Louise Bertrand

Class III β-Tubulin Is Constitutively Coexpressed With Glial Fibrillary Acidic Protein and Nestin in Midgestational Human Fetal Astrocytes: Implications for Phenotypic Identity

Class III &bgr;-tubulin isotype (&bgr;III-tubulin) is widely regarded as a neuronal marker in developmental neurobiology and stem cell research. To test the specificity of this marker protein, we determined its expression and distribution in primary cultures of glial fibrillary acidic protein (GFAP)-expressing astrocytes isolated from the cerebral hemispheres of 2 human fetuses at 18 to 20 weeks of gestation. Cells were maintained as monolayer cultures for 1 to 21 days without differentiation induction. By immunofluorescence microscopy, coexpression of &bgr;III-tubulin and GFAP was detected in cells at all time points but in spatially distinct patterns. The numbers of GFAP+ cells gradually decreased from Days 1 to 21 in vitro, whereas &bgr;III-tubulin immunoreactivity was present in 100% of cells at all time points. &bgr;-III-tubulin mRNA and protein expression were demonstrated in cultured cells by reverse-transcriptase-polymerase chain reaction and immunoblotting, respectively. Glial fibrillary acidic protein+/&bgr;-III-tubulin-positive cells coexpressed nestin and vimentin but lacked neurofilament proteins, CD133, and glutamate-aspartate transporter. Weak cytoplasmic staining was detected with antibodies against microtubule-associated protein 2 isoforms. Confocal microscopy, performed on autopsy brain samples of human fetuses at 16 to 20 gestational weeks, revealed widespread colocalization of GFAP and &bgr;III-tubulin in cells of the ventricular/subventricular zones and the cortical plate. Our results indicate that in the midgestational human brain, &bgr;III-tubulin is not neuron specific because it is constitutively expressed in GFAP+/nestin+ presumptive fetal astrocytes.

Thyroid hormone distribution in the mouse brain: the role of transthyretin

Transthyretin is the major thyroxine-binding protein in the plasma of rodents, and the main thyroxine-binding protein in the cerebrospinal fluid of both rodents and humans. The choroid plexus synthesizes transthyretin and secretes it to the cerebrospinal fluid. Although it was suggested that transthyretin might play an important role in mediating thyroxine transfer from the blood into the brain across the choroid plexus-cerebrospinal fluid barrier, newer findings question this hypothesis. Because thyroid hormone passage across brain barriers is a precondition for its action in the CNS, and because brain is an important target of thyroid hormone action, we investigated the role of transthyretin in mediating thyroid hormone access to and distribution within the brain in a transthyretin-null mouse model system. In this report we describe the results derived from use of film autoradiography, a technique that yields definitive morphological results. Film autoradiograms were prepared at 3 and 19 h after intravenous injection of either high specific activity [(125)I]thyroxine or [(125)I]triiodothyronine. Image analyses were designed to demonstrate regional changes in hormone distribution, and to highlight alterations in iodothyronine delivery from ventricles to brain parenchyma. We find no qualitative or quantitative differences in these parameters between the transthyretin-null and the wild-type mouse brain after either [(125)I]thyroxine or [(125)I]triiodothyronine administration. The data presented here now provide definitive evidence that, under standard laboratory conditions, transthyretin is not required for thyroid hormone access to or distribution within the mouse brain. This study also provides the first map of iodothyronine distribution in the brain of the mouse.

Informatics center for mouse genomics: the dissection of complex traits of the nervous system.

In recent years, there has been an explosion in the number of tools and techniques available to researchers interested in exploring the genetic basis of all aspects of central nervous system (CNS) development and function. Here, we exploit a powerful new reductionist approach to explore the genetic basis of the very significant structural and molecular differences between the brains of different strains of mice, called either complex trait or quantitative trait loci (QTL) analysis. Our specific focus has been to provide universal access over the web to tools for the genetic dissection of complex traits of the CNS—tools that allow researchers to map genes that modulate phenotypes at a variety of levels ranging from the molecular all the way to the anatomy of the entire brain.Our website, The Mouse Brain Library (MBL; http://mbl.org) is comprised of four interrelated components that are designed to support this goal: The Brain Library, iScope, Neurocartographer, and WebQTL. The centerpiece of the MBL is an image database of histologically prepared museum-quality slides representing nearly 2000 mice from over 120 strains—a library suitable for stereologic analysis of regional volume. The iScope provides fast access to the entire slide collection using streaming video technology, enabling neuroscientists to acquire high-magnification images of any CNS region for any of the mice in the MBL. Neurocartographer provides automatic segmentation of images from the MBL by warping precisely delineated boundaries from a 3D atlas of the mouse brain. Finally, WebQTL provides statistical and graphical analysis of linkage between phenotypes and genotypes.

