In Vitro Cell Cultures as a Model of the Basal Forebrain

  • B. H. Wainer
  • H. J. Lee
  • J. D. Roback
  • D. N. Hammond
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 295)


The basal forebrain has attracted considerable attention because of its putative role in complex functions such as learning, memory and behavioral state control as well as its vulnerability in neurological disorders such as Alzheimer’s Disease (AD). The finding that nerve growth factor provides trophic support for the cholinergic basal forebrain neurons has stimulated further interest in understanding trophic interactions of basal forebrain neurons as well as in possible trophic factor therapeutic stategies for disease states. Our laboratory has utilized primary cell cultures and developed immortalized central nervous system cell lines to study the trophic interactions that establish and maintain the septohippocampal pathway, a basal forebrain component which plays an essential role in cognitive function and is prominently affected in AD. The results of our primary cell culture studies have demonstrated the importance of trophic signals elaborated by the hippocampus in mediating the development of septal cholinergic neurons. Nerve growth factor plays an important role in this process, but it cannot account for all of the trophic signals elaborated by authentic hippocampal target cells. The development by this laboratory of clonal cell lines of septal and hippocampal lineage offers the prospect of investigating both the response to and elaboration of neural trophic signals at a more precise level of resolution than can be achieved with primary cultures. The technology and information that is generated from the engineering of such cell lines will also serve as a strategy to study trophic interactions in other brain circuits in future years, and to investigate possible changes or dysfunctions that occur neurological disease.


Nerve Growth Factor Cholinergic Neuron Basal Forebrain Clonal Cell Line ChAT Activity 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Alt, F., Blackwell, K. and Yancopoulos, G.D. (1987) Development of the primary antibody repertoire. Science238:1079.PubMedCrossRefGoogle Scholar
  2. Anderson, K.J., Dam, D., Lee, S. and Cotman, C.W. (1988) Basic fibroblast growth factor prevents death of lesioned cholinergic neurons in vivo. Nature332:360.PubMedCrossRefGoogle Scholar
  3. Appel, S.H. (1981) A unifying hypothesis for the cause of amyotrophic lateral sclerosis, Parkinsonism, and Ahaheimer disease. Ann. Neurol. 10:499.PubMedCrossRefGoogle Scholar
  4. Banker, G. and Goslin, K. (1988) Developments in neuronal cell culture. Nature. 336:185.Google Scholar
  5. Barbin, G., Manthorpe, M. and Varon, S. (1984) Purification of the chick eye ciliary neuronotrophic factor. J. Neu roc hem. 43:1468.Google Scholar
  6. Becker, R.E. and Giacobini, E. (1988) Mechanisms of Cholinesterase inhibition in senile demenda of the Alzheimer type: Clinical, pharmacological, and therapeudc aspects. Drug Dev. Res. 12:163.CrossRefGoogle Scholar
  7. Blusztajn, J.K. and Wurtman, R.J. (1983) Choline and cholinergic neurons. Science221:614.PubMedCrossRefGoogle Scholar
  8. Bostwick, J.R., Appel, S.H. and Perez-Polo, J.R. (1987) Distinct influences of nerve growth factor and a central cholinereic trophic factor on medial septal explants. Brain Res. 422:92.PubMedCrossRefGoogle Scholar
  9. Bottenstein, J.E. (1985) Growth and differentiation of neural cells in defined media. In:“ Cell Culture in the Neurosciences.”eds:Bottenstein, J.E. and Sato, G.,Plenum Press, New York, pp. 3.CrossRefGoogle Scholar
