Figure 2 Protocols for stress-loading and drug administration.

A: Protocol for evaluation of protective activity. Mice were orally given the drug once a day for 21 days (indicated by the arrows) during exposure to the 3 types of CMSs (CMS1, CMS2 or CMS3, indicated as boxes colored white, gray, and black, respectively). Then, the behavioral test was performed at 1 day after the end of the stress-loading (open arrow).

B: Protocol for evaluation of therapeutic activity. Mice were subjected to the 3 different CMSs (CMS1, CMS2 or CMS3, illustrated as boxes as defined in “A.”) for 20 days. Each drug was orally given once a day for 14 or 21 days starting 1 day after the stress-loading, and the behavioral test was performed at 1 day after the final drug administration (open arrows).

Figure 3 Protective effect against the depression-like symptom.

Mice were daily injected orally with vehicle or CAPE (10, 50 or 250 µmol/kg) for 3 weeks with or without exposure to the CMSs and then subjected to the TST 1 day after the final administration (see protocol A in Figure 2). The significance of the difference between the value of the stress-exposed group and that of the non-stress-exposed group was determined by one-way ANOVA with Tukey’s post hoc test as **p< 0.01. Among the stress groups, significant differences from the value of the vehicle-treated group were also determined by one-way ANOVA with Tukey’s test as #p< 0.05.

Figure 4 Therapeutic activity for the depression-like symptom.

Vehicle, CAPE (50 µmol/kg) or fluvoxamine (3 µmol/kg) was daily injected orally for 2 (A) or 3 weeks (B) into mice that had been exposed or not to the CMSs (see protocol B in Figure 2). Then, the mice were evaluated for immobility time in the TST 1 day after the final administration. The significance of the difference between the value of the stress-exposed mice and that of the non-stress-exposed mice was determined by one-way ANOVA with Tukey’s post hoc test as ***p< 0.001. Furthermore, among the CMS-exposed groups, a significant difference from the value of the vehicle-treated mice was similarly determined by one-way ANOVA with Tukey’s post hoc test as #P<0.05, ##p< 0.01.

Figure 5 Protective effect against the anxiety-like symptom evaluated by the EPMT.

Mice were daily injected orally with vehicle or CAPE, as described in the legend of Figure 3, for 3 weeks with or without exposure to the CMSs; and they were then subjected to the EPMT 1 day after the final administration (see protocol A in Figure 2). The time spent in open arms (A) and the number of entries into the arms (B) were then evaluated. The significance of the difference between the value of the stress mice and that of the non-stress mice was determined by one-way ANOVA with Tukey’s post hoc test as **p< 0.01. Among the stress groups, significant differences from the value of the vehicle-treated mice were also determined by one-way ANOVA with Tukey’s test as #p< 0.05.

Figure 6 Protective activity against the anxiety-like symptom evaluated by performing the LDT.

Mice were daily injected orally with vehicle or CAPE, as described in the legend of Figure 3, for 3 weeks with or without exposure to the CMSs; and they were then subjected to the LDT 1 day after the final administration (see protocol A in Figure 2). The time spent in the bright chamber (A) and the number of entries into the bright chamber (B) are indicated. The significance of the difference between the value of the stress mice and that of the non-stress mice was determined by one-way ANOVA with Tukey’s post hoc test as ***p< 0.001. Among the stress groups, significant differences from the value of the vehicle-treated mice were also determined by one-way ANOVA with Tukey’s test as ##p< 0.01.

Figure 7 Therapeutic activity against the anxiety-like symptom evaluated by use of the EPMT.

Vehicle, CAPE (50 µmol/kg) or fluvoxamine (3 µmol/kg) was daily injected orally for 2 (A, C) or 3 weeks (B, D) into mice that had been exposed or not to the CMSs (see protocol B in Figure 2). Then, the mice were tested for the time spent in the open arms in the EPMT (A, B) and the number of entries into arms (C, D) 1 day after the final administration. The significance of the difference between the value of the stress mice and that of the non-stress mice was determined by one-way ANOVA with Tukey’s post hoc test as *p< 0.05. Furthermore, among the stress groups, a significant difference from the value of the vehicle-treated stress mice was examined by one-way ANOVA with Tukey’s post hoc test.

Figure 8 Therapeutic activity against the anxiety-like symptom evaluated by performing the LDT.

Vehicle, CAPE (50 µmol/kg) or fluvoxamine (3 µmol/kg) was daily injected orally for 2 (A, C) or 3 weeks (B, D) into mice that had been exposed or not to the CMSs (see protocol B in Figure 2). Then, the mice were tested for the time spent in the bright chamber in the LDT (A, B) or the number of entries into the bright chamber (C, D) 1 day after the final administration. The significance of the difference between the value of the stress mice and that of the non-stress mice was determined by one-way ANOVA with Tukey’s post hoc test as *p< 0.05, **p< 0.01. Furthermore, among the stress groups, a significant difference from the value of the vehicle-treated stress mice was determined by one-way ANOVA with Tukey’s post hoc test as *p< 0.05.

Figure 9 Ameliorative activity against the CMS-induced decrease in the hippocampal pERK1/2 and pCREB levels.

A, C: After daily oral administration of vehicle or CAPE to the mice for 21 days during CMS exposure (see protocol A in Figure 2), the hippocampi were dissected out 1 day after the final administration. Representative images of Western immunoblots are shown.

B, D: Mice were injected with vehicle or CAPE for 14 days after the end of the CMS exposure (see protocol B in Figure 2), and the hippocampi were dissected out 1 day after the end of the administration. The ratio of the intensity of the pERK1/2 (A, B) or pCREB (C, D) band to that of the total ERK1/2 or CREB band was calculated after Western immunoblotting, and the values were expressed as fold-increase over the value of the vehicle-treated nonstress group taken as “1.” The significance of differences between the values of the stress and the non-stress mice was determined by one-way ANOVA with Tukey’s post hoc test as *P< 0.05, **P< 0.01 or ***P< 0.001. For differences between values of stress groups, the significance was similarly determined by the same statistical treatment as #P< 0.05, ##p< 0.01.