Automated long-term two-photon imaging in head-fixed walking Drosophila

The brain of Drosophila shows dynamics at multiple timescales, from the millisecond range of fast voltage or calcium transients to functional and structural changes occurring over multiple days. To relate such dynamics to behavior requires monitoring neural circuits across these multiple timescales in behaving animals. Here, we develop a technique for automated long-term two-photon imaging in fruit flies, during wakefulness and sleep, navigating in virtual reality over up to seven days. The method is enabled by laser surgery, a microrobotic arm for controlling forceps for dissection assistance, an automated feeding robot, as well as volumetric, simultaneous multiplane imaging. The approach is validated in the fly's head direction system. Imaging in behaving flies over multiple timescales will be useful for understanding circadian activity, learning and long-term memory, or sleep.