Sex differences in stress-induced corticosteroid receptor interaction with the rat brain genome: Gene transcriptional and behavioural implications

Stress strongly influences the lives of both humans and animals. Psychological stress, like marital problems and bullying, is very debilitating for mental health in humans. Our farmed and companion animals can also suffer from psychological stress such as overcrowding, long-distance transportation and abuse. Successful coping with such stressful events involves adaptive processes in the brain to increase the individual's resilience. To help people to cope with stress in their lives, to develop directives to reduce stress and to improve wellbeing of our companion and farmed animals, we need to increase our understanding of how the brain responds to psychologically stressful events. Presently, 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 glucocorticoid (GC) hormones ('stress hormones') from the adrenal glands into the blood. GCs 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. Stress-induced GCs bind to these receptors resulting in translocation to the cell nucleus. The hormone-receptor complex can then bind to certain genes within the DNA 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. Recently, we made significant progress in elucidating the genes and their functions affected by MR and GR after stress. It should be underscored, however, that this work, like most work in the stress/GC field, was conducted on male experimental animals (e.g. rats). Currently, it is completely unknown how MR and GR affect brain function at the gene level in females. This knowledge is however of great importance because females process and respond to stressful events differently than males. Clearly, the present situation in this scientific field is highly surprisingly given that half of humanity and a significant proportion of our companion/farmed animals is female.

Therefore, it is our aim to investigate how GCs via MR and GR affect the activity of genes in the female rat brain after stress to gain insight into why females respond differently to stress than males. Our first step will be to investigate the entire genome of both the female and male brain for the identity of the genes interacting with MR and GR after a stressful challenge and how this interaction alters the activity of these genes. We will conduct this investigation at different stages of the females' oestrus cycle. Furthermore, using a bioinformatics process called pathway analysis, this study will allow us to identify the molecular, cellular and behavioural functions regulated by MRs and GRs after stress comparatively between the two sexes. These analyses will provide us with a list of genes of interest for further study.

Subsequently, we will compare males and females in their response to different forms of stress because they process challenging situations differently. Female sex hormones like oestrogens are thought to be strongly involved in the different responses to stress in males and females. Therefore, in a separate study we will investigate the role of these hormones by inhibiting their synthesis and determine the impact on the molecular action of MR and GR in the brain after stress. Finally, as males and females show differences in emotionality which may be underpinned by differences in GC action, we will study the interaction of MR and GR with genes of interest in both sexes and their anxiety-related behaviour after administration of an anxiety-inducing drug. This work will substantially contribute to closing the gap in knowledge about how stress affects brain function in females.

Glucocorticoid (GC) hormones coordinate adaptive responses to stressful events to maintain health and wellbeing. In the brain, particularly in the hippocampus, GCs bind to two types of corticosteroid receptors: mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs). MRs and GRs are ligand-dependent transcription factors that bind to the same GC-responsive elements (GREs) within GC-target genes (e.g. Fkbp5, Sgk1). Using chromatin immuno-precipitation-sequencing (ChIP-Seq) and RNA-Seq, we recently found that, after stress, MR and GR interact with many genes resulting in significant changes in gene activity. Most work in this field, including these sequencing studies, however, have been conducted exclusively in male experimental animals (e.g. rats). Hence, interaction of MR and GR with genes in the hippocampus and other brain regions of females has never been reported. This is highly surprising as males and females clearly differ in stress and emotional responsiveness. Our project aims to close this gap in knowledge. We hypothesise that: 1. MR and GR show distinct binding patterns to GC-responsive genes and associated gene transcriptional responses in the female and male rat brain under baseline and acute stress conditions; 2. Sex steroids play an important role in the sex differences observed in MR and GR action within the rat brain genome; 3. Female and male rats show distinct changes in MR and GR action within the brain genome to anxiogenic stimuli which is associated with changes in GC secretion and anxiety-related behaviour. To test our hypotheses we will use acute stress models and pharmacological approaches. We will apply state-of-the-art ChIP and (Ribo-Zero) RNA analyses in combination with next-generation sequencing and qPCR to elucidate MR and GR genomic interaction profiles and m/hnRNA responses in relation to GC responses and changes in anxiety-like behaviour.