J. Keelan et al., Quantitative imaging of glutathione in hippocampal neurons and glia in culture using monochlorobimane, J NEUROSC R, 66(5), 2001, pp. 873-884
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.