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

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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.