Motor systems flexibly implement diverse motor programs to pattern behavioral sequences, yet their neural underpinnings remain unclear. Here, we investigated the neural circuit mechanisms of flexible courtship behavior in Drosophila. Courting males alternately produce two types of courtship song. By recording calcium signals in the ventral nerve cord (VNC) in behaving flies, we found that different songs are produced by activating overlapping neural populations with distinct motor functions in a combinatorial manner. Recordings from the brain suggest that song is driven by two descending pathways – one defines when to sing and the other specifies what song to sing. Connectomic analysis reveals that these “when” and “what” descending pathways provide structured input to VNC neurons with different motor functions. These results suggest that dynamic changes in the activation patterns of descending pathways drive different combinations of motor modules, thereby flexibly switching between different motor actions.

To perform most behaviors, animals must send commands from higher-order processing centers in the brain to premotor circuits that reside in ganglia distinct from the brain, such as the mammalian spinal cord or insect ventral nerve cord. How these circuits are functionally organized to generate the great diversity of animal behavior remains unclear. An important first step in unraveling the organization of premotor circuits is to identify their constituent cell types and create tools to monitor and manipulate these with high specificity to assess their function. This is possible in the tractable ventral nerve cord of the fly. To generate such a toolkit, we used a combinatorial genetic technique (split-GAL4) to create 195 sparse driver lines targeting 198 individual cell types in the ventral nerve cord. These included wing and haltere motoneurons, modulatory neurons, and interneurons. Using a combination of behavioral, developmental, and anatomical analyses, we systematically characterized the cell types targeted in our collection. Taken together, the resources and results presented here form a powerful toolkit for future investigations of neural circuits and connectivity of premotor circuits while linking them to behavioral outputs.

Animal sounds are produced by patterned vibrations of specific organs, but the neural circuits that drive these vibrations are not well defined in any animal. Here we provide a functional and synaptic map of most of the neurons in the Drosophila male ventral nerve cord (the analog of the vertebrate spinal cord) that drive complex, patterned song during courtship. Male Drosophila vibrate their wings toward females during courtship to produce two distinct song modes – pulse and sine song – with characteristic features that signal species identity and male quality. We identified song-producing neural circuits by optogenetically activating and inhibiting identified cell types in the ventral nerve cord (VNC) and by tracing their patterns of synaptic connectivity in the male VNC connectome. The core song circuit consists of at least eight cell types organized into overlapping circuits, where all neurons are required for pulse song and a subset are required for sine song. The pulse and sine circuits each include a feed-forward pathway from brain descending neurons to wing motor neurons, with extensive reciprocal and feed-back connections. We also identify specific neurons that shape the individual features of each song mode. These results reveal commonalities amongst diverse animals in the neural mechanisms that generate diverse motor patterns from a single set of muscles.