Long-term culture of mouse cortical neurons as a model for neuronal development, aging, and death.

A long-term cell culture system was used to study maturation, aging, and death of cortical neurons. Mouse cortical neurons were maintained in culture in serum-free medium (Neurobasal supplemented with B27) for 60 days in vitro (DIV). The levels of several proteins were evaluated by immunoblotting to demonstrate that these neurons matured by developing dendrites and synapses and remained continuously healthy for 60 DIV. During their maturation, cortical neurons showed increased or stable protein expression of glycolytic enzyme, synaptophysin, synapsin IIa, alpha and beta synucleins, and glutamate receptors. Synaptogenesis was prominent during the first 15 days and then synaptic markers remained stable through DIV60. Very early during dendritic development at DIV3, beta-synuclein (but not alpha-synuclein) was localized at the base of dendritic growth cones identified by MAP2 and alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptor GluR1. In mature neurons, alpha and beta synucleins colocalized in presynaptic axon terminals. Expression of N-methyl-D-aspartate (NMDA) and AMPA receptors preceded the formation of synapses. Glutamate receptors continued to be expressed strongly through DIV60. Cortical neurons aging in vitro displayed a complex profile of protein damage as identified by protein nitration. During cortical neuron aging, some proteins showed increased nitration, while other proteins showed decreased nitration. After exposure to DNA damaging agent, young (DIV5) and old (DIV60) cortical neurons activated apoptosis mechanisms, including caspase-3 cleavage and poly(ADP)-ribose polymerase inactivation. We show that cultured mouse cortical neurons can be maintained for long term. Cortical neurons display compartmental changes in the localization of synucleins during maturation in vitro. These neurons sustain protein nitration during aging and exhibit age-related variations in the biochemistry of neuronal apoptosis.

[1]  L. Petrucelli,et al.  α-Synuclein Shares Physical and Functional Homology with 14-3-3 Proteins , 1999, The Journal of Neuroscience.

[2]  P. De Camilli,et al.  The distribution of synapsin I and synaptophysin in hippocampal neurons developing in culture , 1991, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[3]  E. Johnson,et al.  Cytosine arabinoside kills postmitotic neurons: evidence that deoxycytidine may have a role in neuronal survival that is independent of DNA synthesis , 1989, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[4]  Gregory J Brewer,et al.  Isolation and culture of adult rat hippocampal neurons , 1997, Journal of Neuroscience Methods.

[5]  R. Hertzberg,et al.  Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. , 1985, The Journal of biological chemistry.

[6]  R. Huganir,et al.  AMPA glutamate receptor subunits are differentially distributed in rat brain , 1993, Neuroscience.

[7]  R. Baughman,et al.  Primary culture of identified neurons from the visual cortex of postnatal rats , 1986, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[8]  Y. Lazebnik,et al.  Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE , 1994, Nature.

[9]  P. Knaus,et al.  Expression of Synaptophysin During Postnatal Development of the Mouse Brain , 1986, Journal of neurochemistry.

[10]  C. Portera-Cailliau,et al.  Excitotoxic neuronal death in the immature brain is an apoptosis‐necrosis morphological continuum , 1997, The Journal of comparative neurology.

[11]  A. Furuta,et al.  AMPA receptor protein in developing rat brain: glutamate receptor-1 expression and localization change at regional, cellular, and subcellular levels with maturation , 1998, Neuroscience.

[12]  Carlos Portera-Cailliau,et al.  Neurodegeneration in Excitotoxicity, Global Cerebral Ischemia, and Target Deprivation: A Perspective on the Contributions of Apoptosis and Necrosis , 1998, Brain Research Bulletin.

[13]  W. Markesbery,et al.  Survival of hippocampal and cortical neurons in a mixture of MEM+ and B27-supplemented neurobasal medium. , 2000, Free Radical Biology & Medicine.

[14]  M. Avoli,et al.  Age‐dependent appearance of synaptic currents in rat neocortical neurons in culture , 1994, Synapse.

[15]  G. Withers,et al.  Delayed localization of synelfin (synuclein, NACP) to presynaptic terminals in cultured rat hippocampal neurons. , 1997, Brain research. Developmental brain research.

[16]  G. Banker.  Trophic interactions between astroglial cells and hippocampal neurons in culture. , 1980, Science.

[17]  L. Martin,et al.  Neuronal cell death in nervous system development, disease, and injury (Review). , 2001, International journal of molecular medicine.

[18]  F. Gage,et al.  Proliferation, differentiation, and long-term culture of primary hippocampal neurons. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[19]  J. Volpe.  Neurology of the Newborn , 1959, Major problems in clinical pediatrics.

[20]  C. Bendixen,et al.  Camptothecin-stabilized topoisomerase I-DNA adducts cause premature termination of transcription. , 1990, Biochemistry.

