Extended Data Fig. 8: ACE statistical analysis for unraveling neuronal ensembles controlling walking.

a. ARA labels at the level of whole brain vs. depth 6 based on the ARA ontology (hierarchy). b. Group-wise heatmaps of neuronal density were obtained by subtracting the average of the voxelized and warped segmentation maps in each group. Panels show different coronal, sagittal, and axial views as an example. c. Using voxelized and warped segmentation maps and ARA labels at 10 µm, we obtained neuronal density per brain region at both whole-brain and depth 6 levels. An independent student t-test (two-sided) was then applied between c-Fos+ cell density per ARA label to perform a whole-brain and depth 6 level ROI-wise statistical test (N: 3/group) Data are presented as mean values ± standard deviation. The upper panel shows the results of the whole-brain analysis with a zoomed version on the right, demonstrating the most significant ROIs. The lower panel shows the results of the depth 6 analysis. The ARA labels are sorted based on p value (***p < 0.001, **p < 0.01, and *p < 0.05). d. ACE cluster-wise analysis with group-wise two-way ANOVA documented several sub-regional and laminar neuronal clusters differentially activated during walking. Left panel shows a coronal view of a cluster-wise p-value map with two zoomed views of significant clusters spanning in the primary and secondary motor area layers 6, 6a and 6b and the primary somatosensory area layers 2/3 and 4. The table summarizes the information of each cluster. Using ACE cluster-wise analysis, the volume (µm3 x1,000²), maximum strength (Cluster-wise TFCE F-statistic x1,000), brain regions each cluster spanned out on, and centroid of each significant cluster on Allen 10 µm atlas was computed.