A comprehensive understanding of nervous system function requires integration of multiple types of data, from behavioral to anatomical. Ultimately function can be reduced to the patterns of connectivity between cells and the activation of single neurons. In this application we propose a set of aims that makes use of a unique organism with optimal qualities for multimodal nervous system investigation: the simple chordate, Ciona intestinalis. The anatomy of the Ciona larval tadpole CNS shows strong conservation with the CNS of the vertebrates, including homologs of the forebrain, midbrain/hindbrain, and spinal cord. Despite these conserved vertebrate-like features the Ciona tadpole is barely 1 mm long and has only ~2500 cells. Moreover the tadpole CNS has only 177 neurons. While the development of the Ciona CNS has been the subject of investigation for many years, new research opportunities have opened with the recent completion of the Ciona tadpole CNS connectome from serial section electron microscopy. The overall goal of this proposal is to build from the connectome data to identify correlates of behavior at the level of single, identifiable neurons. This project will be the combined undertaking of three research groups with expertise in Ciona live imaging and transgenesis, computer vision and image analysis, and electron microscopy and Ciona neuroanatomy. Preliminary data presented here demonstrate that the transparency and small size of Ciona tadpoles make them ideal for live imaging of brain activity using genetically encoded Ca2+ or voltage indicators. The aims of this proposal will be, first, to expand Ciona as a model of nervous system function, including a detailed characterization of behavior, the generation of a brain atlas at cellular resolution, and the quantification of stereotypy in brain cellular anatomy and connectivity; and, second, to apply these new tools in the characterization of patterns of neuronal activity as Ciona larvae respond to sensory cues by taking advantage of state-of-the-art imaging and neural activity detection methods. The connectome makes specific and novel predictions about patterns of neural activity, and the relative simplicity of the Ciona CNS will provide an innovative system for validating such predictions. This project presents challenges in image acquisition, analysis, and multimodal integration that stretch the limits of current technology, but holds the potential to provide an unparalleled view of chordate CNS function spanning multiple levels, from behavioral to synaptic.