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Acute and Long-Term Effects on Gene Expression and Behaviour Western University Scholarship@Western Electronic Thesis and Dissertation Repository 11-16-2015 12:00 AM Ethanol exposure during synaptogenesis in a mouse model of fetal alcohol spectrum disorders: acute and long-term effects on gene expression and behaviour Morgan L. Kleiber The University of Western Ontario Supervisor Shiva M. Singh The University of Western Ontario Graduate Program in Biology A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © Morgan L. Kleiber 2015 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Developmental Neuroscience Commons, and the Genetics and Genomics Commons Recommended Citation Kleiber, Morgan L., "Ethanol exposure during synaptogenesis in a mouse model of fetal alcohol spectrum disorders: acute and long-term effects on gene expression and behaviour" (2015). Electronic Thesis and Dissertation Repository. 3343. https://ir.lib.uwo.ca/etd/3343 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Ethanol exposure during synaptogenesis in a mouse model of fetal alcohol spectrum disorders: acute and long-term effects on gene expression and behaviour (Thesis format: Monograph) by Morgan L. Kleiber Graduate Program in Biology Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy The School of Graduate and Postdoctoral Studies The University of Western Ontario London, Ontario, Canada © Morgan L. Kleiber 2015 Abstract Alcohol is a neuroactive molecule that is able to exert variable and often detrimental effects on the developing brain, resulting in a broad range of physiological, behavioural, and cognitive phenotypes that characterize ‘fetal alcohol spectrum disorders’ (FASD). Factors affecting the manifestation of these phenotypes include alcohol dosage, timing of exposure, and pattern of maternal alcohol consumption; however, the biological processes that are vulnerable to ethanol at any given neurodevelopmental stage are unclear, as is how their disruption results in the emergence of specific pathological phenotypes later in life. The research included in this thesis utilizes a C57BL/6J (B6) mouse model to examine the changes to gene expression and behaviour following a binge-like exposure to ethanol during synaptogenesis, a period of neurodevelopment characterized by the rapid formation and pruning of synaptic connectivity that correlates to brain development during the human third trimester. B6 pups were treated with a high dose (5 g/kg over 2 hours) of ethanol at postnatal day 4 (P4), P7, or on both days (P4+7). Mice were evaluated using a battery of behavioural tests designed to assess FASD-relevant phenotypes, and showed delayed achievement of neuromuscular coordination, hyperactivity, increased anxiety-related traits, and impaired spatial learning and memory. Gene expression analysis identified 315 transcripts that were altered acutely (4 hours) following ethanol exposure. Up-regulated transcripts were associated with cellular stress response, including both pro- and anti-apoptotic molecules, as well as maintenance of cell structural integrity. Down-regulated transcripts were associated with energetically costly processes such as ribosome biogenesis and cell cycle progression. Genes critical to synapse formation were also affected, as well as genes important for the appropriate development of the hypothalamic-pituitary-adrenal axis. ii Additionally, gene expression changes within the adult brain of mice treated with ethanol at P4+7 were examined to evaluate the long-term effects of neurodevelopmental alcohol exposure. Array analysis identified 376 altered mRNA transcripts with roles in synaptic function, plasticity, and stability, as well as epigenetic processes such as folate metabolism and chromatin remodeling. MicroRNA analyses identified changes in the levels of 33 microRNA species, suggesting that that long-term changes to gene expression following may be maintained (at least in part) via epigenetic mechanisms. Taken together, these analyses illustrate the sensitivity of synaptogenesis to ethanol exposure, leading to a ‘molecular footprint’ of gene expression changes that persists into adulthood and may contribute to the emergence of long-term behavioural and cognitive phenotypes associated with FASD. Keywords fetal alcohol spectrum disorder, brain, development, gene expression, ethanol, mouse model, microarray, behaviour iii Co-Authorship Data contained within this thesis includes work from previously published manuscripts: 1. Some of the behavioural data included in this thesis can be found within a manuscript entitled “Neurodevelopmental timing of ethanol exposure may contribute to the observed heterogeneity of behavioural deficits in a mouse model of fetal alcohol spectrum disorders (FASD)” by Katarzyna Mantha, Morgan Kleiber, and Shiva M. Singh, published in the Journal of Behavioral and Brain Science (2013). Morgan Kleiber designed the behavioural battery of tests and performed all behavioural testing and statistical analysis for mice treated during the third trimester-equivalent. Testing and analysis of mice treated during the first and second trimester-equivalent were performed by Katarzyna Mantha, who also wrote the manuscript. All authors contributed to the editing of the final publication. 