(A) Spread of fluorescence labeling following viral injection of AAV2-CRE-eGPF in the superior colliculus of two FLEX-TdTomato mice. 3D objects showing the injection sites were created using custom python scripts following acquisition of a 3D image of the entire brain with serial two-photon tomography and registration of the image data to the atlas’ template (with brainreg, Tyson et al., 2020a). (B) Streamlines visualization of efferent projections from the mouse primary motor cortex following injection of an anterogradely transported virus expressing fluorescent proteins (original data from Oh et al., 2014), downloaded from (Neuroinformatics NL with brainrender). (C) Visualization of the location of several implanted neuropixel probes from multiple mice (data from Steinmetz et al., 2019). Dark salmon colored tracks show probes going through both primary/anterior visual cortex (VISp/VISa) and the dorsal lateral geniculate nucleus of the thalamus. (D) Single periaqueductal gray (PAG) neuron. The PAG and superior colliculus are also shown. The neuron’s morphology was reconstructed by targeting the expression of fluorescent proteins in excitatory neurons in the PAG via an intersectional viral strategy, followed by imaging of cleared tissue and manual reconstruction of the neuron’s morphology with Vaa3D software. Data were registered to the Allen atlas with SHARPTRACK (Shamash et al., 2018). The 3D data was saved as a .stl file and loaded directly into brainrender. (E) Gene expression data. Left, expression of genes ‘brn3c’ and ‘nk1688CGt’ in the tectum of the larval zebrafish brain (gene expression data from fishatlas.neuro.mpg.de, 3D objects created with custom python scripts). Right, expression of gene ‘Gpr161’ in the mouse hippocampus (gene expression data from Wang et al., 2020), downloaded with brainrender (3D objects created with brainrender). Colored voxels show voxels with high gene expressions. The CA1 field of the hippocampus is also shown.