Images of Ca 2+ flux in astrocytes: evidence for spatially distinct sites of Ca 2+ release and uptake

Abstract

In this study, we have developed a mathematical method to derive the Ca 2+ fluxes underlying agonist-evoked Ca 2+ waves in cultured rat cortical astrocytes. Astrocytes were stimulated with norepinephrine (100 nM) to evoke Ca 2+ waves, which were recorded by measuring FIuo-3 fluorescence changes with high spatial and temporal resolution. Normalized fluorescence (ΔF/F) was analyzed in discrete cellular spaces in a series of successive slices along the length of the cell. From these data, Ca 2+ flux was then calculated using a one dimensional reaction-diffusion equation which utilizes the temporal and spatial derivatives of the fluorescence data and the diffusion coefficient of Ca 2+ in the cytosol. This method identified distinct sites of positive flux (Ca 2+ release into the cytosol) and of negative flux (Ca 2+ removal from cytosol) and showed that in astrocytes, sites of Ca 2+ release from stores regularly alternate with sites of Ca 2+ removal from the cytosol. Cross correlation analysis of the two distribution patterns gave positive correlation at 2 μm out of phase and a negative correlation in phase. Thapsigargin-induced Ca 2+ waves were analyzed to determine if the negative flux was due to Ca 2+ uptake via thapsigargin-sensitive Ca 2+ pumps. Negative flux sites were still found under these conditions, suggesting that multiple mechanisms of Ca 2+ removal from the cytosol may contribute to negative flux sites. This method of calculation of flux may serve as a means to describe the distribution of functional ion channels and pumps participating in cellular Ca 2+ signalling.