Scanning Brain Networks with Micro-CT
Ryuta Mizutani , 1 * Rino Saiga , 1 Susumu Takekoshi , 2 Makoto Arai , 3 Akihisa Takeuchi , 4 and Yoshio Suzuki 4 1 Department of Applied Biochemistry , Tokai University , Hiratsuka , Kanagawa 259-1292 , Japan 2 Tokai University School of Medicine , Isehara , Kanagawa 259-1193 , Japan 3 Tokyo Metropolitan Institute of Medical Science , Setagaya , Tokyo 156-8506 , Japan 4 Japan Synchrotron Radiation Research Institute (JASRI/SPring-8) , Sayo , Hyogo 679-5198 , Japan
*
ryuta@tokai-u.jp Introduction
A wide variety of brain functions including cognition, verbal ability, and decision making are performed by the synergy of a huge number of neurons confi gured in a three- dimensional (3D) network in brain tissue. T e fi rst step to understanding how the brain functions is to analyze this 3D structure. Because neurons exert their functions by interacting with each other, a number of neurons should be visualized simultaneously to illustrate the functional mechanisms of the brain. Although structural studies of brain tissues have been reported, the structures of these tissues, which are composed of many neurons, are diffi cult to comprehend. T is is because there is a lack of quantitative descriptions of neuronal networks, which should be represented with 3D Cartesian coordinates, rather than a 3D distribution of intensities. T is article reports the structures of brain tissues belonging
to humans [ 1 ] and the fruit fl y Drosophila melanogaster [ 2 ] that were ascertained using X-ray tomographic microscopy, a microscopic version of medical computed tomography (CT). In order to analyze the brain network, the structure of each neuron should be described in terms of 3D Cartesian coordinates by building skeletonized models of neurons. T e structural components of the fl y brain network were classifi ed on the basis of their 3D structures. T is classifi ed model allowed identifi cation of anatomical segments. T e network of human brain tissue indicated that the neurons form feedback loops. Such feedback loops can be viewed as canonical elements in brain circuits.
Materials and Methods Contrasting brain tissues . T e transparency of biological tissue to hard X rays enables radiographic analysis of tissue structures. Soſt tissues are composed of light elements, which produce little contrast in a hard X-ray image. In clinical diagnosis, luminal structures of a living body are visualized by using X-ray contrast media. T ese contrast media contain high-atomic-number (high-Z) elements that attenuate X rays effi ciently. Tissue-staining methods using high-Z elements, such as Golgi impregnation and reduced-silver impregnation, are known as conventional techniques for the histology of brain tissues. In this study, the tissue structures were visualized with these impregnation methods.
Post-mortem human cerebral tissues were collected with informed consent from the legal next of kin using protocols approved by ethical committees of organizations related to this study. T e frontal cortex tissues were subjected to Golgi impreg- nation, as described previously [ 1 ]. Neurons were visualized with silver deposits in dendrites, axons, and cell bodies with this method. Wild-type fruit fl ies Drosophila melanogaster Canton-S (Drosophila Genetic Resource Center, Kyoto Institute of Technology, Japan) were raised on standard cornmeal- molasses fl y food and kept at 20°C. Adult fl y brains, also referred to as the cephalic ganglion, were dissected and subjected to modifi ed reduced-silver impregnation, as described previously [ 2 ]. It has been shown that neurons were visualized with metal gold particles derived from the aurate toning reagent used in this method. Tissue embedding . Tissue structures should be kept constant during the data collection. However, biological soſt tissues themselves are not rigid and show deformations depending on ambient conditions. T erefore, the tissue samples were embedded in epoxy resin [ 1 , 2 ] as one would do in the case of transmission electron microscopy.
Figure 1 : (a) X-ray tomographic microscopy at BL20XU of SPring-8. The incident X-ray beam comes from the left, and transmission images were acquired with the detector. (b) Samples for tomographic microscopy. Pellet sample (labeled A) was attached to the fl at surface of the brass fi tting using double-stick tape B. Capillary sample C was inserted in clay stuffed in a hole of brass fi tting D. Clay ejected from a side opening can be seen. Capillary sample E sleeved with a brass tube F was fi xed using a wrench G. (c) Schematic drawing of the upper end of the fi tting D. An opening with a diameter of 0.5 mm was made for venting air and clay under pressure in the hole.
12 doi: 10.1017/S1551929515000784
Resin embedding was performed by transferring the tissue sample from an aqueous solution to fi rst ethanol, then to n -butyl glycidyl ether, and fi nally to the resin. In the case of embedding a block sample with dimensions of 5 × 5 × 5 mm 3 , the
www.microscopy-today.com • 2015 September
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