Odorant responses in freely moving C. elegans: Insights into learning and the role of small RNA regulation

Abstract

The ability to sense and respond to dynamic environmental stimuli is a fundamental property of all living organisms. The nematode Caenorhabditis elegans, with its well-defined neural circuitry and genetic tractability, serves as an ideal model for studying the molecular and cellular mechanisms underlying sensory responses and behavioral plasticity. Here, we explore the behavioral responses of C. elegans to two attractive (butanone and benzaldehyde) and one aversive (nonanone) volatile odorant using a refined assay design that minimizes confounding variables by avoiding chemical or physical immobilization and incorporates bidirectional rectangular arenas to control for external gradients. Further, we introduce an information-theory-centric measure of dispersal that is applicable broadly to other model systems. Together with a measure for the responding population, this addresses potential locomotion defects and the presence of uncharacterized gradients within the experimental arena. Using this rigorous methodological and analytical framework, we investigated the role of small RNA regulators in chemotaxis to odorants and starvation-associated memory of pre-exposure to odorants. Defective chemotaxis and learning were observed in worms lacking some of the components of the RNA silencing machinery involved in double stranded RNA (dsRNA) import, primary dsRNA processing, secondary amplification and nuclear silencing, implicating small RNA-mediated regulatory mechanisms in odorant sensation and experience-dependent behavioral plasticity. Additionally, we reveal randomness in starvation-associated memory of butanone pre-exposure even under well-controlled laboratory conditions and speculate that learning is an infrequent occurrence in the dynamic wild. Finally, we highlight the limitations of transgenic models in behavioral assays, demonstrating that NeuroPAL worms, commonly used for neuronal identity mapping, exhibit altered baseline chemotaxis responses. These findings underscore the necessity of careful experimental design and interpretation when utilizing genetically modified strains for behavioral analyses. Together, our results refine behavioral methodologies for C. elegans chemotaxis assays and provide novel insights into small RNA-mediated regulation of sensory behavior. Future research leveraging automated behavioral tracking, whole-brain functional imaging, and transgenerational analyses will further elucidate the mechanistic interplay between small RNA pathways and neural function in behavioral plasticity.