:545–556.PubMedCrossRefGoogle Scholar
  35. Landureau, J. C. 1968. Cultures in vitro de cellules embryonnaires de Blattes (Insectes Dictyoptères). II. Obtention de lingnées cellulaires à multiplication continue. Exptl. Cell Res. 50:323–337.CrossRefGoogle Scholar
  36. Levi-Montalcini, R. 1963. Growth and differentiation in the nervous system, pp. 261–295. In J. M. Allen (ed.). The Nature of Biological Diversity. McGraw-Hill, New York.Google Scholar
  37. Levi-Montalcini, R., and Chen, J. S. 1969. In vitro studies of the insect embryonic nervous system, pp. 277–298. In S. H. Barondes (ed.). Cellular Dynamics of the Neuron. Academic Press, New York.Google Scholar
  38. Levi-Montalcini, R., and Chen, R. S. 1971. Selective outgrowth of nerve fibers in vitro from embryonic ganglia of Periplaneta americana. Arch. Ital. Biol. 109:307–337.PubMedGoogle Scholar
  39. Locke, M. 1958. The formation of tracheae and tracheoles in Rhodnius prolixus. Quart. J. Microscop. Sci. 99:29–46.Google Scholar
  40. Lumsden, C. E. 1968. Nervous tissue in culture, pp. 67–140. In G. H. Bourne (ed.). The Structure and Function of Nervous Tissue, Vol. 1. Academic Press, New York.Google Scholar
  41. Marks, E. P. 1968. Regenerating tissues from the cockroach Leucophaea maderae: Effects of humoral stimulation in vitro. Gen. Comp. Endocrinol. 11:31–42.PubMedCrossRefGoogle Scholar
  42. Marks, E. P. 1970. The action of hormones in insect cell and organ cultures. Gen. Comp. Endocrinol. 15:289–302.PubMedCrossRefGoogle Scholar
  43. Marks, E. P., and Reinecke, J. P. 1965. Regenerating tissues from the cockroach Leucophaea maderae: Effects of endocrine glands in vitro. Gen. Comp. Endocrinol. 5:241–247.CrossRefGoogle Scholar
  44. Marks, E. P., Reinecke, J. P., and Leopold, R. A. 1968. Regenerating tissues from the cockroach Leucophaea maderae: nerve regeneration in vitro. Biol. Bull. 135:520–529.CrossRefGoogle Scholar
  45. Meola, S. M., and Lea, A. O. 1972. The ultrastructure of the corpus cardiacum of Aedes sollicitans and the histology of the cerebral neurosecretory system of mosquitoes. Gen. Comp. Endocrinol. 18:210–234.PubMedCrossRefGoogle Scholar
  46. Mordue, W., and Goldsworthy, G. J. 1969. The physiological effects of corpus cardiacum extracts in locusts. Gen. Comp. Endocrinol. 12:360–369.PubMedCrossRefGoogle Scholar
  47. Muller, H. P., and Engelmann, F. 1968. Studies on the endocrine control of metabolism in Leucophaea maderae (Blattaria). II. Effect of the corpora cardiaca on fat-body respiration. Gen. Comp. Endocrinol. 11:43–50.PubMedCrossRefGoogle Scholar
  48. Natalizi, G. M., and Frontali, N. 1966. Purification of insect hyperglycaemic and heart accelerating hormones. J. Insect Physiol. 12:1279–1287.CrossRefGoogle Scholar
  49. Natalizi, G. M., Pansa, M. C., D’Ajello, V., Casaglia, O., Bettini, S., and Frontali, N. 1970. Physiologically active factors from corpora cardiaca of Periplaneta americana. J. Insect Physiol. 16:1827–1836.CrossRefGoogle Scholar
  50. Normann, T. C. 1965. The neurosecretory system of the adult Calliphora erythrocephala. I. The fine structure of the corpus cardiacum with some observations on adjacent organs. Z. Zellforsch. 67:461–501.PubMedCrossRefGoogle Scholar
  51. Normann, T. C., and Duve, H. 1969. Experimentally induced release of a neurohormone influencing hemolymph trehalose level in Calliphora erythrocephala (Diptera). Gen. Comp. Endocrinol. 12:449–459.PubMedCrossRefGoogle Scholar
