Autofluorescence as a confound in the determination of calcium levels in hippocampal slices using fura-2AM dye

Abstract

Recent publications have reported calcium level determinations in slices of brain using imaging techniques and the dye fura-2AM. In general these studies ignore or deal only perfunctorily with the problem of autofluorescence in slices. This confound, which is a result of the pyridine nucleotides that are normally present in tissue, has been previously reported to interfere with Ca2+ measurements in slices. Because these pyridine compounds are involved in cell metabolism, the fluorescence intensity is labile over time following experimental manipulations. We were studying Ca2+ levels in hippocampal slices using standard imaging techniques. We found significant and variable autofluorescence at the wavelengths used for calcium determination which interfered with data interpretation in fura-treated slices. The intensity of this autofluorescence is an additive effect and is not large enough to be observed when imaging monolayers. In this paper we present a method for conducting experiments and analyzing data that decreases interference from autofluorescence. Experiments were carried out on both slices bulk loaded with fura-2AM and slices loaded with control buffer. A point to point subtraction of the control slice values gave representative calcium fluorescence values. Hippocampal slices were challenged with sodium cyanide or kainic acid. The metabolic response, seen in the fura-free slices, and the calcium response varied within and between these two treatments. Regional differences in the hippocampal sub fields were also demonstrated in response to the two treatments. These corresponded to known regional vulnerabilities to cyanide and kainate. We conclude that autofluorescence in slices need be considered when determining calcium concentrations using fura-2AM.