Fig 1.
Eight-week-old female C57BL/6J mice maintained for 2 weeks after ovariectomy (OVX) or sham operation were fed a standard diet or a 60% high-fat diet for 4 weeks. At the age of 14 weeks, an intracerebroventriclar (ICV) cannula was implanted, and artificial cerebrospinal fluid (ACSF) was continuously infused into the lateral ventricle by an osmotic pump. At the age of 16 weeks, continuous subcutaneous administration of E2 (50 μg/kg/day) was conducted in the E2-SC group, or E2 (1 μg/kg/day) was infused via the ICV cannula in the E2-ICV group. Behavioral tests were conducted during 3 weeks of E2 administration.
Table 1.
Body weight and tissue weights of the mice.
Fig 2.
Effects of E2 administration on anxiety phenotypes in the open field test.
Mice were placed at the center of an open field apparatus. The number of crosses into the central area (A) and the time spent in the central area (B) during a 25-min observation period were determined as indexes of the level of anxiety. Results are shown as Turkey style box plots with data falling outside the lower and upper quartiles plotted as circles. Values are the mean ± SE (control, n = 16; OVX-HF, n = 18; E2-SC, n = 11; E2-ICV, n = 10). P values were determined by the two-tailed Kruskal Wallis H-test with the Mann-Whitney U-test. **P<0.01 vs. the control group; †p<0.05 vs. the OVX-HF group.
Fig 3.
Effects of E2 administration on anxiety phenotypes in the light-dark box test.
Mice were placed in the test apparatus consisting of a black plastic box (dark box) and a transparent plastic box (light box) connected by a small tunnel that allowed mice to move freely between the compartments. The number of traverses of the tunnel and the time spent in the light box during 10 min were analyzed (A). The time spent in the light box is considered to be an index of the level of anxiety and the number of tunnel traverses is an index of general motor activity (B). Results are shown as Turkey style box plots with data falling outside the lower and upper quartiles plotted as circles. Values are the mean ± SE (control, n = 13; OVX-HF, n = 17; E2-SC, n = 11; E2-ICV, n = 10). P values were determined by the two-tailed Kruskal Wallis H-test with the Mann-Whitney U-test. *P<0.05 vs. the control group; †p<0.05 vs. the OVX-HF group.
Fig 4.
Effects of E2 administration on depression phenotypes in the tail suspension test.
Mice were taken from the home cage and a small piece of adhesive tape was attached approximately 2 cm from tip of the tail. Then mice were individually suspended on a hook 5 cm from ceiling in the open box for a period of 10 min. After the mice stopped making efforts to escape after several attempts, the duration of immobility was recorded as an index of depression-like behavior. Results are shown as Turkey style box plots. Values are the mean ± SE (control, n = 9; OVX-HF, n = 7; E2-SC, n = 11; E2-ICV, n = 8). P values were determined by the two-tailed Kruskal Wallis H-test with the Mann-Whitney U-test. *P<0.05 vs. the control group; ††p<0.01, †p<0.05 vs. the OVX-HF group.
Fig 5.
Effects of E2 administration on depression phenotypes in the forced swim test.
On day 1, mice were placed in a plastic cylinder filled with water, forced to swim for 15 min, and then returned to the home cage. On day 2, mice were placed into the water again and forced to swim for 6 min. The duration of immobility was recorded during the last 5 min of the session as an index of depression-like behavior. Results are shown as Turkey style box plots with data falling outside the lower and upper quartiles plotted as circles. Values are the mean ± SE (control, n = 8; OVX-HF, n = 9; E2-SC, n = 11; E2-ICV, n = 8). P values were determined by the two-tailed Kruskal Wallis H-test with the Mann-Whitney U-test. **P<0.01, *P<0.05 vs. the control group; ††p<0.01 vs. the OVX-HF group.
Fig 6.
Biological analysis of the mice after behavioral experiments.
Mice were sacrificed and samples were obtained after conducting all of the behavioral experiments. The serum corticosterone level was determined with an ELISA kit (A). Expression of GR mRNA (B) and 5-TH1A mRNA (C) in the hippocampus was determined by real-time PCR. The level of proBDNF protein in the hippocampus was analyzed by western blotting (D). Values are the mean ± SE (n = 7-11/group). P values were determined by the two-tailed Kruskal Wallis H-test with the Mann-Whitney U-test. *P<0.05 vs. the control group; ††p<0.01 vs. the OVX-HF group.