FIGURE 3.

Memory formation and retention after massed, spaced, or extinction training. (A) Memory retention curve. Animals were conditioned with 0.01% 1-nonanol and 160 mM KCl indicated numbers of cycles by massed or spaced training. Their LIs were measured at indicated time intervals after conditioning. Asterisks indicate statistically significant differences (^∗∗p < 0.01) determined by one-way ANOVA, followed by the Tukey-Kramer test for further comparison of LIs of animals conditioned by spaced training with those of animals conditioned by single conditioning or massed training. However, LIs of animals conditioned by eight-cycle spaced training or massed training were statistically indistinguishable from each other, when analyzed by using a two-sided Student’s t-test. LIs of animals conditioned by the eight-cycle massed training showed statistically significant differences (^§p < 0.05, ^§§p < 0.01) determined by one-way ANOVA, followed by the Tukey-Kramer test for further comparison of LIs of animals conditioned by single conditioning or three-cycle massed training. LI values were calculated from the equation shown in Figure 2A using CI_reference values of animals treated with 100% EtOH and 160 mM KCl. Data are means ± SEM (n = 6–9 assays). (B) Extinction learning after spaced training. Animals were conditioned with 0.01% 1-nonanol and 160 mM KCl by eight-cycle spaced training with a 10-min ITI. They were then transferred to an NGM plate with a bacterial lawn. After 3 h incubation at 20°C, animals were treated eight times (without ITI) only with 0.01% 1-nonanol diluted with EtOH or 100% EtOH. Immediately after the treatment, chemotaxis assays were performed. LIs of animals with and without extinction trials were statistically different (^∗p < 0.05) when analyzed using a two-sided Student’s t-test. LI values were calculated from the equation shown in Figure 2A using CI_reference values of animals treated with 100% EtOH and 160 mM KCl. Data are displayed as means ± SEM (n = 9 assays).