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Clarity for Mapping the Nervous System perspective FOCUS ON MAPPING THE BRAIN CLARITY for mapping the nervous system Kwanghun Chung1,2 & Karl Deisseroth1–4 With potential relevance for brain-mapping work, of irregularly arranged lipid interfaces that character- hydrogel-based structures can now be built from izes this tissue likewise creates an effective scattering within biological tissue to allow subsequent barrier to photon penetration for optical interroga- removal of lipids without mechanical disassembly tion of mammalian brains7, unlike the Caenorhabditis of the tissue. This process creates a tissue-hydrogel elegans (worm) or larval Danio rerio (zebrafish) hybrid that is physically stable, that preserves fine nervous systems, which are more accessible owing structure, proteins and nucleic acids, and that is in part to smaller size and less myelination. Single- permeable to both visible-spectrum photons and photon microscopy can provide optical transmis- exogenous macromolecules. Here we highlight sion of information from only about 50 micrometers relevant challenges and opportunities of this below the mammalian brain surface, and even well- approach, especially with regard to integration optimized two-photon microscopy cannot be used to with complementary methodologies for brain- image deeper than about 800 micrometers, far short mapping studies. of enabling visualization of full projection patterns Mammalian brains are staggeringly complex in terms and global arrangement of cell populations in the of both scale and diversity; many billions of neurons intact brain8. are present, among them likely at least hundreds of Over the past few decades, a great deal of tech- genetically distinct cell types, with each type of cell nological innovation has been stimulated by these represented by many distinct projection patterns. challenges8–21. First, newer automated methods for CLARITY1 is a newly developed technology that can mechanical sectioning of tissue have overcome some be used to transform intact biological tissue into a of the drawbacks of traditional sectioning methods Nature America, Inc. All rights reserved. Inc. Nature America, 3 hybrid form in which tissue components are removed that were laborious, expensive and damaged the and replaced with exogenous elements for increased tissue. Serial block-face mechanical9–13 or optical- © 201 accessibility and functionality. CLARITY has the ­ablative14 methods, in combination with imaging rea- potential to facilitate rapid extraction of anatomical- douts such as two-photon tomography14,15, electron projection information important for many fields of microscopy16 or array tomography17, have been used neuroscience research2,3; such information can be to map macroscopic to nanoscopic brain structures collected along with molecular-phenotype informa- (see Review18 in this issue). In some cases, molecu- tion at the resolution of single cells. Alone or in combi- lar labeling is built into these processes, and ongoing nation with other methods4,5, such an approach could work includes approaches for addressing additional contribute to the study of function and dysfunction in challenges such as generation of contrast in tissue this complex system. before sectioning19,20. Tools for automated analysis, In general, obtaining system-wide detailed informa- efficient reconstruction and error-free alignment also tion from neural tissue is a formidable challenge (to continue to be developed21 as well as for registration say nothing of subsequent data curation and analysis). with activity information, and indeed detailed wiring In the mammalian central nervous system, seamlessly information linked to activity has been obtained from intertwined neural processes leave little extracellular well-defined volumes22,23. space, creating barriers to macromolecule diffusion Second, optical clearing methods have been for in situ hybridization, antibody staining or other ­developed that involve immersion of the specimen in forms of molecular phenotyping deeper than the first medium that matches the refractive index of the tissue, few cellular layers of intact tissue6. The high density thereby reducing light scattering and extending 1Department of Bioengineering, Stanford University, Stanford, California, USA. 2CNC Program, Stanford University, Stanford, California, USA. 3Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, California, USA. 4Howard Hughes Medical Institute, Stanford University, Stanford, California, USA. Correspondence should be addressed to K.C. ([email protected]) and K.D. ([email protected]). RECEIVED 18 MArCH; ACCEPTED 22 APrIL; PUBLISHED ONLINE 30 mAY 2013; COrrECTED AFTEr PrINT 20 JUNE 2013; DOI:10.1038/NmETH.2481 508 | VOL.10 NO.6 | JUNE 2013 | NATURE METHODS FOCUS ON MAPPING