Early-life stress alters sleep structure and the excitatory-inhibitory balance in the nucleus accumbens in aged mice

Abstract

Background: Exposure to adverse experiences in early life may profoundly reshape the neurodevelopmental trajectories of the brain and lead to long-lasting behavioral and neural alterations. One deleterious effect of early-life stress that manifests in later life is sleep disturbance, but this has not been examined in aged mice and the underlying neural mechanisms remain unknown. Considering the important role of the nucleus accumbens (NAc) in the sleep-wake regulation, this study aimed to assess the effects of early-life stress on the sleep behaviors in aged mice and the potential involvement of the NAc in stress-induced sleep abnormalities.

Methods: Twenty aged male C57BL/6 mice (>16 months, n = 10 per group) were used in this study. During post-natal days 2 to 9, dams were provided with either sufficient (control) or a limited nesting and bedding materials (stressed). When the mice were 16 to 17 months old, their sleep-wake behaviors were recorded over 24 h using electroencephalogram and electromyelogram. The amount of each sleep-wake stage, mean duration, and stage transition was analyzed. Then, five animals were randomly chosen from each group and were used to measure the expression levels of vesicular glutamate transporter-1 (VGluT1) and vesicular transporters of γ-aminobutyric acid (VGAT) in the NAc using immunohistochemistry. Group comparisons were carried out using Student t test or analysis of variances when appropriate.

Results: Compared with the control mice, the early-life stressed aged mice spent less time awake over 24 h (697.97 ± 77.47 min vs. 631.33 ± 34.73 min, t17 = 2.376, P = 0.030), accordingly, non-rapid eye movement sleep time was increased (667.37 ± 62.07 min vs. 723.54 ± 39.21 min, t17 = 2.326, P = 0.033) and mean duration of rapid eye movement sleep was prolonged (73.00 ± 8.98 min vs. 89.39 ± 12.69 min, t17 = 3.277, P = 0.004). Meanwhile, we observed decreased VGluT1/VGAT ratios in the NAc in the stressed group (F(1, 16) = 81.04, P < 0.001).

Conclusion: Early adverse experiences disrupt sleep behaviors in aged mice, which might be associated with the excitatory-inhibitory imbalance in the NAc.

Figure 1

The sleep bioassay system for aged mice. (A) SleepSign software analysis window. (B) Typical examples of EEG and EMG over 10 s during wakefulness, NREM, or REM sleep in an aged mouse. EEG: Electroencephalogram; EMG: Electromyogram; NREM: Non-rapid eye movement; REM: Rapid eye movement.

Figure 2

Early-life stress decreased wakefulness and increased NREM sleep in aged mice. (A and B) Early-life stress reduced the amount of wakeful time in aged mice over 24 h (A), especially during the dark phase (B). (C) The amount of time spent awake every 3 h across a light/dark cycle. (D and E) Early-life stress increased the amount of time spent in NREM sleep in aged mice over the 24 h (D) and during the dark phase (E). (F) The amount of time spent in NREM sleep every 3 h across a light/dark cycle. (G–I) Early-life stress did not significantly affect the amount of time spent in REM sleep over 24 h (G), during the light or dark phase (H) or in 3-h bins across a light/dark cycle (I). ^∗P < 0.05, ^†P < 0.01. The data are presented as the mean ± standard error. (n = 9–10 per group). CT: Control; ES: Early-life stress; NREM: Non-rapid eye movement; REM: Rapid eye movement.

Figure 3

Early-life stress increased the mean REM duration in aged mice. (A–C) The number of episodes of wakefulness (A), NREM sleep (B), and REM sleep (C) over 24 h was not significantly affected by early-life stress. (D–F) Early-life stress did not alter the number of episodes of wakefulness (D), NREM sleep (E), or REM sleep (F) during the light or dark phase in aged mice. (G–L) Early-life stress increased the mean duration of REM sleep per episode (I and L), but not wakefulness (G and J) and NREM sleep (H and K), over 24 h and during the light and dark phases. ^∗P < 0.01. The data are presented as the mean ± standard error (n = 9–10 per group). CT: Control; ES: Early-life stress; NREM: Non-rapid eye movement; REM: Rapid eye movement.

Figure 4

Early-life stress did not affect sleep stage transition in aged mice. Early-life stress did not alter stage transitions over 24 h (A) or during the light phase (B) or the dark phase (C) in aged mice. The data are presented as the mean ± standard error (n = 9–10 per group). CT: Control; ES: Early-life stress; R: Rapid eye movement; S: Non-rapid eye movement; W: Wake.

Figure 5

Early-life stress decreased the excitatory/inhibitory ratio in the NAc. (A) A schematic of the two sub-regions of NAc, the core, and shell, used for immunohistochemistry analyses and representative sections immunostained for VGluT1 (left) and VGAT (right). Scale bar = 500 μm. (B and C) Representative immunoreactivity of VGluT1 and VGAT in the CT and ES groups in photographs of 30 μm coronal sections of the two sub-regions of the NAc. Scale bar = 200 μm. (D–F) The relative optical density of VGluT1 (D) and VGAT (E) and their ratio (F) in both groups. ^∗P < 0.001, indicating the significance of the main effect of group. The data are presented as the mean ± standard error (n = 5 per group). CT: Control; ES: Early-life stress; NAc: nucleus accumbens; VGAT: Vesicular GABA transporter; VGluT1: Vesicular glutamate transporter-1.