Electron Microscopy Core The synaptic circuits that underlie vertebrate behavior are of interest to all of the investigators on this grant (and neuroscientists more generally). Generating neural circuits in zebrafish is the principal goal of this core. Getting this connectivity entails revealing four essential features of neural circuitry for each neuron: first, rendering its local input at sufficient resolution to detect all the impinging synaptic connections, second, identifying the sign of these connections, third, identifying the cells giving rise to this presynaptic input and fourth, identifying the postsynaptic cells innervated by this neuron's axon. Ideally, these requirements need to be met for all the neurons in one vertebrate brain because without this information, neuroscientists cannot accurately trace signals, synapse by synapse, from sensory input to motor output -- a profound impediment to deciphering of how a brain's structure relates to its behavioral repertoire. Owing to the rapid pace of technological and computational improvements, it is now feasible for the first time to create a whole vertebrate animal connectional map. The methods we will use automate a serial section electron microscopy pipeline so that high resolution (nanoscale) images can be acquired over large (millimeter scale) volumes. This core will use a modern serial-section multibeam scanning electron microscopy approach that we have been developing for about 10 years 1 2 3 4 5 6 7 8 9 10 11 12 13 14 and combine this acquisition workflow with sophisticated image processing, leveraging machine learning expertise and infrastructure at Google, the Advanced Physics Lab at Johns Hopkins, ground truth and proofreading taking place in the Outreach and Training Core and the proofreading and manual tracing described in Project 3. One major purpose of this Core is to assure that we do this integration successfully. One important aspect of this integration that will make the connectional map more useful analytically, will be to oversee the technical aspects of the overlay of essential fluorescence microscopy data derived from the same fish. This light microscopy-based data reveals whether neurons are excitatory or inhibitory and the activity patterns of neurons during eight behaviors. We believe this Correlated Light and Electron Microscopy (CLEM) will add powerful new dimensions to the analysis of the wiring diagram of a behaving vertebrate.