[21]  A. Schousboe,et al.  UPTAKE AND METABOLISM OF GLUTAMATE IN ASTROCYTES CULTURED FROM DISSOCIATED MOUSE BRAIN HEMISPHERES , 1977, Journal of neurochemistry.

[22]  G. Banker,et al.  Culturing nerve cells , 1998 .

[23]  C. Portera-Cailliau,et al.  Non‐NMDA and NMDA receptor‐mediated excitotoxic neuronal deaths in adult brain are morphologically distinct: Further evidence for an apoptosis‐necrosis continuum , 1997, The Journal of comparative neurology.

[24]  D. Price,et al.  Synaptic pathology and glial responses to neuronal injury precede the formation of senile plaques and amyloid deposits in the aging cerebral cortex. , 1994, The American journal of pathology.

[25]  David F. Clayton,et al.  Characterization of a novel protein regulated during the critical period for song learning in the zebra finch , 1995, Neuron.

[26]  H. M. Geller,et al.  Induction of neuronal apoptosis by camptothecin, an inhibitor of DNA topoisomerase-I: evidence for cell cycle-independent toxicity , 1996, The Journal of cell biology.

[27]  C. Lesuisse,et al.  Regulation of agrin expression in hippocampal neurons by cell contact and electrical activity. , 2000, Brain research. Molecular brain research.

[28]  M. Strong,et al.  Nitration of the low molecular weight neurofilament is equivalent in sporadic amyotrophic lateral sclerosis and control cervical spinal cord. , 1998, Biochemical and biophysical research communications.

[29]  J. Trojanowski,et al.  Synucleins Are Developmentally Expressed, and α-Synuclein Regulates the Size of the Presynaptic Vesicular Pool in Primary Hippocampal Neurons , 2000, The Journal of Neuroscience.

[30]  G H Sato,et al.  Growth of a rat neuroblastoma cell line in serum-free supplemented medium. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[31]  L. Thal,et al.  Expression Pattern of Synucleins (Non‐Aβ Component of Alzheimer's Disease Amyloid Precursor Protein/α‐Synuclein) During Murine Brain Development , 1998 .

[32]  R. Simantov.  Glutamate Neurotoxicity in Culture Depends on the Presence of Glutamine: Implications for the Role of Glial Cells in Normal and Pathological Brain Development , 1989, Journal of neurochemistry.

[33]  L. Martin.  p53 Is Abnormally Elevated and Active in the CNS of Patients with Amyotrophic Lateral Sclerosis , 2000, Neurobiology of Disease.

[34]  B. Halliwell,et al.  Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. , 1986, Archives of biochemistry and biophysics.

[35]  A. Brambrink,et al.  Neuronal Death in Newborn Striatum after Hypoxia-Ischemia Is Necrosis and Evolves with Oxidative Stress , 2000, Neurobiology of Disease.

[36]  H. Sontheimer,et al.  Astrocytes protect neurons from neurotoxic injury by serum glutamate , 1998, Glia.

[37]  J. Yodoi,et al.  Effects of 2-mercaptoethanol on survival and differentiation of fetal mouse brain neurons cultured in vitro , 1993, Neuroscience Letters.

[38]  L. Martin,et al.  Injury‐induced apoptosis of neurons in adult brain is mediated by p53‐dependent and p53‐independent pathways and requires bax , 2001, The Journal of comparative neurology.

[39]  L. Martin,et al.  Motor neuron degeneration after sciatic nerve avulsion in adult rat evolves with oxidative stress and is apoptosis , 1999 .

[40]  A. Furuta,et al.  Laminar segregation of the cortical plate during corticogenesis is accompanied by changes in glutamate receptor expression. , 1999, Journal of neurobiology.

[41]  T. Voigt,et al.  Identification of two distinct populations of γ‐aminobutyric acidergic neurons in cultures of the rat cerebral cortex , 1997 .

[42]  Richard L. Huganir,et al.  Regulation of morphological postsynaptic silent synapses in developing hippocampal neurons , 1999, Nature Neuroscience.

[43]  K. Chen,et al.  Calcium Channel Density and Hippocampal Cell Death with Age in Long-Term Culture , 1997, The Journal of Neuroscience.

[44]  A. Shaikh,et al.  Mechanisms for neuronal degeneration in amyotrophic lateral sclerosis and in models of motor neuron death (Review). , 2000, International journal of molecular medicine.

[45]  B. Pettmann,et al.  Astroglial and fibroblast growth factors have neurotrophic functions for cultured peripheral and central nervous system neurons. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[46]  D. Butterfield,et al.  Aging in a dish: Age‐dependent changes of neuronal survival, protein oxidation, and creatine kinase BB expression in long‐term hippocampal cell culture , 1999, Journal of neuroscience research.

[47]  G. Brewer,et al.  Optimized survival of hippocampal neurons in B27‐supplemented neurobasal™, a new serum‐free medium combination , 1993, Journal of neuroscience research.