  10. Garden, M.J., Trojanowski, J.Q., Schlaepfer, W.W. and Lee, V.M.-Y. (1987) Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns. J. Neurosci. 7:3489.Google Scholar
  11. Cepko, C. (1988) Immortalization of neural cells via oncogene transducdon. TrendsGoogle Scholar
  12. Cepko, C.L. (1989) Immortalization of neural cells via retroviral-mediated oncogene transducdon. Ann. Rev. Neurosci. 12:47.PubMedCrossRefGoogle Scholar
  13. Chan Palay, V. (1988) Galanin hyperinnervates surviving neurons of the human basal nucleus of Meynert in dementias of Alzheimer’s and Parkinson’s disease: a hypothesis for the role of galanin in accentuating cholinergic dysfunction in dementia. J Comp.Neurol.273:543.CrossRefGoogle Scholar
  14. Davies, P., Katzman, R. and Terry, R.D. (1980) Reduced somatostatin-like immunoreactivity in cerebral cortex from cases of Alzheimer disease and Alzheimer senile demenna. Nature288:279.PubMedCrossRefGoogle Scholar
  15. DeLellis, R.A., Merk, F.B., Deckers, P., Warren, S. and Balogh, K. (1973) Ultrastructure and in vitro growth characteristics of a transplantable rat pheochromocytoma. Cancer32:227.PubMedCrossRefGoogle Scholar
  16. Emerit, M.B., Segovia, J., Alho, H., Mastrangelo, M.J. and Wise, B.C. (1989) Hippocampal membranes contain a neurotrophic activity that stimulates cholinergic properties of fetal rat septal neurons cultered under serum-free conditions. L Neurochem. 52:952.CrossRefGoogle Scholar
  17. Frederiksen, K., Jat, P.S., Vallz, N., Levy, D. and McKay, R. (1988) Immortalizadon of precursor cells from the mammalian ens. Neuron1:439.PubMedCrossRefGoogle Scholar
  18. Gahwiler, B.H., Enz, A. and Hefti, F. (1987) Nerve growth factor promotes development of the rat septo-hippocampal cholinergic projection in vitro. Neurosci.Lett.75:6.PubMedCrossRefGoogle Scholar
  19. Garber, B.B. and Moscona, A.A. (1972) Reconstruction of brain tissue from cell suspensions. I. Aggreganon of patterns of cells dissociated from different regions of the developing brain. Dev. Biol. 27:217.Google Scholar
  20. Gnahn, H., Hefn, F., Heumann, R., Schvab, M.E. and Thoenen, H. (1983) NGF- mediated increase of choline acetyltransferase (ChAT) in the neonatal forebrain: Evidence for a physiological role of NGF in the brain?. Dev. Br. Res. 9:45.CrossRefGoogle Scholar
  21. Guroff, G. (1985) PCI2 Cells as a model of neuronal differennation. In:“ Cell Culture in the Neurosciences.” eds:Bottenstein, J.E. and Sato, G., Plenum Press, New York, pp. 245–272.CrossRefGoogle Scholar
  22. Hammond, D.N., Wainer, B.H., Tonsgard, J.H. and Heller, A. (1986) Neuronal properdes of clonal hybrid cell lines derived from central cholinergic neurons.Google Scholar
  23. Hammond, D.N., Lee, H.J. and Wainer, B.H. (1989) A membrane-associated factor influences central cholinergic neuron development. Ann. Neurol. 26:445.Google Scholar
  24. Hammond, D.N., Lee, H.J. and Wainer, B.H. (1999a) A hippocampal cell line expresses a membrane-associated cholinergoc trophic activity not attributable to nerve growth factor. In:“ Alzheimer’s and Parkinson’s Disease II: Basic and therapeutic strategies.” eds:Nagatsu, T., Fisher, A. and Yoshida, M.,Plenum Press, New York, in press.Google Scholar
  25. Hammond, D.N., Lee, H.J., Tonsgard, J.H. and Wainer, B.H. (1999b) Development and characterization of clonal cell lines from septal cholinergic neurons. Brain Res. 512:190.CrossRefGoogle Scholar
