Environmental perturbation of the circadian clock during pregnancy leads to transgenerational mood disorder-like behaviors in mice

Abstract

It remains unknown whether chronic circadian disturbance (CCD) during pregnancy can lead to mood disorders in the offspring. Here we show that pregnant mice in the F0 generation that were exposed to CCD stress displayed depression-like behaviors, and produced offspring in the F1 and F2 generations that also exhibited mood-associated behavioral phenotypes despite the lack of direct stressful experiences during their postnatal or adult period. Prenatal CCD stress was correlated with the elevation of plasma corticosterone levels in F1 mice. Furthermore, the diurnal expression profiles of core circadian clock genes were disrupted in the suprachiasmatic nucleus of F1 mice. Proteomics analysis revealed that prenatal CCD stress resulted in distinct changes in protein expression in the hypothalamus of female F1 mice, in particular proteins that were associated with cellular activities, metabolism, development and diseases. Sex-specific differences in melanocortin 4 receptor expression were apparent in the CCD F1 generation. We conclude that maternal exposure to chronic circadian disturbance during pregnancy can lead to sex-specific mood disorders that persist for at least two filial generations. The underlying mechanisms may depend on transgenerational changes in plasma corticosterone levels, circadian pacemaking, and hypothalamic protein expression.

Figure 1

Experimental design and behavioral analyses of pregnant F0 mice in the CCD, CRS and NS groups. (a,b) Flow charts illustrate the experimental design, including the strategy and time course of animal breeding, stress exposures and behavioral tests in the three generations. (c) Depiction of the CCD paradigm, in which the LD cycle is delayed by 8 hours every two days from G0 to G18. (d–f) Body weight (d), sucrose preference (e) and immobility times in the forced swim test (f) of pregnant F0 mice at G0 and G18 in the CCD, CRS and NS groups. *P < 0.05, ***P < 0.001 vs. NS F0 group, n = 10 per group.

Figure 2

Effects of prenatal stresses on the body weight, sucrose preference and FST immobility times of F1 mice in the CCD, CRS and NS groups. (a,d) Body weight of female (a) and male (d) F1 mice at PND 28, 56 and 84 in the CCD, CRS and NS groups. (b,e) Sucrose preference of female (b) and male (e) F1 mice at PND 28, 56 and 84 in the CCD, CRS and NS groups. (c,f) FST immobility times of female (c) and male (f) F1 mice at PND 28, 56 in the CCD, CRS and NS groups. *P < 0.05, **P < 0.01, ***P < 0.001 vs. sex-matched NS F1 group, n = 33 female mice per group, n = 18 male mice per group.

Figure 3

mRNA and protein expression patterns of Clock and Bmal1 in the SCN of F1 mice in the CCD, CRS and NS groups. (a–h) Diurnal oscillations of Clock (a, e), Bmal1 (b,f), Per1 (c,g) and Per2 (d,h) transcripts in the SCN of NS F1 and CCD F1 mice, n = 3 per time point per group, n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. NS F1 group.

Figure 4

Gestational CCD stress in the F0 generation leads to mood disorder-like phenotypes in the F2 generation. (a,c) FST immobility times of female (a) and male (c) F2 mice at PND28 and 56. (b,d) Sucrose preference of female (b) and male (d) F2 mice at PND28 and 56. *P < 0.05, **P < 0.01, ***P < 0.001 vs. sex-matched NS F2 group, n = 15 per group.

Figure 5

Analysis of the hypothalamic proteome of CCD F1 female mice using Gene Ontology (GO) and IPA databases. (a) Pie chart of biological processes analyzed from the GO analysis database. (b) IPA database showing the associations of the differentially expressed proteins in the hypothalamus of CCD F1 female mice with diseases/disorders (b1), molecular/cellular functions (b2) and physiological system development and functions (b3). (c) Cluster analysis of differentially expressed proteins in the CCD F1 and CRS F1 groups. Differential expression was established relative to NS F1 female mice. (d) The same trend of the protein list of CCD F1 and CRS F1 groups.

Figure 6

Protein interaction networks involving proteins that are differentially expressed in the hypothalamus of CCD F1 female mice. (a) Network of cell-to-cell signaling and interaction, nervous system development and function, and cell morphology. (b) Network of behavior, cardiovascular development and function, and organ development. (c) Network of endocrine disorders, metabolic disease, and endocrine system development and function. In panels a, b and c, red symbols represent up-regulated proteins, and green symbols represent down-regulated proteins. (d) Western blots showing the hypothalamic expression of type 4 melanocortin receptor (MC4-R) in female and male F1 mice from the CCD, CRS and NS groups. MC4-R is indicated by the red circle in network C. (e) Quantitative analysis of MC4-R protein expression based on Western blotting. *P < 0.05, **P < 0.01 vs. NS F1 group.