Abstract

In order to localize the neural circuits involved in generating behaviors, it is necessary to assign activity onto anatomical maps of the nervous system. Using brain registration across hundreds of larval zebrafish, we have built an expandable open-source atlas containing molecular labels and definitions of anatomical regions, the Z-Brain. Using this platform and immunohistochemical detection of phosphorylated extracellular signal–regulated kinase (ERK) as a readout of neural activity, we have developed a system to create and contextualize whole-brain maps of stimulus- and behavior-dependent neural activity. This mitogen-activated protein kinase (MAP)-mapping assay is technically simple, and data analysis is completely automated. Because MAP-mapping is performed on freely swimming fish, it is applicable to studies of nearly any stimulus or behavior. Here we demonstrate our high-throughput approach using pharmacological, visual and noxious stimuli, as well as hunting and feeding. The resultant maps outline hundreds of areas associated with behaviors.

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Acknowledgements

We are grateful to M. Wullimann (Ludwig Maximilian University) for his critical and detailed input regarding the identification of regions for the Z-Brain segmentation, Y. Yoshihara (RIKEN Brain Science Institute) and T. Okuyama (University of Tokyo) for pointing us to the pERK antibody, G. Jefferis (MRC Laboratory of Molecular Biology) for help with brain registrations, M. Nikitchenko (Harvard) for help with computational and web resources, A. Douglass (University of Utah) and J. Wortzman (Harvard) for creation of the Tg(UAS:GCaMP5G) line, D. Prober (Caltech) for sharing the Tg(hcrt:mRFP) and Tg(qrfp:GFP) lines before publication, M. Hasemeyer (Harvard) for many helpful discussions, and the many members of the zebrafish community who shared their transgenic fish lines. Funding was provided by an HFSP Long-Term fellowship (LT000772/2012-L to O.R.); the Agency for Science, Technology and Research, Singapore (C.L.W.); a Marie Curie Fellowship (E.A.N.); the Swartz Foundation (J.E.F.); the Simons Foundation (SCGB award 325207 to F.E.); and NIH grants R01 HL109525, U01 MH105960 (both to A.F.S.), R24 NS086601, U01 NS090449 and DP1 NS082121-02 (to F.E.).

Author information

Author notes

    • Eva A Naumann
    • , David Schoppik
    •  & Ruben Portugues

    Present addresses: Department of Cell and Developmental Biology, University College London, London, UK (E.A.N.); Departments of Otolaryngology and Neuroscience & Physiology, NYU Langone School of Medicine, New York, New York, USA (D.S.); Sensorimotor Control Research Group, Max Planck Institute of Neurobiology, Martinsried, Germany (R.P.).

    • Florian Engert
    •  & Alexander F Schier

    These authors contributed equally to this work.

Affiliations

  1. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Owen Randlett
    • , Eva A Naumann
    • , Onyeka Nnaemeka
    • , David Schoppik
    • , Ruben Portugues
    • , Alix M B Lacoste
    • , Clemens Riegler
    • , Florian Engert
    •  & Alexander F Schier
  2. Program in Neuroscience, Harvard Medical School, Boston, Massachusetts, USA.

    • Caroline L Wee
  3. Center for Brain Science, Harvard University, Cambridge, Massachusetts, USA.

    • James E Fitzgerald
    •  & Alexander F Schier
  4. Department of Neurobiology, Faculty of Life Sciences, University of Vienna, Vienna, Austria.

    • Clemens Riegler
  5. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

    • Alexander F Schier
  6. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA.

    • Alexander F Schier
  7. FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Alexander F Schier

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Contributions

O.R., F.E. and A.F.S. conceived of the project. O.R. performed most experiments and data analysis. C.L.W., E.A.N., D.S. and A.M.B.L. also performed experiments. E.A.N., J.E.F. and R.P. also analyzed data. D.S. and C.R. created new transgenic fish strains. O.N. built the website. O.R., F.E. and A.F.S. wrote the paper, with input from all other authors. F.E. and A.F.S. supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Owen Randlett or Florian Engert.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–6, Supplementary Table 1

Videos

  1. 1.

    Anatomical labels in the Z-Brain.

    The 29 image stacks in the Z-Brain database are show, scrolling from dorsal to ventral. Stacks are in the same order as in Supplementary Table 1, moving right to left and top to bottom of the montage.

  2. 2.

    Segmented anatomical regions in the Z-Brain.

