Monochlorobimane detection of sulfhydryls in cultured hippocampal neurons

The relationship between sulfhydryl content and the response to glutamate overstimulation was examined in cultured hippocampal neurons. The fluorescence of the SH-sensitive dye, monoclorobimane (MBC1), increased with time in a biphasic manner consistent with its interaction with glutathione and other protein sulfhydryls. MBC1 fluorescence was largely associated with the cytosol. No punctate fluorescence remained following digitonin treatment suggesting that mitochondrial sulfhydryls did not constitute an identifiable second fluorescent component. MBC1 fluorescence increased in neurons pretreated with reductants and decreased following buthionine sulfoximine (BSO) or oxidant exposure. Glutamate exposure drastically decreased MBC1 fluorescence, indicating oxidation of cellular SH groups had occurred. These experiments demonstrate that MBC 1 can be used as a marker for neuronal SH redox status and that oxidation of sulfhydryls is associated with glutamate excitotoxicity.