Design and implementation of software for assembly and browsing of 3D brain atlases

Visualization software for three dimensional digital brain atlases present many challenges in design and implementation. These challenges include the design of an effective human interface, management of large data sets, display speed when slicing the data set for viewing/browsing, and the display of delineated volumes of interest (VOI). We present a software design, implementation and storage architecture that addresses these issues, allowing the user to navigate through a reconstructed volume quickly and smoothly, with an easy-to-use human interface. The software (macostat, for use with Macintosh OS) allows the user to rapidly display slices of the digital atlas at any arbitrary slicing angle, complete with delineated VOIs. The VOIs can be assigned colors of the users choosing. The entire atlas, or selected portions, may be resliced with slices stored as individual image files, complete with delineations. These delineations may be transferred to corresponding sections of experimental materials using our analysis program (brain). The software may be obtained from the laboratorys web site: http://www.neuroterrain.org

Surface alignment of an elastic body using a multiresolution wavelet representation

An algorithm for nonlinear registration of an elastic body is developed. Surfaces (outlines) of known anatomic structures are used to align all other (internal) points. The deformation field is represented with a multiresolution wavelet expansion and is modeled by the partial differential equations of linear elasticity. A hierarchical approach that reduces algorithm complexity is adopted. The performance of the algorithm is evaluated by two-dimensional alignment of sections from mouse brains located in the olfactory bulbs. The registration algorithm was guided by manually delineated contours of a subset of brain structures and validated based on another subset of brain structures. The wavelet alignment algorithm produced a twofold to fivefold improvement in accuracy over an affine (linear) alignment algorithm.

The neuroterrain 3D mouse brain atlas

A significant objective of neuroinformatics is the construction of tools to readily access, search, and analyze anatomical imagery. This goal can be subdivided into development of the necessary databases and of the computer vision tools for image analysis. When considering mesoscale images, the latter tools can be further divided into registration algorithms and anatomical models. The models are atlases that contain both bitmap images and templates of anatomical boundaries. We report here on construction of such a model for the C57BL/6J mouse. The intended purpose of this atlas is to aid in automated delineation of the Mouse Brain Library, a database of brain histological images of importance to neurogenetic research.

Glioneuronal phenotype in a diencephalic pilomyxoid astrocytoma.

We report the presence of divergent populations of cells in a hypothalamic/chiasmatic pilomyxoid astrocytoma of an 11-month-old male, exhibiting differential immunohistochemical localizations for glial fibrillary acidic protein (GFAP) and synaptophysin. The tumor cells were negative for Neu-N and neurofilament protein. Ultrastructurally, the tumor comprised 2 cell types, one with features attributable to a neuronal phenotype alongside cells exhibiting an overt astroglial phenotype. This composite organization was confirmed by confocal microscopy, which revealed 2 distinct, albeit tightly interwoven, populations of GFAP and synaptophysin-labeled tumor cells. Our results indicate that a subset of the so-called pilomyxoid astrocytomas of the hypothalamic/chiasmatic region may represent phenotypically mixed glioneuronal neoplasms distinct from the pilocytic astrocytomas.

Localization of sections within the brain via 2D to 3D image registration

The mouse brain library (MBL) is a database of brain images, each consisting of sparse coronal or horizontal sections. To facilitate morphometric research, it is necessary to index each of these sections by its location within a canonical 3D atlas (NeuroTerrain). This is done with a 2D to 3D matching technique which was developed in this study. The registration method is imaged-based and uses a genetic algorithm to find the local-affine transformation that maximizes a mutual information metric. The average distance between the registration results achieved with the proposed method as compared with manual matching by an expert was 250 microns. This compares well with repeated manual trials where inter-trial matching distance was on average 200 microns.

Planar-to-Curved-Surface Image Registration

A 2D-to-3D non-rigid intensity-based registration method is proposed. This planar-to-curved-surface image alignment algorithm matches experimental data of twodimensional images with their corresponding images overlaid on a curved-surface within a volumetric image. This PDE-based 2D-to-3D registration technique allows for inter-modality matching of sectional data with a volumetric image of homologous objects. We demonstrate the feasibility of this method through simulations and in the application of atlas-based segmentation of a mouse brain histological and MRI images

3D atlases - bridges between neurogenomics and neuroanatomy

Neuroscience research in the post-genomic era requires rapid screening and analysis of large tissue expanse. It demands close integration of information derived from histological procedures and genomic data. This is impeded by the absence of technology to automatically extract histological information and to link it to genomic data. We are developing the necessary tools. At the core of these are atlases, visualization tools and registration algorithms. This paper describes a cooperative effort in research and development of these technologies.