Fluoroscopic determination of NAD(P)H/NAD(P)+ ratios in brain slices under perifusion conditions

The adaptation of a commercially available fluorescence microscope for continuous monitoring of endogenous NAD(P)H levels in brain slices is described. A specially designed self-stirring perifusion chamber was used to monitor a stable fluorescence signal for hours, indicating sufficient oxygen and substrate supply under measuring conditions. Profiles and reversibility of the fluorescence signals following changes in NAD(P)H levels in the picomolar range are demonstrated in slices of rat hippocampus. The degree of reduction obtained under normal metabolic conditions in the presence of 10 mM glucose was 29.9% +/- 1.3 (mean +/- S.D.; n = 12). The present technique allows convenient monitoring of the influence of various exogenous factors on metabolic conditions which are accompanied by changes in the redox state of brain nicotinamide nucleotides.