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A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing

Abstract

A three-dimensional single-cell-resolution mammalian brain atlas will accelerate systems-level identification and analysis of cellular circuits underlying various brain functions. However, its construction requires efficient subcellular-resolution imaging throughout the entire brain. To address this challenge, we developed a fluorescent-protein-compatible, whole-organ clearing and homogeneous expansion protocol based on an aqueous chemical solution (CUBIC-X). The expanded, well-cleared brain enabled us to construct a point-based mouse brain atlas with single-cell annotation (CUBIC-Atlas). CUBIC-Atlas reflects inhomogeneous whole-brain development, revealing a significant decrease in the cerebral visual and somatosensory cortical areas during postnatal development. Probabilistic activity mapping of pharmacologically stimulated Arc-dVenus reporter mouse brains onto CUBIC-Atlas revealed the existence of distinct functional structures in the hippocampal dentate gyrus. CUBIC-Atlas is shareable by an open-source web-based viewer, providing a new platform for whole-brain cell profiling.

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Fig. 1: Identification of swelling reagents by comprehensive chemical screening.
Fig. 2: CUBIC-X for whole-brain expansion and hyperhydrative RI matching.
Fig. 3: Whole-brain imaging of a CUBIC-X expanded brain with customized LSFM.
Fig. 4: Construction of a single-cell-resolution mouse brain atlas (CUBIC-Atlas).
Fig. 5: CUBIC-Atlas is applicable to whole-brain cell profiling of mice at various developmental timepoints.
Fig. 6: CUBIC-Atlas revealed a cell number increase in midbrain and decreases in visual area and primary somatosensory area of cerebral cortex during early postnatal development.
Fig. 7: Probabilistic annotation of CUBIC-Atlas revealed a functionally distinct structure in granule cell layer of hippocampal dentate gyrus.

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Acknowledgements

We thank all lab members at The University of Tokyo and RIKEN QBiC, in particular: A. Millius and W. Kylius for editing, E.A. Susaki and A. Kuno for discovering the tissue-swelling phenomenon, S. Shoi for helping with statistical analysis, K. Yoshida for helping with the decomposing transformation matrix, and C. Shimizu for supporting swelling experiments. We also thank H. Hayakawa and S. Jiang for supporting the preparation of a C57BL/6 J mouse brain, D. Perrin and H. Yukinaga for informatics instruction, J. Kaneshiro, T. Watanabe and Olympus Engineering for helping design the microscope, T. Mitani and K. Matsumoto for reproducibility confirmation, S. Takano, S. Yamazoe and T. Tsukuda for the measurements of zeta potentials and Bitplane for instruction of Imaris 8.1.2. This work was supported by a grant from AMED-CREST (AMED/MEXT, grant number JP17gm0610006, to H.R.U.), CREST (JST/MEXT, to H.R.U.), Brain/MINDS (AMED/MEXT, grant number JP17dm0207049, to H.R.U. and H. M.), Basic Science and Platform Technology Program for Innovative Biological Medicine (AMED/MEXT, grant number JP17am0301025, to H.R.U.), Translational Research Network Program from Japan Agency for Medical Research and development (AMED, to H.M.), World Premier International Research Center Initiative (MEXT, to H.R.U), a Grant-in-Aid for Scientific Research (JSPS KAKENHI, grant number 16 J05041, to T.C.M.), a Grant-in-Aid for Scientific Research (S) (JSPS KAKENHI, grant number 25221004, to H.R.U.), Grant-in-Aid for Challenging Exploratory Research (JSPS KAKENHI, grant number 16K15124, to K.T.), a Grant-in-Aid for Scientific Research on Innovative Areas (JSPS KAKENHI, grant number 23115006, to H.R.U., 15H01558, to H.M., 17H05688, to K.T.), and a Grant-in-Aid from the Naito Foundation (to K.T.).

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Contributions

H.R.U., T.C.M., T.M. and K.T. designed the study. T.C.M., T.M. and S.S. performed most of the experiments. S.A.H. contributed to CATMAID data sharing. D.S. designed CAD. K.B. and H.M. prepared C57BL/6 mouse brains. H.S., M.I. and K.F.T. produced PLP-YFP and Mlc1-YFP mice. Y.S. produced recombinant fluorescent proteins. H.K. produced R26-H2B-EGFP mice. H.R.U., T.C.M., T.M. and K.T. wrote the manuscript. All authors discussed the results and commented on the manuscript text.

Corresponding author

Correspondence to Hiroki R. Ueda.

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Competing interests

T.C.M., K.T. and H.R.U. have filed patent application for the CUBIC-X technique. Part of this study was done in collaboration with Olympus Corporation.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–22

Life Sciences Reporting Summary

Supplementary Table 1

The list of 11 chemicals with high swelling ability. Eleven chemicals with high swelling ability selected in Fig. 1d are described. Chemical number, chemical name, CAS number, supplier, catalog number and cost are shown.

Supplementary Table 2

Parts list of the customized LSFM. The parts of customized LSFM are categorized into 10 modules: optical table, laser, illumination rail systems, illumination relay systems, illumination sheet generative systems, sample chamber systems, sample positioning systems, detection systems, microscope control unit, and image processing servers.

Supplementary Table 3

Total cell numbers for 8-week-old C57BL/6N male mice in each brain area. We list graph order in Fig. 4l, Allen Brain Atlas (ABA) ID, name of the area, acronym, RGB color value and cell numbers for three independent brains from 8-week-old C57BL/6N male mice. We referred to the ABA website for the name and acronym. RGB colors originally allocated in ABA were slightly modified to avoid duplication among the different brain areas.

Supplementary Video 1

CAD movie showing the imaging sequences of the customized LSFM.

Supplementary Video 2

Comprehensive cell detection with PI-stained CUBIC-X brain around the third ventricle.

Supplementary Video 3

3D rendering of CUBICAtlas with representative anatomical annotation.

Supplementary Video 4

3D rendering of CUBICAtlas at single-cell resolution.

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Murakami, T.C., Mano, T., Saikawa, S. et al. A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing. Nat Neurosci 21, 625–637 (2018). https://doi.org/10.1038/s41593-018-0109-1

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