Fig. 1: Development of CaMPARI_GFAP as a functional tool to study astrocytic networks.

A Top, CaMPARI1 was expressed within an adeno-associated virus (AAV) vector system, under the glial fibrillary acidic protein (GFAP) astroglial promoter. Bottom, CaMPARI_GFAP was injected in the nucleus accumbens (NAc) for acute slice imaging. B Left, confocal images of CaMPARI_GFAP expression in NAc astrocytes. Fluorescence signals show green-to-red photoconversion after illumination with 405 nm light. Magenta and cyan show inmunolabeling with astrocytic (S100) and neuronal (NeuN) markers (right; scale bar = 100 µm) and their inset (bottom; scale bar = 30 µm). Right, quantification showing S100 and NeuN cell populations with percentages of CaMPARI_GFAP green- and red-positive cells (8 fields, 2 mice). Two-way ANOVA, ***P < 0.001. C Top, scheme of CaMPARI_GFAP green fluorescence as a negative calcium indicator, when CaMPARI_GFAP binds Ca²⁺ its fluorescence decreases. Bottom, time-lapse of astrocytic activity monitoring CaMPARI_GFAP green fluorescence changes in acute slices responding to ATP (20 mM) stimulus in control condition and after perfusion of thapsigargin (1 µM). Scale bar, 50 µm. D Astrocytic Ca²⁺ traces evoked by ATP (20 mM, yellow arrow) in the control condition, in the presence of thapsigargin (1 µM), and after intracellular BAPTA infusion. E Representative heatmaps of ROIs activity, color-coded according to fluorescence change versus time, before and after local ATP application in control (left) and presence of thapsigargin (right; above line) or BAPTA infusion (right; below line). F Quantification of ROIs responding to ATP in control (green; 9 slices, 2 mice), thapsigargin (orange; 6 slices, 2 mice), and BAPTA (purple; 2 slices, 1 mouse) conditions. One-way ANOVA, Holm–Sidak for multiple comparisons, ***P < 0.001. G Top, scheme of CaMPARI_GFAP photoconversion color code: green (low Ca²⁺ activity) to red (high Ca²⁺ activity). Bottom, representative CaMPARI_GFAP green, red, and merged fluorescence of astrocytes after ATP local stimulation (20 mM) and 10 s 405 nm light pulse in control condition and with thapsigargin (1 µM). H CaMPARI_GFAP green and red fluorescence quantification (ΔF/F₀) vs distance from ATP stimulus (0 mm refers to ATP-filled pipette). Error bars express SEM. Source data are provided as a Source Data file.