  26. Hartikka, J. and Hefti, F. (1988a) Comparison of nerve growth factor’s effects on development of septum,striatum, and nucleus basalis cholinergic neurons in vitro. L Neurosci. Res. 21:352.CrossRefGoogle Scholar
  27. Hartikka, J. and Hefn, F. (1988b) Development of septal cholinergic neurons in culture: Pladng density and glial cells modulate effects of NGF on survival, fiber growth, and expression of transmitter-specific enyzmes. J. Neurosci. 8:2967.PubMedGoogle Scholar
  28. Hefti, F., Hardkka, J., Eckenstein, F., Gnahn, H., Heumann, R. and Schwab, M. (1985) Nerve growth factor increases choline acetyltransferase but not survival or fiber outgrowth of cultured fetal septal cholinergic neurons. Neuroscience14:55.PubMedCrossRefGoogle Scholar
  29. Hefti, F. and Weiner, W.J. (1986) Nerve growth factor and Alzheimer’s Disease. Ann. Neurol. 20:275.PubMedCrossRefGoogle Scholar
  30. Hefn, F., Hartikka, J. and Knusel, B. (1989) Function of neurotrophic factors in the adult and aging brain and their possible use in the treatment of neurodegenerative diseases. Neurobiol.Aging10:515.CrossRefGoogle Scholar
  31. Hofer, M.M. and Barde, Y.-A. (1988) Brain-derived neurotrophic factor prevents neuronal death in vivo. Nature331:261.PubMedCrossRefGoogle Scholar
  32. Hoffmann, P.C., Hemmendinger, L.M., Kotake, C. and Heller, A. (1983) Enhanced dopamine cell survival in reaimreiates containing telencephalic target cells. Brain Res. 274:275.PubMedCrossRefGoogle Scholar
  33. Hohn, A., Leibrock, J., Bailey, K. and Barde, Y.-A. (1990) Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family. Nature344:339.PubMedCrossRefGoogle Scholar
  34. Hsiang, J., Wainer, B.H., Shalaby, I.A., Hoffmann, P.C., Heller, A. and Heller, B.R. (1987) Neurotrophic effects of hippocampal target cells on developing septal cholinergic neurons in culture. Neuroscience21:333.PubMedCrossRefGoogle Scholar
  35. Hsiang, J., Price, S.D., Heller, A., Hoffmann, P.C. and Wainer, B.H. (1988) Ultrastructural evidence for hippocampal target cell-mediated trophic effects on septal cholinergic neurons in reaggregating cell cultures. Neuroscience26:417.PubMedCrossRefGoogle Scholar
  36. Hsiang, J., Heller, A., Hoffmann, P.C., Mobley, W.C. and Wainer, B.H. (1989) The effects of nerve growth factor on the development of septal cholinergic neurons in reaggregate cell cultures. Neuroscience29:209.PubMedCrossRefGoogle Scholar
  37. Johnson, E.M.Jr., Gorin, P.D., Brandeis, L.D. and Pearson, J. (1980) Dorsal root ganglion neurons are destroyed by in utero exposure to maternal annbody to nerve growth factor. Science210:916.PubMedCrossRefGoogle Scholar
  38. Johnson, J.E., Barde, Y.-A., Schwab, M. and Thoenen, H. (1990) Brain-derived neurotrophic factor supports the survival of cuUured rat retinal ganglion cells. L. Neurosci. 6:3031.Google Scholar
  39. Kamegai, M., Niijima, K., Kunishita, T., Nishizawa, M., Ogawa, M., Araki, M., Ueki, A., Konishi, Y. and Tabira, T. (1990) Interleukin 3 as a trophic factor for central choHnergic neurons in vitro and in vivo. Neuron 4:429.PubMedCrossRefGoogle Scholar
  40. Keller, F., Rimvall, K., Barbe, M.F. and Levitt, P. (1989) A membrane glycoprotein associated with the limbic system mediates formation of the septo-hippocampal pathway in vitro. Neuron3:551.PubMedCrossRefGoogle Scholar
  41. Knusel, B., Michel, P.P., Schwaber, J.S. and Hefn, P. (1990) Selective and nonselective stimulation of central cholinergic and dopaminergic development in vitro by nerve growth factor, basic fibroblast growth factor, epidermal growth factor, insulin and the insulin-like growth factors I and IL J. Neurosci. 10:558.PubMedGoogle Scholar
