Figure 1.

Rise in basal [Ca ²⁺]_i and attenuation of receptor-evoked Ca ²⁺ signaling during microglial activation. Microglial calcium signaling in response to UTP and C5a application was compared between LPS-activated (100 ng/ml, 24 hr) and untreated cells (CTL). Using fura-2-based imaging, [Ca ²⁺]_i was measured as the ratio of fluorescence at 340 and 380 nm (R 340/380). For eliciting calcium transients, UTP (100 μm), C5a (2 nm), and ATP (100 μm) were added to the bath solution for 30 sec (bars, position indicating application, i.e., switch to the corresponding bath solution). A, B, Representative [Ca ²⁺]_i traces of a control and a LPS-stimulated cell. C, D, Average [Ca ²⁺]_i traces in control and LPS-stimulated cells as summarized from 9 and 10 independent experiments with a total of n = 799 and n = 754, respectively. Data are given as mean ± SEM.E, Overlay of the traces in C and D revealing the reduction in the signal amplitude and the concomitant increase in basal [Ca ²⁺]_i during LPS activation. F, Comparison of the average basal [Ca ²⁺]_i in control and LPS-treated microglia as calculated from the first 10 image frames of the cells in E. Data are mean ± SEM. G, H, Histograms of UTP-evoked [Ca ²⁺]_i signal amplitudes in the control and LPS-stimulated cells (as for C and D). Amplitudes were calculated as the difference between basal (average of the first 10 recorded image frames) and peak R 340/380 values of each individual cell (ΔR). Values of ΔR were classified into 10 ranges (0 ≤ ΔR < 0.05, 0.05 ≤ ΔR < 0.10, etc.). The histogram for the LPS-activated cells clearly shows a shift to smaller values.