产前束缚应激对成年后代长效影响的转录组学分析

Molecular mechanism related to long-lasting effects of prenatal maternal stress on adult offspring behaviors based on transcriptomic analysis

目的通过全转录组测序分析,探究小鼠束缚性应激引起后代与情绪控制中心(内侧前额叶皮层、海马、杏仁核)相关的焦虑样行为的分子机制。方法妊娠期最后1周(胚胎第14天直至分娩),每天上午8:00~9:00将C57BL/6孕鼠放置在束缚器中应激60 min,标记为应激组(STR组),对照组(CTL组)则不做任何处理;STR和CTL组均根据分娩后不同时间点进行处理;出生后第3天(postnatal day 3,P3)收集并提取内侧前额叶皮层、海马和杏仁核的组织总RNA进行全转录组测序分析;出生后第30天(postnatal day 30,P30)子代全脑进行冰冻切片,用于免疫荧光染色;出生后第120天(postnatal day 120,P120)子代进行旷场和提示性恐惧条件反射测试,以评估焦虑样行为。结果在旷场实验中,相比于CTL组,STR组的成年子代在场地中的运动总距离较短,中心驻留时间也明显减少;在条件恐惧记忆测试中,STR组子代的冻结反应率显著高于对照组,说明产前束缚应激诱发了后代小鼠的焦虑样行为;测序结合差异基因,基因表达网络分析和免疫荧光染色表明,产前束缚应激主要减少了内侧前额叶皮层中GABA神经元和黑质中多巴胺神经元的数量,以及影响海马和杏仁核神经发生和神经环路相关基因的表达。结论产前束缚应激影响后代海马神经发生和内侧前额叶皮层中多巴胺能通路和GABA能神经发生,可能与后代小鼠出现的焦虑样行为有关。

Objective To investigate the molecular mechanism related to the long-lasting effects of prenatal maternal stress on adult offspring behaviors with transcriptomic analysis.Methods A prenatal stress model was established by restraining C57BL/6 mice in plastic restrainers 60 min/d from embryonic d 14(E14)to delivery.In control group,the pregnant mice were left undisturbed in their home cages.At postnatal d3(P3),the medial prefrontal cortex(mPFC),the hippocampus,and the amygdala tissues were collected.Total RNA was extracted followed by library construction and Illumina sequencing.immunostaining was performed at P30 to validate mRNA sequencing results.For anxiety-like behavior assessment,open field and cued fear conditioning were carried out at P120.Results In the open field experiment,compared with the control group,prenatally stressed adult offspring made fewer trips in the field and spent significantly less time in the center.In the cued fear conditioning test,the freezing percentage was significantly higher in the stress group than that of in the control group.Behavioral assessment tests indicated that prenatal restraint stress caused anxiety-like behavior in adult offspring.Total RNA sequencing combined with differentially expressed gene and gene-to-gene network analysis and immunostaining revealed that in the mPFC,prenatal stress perturbed the expression of critical components of dopamine signaling and genes related to neurogenesis of GABAergic neurons.In the hippocampus and amygdala,prenatal stress mainly altered the expression level of genes that regulate neurogenesis and ion transportation.Conclusion Prenatal restraint stress can disturb the expression level of genes that regulate neurogenesis in the mPFC and hippocampus,which may result in anxiety-like behaviors later in life.