  42. Kohier, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of defined specifity. Nature256:495.CrossRefGoogle Scholar
  43. Lee, H.J., Elliot, G.J., Hammond, D.N., Lee, V.M.-Y. and Wainer, B.H. (1989) Expression of the mature array of neurofilament protein isoforms by a clonal cell line from the ens. Soc. Neurosci. Abs. 15:327.Google Scholar
  44. Lee, H.J., Hammond, D.N., Large, T.H., Sim, J.A., Brown, D.A., Otten, U.H. and Wainer, B.H. (1990a) Neuronal properties and trophic activines of immortalized hippocampal cells from embryonic and young adult mice. J. Neurosci. 10:1779.PubMedGoogle Scholar
  45. Lee, H.J., Hammond, D.N., Large, T.H. and Wainer, B.H. (1990b) Immortalized young adult cholinegic neurons from the medial septal region: Production and characterization. Dev. Brain Res. 52:219.CrossRefGoogle Scholar
  46. Lendahl, U. and McKay, R.D.G. (1990) The use of cell lines in neurobiology. Trends Neurosci. 13:132.PubMedCrossRefGoogle Scholar
  47. Levi-Montalcini, R. and Booker, B. (1960) Destruction of the sympathedc ganglia in mammals by an antiserum to a nerve-growth protein. Proc. Natl. Acad. Sci. USA46:384.PubMedCrossRefGoogle Scholar
  48. Levi-Montalcini, R. (1987) The nerve growth factor 35 years later. Science237:1154.PubMedCrossRefGoogle Scholar
  49. Lillien, L.E., Sendtner, M., Rohrer, I. L., Hughes, S.M. and Raff, M.C. (1988) Type-2 astrocyte development in rat brain cultures is initiated by a CNTF-like protein produced by type-1 astrocytes. Neuron1:485.PubMedCrossRefGoogle Scholar
  50. Lindholm, D., Heumann, R., Meyer, M. and Thoenen, H. (1987) Interleukin-1 regulates synthesis of nerve growth factor in non-neuronal cells of rat sciatic nerve. Nature330:658.PubMedCrossRefGoogle Scholar
  51. Maisonpierre, P.C., Belluscio, L., Squinto, S., Ip, N.Y., Furth, M.E., Lindsay, R.M. and Yancopoulos, G.D. (1990) Neurotrophin-3: A neurotrophic factor related to NGF and BDNF. Science247:1446.PubMedCrossRefGoogle Scholar
  52. Marx, J. (1990) NGF and Alzheimer’s: Hopes and Fears. Science247:408.PubMedCrossRefGoogle Scholar
  53. Meek, W.H., Church, R.M., Wenk, G.L. and Olton, D.S. (1987) Nucleus basalis magnocellularis and medial septal area lesions differentially impair temporal memory. J Neurosci. 7:3505.Google Scholar
  54. Mobley, W.C., Rutkowski, J.L., Tennekoon, G.I., Buchanan, K. and Johnston, M.V. (1985) Choline acetyltransferase activity in striatum of neonatal rats increased by nerve growth factor. Science229:284.PubMedCrossRefGoogle Scholar
  55. Morris, J.E. and Moscona, A.A. (1970) Induction of glutamine synthetase in embryonic retina: Its dependence on cell interactions. Science167:1736.PubMedCrossRefGoogle Scholar
  56. Morrison, R.S., Kornblum, H.I., Leslie, F.M. and Bradshaw, R.A. (1987) Trophic stimuladon of cultured neurons from neonatal rat brain by EGF. Science238:72.PubMedCrossRefGoogle Scholar
  57. Ojika, K. and Appel, S.H. (1984) Neurotrophic effects of hippocampal extracts on medial septal nucleus in vitro. Proc. Natl. Acad. Sci. USA81:2567.PubMedCrossRefGoogle Scholar
  58. Paige, C.I and Wu, G.E. (1989) The B cell repertoire. Faseb J. 3:1818.PubMedGoogle Scholar
  59. Phelps, C.H., Gage, F.H., Growdon, J.H., Hefti, F., Harbaugh, R., Johnston, M.V., Khachaturian, Z.S., Mobley, W.C., Price, D.L., Raskind, M., Simpkins, J., Thai, L.J. and Woodcock, J. (1989) Potential use of nerve growth factor to treat Alzheimer’s Disease. Neurobiol. Aging10:205.Google Scholar
