Extended Data Fig. 5: Generation of binarized functional maps of stimulus-evoked SF-iGluSnFR responses and additional experiments related to chemogenetic activation of astrocytes in situ.

a–h, Description of the analytical pipeline used to quantify SF-iGluSnFR responses. a, 6 epochs corresponding to short periods before or after drug applications (240 ms after CNO in this example) were used as input for the analytical pipeline. For each epoch, we generated an image representing the pixel-by-pixel colour-coded z-score mean projection map of the SF-iGluSnFR signal in the FOV for the period. b, For each of the epochs, we segmented the FOV by a 32 x 32 grid, in which each of the 1024 spaces represented a 1.13 µm x 1.13 µm ROI. c, As an example, we show at higher magnification the z-scored SF-iGluSnFR signal for epoch 1 in the region of 36 ROIs framed in b. d, To continue the example, we then focus on two nearby individual ROIs (* and **) within this framed region, and perform peak detection across the 6 rounds of CNO application. e, Left, Traces show 6/6 suprathreshold (>2 z-scores) responses to CNO in ROI (*) and only 2/6 in ROI (**). Peak detection is similarly performed in all 1024 ROIs of the 32x32 grid, counting the number of responses to CNO application (maximum of 6) within each ROI to generate a colour-coded map of the entire 37.3 x 37.3 µm FOV, going from yellow ROIs (6/6 suprathreshold responses like in ROI *) to dark blue ROIs (0/6 responses). Right, example of the colour-coded map in the magnified region of 36 ROIs. f, The low-magnification view of the colour-coded map for the entire 37.3 x 37.3 µm FOV, with magnified region in the white square, allows to visually appreciate the ROIs most consistently responding to CNO. g, The same analytical steps used for segmentation and peak detection of the SF-iGluSnFR responses to CNO were applied to the responses evoked by 6 applications of L-Glut in the same FOV. Left: while most ROIs reliably responded to L-Glut application (>4 suprathreshold peaks; not shown), a few of them did not (here depicted as magenta ROIs) and were subtracted from the CNO map to generate a new grid map (Right) containing only CNO responsive ROIs also reliably responsive to L-Glut application. This step helped eliminating false positive, ensuring that the CNO-evoked SF-iGluSnFR response came from a ___location capable of reliably detecting L-glutamate. h, Left, binarized map of the grid map from panel g Right. ROIs with ≥4 CNO-evoked SF-iGluSnFR responses were assigned a value of 1 (yellow) and those with ≤3 responses were assigned a value of 0 (purple). Right, we grouped clusters of suprathreshold recurrently active ROIs (yellow) based on 8-neighbour connectivity (all edges and corners) and excluded active clusters containing <4 ROIs by spatial filtering (see Methods). The final binarized functional map, containing only active clusters (“hotspots”) with ≥4 neighbours, was used to calculate hotspots number and areas. i-j, CNO-dependent Gq-DREADD stimulation evokes Ca²⁺ elevations in all the tested astrocytes. i, Left, timeline of the experiments: TAM-inducible GFAP^creERT2GCaMP6f^fl/fl mice were unilaterally injected with AAV5-hGFAP::hM3D(Gq)-mCherry virus. After 3 days mice received TAM administration for 3 days and after 4 weeks two-photon Ca²⁺ imaging was performed. Right, representative fluorescence image of an astrocyte FOV (red: hM3D(Gq); green: GCaMP6f). (n = 2 mice). j, Traces of cytosolic GCaMP6f Ca²⁺ responses in the ROI (whole astrocyte) for each tested astrocyte (n = 10 cells) in response to a single puff of CNO (100 µM) expressed in z-scores of the raw GCaMP6f signal. Note large Ca2+ elevation in all CNO-stimulated astrocytes. Each trace is accompanied by ROI display as perceptually uniform ‘magma’ colormap. Scale bar: 5 µm. k, Stimulation with vehicle does not reproduce the glutamate-releasing effect of CNO in Gq-DREADD-expressing astrocytes: top, wild-type mice (n = 2) were unilaterally injected in hippocampus with AAV5-hGFAP::SF.iGluSNFR(A184S) and AAV5-hGFAP::hM3D(Gq)-mCherry viruses. Bottom, Binarized functional maps of vehicle- and L-Glut-evoked SF-iGluSnFR responses of 5 individual astrocyte FOVs. In none of them, vehicle induced a significant response, while in all of them L-glut elicited the usual large response. Scale bar: 5 µm. l, CNO does not evoke glutamate release in astrocytes expressing an mCherry scrambled virus instead of Gq-DREADD. Top, wild-type mice (n = 2) were unilaterally injected in hippocampus with AAV5-hGFAP::SF.iGluSNFR(A184S) and AAV5-hGFAP::mCherry viruses. Bottom, binarized functional maps of CNO- and L-Glut-evoked SF-iGluSnFR responses of 5 individual astrocyte FOVs. CNO never evoked a significant response, whereas L-Glut always did. Scale bar: 5 µm.