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Evaluation of oxytocin as a possible treatment for the effects of prenatal alcohol exposure on neurogenesis,… Baglot, Samantha L. 2018

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 EVALUATION OF OXYTOCIN AS A POSSIBLE TREATMENT FOR THE EFFECTS OF PRENATAL ALCOHOL EXPOSURE ON NEUROGENESIS, STRESS REACTIVITY, AND ANXIETY-LIKE BEHAVIOUR IN MALE AND FEMALE RATS  by  Samantha L. Baglot B.A., The University of British Columbia, 2015   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2018  © Samantha Baglot, 2018    ii Abstract The hippocampus provides inhibitory regulation on the hypothalamic-pituitary-adrenal (HPA) axis. Hippocampal neurogenesis (birth of new neurons) has been implicated in the HPA axis ability to mount an appropriate response to stress. Both the hippocampus and HPA axis are highly susceptible to early environmental modulation. Prenatal alcohol exposure (PAE) has been shown to alter HPA axis activity, reduce hippocampal neurogenesis, and increase anxiety-like behaviour. Dysregulated HPA axis activity and altered hippocampal neurogenesis following PAE likely underlie the behavioural expression of anxiety. The neuropeptide, oxytocin (OT), has been shown to dampen HPA axis response to stress, stimulate hippocampal neurogenesis, and act as an anxiolytic. OT is a prime therapeutic candidate to treat the altered stress responsivity and increased anxiety following PAE because it may act on underlying neurogenic and endocrine mechanisms. The objective of this study was to examine whether OT can modulate the effects of PAE on neurogenic, stress-response, and behavioural outcomes. In adulthood, male and female offspring from alcohol-fed, pair-fed, and control dams were treated daily with OT or vehicle for 10 days. OT-treated animals exhibited sedative-like effects and reduced locomotor activity. PAE animals showed fewer sedative-like effects, which may suggest altered OT sensitivity, as well as exhibited hyperactivity. Decreased locomotor activity following OT may preferentially impact hyperactive PAE animals. Interestingly, PAE animals also showed decreased anxiety-like behaviour; however, this effect may be confounded by hyperactivity and reflect impulsivity or inappropriate risk-assessment. PAE males showed attenuated CORT recovery following acute restraint stress, which OT did not modulate. PAE animals exhibited increased density of immature neurons in the dorsal dentate gyrus, whereas OT had no effect on neurogenesis. Utilizing the methods in this study OT was not able to mitigate the effects of PAE on endocrine   iii or neurogenic domains. However, OT may act to reduce hyperactivity following PAE, which may support attenuation of learning and memory deficits, attention problems, and impulsivity. Our findings are an important extension of previous work on altered neurogenic, endocrine, and behavioural responses following PAE. Our results support and extend literature on the use of OT as a therapeutic intervention, with novel utilization following PAE.    iv Preface SL Baglot designed the experiment, with help and guidance from J Weinberg, LAM Galea, and members from both laboratories. SL Baglot was involved in organization and execution of all tasks within the animal experiments, with the assistance of PJ Holman, C Raineki, L Ellis, T Bodnar, SE Lieblich, W Yu, V Lam, N Lan, A Lussier, and many undergraduate students. Major contributions to completion of drug administration, blood sampling, and behavioural testing by undergraduate students: L Wang, R Richardson, and P Ubi. Tissue sectioning was completed with the help of P Ubi, E Morgan, and E Kneller. Immunohistochemistry was completed with guidance from SE Lieblich, as well as assistance from many members of both the Weinberg and Galea laboratories. SL Baglot completed all cell counting. In order to calculate cell density, N Minielley and K Go measured hippocampal areas under the support and supervision of SL Baglot. Radioimmunoassay was completed with assistance from W Yu. All behavioural scoring was completed by C Fung and A Chao under the support and supervision of SL Baglot. All statistical analyses were conducted by SL Baglot, with guidance from LAM Galea, J Weinberg, T Bodnar, and PJ Holman. SL Baglot wrote the entirety of this thesis; prior to submission of the final thesis, J Weinberg and LAM Galea provided critical feedback and suggested edits.  The animal studies presented in this thesis were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, the Canadian Council on Animal Care (CCAC) guidelines, and were approved by the University of British Columbia Animal Care Committee (certificates: A15-0070, A14-0258).    v Table of Contents Abstract ..................................................................................................................................... ii Preface ...................................................................................................................................... iv Table of Contents ...................................................................................................................... v List of Tables........................................................................................................................... vii List of Figures ........................................................................................................................ viii List of Abbreviations ............................................................................................................... ix Acknowledgements .................................................................................................................. xi Dedication .............................................................................................................................. xiii Chapter 1: Introduction ........................................................................................................... 