Drosophila melanogaster has a rich repertoire of innate and learned behaviors. Its 100,000-neuron brain is a large but tractable target for comprehensive neural circuit mapping. Only electron microscopy (EM) enables complete, unbiased mapping of synaptic connectivity; however, the fly brain is too large for conventional EM. We developed a custom high-throughput EM platform and imaged the entire brain of an adult female fly at synaptic resolution. To validate the dataset, we traced brain-spanning circuitry involving the mushroom body (MB), which has been extensively studied for its role in learning. All inputs to Kenyon cells (KCs), the intrinsic neurons of the MB, were mapped, revealing a previously unknown cell type, postsynaptic partners of KC dendrites, and unexpected clustering of olfactory projection neurons. These reconstructions show that this freely available EM volume supports mapping of brain-spanning circuits, which will significantly accelerate Drosophila neuroscience..

Neuronal circuit mapping using electron microscopy demands laborious proofreading or reconciliation of multiple independent reconstructions. Here, we describe new methods to apply quantitative arbor and network context to iteratively proofread and reconstruct circuits and create anatomically enriched wiring diagrams. We measured the morphological underpinnings of connectivity in new and existing reconstructions of Drosophila sensorimotor (larva) and visual (adult) systems. Synaptic inputs were preferentially located on numerous small, microtubule-free 'twigs' which branch off a single microtubule-containing 'backbone'. Omission of individual twigs accounted for 96% of errors. However, the synapses of highly connected neurons were distributed across multiple twigs. Thus, the robustness of a strong connection to detailed twig anatomy was associated with robustness to reconstruction error. By comparing iterative reconstruction to the consensus of multiple reconstructions, we show that our method overcomes the need for redundant effort through the discovery and application of relationships between cellular neuroanatomy and synaptic connectivity.