  52. Palay, S. L. 1957. The fine structure of the neurohypophysis, pp. 31–49. In H. Waelsch (ed.). Ultrastructure and Cellular Chemistry of Neural Tissue, Vol. II of Progress in Neurobiology. Paul B. Hoeber, New York.Google Scholar
  53. Pearson, K. G. 1972. Central programming and reflex control of walking in the cockroach. J. Exptl. Biol. 56:173–193.Google Scholar
  54. Pearson, K. G., and Iles, J. F. 1970. Discharge patterns of coxal levator and depressor motoneurones of the cockroach, Periplaneta americana. J. Exptl. Biol. 52:139–165.Google Scholar
  55. Penzlin, H., and Stölzner, W. 1971. Frontal ganglion and water balance in Periplaneta americana L. Experientia 3:390–391.CrossRefGoogle Scholar
  56. Peterson, E. R., and Crain, S. M. 1970. Innervation in cultures of fetal rodent skeletal muscle by organotypic explants of spinal cord from different animals. Z. Zeilforsch. 106:1–21.CrossRefGoogle Scholar
  57. Pichon, Y., and Callec, J. J. 1970. Further studies on synaptic transmission in insects. I. External recording of synaptic potentials in a single giant axon of the cockroach, Periplaneta americana L. J. Exptl. Biol. 52:257–265.Google Scholar
  58. Pipa, R. L., and Cook, E. F. 1959. Studies on the hexapod nervous system. I. The peripheral distribution of the thoracic nerves of the adult cockroach, Periplaneta americana. Ann. Entomol. Soc. Am. 52(6):695–710.Google Scholar
  59. Scharrer, B. 1962. Neurosecretion. The fine structure of the neurosecretory system of the insect Leucophaea maderae. Mem. Soc. Endocrinol., No. 12, pp. 89–97.Google Scholar
  60. Scharrer, B. 1963. Neurosecretion. XIII. The ultrastructure of the corpus cardiacum of the insect Leucophaea maderae. Z. Zeilforsch. 60:761–796.CrossRefGoogle Scholar
  61. Scharrer, B., and Weitzman, M. 1970. Current problems in invertebrate neurosecretion, pp. 1–23. In W. Bargmann and B. Scharrer (eds.). Aspects of Neuroendocrinology. Springer-Verlag, Berlin, Heidelberg, New York.Google Scholar
  62. Scharrer, E., and Scharrer, B. 1963. Neuroendocrinology. Columbia University Press, New York and London. 289 pp.Google Scholar
  63. Schneider, I. 1967. Insect tissue culture, pp. 543–554. In N. Wilt and N. K. Wessells (eds.). Methods in Developmental Biology. Crowell, New York.Google Scholar
  64. Schneiderman, H. A., and Gilbert, L. I. 1964. Control of growth and development in insects. Science 143:325–333.PubMedCrossRefGoogle Scholar
  65. Seshan, K. R., and Levi-Montalcini, R. 1971. In vitro analysis of corpora cardiaca and corpora allata from nymphal and adult specimens of Periplaneta americana. Arch. Ital. Biol. 109:81–109.PubMedGoogle Scholar
  66. Sidman, R. L. 1970. Cell proliferation, migration and interaction in the developing mammalian central nervous system, pp. 100–107. In F. O. Schmitt, G. C. Quarton, T. Melnechuk, and G. Adelman (eds.). The Neurosciences—Second Study Program. Rockefeller University Press, New York.Google Scholar
  67. Silvana, D. 1971. Cell culture of Diptera, pp. 247–265. In invertebrate Tissue Culture, Vol. 1. Academic Press, New York.Google Scholar
  68. Smith, D. S. 1968a. Insect Cells: Their Structure and function. Oliver and Boyd, Edinburgh.Google Scholar
