Multiphoton imaging of neural structure and activity in Drosophila through the intact cuticle.

Max Jameson Aragon,Jamien Shea,Mengran Wang,Aaron T Mok,Chris Xu,Nilay Yapici,Haein Kim,Nathan Barkdull

doi:10.7554/elife.69094

Abstract

We developed a multiphoton imaging method to capture neural structure and activity in behaving flies through the intact cuticle. Our measurements showed that the fly head cuticle has surprisingly high transmission at wavelengths >900nm, and the difficulty of through-cuticle imaging is due to the air sacs and/or fat tissue underneath the head cuticle. By compressing or removing the air sacs, we performed multiphoton imaging of the fly brain through the intact cuticle. Our anatomical and functional imaging results show that 2- and 3-photon imaging are comparable in superficial regions such as the mushroom body, but 3-photon imaging is superior in deeper regions such as the central complex and beyond. We further demonstrated 2-photon through-cuticle functional imaging of odor-evoked calcium responses from the mushroom body γ-lobes in behaving flies short term and long term. The through-cuticle imaging method developed here extends the time limits of in vivo imaging in flies and opens new ways to capture neural structure and activity from the fly brain.

Highlights

Animal nervous systems across lineages have evolved to solve many of the same problems; foraging for food and water, finding mates to reproduce, and avoiding predators to stay alive
A major technological challenge to revealing how the brain encodes behavioral states in real-time is that even the simplest neural computation involves interactions across the nervous system at various time scales, while our tools for assessing neural activity are restricted in time and space because of the currently available imaging sensors, methods, and preparations (Lerner et al, 2016)
Through-cuticle multiphoton imaging of the fly brain Based on our cuticle transmission results, we developed a cuticle-intact imaging method where we either used head compression to minimize the volume of the air sacs (Figure 2A, Video 1) or removed them completely from the head capsule (Figure 2-figure supplement 1B-D)

Summary

INTRODUCTION

Animal nervous systems across lineages have evolved to solve many of the same problems; foraging for food and water, finding mates to reproduce, and avoiding predators to stay alive. To understand how molecularly defined neural circuits evaluate sensory information in different behavioral states, it is critical to capture the activity of populations of neurons over long time scales as flies are changing their physiological needs (Luo et al, 2018; Simpson and Looger, 2018) These functional imaging experiments require imaging preparations, which should allow chronic neural activity imaging for at least 12 hours. Current methods used in fly optical physiology require the fly head cuticle, trachea, and fat body to be removed by microsurgery to provide optical access to the nervous system (Grover et al, 2016; Minocci et al, 2013; Seelig et al, 2010; Sinha et al, 2013; Wang et al, 2003) These preparations are limited in imaging duration because after some time, the brain tissue starts to degenerate due to damaged circulation resulting from the cuticle removal surgery. The cuticle-intact imaging method developed here allows multiphoton imaging of the fly brain through the head cuticle opening new ways to capture neural structure and activity from the fly brain at long time scales and potentially through the entire lifespan of flies

RESULTS

DISCUSSION

MATERIALS AND METHODS