Fig 2: Activation of the RhoA pathway changes astrocyte morphology in vitro. (A) Increasing RhoA activity leads to retraction of astrocyte processes and a rounder cell shape. Representative images of astrocytes infected with rAAVs expressing variants of the small GTPase RhoA (dominant-negative, RhoA-DN; wildtype, RhoA-WT; constitutively active, RhoA-CA) tagged with mCherry for visualization or the far-red fluorescent protein TurboFP650 (Ctrl) under a mouse GFAP (mGFAP) promoter. White lines depict the region of interest (ROI) used for analysis (scale bars 20 µm). (B) Evaluation of astrocyte morphology shows a reduction of astrocyte area, perimeter, convex area, and the perimeter of the convex area with increasing RhoA activity (p < 0.001, Kruskal–Wallis test; Dunn’s multiple comparisons post hoc test, * p = 0.05, *** p = 0.001; cells from 4 individual cultures; n = 29, 28, 28 and 39 cells for Ctrl, RhoA-DN, RhoA-WT, and RhoA-CA, respectively). Box and Whisker plots: box indicates the 25th and 75th percentiles, the whiskers the minimum and maximum values, the horizontal line in the box the median, and hollow circles on top the individual data points.

Fig 3: Microglia form direct purinergic contact with cells in the NVU that regulate CBF. (a) 3D reconstruction of in vivo two-photon Z-stacks down to 600 µm below the dura mater in the cerebral cortex of CX3CR1tdTomato mice. Note contacting microglia (arrowheads) at meningeal (m), penetrating (p), and first- to third-order capillaries. Scale bar, 50 µm. (b) Microglial processes (arrowheads) dynamically contact different segments of the vascular tree (visualized by i.v. FITC-dextran). Scale bar, 20 µm. (c) Microglial processes are extended beyond the perivascular glial endfeet and form direct contact with smooth muscle cells (arrows) at the level of penetrating arteries. (d) CLSM images show microglia (P2Y12R, cyan) contacting endothelial cells (tomato lectin, magenta) in the cerebral cortex. (e) EM images show microglia (m, P2Y12R-immunogold labeling, cyan) directly contacting endothelial cells (e, magenta) and pericytes (p, purple). (f) Frequency of vessels receiving microglial contact, and microglial process coverage of endothelial cell surface. (g) EM images show microglia (m, P2Y12R-immunogold labeling, cyan) directly contacting pericytes (p, purple). (h) 83.4 ± 1.4% of pericytic cell bodies are contacted by microglial processes. (i) 3D reconstruction of electron tomogram shows clustering of anti-P2Y12R-immunogold on microglial processes (m) directly contacting the endothelium (e) of an arteriole/postcapillary venule. The left two panels are conventional EM images of the same area on the adjacent ultrathin section. The right panels show a tomographic virtual section and 3D reconstruction of the direct contact. (j) Unbiased anatomic analysis reveals enrichment of endothelial mitochondria (TOM20+, green), at sites of microglial contacts (P2Y12R+, cyan). ****, P < 0.0001, Mann–Whitney U test. See analysis details in Fig. S1 d. (k) EM images show microglia (m, P2Y12R-immunogold, cyan) directly contacting endothelial cells (e, magenta) in human neocortex. z1–z3 panels show the contact on three consecutive ultrathin sections; arrows mark the edges of direct membrane contact. (l) CLSM image shows microglia (P2Y12R, cyan) contacting the cell body of an astrocyte (GFAP labeling, green) and astrocytic endfeet (arrowheads). (m) EM images show direct contact between microglial (m, cyan) and astrocytic (a, green) processes. e, endothelial cell, magenta; lum, lumen. (n) CLSM images in human neocortex reveal P2Y12R+ microglial processes (cyan) contacting perivascular astrocytes (GFAP, green) on astrocyte endfeet (white arrowheads) and endothelial cells (tomato-lectin, magenta, white arrows), with astrocytic endfeet directly touching the endothelial monolayer (empty arrowheads). (o) CLSM image and fluorescent intensity plots show microglial process (m) contacting the endothelial layer (e) within the astrocytic layer (a). (p) To reveal 3D connections of individual microglial cells, a CLSM maximum-intensity plot was generated. Microglia (cyan) contact several microvessels (lectin, magenta; AQP4, green) and neurons (Kv2.1, ochre) simultaneously. For appropriate visualization of neuronal cell bodies, Kv2.1 is shown in yellow pseudocolor only with the maximal diameter planes included. Scale bars: (c) 50 µm; (d) 3 µm; (e) 2 µm; (i) 200 nm; (j) 2 µm; (k) left, 1 µm, and z3, 400 nm; (l) 10 µm; (m) 200 nm; (n) 10 µm; (o) 5 µm; (p) 10 µm.

