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FIGURE 1 | Identification and reconstruction of pial surface vessels. (A,C) Reconstructed 3D mouse brain surface with surface rendering on sagittal and horizontal views, respectively (data resolution is 5 × 5 × 5 µm 3 ). (B,D) Tracing results of the pial surface arteries and veins. Arteries, including Mcer, Acer, and Pcer, are presented in red, whereas veins, including Sss, Rrhv, Crhv, and Trs, are in blue. Mcer, Middle cerebral artery; Acer, Anterior cerebral artery; Pcer, Posterior cerebral artery; Sss, Superior sagittal sinus; Rrhv, Rostral rhinal vein; Crhv, Caudal rhinal vein; Trs, Transverse sinuses.

FIGURE 2 | Segmentation of the brain regions. (A–C) The representative coronal sections in the same mouse brain. The blue lines represent the contour of the brain regions segmented manually. (D,E) Horizontal sections with a distance of 300µm to present contour indicated by cytoarchitecture of the CP and coronal sections with a distance of 500µm to present contour indicated by cytoarchitecture of the HIP are presented. The images in the blue and green boxes are local, enlarged views of the CP and HIP, respectively. The 3D structures in yellow and violet are the constructed contours of the CP and HIP, respectively. A, anterior; P, posterior; D, dorsal; V, ventral. A list of abbreviations and the full names of the brain regions is presented in Supplementary Table 1.

FIGURE 3 | The structure, distribution and labeling of the whole brain vascular system of different arteries and veins in 3D. Red indicates arteries, and blue indicates veins. The vascular names are all marked in black. The names of brain regions are marked in gray. (A) The distributions of Acer and its major branches are mainly located between the CP. (B) The distributions of Mcer and its major branches, which mainly extend into the CP and the outside of the pial surface. (C) The distributions of Pcer and its major branches, which mainly penetrate the HIP and TH below the HIP. (D) The distributions of Pcer and Vert and their major branches. The annotated branches of Vert mainly extend to both the surface and interior of the CB, P, and MY. (E) The distributions of Ach and Achv and their major branches, which are located in the rostral area of the HIP. (F) The distributions of Lhiv and its branches, which penetrate into the upper portion of the HIP and TH below the HIP. (G) The distributions of the thalamostriate vein (Thsv) and Gcv inside the brain, which extend to the caudal part of the CP and the rostral part of the TH along the middle area of the HIP. (H) The distributions of Mcolv and Lcolv and their branches. Mcolv is mainly located in the rostral aspect of the CB and the lateral aspect of the MB below the HIP, whereas Lcolv penetrates the lower portion of the HIP. (I) The distributions of the pial surface veins and the connected cortical penetrating veins. The penetrating veins presented here extend to the subcortical area. The list of abbreviations and full names of the brain regions is presented in Supplementary Table 1, and the tree of abbreviations and full names of the vessels is presented in Figure 4. D, Dorsal; V, ventral; L, left; R, right; A, anterior; P, posterior.

FIGURE 4 | Complete tree-like structures and annotations of the arterial and venous vascular system in the whole mouse brain. To ensure readability, the tree-like structure is arranged in the sagittal direction, in which left represents rostral, right represents caudal, up represents dorsal, and down represents ventral. The full names of vessels are presented in each box, and abbreviations are presented in brackets. (A) Tree-like structure of the arterial system. (B) Tree-like structure of the venous system. Vessels in black indicate the reported branches of the mouse brain vascular system study. Vessels in blue indicate the branches reported in the rat brain vascular system study but not reported in the mouse brain. Vessels in red indicate the branches not reported in the rat or mouse brain. Vessels in gray indicate the missing branches in our data.

FIGURE 5 | Vascular conserved distribution results across five mouse brains. (A) Sagittal slice used for localization; the gray box shows the range in the rostral to the caudal direction of the 3D datasets in (B–F). (B–F) Three-dimensional reconstructed results for Lhiv, Lcolv, and Pcer in five mouse brains. To identify the position and direction of the vessels in 3D, the contour of the HIP is shown as well. In (B), the main branches of Pcer and Scba are marked in red, while the branches of Lcolv and Lhiv are marked in blue, and brain regions are marked in gray. A list of abbreviations and full names of the brain regions is presented in Supplementary Table 1, while the tree of abbreviations and the full names of vessels is presented in Figure 4.

FIGURE 6 | Distribution map of the arterial and venous vascular system in the whole C57 mouse brain. Abbreviations and trending curves of the arteries and veins are presented in red and blue, respectively. Both the arteries and veins are presented in 3 levels using 3 different sizes from large to small of the font, sphere and link curves, which respectively represent the size of the vessel diameter, original/terminal points, and extending directions. The dotted curves and full curves respectively represent the vascular branches in the pial surface and inside the brain. A tree of abbreviations and full names of the vessels is presented in Figure 4.

FIGURE 7 | Typical distribution patterns of arteries and veins. Arteries are presented in red, and veins are in blue. The arrows in red and blue indicate the characteristics of the extending direction. D, dorsal; V, ventral; L, left; R, right; A, anterior; P, posterior. A tree of abbreviations and the full names of the vessels is presented in Figure 4. (A) The distribution patterns of vessels on the brain surface. (B–F) The distribution patterns of vessels inside the brain.

FIGURE 8 | Distributing patterns between the arterial and venous vascular system and brain regions in the whole mouse brain. (A,B) The statistical results for the volume distributions of Mcer and Lhiv within different brain regions. The segmented brain regions are presented on the horizontal axis, and the distribution proportions (after log10 transformation) of vessels in the corresponding brain region are presented on the vertical axis. (C,D) Distributing patterns of arteries and veins with the main brain regions. The lines and arrows between the vessels and brain regions indicate the branches that extend to the brain region, while the line thickness represents the values of distribution proportions. A list of abbreviations and the full names of the brain regions is presented in Supplementary Table 1, while a tree of abbreviations and the full names of vessels is presented in Figure 4.

FIGURE 9 | Quantitative statistical results for the capillary density in the cortical and subcortical areas of the mouse brain. (A) Maximum projection of 200µm to show the vessels in HIP (image size of 400 × 400µm). (B) The reconstructed fine vascular network in HIP (stack size of 400 × 400 × 400µm). (C) Separated capillaries in HIP with diameter size of 8µm (stack size of 400 × 400 × 400µm). (D,E) Quantitative statistical results for the fractional vascular volume (Fv) and normalized vascular length (Nl) in the S1BF, SC, HIP, TH, and HY. SD, small-sized vessel (D < 8µm) density; MD, medium-sized vessel (8 < D < 20µm); LD, large-sized vessel (D > 20µm) density. (F–H) The detailed diversity distributions (P-values) among 5 brain regions of fv in SD, MD, and LD. (I–K) The detailed diversity distributions among 5 brain regions of nl in SD, MD, and LD; S1BF, primary somatosensory cortex, barrel field; SC, superior colliculus; HIP, hippocampus; TH, thalamus; HY, hypothalamus.

 Movie S1. GFP-labeled cholinergic neurons distributed throughout the entire cerebral cortex

Movie S2. The arborization of 50 well-separated cholinergic neurons in the MS/VDB throughout the brain

Movie S3. Individual cholinergic neurons can project long distances to distinct brain regions through different projection routes