Associating stimuli with positive or negative reinforcement is essential for survival, but a complete wiring diagram of a higher-order circuit supporting associative memory has not been previously available. Here we reconstruct one such circuit at synaptic resolution, the Drosophila larval mushroom body. We find that most Kenyon cells integrate random combinations of inputs but that a subset receives stereotyped inputs from single projection neurons. This organization maximizes performance of a model output neuron on a stimulus discrimination task. We also report a novel canonical circuit in each mushroom body compartment with previously unidentified connections: reciprocal Kenyon cell to modulatory neuron connections, modulatory neuron to output neuron connections, and a surprisingly high number of recurrent connections between Kenyon cells. Stereotyped connections found between output neurons could enhance the selection of learned behaviours. The complete circuit map of the mushroom body should guide future functional studies of this learning and memory centre.AL-K was supported by NIH grant #F32DC014387. AL-K and LFA were supported by the Simons Collaboration on the Global Brain. LFA was also supported by the Gatsby, Mathers and Kavli Foundations. CEP and YP were supported by the DARPA XDATA program (AFRL contract FA8750-12-2-0303) and the NSF BRAIN EAGER award DBI-1451081. KE and AST thank the Deutsche Forschungsgemeinschaft, TH1584/1-1, TH1584/3- 1; the Swiss National Science Foundation, 31003A 132812/1; the Baden Wurttemberg Stiftung; Zukunftskolleg of the University of ¨ Konstanz and DAAD. BG and TS thank the Deutsche Forschungsgemeinschaft, CRC 779, GE 1091/4-1; the European Commission, FP7-ICT MINIMAL. We thank the Fly EM Project Team at HHMI Janelia for the gift of the EM volume, the Janelia Visiting Scientist program, the HHMI visa office, and HHMI Janelia for funding
