Fig. 1
Brain-wide inputs to the PPN and collateralization of PPN-targeting cortical pyramidal neurons. a Horizontal view from whole-brain VISoR imaging of excitatory neurons labeled by retrograde tracing virus (rAAV2-retro-EF1α-DIO-EGFP) injected into the PPN of a CaMKIIα-Cre transgenic mouse. Solid lines indicate cortical and subcortical areas to be examined in b and c-d, respectively. A: anterior; P: posterior; L: left; R: right; D: dorsal; V: ventral. b Maximum‑intensity projection of part of a 100 μm-thick coronal section encompassing the MOs, ACA, and PL (b1). Enlarged views of the boxed areas in b1 show EGFP-expressing PPN-targeting neurons in the MOs (b2), ACA (b3) and PL (b4). c, d Maximum-intensity projection of part of a coronal section encompassing the PAG (c1) and the PARN (d1). Enlarged views of the boxed areas in (c2) and (d2), showing labeled neurons in the PAG and PARN, respectively. e Diagrams of the cell type specific and target-specific sparse labeling of PPN-projecting cortical pyramidal neurons. f–h Imaging and 3D reconstruction of individual PPN-projecting neurons from the ACA (f), PL (g), and MOs (h), showing brain-wide axonal projections (f1, g1, h1) and dendritic morphologies (f2, f3, g2, g3, h2, h3) of sparsely labeled cortical neurons. Enlarged views of subcortical areas within the PAG (f4, f5), VTA (g4, g5), STR (h4, h5), and PPN (f6, f7, g6, g7, h6, h7) show termini of axonal collaterals from the traced cortical neurons. i Collateralization profile of individual pyramidal neurons in the ACA (n = 4), PL (n = 4) and MOs (n = 3). The heatmap shows the number of axon terminals made by each neuron. j Qualitative summary of subcortical collateralization from the three cortical areas. The collateralization ratio (CR, defined as the number of PPN-projecting neurons from a given cortical area making at least 2 terminals in a specified subcortical target area divided by the total number of neurons traced for this cortical area) is separated into 3 categories: “−” for CR = 0; “+” for CR ≤ 33.3%; “++” for 33.3% < CR < 66.7%; “+++” for CR ≥ 66.7%