Ca2+ entry via AMPA/KA receptors and excitotoxicity in cultured cerebellar Purkinje cells

James R. Brorson, Patricia A. Manzolillo, Richard J. Miller*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

196 Scopus citations

Abstract

Initial studies of glutamate receptors activated by kainate (KA) found them to be Ca2+ impermeable. Activation of these receptors was thought to produce Ca2+ influx into neurons only indirectly by Na+-dependent depolarization. However, Ca2+ entry via AMPA/KA receptors has now been demonstrated in several neuronal types, including cerebellar Purkinje cells. We have investigated whether such Ca2+ influx is sufficient to induce excitotoxicity in cultures of cerebellar neurons enriched for Purkinje cells. Agonists at non-NMDA receptors induced Ca2+ influx in the majority of these cells, as measured by whole-cell voltage clamp and by fura-2[Ca2+](i) microfluorimetry. To assess excitotoxicity, neurons were exposed to agonists for 20 min and cell survival was evaluated by a fluorescence assay 24 hr later. KA (100 μM) reduced neuronal survival relative to controls to 43 ± 3% when applied in Na+-containing solution and to 45 ± 3% in Na+-free solution. This toxicity was blocked completely by CNQX but only slightly by 100 μM Cd2+ and 50 μM D-(-)-2-amino-5-phosphonovaleric acid. Both Purkinje neurons and non-Purkinje cell types present in the cultures were similarly vulnerable to toxic KA exposure, but the population marked by KA- induced Co2+ uptake was selectively diminished by the excitotoxicity. Na+- independent excitotoxicity could also be induced by domoate, AMPA, or glutamate. Compared to KA, NMDA was relatively ineffective in inducing cell death. Most of the KA-induced excitotoxicity could be blocked by removal of extracellular Ca2+ during the KA exposure and for a 5 min period thereafter. Furthermore, antagonists of the Ca2+-activated enzymes nitric oxide synthase and calpain significantly reduced the KA-induced cell death. These results show that non-NMDA receptor activation can cause excitotoxicity in cerebellar Purkinje neurons by mechanisms not involving Na+ influx, but rather depending on direct Ca2+ permeation and activation of Ca2+- dependent enzymatic processes.

Original languageEnglish (US)
Pages (from-to)187-197
Number of pages11
JournalJournal of Neuroscience
Volume14
Issue number1
DOIs
StatePublished - 1994

Keywords

  • AMPA
  • Purkinje cells
  • calcium
  • calpain I
  • excitotoxicity
  • glutamate receptors
  • kainate
  • nitric oxide

ASJC Scopus subject areas

  • Neuroscience(all)

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