Fig. 1: Anatomic delineation and cell type organization of the DP.
a, DP delineation based on Nissl cytoarchitecture. Two DP sublayers were identified: the DPs and DPd. Connectivity confirms DPs and DPd distinction: the BLAal (PHAL, magenta) targets the DPs, while the BLAam (AAV-GFP) innervates the ILA and DPd. b, Inputs from the AUDv and RE terminate in DP layer 1, defining the DP boundaries. c, Connectivity-based characterization of DP sublayers. FG-labelled DP to ACAd neurons are confined to DPs, whereas MPO- and thalamus-projecting neurons localize in the DPd. LHA-projecting neurons span the DPd and deep DPs. ProSUB axons densely innervate the DPs. d, Cell-type-specific gene expression supports regional and laminar DP organization. The RNAscope analysis shows that Cux2 is absent in the DP, Etv1 is enriched in the DPs and Tle4 is expressed in the DPd. Cacna1h is expressed in the neocortex and DPs. DPd is distinguished with Vglut2 expression in VGLUT2 Cre × Ai14 mice and via in situ hybridization. Bright-field in situ hybridization images from the ABA (https://mouse.brain-map.org/). e, Schematic of neuron types in the DPs and DPd. BS, brainstem; d, deep; s, superficial. f, Dendritic morphology of reconstructed DP neurons (left). Morphological clustering (right, top) based on the size (x axis) and complexity (y axis) of dendritic arbours (n = 47) identifies two morphological groups: one with smaller, simpler dendrites (n = 32; dendritic length, 3,024 ± 833 µm; number of branches, 38 ± 12), and another with larger, more complex dendrites (n = 15; dendritic length, 5,821 ± 958 µm; number of branches, 76 ± 13). The 47 DP neurons were pooled from four MORF3 mice. On the basis of the somatic distance from the midline, neurons also were clustered as superficial-layer (n = 26; distance from the midline, 288 ± 81 µm) or deep-layer (n = 21; distance from the midline, 605 ± 106 µm) neurons and are plotted in the size and complexity dimensions (middle right). Examples of superficial and deep neurons are presented from a single brain (bottom left). The box plot (bottom right) demonstrates that superficial neurons have a greater dendritic length (superficial, 4,275 ± 1,717 µm; deep, 3,472 ± 1285 µm; one-tailed Wilcoxon signed-rank test, FDR-corrected P = 0.0465), a higher number of branches (superficial, 56 ± 24; deep, 42 ± 16; FDR-corrected P = 0.0317) and contain a greater proportion of dendrites between soma and midline (superficial, 65 ± 17%; deep, 53 ± 17%; FDR-corrected P = 0.026). The smaller–simpler neurons were more distant from the midline compared with the larger–complex neurons (small–simple distance, 472 ± 184 µm; large–complex distance, 340 ± 153 µm; FDR-corrected P = 0.026). The line inside the box plot shows the median, the top and bottom edges are the upper (0.75) and lower (0.25) quartiles, the top whisker connects the upper quartile to the non-outlier maximum, and the bottom whisker connects the lower quartile to the non-outlier minimum. Scale bars, 200 μm (a (middle), b, c and d (left)), 300 μm (d (right) and f) and 500 μm (a (left and right)). A full list of abbreviations is provided in Supplementary Table 1.
