Focus on Mapping the Brain e are entering a new era in neuroscience in which techno- CONTENTS logical development will allow us to obtain full anatomi- 483 From the connectome Wcal, high-resolution renderings of entire brain circuits and to brain function to map the activity of ever larger cellular populations as an animal C I Bargmann & E Marder performs specific behaviors. Assembling anatomical, molecular and 491 Making sense of brain functional maps has the potential to greatly advance our under- network data standing of how brains work. O Sporns In this Focus, experts outline the technologies needed to obtain 494 Why not these maps and discuss what will be needed beyond them to under- connectomics? J L Morgan & stand brain function. J W Lichtman Visualization of functional In a Historical Perspective, Cornelia Bargmann and Eve Marder connectivity in the human 501 Cellular-resolution discuss what has been learned from invertebrate circuits whose connectomics: cerebral cortex based on magnetic connectivity patterns are known and what will be needed beyond challenges of dense resonance imaging data. Brain neural circuit image by Joachim Böttger and anatomical maps to understand brain function in other organisms. reconstruction Daniel Margulies (Max Planck In a Commentary, Jeff Lichtman and Joshua Morgan express their M Helmstaedter Institute for Human Cognitive and views about why obtaining detailed, high-resolution structural Brain Sciences, Leipzig, Germany) maps should be an essential part of this endeavor. To understand 508 CLARITY for mapping with compositing help by Tobias S. the nervous system Hoffmann. Cover composition by the deluge of data these maps will engender once generated, Olaf K Chung & K Deisseroth Erin Dewalt. Sporns argues in another Commentary that data representation and 515 Mapping brain modeling will be critical. circuitry with a light Other experts discuss the newest technologies available for microscope obtaining brain maps. Moritz Helmstaedter presents the state of P Osten & T W Margrie the art and current challenges of electron microscopy–based cir- 524 Imaging human © 2013 Nature America, Inc. All rights reserved. America, Inc. © 2013 Nature cuit reconstruction. In three papers, the potential of using light connectomes at the macroscale to unveil the function and anatomy of brain circuits is presented. R C Craddock, S Jbabdi, Karl Deisseroth and Kwanghun Chung discuss their newly devel- C-G Yan, J T Vogelstein, npg oped method named CLARITY for rendering mammalian brains F X Castellanos, permeable to visible photons and molecules. Pavel Osten and Troy A Di Martino, C Kelly, Margrie review light-microscopy methods available for large-scale K Heberlein, S Colcombe & anatomical tracing and discuss ways to integrate molecular identity, M P Milham activity recording and anatomical information. In a Resource, Josh 540 Improved tools for the Brainbow toolbox Sanes and colleagues present improved tools for mapping the mouse Dawen Cai, K B Cohen, brain using the Brainbow technology. Finally, Michael Milham, Stan T Luo, J W Lichtman & Colcombe and their colleagues review methods for functional and J R Sanes anatomical analysis of human brains at the macroscale. We are pleased to acknowledge the financial support of Carl Zeiss Microscopy, Hamamatsu Corporation, LaVision BioTec, TissueVision, Inc. and Chroma Technology Corp. Nature Methods carries sole responsibility for all editorial content and peer review. Erika Pastrana Editor, Nature Methods Daniel Evanko Copy Editor Odelia Ghodsizadeh Carol Evangelista, Ivy Robles Focus Editor Erika Pastrana Managing Production Editor Sponsorship David Bagshaw, Publisher Stephanie Diment Renee Lucas Yvette Smith, Reya Silao Senior Copy Editor Irene Kaganman Production Editors Brandy Cafarella, Marketing Nazly De La Rosa NATURE METHODS | VOL.10 NO.6 | JUNE 2013 | 481 FOCUS ON MAPPING THE BRAIN HISTORICAL PERSPECTIVE From the connectome to brain function Cornelia I Bargmann1 & Eve Marder2,3 In this Historical Perspective, we ask what behaviors5–7. In association with the recordings of information is needed beyond connectivity these individually recognizable, identified neurons, the diagrams to understand the function of nervous cells were filled with dye to visualize their structures systems. Informed by invertebrate circuits and projection patterns via light microscopy8–10. In whose connectivities are known, we highlight some cases, electron microscopy was used to observe the importance of neuronal dynamics and the anatomical synapses in these small circuits11–13. neuromodulation, and the existence of parallel But until the publication of the heroic electron micros- circuits. The vertebrate retina has these features in copy reconstruction of the full