Superoxide production in rat hippocampal neurons: Selective imaging with hydroethidine

Vytautas P. Bindokas, Joaquín Jordán, Chong C. Lee, Richard J. Miller*

*Corresponding author for this work

Research output: Contribution to journalArticle

750 Scopus citations

Abstract

Digital-imaging microfluorimetry of the oxidation of hydroethidine (HEt) to ethidium can be used to monitor superoxide (O2) production selectively within individual rat hippocampal pyramidal neurons in culture and in brain slices. Under assay conditions, oxidation was not accomplished by hydroxyl radical, singlet O2, H2O2, or nitrogen radicals. Neuronal O2 production varied with metabolic activity and age. O2 generation increased after treatment with AMPA, kainic acid, and NMDA, and the mitochondrial uncoupler carbonylcyanide p-(trifluoromethoxy)phenyl hydrazone, but usually not after depolarization (50 mM K+). O2 concentrations were sensitive to scavengers and nitric oxide. HEt oxidation was higher in Ca2+-containing versus Ca2+-free saline. However, Ca2+ ionophores did not increase oxidation greatly. H2O2 application produced a secondary increase in O2. The major source of O2 under basal and stimulated conditions appeared to be the mitochondria. Consistent with this, ethidium staining in dendrites was punctate, colocalized with mitochondria, and blocked by CN.

Original languageEnglish (US)
Pages (from-to)1324-1336
Number of pages13
JournalJournal of Neuroscience
Volume16
Issue number4
DOIs
StatePublished - Feb 15 1996

Keywords

  • HO
  • NMDA
  • NOS
  • dihydro ethidium
  • excitotoxicity
  • free radicals
  • microfluorimetry
  • mitochondria
  • reactive oxygen species

ASJC Scopus subject areas

  • Neuroscience(all)

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