Technical Report | Published:

SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction

Nature Neuroscience volume 16, pages 11541161 (2013) | Download Citation

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Abstract

We report a water-based optical clearing agent, SeeDB, which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. Our method maintained a constant sample volume during the clearing procedure, an important factor for keeping cellular morphology intact, and facilitated the quantitative reconstruction of neuronal circuits. Combined with two-photon microscopy and an optimized objective lens, we were able to image the mouse brain from the dorsal to the ventral side. We used SeeDB to describe the near-complete wiring diagram of sister mitral cells associated with a common glomerulus in the mouse olfactory bulb. We found the diversity of dendrite wiring patterns among sister mitral cells, and our results provide an anatomical basis for non-redundant odor coding by these neurons. Our simple and efficient method is useful for imaging intact morphological architecture at large scales in both the adult and developing brains.

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References

  1. 1.

    et al. Correlations of neuronal and microvascular densities in murine cortex revealed by direct counting and colocalization of nuclei and vessels. J. Neurosci. 29, 14553–14570 (2009).

  2. 2.

    & Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).

  3. 3.

    Tissue Optics: Light Scattering Methods and Instruments for Medical Diagnosis, 2nd edn. (SPIE Press, 2007).

  4. 4.

    , & A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus. Development 105, 61–74 (1989).

  5. 5.

    et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat. Methods 4, 331–336 (2007).

  6. 6.

    , , , & 2,2′-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy. Microsc. Res. Tech. 70, 1–9 (2007).

  7. 7.

    & Labeling and confocal imaging of neurons in thick invertebrate tissue samples. Cold Spring Harb. Protoc. published online, (1 September 2012).

  8. 8.

    , , , & Chemical clearing and dehydration of GFP expressing mouse brains. PLoS ONE 7, e33916 (2012).

  9. 9.

    et al. Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury. Nat. Med. 18, 166–171 (2012).

  10. 10.

    An enzyme method of clearing and staining small vertebrates. Proc. USA Natl. Museum 122, 1–17 (1967).

  11. 11.

    & Enzyme clearing of alcian blue stained whole small vertebrates for demonstration of cartilage. Stain Technol. 52, 229–232 (1977).

  12. 12.

    et al. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat. Neurosci. 14, 1481–1488 (2011).

  13. 13.

    & Whole-mount staining of Xenopus and other vertebrates. Methods Cell Biol. 36, 419–441 (1991).

  14. 14.

    & Theory and Practice of Histological Techniques (Churchill Livingstone, 2007).

  15. 15.

    Protein fructosylation: fructose and the Maillard reaction. Am. J. Clin. Nutr. 58, 779S–787S (1993).

  16. 16.

    et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).

  17. 17.

    & Topography of the human corpus callosum revisited–comprehensive fiber tractography using diffusion tensor magnetic resonance imaging. Neuroimage 32, 989–994 (2006).

  18. 18.

    et al. Pre-target axon sorting establishes the neural map topography. Science 325, 585–590 (2009).

  19. 19.

    , , , & Segregation and pathfinding of callosal axons through EphA3 signaling. J. Neurosci. 31, 16251–16260 (2011).

  20. 20.

    & Glomerulus-specific synchronization of mitral cells in the olfactory bulb. Neuron 31, 639–651 (2001).

  21. 21.

    Olfactory maps in the brain. Annu. Rev. Neurosci. 34, 233–258 (2011).

  22. 22.

    et al. In vivo simultaneous tracing and Ca2+ imaging of local neuronal circuits. Neuron 53, 789–803 (2007).

  23. 23.

    et al. Structure and emergence of specific olfactory glomeruli in the mouse. J. Neurosci. 21, 9713–9723 (2001).

  24. 24.

    , & Dendritic and axonal organization of mitral and tufted cells in the rat olfactory bulb. J. Comp. Neurol. 226, 346–356 (1984).

  25. 25.

    et al. Sensory maps in the olfactory cortex defined by long-range viral tracing of single neurons. Nature 472, 217–220 (2011).

  26. 26.

    et al. Parallel mitral and tufted cell pathways route distinct odor information to different targets in the olfactory cortex. J. Neurosci. 32, 7970–7985 (2012).

  27. 27.

    , & Distribution of dendrites of mitral, displaced mitral, tufted, and granule cells in the rabbit olfactory bulb. J. Comp. Neurol. 219, 339–355 (1983).

  28. 28.

    et al. Differential axonal projection of mitral and tufted cells in the mouse main olfactory system. Front. Neural Circuits 4, 120 (2010).

