(Taken from application abstract): New methods have led to rapid expansion of information on the molecular makeup of nerve cells and glia. Previously unknown proteins are being discovered at a rapid rate. Antibodies are now readily available to map the cellular and subcellular localization of these proteins, but tools for organizing and integrating these data with new and existing information are lacking. Computerized databases for accessing and visualizing the 3 dimensional (3-D) distributions of various macromolecules will provide important tools for the neurobiologist interested in modeling the brain's molecular complexity in health and disease. In this project we will develop techniques and software tools for the acquisition and representation of immunocytochemical data on the 3-D distribution of cellular constituents within realistic compartmental neuronal models. Macromolecules involved in inter- and intra-cellular signaling such as ion channels, neurotransmitter receptors and second messenger proteins will be localized using light microscopic (LM) and electron microscopic (EM) immunocytochemistry. Concurrently, realistic morphological representations of the neurons and neuronal compartments expressing these proteins will be obtained from intra cellularly injected neurons. The immunolocalization data will be combined with compartmental neuronal models based on these identified neurons and used to populate an atlas-based database for representation, storage and retrieval of information on neurons and the locations of their constituents. Initial studies will concentrate on the cerebellum with plans to study other brain regions later (e.g., neostriatum, hippocampus, olfactory bulb, cortex). The techniques and software developed will be made available to other neuroscientists to use in adding results from similar studies to the database. To accomplish these goals we will develop or implement: 1) a standard scheme for acquiring 3-D immunolocalization data by using double and triple label immunofluorescence and confocal LM; 2) methods for acquiring large 3-D datasets on neuronal form using intra cellularly filled neurons imaged with confocal LM and EM; 3) methods for combining immunocytochemical localization with intracellular filling to aid in determining the distribution of target molecules relative to cellular compartments; 4) software tools and methods for segmentation, editing, and data entry into a database for 3-D data on neuronal form acquired using LM and EM datasets; 5) software and methods for acquiring and merging multi resolution datasets; 6) a multi resolution database and visualization system integrating these data; and 7) software to access and query this database over the Internet.
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