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Detection of high molecular weight DNA fragments characteristic of early stage apoptosis in cerebellar granule cells exposed to glutamate

Experimental Brain Research volume 135, pages 173–178 (2000)Cite this article

Abstract

In cerebellar granule cells a rapid necrotic cell death has been observed during and immediately after glutamate exposure, followed by a delayed apoptotic type of neuronal death in a subpopulation of the surviving neurons. In some experimental models the DNA fragmentation characteristic of apoptosis is readily detected. In other systems apoptosis may occur only in a limited number of cells, rendering DNA fragmentation undetectable using conventional DNA-staining techniques (e.g., ethidium bromide). We have used a sensitive and non-radioactive method for labeling, detection, and quantification of high molecular weight (HMW) DNA fragments. This method is based on the introduction of thymine dimers into DNA after separation by pulse field gel electrophoresis, followed by detection with thymine dimer specific antibodies. Applying this method to cerebellar granule cells in culture, we detected an increase in the amount of HMW DNA fragments characteristic of apoptosis as early as 4 h after glutamate exposure. The N-methyl-D-aspartic acid (NMDA)-receptor antagonist MK801 protected against the fragmentation, whereas no protection was observed using the non-NMDA-receptor antagonist CNQX.

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Affiliations

  1. Institute of Pharmacy, University of Oslo, PO Box 1068 Blindern, 0316|, Oslo, Norway

    Hege H. Slagsvold, Guro Eidem, Nina Kristoffersen & Ragnhild E. Paulsen

  2. Affitech AS, Gaustadalléen 21, 0371, Oslo, Norway

    Ole J. Marvik

Authors
  1. Hege H. Slagsvold
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  2. Ole J. Marvik
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  3. Guro Eidem
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  4. Nina Kristoffersen
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  5. Ragnhild E. Paulsen
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Correspondence to Ragnhild E. Paulsen.

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Published online: 30 August 2000

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Slagsvold, H.H., Marvik, O.J., Eidem, G. et al. Detection of high molecular weight DNA fragments characteristic of early stage apoptosis in cerebellar granule cells exposed to glutamate. Exp Brain Res 135, 173–178 (2000). https://doi.org/10.1007/s002210000521

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  • DOI: https://doi.org/10.1007/s002210000521

Key words

  • Antibody
  • DNA fragmentation
  • DNA labeling
  • Glutamate toxicity
  • Cerebellar granule cells