Oxygen‐induced superoxide production in solitary complex neurons in rat medullary slices

Abstract

We previously reported that the rate of superoxide (•O2−) production in CA1 neurons in hippocampal slices decreases as O2 is decreased from 95% to 60, 40 or 20% (J. Neurophysiol. 98:1030‐1041, 2007). Here we report the effects of continuous exposure to O2 (95, 40 & 20%) on •O2− production in solitary complex (SC) neurons in medullary slices (400μm, weaned rats). The rate of intracellular •O2− production was measured using the fluorogenic probe, DHE (2.5μM), added to the superfusate (35–37°C). Fluorescence imaging initially classified cells at the start of the experiment as dim versus bright. Dim SC neurons incubated in 95%O2 had a similar rate of •O2− production (increasing fluorescence) over time as that reported previously for CA1 hippocampal neurons. Continuous exposure of dim cells to 20 or 40%O2, however, revealed slightly lower but similar rates of •O2− production that were significantly greater than that reported for CA1 neurons. SC neurons initially identified as bright cells increased their rate of fluorescence during the first 60–90 minutes but thereafter fluorescence reached a plateau indicative of a decreased rate of •O2− production. In bright SC cells, 95% and 20%O2 caused the lowest and highest rate of •O2− production, respectively. We conclude the following: 1) dim SC neurons have a higher capacity for •O2− production during exposure to 20–40%O2 than CA1 hippocampal neurons. 2) Neurons with initially bright DHE fluorescence may be damaged from slicing and lose their •O2− generating mechanisms rapidly, especially in the presence of 95%O2 (ONR N000140110179, NIH R01HL56683).