[CA2-2(](I) RECORDINGS FROM NEURAL CELLS IN ACUTELY ISOLATED CEREBELLAR SLICES EMPLOYING DIFFERENTIAL LOADING OF THE MEMBRANE PERMEANT FORMOF THE CALCIUM INDICATOR FURA)

This paper contains a description of the procedure for monitoring the cytoplasmic free calcium concentration ([Ca2+](i)) from intact neurone s and glial cells in acutely isolated cerebellar slices. The loading o f cells with the calcium indicator fura-2. was achieved by slice incub ation in Tyrode solution containing 5 mu M fura-2 acetoxymethylester ( fura-2/AM) and 0.02% (w/v) pluronic-F127 under a controlled (temperatu re, 35 degrees C; humidity, 98%; and gas, 5% O-2 + 95% CO2) environmen t. In such conditions, different cellular elements of the cerebellum ( namely granule neurones, Bergmann glial cells and Purkinje neurones) a cquired fura-2 at different rates. Ten minutes of slice incubation gav e adequate staining of granule neurones only, 20 min of incubation all owed 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 deesterifi cation, slices were kept in continuously gassed bicarbonate-buffered s olution for not less than 1 h thereafter. The fluorescence signals (ex cited at 360 and 380 nm) were collected from either a 25-mu m or 40-mu m area limited by fixed diaphragm inserted in front of the photomulti plier tube; an individual cell was positioned in approximately the cen tre of the fluorescence measurement area. These signals were comprised of [Ca2+](i)-related changes in fura-2 fluorescence recorded from a c ell of interest and background fluorescence. The latter resulted from the summation of slice autofluorescence, signals from the fura-2 acqui red by neighbouring tissue and signals from fura-2 compartmentalized b y 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 i dentification of cell type and viability. The background fluorescence values were then used to correct fluorescence recordings by subtractio n from original traces. Corrected records were used for [Ca2+](i) calc ulation.