Indo-1 measurements of intracellular free calcium in the hippocampal slice: complications of labile NADH fluorescence

Abstract

The use of the fluorescent calcium indicator, Indo-1, was evaluated for measuring changes in intracellular free calcium during electrical stimulation and anoxia in hippocampal slices. Fluorescence was measured from slices illuminated with brief (3 ns) light pulses (337 nm wavelength) from a nitrogen laser. This method of illumination produced more intense fluorescence than illumination with light from filtered xenon or mercury arc lamps, and prevented loss of electrical excitability encountered following continuous UV illumination. Background fluorescence in control slices (without Indo-1) was considerable, often approaching 50% of that obtainable after dye loading. A more serious concern, however, was that a large fraction (approximately 10%) of the background fluorescence was labile to both electrical stimulation and anoxia. This fluorescence results from changes in the reduction/oxidation (redox) state of nicotinamide adenine dinucleotide (NAD), which fluoresces in its reduced (NADH) but not oxidized (NAD +) form. Qualitative changes in free calcium could be measured by first determining the ratio of change in NADH fluorescence at 405 and 485 nm (the wavelengths of light used to measure calcium with Indo-1) prior to dye loading. Any arbitrary baseline could be selected as long as the ratio for the baseline at 405 and 485 nm was identical to that determined for labile NADH. By application of this compensation procedure, it was determined that intracellular calcium rose abruptly during the onset of anoxia and again during the spreading depression-like loss of ion homeostasis which inevitably occurred during anoxia in these slices. However, quantitation of changes in intracellular free calcium concentration during anoxia is not yet possible, since the contribution of labile NADH fluorescence cannot be subtracted reliably from the total fluorescence signal.