Early life stress confers lifelong stress susceptibility in mice via ventral tegmental area OTX2

Abstract

Early life stress increases risk for depression. Here we establish a "two-hit" stress model in mice wherein stress at a specific postnatal period increases susceptibility to adult social defeat stress and causes long-lasting transcriptional alterations that prime the ventral tegmental area (VTA)-a brain reward region-to be in a depression-like state. We identify a role for the developmental transcription factor orthodenticle homeobox 2 (Otx2) as an upstream mediator of these enduring effects. Transient juvenile-but not adult-knockdown of Otx2 in VTA mimics early life stress by increasing stress susceptibility, whereas its overexpression reverses the effects of early life stress. This work establishes a mechanism by which early life stress encodes lifelong susceptibility to stress via long-lasting transcriptional programming in VTA mediated by Otx2.

Fig. 1. Early life stress enhances susceptibility to depression-like behavior

(A) Social interaction ratio and (B) exploration heat maps with social target present within the outlined zone. (C) Proportions of susceptible, indifferent, and resilient mice in each group. (D) Percentage preference for 1% sucrose solution in a two-bottle choice test. (E) Time immobile in a forced swim test (left) at each min and (right) total. (F) Open field center (left) exploration time and (right) total distance traveled. See table S2 for group sizes. Bars show means ± SEM overlaid with individual data points. *P < 0.05, **P < 0.01 indicate post hoc differences between socially defeated groups; α and δ indicate main effects (P < 0.05) of late postnatal stress (Late) or adult social defeat stress (Defeat), respectively.

Fig. 2. RNA-seq reveals long-lasting transcriptional alterations in VTA that predict OTX2 as an upstream regulator

(A) Union heat map of late postnatal stress and social defeat stress DEGs in VTA compared with standard-reared (Std)–Control (|fold-change| >1.3, P < 0.05). (B) Threshold-free comparison of DEGs by rank-rank hypergeometric overlap. Pixels represent the overlap between the transcriptome of each comparison (versus Std-Control), with the significance of overlap [−log₁₀(P value) of a hypergeometric test] color coded. Genes along each axis are sorted from most significantly upregulated to most down-regulated from the lower left corner. (C) Top predicted upstream regulators of DEGs by late postnatal stress. (D) Log₂ fold-change of OTX2-predicting genes and Otx2 by late postnatal stress. (E) Relative Otx2 mRNA levels across postnatal development. (F) OTX2-immunoreactive cells in VTA at PND17 and PND70 among Std (white) and late postnatal stress (blue) mice. (G) Mouse VTA (diagram, left) imaged at PND17. Scale bars, 50 μm. Data are means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 between Std and Late Control groups.

Fig. 3. Otx2 expression in VTA bidirectionally controls depression-like behavior

Depression-like behavior indicated by social interaction ratio (top), and VTA mRNA levels of Otx2 and OTX2-predicting genes (bottom), after each manipulation. HSV-Gfp (solid green) or HSV-Otx2 (solid purple) were overexpressed at (A) juvenile PND15 to 17 or (B) adult PND60 to 70 time points. HSV-miR-LacZ (open green) or HSV-miR-Otx2 (open purple) were injected for knockdown at (C) juvenile or (D and E) adult time points. Social defeat and behavioral testing was begun in adulthood 6 weeks after (A and C) juvenile or (E) adult manipulation or (B and D) <1 week after adult manipulation. Hashing indicates postdefeat quantification. Bars are means ± SEM, t < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001; horizontal bars indicate main effect of condition [two-way analysis of variance (ANOVA)] (see table S3).

Fig. 4. Late postnatal stress transiently alters OTX2 binding at target genes

ChIP for OTX2 (+) and IgG (−) in VTA at PND21 and PND70 for representative OTX2 target genes, Sema3c and Wnt1, after standard rearing (white/gray) or late postnatal stress (blue). Binding at Col8a1, Pax6, Thbs4, and Rbp3 shows a similar pattern (see fig. S4). (Top) Regulatory regions illustrated by H3K4me1 ChIP-seq enrichment, above gene features. OTX2 binding motif sites (purple), and primer pairs for qChIP amplification, are indicated (red bar, H3K4me1+ region corresponding to data below; gray bar, H3K4me1– region) (see fig. S4). (Bottom) Bars (means ± SEM) represent percentage input (n = 4 to 6), *P < 0.05.