Goal-directed animal behaviors are typically composed of sequences of motor actions whose order and timing are critical for a successful outcome. Although numerous theoretical models for sequential action generation have been proposed, few have been supported by the identification of control neurons sufficient to elicit a sequence. Here, we identify a pair of descending neurons that coordinate a stereotyped sequence of engagement actions during Drosophila melanogaster male courtship behavior. These actions are initiated sequentially but persist cumulatively, a feature not explained by existing models of sequential behaviors. We find evidence consistent with a ramp-to-threshold mechanism, in which increasing neuronal activity elicits each action independently at successively higher activity thresholds.

Rapidly and selectively modulating the activity of defined neurons in unrestrained animals is a powerful approach in investigating the circuit mechanisms that shape behavior. In Drosophila melanogaster, temperature-sensitive silencers and activators are widely used to control the activities of genetically defined neuronal cell types. A limitation of these thermogenetic approaches, however, has been their poor temporal resolution. Here we introduce FlyMAD (the fly mind-altering device), which allows thermogenetic silencing or activation within seconds or even fractions of a second. Using computer vision, FlyMAD targets an infrared laser to freely walking flies. As a proof of principle, we demonstrated the rapid silencing and activation of neurons involved in locomotion, vision and courtship. The spatial resolution of the focused beam enabled preferential targeting of neurons in the brain or ventral nerve cord. Moreover, the high temporal resolution of FlyMAD allowed us to discover distinct timing relationships for two neuronal cell types previously linked to courtship song.