2. Data pertaining to acute gene expression changes in neonates and microRNA expression changes in adult mice following third trimester-equivalent ethanol exposure are published in the manuscript “Third trimester-equivalent ethanol exposure is characterized by an acute cellular stress response and an ontogenetic disruption of genes critical for synaptic establishment and function in mice” by Morgan L. Kleiber, Benjamin I. Laufer, Randa L. Stringer, and Shiva M. Singh, published in Developmental Neuroscience (2014). Morgan Kleiber performed all animal and mRNA work, including analysis of array data. Analysis of microRNA data was performed by Benjamin Laufer and Morgan Kleiber. Quantitative PCR confirmation of mRNA levels was conducted by Randa Stringer and Morgan Kleiber. Morgan Kleiber wrote the manuscript with contributions from Shiva M. Singh. iv 3. Array data and analyses related to the gene expression changes in adult mice are published in the manuscript “Neurodevelopmental alcohol exposure elicits long- term changes to gene expression that alter distinct molecular pathways dependent on timing of exposure” by Morgan L. Kleiber, Katarzyna Mantha, Randa L. Stinger, and Shiva M. Singh, within the Journal of Neurodevelopmental Disorders (2013). Morgan Kleiber performed all animal and mRNA experiments for mice treated during the third trimester-equivalent. Katarzyna Mantha performed animal and mRNA experiments for the first and second-trimester equivalent treatment groups. Randa Stringer performed PCR confirmation of gene expression for the first trimester-equivalent samples. Morgan Kleiber wrote the manuscript, with contributions from Katarzyna Mantha and Shiva M. Singh. In all studies, Shiva M. Singh also provided intellectual contributions towards project development, manuscript revisions, and supervision. v Acknowledgements A thesis is never a solitary pursuit. I would like to thank the administrative staff and the many faculty members, particularly those in the Molecular Genetics Unit, that have supported my academic career. I have learned much from those that took interest in helping to further my training and I do not take their time and commitment for granted. Thank you to all the graduate students of the Department of Biology who have become great friends and colleagues, wherever in the world we may find ourselves. In particular, thank you to all the members of the Singh lab (alumni and current), including those that decided to adopt the fetal alcohol spectrum disorder project and run with it – I know I’m leaving it in good hands. I would like to thank my advisors Dr. Sashko Damjanovski and Dr. Kathleen Hill. Sash – thank you for your valuable input, especially during the preparation of this thesis. Kathleen – you have been both a mentor and a role model, and your generosity with your time and advice has contributed immeasurably to my development as both a researcher and a teacher. I give my sincerest thanks to Dr. Shiva Singh, my supervisor and mentor for many valuable years. His enthusiasm for genetic and mental health research and his desire to foster that enthusiasm in his students is undeniable. I am incredibly grateful for his mentorship, patience, support, and willingness to let me pursue research as my interests and the questions led. Thank you, Haroon Sheikh. I never expected that under the gruff exterior of the man that walked into the lab that day I would find such a sincere and generous friend, partner, and one of the best men I’ve ever known. Finally, my deepest thanks go to my family who traveled this road with me from the beginning and felt every obstacle and success as deeply as I. This thesis is dedicated to you. vi Table of Contents Abstract……………………………………………………………………………………………………………….ii Co-Authorship…………………………………………………………………………………………………….iv Acknowledgements…………………………….………………………………………………………………vi Table of Contents……………………………………………………………………………………………….vii List of Tables .........................................................................................................................................................
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