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

Highlights

• 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.

Abstract

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.

Introduction

The brain of Drosophila shows dynamics at multiple timescales, from single action potentials to functional and structural changes across multiple days. Such slow changes occur for example due to sleep Kirszenblat and van Swinderen (2019), circadian rhythms Liang (2019); Liang et al. (2016), or memory consolidation Chen (2012); Dag (2019). To relate fast and slow activity changes across these conditions therefore requires monitoring neural circuits over multiple timescales in behaving animals Yang et al. (2010).

While head-fixed walking preparations allow imaging of neural activity during behavior Seelig (2010), experiments typically last on the order of an hour, and two-photon imaging Denk and Strickler (1990) over multiple timescales, including naturally occurring extended epochs of immobility, a prerequisite for sleep, has so far not been achieved. A preparation for widefield bioluminescence imaging for over up to 12 h through a resealed opening in the fly’s head was developed in Minocci et al. (2013). Repeated imaging over up to 50 days was achieved with the help of a transparent window in Huang (2018) and allowed functional imaging in an epifluorescence widefield microscope after anesthetizing and reintroducing the fly into the setup for each experiment. Three-photon imaging of neural activity in intact walking flies over 12 h was performed for bright and large neurons close to the surface of the brain Aragon et al. (2019).

Here, we describe a method for multi-day two-photon calcium imaging in behaving fruit flies over up to seven days. Continuous monitoring of behavior, which included sleep and circadian modulation, in a virtual reality setup was combined with intermittent, fast volumetric imaging of calcium activity during between 8% and 16% of the time (imaging every 5 min in trials of either 30 or 60 s throughout the duration of the experiments).

For this, a transparent window was inserted into the fly’s head Huang (2018), using a cost effective laser surgery approach combined with a microrobotic arm for operating forceps under a dissection microscope. The imaging and behavior experiments did not require supervision, with an automated feeding system maintaining the fly over the course of the experiment. Simultaneous two-plane imaging Amir (2007) allowed volumetric recording of neural activity at high frame rates. We validated the method in wedge neurons, also known as EPG neurons, in the head direction system of the fly Seelig and Jayaraman (2015); Hulse and Jayaraman (2020).