24-hour-restraint stress induces long-term depressive-like phenotypes in mice

Abstract

There is an increasing risk of mental disorders, such as acute stress disorder (ASD), post-traumatic stress disorder (PTSD) and depression among survivors who were trapped in rubble during earthquake. Such long-term impaction of a single acute restraint stress has not been extensively explored. In this study, we subjected mice to 24-hour-restraint to simulate the trapping episode, and investigated the acute (2 days after the restraint) and long-term (35 days after the restraint) impacts. Surprisingly, we found that the mice displayed depression-like behaviors, decreased glucose uptake in brain and reduced adult hippocampal neurogenesis 35 days after the restraint. Differential expression profiling based on microarrays suggested that genes and pathways related to depression and other mental disorders were differentially expressed in both PFC and hippocampus. Furthermore, the depression-like phenotypes induced by 24-hour-restraint could be reversed by fluoxetine, a type of antidepressant drug. These findings demonstrated that a single severe stressful event could produce long-term depressive-like phenotypes. Moreover, the 24-hour-restraint stress mice could also be used for further studies on mood disorders.

Figure 1. The 24-hour-restraint stress induced depressive-like behaviors.

(A) Experiment designs to test the short-term (S group) and long-term (L group) impact of the restraint on mice. (B–C) Sucrose preference percentage of 24 hours in sucrose preference test of S group (B) and L group (C). The p-value (t-test) for S group is <0.05 (N = 24 vs. 25) and L group is <0.05 (N = 23 vs. 25). (D–E) The struggling time before the first abandon and total time of immobility in the forced swimming test of S group (D) and L group (E). The p-values of t-test of first abandon are <0.05 (N = 24 vs. N = 25) and <0.01(N = 25 vs. 28) respectively. And the p-values of t-test of immobility are <0.01 (N = 24 vs. N = 25) and <0.01 (N = 26 vs. 28) respectively. (F,G) The percentage of freezing time in the contextual fear conditioning test for the S group (F) and L group (G). The p-values of t-test are 0.30 (N = 21 vs. 23) and <0.001 (N = 20 vs. 21) respectively. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. Alternations of glucose metabolism by small animal PET scanning.

Glucose uptake is measured as %ID/cc (%injected dose per mL of tissue). (A) Experiment design of the PET scanning. (B) The average glucose uptake of the whole brain of S group shows no difference between control and restraint mice (N = 7 vs. 9). (C) The changes of glucose uptake in brain regions of S group. Glucose uptake of brain regions including STR, CTX, BFS, LAMY, SC, OLF, RIC is significantly increased in restraint mice. (D) The average glucose uptake of the whole brain for L group is significantly decreased in restraint mice (N = 8 vs. N = 7), p value of student t-test is<0.01. (E) The glucose uptake of different brain regions of L group. Brain regions such as STR, CTX, HIP, THA, CG, SC, MID and IC were significantly decreased in restraint mice. (F) Representatives of the glucose uptake of S and L group. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01. Abbreviations: RSTR, right striatum; LSTR, left striatum; CTX, cortex; RHIP, right hippocampal region; LHIP, left hippocampal region; THA, thalamus; CB, cerebellum; BFS, basal forebrain/septum; HYP, hypothalamus; RAMY, right amygdala; LAMY, left amygdala; CG, cingulate gyrus; SC, superior colliculus; OLF, olfactory areas; RMID, right midbrain; LMID, left midbrain; LIC, left inferior colliculus; RIC, right inferior colliculus.

Figure 3. Effects of the 24-hour-restraint stress on adult neurogenesis.

(A) Representative staining of BrdU (green), Ki67 (red) and DAPI (blue) in DG of hippocampus. (B–C) The numbers of BrdU⁺ (B) and Ki67⁺ (C) cells of S group display no difference between control and restraint mice. The p-values are 0.76 (N = 6 vs. 7) and 0.61 (N = 4 vs. 4) respectively. (D–E) The numbers of BrdU⁺ (D) and Ki67⁺ (E) cells of L group are decreased in the restraint mice. The p-values are <0.01 (N = 7 vs. 7) and <0.05 (N = 4 vs. 4) respectively. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01.

Figure 4. High-throughput gene expression analysis in short-term (S) and long-term (L) brain tissues (PFC and hippocampus) by microarrays.

(A) Number of detected differentially expressed genes (DEGs). (B) Number of overlapping DEGs among short-term PFC, long-term PFC, short-term hippocampus and long-term hippocampus. (C) Enriched KEGG pathways revealed by the DEGs. Enrichments from up-regulated DEGs and down-regulated DEGs are shown in “red” and “green” respectively. (D–E). Fold changes of selected genes in PFC (D) and hippocampus (E) of S group measured by quantitative RT-PCR. F-G. Fold changes of selected genes in PFC (F) and hippocampus (G) of L group. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. Fluoxetine treatment reverses the depressive-like phenotypes induced by 24-hour-restraint.

(A) Experiment design in order to test the effect of fluoxetine (flx) treatment. (B) The sucrose consumption in the sucrose preference test. (2-way ANOVA, stress × durg: p < 0.01, drug: p = 0.03; stress: p < 0.01, N = C:23, R:28, flx-C:28, flx-R:28). (C) The struggling time before the first abandon in the forced swim test (2-way ANOVA, stress×durg: p = 0.2271; drug: p = 0.092; stress: p < 0.01; N = C:30, R:30, flx-C:29, flx-R:30). (D) The total time of immobility in the forced swim test (2-way ANOVA, stress×durg: p < 0.01; drug: p = 0.095; stress: p < 0.05, N = C:30, R:30, flx-C:29, flx-R:30). (E) The percentage of freezing in the fear condition test (2-way ANOVA, stress × durg: p = 0.25; drug: p < 0.001; stress: p < 0.01, N = C:30, R:30, flx-C:29, flx-R:27). (F) The number of Brdu⁺ cells (2-way ANOVA, stress × durg: p = 0.62; drug: p < 0.01; stress: p = 0.09, N = C:4, R:4, flx-C:3, flx-R:3). (G,H) Changes of ATP concentration level in the PFC (G) (2-way ANOVA, stress × durg: p = 0.945; drug: p < 0.05; stress: p = 0.436, N = 5 for each group) and hippocampus (H) (2-way ANOVA, stress × durg: p < 0.01; drug: p = 0.25; stress: p = 0.73, N = 5 for each group). The asterisks in the figures showed the significant interactions of subgroups detected by post-hoc tests (Tukey Honest Significant Differences): *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± SEM.