1 SCIENTIFIC REPORTS | (2019) 9:764 | DOI:10.1038/s41598-018-37088-3 www.nature.com/scientificreports Antenatal Glucocorticoid Exposure Results in Sex-Specifc and Transgenerational Changes in Prefrontal Cortex Gene Transcription that Relate to Behavioural Outcomes Andrea Constantinof 1 , Vasilis G. Moisiadis 1 , Alisa Kostaki 1 , Moshe Szyf 2 & Stephen G. Matthews 1,3,4,5 Synthetic glucocorticoids (sGC) are administered to women at risk for pre-term delivery to reduce respiratory distress syndrome in the newborn. The prefrontal cortex (PFC) is important in regulating stress responses and related behaviours and expresses high levels of glucocorticoid receptors (GR). Further, antenatal exposure to sGC results in a hyperactive phenotype in frst generation (F 1 ) juvenile male and female ofspring, as well as F 2 and F 3 juvenile females from the paternal lineage. We hypothesized that multiple courses of antenatal sGC modify gene expression in the PFC, that these efects are sex-specifc and maintained across multiple generations, and that the gene sets afected relate to modifed locomotor activity. We performed RNA sequencing on PFC of F 1 juvenile males and females, as well as F 2 and F 3 juvenile females from the paternal lineage and used regression modelling to relate gene expression and behavior. Antenatal sGC resulted in sex-specifc and generation-specifc changes in gene expression. Further, the expression of 4 genes (C9orf116, Calb1, Glra3, and Gpr52) explained 20–29% of the observed variability in locomotor activity. Antenatal exposure to sGC profoundly infuences the developing PFC; efects are evident across multiple generations and may drive altered behavioural phenotypes. Te prefrontal cortex (PFC) is essential for top-down regulation of neuroendocrine and behavioural processes 1,2 . Glutamatergic eferents project from the PFC to forebrain regions that then project GABAergic eferents to the paraventricular nucleus (PVN), decreasing the hypothalamic-pituitary-adrenal (HPA) axis response to stress 2,3 . Te PFC is also highly sensitive to environmental stimuli (e.g. stress, sleep, diet), in particular, stimuli present during fetal and/or early postnatal life 4 . For example, antenatal exposure to high levels of glucocorticoids (GCs) programs changes in gene expression in the PFC that persist through adulthood 5 . Furthermore, altered signaling of key pathways in the PFC, such as the GABAergic signaling pathway, have been implicated in many psychiatric disorders that have developmental origins, including Attention Defcit Hyperactivity Disorder (ADHD) 6 , post- traumatic stress disorder (PTSD), major depressive disorder (MDD), and bipolar personality disorder (BPD) 7 . Tus, the PFC is a critical brain region of interest to the study of the impact of fetal exposures. Antenatal synthetic glucocorticoids (sGC) are administered to women at risk for preterm delivery to decrease the morbidity and mortality in the newborn associated with preterm birth (e.g. respiratory distress syndrome) 8–10 . 1 Departments of Physiology, University of Toronto, Toronto, ON, M5S1A8, Canada. 2 Departments of Pharmacology & Therapeutics, Sackler Program for Epigenetics & Psychobiology, McGill University, Montreal, QC, H3G1Y6, Canada. 3 Departments of Obstetrics and Gynecology, University of Toronto, Toronto, ON, M5S1A8, Canada. 4 Departments of Medicine, University of Toronto, Toronto, ON, M5S1A8, Canada. 5 Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON, M5G1X5, Canada. Correspondence and requests for materials should be addressed to S.G.M. (email: stephen.matthews@utoronto.ca) Received: 28 February 2018 Accepted: 28 November 2018 Published: xx xx xxxx OPEN