Fig. 5: Whole-brain cell organization patterns revealed by unsupervised clustering.

a Schematic of grid analysis. The CCFv3 3D space was divided into 10 μm cubes; distance parameters for 19 neuron types within each cube served as features (Methods). b UMAP visualization of whole-brain cubic data, downsampled to 20 μm for computation. Each dot represents a cube, colored by its brain region. c The first-stage clustering result of the whole-brain cubic data. d Visualization of clustering results in coronal, horizontal, and sagittal planes. Distances from bregma for coronal planes, from the midline for sagittal planes, and from the dorsal top for horizontal planes of the mouse brain are indicated below, with all dimension units in millimeters. e Enlarged views comparing of clustering results with anatomical boundaries. f Volume proportion of 18 clusters in 12 big brain regions. g 3D views of C6 in the first-stage and coronal visualization of sC13 and sC17 in the secondary-stage. Distances from bregma are shown below (mm). h Heatmaps indicating heterogeneous distributions of Camk2a⁺, GAD2⁺ and VGAT⁺ neurons in CPdl and CPvl. Color-coded distance parameters represent neuron density. i Fiber photometry recording of glutamatergic or GABAergic neurons in CPdl or CPvl. Left, experimental strategy for virus injection with fiber implantation for calcium recording (top) and schematic of open-field test (bottom). Right, average calcium responses of CPdl (blue) and CPvl (red) aligned to locomotion onset (pink line) and end (azure line). Quantification of peak time or area under the curve (AUC) highlights the functional differences between CPdl and CPvl (two-tailed unpaired t-test; mean ± SEM). In Camk2a group, trials = 11, mice n = 7 for CPdl and trials = 5, n = 4 for CPvl; in GAD2/VGAT group, trials = 8, n = 4 for CPdl and trials = 6, n = 4 for CPvl. j Optogenetic stimulation in CPdl and CPvl. Left, behavioral test procedure. Middle, cumulative positions of mice during control and stimulation periods. Right, statistical plots of mean velocity during control (gray) and stimulation (blue) periods (two-tailed paired t-test). In Camk2a group, trials = 6, n = 4 for CPdl, trials = 9, n = 5 for CPvl; in GAD2/VGAT group, trials = 5, n = 5 for CPdl and trials = 6, n = 6 for CPvl. Source data are provided as a Source Data file.