Optical Imaging Reveals Elevated Intracellular Chloride in Hippocampal Pyramidal Neurons after Oxidative Stress

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Abstract
The accumulation of reactive oxygen species (ROS) in the brain is associated with several neurodegenerative conditions. ROS can affect ionic homeostasis leading to impaired neurotransmission. Here, we determined the ability of H2O2, a membrane permeant ROS, to alter intraneuronal Cl−, an important regulator of neuronal excitability. Real-time alterations in intracellular chloride, [Cl−]i, were measured with UV laser scanning confocal microscopy in hippocampal slices loaded with the cell-permeant form of 6-methoxy-N-ethylquinolium iodide (MEQ), a Cl−-sensitive fluorescent probe. In slices superfused with H2O2for 10 min, there was a significant decrease in MEQ fluorescence (elevation in [Cl−]i) in area CA1 pyramidal cell soma but not in interneurons located in stratum radiatum. Alterations in [Cl−]i induced by H2O2were prevented by the iron chelator deferoxamine and the vitamin E analog Trolox, suggesting the involvement of free radicals. The influx of Cl−probably occurred through the GABA-gated Cl−channel because the effects of H2O2were blocked by picrotoxin. In addition, HPLC analysis of the superfusates indicated that GABA and glutamate accumulated extracellularly after H2O2exposure. Excitatory amino acid receptor antagonists 2-amino-5-phoshopentanoic acid and 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-sulfonamide also attenuated the effect of H2O2on MEQ fluorescence. The changes in [Cl−]i induced by H2O2were Ca2+-dependent and Na+-independent. After exposure of slices to H2O2, the ability of the GABA agonist muscimol to increase [Cl−]i was attenuated. Thus, ROS, like H2O2, may impair transmembrane Cl−gradients and reduce inhibitory neurotransmission, further promoting neuronal damage in oxidative stress-related disease and in aging.

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