神经科学

C. elegans shows robust and reproducible behavioral responses to oxygen. Specifically, worms prefer O₂ levels of 5–10% and avoid too high or too low O₂. Their O₂ preference is not fixed but shows plasticity depending on experience, context, or genetic background. We recently showed that this experience-dependent plasticity declines with age, providing a useful behavioral readout for studying the mechanisms of age-related decline of neural plasticity. Here, we describe a technique to visualize behavioral O₂ preference and its plasticity in C. elegans, by creating spatial gradients of [O₂] in a microfluidic polydimethylsiloxane (PDMS) chamber and recording the resulting spatial distribution of the animals.

Caenorhabditis elegans is a simple metazoan that is often used as a model organism to study various human ailments with impaired motility phenotypes, including protein conformational diseases. Numerous motility assays that measure neuro-muscular function have been employed using C. elegans. Here, we describe “time-off-pick" (TOP), a novel assay for assessing motility in C. elegans. TOP is conducted by sliding an eyebrow hair under the mid-section of the worm and counting the number of seconds it takes for the worm to crawl completely off. The time it takes for the worm to crawl off the eyebrow hair is proportional to the severity of its motility defect. Other readouts of motility include crawling or swimming phenotypes, and although widely established, have some limitations. For example, worms that are roller mutants are less suitable for crawling or swimming assays. We demonstrated that our novel TOP assay is sensitive to age-dependent changes in motility, thus, providing another more inclusive method to assess motor function in C. elegans.

Graphical abstract:

Conceptual overview of the “time-off-pick” (TOP) assay.

Various C. elegans models exhibit age-dependent defects in motility. The time it takes for a worm to crawl off of an eyebrow pick that is slid under its mid-section is measured in TOP seconds. A greater TOP is indicative of a greater motility defect. Eventually, worms with phenotypes that lead to paralysis will not be able to leave the pick.

Odor is the most fundamental chemical stimulus that delivers information regarding food, mating partners, enemies, and danger in the surrounding environment. Research on odor response in animals is widespread, although studies on experimental systems in which the gradient of odor concentration is quantitatively measured has been quite limited. Here, we describe a method for measuring a gradient of odor concentration established by volatilization and diffusion in a relatively small enclosed space, which has been used widely in laboratories to analyze small model animals such as the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. We first vaporized known amounts of a liquid odorant 2-nonanone in a tank and subjected them to gas chromatographic analysis to obtain a calibration curve. Then, we aspirated a small amount of gas phase from a small hole on an agar plate and measured the odor concentration. By repeating this at different spatial and temporal points, we were able to detect a gradient of the odor concentration that increased over time. Furthermore, by applying these measured values to mathematical models of volatilization and diffusion, we were able to visualize an estimated dynamic change in odor concentration over an agar plate. Combining monitoring of odor concentration change in an agar plate with behavioral monitoring by machine vision will allow us to estimate how the brain computes information regarding odor concentration change in order to regulate behavior.

The nematode Caenorhabditis elegans is widely used for behavioral studies ranging from simple chemosensation to associative learning and memory. It is vital for such studies to determine optimal concentrations of attractive and aversive chemicals that C. elegans can sense. Here we describe a resource localization assay in which a chemical compound of interest is placed in two compartments of a quadrant plate in order to determine optimal concentrations of the chemical in behavioral studies. Using the assay, we determined the optimal concentration of a water-soluble attractant, KCl, as an unconditioned stimulus for the study of associative learning and memory. In this protocol, we also describe a chemotaxis assay using a square agar plate spotted with an aversive olfactory cue, 1-nonanol, as a conditioned stimulus.

Nematodes have sensitive olfactory perception, which is used to detect and differentiate many volatile odorants. Some odorants are attractive, others repulsive, and yet others evoke no particular response. Chemotaxis assays can be used to determine the role of certain odors in many different behaviors including foraging, predator avoidance, and mate attraction. In addition to chemotaxis, some species of nematodes in the entomopathogenic genus Steinernema can jump, which is thought to play an important role in host-seeking and dispersal (Dillman and Sternberg, 2012). Jumping and chemotaxis assays have been successfully used to identify odorants that stimulate these behaviors in a variety of nematodes (Bargmann et al., 1993; Campbell and Kaya, 1999; Hallem et al., 2011; Dillman et al., 2012; Castelletto et al., 2014). Here a detailed protocol for chemotaxis and jumping assays is provided based on the growing body of literature.

Olfaction is a well-studied sensory mechanism in Caenorhabditis elegans (C. elegans). The nematodes respond to a wide range of chemicals by either attraction, repulsion or a mixture thereof (Bargmann et al., 1993). We have used olfaction to characterize behavioural and molecular circadian rhythms in C. elegans. The circadian clock is a biological oscillator that provides an endogenous temporal structure that approximately matches the 24-hour periodicity in the environment (due to the rotational movement of the Earth). Circadian rhythms are present in most organisms from cyanobacteria to humans and they typically regulate sensory functions among many other processes. Olfaction is under circadian control in many animals (Granados-Fuentes et al., 2006; Granados-Fuentes et al., 2011; Tanoue et al., 2008; Krishnan et al., 1999). This protocol was designed to allow the assessment of olfaction for a population of worms within a short time interval, in the same plate where the worms grew (to avoid washing steps that may disturb the rhythms), and in the presence of food.