A FLOW CYTOMETRIC STUDY OF N-METHYL-D-ASPARTATE EFFECTS ON DISSOCIATED CEREBELLAR CELLS

The effects of N-merhyl-D-aspartate (NMDA) on the generation of intrac ellular reactive oxygen species (ROS) and intracellular calcium in rat dissociated cerebellar cells were examined by flow cytometry. Flow cy tometry allows the selection of a specific viable neuronal population with high sensitivity. We used 2',7'-dichlorofluorescin diacetate (DCF H-DA) as a marker of intracellular oxidative stress, and intracellular calcium was measured using Indo-1 as a calcium-sensitive indicator. T he cerebellar cell population was isolated by size, granularity and NM DA-sensitivity by cell-sorting. In this cerebellar cell preparation, i n which no glial cells were found, NMDA induced a concentration-depend ent increase in ROS and intracellular calcium levels. These effects we re inhibited by the non-competitive NMDA antagonist (+)MK-801. These r esults indicate that flow cytometry could be a useful tool to study th e effect of neuroprotective drugs on NMDA receptor in isolated cerebel lar neurons. Moreover, due to its high speed of analysis and the possi bility to detect simultaneously a variety of fluorescent markers, we s tated the utility of this technique in the pharmacology and physiology of the central nervous system.