Despite the wide use of the zebrafish embryo as a model system for neuroscience research, neuroanatomical resources are scarce and most detailed neuroanatomical data has to be divined from diffuse information in the literature and the two available histological atlases of larval and adult brains. Although these paper atlases are an excellent, accurate and indispensable resource, they are not easy to use unless one is already expert in fish neuroanatomy. Traditional atlases standardly use annotated images from serially cut histological sections and so to fully understand the information presented, the user must already have a good understanding of the anatomical relationships and connectivity of the brain as this is difficult to interpret from 2D sections.
zebrafishbrain.org will be unlike traditional neuroanatomy atlases, because it will use a mix of media types to present data, including confocal sections and reconstructions, schematic models and ultimately 3D and 4D movies. Thus far, our approach has been to collect high-resolution confocal stacks from many different transgenic lines at multiple developmental stages and reconstruct images into flattened projections using Improvision volocity software.
An introduction to the zebrafish brain
Brain subdivisions. This video highlights the various major subdivisions of the 4dpf zebrafish brain. The embryos have been labelled with anti-acetylated tubulin and anti-SV2 antibodies
Zebrafish Properties
The embryonic zebrafish has outstanding optical properties that permit imaging of the entire intact brain at high resolution by confocal microscopy. This property, coupled with the availability of large numbers of transgenic lines in which subsets of neurons are labelled by fluorescent reporters make the zebrafish a fantastically powerful model for neuroanatomical studies. Transgenic animals are an excellent resource for resolving neuroanatomy.
Traditional neuroanatomical tracing techniques, such as lipophilic dye labelling of axons, suffer from the problem that it is hard and time consuming to restrict labelling to unique subsets of neurons. Genetic approaches get around this problem by enabling the cell-type specific expression of transgenes encoding proteins that label the entire morphologies of the expressing cells. This approach can be used to label entire classes of neurons or indeed individual neurons.
Imaging Techniques
We have also developed a single cell focal electroporation technique that enables us to express transgenes in individual neurons in the intact brain, providing exceptionally high-resolution information on the morphologies of specific neuronal sub-types. In concert, these techniques allow us to visualise different neuronal subgroups within the CNS and characterize their connectivity.
Future Perspectives
Aside from finding funding to support the project, one of our biggest challenges in the future is the annotation of 3D models. All of the high-resolution confocal stacks we have collected of the various transgenic lines and immunolabelling have the potential to be rendered in 3D as interactive models. Interactive 3D models will allow the user to see structures in any and all orientations. If we are able to properly annotate and integrate these models in a user friendly manner into the atlas, they would enhance it greatly.
Traditional atlases relying on a single media type usually present images in strictly transverse, sagittal or horizontal views that are not so easily interpretable. By using a mixture of media types including confocal sections and reconstructions, 3D and 4D movies and schematic models, we can present information in whatever format is most helpful and we are not constrained by standardised orientations for viewing. Our intention is to present data from whole brain reconstructions down to high-resolution reconstructions of typical morphologies of individual neurons of specific classes. We hope that in addition to being a useful and intuitive tool for the community, the atlas will provide a showcase for the beautiful images and movies being produced by the zebrafish community using cutting edge imaging techniques and rendering software.
Funding
Our progress on this project so far has been funded by the ZF-Models integrated project (with additional Wellcome Trust support to Steve Wilson). This has allowed us to build a beta-version of the atlas and generate tools, resources and datasets to facilitate the development of the zebrafish as a model for human diseases. We have gathered pilot data to populate the atlas from various transgenic lines.
We have recently secured funding for the next three years from the BBSRC for further development of the resource. The project also receives support from the EU via the FP7 projects ZF-Health and NeuroXsys.