Holmes, Megan

Wang, Szu-Han

Shearer, Fraser John George

2022-03-18T11:03:43Z

2022-03-18T11:03:43Z

2022-03-18

The early life environment has a profound effect on later-life health and there is a robust positive correlation between stress (elevated glucocorticoids) during pregnancy and neuropsychiatric disorders in the adult offspring. However, the mechanisms by which inappropriate prenatal glucocorticoid exposure programmes mental health in later life are not fully understood. Animal models have demonstrated several possibilities but are confounded by the systemic nature of prenatal stress, which makes it hard to determine the site of glucocorticoid action, the mother, the fetus or both. The enzyme 11β-hydroxysteroid dehydrogenase type 2 (HSD2) is expressed in the placenta and throughout the fetus in early gestation. HSD2 metabolises glucocorticoids to their inert 11-keto forms. To determine whether the loss of brain expression of HSD2 alone is sufficient to programme the neuropsychiatric effects of prenatal glucocorticoid exposure, a mouse model was created lacking HSD2 only in developing fetal neurons (HSD2BKO). These mice have a memory deficit, display depression-like behaviour and have downregulated 5HT1A receptor (Htr1a) expression in the hippocampus as young adults. In this project, we aimed to determine the consequences of HSD2BKO on the brain in development, mid-life and old age and correlate these with behavioural outcomes. To determine the molecular changes that may lead to the observed phenotype in the model, a developmental time course of gene expression was performed (key genes involved in neuronal and neurovascular development). Additionally, we aimed to test the effect of maternal stress on the HSD2BKO model to determine the differential effects of elevated maternal glucocorticoids on the HSD2BKO model. We then looked at the adult brain with a hypothesis-free approach (hippocampal RNA-seq). Finally, behavioural outcomes were determined by novel object recognition (NOR), water maze tests and fear conditioning. Functional changes in the brains of HSD2BKO were determined by task-based functional magnetic imaging (fMRI). Developmental time course of gene expression demonstrated that Htr1a was downregulated in the sub-cortical regions of HSD2BKO fetuses at E17.5 but not E15.5 nor P1. Genes related to vascular development, vascular endothelial growth factor A (Vegfa) and neuropillin 1 (Nrp1), were not affected by genotype, nor was doublecortin (Dcx). At E17.5, control fetuses from stressed dams had significantly lower body weights compared with non-stressed controls; there was no significant difference in the weights of HSD2BKO mice based on stress condition. No difference was found in brain weights between genotypes nor between stress conditions, however, the ratio of brain weight to fetus weight was significantly lower in the stressed HSD2BKO offspring compared with the non-stressed offspring. RNA-seq analysis found no significant differences between the HSD2BKO and control mice, nor between the stress groups. The greatest source of genetic variance was between the brain regions (cortex, sub-cortex, hindbrain). Investigation of gene expression via RNA-seq did not reveal substantial differences in the adult hippocampus between the groups. However, RT-qPCR validation confirmed upregulation of protein kinase B beta (Akt2) in the hippocampus of 3–5-month-old HSD2BKO mice. Examination of the expression of genes upstream and downstream of Akt2 found upregulated expression of glycogen-synthase kinase 3 beta (Gsk3β) in the HSD2BKO cerebellum. In an object-location configuration of NOR, both control and HSD2BKO could complete the task at 3–5 months, but by 10–11 months, both genotypes significantly declined in their ability to recognise when the location of an object had been changed. In the Morris water maze, the aged HSD2BKO mice took longer to significantly reduce their latency to the platform, compared to controls. A similarly subtle difference was seen in examination of conditioned fear, where freezing behaviour had reduced in the HSD2BKO mice by the final presentation of the conditioned stimulus (CS), but not in controls. This was associated with greater activation of the BOLD fMRI signal in the infralimbic cortex at the final CS presentation of HSD2BKO mice versus control mice. Overall, we found subtle changes in behaviour of the HSD2BKO mice in the water maze. These may reflect differences in the connections between brain regions, particularly to the prefrontal cortex, which might relate to the altered activity in the infralimbic cortex observed in fMRI of conditioned fear. Further, the discovery of altered Htr1a expression early in development suggests a long-lasting effect of glucocorticoids on the serotonin system and could influence the development of the connectome, leading to the phenotypes observed.

https://hdl.handle.net/1842/38749

http://dx.doi.org/10.7488/era/2003

en

The University of Edinburgh

Effects of inappropriate glucocorticoid exposure of the fetal brain in mice lacking 11β-hydroxysteroid dehydrogenase type 2: from development to behaviour and ageing

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy