Journal of Neurocytology

, Volume 18, Issue 3, pp 311–318 | Cite as

Bromodeoxyuridine immunohistochemical determination of the lengths of the cell cycle and the DNA-synthetic phase for an anatomically defined population

  • R. S. Nowakowski
  • S. B. Lewin
  • M. W. Miller


A cumulative labelling protocol using 5-bromo-2′-deoxyuridine (BUdR) was followed to determine: (1) the growth fraction (i.e., the proportion of cells that comprise the proliferating population), (2) the length of the cell cycle, and (3) the length of the DNA-synthetic phase (S-phase) for proliferative cells in the dentate gyrus of the mouse. On postnatal day 20 (P20), C57BL/6J mice were injected with BUdR at two hour intervals for a total period of 12 hours. Animals were sacrificed at selected intervals, and the brains were processed for immunohistochemistry using a monoclonal antibody directed against single-stranded DNA containing BUdR. The numbers of BUdR-labelled and unlabelled cells in sections through the hilus of the dentate gyrus were counted. The number of BUdR-labelled cells increased linearly from an initial value of about 12% of the total number of cells to a maximum value of just over 24% of the total. These findings indicate that, at P20, a maximum of 24.2 ± 1.2% of the cells in the dentate hilus are part of the proliferating population. The calculated length of the cell cycle of the cells comprising the intrahilar proliferative zone was estimated to be 16.1 ± 0.8h. The length of the S-phase was estimated at 8.0 ± 0.4 h. In addition, mathematical analysis, using one and two population models, indicates that over 90% of the proliferating cells in the dentate hilus at this age comprise a single population at least in terms of the lengths of the cell cycle and the S-phase. This protocol provides a convenient method for thein situ analysis of the cell cycle for anatomically defined proliferative populations.


