Early-Life Stress Induces Depression-Like Behavior and Synaptic-Plasticity Changes in a Maternal Separation Rat Model: Gender Difference and Metabolomics Study

Abstract

More than 300 million people suffer from depressive disorders globally. People under early-life stress (ELS) are reportedly vulnerable to depression in their adulthood, and synaptic plasticity can be the molecular mechanism underlying such depression. Herein, we simulated ELS by using a maternal separation (MS) model and evaluated the behavior of Sprague-Dawley (SD) rats in adulthood through behavioral examination, including sucrose preference, forced swimming, and open-field tests. The behavior tests showed that SD rats in the MS group were more susceptible to depression- and anxiety-like behaviors than did the non-MS (NMS) group. Nissl staining analysis indicated a significant reduction in the number of neurons at the prefrontal cortex and hippocampus, including the CA1, CA2, CA3, and DG regions of SD rats in the MS group. Immunohistochemistry results showed that the percentages of synaptophysin-positive area in the prefrontal cortex and hippocampus (including the CA1, CA2, CA3, and DG regions) slice of the MS group significantly decreased compared with those of the NMS group. Western blot analysis was used to assess synaptic-plasticity protein markers, including postsynaptic density 95, synaptophysin, and growth-associated binding protein 43 protein expression in the cortex and hippocampus. Results showed that the expression levels of these three proteins in the MS group were significantly lower than those in the NMS group. LC-MS/MS analysis revealed no significant differences in the peak areas of sex hormones and their metabolites, including estradiol, testosterone, androstenedione, estrone, estriol, and 5β-dihydrotestosterone. Through the application of nontargeted metabolomics to the overall analysis of differential metabolites, pathway-enrichment results showed the importance of arginine and proline metabolism; pantothenate and CoA biosyntheses; glutathione metabolism; and the phenylalanine, tyrosine, and tryptophan biosynthesis pathways. In summary, the MS model caused adult SD rats to be susceptible to depression, which may regulate synaptic plasticity through arginine and proline metabolism; pantothenate and CoA biosyntheses; glutathione metabolism; and phenylalanine, tyrosine, and tryptophan biosyntheses.

Keywords: depression; early-life stress; maternal separation; metabolomics; synaptic plasticity.

Figure 1

MS reduces the body weight of SD rats. (A) The tendency of weight gain from PND28 to PND63. (B) Weight gain from PND28 to PND63 after MS. Statistical analyses are performed by two-way ANOVA followed by t-test. Values are presented as mean ± SEM, *p < 0.05. n = 15 per group.

Figure 2

MS causes depression-like and anxiety-like behavior in SD rats. (A) Effect of MS on sucrose preference (%) in the sucrose-preference test on SD rats. (B) Effect of MS on immobility time(s) in the forced-swimming test on rats. (C–E) Effect of MS on central region time (s), central region distance (mm), and activity in the open-field test on rats. Statistical analyses are performed by two-way ANOVA followed by t-test. Values are presented as mean ± SEM. *p < 0.05, **p < 0.01 (compared with the NMS group), n = 15 per group.

Figure 3

MS decreases the number of neurons. (A) Schematic of the coronal section from rat hippocampus and the locations of CA1, CA2, CA3, and DG regions. (B) The red frame area indicates the field of view of the prefrontal cortex. (C) Representative 400× photomicrographs of Nissl staining in the hippocampal of CA1, CA2, CA3, and DG regions. Results of the number of Nissl staining positive cells in the hippocampal CA1, CA2, CA3, and DG regions are statistically significant, except for the decreasing trend of the M-MS group in the DG region. (D) Representative 400× photomicrographs of Nissl staining in the prefrontal cortex. Statistical results of the number of Nissl-positive cells in the prefrontal cortex. Statistical analyses are performed by two-way ANOVA followed by t-test. Data are presented as mean ± SEM, *p < 0.05, **p < 0.01, n = 3 per group, scale bar = 50 µm.

Figure 4

MS reduces SYN protein expression in immunohistochemistry. (A) Schematic of the coronal section from rat hippocampus and the locations of CA1, CA2, CA3, and DG regions. (B) The red frame area indicates the field of view of the prefrontal cortex. (C) Representative 400× photomicrographs of SYN protein expression of the CA1, CA2, CA3, and DG regions of the hippocampus. Results of the percentage of SYN-positive area in the hippocampal CA1, CA2, CA3, and DG regions are statistically significant. (D) Representative 400× photomicrographs of SYN protein expression in the prefrontal cortex. Statistical results of the percentage of SYN-positive area in the prefrontal cortex. Statistical analyses are performed by two-way ANOVA followed by t-test. Data are presented as mean ± SEM, *p < 0.05, **p < 0.01, n = 3 per group, scale bar = 50 µm.

Figure 5

MS reduces the expression of synaptic-plasticity protein. (A) The bands of synaptic-plasticity proteins of SYN, PSD-95, and GAP-43 in the hippocampus by WB. Statistical results indicate the relative protein levels expressed by SYN, GAP-43, and PSD-95. (B) The bands of synaptic-plasticity proteins of SYN, PSD-95, and GAP-43 in cortex by WB. Statistical results indicate the relative protein levels expressed by SYN, GAP-43, and PSD-95. Statistical analyses are performed by two-way ANOVA followed by t-test. Data are presented as mean ± SEM, *p < 0.05, **p < 0.01, n = 3 per group.

Figure 6

MS shows no gender difference in depression-like behavior in male and female rats. (A) PCA score plot of the F-MS and M-MS groups in negative ion mode. (B) PCA score plot of F-MS and M-MS groups in positive-ion mode. (C) Peak area of sex hormones and their metabolites detected by LC–MS/MS.

Figure 7

Model analysis of nontargeted metabolomics of brain tissues in SD rats. (A) TIC mass spectrum in negative ion mode. (B) PLS-DA 3D score plot in negative ion mode of the NMS and MS groups. (C) OPLS-DA score plot in negative ion mode of the NMS and MS groups. (D) The corresponding OPLS (V+S) plot of the NMS and MS groups in negative ion mode. (E) TIC mass spectrum in positive-ion mode. (F) PLS-DA 3D score plot in positive-ion mode of the NMS and MS groups. (G) OPLS-DA score plot in positive-ion mode of the NMS and MS groups. (H) The corresponding OPLS (V+S) plot of the NMS and MS groups in positive-ion mode.

Figure 8

Analysis of differential metabolites. (A) Peak area detected by LC–MS/MS for differential metabolites in the brain tissue. (B) The relative content of differential metabolites from the heat map of brain tissues. Class 1: NMS group. Class 2: MS group. The ribbon −3~3: represents the content of differential metabolites from low to high. (C) Pathway analysis of differential metabolites in the NMS and MS groups. a: pantothenate and CoA biosynthesis, b: arginine and proline metabolism, c: glutathione metabolism, d: phenylalanine, tyrosine, and tryptophan biosynthesis.