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

  1. Christian Lesuisse1,
  2. Lee J. Martin1,2,*

Article first published online: 8 MAR 2002

DOI: 10.1002/neu.10037

Issue

Journal of Neurobiology

Journal of Neurobiology

Volume 51, Issue 1, pages 9–23, April 2002

Additional Information

How to Cite

Lesuisse, C. and Martin, L. J. (2002), Long-term culture of mouse cortical neurons as a model for neuronal development, aging, and death. J. Neurobiol., 51: 9–23. doi: 10.1002/neu.10037

Author Information

  1. 1

    Department of Pathology, Division of Neuropathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

  2. 2

    Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

*Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Publication History

  1. Issue published online: 8 MAR 2002
  2. Article first published online: 8 MAR 2002
  3. Manuscript Accepted: 9 NOV 2001
  4. Manuscript Received: 4 MAY 2001

Funded by

  • U.S. Public Health Service
  • National Institutes of Health
  • National Institute of Neurological Disorders and Stroke. Grant Number: NS34100
  • National Institute on Aging. Grant Number: AG16282
  • U.S. Army Medical Research and Materiel Command. Grant Number: DAMD17-99-1-9553

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Keywords:

  • Alzheimer's disease;
  • amyotrophic lateral sclerosis;
  • apoptosis;
  • NMDA receptor;
  • Parkinson's disease;
  • synucleins

Abstract

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, α and β 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, β-synuclein (but not α-synuclein) was localized at the base of dendritic growth cones identified by MAP2 and α-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptor GluR1. In mature neurons, α and β 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. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 9–23, 2002

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