Summary The mechanisms by which synaptic partners recognize each other and establish appropriate numbers of connections during embryonic development to form functional neural circuits are poorly understood. We combined electron microscopy reconstruction, functional imaging of neural activity, and behavioral experiments to elucidate the roles of (1) partner identity, (2) location, and (3) activity in circuit assembly in the embryonic nerve cord of Drosophila . We found that postsynaptic partners are able to find and connect to their presynaptic partners even when these have been shifted to ectopic locations or silenced. However, orderly positioning of axon terminals by positional cues and synaptic activity is required for appropriate numbers of connections between specific partners, for appropriate balance between excitatory and inhibitory connections, and for appropriate functional connectivity and behavior. Our study reveals with unprecedented resolution the fine connectivity effects of multiple factors that work together to control the assembly of neural circuits.

Neural circuit assembly occurs with subcellular precision, yet the mechanisms underlying this precision remain largely unknown. Subcellular synaptic specificity could be achieved by molecularly distinct subcellular domains that locally regulate synapse formation, or by axon guidance cues restricting access to one of several acceptable targets. We address these models using two Drosophila neurons: the dbd sensory neuron and the A08a interneuron. In wild-type larvae, dbd synapses with the A08a medial dendrite but not the A08a lateral dendrite. dbd-specific overexpression of the guidance receptors Unc-5 or Robo-2 results in lateralization of the dbd axon, which forms anatomical and functional monosynaptic connections with the A08a lateral dendrite. We conclude that axon guidance cues, not molecularly distinct dendritic arbors, are a major determinant of dbd-A08a subcellular synapse specificity.

14Neural circuit assembly occurs with subcellular precision, yet the mechanisms underlying this 15 precision remain largely unknown. Subcellular synaptic specificity could be achieved by 16 molecularly distinct subcellular domains that locally regulate synapse formation, or by axon 17 guidance cues restricting access to one of several acceptable targets. We address these 18 models using two Drosophila neurons: the dbd sensory neuron and the A08a interneuron. In 19 wild-type larvae, dbd synapses with the A08a medial dendrite but not the A08a lateral dendrite. 20 dbd-specific overexpression of the guidance receptors Unc-5 or Robo-2 results in lateralization 21 of the dbd axon, which forms anatomical and functional monosynaptic connections with the 22A08a lateral dendrite. We conclude that axon guidance cues, not molecularly distinct dendritic 23 arbors, are a major determinant of dbd-A08a subcellular synapse specificity. 25Nervous system function is determined by the precise connectivity of neurons. From the 26 Drosophila larva with 10,000 neurons to the human with 80 billion neurons, all neurons are 27 faced with the challenge of identifying the correct subset of synaptic partners among many 28 potential target neurons. In addition to specificity at a cellular level, neural circuits also exhibit 29 synaptic specificity at the subcellular level (reviewed in Yogev and Shen, 2014). In Drosophila, 30