1 1.1 General overview and study objectives ........................................................................ 1 1.2 Overview of fetal alcohol spectrum disorders (FASD) ................................................. 3 1.3 Interaction of the hypothalamic-pituitary-adrenal axis and hippocampus ..................... 4 1.4 Prenatal alcohol exposure effects on hypothalamic-pituitary-adrenal axis, hippocampal neurogenesis, and anxiety-like behaviour ................................................................................ 8 1.5 Modulation of hypothalamic-pituitary-adrenal axis and hippocampal neurogenesis by oxytocin ................................................................................................................................ 10 1.6 Study hypothesis ....................................................................................................... 12 Chapter 2: Methods ................................................................................................................ 14 2.1 Animals and breeding ................................................................................................ 14 2.2 Diets and feeding ...................................................................................................... 14 2.3 Birth and weaning ..................................................................................................... 15 2.4 Adult drug administration .......................................................................................... 16 2.5 Acute stress exposure & radioimmunoassay for corticosterone .................................. 17 2.6 Behavioural test – novelty suppressed feeding (NSF) ................................................ 18 2.7 Tissue collection and processing................................................................................ 19 2.8 Doublecortin immunohistochemistry ......................................................................... 20 2.9 Microscopy ............................................................................................................... 21 2.10 Statistical analyses .................................................................................................... 21 Chapter 3: Results .................................................................................................................. 24 3.1 Alcohol-consuming dams showed reduced weight gain across gestation and early lactation ................................................................................................................................ 24   vi 3.2 Oxytocin reduced weight gain in males, but not females ............................................ 25 3.3 Oxytocin administration resulted in short-term sedative-like effects .......................... 25 3.4 Oxytocin administration resulted in long-term decreases in locomotor activity; prenatal alcohol exposure resulted in hyperactivity ............................................................................. 26 3.5 Prenatal alcohol exposure altered latency to feed, but not to investigate, in both males and females the novelty suppressed feeding task ................................................................... 28 3.6 Prenatal alcohol exposure reduced rearing in both sexes, whereas, oxytocin and prenatal alcohol exposure differentially increased self-grooming in males and females ......... 30 3.7 Prenatally alcohol exposed males showed differential corticosterone levels across time, whereas low dose oxytocin reduced corticosterone response to acute stress in males ............. 32 3.8 Both males and females prenatally exposed to alcohol showed elevated doublecortin expression in the dorsal hippocampus ................................................................................... 34  Chapter 4: Discussion ............................................................................................................. 36 4.1 Body weight .............................................................................................................. 37 4.2 Sedative-like effects of oxytocin................................................................................ 38 4.2.1 Prenatal alcohol exposure alters sedative-like response to oxytocin.................... 39 4.3 Locomotor activity .................................................................................................... 40 4.3.1 Hyperactivity following prenatal alcohol exposure ............................................ 40 4.3.2 Reduced locomotor activity following oxytocin ................................................. 40 4.4 Novelty suppressed feeding ....................................................................................... 41 4.4.1 Altered anxiety-like and stress-coping behaviour following prenatal alcohol exposure................................................................................................................................ 41 4.4.2 Oxytocin increased self-grooming in adult males but not females ...................... 43 4.5 Basal and stress-induced corticosterone levels ........................................................... 44 4.5.1 Prenatal alcohol exposure dysregulates corticosterone levels in adult males but not female.................................................................................................................................... 44 4.5.2 Low dose oxytocin reduces corticosterone response to acute stress in males ...... 45 4.6 Hippocampal neurogenesis ........................................................................................ 