Pavel Osten - US grants

DESCRIPTION (provided by applicant): Autism comprises a spectrum of highly heritable disorders and today the relevant susceptibility genes are being identified by large scale genomics projects. An important outcome of the identification of autism genes is the possibility to use genetic mouse models to study circuit, cellular and molecular mechanisms by which these genes affect brain development and function. The current project is focused on three mouse models carrying mutations in cell adhesion molecules (CAMs): the neuroligin 3 R451C, neuroligin 4 null, and Cntnap2 null mice. These mice were selected as representatives of a larger family of synaptic genes linked to autism, which includes neuroligins, neurexins, Cntnap, cadherin, contactin, and Shank proteins. The identification of rare mutations in these genes provides a strong support for the role of synaptic maturation and function in autism. The current proposal aims to begin to dissect the underlying circuit and cellular mechanisms in the three mouse models. In order to be able to compare brain functions in different autism mouse models, we have developed a novel method for high-throughput imaging of whole mouse brains. This method, which we call serial two- photon (STP) tomography, integrates two-photon laser-scanning microscopy and tissue sectioning. To study brain functions by STP tomography, we use transgenic c-fos-GFP mice that express green fluorescent protein (GFP) as a reporter for the induction of the immediate early gene c-fos. This allows us to identify brain regions with abnormal c-fos induction, and by extension neural activation, evoked during behavioral tasks or by systemic drug applications. Such abnormal regions-candidate brain areas for autism-related pathology-then become the focus of detailed electrophysiological and anatomical studies, which aim to determine the exact underlying circuit and cellular mechanisms. The Specific Aims are: 1. To study how CAM mutations affect brain circuits mediating social behavior. 2. To study how CAM mutations affect oscillatory cortical activity and the balance of brain excitation and inhibition. 3. To study anatomical connectivity and cellular physiology of candidate brain regions. We believe that a successful completion of the proposed experiments will provide mechanistic insights into neurodevelopmental changes in brain functions that lie downstream of the synaptic genes in autism. Our ultimate goal is to use such results to formulate hypotheses for the development and testing of therapeutic strategies in the future. PUBLIC HEALTH RELEVANCE: Autism spectrum disorders (ASDs) are among the most heritable human diseases. Our goal is to identify causal links between autism susceptibility genes and neural circuit deficits in genetic mouse models of autism. We hope that this work will identify candidate brain regions and circuits for detailed mechanistic studies and therapeutic development.

? DESCRIPTION (provided by applicant): The mouse brain comprises ~70 million neurons and ~30 million glia and other cells. Neurons have been traditionally classified based on their morphology, connectivity, stimulus-response, gene expression, and location in the brain. While we know reasonably well the main cell types that are present at different brain locations, we have little quantitative knowledge about brainwide cell type distribution. In addition, cell type-based brainwide connectivity, especially at the level of projection patterns of single neurons, also remains largely unmapped. This knowledge gap prevents us from incorporating the accumulated cell type-based cellular data into comprehensive circuit models of mammalian brain function. Here we propose to develop a largely automated methodology for 1) quantitative atlasing of neuronal and glia cell types on the basis of their complete cell counts per anatomical regions in the whole mouse brain, and 2) complete tracing of axonal arborizations of identified neuronal cell types. The specific Aims are: Specific Aim 1 (FOA goals 1, 4-6): To develop a versatile platform for brainwide atlasing of molecularly defined cell types. Computational and statistical methods will be developed to obtain accurate cell numbers in whole brains of cell type-specific reporter mice imaged by serial two-photon (STP) tomography and registered to reference brain atlases. These methods will then be used to map the distribution of five different GABAergic cell types during development, in the adult male and female brain, and during aging. Specific Aim 2 (FOA goal 6): To enhance the throughput of cell type-based atlasing and connectivity mapping through the development of a second-generation imaging platform. A light-sheet fluorescent microscopy-based method, named Oblique Plane Tomography (OPT), will be developed for fast high- resolution imaging of CLARITY-treated mouse brains. Computational methods will be established to allow the use of OPT in cell type atlasing and projection tracing in the whole mouse brain. Specific Aim 3 (FOA goals 2-3): Neuroinformatics infrastructure: an integrated web portal for cell-type specific data, tools & analytics. The developed methods, including a statistical toolbox for online and offline analytics, and the data generated will be distributed via a public website that will also serve to integrate the relevant information on the molecular identity, location, connectivity and other cell type characteristics. Relevance to public health: We will establish an automated and quantitative approach to the study of cell type distribution and connectivity in the mouse brain. Our methods can be readily scaled up and applied to a broad range of cell type-specific studies of both normal brain functions and pathological conditions related to human brain disorders.

Even though scientists have been captivated by the diversity of brain cells for well over a century, since the initial anatomical descriptions by Santiago Ramón y Cajal, it is only recently that the developments in new technologies allowed us to truly appreciate the astonishing complexity of cell types in the mammalian brain. Yet, even today, our knowledge of cell type anatomy and function is largely limited to a few brain areas, such as the sensory cortex or the hippocampus. This lack of whole-brain data has led to generalizations from one brain area to other regions with similar architecture. However, our preliminary studies applying quantitative whole brain analyses have revealed unexpected region specific differences in cell type ratios and uncovered new gender-based differences in cellular anatomy that can inform our understanding of brain development, wiring and function. We thus believe that closing the gaps in our knowledge of the cellular architecture of the brain will be a major contribution to enable a modern quantitative understanding of the brain as a system. Here we propose to establish an Anatomical Collaboratory for Systematic Atlasing of Cell-Type Distribution and Morphology in female and male brain. This Collaboratory will apply standardized, unbiased and largely automated methods developed in our laboratories to atlas the distribution and morphology of >80 molecularly defined cell classes and cell type across all regions of the female and male brain. This work will yield the most comprehensive characterization of cell type anatomy in the mammalian brain to date, establishing a structural basis for building an integrated Cell Type Brain Atlas. Specific Aim 1: Quantify and atlas the brainwide distribution of molecularly defined cell types. This work will generate a quantitative brain atlas of the distributions of molecularly defined cell classes and cell types within the Common Coordinated Framework (CCF) of the adult brain. Specific Aim 2: Quantify and atlas the brainwide morphologies of molecularly defined cell types. This work will generate a comprehensive structural brain atlas of somatodendritic cell-type morphologies across all CCF anatomical areas in the female and male brain. Specific Aim 3: Develop and deploy a neuroinformatics infrastructure: an integrated web portal for cell-type specific data, tools, and analytics. This work will disseminate the generated data via CSHL cell type-dedicated BrainArchitecture web portal and through the BRAIN Cell Data Center (BCDC). !