  60. Piddington, R. and Moscona, A.A. (1967) Precocious induction of retinal glutamine synthetase by hydrocortisone in the embryo and in culture. Age-dependent differences in nssue response. Biochim. Biophvs. Acta141:429.CrossRefGoogle Scholar
  61. Price, D.L. (1986) New perspectives on Alzheimer’s disease. Annu.Rev.Neurosci.9:489.PubMedCrossRefGoogle Scholar
  62. Prince, D.A. and Connors, B.W. (1986) Mechanisms of interictal epileptogenesis. In:“ Basic Mechanisms of the Epilepsies, Molecular and Cellular Approaches: Advances in Neurology Vol. 44.” eds:Delgado-Escueta, A.V., Woodbury, D.M., Ward Jr., A.A. and Porter, R.J., Raven Press, New York, pp. 275–299.Google Scholar
  63. Prochiantz, A., diPorzio, U., Kato, A., Berger, B. and Glowinski, J. (1979) In vitro maturation of mesencephalic dopaminergic neurons from mouse embryos is enhanced in the presence of their striatal target cells. Proc. Natl. Acad. Sci. USA76:5387.PubMedCrossRefGoogle Scholar
  64. Purves, D. (1988)“Body and Brain: A Trophic Theory of Neural Connections.” Harvard University Press, Cambridge, Massachusetts.Google Scholar
  65. Roback, J.D., Large, T.H., Otten, U. and Wainer, B.H. (1990) Nerve growth factor expression in the isolated hippocampus in vitro. Dev. Biol. 137:451.PubMedCrossRefGoogle Scholar
  66. Ronnett, G.V., Hester, L.D., Nye, J.S., Connors, K. and Snyder, S.H. (1990) Human cortical neuronal cell line: Establishment from a patient with unilateral megalencephaly. Science248:603.PubMedCrossRefGoogle Scholar
  67. Rosenberg, M.B., Friedmann, T., Robertson, R.C., Tuszynski, M., Wolff, J.A., Breakefield, X.O. and Gage, F.H. (1988) Grafting genetically modified cells to the damaged brain restorative effects of ngf expression. Science242:1575.PubMedCrossRefGoogle Scholar
  68. Rye, D.B., Wainer, B.H., Mesulam, M.-M., Mufson, E.J. and Saper, C.B. (1984) Cortical projections arising from the basal forebrain: a study of cholinergic and noncholinergic components employing combined retrograde tracing and immunohistochemical localization of choline acetyltransferase. Neuroscience13:627.PubMedCrossRefGoogle Scholar
  69. Rye, D.B., Saper, C.B., Lee, H.J. and Wainer, B.H. (1987) Pedunculopontine tegmental nucleus of the rat: cytoarchitecture, cytochemistry, and some extrapyramidal connections of the mesopontine tegmentum. J Comp.Neurol.259:483.PubMedCrossRefGoogle Scholar
  70. Saper, C.B., Wainer, B.H. and German, D.C. (1987) Axonal and transneuronal transport in the transmission of neurological disease: potential role in system degenerations, including Alzheimer’s disease. Neuroscience23:389.PubMedCrossRefGoogle Scholar
  71. Saper, C.B. (1988) Chemical neuroanatomy of Alzheimer’s disease. In:“ Handbook of Psychopharmacology Vol. 20.” eds:Iversen, L.L., Iversen, S.D. and Snyder, S.H., Plenum, New York, pp. 131–156.CrossRefGoogle Scholar
  72. Snider, W.D. and Johnson, E.M.Jr. (1989) Neurotrophic molecules. Ann.Neurol.26:489.PubMedCrossRefGoogle Scholar
  73. Springer, J.E. and Loy, R. (1985) Intrahippocampal injections of antiserum to nerve growth factor inhibit sympathohippocampal sprouting. Brain Res.Bull.15:629.PubMedCrossRefGoogle Scholar
  74. Thoenen, H., Bandtlow, C. and Heumann, R. (1987) The physiological function of nerve growth factor in the central nervous system: comparison with the periphery. Rev. Physiol. Biochem. Pharmacol. 109:146.Google Scholar
  75. Tishler, A.S. and Greene, L.A. (1975) Nerve growth factor-induced process formation by cultured rat pheochromocytoma cells. Nature258:341.CrossRefGoogle Scholar
