Fig. 5.

ROI-based laminar analysis of Ca²⁺ activity during stimulation localizes origin of fast signals to active astrocytic cell bodies, uncontaminated by neuropil signals. (A) OGB- and SR101-loaded 2-PM images were taken during stimulation in separate experiments in which the focal plane was changed at 4-µm intervals. The double stain allowed us to record localized activity in the same ROI as the focus changed from the neuropil (n.p.) to astrocyte border, to astrocytic soma (a.s.), to astrocyte border, and back to the neuropil (n = 16 ROIs from three animals). Leftmost shows the raw fluorescent Ca²⁺ signal at neuropil and astrocyte locations in arbitrary units (a.u.) whereas Center shows the images. In Right are the mean values from different levels from all 16 ROIs included in this study. The baseline fluorescence (●) was higher in astrocytic soma than in the neuropil, and, during stimulation, the Ca²⁺ response (Δ) was larger in the astrocyte soma than in the neuropil. (B) Fast Ca²⁺ signals from astrocytes along the z axis. The stack is recorded at different depths of z. The activity is shown in pixels defined as astrocytic by a > 2 SD SR101. The Ca²⁺ response is shown in percentage of baseline (ΔF/F₀), summed from 10 to 120 ms after stimulation. To the Right is the same image in OGB/SR101. The arrow points to interpolated fast Ca²⁺ signal in ΔF/F₀ from an ROI inside and above the astrocyte. (C) The baseline fluorescent level of activity was higher in astrocytes than in neuropil. The baseline Ca²⁺ activity was estimated as the mean of the SD in the fluorescence signal (F_SD). F_SD was much higher in astrocyte somas (n = 151) than in neuropil (n = 115) in 32 animals. Error bars in SEM. (D) The influence of neuropil fluorescence on astrocyte Ca²⁺ signals during evoked activity is negligible. For each focus level of experiment, areas without a clear view of the astrocyte soma were considered to be neuropil. The contribution from each such area to the center of the astrocyte was estimated by Gaussian distribution (green arcs), based on size of the point spread function of the system. The arrow indicates the distance between summed fluorescence contributed from neuropil (black stippled line) due to point spread of fluorescence along the z axis and the level of fluorescence inside the astrocyte soma (red). (E) The width of the point spread function was found to be σ = 1.46 µm as indicated by imaging of 1-µm beads under the same conditions as during the experiments (n = 20 beads). (F) The averaged difference in raw fluorescence levels (n = 28 ROIs), error bars in SD; the black squares show the max and min values and the white square the mean difference in fluorescence.