(A) Experimental schematic outlining the intersectional viral strategy, brain clearing, automated 3D axon segmentation, and alignment to the Allen Brain Institute Common Coordinate Framework. (B) Axonal innervation in a 3D view of the left hemisphere of one representative brain each from the intersection of Sert-Flp and either Vglut3-Cre or Trh-Cre. (C) Coronal Z-projection (500 µm of depth) showing axonal innervation patterns of 6 aligned brains. The schematic reference image is one 5 µm thick plane in the middle of the 500 µm stack. (D) Coronal heatmaps of axonal innervation patterns at 12 positions along the rostral–caudal axis for the same six brains as seen in (C). Weightings for individual voxels represent axonal content within a radius of 225 µm. (E) Top, bar plot shows the quantification of total axonal content in each of 6 brains prior to normalization. Bottom, heatmap breaks out the total content into each of 282 individual brain regions using boundaries from the Allen Institute CCF. Values are normalized to both target region volume and total axon content per brain. Display order is grouped by mean normalized prevalence of axons in each genotype and ordered by the second principal component. P-values for individual t-tests are uncorrected; those that survive FDR-testing at 10% are indicated with a black bar. See Supplementary file 5 for full list of regions. EP, Endopiriform nucleus; M1, Primary motor area; S1-B, Primary somatosensory area, barrel field; CLI, Central linear nucleus raphe; AHN, Anterior hypothalamic nucleus; DMH, Dorsomedial nucleus of the hypothalamus; MG, Medial geniculate nucleus; ZI, Zona incerta.