This proposal is a continuation of our efforts to create a three-dimensional, Google Earth-like, digital Connectome atlas of the C57Black/6J mouse brain. Currently, we have established the entire pipeline for manufacturing, collecting, and processing large-scale, multi-fluorescent connectivity data in a high-throughput, industrialized manner. To track and organize these large-scale data, we have developed the Laboratory Information Management System (LIMS), which expedites data management and reduces human error. Following an unbiased, systematic, stereotaxic grid-based approach using double coinjections of proven neural circuit tracing technology, we have been constructing a skeleton Connectome map (Phase I), which will constitute about 1600 afferent and 1600 efferent pathways from 400 animals. In Phase II, proposed here, we will continuously generate large-scale, ultra-high-resolution connectivity data following a refined grid- based injection strategy using 1200 animals. The collection of these injections will generate a total of 4800 afferent and 4800 efferent pathways, enabling whole brain coverage. Further, in Phase I, we developed the revolutionary iConnectome visualization program (www.MouseConnectome.org) that features a searchable catalog of multi-fluorescent neural tracer injections available to view at high-resolution within their own bright- field Nissl-stained cytoarchitectural background. The iConnectome is accompanied by a comprehensive connectivity online database (BAMS) that enables users to map complex neuronal networks in a matrix format and to correlate them with connectivity data available for the rat, monkey, and human. In addition, the iConnectome Annotation Reporting System (iCARS), developed by our informatics team, will provide level by level annotation information for each injection site. Powerful informatics tools eventually will enable users to compare connectivity patterns of any injection site within a standard 3D anatomic frame. To do this, in Phase II, we will graphically reconstruct neural pathways that link all brain structures and will deliver four versions of the Mouse Connectome Atlas (MCA): (1) MCA1.0 (Google satellite and Google street view) for displaying high- resolution multi-fluorescent labeled raw imaging data; (2) MCA2.0 (Google Map) will provide graphically reconstructed axonal pathways and retrogradely labeled neurons; (3) MCA3.0 (Google driving direction Roadmap) will be the schematic connectivity route map that links brain structures; and (4) Connectome matrix and table-like graphic representation of neural networks generated by BAMS, which will also provide the text version navigation instructions for MCA3.0. All four versions will be synchronized and integrated into the same framework, which will enable us to assemble the ultimate version Google Earth-like Connectome atlas. Although an enormous undertaking, our Phase I progress and our team with world-class expertise in neuroanatomy, brain imaging, and neuroinformatics is a testament to our ability to deliver the completed three- dimensional digital mouse Connectome atlas in Phase III.