BDNF-Related Imbalance of Copine 6 and Synaptic Plasticity Markers Couples With Depression-Like Behavior and Immune Activation in CUMS Rats

Abstract

Chronic stress is a contributing risk factor in the pathogenesis of depression. Although the mechanisms are multifaceted, the relationship can be ascribed partly to stress-related alterations in immune activation and brain plasticity. Considering the increasing evidence regarding the role of Copine 6 in the regulation of synaptic plasticity, the aim of the present study is to investigate Copine 6 expression in the hippocampus and the prefrontal cortex (PFC) in a stress-induced depression rat model. The behavior of the rats was evaluated via the open field test, saccharin preference test, elevated plus maze test, tail suspension test, Morris water maze, and forced swimming test. The plasma concentrations of C-reactive protein (CRP) and interleukin-6 (IL-6) were measured, and the protein expressions of brain-derived neurotrophic factor (BDNF), Copine 6, and synaptic plasticity markers in the hippocampus and the PFC were also detected. The results showed that chronic unpredictable mild stress (CUMS) induces depression-like behavior in rats, accompanied by increased plasma concentrations of CRP and IL-6. Moreover, the protein expressions of BDNF, Copine 6, and synapsin I were decreased in both the hippocampus and the PFC of CUMS rats, and the protein expression of synaptotagmin I was decreased in the hippocampus. Furthermore, Pearson's test revealed a potential relationship between the depression-like behavior, the plasma CRP concentration, and the protein expressions of BDNF, Copine 6, synapsin I, or synaptotagmin I in the hippocampus or the PFC. Together with our previous results, the current findings suggest that apart from immune activation, the BDNF-related imbalance of Copine 6 expression in the brain might play a crucial role in stress-associated depression-like behaviors and synaptic plasticity changes.

Keywords: BDNF; Copine 6; chronic unpredictable mild stress (CUMS); depression; saccharin preference; synapsin I; synaptotagmin I.

FIGURE 1

Schedule of the experimental design. CUMS, chronic unpredictable mild stress; OFT, open field test; SPT, saccharin preference test; EPM, elevated plus maze; YM, Y-maze; MWM, Morris water maze; TST, tail suspension test; FST, forced swimming test; CRP, C-reactive protein; BDNF, brain-derived neurotrophic factor; PFC, prefrontal cortex.

FIGURE 2

Body-weight gain in the control and CUMS rats. The data are presented as the mean ± SEM, with 10 rats in each group. The net body-weight gain was lower in the CUMS group than in the control group during the latter 2-week stress period. ^∗P < 0.05, ^∗∗P < 0.001; compared with control group.

FIGURE 3

Behavior of the control and CUMS rats in the OFT and the EPM tests. The data are presented as the mean ± SEM, with 10 rats in each group. In the OFT (A–E), repeated measures ANOVA show that both time and stress had a significant effect on total moving distance (A), center duration (B), and rearing number (C). The time effect on the grooming number (D) and the stress effect on the defecation number (E) are also shown. In the EPM test (F–H), the total distance (F), distance in the closed arm (F), and duration in the junction (H) in the CUMS group were less than those in the control group, although there is no significant difference in the distance (F), frequency (G), and duration (H) in the open arm between groups, ^∗P < 0.05, ^∗∗P < 0.001; compared with control group.

FIGURE 4

Behavior of the control and CUMS rats in the SPT, TST, FST, and MWM test. The data are presented as the mean ± SEM, with 10 rats in each group. In the SPT (A), the results of repeated measures ANOVA show an effect of time but not stress on the saccharin preference index from week 0 to stress exposure week 2. From the second to the fourth stress exposure weeks, both time and stress had a significant effect on the saccharin preference index. Compared with the control rats, the CUMS rats spent more time immobile in the FST (B) and TST (C) but less time in the probe phase of the MWM test (E). In the place navigation of the MWM test, the escape latency of both groups in the consecutive three days’ place navigation test gradually declined (D), with no significant difference between groups.

FIGURE 5

Protein expressions of BDNF, Copine 6, synapsin I, and synaptotagmin I in the hippocampus and the PFC of the control and CUMS rats. Typical graph (A) and statistical analyses (B,C) of the western blot results. Data in (B,C) are presented as the mean ± SEM (n = 4 for each group). In the hippocampus (A,B), the expression of all four proteins was all decreased in the CUMS group. In the PFC (A,C), remarkable decreases in the BDNF, Copine 6 and synapsin I protein expressions were observed, with an insignificant change in synaptotagmin I expression. ^∗P < 0.05, ^∗∗P < 0.001; compared with control group.

FIGURE 6

Correlation analysis of the protein expressions of BDNF, Copine 6, synapsin I, and synaptotagmin I in the hippocampus and the PFC. In the hippocampus, apart from the positive correlation between synapsin I and synaptotagmin I (A) expression, BDNF expression was positively related to the expression of Copine 6 (B) and synaptotagmin I (C). Additionally, the protein expression of Copine 6 was positively correlated with that of synaptotagmin I (D) and synapsin I (E).

FIGURE 7

Correlation analysis of the depression-like behavior and the protein expressions of BDNF, Copine 6, or synapsin I in the hippocampus and the PFC. A positive relationship was found between the saccharin preference index after four consecutive weeks of CUMS and the hippocampal expression of BDNF (A). A negative relationship was seen between immobility in the FST and the expression of Copine 6 (B) and synapsin I (C) in the hippocampus and the expression of BDNF in the PFC (D). Immobility in the TST was negatively related to the hippocampal expression of synapsin I (E) and the expression of BDNF in the PFC (F).

FIGURE 8

Plasma IL-6 and CRP concentrations of the control and CUMS rats, and correlation analysis of the plasma CRP concentration and immobility in the FST and the protein expressions in the hippocampus and the PFC. The data are presented as the mean ± SEM in (A,B), with 10 rats in each group. The plasma IL-6 (A) and CRP (B) concentrations were both notably increased in the CUMS group. The plasma CRP concentration was positively related to immobility in the FST (C), and negatively related to the protein expressions of BDNF (D) and synaptotagmin I (E) in the hippocampus and BDNF in the PFC (F).