PRENATAL EXPOSURE TO CORTICOSTEROIDS: HYPOTHALAMIC CHANGES RELEVANT FOR POSTNATAL BEHAVIORAL IMPAIRMENTS

Prenatal exposure to synthetic corticosteroids can significantly alter postnatal development through changes in neurotransmitters, peptides and their receptors, and thus having long-lasting behavioral effects. Some of these changes have been observed in animal experiments, others also in humans prenatally exposed to synthetic corticosteroids. Here, we focused on transcriptomic changes within the ARC of rats prenatally exposed to either betamethasone or saline. The expression of transcriptome has been assessed by novel computational tools to determine complex changes that may have life-long effects on phenotype, i.e., behavior. Total of 18,094 unigenes were quantified in the hypothalamic ARC of P14 male and female rats prenatally exposed to betametasone used in this experiment. Out of these genes, Kyoto Encyclopedia for Genes and Genomes (http://www.genome.jp) selected 112 for the dopaminergic synapse, 75 for the GABAergic and 97 for the glutamatergic synapse. We further analyzed composition, topology and modulatory networks of the genomic fabric of the dopaminergic, GABAergic, and glutamatergic synapse (the transcriptome of the most interconnected and stably expressed gene network responsible for specific transmission). Finally we investigated the “transcriptomic landscape” of the GSF in the ARC of P14 males (M) and females (F) prenatally (G15) exposed to betamethasone (B) or saline (S). We combined in one measure (PWR = Pair-Wise Relevance) expression levels, controls and coordination of all pairs that can be formed by synapse genes with the other synapse genes, higher PWRs indicating larger influence of that gene pair to the fabric modulation. We found that prenatal exposure to betamethasone caused sex-dependent changes in the dopaminergic/GABA/glutamatergic synapse genes:. In males, 10 dopaminergic (9%), 4 GABAergic (5%) and 5 glutamatergic synapse genes (5%) were down-regulated. While in females, 9 dopaminergic (8%), 3 GABAergic (4%) and 6 glutamatergic (6%) synapse genes were downregulated. The data indicate that in both sexes the dopaminergic synapse was the most affected. In contrast, in control animals, no significant differences between male and female were present in these synapse genes. Since the most noticeable transcritpomic changes were found in the transcriptome of DA glutamatergic synapse, we investigated the expression of tyrosine-hydroxylase (TH) NMDA receptor subunits in the ARC. The western blot analyses and immunohistochemistry confirmed the sex-specific differences between prenatally betamethasone-exposed and saline-exposed P15 rats. Accordingly to the changes in gene expression, prenatal exposure to synthetic corticosteroids was associated with postnatal changes in behavior and susceptibility to certain types of seizures. While we did not find any significant impairements in normal behavioral patterns (open field activity), there was a sex-specific change in the novel object recognition test. We found that behavioral lateralization in females is lost after prenatal betamethasone exposure and both male and female prenatally betamethasone exposed rats were avoiding novelty. This trait is similar to children with autism and suggests that certain elements of autistic behaviors can be present after prenatal exposure to synthetic corticosteroids. Additionally, there were changes in the search patterns in the Morris water maze as well as in the Barnes maze. In conclusion, our work is consistent with findings of profound reprogramming changes in the brain after prenatal corticosteroid exposure associated with alterations cognitive functions and seizure susceptibility.产前暴露于合成糖皮质激素，可通过改变神经递质、肽类及其受体的表达，显著影响子代出生后发育，进而产生持久的行为学影响。此类改变已有部分在动物实验中被观测到，另有部分在产前暴露于合成糖皮质激素的人类子代中得到证实。本研究聚焦于产前暴露于倍他米松或生理盐水的大鼠下丘脑弓状核（Arcuate Nucleus, ARC）内的转录组改变。本研究借助新型计算工具对转录组表达谱进行分析，以甄别可能对子代表型（即行为表现）产生终身影响的复杂调控变化。本实验中，研究人员对产前暴露于倍他米松的14日龄（Postnatal Day 14, P14）雌雄大鼠的下丘脑弓状核内的18094个单基因（unigene）进行了定量分析。在上述基因中，京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG，http://www.genome.jp）筛选出112个与多巴胺能突触相关、75个与γ-氨基丁酸能突触相关，以及97个与谷氨酸能突触相关的基因。本研究进一步分析了多巴胺能、γ-氨基丁酸能及谷氨酸能突触相关基因组调控网络的组成、拓扑结构与调控模式——该网络即负责特异性突触传递、连接度最高且表达最稳定的基因转录组网络。最后，本研究对产前（妊娠第15日，G15）暴露于倍他米松（B）或生理盐水（S）的14日龄雌雄大鼠下丘脑弓状核内突触基因组调控网络（Genomic Synaptic Fabric, GSF）的"transcriptomic landscape"进行了分析。本研究将突触基因间所有可能配对的表达水平、表达调控及协同模式整合为一项综合指标（成对相关性，Pair-Wise Relevance, PWR）：PWR值越高，代表该基因配对对突触基因组调控网络的调控影响越大。研究发现，产前暴露于倍他米松可导致多巴胺能/γ-氨基丁酸能/谷氨酸能突触相关基因出现性别依赖性表达改变：雄性大鼠中，共有10个多巴胺能突触相关基因（占比9%）、4个γ-氨基丁酸能突触相关基因（占比5%）及5个谷氨酸能突触相关基因（占比5%）出现表达下调；雌性大鼠中则分别有9个（8%）、3个（4%）及6个（6%）上述突触相关基因发生表达下调。数据显示，在雌雄大鼠中，多巴胺能突触相关基因均为受影响最显著的类别；而在对照组动物中，上述突触相关基因的表达未出现显著的性别差异。鉴于多巴胺能与谷氨酸能突触的转录组变化最为显著，本研究对下丘脑弓状核内酪氨酸羟化酶（Tyrosine Hydroxylase, TH）及N-甲基-D-天冬氨酸（NMDA）受体亚基的表达进行了检测。蛋白质免疫印迹（Western Blot）与免疫组化实验结果证实，产前暴露于倍他米松与暴露于生理盐水的15日龄大鼠之间存在性别特异性表达差异。结合基因表达变化结果，产前暴露于合成糖皮质激素与子代出生后的行为改变及特定类型癫痫易感性升高存在关联。尽管本研究未观测到正常行为模式（如旷场活动）出现显著损伤，但新物体识别实验结果显示出性别特异性改变。研究发现，产前暴露于倍他米松的雌性大鼠行为侧化特征消失，且雌雄两组暴露大鼠均表现出对新异事物的回避行为。该特征与自闭症儿童的行为表现相似，提示产前暴露于合成糖皮质激素可能会引发自闭症样行为特征。此外，莫里斯水迷宫（Morris Water Maze）与巴恩斯迷宫（Barnes Maze）实验结果显示大鼠的搜索模式出现改变。综上，本研究结果与此前报道一致：产前暴露于糖皮质激素可引发大脑发生显著的重编程改变，进而导致认知功能与癫痫易感性出现异常。