Extended Data Fig. 7: Brain-wide identification of local neuronal activity changes underlying walking.

a. The overview of experimental design to analyze c-Fos+ cell distribution in whole-brain LSFM data during walking (n = 3/group). b. Automated segmentation of c-Fos+ cell distribution using ACE’s segmentation module. Panels show a 3D rendering of a maximum intensity projection of raw data from the walking group, ACE output (blue), and raw data overlaid on ACE’s output. c. Segmentation maps were voxelized to the ARA 10um resolution. Subsequently, the voxelized segmentation maps were warped to ARA space. Left panels show an example of a downsampled subject overlaid on ARA labels after registration from each group. Right panels show a 3D rendering of voxelized and warped segmentation maps color-coded based on 6 ARA regions: CTX, Cerebral Cortex; CNU, Cerebral Nuclei; MB, Midbrain; HB, Hindbrain; IB, Interbrain; and CB, Cerebellum. d. To identify neural activity hotspots, group-wise heatmaps of neuronal density were obtained by subtracting the average of the voxelized and warped segmentation maps in each group. Panels show two different coronal views as an example. e. Result of ACE cluster-wise threshold-free cluster enhancement permutation analysis, using a group-wise two-way ANOVA. The panels demonstrate the resulting p-value map representing the clusters showing significant differences between groups and corresponding to the coronal sections in d. Zoomed views show two significant clusters in MOp (left panel) and Retrosplenial area (right panel). See Supplementary Table 3 for strength (effect size), volume, and brain regions each cluster spanned. f. Lateral Hypothalamic Area (LHA) label was warped back into native space using the deformation matrix obtained by registration. Left and right panels show two example subjects from the walking and homecage groups respectively with a zoomed version of LHA, showcasing higher c-Fos+ activity in the walking vs. homecage condition. Section a is created in BioRender.com.