    Three slice views of the Z-Brain are shown in the left panel (blue box = x/y, green = x/z, red =y/z). Shown are the outlines of all the anatomical regions contained in the Z-Brain, overlayed on the Tg(HuC:H2B-RFP) mean stack label. The slice region for each box is shown by the colored tick marks. The right panel displays a rotating 3D reconstruction of the Z-Brain region outlines. Colours are assigned pseudo-randomly, biased such that the major anatomical regions are enriched for the following colours: Telencephalon \xA0 Green, Diencephalon \xA0 Cyan, Mesencephalon \xA0Yellow, Rhombencephalon \x96 Red, Spinal Cord \xA0 Magenta, Ganglia/Other (including eyes and olfactory epithelium) \xA0 Blue.\xA0

  3. 3.

    MAP-Map: exposure to pentylenetetrazol (PTZ), related to Fig. 2f.

    Green and magenta signals represent increased and decreased neural activity (pERK levels) over controls, respectively. Blue, green and red boxed regions show the x/y, x/z and y/z slice views. Tick marks depict the slice view position.

  4. 4.

    MAP-Map: exposure to MS-222, related to Fig. 2g.

    Green and magenta signals represent increased and decreased neural activity (pERK levels) over controls, respectively. Blue, green and red boxed regions show the x/y, x/z and y/z slice views. Tick marks depict the slice view position.

  5. 5.

    MAP-Map: exposure to MS-222, related to Fig. 2h.

    Green and magenta signals represent increased and decreased neural activity (pERK levels) over controls, respectively. Blue, green and red boxed regions show the x/y, x/z and y/z slice views. Tick marks depict the slice view position.

  6. 6.

    MAP-Map: the optomotor response, related to Fig. 3c.

    Green and magenta signals represent increased and decreased neural activity (pERK levels) over controls, respectively. Blue, green and red boxed regions show the x/y, x/z and y/z slice views. Tick marks depict the slice view position.

  7. 7.

    MAP-Map: exposure to mustard oil, related to Fig. 4a.

    Green and magenta signals represent increased and decreased neural activity (pERK levels) over controls, respectively. Blue, green and red boxed regions show the x/y, x/z and y/z slice views. Tick marks depict the slice view position.

  8. 8.

    MAP-Map: tap stimuli, related to Fig. 4b.

    Green and magenta signals represent increased and decreased neural activity (pERK levels) over controls, respectively. Blue, green and red boxed regions show the x/y, x/z and y/z slice views. Tick marks depict the slice view position.

  9. 9.

    MAP-Map: exposure to noxious heat, related to Fig. 4c.

    Green and magenta signals represent increased and decreased neural activity (pERK levels) over controls, respectively. Blue, green and red boxed regions show the x/y, x/z and y/z slice views. Tick marks depict the slice view position.

  10. 10.

    MAP-Map: electric shock stimuli, related to Fig. 4d.

    Green and magenta signals represent increased and decreased neural activity (pERK levels) over controls, respectively. Blue, green and red boxed regions show the x/y, x/z and y/z slice views. Tick marks depict the slice view position.

  11. 11.

    Intersection of aversive MAP-Maps, related to Fig. 4e.

    Green and magenta signals represent increased and decreased neural activity (pERK levels) over controls, respectively. Blue, green and red boxed regions show the x/y, x/z and y/z slice views. Tick marks depict the slice view position.

  12. 12.

    MAP-Map: exposure to paramecia, related to Fig. 4i.

    Green and magenta signals represent increased and decreased neural activity (pERK levels) over controls, respectively. Blue, green and red boxed regions show the x/y, x/z and y/z slice views. Tick marks depict the slice view position.

Excel files

  1. 1.

    Supplementary Data 1

    Anatomical analyses of MAP-Maps using the Z-Brain. For each MAP-Map (Fig. 2f-h, 3c, 4a-e,i) there are two table outputs as separate sheets in the .xls file, reflecting green (activation) signals, and magenta (suppressive) signals, relative to controls. In each table, column 1 is the name of the ranked Z-Brain region, column 2 is the mean signal within that region, where 65535 would represent a fully saturated signals with all voxels having greater than or equal to a 0.5 delta median value, and 0 would represent no signal. In columns 3 through 12 we list the top five candidate anatomical labels that overlap with the MAP-Map signal in the region. Odd columns list the label's name, even columns indicate the enrichment for that label, which is calculated as the mean label signal within the pixels that show activity, divided by the mean label signal in the 50 pixels surrounding the region. Therefore, numbers greater than 1 indicate signal enrichment over the surrounding.

  2. 2.

    Supplementary Data 2

    Quantification and ranking of label signals within Z-Brain regions. For each region the labels are ranked according to either the local signal enrichment calculated as the mean signal within the region, divided by the mean signal in a 50 voxel radius surrounding the region (Brightness Ratio to Surrounding sheet), or the mean signal within the region (Average Brightness sheet).

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DOI

https://doi.org/10.1038/nmeth.3581

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