  29. 29.

    , , , & Distinct representations of olfactory information in different cortical centres. Nature 472, 213–216 (2011).

  30. 30.

    et al. Lateral connectivity in the olfactory bulb is sparse and segregated. Front. Neural Circuits 5, 5 (2011).

  31. 31.

    , & Different granule cell populations innervate superficial and deep regions of the external plexiform layer in rat olfactory bulb. J. Comp. Neurol. 217, 227–237 (1983).

  32. 32.

    , & Rat olfactory bulb mitral cells receive sparse glomerular inputs. Neuron 59, 802–814 (2008).

  33. 33.

    et al. Structural and molecular interrogation of intact biological systems. Nature 497, 332–337 (2013).

  34. 34.

    & Origins of correlated activity in an olfactory circuit. Nat. Neurosci. 12, 1136–1144 (2009).

  35. 35.

    , & Odor coding by modules of coherent mitral/tufted cells in the vertebrate olfactory bulb. Proc. Natl. Acad. Sci. USA 106, 2401–2406 (2009).

  36. 36.

    , & Population dynamics of adult-formed granule neurons of the rat olfactory bulb. J. Comp. Neurol. 239, 117–125 (1985).

  37. 37.

    & Aging of the rat olfactory bulb: growth and atrophy of constituent layers and changes in size and number of mitral cells. J. Comp. Neurol. 72, 345–367 (1977).

  38. 38.

    , , , & Non-redundant odor coding by sister mitral cells revealed by light addressable glomeruli in the mouse. Nat. Neurosci. 13, 1404–1412 (2010).

  39. 39.

    , , , & Odorant response properties of individual neurons in an olfactory glomerular module. Neuron 77, 1122–1135 (2013).

  40. 40.

    et al. From the olfactory bulb to higher brain centers: genetic visualization of secondary olfactory pathways in zebrafish. J. Neurosci. 29, 4756–4767 (2009).

  41. 41.

    et al. Cortical representations of olfactory input by trans-synaptic tracing. Nature 472, 191–196 (2011).

  42. 42.

    et al. Preferential electrical coupling regulates neocortical lineage-dependent microcircuit assembly. Nature 486, 113–117 (2012).

  43. 43.

    et al. Clonally related visual cortical neurons show similar stimulus feature selectivity. Nature 486, 118–121 (2012).

  44. 44.

    et al. Similarity of visual selectivity among clonally related neurons in visual cortex. Neuron 75, 65–72 (2012).

  45. 45.

    et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).

  46. 46.

    , , & Targeting single neuronal networks for gene expression and cell labeling in vivo. Neuron 67, 562–574 (2010).

  47. 47.

    & Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons. Neuron 72, 938–950 (2011).

  48. 48.

    et al. mGRASP enables mapping mammalian synaptic connectivity with light microscopy. Nat. Methods 9, 96–102 (2012).

  49. 49.

    et al. Formation of precise connections in the olfactory bulb occurs in the absence of odorant-evoked neuronal activity. Neuron 26, 69–80 (2000).

  50. 50.

    In vivo electroporation in the embryonic mouse central nervous system. Nat. Protoc. 1, 1552–1558 (2006).

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Acknowledgements

We thank J.R. Sanes (Harvard) (Thy1-YFP-G, H) and P. Mombaerts (Max Planck Institute) (OMP-GFP) for providing mouse strains. We are also grateful to M. Eiraku and K. Muguruma for assistance with two-photon microscopy setup, Olympus for the customized objective lens, Y. Mimori-Kiyosue for equipment, J. Nabekura and T. Nemoto for instructions on in vivo two-photon imaging, and R. Iwata and H. Hiraga for valuable comments on the manuscript. This work was supported by grants from the PRESTO program of the Japan Science and Technology Agency, the Sumitomo Foundation, the Nakajima Foundation, the Mitsubishi Foundation, the Strategic Programs for R&D (President's Discretionary Fund) of RIKEN, and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to T.I.). The imaging experiments were supported by the RIKEN Center for Developmental Biology Imaging Facility and the Four-dimensional Tissue Analysis Unit. Animal experiments were supported by the Laboratory for Animal Resources and Genetic Engineering at the RIKEN Center for Developmental Biology.

Author information

Affiliations

  1. Laboratory for Sensory Circuit Formation, RIKEN Center for Developmental Biology, Kobe, Japan.

    • Meng-Tsen Ke
    • , Satoshi Fujimoto
    •  & Takeshi Imai
  2. Graduate School of Biostudies, Kyoto University, Kyoto, Japan.

