Journal of Neurocytology

, Volume 30, Issue 5, pp 391–401

Proliferation and death of cultured fetal neocortical neurons: effects of ethanol on the dynamics of cell growth



Neuronal number in the mature CNS is determined by the balance of cell proliferation and death. The effects of ethanol on cell proliferation and death were examined in primary cultures of neocortical neurons derived from 16-day-old rat fetuses. The cells were treated with ethanol (0 or 400 mg/dl) and examined for (1) immunohistochemical identity, (2) cell cycle kinetics using a cumulative bromodeoxyuridine labeling technique, (3) viable cell number via a trypan blue assay, and (4) the incidence of cell death with terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase 3 immunhistochemistry. After two days in culture, most (>85%) cells expressed a neuron-specific antigen(s) whether or not ethanol was added to the culture medium. Ethanol affected the proliferation of the cultured cells, e.g., the length of the cell cycle was greater in the ethanol-treated cells than in controls. The number of trypan blue-negative (viable) cells was profoundly decreased by ethanol exposure. This decrease was accompanied by increases in the frequencies of TUNEL- and caspase 3-positive cells and of cells exhibiting nuclear condensations. Thus, ethanol decreases the number of viable cells in vitro by slowing cell proliferation and increasing the incidence of cell death. The expression of the death indices in untreated cultures is most consistent with a single (apoptotic) pathway of cell death, rather than simultaneous apoptotic and necrotic modes of death. Furthermore, it appears that ethanol initiates an apoptotic death among cultured cortical neurons.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Adickes, E. D., Mollner, T. J. & Lockwood, S. K. (1988) Closed chamber system for delivery of ethanol to cell cultures. Alcohol and Alcoholism 23, 277–281.Google Scholar
  2. Al-Ghoul, W. M. & Miller, M. W. (1989) Transient expression of AIz-50-immunoreactivity in developing rat neocortex: A marker for naturally occurring neuronal death? Brain Research 481, 361–367.Google Scholar
  3. Baraona, E. & Lieber, C. S. (1982) Effects of alcohol on hepatic transport of proteins. Annual Review of Medicine 33, 281–292.Google Scholar
  4. Bauer-Moffett, C. & Altman, J. (1977) Effect of ethanol chronically administered to preweanling rats on cerebellar development: A morphological study. Brain Research 119, 249–268.Google Scholar
  5. Bhave, S. V. & Hoffman, P. L. (1997) Ethanol promotes apoptosis in cerebellar granule cell by inhibiting the trophic effect of NMDA. Journal of Neurochemistry 68, 578–586.Google Scholar
  6. Blaschke, A. J., Staley, K. & Chun, J. (1996) Widespread programmed cell death in proliferative and postmitotic regions of the fetal cerebral cortex. Development 122, 1165–1174.Google Scholar
  7. Blaschke, A. J., Weiner, J. A. & Chun, J. (1998) Programmed cell death is a universal feature of embryonic and postnatal neuroproliferative regions throughout the central nervous system. Journal of Comparative Neurology 396, 39–50.Google Scholar
  8. Busser, J., Geldmacher, D. S. & Herrup, K. (1998) Ectopic cell cycle proteins predict the sites of neuronal cell death in Alzheimer's disease brain. Journal of Neuroscience 18, 2801–2807.Google Scholar
  9. Cartwright, M. M., Tessmer, L. L. & Smith, S. M. (1998) Ethanol-induced neural crest apoptosis is coincident with their endogenous death, but is mechanistically distinct. Alcoholism: Clinical and Experimental Research 22, 142–149.Google Scholar
  10. Cheema, Z. F., West, J. R. & Miranda, R. C. (2000) Ethanol induces Fas/Apo [Apoptosis]-1 mRNA and cell suicide in the developing cerebral cortex. Alcoholism: Clinical and Experimental Research 24, 535–543.Google Scholar
  11. Clarren, S. K., Alvord, E. C., JR., Sumi, S. M., Streissguth, A. P. & Smith, D. W. (1978) Brain malformations related to prenatal exposure to ethanol. Journal of Pediatrics 92, 64–67.Google Scholar
  12. Collins, M. A., Zou, J. Y. & Neafsey, E. J. (1998) Brain damage due to episodic alcohol exposure in vivo and in vitro: Furosemide neuroprotection implicates edemabased mechanism. FASEB Journal 12, 221–230.Google Scholar
  13. Dutton, G. R. (1990) Isolation, culture and use of viable central nervous system perikarya. In Methods in Neurosciences, Vol 2., pp. 87–102. New York: Academic Press.Google Scholar
