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.
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References[977-983]
Berridge MJ (1993) Inositol trisphosphate and calcium signalling. Nature 361:315–325
Clapham DE (1995) Calcium signaling. Cell 80:259–268
Connor JA, Miller LD, Petrozzino J, Muller W (1994) Calcium signaling in dendritic spines of hippocampal neurons. J Neurobiol 25:234–242
Edwards FA, Konnerth A, Sakmann B, Takahashi T (1989) A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system. Pflügers Arch 414:600–612
Finkbeiner SM (1993) Glial calcium. Glia 9:83–104
Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch clamp techniques for high-resolution current recording from cell and cell-free membrane patches. Pflügers Arch 391:85–100
Kano M, Rexhausen U, Dreessen J, Konnerth A (1992) Synaptic excitation produces a long-lasting rebound potentiation of inhibitory synaptic signals in cerebellar Purkinje cells. Nature 356:601–604
Kano M, Garaschuk O, Verkhratsky A, Konnerth A (1995) Ryanodine receptor-mediated intracellular calcium release in rat cerebellar Purkinje neurones. J Physiol (Lond) 487:1–15
Kirischuk S, Scherer J, Kettenmann H, Verkhratsky A (1995) Activation of P2 purinoreceptors triggers Ca2+ release from InsP3-sensitive internal stores in mammalian oligodendrocytes. J Physiol, (Lond) 483:41–57
Kirischuk S, Möller T, Voitenko N, Kettenmann H, Verkhratsky A (1995) ATP-induced cytoplasmic calcium mobilization in Bergmann glial cells. J Neurosci 15:7861–7871
Kostyuk P, Verkhratsky A (1994) Calcium stores in neurones and glia. Neuroscience 63:381–404
Kostyuk PG, Verkhratsky AN (1995) Calcium signalling in the nervous system. Wiley, Chichester, p 220
Llano I, DiPolo R, Marty A (1994) Calcium-induced calcium release in cerebellar Purkinje neurones. Neuron 12:663–673
Müller T, Möller T, Berger T, Schnitzer J, Kettenmann H (1992) Calcium entry through kainate receptors and resulting potassium-channel blockade in Bergmann glial cells. Science 256:1563–1566
Petersen OH, Petersen CCH, Kasai H (1994) Calcium and hormone action. Annu Rev Physiol 56:297–319
Pozzan T, Rizzuto R, Volpe P, Meldolesi J (1994) Molecular and cellular physiology of intracellular calcium stores. Physiol Rev 74:595–636
Zirpel L, Lachica EA, Rubel EW (1995) Activation of a metabotropic glutamate receptor increases intracellular calcium concentrations in neurones of the avian cochlear nucleus. J Neurosci 15:214–222
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Kirischuk, S., Verkhratsky, A. [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. Pflügers Arch. 431, 977–983 (1996). https://doi.org/10.1007/s004240050094
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DOI: https://doi.org/10.1007/s004240050094