47 4.6.1 Prenatal alcohol exposure increased doublecortin-density in dorsal hippocampus in adult males and females ................................................................................................ 47 4.6.2 Oxytocin did not increase doublecortin-density in the hippocampus .................. 49 4.7 The effects of pair-feeding......................................................................................... 50 4.8 Oxytocin as a possible intervention following prenatal alcohol exposure: limitations and future directions ............................................................................................................. 51  Bibliography ........................................................................................................................... 54 Appendices .............................................................................................................................. 73 Appendix A: Supplementary figures ..................................................................................... 73   vii List of Tables Table 1 Pregnancy outcomes and maternal body weights throughout gestation and lactation..... 24 Table 2 Adult weights throughout drug treatment ..................................................................... 25    viii List of Figures Figure 1 Hypothalamic-pituitary-adrenal axis response to stress and negative feedback .............. 6 Figure 2 Experimental timeline ................................................................................................. 23 Figure 3 Short-term sedative-like effects of oxytocin and long-term locomotor activity ............ 27 Figure 4 Feeding behaviour in the novelty suppressed feeding task ........................................... 29 Figure 5 Rearing and self-grooming in the novelty suppressed feeding task .............................. 31 Figure 6 Corticosterone profile before and after acute restraint stress ........................................ 33 Figure 7 Density of doublecortin expressing cells in the dorsal and ventral dentate gyrus of the hippocampus ............................................................................................................................ 35    ix List of Abbreviations ACTH      Adrenocorticotropic Hormone ADHD      Attention Deficit Hyperactivity Disorder ANOVA     Analysis of Variance ASD      Autism Spectrum Disorder BAC      Blood Alcohol Content BAS      Basal Sample of Corticosterone BOLD      Blood Oxygen Level Dependent BrdU      Bromodeoxyuridine C      Control CCAC      Canadian Council on Animal Care CORT      Corticosterone  CRH      Corticotropin Releasing Hormone d      Day DCX      Doublecortin FAS      Fetal Alcohol Syndrome FASD      Fetal Alcohol Spectrum Disorder FL      Kellogg's® Froot Loops® GCL      Granule Cell Layer GD      Gestation Day GR      Glucocorticoid Receptors HPA      Hypothalamic-Pituitary-Adrenal i.p.      Intraperitoneal   x IHC      Immunohistochemistry LTP      Long-term Potentiation MR      Mineralocorticoid Receptor NIH      National Institute of Health NSF      Novelty Suppressed Feeding OT      Oxytocin OT.5      Oxytocin at a dose of 0.5mg/kg OT1      Oxytocin at a dose of 1.0 mg/kg PAE      Prenatal Alcohol Exposure PBS      Phosphate Buffered Saline PD      Postnatal Day PF      Pair-Fed PFA      Paraformaldehyde  RIA      Radioimmunoassay REC      Recovery Sample of Corticosterone RES      Response Sample of Corticosterone RT      Room Temperature SAL      Saline SGZ      Subgranular Zone       xi Acknowledgements Firstly, I would like to express my utmost gratitude to my supervisors, Dr. Joanne Weinberg and Dr. Liisa Galea. Thank you for providing unlimited mentorship and support throughout my graduate degree, and for believing in my potential from the beginning of my start as an undergraduate lab volunteer. You have both passed on your immense love of research and have given me every opportunity to grow into a better scientist. Thank you for teaching the value of being critical, the importance of preserving, and encouraging me in all my endeavors. I will forever be grateful for your mentorship. It has been a privilege to learn from you both. To my supervisory committee – Drs. Cathy Rankin and Jason Snyder – thank you for sharing your knowledge, providing valuable feedback and guidance throughout my training, and for pushing me to think critically and challenge myself. I would also like to thank Dr. Brian Christie for taking the time to support my project as an external examiner.  Secondly, I would like to express my sincerest gratitude to all members (past and present) of the Weinberg and Galea lab families – without all of you none of this work would have been possible. Lab managers Linda Ellis, Stephanie Lieblich, and Wayne Yu, thank you for your contributions to this thesis, for sharing your technical expertise, and for your friendships over the years. Thank you to Jess Chaiton, Dr. Paula Duarte-Guterman, and Dr. Aarthi Gobinath for sharing your methodological expertise, providing invaluable support and friendship, and engaging in both scientific and otherwise hilarious conversations. Thank you to the team of enthusiastic undergraduate students who contributed to this work, with special thanks to Pushpkiran Ubi, Cecilia Fung, Nicole Minielley, and Lisa Wang. Also, thank you to Erin Morgan for your contribution to this project, and for your years of friendship and support.    