Eviatar Yemini

A database of Caenorhabditis elegans behavioral phenotypes

Using low-cost automated tracking microscopes, we have generated a behavioral database for 305 Caenorhabditis elegans strains, including 76 mutants with no previously described phenotype. The growing database currently consists of 9,203 short videos segmented to extract behavior and morphology features, and these videos and feature data are available online for further analysis. The database also includes summary statistics for 702 measures with statistical comparisons to wild-type controls so that phenotypes can be identified and understood by users.

A dictionary of behavioral motifs reveals clusters of genes affecting Caenorhabditis elegans locomotion

Visible phenotypes based on locomotion and posture have played a critical role in understanding the molecular basis of behavior and development in Caenorhabditis elegans and other model organisms. However, it is not known whether these human-defined features capture the most important aspects of behavior for phenotypic comparison or whether they are sufficient to discover new behaviors. Here we show that four basic shapes, or eigenworms, previously described for wild-type worms, also capture mutant shapes, and that this representation can be used to build a dictionary of repetitive behavioral motifs in an unbiased way. By measuring the distance between each individuals behavior and the elements in the motif dictionary, we create a fingerprint that can be used to compare mutants to wild type and to each other. This analysis has revealed phenotypes not previously detected by real-time observation and has allowed clustering of mutants into related groups. Behavioral motifs provide a compact and intuitive representation of behavioral phenotypes.

The Multilayer Connectome of Caenorhabditis elegans

Connectomics has focused primarily on the mapping of synaptic links in the brain; yet it is well established that extrasynaptic volume transmission, especially via monoamines and neuropeptides, is also critical to brain function and occurs primarily outside the synaptic connectome. We have mapped the putative monoamine connections, as well as a subset of neuropeptide connections, in C. elegans based on new and published gene expression data. The monoamine and neuropeptide networks exhibit distinct topological properties, with the monoamine network displaying a highly disassortative star-like structure with a rich-club of interconnected broadcasting hubs, and the neuropeptide network showing a more recurrent, highly clustered topology. Despite the low degree of overlap between the extrasynaptic (or wireless) and synaptic (or wired) connectomes, we find highly significant multilink motifs of interaction, pinpointing locations in the network where aminergic and neuropeptide signalling modulate synaptic activity. Thus, the C. elegans connectome can be mapped as a multiplex network with synaptic, gap junction, and neuromodulator layers representing alternative modes of interaction between neurons. This provides a new topological plan for understanding how aminergic and peptidergic modulation of behaviour is achieved by specific motifs and loci of integration between hard-wired synaptic or junctional circuits and extrasynaptic signals wirelessly broadcast from a small number of modulatory neurons.

A consistent muscle activation strategy underlies crawling and swimming in Caenorhabditis elegans

Although undulatory swimming is observed in many organisms, the neuromuscular basis for undulatory movement patterns is not well understood. To better understand the basis for the generation of these movement patterns, we studied muscle activity in the nematode Caenorhabditis elegans. Caenorhabditis elegans exhibits a range of locomotion patterns: in low viscosity fluids the undulation has a wavelength longer than the body and propagates rapidly, while in high viscosity fluids or on agar media the undulatory waves are shorter and slower. Theoretical treatment of observed behaviour has suggested a large change in force–posture relationships at different viscosities, but analysis of bend propagation suggests that short-range proprioceptive feedback is used to control and generate body bends. How muscles could be activated in a way consistent with both these results is unclear. We therefore combined automated worm tracking with calcium imaging to determine muscle activation strategy in a variety of external substrates. Remarkably, we observed that across locomotion patterns spanning a threefold change in wavelength, peak muscle activation occurs approximately 45° (1/8th of a cycle) ahead of peak midline curvature. Although the location of peak force is predicted to vary widely, the activation pattern is consistent with required force in a model incorporating putative length- and velocity-dependence of muscle strength. Furthermore, a linear combination of local curvature and velocity can match the pattern of activation. This suggests that proprioception can enable the worm to swim effectively while working within the limitations of muscle biomechanics and neural control.

Tracking Movement Behavior of Multiple Worms on Food

Neurobiological research in genetically tractable organisms relies heavily on robust assays for behavioral phenotypes. The simple body plan of the nematode Caenorhabditis elegans makes it particularly amenable to the use of automated microscopy and image analysis to describe behavioral patterns quantitatively. Forward genetic screens and screens of drug libraries require high-throughput phenotyping, a task traditionally incompatible with manual scoring of quantitatively varying behaviors. High-throughput automated analysis of C. elegans movement behavior is now possible with several different tracking software packages. The Multiworm Tracker (MWT) described here is designed for high-throughput analysis: it can record dozens of worms simultaneously at 30 frames per second for hours or days at a time. This is accomplished by performing all image analysis in real time, saving only the worm centroid, bearing, and outline data to the disk. To simplify image processing, the system focuses only on worms that have moved, and detects and discards worms that are touching rather than trying to isolate them computationally. Because the software is entirely automated, protocols can run unattended once the worms have been placed and the software has been started. The MWT does not save images for later analysis, but behavior can be validated manually with a companion analysis tool that replays recorded body postures. This protocol describes a basic basal movement assay on food using the MWT; similar protocols apply to related assays and to similar multiple animal trackers. The protocol can be extended to a variety of assays ranging from tap response to chemotaxis.

Preparation of samples for single-worm tracking.

Neurobiological research in genetically tractable organisms relies heavily on robust assays for behavioral phenotypes. The simple body plan of the nematode Caenorhabditis elegans makes it particularly amenable to the use of automated microscopy and image analysis to describe behavioral patterns quantitatively. This protocol first describes the preparation and use of media for growing and maintaining worms for tracking. The second part of the protocol describes how to prepare a single young adult worm for recording during video analysis. Although the protocol was developed for use in a single-worm tracker, it addresses factors important for the generation of reproducible, standardized images in all systems.

Locomotion analysis identifies roles of mechanosensory neurons in governing locomotion dynamics of C. elegans

SUMMARY The simple and well-characterized nervous system of C. elegans facilitates the analysis of mechanisms controlling behavior. Locomotion is a major behavioral output governed by multiple external and internal signals. Here, we examined the roles of low- and high-threshold mechanosensors in locomotion, using high-resolution and detailed analysis of locomotion and its dynamics. This analysis revealed a new role for touch receptor neurons in suppressing an intrinsic direction bias of locomotion. We also examined the response to noxious mechanical stimuli, which was found to involve several locomotion properties and to last several minutes. Effects on different locomotion properties have different half-lives and depend on different, partly overlapping sets of sensory neurons. PVD and FLP, high-threshold mechanosensors, play a major role in some of these responses. Overall, our results demonstrate the power of detailed, prolonged and high-resolution analysis of locomotion and locomotion dynamics in enabling better understanding of gene and neuron function.

Illumination for Worm Tracking and Behavioral Imaging

Neurobiological research in genetically tractable organisms relies heavily on robust assays for behavioral phenotypes. The simple body plan of the nematode Caenorhabditis elegans makes it particularly amenable to the use of automated microscopy and image analysis to describe behavioral patterns quantitatively. This protocol provides an approach for obtaining uniform illumination during worm tracking. Good lighting can be more of an art than a science. Once the system is set up, it will be necessary to play with it, testing the results after each adjustment to ensure that the analysis software is able to clearly identify the worm and its boundaries. Although the protocol was developed for use in a single-worm tracker, it addresses factors important for the generation of reproducible, standardized images in all systems.