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Mapping the cytoarchitecture of the whole brain can reveal the organizational logic of neural systems. However, this remains a significant challenge, especially for gyrencephalic brains with a large volume. Here we propose an integrated pipeline for generating a cytoarchitectonic atlas with single-cell resolution of the whole brain. To analyze a large-volume brain, we used a modified en-bloc Nissl staining protocol to achieve uniform staining of large-scale brain specimens from ferret (Mustela putorius furo). By combining whole-brain imaging and big data processing, we established strategies for parsing cytoarchitectural information at a voxel resolution of 0.33 μm × 0.33 μm × 1 μm and terabyte-scale data analysis. Using the cytoarchitectonic datasets for adult ferret brain, we identified giant pyramidal neurons in ferret brains and provide the first report of their morphological diversity, neurochemical phenotype, and distribution patterns in the whole brain in three dimensions. This pipeline will facilitate studies on the organization and development of the mammalian brains, from that of rodents to the gyrencephalic brains of ferret and even primates.

Mapping the cytoarchitecture of the whole brain can reveal the organizational logic of neural systems. However, this remains a significant challenge, especially for gyrencephalic brains with a large volume. Here we propose an integrated pipeline for generating a cytoarchitectonic atlas with single-cell resolution of the whole brain. To analyze a large-volume brain, we used a modified en-bloc Nissl staining protocol to achieve uniform staining of large-scale brain specimens from ferret ( Mustela putorius furo ). By combining whole-brain imaging and big data processing, we established strategies for parsing cytoarchitectural information at a voxel resolution of 0.33 μm × 0.33 μm × 1 μm and terabyte-scale data analysis. Using the cytoarchitectonic datasets for adult ferret brain, we identified giant pyramidal neurons in ferret brains and provide the first report of their morphological diversity, neurochemical phenotype, and distribution patterns in the whole brain in three dimensions. This pipeline will facilitate studies on the organization and development of the mammalian brains, from that of rodents to the gyrencephalic brains of ferret and even primates., (Copyright © 2020 Long, Jiang, Zhang, Chen, Jia, Xu, Luo, Gong, Li and Li.)