xii To Dr. Charlis Raineki – thank you for sharing your knowledge and passion for science, for providing feedback on an uncountable number of rough drafts, and for offering mentorship since my earliest research as an undergraduate. To Dr. Tamara Bodnar and Parker Holman – thank you for always engaging my scientific questions and discussions, for your guidance in all aspects of my graduate degree, for your mentorship in the lab and in the classroom, and for always encouraging me. To Rand Mahmoud – thank you for entertaining both scientific and personal conversations, for your research guidance, for always uplifting and reassuring me, and for being my bus party partner. The four of you have become an invaluable source of strength, inspiration, and guidance. Thank you for being a part of some of my most fun experiences. I will always be so extremely grateful for your mentorship and friendship. Thirdly, I would like to acknowledge the funding support that I have received over the years including the UBC Aboriginal Fellowship and the Natural Sciences and Engineering Research Council of Canada (NSERC) Alexander Graham Bell Canada Graduate Scholarship (CGS-M). I would also like to acknowledge the funding agencies that provided the grant support that made this work possible: NIH/NIAAA RO1 AA022460 and R37 AA007789 to JW, NeuroDevNet 20R64153 to JW, and CIHR MOP142308 to LAMG.  Finally, thank you to my family and friends for their enthusiasm and support. To my mother, Susan Coles – I owe my ambition, perseverance, and personal strength to you. From cheering at every Karate accomplishment, school event, and art contest as a child to encouraging my passion for science and always being there to listen, your support has been invaluable. To my partner, Justin Bennet – I owe my ability to be outspoken and some of my sense of humor to you. Thank you for making me laugh, balancing out my worrisome and rigidly organized personality, and constantly encouraging me throughout the challenges of graduate school and life.   xiii Dedication This thesis is dedicated to my grandmother, Catherine Coles, and my mother, Susan Coles. Thank you for your countless sacrifices, as well as endless love, comfort, and guidance. Both my undergraduate and graduate degrees could not have been completed without your continuous support and encouragement.                     1 Chapter 1: Introduction 1.1 General overview and study objectives  The endocrine and nervous systems have a complex and reciprocal relationship with shared connections, regulatory feedback, and signaling molecules (reviewed in Hiller-Sturmhöfel and Bartke, 1998). At the forefront of this relationship is the interaction between the hypothalamic-pituitary adrenal (HPA) axis and limbic brain regions, often referred to as the limbic-HPA. The HPA axis is part of the endocrine system responsible for stress-response; however, the HPA axis does not act alone and many limbic brain regions, including the hippocampus, amygdala, and medial prefrontal cortex, are involved in modulation of the stress-response (reviewed in Jankord and Herman, 2008). One of the key limbic structures involved in inhibition of the stress response is the hippocampus, which is widely known to be involved in learning and memory, and emotional behaviour. Interestingly, the hippocampus is a unique brain region with the ability to produce new neurons across the lifespan (i.e. neurogenesis), and these new neurons have been implicated in stress and emotional regulation. The interaction of the limbic-HPA axis underlies the physiological and behavioural responses to stress (reviewed in Jankord and Herman, 2008); even slight alterations in one part of this network can have severe concomitant effects on the other parts, as well as on physiological and behavioural output. Dysfunction of the HPA axis and limbic brain regions, such as the hippocampus, are related to psychopathology (reviewed in Frodl and O’Keane, 2013). In fact, the neurocircuitry underlying mood disorders, such as anxiety and depression, has significant overlap with the neurocircuitry involved in the stress response (reviewed in Shin and Liberzon, 2010; Frodl and O’Keane, 2013). Anxiety is a typical and often adaptive response to real or perceived threat; the stress response often precedes or is active during anxiety responses. However, anxiety disorders include pathologically excessive anxiety   2 and fear, and are some of the most common psychiatric disorders with an estimated global lifetime prevalence of ~16% (Somers et al., 2006; Remes et al., 2016).   A plethora of literature demonstrates that susceptibility to anxiety and other mental health problems can be determined early in life. In other words, early environment impacts the endocrine and nervous systems and can program these systems towards expression of high levels of anxiety, particularly in response to stress (reviewed in Gross and Hen, 2004; Lupien et al., 2009). Human and animal studies have shown that maternal stress, maternal depression, and early-life adversity (such as deprivation, neglect, or abuse) alter development of the HPA axis and the hippocampus, leading to increased susceptibility to anxiety and depression (Vallée et al., 1997; Mirescu et al., 2004; O’Connor et al., 2005; Halligan et al., 2007; Moriceau et al., 2009; Raineki et al., 2012). A growing body of literature suggests that exposure to alcohol in utero re-programs the fetal HPA axis, (Weinberg, 1988, 1989; Haley et al., 2006; Glavas et al., 2007; Uban et al., 2013), alters the development of the hippocampus (Redila et al., 2006; Willoughby et al., 2008; Sliwowska et al., 2010; Uban et al., 2010; Gil-Mohapel et al., 2014; Raineki et al., 2014), and increases susceptibility to anxiety (Roebuck et al., 1999; Hofmann et al., 2005; Hellemans et al., 2008; Denys et al., 2011; Cullen et al., 2013). Alcohol is a teratogen that disrupts normal development of the brain and body; prenatal alcohol exposure (PAE) produces a range of adverse alterations, together known as Fetal Alcohol Spectrum Disorders (FASD) (Stratton et al., 1996; Astley, 2000). The deficits following PAE often manifest early in childhood and persists into adulthood; for example, approximately 90% of individuals with FASD will have an additional mental health diagnosis across their lifetime (Streissguth et al., 1997; Clark et al., 2004). The most common additional diagnoses include depression, attention deficit hyperactivity disorder (ADHD), and anxiety (Denys et al., 2011).    