An integrated epigenomic/transcriptomic approach to elucidate glucocorticoid-regulated gene networks in stress-related cognitive behaviour

压力影响着我们社会中人类和动物的生活。成功应对这种压力事件涉及大脑中的适应和认知过程，使个体在未来对类似的压力源更具弹性。然而，目前我们还不完全了解健康的大脑如何对压力事件产生生理和行为反应，并长期适应这些事件。应激事件导致肾上腺分泌“应激激素”或糖皮质激素（GC）进入血液。这些激素在大脑中起作用，通过与两种不同的GC酶结合“受体”结合来协调对压力的生理和行为反应。这些受体，称为MR和GR，是位于神经细胞中的蛋白质分子。作为压力挑战的结果，GC激素被分泌并与这些受体结合。然后，受体复合物在特定的对接序列（所谓的GRES）处与DNA内的某些基因结合，并调节这些基因的表达。这些基因被认为是重要的改变神经细胞的功能，以响应和适应适当的挑战。目前，关于其活性由于MR或GR结合而改变的基因的信息非常有限。我们的目标是深入了解由MR和/或GR调控的基因，并在学习科普不利的压力情况中发挥关键作用。我们将使用一种称为Morris水迷宫（MWM）的行为动物模型。这是一个圆形水池（直径1.8米），里面有水，老鼠可以通过找到隐藏在水面下的小平台逃离。利用游泳池周围墙壁上的标志（“空间线索”），老鼠很快学会找到平台。当大鼠被放入水池时，GC会分泌，因为这种情况对动物来说是有压力的。然而，这些激素是极其重要的，因为它们通过海马体中的MR和GR起作用，在那里它们刺激平台位置的学习。海马体是空间学习的关键大脑区域。我们的目标是揭示MR和GR调节基因的身份，通过两种方法的组合：1。通过染色质免疫沉淀（ChIP）和下一代测序，我们将确定在MWM训练的不同阶段，与未受干扰的“基线”条件相比，海马MR和GR中的哪些基因与整个大鼠基因组（> 20，000个基因）中的GR结合。我们纳入了一个所谓的游泳对照（SC）组，该组由与MWM训练（即有平台的游泳池）动物相同时间放置在无平台游泳池中的大鼠组成。因此，这些（SC）大鼠将经历在游泳池中的压力，但不会学会寻找平台。因此，包括SC组将有助于区分与压力影响相关的MR和/或GR结合的基因和与空间学习相关的基因，以找到平台。由于目前仍不清楚基因内MR和GR的结合是否确实改变这些基因的（mRNA）表达，我们将应用第二种技术：使用RNA测序，我们将评估在整个海马基因组的MWM训练，SC和基线大鼠的mRNA浓度的变化。两个数据集的计算比较（生物信息学）将使我们能够确定其活性因MR和/或GR结合而改变的基因，特别是空间学习的结果。随后，将进行实验，其中MR，GR或特定基因将被抑制，并对MR/GR结合，基因表达和MWM性能的影响将被研究，以深入了解这些受体和选定的高兴趣基因在空间学习中的特定作用。这些研究将增加我们对应激后分泌的GC如何帮助积极科普这种挑战的理解，并在类似事件再次发生时做好更好的准备。

Stress affects the lives of both humans and animals in our society. Successful coping with such stressful events involves adaptive and cognitive processes in the brain that make the individual more resilient to similar stressors in the future. Currently, however, we do not fully understand how the healthy brain generates physiological and behavioural responses to stressful events and adapts in the long-term to such events. Stressful events result in the secretion of 'stress hormones' or glucocorticoid (GC) hormones from the adrenal glands into the blood stream. These hormones act in the brain to coordinate physiological and behavioural responses to stress through binding to two different GC hormone-binding 'receptors'. These receptors, called MRs and GRs, are protein molecules located in nerve cells. As a result of a stressful challenge, GC hormone is secreted and binds to these receptors. The hormone-receptor complex then binds to certain genes within the DNA at specific docking sequences (so-called GREs) and regulate the expression of those genes. These genes are thought to be important to change the function of nerve cells in order to respond and adapt properly to the challenge. Presently, there is only very limited information about the genes whose activity is altered due to MR or GR binding. We aim to obtain insight into the genes that are regulated by MRs and/or GRs and play a critical role in learning to cope with an adverse, stressful situation. We will use a behavioural animal model called the Morris water maze (MWM). This is a circular pool (diameter 1.8 meter) containing water from which a rat can escape by finding a small platform hidden underneath the water surface. Using signs ('spatial cues') on the walls around the pool, the rat learns quickly to find the platform. When the rat is put in the pool, GCs are secreted because the situation is stressful for the animals. These hormones are however extremely important as they act via MRs and GRs in the hippocampus where they stimulate learning of the platform location. The hippocampus is a brain region critical for spatial learning. We aim to reveal the identity of the MR- and GR-regulated genes by combination of two methods: 1. With chromatin immuno-precipitation (ChIP) and next-generation sequencing we will determine in which genes in the hippocampus MRs and GRs are binding to GREs throughout the entire rat genome (>20,000 genes) at different stages of MWM training compared to the undisturbed 'baseline' condition. We include a so-called swim control (SC) group consisting of rats placed in the pool without a platform for the same time as the MWM-trained (i.e. pool with platform) animals. Thus, these (SC) rats will experience the stress of being in the pool but not learn to find a platform. Therefore, including the SC group will help to differentiate between genes bound by MRs and/or GRs involved in the effects of stress and those involved in spatial learning to find the platform. As it is presently still unclear whether binding of MRs and GRs within genes indeed changes (mRNA) expression of these genes, we will apply a second technique: 2. Using RNA sequencing we will assess changes in mRNA concentrations across the entire hippocampal genome of MWM-trained, SC and baseline rats. Computational comparison (bioinformatics) of the two data sets will allow us to determine the genes whose activity is altered as a result of MR and/or GR binding specifically as a result of spatial learning. Subsequently, experiments will be conducted in which MRs, GRs or specific genes will be inhibited and effects on MR/GR binding, gene expression and MWM performance will be studied to obtain insight into the specific roles of these receptors and selected high-interest genes in spatial learning. These studies will increase our understanding about how GCs secreted after stress help to cope actively with such a challenge and to be better prepared if a similar event would reoccur.