Adolescent stress increases depression-like behaviors and alters the excitatory-inhibitory balance in aged mice

Abstract

Background: Depression affects approximately 5% of elderly people and its etiology might be related to chronic stress exposure during neurodevelopmental periods. In this study, we examined the effects of adolescent chronic social stress in aged mice on depressive behaviors and the excitatory-inhibitory (E/I) balance in stress-sensitive regions of the brain.

Methods: Sixty-four adolescent, male C57BL/6 mice were randomly assigned to either the 7-week (from post-natal days 29 to 77) social instability stress (stress group, n = 32) or normal housing conditions (control group, n = 32). At 15 months of age, 16 mice were randomly selected from each group for a series of behavioral tests, including two depression-related tasks (the sucrose preference test and the tail suspension test). Three days following the last behavioral test, eight mice were randomly selected from each group for immunohistochemical analyses to measure the cell density of parvalbumin (PV)- and calretinin (CR)-positive gamma-aminobutyric-acid (GABA)ergic inhibitory inter-neurons, and the expression levels of vesicular transporters of glutamate-1 (VGluT1) and vesicular GABA transporter (VGAT) in three stress-sensitive regions of the brain (the medial pre-frontal cortex [mPFC], hippocampus, and amygdala).

Results: Behaviorally, compared with the control group, adolescent chronic stress increased depression-like behaviors as shown in decreased sucrose preference (54.96 ± 1.97% vs. 43.11 ± 2.85%, t(22) = 3.417, P = 0.003) and reduced latency to immobility in the tail suspension test (92.77 ± 25.08 s vs. 33.14 ± 5.95 s, t(25) = 2.394, P = 0.025), but did not affect anxiety-like behaviors and pre-pulse inhibition. At the neurobiologic level, adolescent stress down-regulated PV, not CR, inter-neuron density in the mPFC (F(1, 39) = 19.30, P < 0.001), and hippocampus (F(1, 42) = 5.823, P = 0.020) and altered the CR, not PV, inter-neuron density in the amygdala (F(1, 28) = 23.16, P < 0.001). The VGluT1/VGAT ratio was decreased in all three regions (all F > 10.09, all P < 0.004), which suggests stress-induced hypoexcitability in these regions.

Conclusions: Chronic stress during adolescence increased depression-like behaviors in aged mice, which may be associated with the E/I imbalance in stress-sensitive brain regions.

Figure 1

Schematic illustration of sub-regions analyzed in the stress-sensitive areas, including the medial pre-frontal cortex (A), hippocampus (B), and amygdala (C) of aged mice for immunohistochemical analyses.

Figure 2

The behavioral effects of adolescent chronic social instability stress in aged mice. (A) Sucrose preference test. (B) Tail suspension test. (C) Open field test. (D) Pre-pulse inhibition test. ∗P < 0.01, ^†P < 0.05, compared between control and stressed group. Data are presented as mean ± standard error (n = 12–16 per group). CT: Control; ST: Stress.

Figure 3

Effects of adolescent chronic social instability stress on the density of PV⁺ and CR⁺ inter-neurons, the expression levels of VGluT1 and VGAT, and the VGluT1/VGAT ratio in the mPFC in aged mice. (A, B) Representative for immunoreactivity of parvalbumin and calretinin in the control and stressed groups in the mPFC. Immunohistochemical staining. Scale bar = 200 μm. (C, D) The density of PV⁺ and CR⁺ inter-neurons in three sub-regions of the mPFC in both groups. (E, F) Representative for immunoreactivity of VGluT1 and VGAT in the mPFC in both groups. Immunohistochemical staining. Scale bar = 200 μm. (G–I) The relative optical density of VGluT1 and VGAT, and their ratio in three sub-regions of the mPFC in both groups. ∗ P < 0.001, indicating the significance of the main effect of group. Data are presented as mean ± standard error (n = 7–8 per group). Cg: Cingulate cortex; CR: Calretinin; CT: Control; IL: Infralimbic cortex; mPFC: Medial pre-frontal cortex; PrL: Pre-limbic cortex; PV: Parvalbumin; ST: Stress; VGAT: Vesicular gamma-aminobutyric acid transporter; VGluT1: Vesicular glutamate transporter-1.

Figure 4

Effects of adolescent chronic social instability stress on the density of PV⁺ and CR⁺ inter-neurons, the expression levels of VGluT1 and VGAT, and the VGluT1/VGAT ratio in the hippocampus in aged mice. (A, B) Representative for immunoreactivity of parvalbumin and calretinin in the control and stressed groups in the hippocampus. Immunohistochemical staining. Scale bar = 200 μm. (C, D) The density of PV⁺ and CR⁺ inter-neurons in three sub-regions of the hippocampus in both groups. (E, F) Representative for immunoreactivity of VGluT1 and VGAT in the hippocampus in both groups. Immunohistochemical staining. Scale bar = 200 μm. (G–I) The relative optical density of VGluT1 and VGAT, and their ratio in three sub-regions of the hippocampus in both groups. ∗ P < 0.05, ^†P < 0.01, ^‡P < 0.001, indicating the significance of the main effect of group. Data are presented as mean ± standard error (n = 7–8 per group). CA1, Cornu ammonis 1; CA3, Cornu ammonis 3; CT, Control; DG, Dentate gyrus; PV, Parvalbumin; ST, Stress; VGAT, Vesicular gamma-aminobutyric acid transporter; VGluT1, Vesicular glutamate transporter-1.

Figure 5

Effects of adolescent chronic social instability stress on density of PV⁺ and CR⁺ inter-neurons, the expression levels of VGluT1 and VGAT, and the VGluT1/VGAT ratio in the amygdala in aged mice. (A, B) Representative for immunoreactivity of parvalbumin and calretinin in the control and stressed groups in the amygdala. Immunohistochemical staining. Scale bar = 200 μm. (C, D) The density of PV⁺ and CR⁺ inter-neurons in two sub-regions of the amygdala in both groups. (E, F) Representative for immunoreactivity of VGluT1 and VGAT in the amygdala in both groups. Immunohistochemical staining. Scale bar = 200 μm. (G–I) The relative optical density of VGluT1 and VGAT, and their ratio in two sub-regions of the amygdala in both groups. P < 0.01, compared between control and stressed group. ^†P < 0.001, the group × sub-region interaction. ^‡P < 0.01, indicating the significance of the main effect of group. Data are presented as mean ± standard error (n = 7–8 per group). BLA: Basolateral amygdala; CeA: Central amygdala; CR: Calretinin; CT: Control; PV: Parvalbumin; ST: Stress; VGAT: Vesicular gamma-aminobutyric acid transporter; VGluT1: Vesicular glutamate transporter-1.