Project Abstract/Summary Elucidating the structure and function of brain circuits is arguably this century's greatest scientific challenge. Pioneering studies in invertebrates have established that this challenge can be met by combining anatomical circuit diagrams ("connectomes"), neuronal activity maps and defined behavioral assays. Recent technological progress now makes it possible to apply this approach to the vertebrate nervous system. The main goal of this proposal is to generate image data sets that are based on serial electron microscopy and have sufficient scale and resolution to create maps of the full set of neurons and synaptic connections in the larval zebrafish brain. The ability to reconstruct local circuits within that data set will be tested for small and well- described sub-networks, the trigeminal sensory ganglion and the optic tectum. To relate structure and function in these dedicated sub-circuits, functional calcium imaging will be combined with EM reconstruction. The combination of the translucence, relative simplicity and small size of the larval zebrafish's nervous system brings this otherwise daunting task into the realm of feasibility. We have assembled a team of four groups: Drs. Reid and Lichtman provide expertise and facilities for serial EM reconstruction. Drs. Engert and Schier provide the functional imaging technology based on in vivo 2-photon laser scanning. The experiments proposed here will provide the foundation to reconstruct the circuit diagram of an entire vertebrate brain and combine sub-circuit anatomy and function in the context of a behaving animal. The EM datasets will be available through open access via a web-based data storage and retrieval system. PUBLIC HEALTH RELEVANCE: Project Narrative We propose to examine the structure and function of the larval zebrafish brain at sub- cellular resolution. We will combine functional imaging using calcium indicators with serial electron microscopy-assisted reconstruction of the zebrafish brain.