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BackgroundUnderstanding the neurobiological basis of cognition and behavior, and disruptions to these processes following injury and disease, requires a large-scale assessment of neural populations, and knowledge of their patterns of connectivity.New methodWe present an analysis platform for large-scale investigation of functional and neuroanatomical connectivity in rodents. Retrograde tracers were injected and in a subset of animals behavioral tests to drive immediate-early gene expression were administered. This approach allows users to perform whole-brain assessment of function and connection in a semi-automated quantitative manner. Brains were cut in the coronal plane, and an image of the block face was acquired. Wide-field fluorescent scans of whole sections were acquired and analyzed using Matlab software.ResultsThe toolkit utilized open-source and custom platforms to accommodate a largely automated analysis pipeline in which neuronal boundaries are automatically segmented, the position of segmented neurons are co-registered with a corresponding image acquired during sectioning, and a 3-D representation of neural tracer (and other products) throughout the entire brain is generated.Comparison with existing methodsCurrent whole brain connectivity measures primarily target mice and use anterograde tracers. Our focus on segmented units of interest (e.g., NeuN labeled neurons) and restricting measures to these units produces a flexible platform for a variety of whole brain analyses (measuring activation, connectivity, markers of disease, etc.).ConclusionsThis open-source toolkit allows an investigator to visualize and quantify whole brain data in 3-D, and additionally provides a framework that can be rapidly integrated with user-specific analyses and methodologies.