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Ethanol-mediated effects on astroglial cells in primary culture. With a focus on acute changes in intracellular Ca2+, cell volume, actin filaments, and gap junction coupling

Doctoral thesis
Authors Louise Adermark
Date of public defense 2003-10-03
ISBN 91-628-5820-3
Publication year 2003
Published at Institute of Clinical Neurosciences
Language en
Keywords astrocyte, BaCl2, cell swelling, fura-2, furosemide, ouabain, scrape loading, volume estimation
Subject categories Physiology

Abstract

The astroglial cells are abundant in the central nervous system (CNS), where they control and regulate the homeostasis of the extracellular milieu. A change in astroglial cell volume, mediated through activation of neuroactive substances or changes in osmolarity, leads to changes in the size and geometry of the extracellular space volume, which could affect the diffusion and concentration of neuroactive substances. The astroglial cells are connected through gap junctions, which are small pores that allow bidirectional communication. The intercellular coupling through gap junctions has been suggested to allow the astroglial network to function as a unit, and to equalize the concentration of intracellular ions.To enable studies of signal transduction systems involved in astroglial cell swelling and volume regulation, we developed a new method for quantifying changes in cell volume at the single-cell level (Paper I). The method, which was based on optical sectioning in combination with image analysis, was used to study changes in astroglial cell volume mediated by ethanol (10 200 mM) in three dimensions (3D) over time (Papers II and IV). The method was proven to be capable of properly reconstructing and segmenting individual cells. Exposure for 5 min to 25 mM or 150 mM ethanol increased astroglial cell volume in 19% and 36% of analyzed cells, while cell shrinkage was detected in 22% and 35%, respectively (Paper IV). The ethanol-mediated volume changes were partially inhibited during incubation in low extracellular sodium ([Na+]o), during blockage of the Na+/K+/2Cl- cotransporter with furosemide, during blockage of the Na+/K+-adenosine triphosphatase (ATPase) with ouabain, or during blockage of the inward-rectifying potassium channels (K+IR) with BaCl2 (Paper IV). Exposure to ethanol-induced morphological changes and ethanol-mediated transformation in filamentous actin led to a more dispersed appearance of the filaments and an increased number of cells with actin in ring formations (Paper II). Ethanol decreased cell-to-cell communication through gap junctions in astroglial cultures from the cerebral cortex, brain stem, or hippocampus, while cultures from the olfactory bulb or hypothalamus remained unaffected (Paper III). The decrease was not seen during incubation in low [Na+]o, or during blockage with furosemide, and ethanol had no additional effect on the decrease in gap junction coupling mediated by ouabain (Paper III). The results presented in Papers II IV suggest that astroglial cells could be important targets during ethanol exposure, and that possible changes in cell volume and gap junction coupling could be of toxicological relevance.

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