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

Background 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. New method Here, we develop a technique for automated long-term two-photon imaging in fruit flies, during wakefulness and extended bouts of immobility, as typically observed during 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. Results The approach is validated in the fly’s head direction system and walking behavior as well a neural activity are recorded. The head direction system tracks the fly’s walking direction over multiple days. Comparison with existing methods In comparison with previous head-fixed preparations, the time span over which tethered behavior and neural activity can be recorded at the same time is extended from hours to days. Additionally, the reproducibility and ease of dissections are improved compared with manual approaches. Different from previous laser surgery approaches, only continuous wave lasers are required. Lastly, an automated feeding system allows continuously maintaining the fly for several days in the virtual reality setup without user intervention. Conclusions Imaging in behaving flies over multiple timescales will be useful for understanding circadian activity, learning and long-term memory, or sleep.