  14. Finlay, B. L. & Slattery, M. (1983) Local differences in the amount of early cell death in neocortex predict adult local specializations. Science 19, 1349–1351.Google Scholar
  15. Freeman, R. S., Estus, S. & Johnson, E. M., Jr. (1994) Analysis of cell cycle-related gene expression in postmitotic neurons: Selective induction of cyclin D1 during programmed cell death. Neuron 12, 343–355.Google Scholar
  16. Guergan, C., Levy, V. & David, J. P. (1997) c-Jun and cyclin D1 proteins as mediators of neuronal death after focal ischaemic insult. Neuroreport 8, 1003–1007.Google Scholar
  17. Green, D. R. & Reed, J. C. (1998) Mitochondria and apoptosis. Science 281, 1309–1312.Google Scholar
  18. Gressens, P., Lammens, M., Picard, J. J. & Evrard, P. (1992) Ethanol-induced disturbances of gliogenesis and neuronogenesis in the developing murine brain: An in vitro and in vivo immunohistochemical and ultrastructural study. Alcohol and Alcoholism 27, 219–226.Google Scholar
  19. Guerri, C., SÁez, R., Sancho-Tello, M., Martin de Aquilera, E. & Renau-Piqueras, J. (1990) Ethanol alters astrocyte development: A study of critical periods using primay cultures. Neurochemistry Research 15, 559–565.Google Scholar
  20. Guizzetti, M. & Costa, L. G. (1996) Inhibition of muscarinic receptor-stimulated glial cell proliferation by ethanol. Journal of Neurochemistry 67, 2236–2245.Google Scholar
  21. Hamre, K. M. & West, J. R. (1993) The effects of the timing of ethanol exposure during the brain growth spurt on the number of cerebellar Purkinje and granule cell nuclear profiles. Alcoholism: Clinical and Experimental Research 17, 610–622.Google Scholar
  22. Hannigan, J. H., Spear, L. P., Spear, N. E. & Goodlett, C. R. (1999) Alcoholism: Effects on brain and development, Hillsdale NJ: Lawrence Erlbaum Associates.Google Scholar
  23. Hoffman, P. L. & Tabakoff, B. (1996) To be or not to be: How ethanol can affect neuronal death during development. Alcoholism: Clinical and Experimental Research 20, 193–194.Google Scholar
  24. Jacobs, J. S. & Miller, M. W. (2000) Cell cycle kinetics and immunohistochemical characterization of dissociated fetal cortical cultures: Evidence that differentiated neurons have mitotic capacity. Developmental Brain Research 122, 67–80.Google Scholar
  25. Jacobson, M. (1991) Histogenesis and morphogenesis of cortical structures. In Developmental Neurobiology (edited by Jacobson, M.) pp. 401–451. New York: Plenum.Google Scholar
  26. Johnson, G. V. W. & Jope, R. S. (1992) The role of microtubule-associated protein 2 (MAP-2) in neuronal growth, plasticity, and degeneration. Journal of Neuroscience Research 33, 505–512.Google Scholar
  27. Kane, C. J., Berry, A., Boop, F. A. & Davies, D. L. (1996) Proliferation of astroglia from the adult human cerebrum is inhibited by ethanol in vitro. Brain Research 731, 39–44.Google Scholar
  28. Kennedy, L. A. & Elliott, M. J. (1985) Cell proliferation in the embryonic mouse neocortex following acute maternal alcohol intoxication. International Journal of Developmental Neuroscience 3, 311–315.Google Scholar
  29. Kennedy, L. A. & Mukerji, S. (1996) Ethanol neurotoxicity. I. Direct effects on replicating astrocytes. Neurobehavioral and Taxicology Teratology 8, 11–17.Google Scholar
  30. Kuhn, P. E. & Miller, M. W. (1998) Expression of p53 and ALZ-50 immunoreactivity in rat cortex: Effect of prenatal exposure to ethanol. Experimental Neurology 154, 418–429.Google Scholar
  31. Ledig, M., Kopp, P. & Mandel, P. (1985) Effect of ethanol on adenosine triphosphatase and enolase activities in rat brainandin cultured nerve cells. Neurochemistry Research 10, 1311–1324.Google Scholar
  32. Liesi, P. (1997) Ethanol-exposed central neurons fail to migrate and undergo apoptosis. Journal of Neuroscience Research 48, 439–448.Google Scholar
  33. Luo, J. & Miller, M. W. (1997a) Basic fibroblast growth factor-and platelet-derived growth factor-mediated cell proliferation in B104 neuroblastoma cells: Effect of ethanol on cell cycle kinetics. Brain Research 770, 139–150.Google Scholar
  34. Luo, J. & Miller, M. W. (1997b) Differential sensitivity of human neuroblastoma cell lines to ethanol: Correlations with their proliferative responses to mitgenic growth factors and expression of growth factor receptors. Alcoholism: Clinical and Experimental Research 21, 1186–1194.Google Scholar
