title: A novel optical intracellular imaging approach for potassium dynamics in astrocytes.
abstract: Astrocytes fulfill a central role in regulating K+ and glutamate, both released by neurons into the extracellular space during activity. Glial glutamate uptake is a secondary active process that involves the influx of three Na+ ions and one proton and the efflux of one K+ ion. Thus, intracellular K+ concentration ([K+]i) is potentially influenced both by extracellular K+ concentration ([K+]o) fluctuations and glutamate transport in astrocytes. We evaluated the impact of these K+ ion movements on [K+]i in primary mouse astrocytes by microspectrofluorimetry. We established a new noninvasive and reliable approach to monitor and quantify [K+]i using the recently developed K+ sensitive fluorescent indicator Asante Potassium Green-1 (APG-1). An in situ calibration procedure enabled us to estimate the resting [K+]i at 133±1 mM. We first investigated the dependency of [K+]i levels on [K+]o. We found that [K+]i followed [K+]o changes nearly proportionally in the range 3-10 mM, which is consistent with previously reported microelectrode measurements of intracellular K+ concentration changes in astrocytes. We then found that glutamate superfusion caused a reversible drop of [K+]i that depended on the glutamate concentration with an apparent EC50 of 11.1±1.4 µM, corresponding to the affinity of astrocyte glutamate transporters. The amplitude of the [K+]i drop was found to be 2.3±0.1 mM for 200 µM glutamate applications. Overall, this study shows that the fluorescent K+ indicator APG-1 is a powerful new tool for addressing important questions regarding fine [K+]i regulation with excellent spatial resolution.
authors.affiliation: Department of Fundamental Neurosciences, University of Lausanne, Lausanne, Switzerland.
authors.forename: Theresa S
authors.initials: TS
authors.lastname: Rimmele
authors.affiliation: Department of Fundamental Neurosciences, University of Lausanne, Lausanne, Switzerland; Cellular Imaging Facility, University of Lausanne, Lausanne, Switzerland.
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Chatton
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology* / Q000378:metabolism; D002414:Cations, Monovalent / Q000032:analysis / Q000378:metabolism; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D061848:Optical Imaging; D011188:Potassium / Q000032:analysis* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D002414:Cations, Monovalent; D005456:Fluorescent Dyes; D018698:Glutamic Acid; D011188:Potassium