Cell Cycle Dentate Gyrus Population Model Convenient Method Single Population 
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. Al-Ghoul, W. &Miller, M. W. (1989) Transient expression of Alz-50 immunoreactivity in developing rat neocortex: a marker for naturally occurring neuronal death?Brain Research 481, 361–7.Google Scholar
  2. Angevine, J. B., Jr. (1965) Time of neuron origin in the hippocampal region: an autoradiographic study in the mouse.Experimental Neurology Supplement 2, 1–71.Google Scholar
  3. Atlas, M. &Bond, V. A. (1965) The cell generation cycle of the eleven-day mouse embryo.Journal of Cell Biology 26, 19–24.Google Scholar
  4. Bannigan, J. G. (1987) Autoradiographic analysis of effects of 5-bromodeoxyuridine on neurogenesis in the chick embryo spinal cord.Developmental Brain Research 36, 161–70.Google Scholar
  5. Bannigan, J. &Langman, J. (1979) The cellular effect of 5-bromodeoxyuridine on the mammalian embryo.Journal of Embryology and Experimental Morphology 50, 123–35.Google Scholar
  6. Bayer S. A. (1980) Development of the lippocampal region in the rat. I. Neurogenesis examined with 3H-thymidine autoradiography.Journal of Comparative Neurology 190 87–114.Google Scholar
  7. Eckenhoff, M. F. &Rakic, P. (1988) Nature and fate of proliferative cells in the hippocampal dentate gyrus during the life span of the rhesus monkey.Journal of Neuroscience 8, 2729–47.Google Scholar
  8. Fujita, S. (1966) Applications of light and electron microscopic autoradiography to the study of cytogenesis of the forebrain. InEvolution of the Forebrain (edited byHassler, R. &Stephan, H.), pp. 180–96. Stuttgart: Georg Thieme Verlag.Google Scholar
  9. Gratzner, H. G. (1982) Monoclonal antibody to 5-bromo and 5-iododeoxyuridine: a new reagent for detection of DNA replication.Science 218, 474–8.Google Scholar
  10. Gray, J. W., Dolbeare, F., Pallavicini, M. G., Beisker, W. &Waldman, F. (1986) Cell cycle analysis using flow cytometry.International Journal of Radiation Biology 49, 237–55.Google Scholar
  11. Guéneau, G., Privat, A., Drouet, J. &Court, L. (1982) Subranular zone of the dentate gyrus of young rabbits as a secondary matrix.Developmental Neuroscience 5, 345–58.Google Scholar
  12. Lorente DE Nó, R. (1934) Studies on the structure of the cerebral cortex. II. Continuation of the study of the ammonic system.Journal fuer Psychologie und Neurologie 46, 113–17.Google Scholar
  13. Mareš, V. &Lodin, Z. (1970) The cellular kinetics of the developing mouse cerebellum. II. The function of the external granular layer in the process of gyrification.Brain Research 23, 343–52.Google Scholar
  14. Miller, M. W. (1986) Effects of alcohol on the generation and migration of cerebral cortical neurons.Science 233, 1308–11.Google Scholar
  15. Miller, M. W. (1988) Effect of prenatal exposure to ethanol on the development of cerebral cortex. I. Neuronal generation.Alcoholism: Clinical Experimental Research 12, 440–9.Google Scholar
  16. Miller, M. W. &Nowakowski, R. S. (1988) Use of bromodeoxyuridine-immunohistochemistry to examine the proliferation, migration, and time of origin of cells in the central nervous system.Brain Research 457, 44–52.Google Scholar
  17. Nowakowski, R. S. &Rakic, P. (1981) The site of origin and route and rate of migration of neurons to the hippocampal region of the rhesus monkey.Journal of Comparative Neurology 196, 129–54.Google Scholar
  18. Nuesse, M., Afzal, S. M., Carr, B. &Kavanau, K. (1985) Cell cycle kinetic measurements in an irradiated rat rhabdomyosarcoma using a monoclonal antibody to bromodeoxyuridine.Cytometry 6, 611–19.Google Scholar
  19. Packard, D. S., Menzies, R. A. &Skalko, R. G. (1973) Incorporation of thymidine and its analog, bromodeoxyuridine, into embryos and maternal tissues of the mouse.Differentiation 1, 397–405.Google Scholar
  20. Rakic, P. &Nowakowski, R. S. (1981) The time of origin of neurons in the hippocampal region of the rhesus monkey.Journal of Comparative Neurology 196, 99–128.Google Scholar
  21. Raza, A., Ucar, K. &Preisler, H. D. (1985) Double labelling andin vitro versusin vivo incorporation of bromodeoxyuridine in patients with acute nonlymphocytic leukemia.Cytometry 6, 633–40.Google Scholar
  22. Shackney, S. E. &Ritch, P. S. (1987) Percent labelled mitosis curve analysis. InTechniques in Cell Cycle Analysis (edited byGray, J. W. &Darzynkiewicz, Z.), pp. 31–46. New York: Humana Press.Google Scholar
  23. Sidman, R. L. (1970) Autoradiographic methods and principles for study of the nervous system with thymidine-H3. InContemporary Research Methods in Neuroanatomy (edited byNauta, W. J. H. &Ebbesson, S. O. E.), pp. 252–74. New York: Springer.Google Scholar
  24. Sidman, R. L. &Rakic, P. (1982) Development of the human central nervous system. InHistology and Histopathology of the Nervous System (edited byHaymaker, W. &Adams, R. D.), pp. 3–45. Springfield: C. C. Thomas.Google Scholar
  25. Simpson-Herren, L. (1987) Autoradiographic techniques for measurement of the labelling index. InTechniques in Cell Cycle Analysis (edited byGray, J. W. &Darzynkiewicz, Z.), pp. 1–30, New York: Humana Press.Google Scholar
  26. Stanfield, B. B. &Cowan, W. M. (1979) The development of the hippocampus and dentate gyrus in normal and reeler mice.Journal of Comparative Neurology 185, 423–59.Google Scholar
  27. Trent, J. M., Gerner, E., Broderick, R. &Crossen, P. E. (1986) Cell cycle analysis using bromodeoxyuridine: comparison of methods for analysis of total cell transit time.Cancer Genetics and Cytogenetics 19, 43–50.Google Scholar
  28. Waechter, R. V. &Jaensch, B. (1972) Generation times of the matrix cells during embryonic brain development: an autoradiographic study in rats.Brain Research 46, 235–50.Google Scholar
  29. Webster, W., Shimada, M. &Langman, J. (1973) Effect of fluorodeoxyuridine, colcemid, and bromodeoxyuridine on developing neocortex of the mouse.American Journal of Anatomy 137, 67–85.Google Scholar
  30. Williams, R. W. &Herrup, K. (1988) The control of neuron number.Annual Reviews of Neuroscience 11, 423–53.Google Scholar
  31. Wimer, R. E., Wimer, C. C. &Alameddine, L. (1988) On the development of strain and sex differences in granule cell number in the area dentata of house mice.Developmental Brain Research 42, 191–8.Google Scholar

Copyright information

© Chapman and Hall Ltd. 1989

Authors and Affiliations

  • R. S. Nowakowski
    • 1
    • 2
  • S. B. Lewin
    • 1
  • M. W. Miller
    • 1
    • 2
    • 3
  1. 1.Department of AnatomyUMDNJ-Robert Wood Johnson Medical SchoolPiscatawayUSA
  2. 2.Physiology/Neurobiology ProgramRutgers UniversityPiscatawayUSA
  3. 3.Department of AnatomyUMDNJ-School of Osteopathic MedicinePiscatawayUSA

Personalised recommendations