  35. Luo, J. & Miller, M. W. (1999) Platelet-derived growth factor-mediated signal transduction underlying astrocyte proliferation: Site of ethanol action. Journal of Neuroscience 19, 10014–10025.Google Scholar
  36. Luo, J., West, J. R., Cook, R. T. & Pantazis, N. J. (1999) Ethanol induces cell death and cell cycle delay in cultures of pheochromocytoma PC12 cells. Alcoholism: Clinical and Experimental Research 23, 644–656.Google Scholar
  37. Miller, M. W. (1986) Fetal alcohol effects on the generation and migration of cerebral cortical neurons. Science 233, 1308–1311.Google Scholar
  38. Miller, M. W. (1998) Development of projection and local circuit neurons in neocortex. In Cerebral Cortex Vol. 7 (edited by Peters, A. & Jones, E. G. eds) pp. 133–175. New York: Plenum.Google Scholar
  39. Miller, M. W. (1989) Effect of prenatal exposure to ethanol on the development of the cerebral cortex: II Cell proliferation in the ventricular and subventricular zones of the rat. Journal of Comparative Neurology 287, 326–338.Google Scholar
  40. Miller, M. W. (1992a) Development of the Central Nervous System: Effects of Alcohol and Opiates. New York: Wiley-Liss.Google Scholar
  41. Miller, M. W. (1992b) Effects of prenatal exposure to ethanol on cell proliferation and neuronal migration. In Development of the Central Nervous System: Effects of Alcohol and Opiates (edited by Miller M.W. ed) pp. 47–69. New York: Wiley-Liss.Google Scholar
  42. Miller, M. W. (1993) Migration of cortical neurons is altered by gestational exposure to ethanol. Alcoholism: Clinical and Experimental Research 17, 304–314.Google Scholar
  43. Miller, M. W. (1995a) Relationship of time of origin and death of neurons in rat somatosensory cortex: Barrel versus septal cortex and projection versus local circuit neurons. Journal of Comparative Neurology 355, 6–14.Google Scholar
  44. Miller, M. W. (1995b) Effect of pre-or postnatal exposure to ethanol on the total number of neurons in the principal sensory nucleus of the trigeminal nerve: Cell proliferation and neuronal death. Alcoholism: Clinical and Experimental Research 19, 1359–1363.Google Scholar
  45. Miller, M. W. (1995c) Generation of neurons in the rat denate gyrus and hippocampus: Effects of prenatal and postnatal treatment with ethanol. Acoholism: Clinical and Experimental Research 19, 1500–1509.Google Scholar
  46. Miller, M. W. (1999a) A longitudinal study of the effects of prenatal ethanol exposure on neuronal acquisition and death in the principal sensory nucleus of the trigeminal nerve: Interaction with changes induced by transection of the infraorbital nerve. Journal of Neurocytology 28, 999–1015.Google Scholar
  47. Miller, M. W. (1999) Kinetics of the migration of neurons to rat somatosensory cortex. Developmental Brain Research 115, 111–122.Google Scholar
  48. Miller, M. W. & Kuhn, P. E. (1995) Cell cycle kinetics in fetal rat cerebral cortex: Effects of prenatal exposure to ethanol assessed by a cumulative lableling technique with flow cytometry. Alcoholism: Clinical and Experimental Research 19, 233–237.Google Scholar
  49. Miller, M. W. & Nowakowski, R. S. (1991) Effect of prenatal exposure to ethanol on the cell cycle kinetics and growth fraction in the proliferative zones of the fetal rat cerebral cortex. Alcoholism: Clinical and Experimental Research 15, 229–232.Google Scholar
  50. Miller, M. W. & Potempa, G. (1990) Numbers of neurons and glia in mature rat somatosensory cortex: Effects of prenatal exposure to ethanol. Journal of Comparative Neurology 293, 92–102.Google Scholar
  51. Miller, M. W. & Robertson, S. (1993) Prenatal exposure to ethanol alters the postnatal development and transformation of radial glia to astrocytes in the cortex. Journal of Comparative Neurology 337, 253–266.Google Scholar
  52. Miller, M. W., Wharton, S. B., Peter, A. & Wyllie, A. H. Proliferation and death of conditionally immortalized neural cells from murine neocortex: p53 blocks the ability of neurons to re-enter the cell cycle. Neuroscience (submitted).Google Scholar
  53. Mooney, S. & Miller, M. W. (1999) Processes associated with naturally occurring neuronal death are exacerbated by ethanol treatment. Recent Research Developments in Neurochemistry 2, 573–586.Google Scholar
  54. Mooney, S. M. & Miller, M. W. (2000) Expression of bcl-2, bax, and caspase-3 in the brain of the developing rat. Brain Research 123, 103–117.Google Scholar