    • Meng-Tsen Ke
    •  & Takeshi Imai
  3. PRESTO, Japan Science and Technology Agency, Saitama, Japan.

    • Takeshi Imai

Authors

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Contributions

M.-T.K. performed most of the experiments. S.F. performed in utero electroporation. T.I. conceived the experiments, performed the initial phase of experiments, supervised the project and wrote the manuscript.

Competing interests

M.-T.K. and T.I. hold a patent (pending) for the SeeDB technique.

Corresponding author

Correspondence to Takeshi Imai.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Tables 1–5, Supplementary Figures 1–13, Supplementary Videos 1–14

Videos

  1. 1.

    Supplementary Video 1

    Adult Thy1-YFP-H line (P70) imaged using confocal microscopy. Serial x-y images were taken at an interval of 10 μm. Depths (z) indicated in the movie are non-calibrated values. The real depths are 1.49× larger. See legend for Figure 3a.

  2. 2.

    Supplementary Video 2

    Volume rendering of adult Thy1-YFP-H mouse (P72) cleared with SeeDB37 and imaged using two-photon microscopy. Images were taken from dorsal side, and 14×1 blocks were tiled. See legend for Figure 3c.

  3. 3.

    Supplementary Video 3

    Serial horizontal optical sections of Thy1-YFP-H line (P72). Hemi-brain sample was cleared with SeeDB37 and imaged using two-photon microscopy. Images were taken from dorsal side every 500 μm. See legend for Figure 3d.

  4. 4.

    Supplementary Video 4

    Thy1-YFP-H mouse (P72) imaged using two-photon microscopy. Serial x-y images were taken at an interval of 25 μm. See legend for Figure 3e.

  5. 5.

    Supplementary Video 5

    Volume rendering of serial optical sections shown in Supplementary Video 2. See legend for Figure 3e.

  6. 6.

    Supplementary Video 6

    Volume rendering of adult Thy1-YFP-H line (P72) cleared with SeeDB37 and imaged using two-photon microscopy. Images were taken from medial side, and 14×1 blocks were tiled. See legend for Suplementary Figure 10a.

  7. 7.

    Supplementary Video 7

    Serial optical sections of Thy1-YFP-H line (P72) hemi-brain imaged from medial face using two-photon microscopy. Images were taken from the medial side every 300 μm, and 35×17 blocks were tiled. See legend for Supplementary Figure 10b.

  8. 8.

    Supplementary Video 8

    Thy1-YFP-H line (P21) cleared with SeeDB37 and imaged using two-photon microscopy. Images were taken from the dorsal side, and serial x-y images are shown. See legend for Supplementary Figure 11c.

  9. 9.

    Supplementary Video 9

    Volume rendering of serial optical sections shown in Supplementary Video 8. See legend for Supplementary Figure 11c.

  10. 10.

    Supplementary Video 10

    The entire olfactory bulb of a Thy1-YFP-G mouse (P21) imaged using two-photon microscopy. Serial tiled x-y images were taken from the medial side. See legend for Supplementary Figure 12.

  11. 11.

    Supplementary Video 11

    Callosal axons imaged using two-photon microscopy. Serial x-y images were taken at an interval of 10 μm. See legend for Figure 4. Left, anterior; Right, posterior.

  12. 12.

    Supplementary Video 12

    Topographic organization of corpus callosum imaged using two-photon microscopy. Anterior and posterior regions of the cerebral cortex layer II-III neurons were labeled with tdTomato (magenta) and EGFP (green), respectively, using in utero electroporation. Serial x-y images were taken at an interval of 15 μm. InSight DeepSee Dual (Spectra Physics) was used to excite tdTomato and EGFP at 1,040 nm and 920 nm, respectively. Our customized 25× objective lens was used. Left, anterior; Right, posterior.

  13. 13.

    Supplementary Video 13

    Reconstruction of individual callosal axons sparsely labeled by Cre-loxP system. See legend for Figure 4b.

  14. 14.

    Supplementary Video 14

    Tracing lateral dendrites of sister mitral cells. Serial confocal images to reconstruct Figure 6a. Depths (z) in the movie are non-calibrated values. The real depths are 1.49× larger. Only presumptive mitral cells were reconstructed in Figure 6a. See legend for Figure 6. Green, OMP-GFP; magenta, Alexa-647 dextran.

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DOI

https://doi.org/10.1038/nn.3447

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