nervous system of common with invertebrate circuits, suggesting that C. elegans14 in the mid-1980s, it was unimaginable they are general across animals. Comparisons across that the electron microscope could be used to deter- these systems suggest approaches to study the mine circuit connectivity rather than providing functional organization of large circuits based on ultrastructural detail to connectivity determined existing knowledge of small circuits. either with physiological or light microscopy–based An animal’s behavior arises from the coordinated activ- anatomical methods. ity of many interconnected neurons—“many” meaning Recent advances in electron microscopy and image 302 for Caenorhabditis elegans, 20,000 for a mollusc, analysis have made it possible to scale up this ultrastruc- several hundred thousand for an insect or billions for tural approach: to serially section and reconstruct pieces humans. Determining the connectivity of these neu- of both vertebrate and invertebrate nervous systems, rons, via combined anatomical and electrophysiologi- with the stated purpose of using detailed connectomes cal methods, has always been a part of neuroscience. As to reveal how these circuits work4,15–18. Such large- we were writing this, these ideas were being revisited scale projects will provide new anatomical data that will © 2013 Nature America, Inc. All rights reserved. America, Inc. © 2013 Nature from the perspective of massively parallel methods for offer invaluable insights into the functional organiza- dense reconstruction, or ‘connectomics’. One thread of tion of the structures studied. An unbiased approach this analysis involves the detailed, high-density map- to data acquisition always reveals surprises and new npg ping of point-to-point connections between neurons insights. Moreover, because of the scope and size of at synapses1–4. The specialized membrane structures these projects, such efforts will generate unprecedented and synaptic vesicles of synapses can be visualized amounts of data to be analyzed and understood. with an electron microscope, and consequently dense Here we ask what additional information is needed reconstructions of nervous-system connectomes rely beyond connectivity diagrams to understand circuit on electron microscopy of serial brain sections. In a function, informed by the invertebrate circuits whose complementary approach, detailed electrophysiologi- connectivity is known. For the prototypical case, the cal analysis shows how synapses and circuits function complete C. elegans nervous system, the anatomical at high resolution, and is increasingly being applied to connectome was largely established over 25 years large numbers of interconnected neurons. ago14. In a variety of other invertebrate preparations, The first approaches used to map complete circuits connectivity was established using combinations of came from studies of the smaller nervous systems electrophysiological recordings and neuronal tracing 30– of invertebrates. In the 1960s and 1970s, systematic 40 years ago, which enabled researchers to generate electrophysiological recordings from neurons in a wiring map that incorporates activity information. discrete ganglia enabled the identification of neu- Despite their different starting points from anatomy and ronal components of circuits that generate specific electrophysiology, these two approaches have uncovered 1Howard Hughes Medical Institute, The Rockefeller University, New York, New York, USA. 2Volen Center, Brandeis University, Waltham, Massachusetts, USA. 3Department of Biology, Brandeis University, Waltham, Massachusetts, USA. Correspondence should be addressed to C.I.B. ([email protected]). RECEIVED 27 FEBRUARY; ACCEPTED 5 APRIL; PUBLISHED ONLINE 30 MAY 2013; DOI:10.1038/NMETH.2451 NATURE METHODS | VOL.10 NO.6 | JUNE 2013 | 483 HISTORICAL PERSPECTIVE FOCUS ON MAPPING THE BRAIN Figure 1 | Connectivity of two a well-studied invertebrate circuits. (a) Connectivity diagram of the crab STG based on electrophysiological AB PD LPG recordings. Red and blue background Electrical synapse shading indicates neurons that are LP IC LG MG GM primarily part of the pyloric and Chemical inhibitory synapses gastric circuits, respectively. Purple PY VD Int1 DG AM shading indicates that some neurons switch between firing in pyloric and gastric time, and that there is no fixed boundary between the pyloric and gastric circuits. Yellow ASJL b PHAR AIMR highlights two neurons that are both ASJR ASIR AINL electrically coupled and reciprocally ALMR PVM PHAL ASIL AIML HSNL PHCR inhibitory. Green highlights one AWALAINRAWAR ASGL AVFL ADLR IL2R ASEL AWBL ADLL of many examples of neurons that IL2L AWBR PVQL ALML PLNR FLPR ADFL VC05AVM PVDR IL2VRIL2VL CEPVRCEPDR ASGR SDQL LUAR ASER ASHR