Animal behaviors reflect their nervous system activity, and are modified by multiple factors including environmental changes and memories of past experience. In this study, to understand cellular and molecular bases for learning and memory in C. elegans, we developed a paradigm for classical conditioning of worms with propanol, as a conditioned stimulus, and hydrochloride (HCl), as an unconditioned stimulus. Before the training, worms were attracted to propanol, an appetitive olfactory stimulus, and avoid HCl, an aversive gustatory stimulus, in chemotaxis assay. After ten-cycle massed (without intertrial intervals, ITI) or spaced (with 10-min ITI) training, in contrast, worms avoided propanol on the assay plate. Furthermore, interstimulus intervals (ISI) between the two stimuli were crucial for the conditioning. The memory after the massed training was extinguished within a few hours, while the memory after the spaced training was retained for 24 hours. Worms treated with cycloheximide or anisomycin failed to form the memory by the spaced training, whereas the memory after the massed training was not significantly affected. Furthermore, the memory after the spaced training was resistant to cold shock, while the memory after the massed training was disrupted by cold shock. Since the memory after the spaced training was consolidated by protein synthesis and was resistant to cold shock, it is classified as long-term memory. In contrast, the memory after the massed training is classified as short-term memory since it did not require protein synthesis and was sensitive to cold shock. Moreover, C. elegans mutants defective in
nmr-1 encoding an NMDA receptor subunit failed to form both of the short-term and long-term memory, while mutations in
crh-1 encoding the CREB transcription factor affected only on the formation of the long-term memory. These results are consistent with results previously observed in other model organisms such as Aplysia, Drosophila, and mice.