Extended Data Fig. 8: Patch-seq experiments on individual DGML astrocytes: astrocyte clusters prediction from scRNAseq and from combined glutamate imaging and transcriptomic information.
From: Specialized astrocytes mediate glutamatergic gliotransmission in the CNS
a,b, Patch-seq experiment on DGML astrocytes: workflow of the experimental procedure. a, Left, patch-seq in red-fluorescent astrocytes expressing tdTomato from GFAPcreERT2tdTomlsl/lsl mice. Right, patch-seq preceded by SF-iGluSnFR imaging in astrocytes virally injected to express GqDREADD-mCherry and SF-iGluSnFR. Stimulations with CNO and L-Glut are like in Fig. 2a–e. The whole-brain image is from the Allen Mouse Brain Connectivity Atlas (https://mouse.brain-map.org/). b, schematic representation of the patch-seq procedure. c, Representative tdTomato-positive astrocyte before and after cell body collection by gentle aspiration (n = 65 cells), here imaged with two-photon microscope (n = 2 cells; see also Supplementary Video 2). d, Electrophysiological properties of individual patch-seq astrocytes recorded before collection: all cells showed linear current/voltage (I/V) curve, low input resistance and very negative membrane potential typical of astrocytes. e, UMAP representation of 85 patch-seq astrocytes predicted according to astrocyte reference annotation (cluster 0 to cluster 8 from integrated astrocytic database) and pie-chart distribution of the patch-seq astrocytes among each predicted cluster. Number of cells predicted per cluster were: cluster 0: 7; cluster 1: 1; cluster 2: 39; cluster 3: 0; cluster 4: 2; cluster 5: 8; cluster 6: 0; cluster 7: 28; cluster 8: 0. f, Top, Dot plot of selected marker genes related to astrocyte identity, vesicular trafficking, and glutamate regulated exocytosis for predicted cluster 7; Bottom, expression level for Slc1a2, Slc17a7 and Slc17a6 in the predicted astrocyte clusters. Note Slc17a7 and Slc17a6 enrichment in cluster 7. Noteworthy, cells assigned to cluster 7 had electrophysiological properties within the average of the whole patch-seq population (resting membrane potential: −79.4 ± 0.87 mV; input resistance: 9.9 ± 0.32 MΩ; linear I/V curve). g, Binarized functional maps of the SF-iGluSNFr signal response to CNO and L-Glut applications for the four astrocytes functionally identified as “responder” (brown), and for four representative astrocytes identified as “non responder” (sand), associated with the cluster prediction for each individual cell. h, Top, UMAP representation of the predicted cluster 7 for “responder” and “non responder” astrocytes, according to the astrocyte reference annotation from the integrated astrocytic database in Fig. 1b. Bottom, corresponding histogram quantification showing statistical significance (two tails Fisher exact test, P = 0.0320) for correct prediction of cluster 7 for “responder” astrocytes and of other clusters for “non responder” astrocytes. Overall, 3 out of 4 responders were correctly attributed to cluster 7, and one to cluster 2. Of the 16 non-responders, 14 were correctly attributed to non-glutamatergic clusters (9 to cluster 2; 3 to cluster 4 and 2 to cluster 5) and two to cluster 7.
