Pillar_1993_Toxicol.In.Vitro_7_69

The toxicity was studied of ethylcholine mustard aziridinium (ECMA) in rat brain reaggregate cultures. The objective was to define optimum conditions for selective effects on cholinergic neurones. Single treatments with 12.5-50 mum-ECMA caused approximately 35% loss of choline acetyltransferase (ChAT) activity in 2 hr, increasing to 70-80% in 72-120 hr. The 2-hr, but not the longer-term loss of activity was prevented by the choline transport blocker hemicholinium-3 (40 mum). Significant loss of ChAT activity could not be obtained with less than 12.5 mum-ECMA. Approximately 55% irreversible inhibition of muscarinic receptor binding also occurred in 2 hr. However, after 12.5 mum-ECMA, binding recovered to control values within 48 hr. These persisted throughout the longer-term more extensive loss of ChAT activity, which suggests that receptor recovery localized to cells other than cholinergic neurones. After 25-50 mum-ECMA, muscarinic receptor binding did not recover, which suggests more widespread cytotoxicity destroying cells other than the cholinergic neurones. More pronounced leakage of lactate dehydrogenase and reduced reaggregate concentrations of total and neurofilament protein were consistent with more generalized cytotoxicity after 25 or 50 mum-ECMA. Examination of ECMA treated reaggregates or cerebellar monolayer cultures by light microscopy confirmed this. Concentrations of 12.5 mum-ECMA therefore represent the optimum for achieving selective toxic effects on cholinergic neurones in the cultures. After ECMA, reaggregate concentrations of 5-hydroxyindole acetic acid were markedly increased (100-300%), which suggests increased activity of 5HT neurones. This demonstrates that reaggregate cultures might be used to study trans-synaptic neurochemical sequelae of brain cholinergic neurone loss.