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Fig. 2. Quantitation of the distribution of cholinergic neurons in whole mouse brain. (A) Horizontal view of genetically labeled cholinergic neurons in the whole brain. Maximum-intensity projections were reconstructed at a resolution of 2 × 2 × 2 μm. Green points indicate GFP-positive cholinergic neurons. Scale bar represents distance from Bregma. A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral. (B) Merged image of coronal sections (GFP and PI signals) at the position indicated in A shows cholinergic neurons in the motor nucleus of trigeminal (V). (Scale bar: 500 μm.) (C) Overview of the 3D distribution of cholinergic neurons in V (Left). The neuron soma is distinguished using Imaris software and reconstructed in 3D space (Right). (Scale bar: 100 μm.) (D) Visualization of the anatomical localization and neuronal distribution of 3D-reconstructed subdivisions in midbrain and hindbrain. (E) Numbers of cholinergic neurons in brain regions of a single hemisphere [from five brains, one-way ANOVA, P < 0.0001, F (12, 104) = 59.99]. (F) Density of cholinergic neurons in midbrain and hindbrain of the single hemisphere shown in C [from five brains, one-way ANOVA, P < 0.0001, F (11, 88) = 12.79].

Fig. 3. Mesoscopic projectome of cholinergic neurons in the basal forebrain. (A) The 3D view of cholinergic neurons in the MS/VDB labeled with AAV-CAG- FLEX-GFP virus. The resolution is 2 × 2 × 2 μm. (B) Diagram of the cholinergic projection from MS/VDB showing different routes to the olfactory bulb, frontal cortex, somatosensory cortex, hippocampal formation, and subcortical regions. Maximum-intensity projection of the coronal section showing the cholinergic terminals in mPFC (C), SSp (D), HPF (E), VISp (F), and ENTm (G). The length of the z stack is 100 μm. (H) A total of 50 cholinergic neurons were reconstructed from the whole-brain database. The soma of these reconstructed neurons focused in MS/VDB are shown in 3D. (I) Distribution of the 50 reconstructed neurons with their target areas. The numbers of neurons are labeled. (J) Schematic diagram illustrating the major projection patterns of the cholinergic neurons in VDB. The numbers of neurons are labeled. (Scale bar: 100 μm.)

Fig. 4. Illustration of cortical cholinergic neurons at single-cell resolution. (A) Cholinergic neurons distributed in the cortex, with the green points indicating the neurons. The flattened view of the mouse cortex in the reference atlas (18) showed the anatomical locations of the mPFC, MOp, MOs, SSp, and VISp. (B) Inverted image of the PI-staining figure showed the layer of the motor cortex. (C) The cholinergic neurons distributed in different layers of the motor cortex. (D) Percentage of cholinergic neurons in layer 2/3 and layer 4–6 of the mPFC, MOp, MOs, VISp, and SSp from five mice. (E) Reconstructed cholinergic neurons in the MOp exhibited bipolar cell (BPC), bitufted cell (BTC), and multipolar cell (MPC) patterns. (F) Statistical results showing various percentages of three morphological neurons distributed in the motor cortex, sensory cortex, and visual cortex (n = 63, 20, and 30, respectively). Reconstruction of cholinergic neurons in the SSp cortex (G) and VISp cortex (H). (I) The dendritic length of cholinergic neurons in layer 2/3 of the SSp cortex showed differences compared with those in the MOp and VISp cortices (unpaired t test, P = 0.0031, t = 3.066).

 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