3 Here, utilizing an animal model, we investigate the effects of PAE on HPA axis response to acute stress, neurogenesis in the hippocampus, and the expression of anxiety-like behaviour in adulthood. Furthermore, we aim to examine whether peripheral oxytocin can attenuate the effects of PAE on these endocrine, neurogenic, and behavioural domains. Oxytocin (OT) is a neuropeptide capable of decreasing HPA axis activity in response to stress (Windle et al., 1997, 2004; Parker et al., 2005; Cohen et al., 2010), increasing hippocampal neurogenesis (Leuner et al., 2012), and acting as an anxiolytic (Windle et al., 1997; Ring et al., 2006; Ayers et al., 2011; De Oliveira et al., 2012).  1.2 Overview of fetal alcohol spectrum disorders (FASD)  Fetal Alcohol Spectrum Disorders (FASD) encompasses a heterogeneous group of disorders resulting from exposure to alcohol during prenatal development (Streissguth et al., 1991; Astley, 2000). These adverse effects occur across a wide range of severities. At the most severe end of the spectrum, fetal alcohol syndrome (FAS) includes distinct facial dysmorphologies, growth retardation, and central nervous system abnormalities, whereas at the opposing end, alcohol-related birth defects and alcohol-related neurodevelopmental disorder includes congenital anomalies or behaviour, motor, and cognitive difficulties respectively (Streissguth et al., 1991; Stratton et al., 1996; Larkby et al., 1997; Astley, 2000). Despite differing levels of alcohol exposure and severity of deficits, individuals across the entire spectrum exhibit poor attention, hyperactivity, impulsivity, learning and memory difficulties, language deficits, problems with adaptive functioning, inappropriate social behaviour, and emotional dysregulation (Stratton et al., 1996; Astley, 2000; Mattson et al., 2001). FASD is the number one preventable developmental disorder in North America, with the annual cost of health   4 care problems associated with FASD over five billion dollars (May et al., 2014).  Since the first description of FAS by Jones and Smith (1973) there has been widespread research on the effects of PAE on the development and function of the endocrine and nervous system. Animal models report similar deficits as those seen in human FASD, including central nervous system abnormalities, physical malformation, and cognitive and behavioural changes (reviewed in Weinberg et al., 2008; Patten et al., 2014). Importantly, animal models provide the opportunity to study the mechanisms of alcohol’s effect on the developing brain without the confounds associated with human studies; specifically, animal models can control for the timing and amount of alcohol exposure. Two such mechanisms likely involved in the emotional dysregulation and increased susceptibility to anxiety following PAE include altered development and functioning of the HPA axis and the hippocampus.   1.3 Interaction of the hypothalamic-pituitary-adrenal axis and hippocampus The HPA axis response involves a cascade of hormones that is activated upon exposure to a stressor (see Figure 1). At the time of stress exposure, the paraventricular nucleus of the hypothalamus releases corticotropin-releasing hormone (CRH), which binds to its receptors in the anterior pituitary gland and stimulates release of adrenocorticotropic hormone. Adrenocorticotropic hormone (ACTH) enters the circulatory system and binds to its receptors in the adrenal glands. ACTH stimulates release of glucocorticoids, which are the main steroid hormones in response to stress; the primary glucocorticoid in humans is cortisol and in rats is corticosterone (CORT). Glucocorticoids have receptors throughout the brain and body promoting the well-known metabolic, cardiovascular, and immune responses following stress (reviewed in Frodl and O’Keane, 2013).    5 Once released into the circulatory system, glucocorticoids terminate further release by binding to glucocorticoid receptors (GR) in the hypothalamus and pituitary gland suppressing release of CRH and ACTH, a process known as negative feedback (see Figure 1). Circulating glucocorticoids also bind to other areas of the brain, including the hippocampus and amygdala, which differentially modulate the HPA axis. The hippocampus contains one of the highest densities of GR in the brain and has primarily GABAergic (i.e. inhibitory) connections to the hypothalamus (reviewed in Jankord and Herman, 2008). Multiple animal experiments utilizing hippocampal lesions, GR antagonism, and glucocorticoid overexpression have described a role of the hippocampus in inhibition of the HPA axis (Fendler et al., 1961; Slusher, 1966; Casady and Taylor, 1976; Herman et al., 1998; Feldman and Weidenfeld, 1999).    