Imaging brain activity during complex social behaviors in Drosophila with Flyception2

Optical in vivo recordings from freely walking Drosophila are currently possible only for limited behaviors. Here, we expand the range of accessible behaviors with a retroreflective marker-based tracking and ratiometric brain imaging system, permitting brain activity imaging even in copulating male flies. We discover that P1 neurons, active during courtship, are inactive during copulation, whereas GABAergic mAL neurons remain active during copulation, suggesting a countervailing role of mAL in opposing P1 activity during mating.