[Ca²⁺]_i recordings from neural cells in acutely isolated cerebellar slices employing differential loading of the membrane-permeant form of the calcium indicator fura-2

Abstract

This paper contains a description of the procedure for monitoring the cytoplasmic free calcium concentration ([Ca²⁺]_i from intact neurones and glial cells in acutely isolated cerebellar slices. The loading of cells with the calcium indicator fura-2 was achieved by slice incubation in Tyrode solution containing 5 µM fura-2 acetoxymethylester (fura-2/AM) and 0.02% (w/v) pluronic-F127 under a controlled (temperature, 35° C; humidity, 98%; and gas, 5% O₂ + 95% CO₂) environment. In such conditions, different cellular elements of the cerebellum (namely granule neurones, Bergmann glial cells and Purkinje neurones) acquired fura-2 at different rates. Ten minutes of slice incubation gave adequate staining of granule neurones only, 20 min of incubation allowed calcium-dependent changes of fluorescence signal measurements in Bergmann glial cells, whereas loading of Purkinje neurones required 40 min of slice exposure to fura-2/AM. In order to assure dye deesterification, slices were kept in continuously gassed bicarbonate-buffered solution for not less than 1 h thereafter. The fluorescence signals (excited at 360 and 380 nm) were collected from either a 25-µm or 40-µm area limited by fixed diaphragm inserted in front of the photomultiplier tube; an individual cell was positioned in approximately the centre of the fluorescence measurement area. These signals were comprised of [Ca²⁺]i-related changes in fura-2 fluorescence recorded from a cell of interest and background fluorescence. The latter resulted from the summation of slice autofluorescence, signals from the fura-2 acquired by neighbouring tissue and signals from fura-2 compartmentalized by intracellular organelles. After the end of fluorescence recordings, the cell was internally dialysed with dye-free intracellular solution in order to determine the actual levels of background fluorescence. In parallel, electrophysiological properties were determined, allowing identification of cell type and viability. The background fluorescence values were then used to correct fluorescence recordings by subtraction from original traces. Corrected records were used for [Ca²⁺]_i calculation.