Extended Data Fig. 4: Brain-wide multicolor labeling of astrocyte morphology.
a, To enhance the labeling of fine astrocytic processes, smFPs were targeted to the plasma membrane using the Lck domain and packaged into AAV.PhP.eb serotype, which was delivered via the retroorbital route into P1 mice. Brains were harvested at P21. b, Multicolor labeling of astrocytes in the brain. WT mice were injected with AAV expressing Lck-smV5, Lck-smMyc, and Lck-GFP driven by the astrocyte-specific GfaABC1D promoter. Stochastic multicolor labeling of astrocytes is observed throughout the brain, including the hippocampus, thalamus, and visual cortex. The right panel shows a 3D reconstruction of neighboring astrocytes in layer 6 of V1. Scale bars 1 mm (hippocampus), 40 µm (thalamus), 10 µm (V1). c, Astrocyte volumes were computed through surface reconstruction. The voxels inside each surface that overlap with each other were calculated and highlighted in yellow, generating a new surface from the overlapping regions. d,e, Astrocyte tiling index was calculated by dividing the overlapping volume between adjacent astrocytes by the volumes of a single astrocyte. Both WT and γC3 KO astrocytes exhibited minimal overlap with adjacent astrocytes. Two-sided unpaired t-tests with Welch’s correction was used to compare WT and γC3KO groups. WT, n = 44 astrocytes from three mice; γC3KO, n = 21 astrocytes from three mice. Error bars, s.e.m. Scale bars 10 µm. ****p < 0.0001. Nested analysis was performed for all statistical comparisons to confirm the results, and details are provided in Supplementary Table 4.