6   Figure 1 Hypothalamic-pituitary-adrenal axis response to stress and negative feedback The hypothalamic-pituitary-adrenal (HPA) axis response involves a cascade of hormones that is activated upon exposure to a stressor. Ultimately the adrenal glands synthesize and release glucocorticoids, which are the main steroid hormones in response to stress. The primary glucocorticoid in humans is cortisol and in rats is corticosterone. Once released into the circulatory system, glucocorticoids bind to glucocorticoid receptors (GR) throughout the brain and body promoting the well-known metabolic, cardiovascular, immune, and behavioural responses following stress. Further, through the process of negative feedback glucocorticoids bind to GR in the hypothalamus and pituitary gland to suppress their own further release and terminate the stress response. Circulating glucocorticoids also bind to other areas of the brain, including the hippocampus and amygdala, which differentially modulate the HPA axis. The hippocampus plays a primarily inhibitory role on the HPA axis.      7 More recently, the role of hippocampal neurogenesis in modulation of the HPA axis has been studied. Neurogenesis refers to the growth and development of new neurons in the brain, and roughly includes: 1) cell proliferation, the production of new cells, 2) cell migration, the movement of new cells to their appropriate location, 3) cell differentiation, the maturation of new cells into neuronal or glial phenotypes, and 4) cell survival, the point at which cells reach full maturity (Christie and Cameron, 2006). Neurogenesis occurs at the greatest level during development; however, the hippocampus possesses neurogenic abilities into adulthood. Importantly, newly generated neurons in the dentate gyrus of the hippocampus that survive to maturity have synaptic inputs and display appropriate resting membrane properties and action potential responses (van Praag et al., 2002); this suggests functional integration of these new neurons into existing hippocampal circuitry. Multiple studies in rodents have shown that ablation or suppression of hippocampal neurogenesis leads to alterations in HPA response and recovery to stress, as well as abnormal negative feedback (Schloesser et al., 2009; Snyder et al., 2011; Tsai et al., 2015). Although the specific direction of HPA axis change depends on the type, timing, and severity of stressor utilized, these studies provide evidence that adult hippocampal neurogenesis is important to mount an appropriate response to stress.  Given the high density of GR in the hippocampus, as well as its role in HPA negative feedback, it is not surprisingly that chronic stress or prolonged elevation of glucocorticoids are among the strongest factors associated with hippocampal impairment. Unlike acute stress, in which the hippocampus promotes glucocorticoid mediated negative feedback and inhibits the HPA axis, in times of chronic stress the high levels of glucocorticoids actually damage the hippocampus, causing alterations in synaptic plasticity, neuroplasticity, and cell death (reviewed in Goosens and Sapolsky, 2007). This damage reduces hippocampal inhibition of the HPA axis,   8 ultimately leading to further glucocorticoid release and additional hippocampal damage. Alterations in hippocampal neurogenesis following chronic stress occur across the lifespan. Most relevant to this study, chronically elevated glucocorticoids during prenatal development, such as through PAE, disrupts the development and functioning of the HPA axis and the hippocampus.   1.4 Prenatal alcohol exposure effects on hypothalamic-pituitary-adrenal axis, hippocampal neurogenesis, and anxiety-like behaviour Ethanol has direct and indirect effects on fetal development. Ethanol indirectly disrupts fetal development through alteration of the interaction between maternal-fetal systems, and directly through crossing the placenta and affecting developing fetal tissues (reviewed in Weinberg et al., 2008). For example, ethanol stimulates maternal HPA causing release of maternal glucocorticoids, which inhibit fetal HPA axis; alternatively, ethanol also crosses the placenta and directly stimulates fetal HPA axis. These conflicting messages impair development and subsequent functioning of the HPA axis. In fact, human studies have shown that PAE leads to greater cortisol reactivity in children (Haley et al., 2006). Moreover, animal models have similarly shown that PAE alters the development and functioning of all parts of the HPA axis including hypothalamus, anterior pituitary, and adrenal glands (reviewed in Weinberg et al., 2008). PAE often leads to hyperresponsivity following both acute and chronic stress, as well as altered HPA negative feedback and prolonged recovery from stress (Weinberg, 1988, 1989, 1992; Osborn et al., 1996; Weinberg et al., 1996; Ogilvie and Rivier, 1997; Glavas et al., 2007; Uban et al., 2013). The effects of PAE on HPA axis are sexually dimorphic and rely on the interactions with and modulation by gonadal hormones (reviewed in Weinberg et al., 2008).   9 Not surprisingly, given the effects of ethanol on all aspects of the developing HPA axis the hippocampus is also particularly vulnerable to the teratogenic effects of alcohol exposure. Studies in clinical literature reveal reduced hippocampal volume following PAE (Riikonen et al., 1999; Willoughby et al., 2008). Furthermore, in rats, PAE results in decreased neural activity in the hippocampus of males (Raineki et al., 2014), altered hippocampal mineralocorticoid receptor (MR) regulation in females and GR regulation in males (Glavas et al., 2007), decreased hippocampal long-term potentiation (LTP) in males but enhanced LTP in females (Titterness and Christie, 2012; Patten et al., 2013; Sickmann et al., 2014), and reduced hippocampal dendritic spines (Abel et al., 1983). Volume reductions in humans suggests gross morphological changes, such as decreased production of cells in the dentate gyrus of the hippocampus. Importantly, studies in rodents have shown that PAE alters cell proliferation, neuronal survival, and cell death across the lifespan (reviewed in Gil-Mohapel et al., 2010). Specific to adulthood, PAE decreases cell proliferation and neuronal survival in males (Christie et al., 2005; Redila et al., 2006; Sliwowska et al., 2010) and reduces the proportion of new neurons and glia cells in females (Uban et al., 2010). The effects of PAE on cell differentiation or the quantity of immature neurons in adulthood remains unknown.  