  69. Smith, D. S. 1968b. The trophic role of glial cells in insect ganglia, pp. 189–198. In J. W. L. Beament and J. E. Treherne (eds.). Insects and Physiology. American Elsevier, New York.Google Scholar
  70. Smith, U., and Smith, D. S. 1966. Observations on the secretory processes in the corpus cardiacum of the stick insect, Carausius morosus. J. Cell. Sci. 1:59–66.Google Scholar
  71. Sperry, R. W. 1963. Chemoaffinity in the orderly growth of nerve fiber patterns and connections. Proc. Natl. Acad. Sci. 50:703–710.PubMedCrossRefGoogle Scholar
  72. Sperry, R. W. 1965. Embryogenesis of behavioural nerve nets, pp. 161–186.In R.L. DeHaan and H. Ursprung (eds.). Organogenesis. Holt, Rinehart & Winston, New York.Google Scholar
  73. Steele, J. E. 1961. Occurrence of a hyperglycaemic factor in the corpus cardiacum of an insect. Nature 192:680–681.CrossRefGoogle Scholar
  74. Thomsen, E. 1952. Functional significance of the neurosecretory brain cells and the corpus cardiacum in the female blow-fly, Calliphora erythrocephala Meig. J. Exptl. Biol. 29: 137–172.Google Scholar
  75. Treherne, J. E. 1968. Axonal function and ionic regulation in insect central nervous tissue, pp. 175–188. In J. W. L. Beament and J. E. Treherne (eds.). Insects and Physiology. American Elsevier, New York.Google Scholar
  76. Unnithan, G. C., Bern, H. A., and Nayar, K. K. 1971. Ultrastructural analysis of the neuroendocrine apparatus of Oncopeltus fasciatus (Heteroptera). Acta Zool. 52:117–143.CrossRefGoogle Scholar
  77. Wigglesworth, V. B. 1954. The Physiology of Insect Metamorphosis. Cambridge University Press, London. 152 pp.Google Scholar
  78. Wigglesworth, V. B. 1959. The histology of the nervous system of an insect, Rhodnius prolixus (Hemiptera). II. The central ganglia. Quart. J. Microscop. Sci. 100:299–313.Google Scholar
  79. Wigglesworth, V. B. 1964. The hormonal regulation of growth and reproduction ininsects. pp. 247–335. In J. W. L. Beament, J. E. Treherne, and V. B. Wigglesworth (eds.) Advances in Insect Physiology. Academic Press, London and New York.Google Scholar
  80. Wigglesworth, V. B. 1965. Cell associations and organogenesis in the nervous system of insects, pp. 199–217. In R. L. DeHaan and H. Ursprung (eds.). Organogenesis. Holt, Rinehart & Winston, New York.Google Scholar
  81. Willey, R. B., and Chapman, G. B. 1960. The ultrastructure of certain components of the corpora cardiaca in orthopteroid insects. J. Ultrastruct. 4:1–14.CrossRefGoogle Scholar
  82. Williams, C. M. 1963. Differentiation and morphogenesis in insects, pp. 243–260. In J. M. Allen (ed.). The Nature of Biological Diversity. McGraw-Hill, New York.Google Scholar
  83. Williams, C. M. 1969. Nervous and hormonal communication in insect development, pp. 133–150. In A. Land (ed.). Developmental Biology. Suppl. 3, Communication in Development. Academic Press, New York and London.Google Scholar
  84. Williams, C. L., and Kambysellis, M. P. 1969. In vitro action of ecdysone. Proc. Natl. Acad. Sci. 63:231.Google Scholar
  85. Young, D. 1969. The motor neurons of the mesothoracic ganglion of Periplaneta americana. J. Insect Physiol. 15:1175–1179.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1973

Authors and Affiliations

  • R. Levi-Montalcini
    • 1
  • K. R. Seshan
    • 2
  1. 1.Laboratorio di Biologia CellulareC. N. R.RomeItaly
  2. 2.Department of BiologyWashington UniversitySt. LouisUSA

Personalised recommendations