To show this, we put adult animals on a lawn of OP50 while exposing them for 6 hr to the smell of a PA14 lawn, which was grown on the lid of the plate. In this experiment, trained animals were exposed to the smell of PA14, but were fed on OP50. These trained animals exhibited olfactory preference comparable to that of the control animals that fed on OP50 without exposure to the smell of PA14 ( Figure S1F). Previously, we used a two-choice assay that quantified the overall movements of populations of crawling worms to elucidate the role of serotonergic neurotransmission in aversive olfactory learning (Zhang et al.,

2005). Importantly, this website the automated microdroplet assay that we utilized in this study recapitulates the phenotypes that were obtained using the two-choice assay and supports the role of serotonin in aversive olfactory learning. The learn more cat-1 mutation, which disrupts both dopamine and serotonin neurotransmission ( Duerr et al., 1999), greatly reduced olfactory learning quantified using the microdroplet assay, whereas the cat-2 mutation, which specifically disrupts dopamine production ( Lints and Emmons, 1999), had no effect on learning ( Figure 1E). The tph-1(mg280) mutant, which is deficient in the

only C. elegans tryptophan hydroxylase required for biosynthesis of serotonin ( Sze et al., 2000), was completely defective in olfactory learning in the microdroplet assay Dipeptidyl peptidase ( Figure 1E). In addition,

the mod-1(ok103) mutant, which is defective in a serotonin-gated chloride channel ( Ranganathan et al., 2000), also showed greatly reduced learning in the microdroplet assay ( Figure 1F). Thus, the microdroplet assay for swimming animals assigns phenotypes that are consistent with the two-choice assay that we previously used. The important advantage of the microdroplet assay is that it allows us to quantify olfactory preference with small numbers of animals. To characterize the neuronal network that regulates the switch of olfactory preference, we began by identifying chemosensory neurons required for olfactory plasticity. We first tested an osm-6 mutant, which is defective in development and sensory function of all ciliated chemosensory neurons ( Collet et al., 1998). The osm-6 mutant showed significantly reduced learning to avoid the smell of PA14 ( Figure 2A). By comparing choice indexes before and after training, we found that the osm-6 mutant was unable to reduce its olfactory preference for the smell of PA14 after training ( Figure S2A). These results indicate a requirement for the function of chemosensory neurons in generating a learned preference. The residual learning ability of the osm-6 mutant likely results from its residual olfactory sensory ability in the microdroplet assay ( Figure S2B).