Curran, Rachel Elizabeth Anne; (1998) An investigation of glial cell signalling in the rat cerebellum. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This investigation focuses on the physiological role of glial cell signalling by examining InsPs induced calcium release from the stores of cerebellar astrocytes. 1. InsPa type 3 receptor protein was identified in rat cerebellar type 1 astrocytes in vitro by immunohistochemical techniques. Cultured astrocytes do not appear to express detectable levels of type 1 but expression of InsPa subtype 2 receptor protein was detected at a low density. Protoplasmic astrocytes in the molecular layer expressed type 3 receptor but no expression of type 3 was found in the Bergmann glial cells. Receptor protein was located in and around the nuclear envelope within protoplasmic astrocytes.2. The kinetic properties of calcium release by InsPs in single astrocytes were studied with the whole cell patch clamp technique, combined with microspectrofluorimetry of a low affinity calcium indicator furaptra and flash photolysis of caged InsPs. InsPs concentrations (0.1-25µM) evoked [Ca2+]i rises in type 1 astrocytes in vitro and in situ. The [Ca2+]i responses comprised an initial delay, a fast rise of free [Ca2+]i, a [Ca2+]i peak where net flux is zero and a slow declining phase. InsPa evoked Ca2+ flux was measured as the rate of change of free [Ca2+]i (d[Ca2+i/dt) in units of moles/litre of cytosol/second (µMs-1). 3. The initial delay to the [Ca2+]i rise decreased with increasing InsPs concentrations from 111±15ms at high to 267±66ms at low. The rate of change of free [Ca2+i and peak free [Ca2+]i appeared to be unrelated to concentration of InsP3 released. The termination of the InsP3 induced [Ca2+]i rise was not due to the metabolism of InsP3. The rate of termination of InsP3 evoked [Ca2+]i rise was linearly related to the Ca2+ flux through the InsP3 receptor channel in both in vitro and in situ experiments, in agreement with previous studies. This result implies a common mechanism for the termination of Ca2+ flux for all InsP3 receptor isoforms, possibly by calcium inhibition of the receptor or store depletion. 4. Furaptra injected into coupled astrocytes diffused from the patched cell into adjacent cells, presumably via gap junctions. U.V. illumination evoked a rapid calcium rise in adjacent cells, indicating that caged InsP3 had also diffused from the patched cell loaded with caged InsP3. This result: would suggest that InsP3 itself could act as a diffusible messenger between neighbouring cells during astrocytic [Ca2+]i wave propagation. 5. Whole cell patched type 1 astrocytes did not respond consistently to bath applied agonists such as glutamate and ATP, in contrast to ester loaded astrocytes in vitro. 6. Calcium release in glial cells was examined in physiological conditions, using rat cerebellar slices (P12). Glial cells were selectively loaded with acetoxy methyl ester form of a low affinity calcium indicator furaptra. 7. During neuronal stimulation the fluorescence signal from Bergmann glial cells was recorded using an imaging system. Preliminary results suggest that [Ca2+]i did not change in somal or filamentous regions of furaptra loaded Bergmann glial cells (with a [Ca2+]i range sensitive >0.5µM) as a result of Purkinje neurone climbing fibre stimulation.

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