THE brain PERSPECTIVE Figure 1 | Imaging of nervous system projections in the intact mouse brain with CLARITY. Adult Thy1-EYFP H line mice (4 months old) were perfused transcardially followed by hybridization of biomolecule-bound monomers into a hydrogel mesh and lipid removal as described1. The clarified mouse brain was imaged from the dorsal region to the ventral region (3.4 mm to the midpoint) using a 10× water-immersion objective. Image adapted from ref. 1. the range of optical imaging24–26. For example, benzyl alcohol−benzyl ben- zoate is an organic solvent that effectively renders biological specimens transpar- ent but reduces the stability of fluores- cent protein signals24,25; in Scale, another clearing method, an aqueous solution is used to preserve fluorescence signals, but the rate and the extent of clearing remain 26 limiting . Notably all current tissue- 1 mm clearing methods leave the densely packed lipid bilayers intact and therefore still face challenges with regard to penetration by visible-spectrum infrastructure (in a process conceptually akin to petrification or light and molecules, making these methods largely incompatible fossilization, except that not only structure but also native bio- with whole-tissue molecular phenotyping. molecules such as proteins and nucleic acids are preserved). This The CLARITY approach1 helps address ongoing challenges outcome is achieved by first infusing small organic hydrogel- by enabling molecular and optical interrogation of large assem- monomer molecules into the intact brain along with cross- bled biological systems, such as the entire adult mouse brain. linkers and thermally triggered polymerization initiators; Light-microscopy (Fig. 1) and biochemical-phenotyping (Fig. 2) subsequent temperature elevation triggers formation, from within techniques can be used to rapidly access the entire intact clari- the brain, of a hydrogel meshwork covalently linked to native pro- fied mouse brain with fine structural resolution and molecular teins, small molecules and nucleic acids but not to lipids, which detail (to the level of spines, synapses, proteins, single-amino-acid lack the necessary reactive groups (Fig. 3a). Subsequent whole- Nature America, Inc. All rights reserved. Inc. Nature America, neurotransmitters and nucleic acids) while in the same prepara- brain electrophoresis in the presence of ionic detergents actively 3 tion maintaining global structural information including brain- removes the lipids (Fig. 3b), resulting in a transparent brain- wide macroscopic connectivity. To clarify tissue (Fig. 3), lipid hydrogel hybrid that both preserves, and makes accessible, struc- © 201 bilayers are replaced with a more rigid and porous hydrogel-based tural and molecular information for visualization and analysis. a 3D rendering b DR Figure 2 | Intact mouse brain molecular DR phenotyping and imaging with CLARITY. RR (a) Three-dimensional (3D) visualization of immunohistology data, showing tyrosine hydroxylase (TH)-positive neurons and fibers in the mouse brain. The intact clarified brain was 1 z = 2,460 µm stained for 6 weeks as described , with primary antibody for 2 weeks, followed by a 1-week wash, SNR SNR c VTA then stained with secondary antibody for 2 weeks followed by a 1-week wash and imaged 2,500 µm VTA from ventral side using the 10× water-immersion objective. D, V, A and P indicate dorsal, ventral, anterior and posterior, respectively. (b–d) Optical PO sections at different depths, corresponding z = 1,005 µm respectively to the upper, middle and lower PO dashed box regions in a. Note that TH-positive d neurons are well-labeled and clearly visible even at a depth of 2,460 m in the intact brain. CPu CPu µ P CPu, caudate putamen; PO, preoptic nucleus; V VTA, ventral tegmental area; SNR, substantia D nigra; RR, retrorubral nucleus; DR, dorsal raphe. A Scale bars, 700 µm (a) and 100 µm (b–d). z = 420 µm Image adapted from ref. 1. NATURE METHODS | VOL.10 NO.6 | JUNE 2013 | 509 perspective FOCUS ON MAPPING THE BRAIN Figure 3 | CLARITY technology and instrumentation. (a) Tissue is cross- a Step 1: hydrogel monomer infusion (days 1–3) linked with formaldehyde in the presence of infused hydrogel monomers. Proteins DNA + + Thermally triggered polymerization then results in a hydrogel-tissue hybrid ER which physically supports tissue structure and chemically incorporates 4 °C Formaldehyde Hydrogel Vesicle native biomolecules into the hydrogel mesh. (b) In electrophoretic tissue monomer clearing (ETC), an electric field is applied across the hybrid immersed in an ionic detergent solution to actively transport ionic micelles into the Plasma hybrid and extract membrane lipids out of the tissue, leaving structures membrane and cross-linked biomolecules in place and available
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