Optical imaging reveals elevated intracellular chloride in hippocampal pyramidal neurons after oxidative stress

The accumulation of reactive oxygen species (ROS) in the brain is associate d with several neurodegenerative conditions. ROS can affect ionic homeostas is leading to impaired neurotransmission. Here, we determined the ability o f H2O2, a membrane permeant ROS, to alter intraneuronal Cl-, an important r egulator of neuronal excitability. Real-time alterations in intracellular c hloride, [Cl-]i, were measured with UV laser scanning confocal microscopy i n hippocampal slices loaded with the cell-permeant form of 6-methoxy-N-ethy lquinolium iodide (MEQ), a Cl--sensitive fluorescent probe. In slices super fused with H2O2 for 10 min, there was a significant decrease in MEQ fluores cence (elevation in [Cl-]i) in area CA1 pyramidal cell soma but not in inte rneurons located in stratum radiatum. Alterations in [Cl-]i induced by H2O2 were prevented by the iron chelator deferoxamine and the vitamin E analog Trolox, suggesting the involvement of free radicals. The influx of Cl- prob ably occurred through the GABA-gated Cl- channel because the effects of H2O 2 were blocked by picrotoxin. In addition, HPLC analysis of the superfusate s indicated that GABA and glutamate accumulated extracellularly after H2O2 exposure. Excitatory amino acid receptor antagonists 2-amino-5-phoshopentan oic acid and 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-su lfonamide also attenuated the effect of H2O2 on MEQ fluorescence. The chang es in [Cl-]i induced by H2O2 were Ca2+-dependent and Na+-independent. After exposure of slices to H2O2, the ability of the GABA agonist muscimol to in crease [Cl 2]i was attenuated. Thus, ROS, like H2O2, may impair transmembra ne Cl 2 gradients and reduce inhibitory neurotransmission, further promotin g neuronal damage in oxidative stress-related disease and in aging.