Technical Reference #99

99.

Mitochondrial Production Of Reactive Oxygen Species In Cortical Neurons Following Exposure To N-Methyl-D-Aspartate Dugan; L.L.; Sensi; S.L.; Canzoniero; L.M.T.; Handran; S.D.; Rotham; S.M.; Lin; T.S.; and Goldberg; M.P., Washington University, The Journal of Neuroscience, 15(99), (1995)
Link To Paper

Abstract:
Increasing evidence suggests that glutamate neurotoxicity is partly mediated by reactive oxygen species formed as a consequence of several processes including arachidonic acid metabolism and nitric oxide production. Here we used an oxidation-sensitive indicator dihydrorhodamine 123 in combination with confocal microscopy to examine the hypothesis that electron transport by neuronal mitochondria may be an important source of glutamate-induced reactive oxygen species (ROS). Exposure to NMDA but not kainate ionomycin or elevated potassium stimulated oxygen radical production in cultured murine cortical neurons demonstrated by oxidation of nonfluorescent dihydrorhodamine 123 to fluorescent rhodamine 123. Electron paramagnetic resonance spectroscopy studies using 55-dimethyl-1- pyrroline-N-oxide (DMPO) as a radical-trapping agent also showed production of ROS by cortical neurons after NMDA but not kainate exposure. NMDA-induced ROS production depended on extracellular Ca2+ and was not affected by inhibitors of nitric oxide synthase or arachidonic acid metabolism. The increased production of ROS was blocked by inhibitors of mitochondrial electron transport rotenone or antimycin and mimicked by the electron transport uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These data support the possibility that NMDA receptor-mediated Ca(2+)-dependent uncoupling of neuronal mitochondrial electron transport may contribute to the oxidative stress initiated by glutamate exposure.

Keywords:
glutamate; excitotoxicity; neurotoxicity; free radicals; mitochondria; dihydrorhodamine; oxidation; electron transport

Materials & Methods:
Swiss-Webster mice (Simonson) as described previously (Rose et al. 1993). Cortical hemispheres were dissected away from the rest of the brain and placed in trypsin (0.25% GIBCO) for 15 min. The cortices were briefly centrifuged the trypsin was removed and the hemispheres resuspended in plating medium which consisted of media stock (Eagle’s t Minimal Essential Media minus L-glutamine GIBCO 11430-022) with 20 mu glucose 26.2 ~IUVNI aHCO) supplemented with L-glutamine (2 mu) 5% fetal calf serum and 5% horse serum (Hyclone). After trituration cell suspensions were diluted and plated onto a preexisting bed of mouse cortical astrocytes on 35 mm culture dishes (Mat-Tek) possessing an oval cut-out sealed by a glass coverslip. Cells were fed biweekly with growth medium (media stock with 10% horse serum 2 mu L-glutamine) until the final feeding at day 11 or 12 in vitro when cultures were fed with media stock supplemented with 2 mu L-glutamine. After 12-17 d in culture cells were used for experiments. Confocal microscopy. Cultures were washed into HEPES bicarbonate

Microscopic Technique
Confocal Microscopy

Cell Type(s)
Neocortical