Increased vulnerability of hippocampal neurons with age in culture: Temporal association with increases in NMDA receptor current, NR2A subunit expression and recruitment of L-type calcium channels

Lawrence D. Brewer, Olivier Thibault, Jeanise Staton, Veronique Thibault, Justin T. Rogers, Gisela Garcia-Ramos, Susan Kraner, Philip W. Landfield, Nada M. Porter

Research output: Contribution to journalArticlepeer-review

76 Scopus citations

Abstract

Excessive glutamate (Glu) stimulation of the NMDA-R is a widely recognized trigger for Ca2+-mediated excitotoxicity. Primary neurons typically show a large increase in vulnerability to excitotoxicity with increasing days in vitro (DIV). This enhanced vulnerability has been associated with increased expression of the NR2B subunit or increased NMDA-R current, but the detailed age-courses of these variables in primary hippocampal neurons have not been compared in the same study. Further, it is not clear whether the NMDA-R is the only source of excess Ca2+. Here, we used primary hippocampal neurons to examine the age dependence of the increase in excitotoxic vulnerability with changes in NMDA-R current, and subunit expression. We also tested whether L-type voltage-gated Ca2+ channels (L-VGCCs) contribute to the enhanced vulnerability. The EC50 for Glu toxicity decreased by approximately 10-fold between 8-9 and 14-15 DIV, changing little thereafter. Parallel experiments found that during the same period both amplitude and duration of NMDA-R current increased dramatically; this was associated with an increase in protein expression of the NR1 and NR2A subunits, but not of the NR2B subunit. Compared to MK-801, ifenprodil, a selective NR2B antagonist, was less effective in protecting older than younger neurons from Glu insult. Conversely, nimodipine, an L-VGCC antagonist, protected older but not younger neurons. Our results indicate that enhanced excitotoxic vulnerability with age in culture was associated with a substantial increase in NMDA-R current, concomitant increases in NR2A and NR1 but not NR2B subunit expression, and with apparent recruitment of L-VGCCs into the excitotoxic process.

Original languageEnglish
Pages (from-to)20-31
Number of pages12
JournalBrain Research
Volume1151
Issue number1
DOIs
StatePublished - Sep 4 2007

Bibliographical note

Funding Information:
We thank Elsie Barr for expert technical assistance with the cell culture preparation and Dr. Amy L.S. Dowling for commenting on an earlier version of the manuscript. We also thank Dr. Derik Castillo for assistance with mathematical analyses (Dept. of Biology, University of Kentucky). This work was supported by National Institutes of Health Grants AG10836 and AG020251.

Keywords

  • Age in culture
  • Concentration-response
  • Excitotoxicity
  • Ifenprodil
  • NMDA receptor
  • Patch clamp

ASJC Scopus subject areas

  • Neuroscience (all)
  • Molecular Biology
  • Clinical Neurology
  • Developmental Biology

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