A Novel Optical Intracellular Imaging Approach for Potassium Dynamics in Astrocytes

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 13361 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 310 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 mu M, corresponding to the affinity of astrocyte glutamate transporters. The amplitude of the [K+](i) drop was found to be 2.3 +/- 0.1 mu M for 200 mM 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.