  76. Trojanowski, J.Q. (1987) Neurofilament proteins and human nervous system tumors. L Histochem. Cytochem. 35:999.CrossRefGoogle Scholar
  77. Vantini, G., Schiavo, N., Di Martino, A., Polato, P., Triban, C., Callegaro, L., Toffano, G. and Leon, A. (1989) Evidence for a physiological role of nerve growth factor in the central nervous system of neonatal rats. Neuron3:267.PubMedCrossRefGoogle Scholar
  78. Wainer, B.H., Levey, A.I., Mufson, E.J. and Mesulam, M.-M. (1984) Cholinergic systems in mammalian brain identified with antibodies against choline acetyltransferase. Neurochem. int.6:163.PubMedCrossRefGoogle Scholar
  79. Wainer, B.H., Levey, A.I., Rye, D.B., Mesulam, M.-M. and Mufson, E.J. (1985) Cholinergic and non-cholinergic septohippocampal pathways. Neurosci.Lett.54:45.PubMedCrossRefGoogle Scholar
  80. Wainer, B.H., Lee, H.J., Roback, J.D. and Hammond, D.N. (1989a) The use of reaggregating cell cultures and immortalized central nervous system cells to study cholinergic trophic mechanisms. In:“ Novel Approaches to the Treatment of Alzheimer’s Disease.” eds:Meyer, E.M. and Simpkins, J., Plenum Press, New York, pp. 71–94.Google Scholar
  81. Wainer, B.H. (1989b) Neurotrophic factors and Alzheimer’s disease. Neurobiol. Aging10:540.Google Scholar
  82. Wainer, B.H. and Mesulam, M.-M. (1990) Ascending cholinergic pathways in the rat brain. In:“ Brain Cholinergic Systems.” eds:Steriade, M. and Biesold, D., Oxford University Press, New York, in press.Google Scholar
  83. Walicke, P.A. (1988) Basic and acidic fibroblast growth factors have trophic effects on neurons from multiple ens regions. J. Neurosci. 8:2618.PubMedGoogle Scholar
  84. Walicke, P.A. (1989) Novel neurotrophic factors, receptors, and oncogenes. Ann. Rev. Neurosci. 12:103.PubMedCrossRefGoogle Scholar
  85. Warren, S. and Chute, R.N. (1972) Pheochromocytoma. Cancer29:327.CrossRefGoogle Scholar
  86. Werner, M.H., Nanney, L.B., Stoscheck, C.M. and King, L.E. (1988) Localization of immunoreactive EGF receptors in human nervous system. J. Histochem. Cytochem. 36:81.PubMedCrossRefGoogle Scholar
  87. Whittemore, S.R. and Sciger, A. (1987) The expression, localization and functional significance of ß-nerve growth factor in the central nervous system. Brain Res. Rev. 12:439.CrossRefGoogle Scholar
  88. Whittemore, S.R., Holets V.R., Gonzalez-Carvajal, M. and Levy, D. (1988) Transplantation of a hippocampally-derived immortal, temperature-sensitive, neuronal cell line into adult rat ens. Soc. Neurosci. Abs. 14:586.Google Scholar
  89. Williams, L.R., Varon, S., Peterson, G.M., Wictorin, K., Fischer, W., Bjorklund, A. and Gage, F.H. (1986) Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fornix transection. Proc.Natl.Acad.Sci.USA.83:9231.PubMedCrossRefGoogle Scholar
  90. Zola Morgan, S., Squire, L.R. and Amaral, D.G. (1986) Human amnesia and the medial temporal region: enduring memory impairment following a bilateral lesion limited to field CAl of the hippocampus. J Neurosci. 6:2950.Google Scholar

Copyright information

© Plenum Press, New York 1991

Authors and Affiliations

  • B. H. Wainer
    • 1
    • 2
    • 4
  • H. J. Lee
    • 1
  • J. D. Roback
    • 2
  • D. N. Hammond
    • 3
    • 4
  1. 1.Department of Pharmacological and Physiological SciencesThe University of ChicagoChicagoUSA
  2. 2.Department of PathologyThe University of ChicagoChicagoUSA
  3. 3.Department of NeurologyThe University of ChicagoChicagoUSA
  4. 4.Department of PediatricsThe University of ChicagoChicagoUSA

Personalised recommendations