Quantitative imaging of glutathione in hippocampal neurons and glia in culture using monochlorobimane

Glutathione (GSH) is a major antioxidant system in the mammalian central ne rvous system (CNS). Abnormalities of GSH metabolism have been associated wi th many disorders of the CNS, including Parkinson's, Alzheimer's, and Hunti ngdon's diseases and ischaemic/reperfusion injury. Investigation of GSH lev els in the CNS generally relies on biochemical assays from cultures enriche d for different cell types. Because glia influence neuronal metabolism, we have studied cultures in which neurons and glia are cocultured. This approa ch demands fluorescence imaging to differentiate between the different cell types in the culture, permitted by the use of monochlorobimane (MCB), whic h reacts with GSH to produce a fluorescent product. We have defined the con ditions required to ensure steady-state MCB loading and show the specificit y of MCB for GSH through a reaction catalysed by glutathione-S-transferase (GST). [GSH] was consistently higher in glia than in neurons, and [GSH] in both cell types decreased with time in culture. Inhibition of GSH synthesis by buthionine sulfoximine (BSO) caused a greater proportional depletion of GSH in glia than in neurons. The depletion of GSH induced by BSO was signi ficantly greater in cells cultured for > 10 days. Furthermore, release of G SH from glia and its breakdown by the ectoenzyme gamma -glutamyltranspeptid ase (gamma GT) maintains [GSH] in neurons. In older cultures, inhibition of gamma GT by acivicin caused significant depletion of neuronal GSH. After i nhibition of GSH synthesis by BSO, inhibition of the glia-neuron traffickin g pathway by acivicin caused widespread neuronal death. Such neurotoxicity was independent of the endogenous glutamate and nitric oxide synthase, sugg esting that it is not due to secondary excitotoxicity. (C) 2001 Wiley-Liss, Inc.