Behavioral study of C. elegans in 3-D environments

Abstract

The goal of behavioral science is to understand biological mechanisms underlying animal behaviors. To bridge the gaps between genes, proteins, neurons, neural circuits and behaviors in a single animal model, Caenorhabditis elegans has been a most widely used model animal. C. elegans genome was wholly sequenced and further, the C. elegans is the only animal of which nervous system is totally mapped. Its simple motor outputs also allows us to easily quantify the behaviors of the worm. For the C. elegans behavioral studies, many behavioral quantification methods, which are called as worm trackers, have been developed. However, the worm trackers are designed for worms that move in 2-D environments, such as agar plates or microfluidic systems. Since the 2-D environments are far from 3-D natural habitats, such as soil or rotting fruits, of the worms, it has long been asked how the worm behaves in 3-D natural environments. Therefore, here I first developed 3-D worm tracker for worms moving freely in 3-D environments. I designed a dual-view imaging system and programmed a software that extracts 3-D postures and trajectories of worms by stereoscopic merging of information extracted from the two views. For quantitative analysis of the postures, I also suggested a parameter, bending vector, that represents a direction and an amount of each bend generated at each body part of the worms. I found that movements of the worms were not restricted in any plane but free in 3-D space. The 3-D worm tracker enabled us to quantitatively analyze locomotion of C. elegans freely moving in 3-D environments. The 3-D worm tracker still had two limitations in analyzing C. elegans behaviors; i) Its reconstruction algorithm cannot be applied to images having overlaps between body parts and ii) manual 3-D tracking is too labor intensive to obtain sufficient data sets for behavioral quantification. Therefore, I improved the 3- D worm tracker to be capable of versatile reconstruction and automatic 3-D tracking. Using the new 3-D worm tracker, I studied roles of head muscular and mechanosensory systems in behavioral regulation under 3-D environments. I found that the head muscular system controls moving directions of worms to be straight in 3-D environments. I also revealed that several mechanotransduction channels play special roles in 3-D environments, which were not shown in 2-D. In particular, mec- 4 gene regulates curving during forward runs. oms-9 and trp-4 genes, which encode mechanotransduction channels mediating head touch sensation, are involved in regulation of reorientation behaviors in 3-D environments. Furthermore, I found that trp-4, specifically expressed in dopaminergic neurons, regulates the reorientation behavior. The 3-D worm trackers, I developed, are powerful methods for behavioral analysis of C. elegans in 3-D conditions. Our finding showed that roles of biological systems of the animal are different in 3-D environments, which are close to natural environments, than in 2-D. These results suggest that behavioral studies in 3-D environments are required for understanding accurate mechanisms regulating animal behaviors. I expected that the 3-D worm trackers can be applied to behavioral studies of C. elegans in various conditions and will provide opportunities to discover previously unexplored biological mechanisms underlying animal behaviors.