© 2012 Nature America, Inc. All rights reserved. NATURE NEUROSCIENCE VOLUME 15 | NUMBER 1 | JANUARY 2012 107 ARTICLES How animal behaviors are regulated by the nervous system is a difficult question to answer because the complexity of the nervous system is so profound in most animals. It can be possible to dissect the regulatory mechanism of how neurons regulate behaviors if a simple model organism is studied. The nematode C. elegans is one such model organism; its nervous system is composed of mere 302 neurons, whose connectivity is almost completely known anatomically. In this study, we decided to elucidate the mechanism of nictation, a stage-specific behavior only shown in the dauer stage. Nictation was originally reported in parasitic nematodes 1 . Specifically, parasitic nematodes are thought to perform nictation to increase their infection efficiency in host animals 2 . Parasitic nematodes nictate in their infective larval stage, which is similar to the dauer stage in C. elegans 3 . Unlike the infective larval stage of parasitic nematodes, however, which is a part of normal development, the dauer larval stage in C. elegans is an alternative developmental stage, specialized for long-term survival. Thus, C. elegans develops into the dauer stage only when it encounters harsh external conditions. Nictation was first described for C. elegans dauers more than 30 years ago 4 , and conserved mechanisms are likely to be responsible for this conserved behavior. So far, the mechanism of nictation has not been discernible with molecular and genetic approaches for two main reasons. First, genetics is not avail- able for parasitic nematodes. Second, nictation is usually observed in C. elegans on old plates with contaminating fungi, the hyphal tips of which serve as the projections necessary for adherence and nicta- tion of the dauers. On standard agar plates, C. elegans movement is limited to two dimensions, preventing the observation of a three- dimensional activity such as nictation. In this study, we developed two efficient assays for nictation and dissected the mechanism of nictation using genetics and other molecular tools available for C. elegans. We show that IL2 ciliated head neurons are essential for nictation. We also discuss evolutionary aspects of the nictation behavior in the nematode. RESULTS Establishment of assays for nictation We first identified materials that would substitute for fungal hyphae as projections on which dauers could nictate in the lab condition so that one could systematically analyze nictation. Among our tested materials, cotton medical gauze, which morphologically mimics fungal hyphae, was the best substrate on which to observe nictat- ing dauers (Fig. 1a and Supplementary Movie 1). Medical gauze could cover the surface area of a plate on which several hundred dauers were grown, making it possible to analyze the behavior at the population level. Although medical gauze is a simple and convenient tool for the basic observation of nictation, it is inadequate for individual track- ing and quantitative behavioral analysis. To overcome this drawback, we developed another assay that uses polydimethylsiloxane (PDMS)- based microfluidic structures that provide the necessary architecture for nictating dauers 5 . Considering their three-dimensional shape and microscopic size, the structures can be thought of as mimicking the natural dirt environment of soil nematodes 5 . Using PDMS as a mold, we produced assay plates made of agar. The structures, which we named micro-dirt chips because of their resemblance to the natural soil environment, were composed of posts of a fixed size with regular gaps. 1 Research Center for Functional Cellulomics, Institute of Molecular Biology and Genetics, Seoul National University School of Biological Sciences, Seoul, Korea. 2 Department of Chemistry and Nano Sciences, Ewha Womans University, Seoul, Korea. 3 Department of Biochemistry, College of Life Sciences and Biotechnology, Yonsei Proteome Research Center, Yonsei University, Seoul, Korea. 4 World Class University Department of Biophysics and Chemical Biology, Seoul National University, Seoul, Korea. 5 These authors contributed equally to this work. Correspondence should be addressed to J.L. (elegans@snu.ac.kr). Received 25 April; accepted 21 September; published online 13 November 2011; corrected after print 10 August 2012; doi:10.1038/nn.2975 Nictation, a dispersal behavior of the nematode Caenorhabditis elegans, is regulated by IL2 neurons Harksun Lee 1,5 , Myung-kyu Choi 1,5 , Daehan Lee 1 , Hye-sung Kim 1 , Hyejin Hwang 2 , Heekyeong Kim 3 , Sungsu Park 2 , Young-ki Paik 3 & Junho Lee 1,4 Many nematodes show a stage-specific behavior called nictation in which a worm stands on its tail and waves its head in three dimensions. Here we show that nictation is a dispersal behavior regulated by a specific set of neurons, the IL2 cells, in C. elegans. We established assays for nictation and showed that cholinergic transmission was required for nictation. Cell type–specific rescue experiments and genetic ablation experiments revealed that the IL2 ciliated head neurons were essential for nictation. Intact cilia in IL2 neurons, but not in other ciliated head neurons, were essential, as the restoration of the corresponding wild-type gene activity in IL2 neurons alone in cilia-defective mutants was sufficient to restore nictation. Optogenetic activation of IL2 neurons induced nictation, suggesting that signals from IL2 neurons are sufficient for nictation. Finally, we demonstrated that nictation is required for transmission of C. elegans to a new niche using flies as artificial carriers, suggesting a role of nictation as a dispersal and survival strategy under harsh conditions.