Fig 4: Microglia form direct contacts with cells in the NVU, but microglia depletion does not disrupt cerebal perfusion or metabolism. (a) 3D reconstruction of a high-resolution CLSM Z-stack shows microglia (P2Y12R, cyan) contacting both the cell body of an astrocyte (GFAP labeling, green, arrow) and astrocytic endfeet (arrowheads) ensheathing a capillary. Pericytes are visualized by anti-PDGFRb labeling (blue). (b) Microglia (CX3CR1, magenta) are able to form direct contact by their cell body with astrocytes (GFAP labeling, green) in the cerebral cortex. Scale bar, 10 µm. (c) CLSM image shows P2Y12R-positive microglial process (cyan) contacting perivascular AQP4-positive astrocyte endfeet (green) and also extends to the endothelial layer (lectin, magenta) where astrocytic coverage is not present (arrows). z1–z3 panels show the contact area on three consecutive confocal sections. Scale bar, 3 µm. (d) The process of semiautomated unbiased analysis of fluorescent intensity area for the graph presented in Fig. 1 j is depicted. White dashed lines represent the outer and the inner profiles, based on the outline of the endothelial cell. P2Y12R intensity was measured along the outer profile and TOM20 intensity along the inner profile, starting from the arrow. The intensity values are plotted (right) along the perimeter of the vessel. Contact site (marked by the gray column in the plots) was defined automatically. Scale bar, 2 µm. (e) CLSM image and fluorescent intensity plots show microglial process extending beyond perivascular astrocytic endfeet to interact with the endothelium. The fluorescent intensity profile plot (measured along the 3.5-µm long white arrow) clearly shows the presence of the microglial process under the astrocytic endfeet. (f) CLSM image and fluorescent intensity plots show microglial processes interacting with GFAP- and AQP4-positive astrocytes in the human brain. The fluorescent intensity profile plot (measured along the 15-µm-long white arrow) clearly shows the presence of the microglial process between the endothelium and the endfeet of perivascular astrocytes. (g) Schematic summary of contacts formed between microglia (m; cyan) and different cell types of the NVU. Neurons (n; red), astrocytes (ast.; green), pericytes (p; purple), endothelial cells (e; pale crimson), and vascular smooth muscle cells (s.m.; dark crimson) are shown. (h) Characterisation of CX3CR1tdTomato mice. Parenchymal tdTomato-positive cells coexpress Iba1 and P2Y12R in the cerebral cortex. Cell nuclei stained with DAPI appear in yellow pseudocolor in the merged middle image. Scale bars, 25 µm (left), 10 µm (middle). (i) Feeding C57BL/6J mice with a diet containing PLX5622 results in an almost complete (97%) elimination of resident microglia as evidenced by the numbers of P2Y12R-positive cells in the cerebral cortex. Scale bar, 100 µm. n = 12 control and n = 9 depleted mice per group; ****, P < 0.0001 control versus depleted, unpaired t test with Welch’s correction. (j and k) HMPAO-SPECT and FDG-PET images of control and microglia-depleted mice. Proportion of measured and injected HMPAO activity (Inj. Act.) and standard uptake values (SUVs) of FDG are shown. Atlas-based ROI analysis (j) shows no significant differences between the normalized regional uptake values (k) of the two groups. n = 5 and 5 mice. Data are expressed as mean ± SEM.

Fig 5: Increasing astrocytic RhoA activity does not affect larger, GFAP-positive processes. (A) Illustration of the experimental approach. Astrocytes in mice with sparse hippocampal EGFP expression were transduced using control and RhoA-CA–expressing rAAVs (hGFAP-tdTomato and hGFAP-tdTomato-RhoA-CA) (left panel). Isolated transduced and EGFP-expressing astrocytes were identified, and their boundaries were determined using their EGFP-fluorescence throughout an image stack (middle panel, single plane example, boundary indicated by dotted red line). Right panel: Example of GFAP immunohistochemistry of the section shown in the left and middle panel (single plane) illustrating GFAP staining of several astrocytes. The boundary of the single astrocyte selected for analysis is indicated by the dotted red line. (B) The GFAP immunofluorescence was masked outside of the single astrocyte under study (left panel, maximum intensity projection of the entire image stack). Right panel: reconstruction of GFAP-positive astrocyte processes obtained using MotiQ as described in [40]. (C) Numerical characterisation of GFAP reconstructions of 52 control and 40 RhoA-CA–expressing astrocytes from 4 and 3 virus-injected mice, respectively. Top left panel: number of GFAP segments (Mann–Whitney U-test, p = 0.46). Top right panel: number of branch points in the reconstruction (Mann–Whitney U-test, p = 0.47). Bottom left panel: average lengths of GFAP segments (Student’s t-test, p = 0.43). Bottom right panel: cumulative length of all GFAP segments per cell (Mann–Whitney U-test, p = 0.28). Box and Whisker plots: box indicates the 25th and 75th percentiles, whiskers the 5th and 95th percentiles, the horizontal line in the box the median, the filled circle the mean, and hollow circles the individual data points.