Harvey J. Karten 1 , Agnieszka Brzozowska-Prechtl 1 , James Prechtl 1 , Haibin Wang 2 , and Partha P. Mitra 2

1. Digital Atlas of the Zebra Finch Brain (Taeniopygia Guttata): a Dimensional and High Resolution Photo Atlas Harvey J. Karten1, Agnieszka Brzozowska-Prechtl1, James Prechtl1, Haibin Wang2, and Partha P. Mitra2 1) Department of Neuroscience, University of California at San Diego, La Jolla, CA 92093, USA 2) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA Introduction One important feature is the fissure where the tectum and forebrain come together. That fissure is not an unambiguous straight line, but on average, it should be about 60° with respect to the plane of section as shown in the left panel above. As the first step in preparing the atlas, we photographed the block face of serial images of frozen brain. Sections were cut at 30 micrometers, and every other section was photographed with a digital camera mounted above the microtome. These images provide a dimensionally accurate set of registered images that were used to construct the initial phase of the atlas. Serial sections were collected, mounted on glass slides, stained with either Nissl stain or myelin stains. • Neuroanatomical research is undergoing major change, driven by the availability of automated scanning microscopes, ability to digitally store and analyze tera/petabyte scale data sets, and to make these high resolution images available through the internet. Here we present the first high resolution Nissl stained digital images of the brain of the zebra finch, which is the mainstay of songbird research. • The zebra finch has proven to be the most widely used model organism for the study of the neurological and behavioral development of birdsong. A unique strength of this research area is its integrative nature, encompassing field studies and ethologically grounded behavioral biology, as well as neurophysiological and molecular levels of analysis. The availability of dimensionally accurate and detailed atlases and photographs of the brain of male and female animals, as well as of the brain during development, can be expected to play an important role in this research program. Traditionally, atlases for the zebra finch brain have only been available in printed format, with the limitation of low image resolution of the cell stained sections. • The cytoarchitectural high resolution photographs and atlas presented here aim at facilitating electrode placement, connectional studies, and cytoarchitectonic analysis. This initial atlas is not in stereotaxic coordinate space. It is intended to complement the stereotaxic atlases of Akutegawa and Konishi [1], and that of Nixdorf and Bischof [2]. • The advantages of a digital atlas over a traditional paper-based atlas are three-fold. • The digital atlas can be viewed at multiple resolutions. At low magnification, it provides an overview of brain sections and regions, while at higher magnification, it showsexquisite details of the cytoarchitectural structure. • It allows digital “re-slicing” of the brain. The original photographs of brain were taken in certain selected planes of section. However, the brains are seldom sliced in exactly the same planein real experiments. Re-slicing provides a useful atlas inuser-chosen planes, which are otherwise unavailable in the paper-based version. • It can be made available on the internet.High resolution histological datasets can be independently evaluated in light of new experimental anatomical, physiological and molecular studies. The staining pattern for Tyrosine Hydroxylase in the transverse plane is shown below. TH is a rate limiting enzyme in the catecholamine synthesis pathway and is therefore used as an indicator for catecholamines (dopamine, norepinephrine, and epinephrine). We have also obtained connectional data in a series showing retinal projections in the zebra finch. This work is still preliminary and not shown here. But a data set on chick using the same technique provides some excellent examples of single axon tracing of retinal efferent terminals [4], which is available at www.brainmaps.org. Discussion The outer border of the brain, as well as large numbers of internal structures, could be identified in photographs of the frozen block face. The Nissl stained sections can thus be registered to the matching block face photographs. Finally, the complete series of the Nissl and Myelin stained tissue were photographed with a scanning digital Aperio scanner at a resolution of 0.5 micrometers per pixel. The resulting RGB images were typically about 1.4 Gigabytes per section, depending upon the area of individual sections. These images are available at www.zebrafinch.org/atlas, where they can be viewed in multiple resolutions. The images were partitioned into stacks of JPEG files. When being delivered over internet, they were stitched together at client side via Brain Maps API [3] that allows zooming to various resolution levels. Although the results presented here are still very preliminary, the high quality of the images clearly demonstrates the feasibility and benefits of the encompassing cytoarchitectural study.The variance across brains may be reduced by selecting all animals within a limited range of body weight. Extending these methods to experimental studies of molecular anatomy, connectional studies and gene knockouts will provide an invaluable tool for data assessment. On the computational side, we plan to segment and register the brain sections automatically, as an intermediate step to preparing outline drawings and annotations. The algorithms and programs used here can be applied to other anatomy projects, such as high-resolution digital mouse brain atlas that is currently in progress in our lab and that of E.G. Jones at UC Davis (www.brainmaps.org). The lower right panel is enlarged below to reveal greater details of the image with high resolution and contrast. Method Acknowledgement We have developed a method for reproducibly sectioning the brain transversely in approximation of the “Frankfurt” plane of section. Although the method is not perfect, it adds substantial value. It involves orienting brain on the microtome according to a few anatomical features on the lateral surface of the brain. Results This work is supported by the NIH (NS50436). PPM is also supported by the W. M. Keck Foundation and the Crick-Clay Professorship. The brains are sectioned in a standardized “Frankfurt”plane that, for anatomical and cytoarchitectural purpose, is preferable to that used in the Stereotaxic plane of the Nixdorf and Bischof atlas. Using “video template” imaging,we have been able to orient andsection the brain in a replicable plane in six cases. Some variance inevitably exists in light of the variation in brain size even within a single cohort of male zebra finch. The following figure shows a Nissl stained brain section at increasingly higher resolutions. The cell bodies are clearly visible at the highest resolution level (0.5 µm/pixel). References [1] Akutagawa E. and Konishi M., stereotaxic atalas of the brain of zebra finch, unpublished. [2] Nixdorf-Bergweiler B. E. and Bischof H. J., A Stereotaxic Atlas of the Brain Of the Zebra Finch, Taeniopygia Guttata, http://www.ncbi.nlm.nih.gov. [3] Brain Maps API, www.brainmaps.org. [4] Karten H. J., single axon tracing of retinal efferent terminals in chick, www.brainmaps.org. Next, we show a screenshot from a web browser with an image of a brain section opened. Images at various resolution levels can be displayed using the zoom-in and -out buttons at the upper left corner. The small window at the lower right corner displays the location of the image within the brain section.