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...

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Bibliographic Details
Published in:Journal of neuroscience methods 2022-02, Vol.368, p.109432-109432, Article 109432
Main Authors: Flores-Valle, Andres, Honnef, Rolf, Seelig, Johannes D.
Format: Article
Language:English
Subjects:
Animals
Behavior
Brain - diagnostic imaging
Calcium imaging
Dissection
Drosophila
Drosophila melanogaster
Feeding
Imaging
Reproducibility of Results
Virtual reality
Volumetric imaging
Walking
Walking on ball
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Summary: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 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. 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. 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. Imaging in behaving flies over multiple timescales will be useful for understanding circadian activity, learning and long-term memory, or sleep. •Long-term, automated two-photon imaging in walking Drosophila for up to 7 days.•Robotically controlled forceps and visible light laser surgery for fly dissection.•Automated feeding during tethered walking in virtual reality.•Simultaneous two-plane imaging with axially extended foci for volume recordings.
ISSN:0165-0270
1872-678X
DOI:10.1016/j.jneumeth.2021.109432