  55. Mooney, S. M., Napper, R. M. A. & West, J. R. (1996) Long-term effect of postnatal alcohol exposure on the number of cells in the neocortex of the rat: Stereological study. Alcoholism: Clinical and Experimental Research 20, 615–623.Google Scholar
  56. Nowakowski, R. S., Lewin, S. B. & Miller, M. W. (1989) Bromodeoxyuridine immunohistochemical determination of the lengths of the cell cycle and the DNAsynthetic phase for an anatomically defined population. Journal of Neurocytology 18, 311–318.Google Scholar
  57. Oberdoerster, J., Kamer, A. R. & Rabin, R. A. (1998) Differential effect of ethanol on PC12 cell death. Journal of Pharmacology and Experimental Therapeutics 287, 359–365.Google Scholar
  58. Oppenheim, R. (1991) Cell death during development of the nervous system. Annual Review of Neuroscience 14, 453–501.Google Scholar
  59. Pantazis, N. J., Dohrman, D. P., Luo, J., Thomas, J. D., Goodlett, C. R. & West, J. R. (1995) NMDA prevents alcohol-induced neuronal cell death of cerebellar granule cells in culture. Alcoholism: Clinical and Experimental Research 19, 846–853.Google Scholar
  60. Pantazis, N. J., West, J. R. & Dai, D. (1998) The nitric oxide-cyclicGMPpathway plays an essential role in both promoting cell survival of cerebellar granule cells in culture and protecting cells against ethanol neurotoxicity. Journal of Neurochemistry 70, 1826–1838.Google Scholar
  61. Polleux, F., Dehay, C., Moraillon, B. & Kennedy, H. (1997) Regulation of neuroblast cell-cycle kinetics plays a crucial role in the generation of unique features of neocortical areas. Journal of Neuroscience 17, 7763–7783.Google Scholar
  62. Price D. J., Aslam, S., Tasker, L. & Gillies, K. (1997) Fates of the earliest generated cells in the developing murine neocortex. Journal of Comparative Neurology 377, 414–422.Google Scholar
  63. Rubin, L. L. (1997) Neuronal cell death: When, Why and how. British Medical Bulletin 53, 617–631.Google Scholar
  64. SÁez, R., Burgal, M., Renau-Piqueras, J., MarquÉs, A. & Guerri, C. (1991) Evolution of several cytoskeletal proteins of astrocytes in primary culture: Effect of prenatal alcohol exposure. Neurochemistry Research 16, 737–747.Google Scholar
  65. Seabold, G., Luo, J. & Miller, M. W. (1998) Effect of ethanol on neurotrophin-mediated cell survival and receptor expression in cortical neuronal cultures. Developmental Brain Research 128, 139–145.Google Scholar
  66. Snyder, A. K., Singh, S. P. & Ehmann, S. (1992) Effects of ethanol DNA, RNA, and protein synthesis in rat astrocyte cultures. Alcoholism: Clinical and Experimental Research 16, 295–300.Google Scholar
  67. Takahashi, T., Nowakowski, R. S. & Caviness, V. S., Jr. (1995) The cell cycle of the pseudostratified ventricular epithelium of the murine cerebral wall. Journal of Neuroscience 15, 6046–6057.Google Scholar
  68. Takahashi, T., Nowakowski, R. S. & Caviness. V. S., Jr. (1996) The leaving or Q fraction of the murine cerebral proliferative epithelium: A general model of neocortical neuronogenesis. Journal of Neuroscience 16, 6183–6196.Google Scholar
  69. Takahashi, T., Bhide, P. G., Goto, T., Miyama, S. & Caviness, V. S., Jr. (1999) Proliferative behavior of the murine cerebral wall in tissue culture: Cell cycle kinetics and checkpoints. Experimental Neurology 156, 407–417.Google Scholar
  70. Thomaidou, D., Mione, M. C., Cavanagh, J. F. R. & Parnavelas, J. G. (1997) Apoptosis and its relation to the cell cycle in the developing cerebral cortex. Journal of Neuroscience 17, 1075–1085.Google Scholar
  71. Waechter, R. V. & Jaensch, B. (1972) Generation times of the matrix cells during embryonic brain development: An autoradiographic study in rats. Brain Research 46, 235–250.Google Scholar
  72. West, J. R., Goodlett, C. R., Bonthius, D. J., Hamre, K. M. & Marcussen, B. L. (1990) Cell population depletion associated with fetal alcohol brain damage: Mechanisms of BAC-dependent cell loss. Alcoholism: Clinical and Experimental Research 14, 813–818.Google Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  1. 1.Neuroscience ProgramUniversity of IowaIowa CityUSA
  2. 2.Department of Neuroscience and PhysiologyState University of New York—Upstate Medical UniversitySyracuseUSA;
  3. 3.Research ServiceVeterans Affairs Medical CenterSyracuseUSA

Personalised recommendations