Dysregulated HPA axis response to stress, as well as altered hippocampal neurogenesis, likely underlie the effects of PAE on the behavioural expression of anxiety. Clinical studies have shown that children with FASD score high on anxiety checklists (Roebuck et al., 1999) and individuals with FASD are extremely likely to be diagnosed with at least one type of anxiety disorder (Famy et al., 1998; Clark et al., 2004; Denys et al., 2011). Studies in rats confirm these findings, with an increase in anxiety-like behaviour on a variety of tasks following PAE (Hofmann et al., 2005; Dursun et al., 2006; Weinberg et al., 2008; Cullen et al., 2013).   10 1.5 Modulation of hypothalamic-pituitary-adrenal axis and hippocampal neurogenesis by oxytocin Oxytocin (OT) is a neuropeptide widely known for its role in parturition, lactation, and social behaviour; however, OT is also strongly implicated in stress and emotional regulation (reviewed in Neumann, 2008; Neumann and Landgraf, 2012). Animal studies reveal OT production in the paraventricular and supraoptic nuclei of the hypothalamus, as well as receptor  expression in peripheral tissues and throughout the brain within the cortex, basal ganglia, limbic system, thalamus, hypothalamus, and brainstem (reviewed in Gimpl and Fahrenholz, 2009). Antibodies for OT receptor localization in humans lacks specificity, however a few studies have shown relatively similar distribution patterns within brainstem, hypothalamic, and limbic regions (Loup et al., 1989; Boccia et al., 2013).  Studies using rodents show OT release in response to a wide range of stressors, taking action within various brain regions (including the hypothalamus and hippocampus) as well as release into the blood stream (Wotjak et al., 1998; Neumann, 2008; Neumann and Landgraf, 2012). Given the role of the hippocampus in HPA axis negative feedback, it is thought that OT may help terminate the stress response. In support of this, both acute and chronic stress exposure increase OT receptor binding in the hippocampus of male rats, whereas removal of glucocorticoids downregulates OT receptor binding (Liberzon et al., 1994; Liberzon and Young, 1997). Further, intracerebroventricular (central) administration of OT reduces not only ACTH and CORT responses to stress (Windle et al., 1997, 2004), but also reduces the stress-induced brain activation in the hypothalamus and hippocampus in female rats (Windle et al., 2004). Hippocampal administration of OT also reduces CORT response to stress, and decreases hippocampal GR expression in male rats (Cohen et al., 2010).    11 Studies utilizing central administration are vital in determining the mechanisms of OT within the brain on regulation of the HPA axis. However, central administration is invasive and in order to develop treatments for mental health disorders in humans the action of peripheral administration must also be examined. It is thought that administration of peripheral OT alters physiology and behaviour through either crossing the blood-brain-barrier, diffusion into the cerebrospinal fluid, and/or upregulation of the endogenous central OT system (Neumann et al., 2013; Lee et al., 2017). Importantly, both nasal and i.p. administration of OT causes rapid elevation in brain and plasma OT content in rodents, with peak levels following i.p. administration within 10-min in plasma and 30-min in the brain (Neumann et al., 2013). Further, although the exact patterns differ, both central and peripheral OT significantly change blood-oxygen level dependent (BOLD) signals within the brain of awake rats in the first 10 min of administration (Ferris et al., 2015). Notably, similar to central administration, chronic nasal administration of OT decreases ACTH levels in female monkeys (Parker et al., 2005) and chronic subcutaneous injection of OT decreases GR mRNA levels in the hippocampus of male rats (Petersson and Uvnäs-Moberg, 2003).  Given the aforementioned modulation of the HPA axis by OT, as well as the role of hippocampal neurogenesis in mounting an appropriate stress response, it is not surprising that peripheral and central administration of OT also increase hippocampal cell proliferation and neuronal survival in adult male rats (Leuner et al., 2012). However, despite studies suggesting differences in development and functioning of OT system in males and females (Neumann, 2008; Tamborski et al., 2016), there is little research analyzing modulatory effects of OT on hippocampal neurogenesis in females.    12 A growing body of literature demonstrates that OT has a well-established anxiolytic effect. Specifically, rodent studies have illustrated that: OT-deficient female mice display more anxiety-like behaviour (Mantella et al., 2003; Amico et al., 2004a); central and peripherally administered OT decreases anxiety-like behaviour in male and female rodents (Windle et al., 1997; Ring et al., 2006; Blume et al., 2008; Ayers et al., 2011; Bowen et al., 2011); and OT-antagonism prevents the anxiolytic effects of peripheral administration (Mantella et al., 2003). Moreover, plasma and cerebral spinal fluid concentrations of OT are negatively related to trait anxiety scores in children (Carson et al., 2015), and intranasal OT in adult men reduces self-assessment of anxiety before a public speaking test (De Oliveira et al., 2012).   1.6 Study hypothesis Human and animal studies have shown that PAE alters the development and long-term functioning of the limbic-HPA axis, including the hippocampus; specifically, PAE causes persistent dysfunction in stress response and recovery, as well as expression of hippocampal neurogenesis. These mechanisms likely underlie the increased susceptibility to anxiety disorders in individuals with FASD, as well as the expression of anxiety-like behaviour in PAE animals. Sex differences not only exist in the development and functioning of the HPA axis and the hippocampus, but also in the type and severity of deficits observed following PAE in animal models and human disorder. We aim to extend research on the effects of PAE on neurogenic, endocrine, and anxiety-like behaviour in adult male and females. Further, the effects of PAE on the level of immature neurons, measured by doublecortin (DCX) expression, in adulthood remains unknown.     13 FASD is a neurodevelopmental disorder, however most deficits that occur following PAE persist well into adulthood. Individuals with FASD exhibit a wide range of deficits and are often treated with anti-psychotics, antidepressants, anxiolytics, and stimulants aimed at alleviating specific symptoms of their additional diagnoses. However, there is a serious lack of treatment targeted towards the specific mechanisms underlying the symptomology of FASD. OT may provide a more targeted approach by acting on some of the mechanisms, specifically the HPA axis and hippocampal neurogenesis, that underlie dysfunctions in stress and emotional regulation following PAE. Further, OT is a prime therapeutic candidate for treatment during adulthood as it promotes neural plasticity of the adult brain. In fact, the modulatory effects of OT on stress response and hippocampal neurogenesis have been shown in non-manipulated animals and under conditions of chronic stress in adulthood (Windle et al., 1997; Cohen et al., 2010; Leuner et al., 2012). Importantly, OT administration has also been shown to reverse anxiety-like behaviour and social deficits in animals following prenatal stress (Lee et al., 2007; Rault et al., 2013). OT has been examined in clinical trials as a treatment for autism spectrum disorder (ASD) (reviewed in Preti et al., 2014). Whether OT can stimulate similar changes in PAE males and females whose previous life history (i.e. exposure to alcohol in utero) has caused altered brain plasticity and HPA axis function has not been demonstrated. Furthermore, the effects of peripheral OT administration on stress response and hippocampal neurogenesis are not as well-known as the effects of central administration, especially in females. We aim to extend research on the effects of OT on neurogenic, endocrine, and anxiety-like behaviour in adult male and females; as well as determine whether OT can modulate the effects of PAE. We hypothesize that OT administration will attenuate the stress-induced hyperresponsivity of the HPA axis, the decreased hippocampal neurogenesis, and the expression of anxiety-like behaviour following PAE in adulthood.   14 Chapter 2: Methods 2.1 Animals and breeding Male (n=17) and nulliparous female (n=36) Sprague-Dawley rats were obtained from Charles Rivers Laboratories (St. Constant, QC, Canada). Rats were pair-housed by sex in clear polycarbonate cages with beta-chip bedding, and ad libitum access to water and standard laboratory chow (Harlan #2918). Colony rooms were maintained on a 12:12 h light/dark cycle (lights on at 0700 h) at a constant temperature of 21 ± 1°C. After a 2-week acclimation period, in which all animals were handled, each female was pair-housed with a single male and vaginal lavage samples were collected each morning. The presence of sperm indicated gestation day 1 (GD1). All animal care procedures were performed in accordance with the National Institutes of Health (NIH) and Canadian Council on Animal Care (CCAC) guidelines and were approved by the University of British Columbia Animal Care Committee.   2.2 Diets and feeding On GD1, females were single-housed into cages on ventilated racks and randomly assigned to one of three conditions: 1) Prenatal alcohol exposure (PAE) – ad libitum access to a liquid ethanol diet with 36% of total calories derived from ethanol (n = 12); 2) Pair-fed (PF) – liquid control diet with maltose-dextrin isocalorically substituted for ethanol, in the amount consumed by a PAE partner (g/kg body weight/day of gestation), to control for the reduced food intake of PAE dams (n = 9); and 3) Control (C) – ad libitum access to a pelleted version of the liquid control diet (n = 10). All dams had ad libitum access to water and were left undisturbed except for weekly weighing (GD1, GD7, GD14, and GD21) and cage changing (GD1, GD7, and GD14). All diets were formulated to provide optimal nutrition to pregnant rats (Weinberg/Keiver   15 High Protein Experimental (PAE) Diet #710324, Control (PF) Diet #710109, and Pelleted Control Diet #102698, Dyets Inc. Bethlehem, PA, USA). Ethanol diet was introduced gradually over the first three days; bottles contained: day 1 – 66% control diet, 34% ethanol diet; day 2 – 34% control diet, 66% ethanol diet; and day 3 – 100% ethanol diet. All diets were presented daily one-hour prior to lights off (1800-1900h) and continued until GD21. On GD17 a blood sample was collected from the tail vein of a subset of PAE, PF, and C dams (n = 1-3) at lights on. Blood alcohol content (BAC) were measured as previously reported (Hellemans et al., 2010; Uban et al., 2010) and determined to be 164 ± 55 mg/dL in alcohol-consuming dams.   2.3 Birth and weaning Beginning on GD22, and continuing throughout lactation, all dams were offered ad libitum access to standard breeder chow (Harlan #2919) and water. On the day of birth (postnatal day 1, PD1) litters were culled to 12 pups (7 males, 5 females when possible). If necessary, cross fostering of pups from the same prenatal condition born on the same day occurred to maintain litter size. Dams and pups were weighed on PD1, PD8, PD15, and PD22. Pups were weaned on PD22 and group housed by litter and sex. At weaning, all pups were ear-notched to indicate litter number. Starting around PD25 pups were pair-housed (into same sex and prenatal condition pairs, but with pups from different litters) in standard cages with filterless lids, enrichment tube, and beta-chip bedding, with ad libitum access to standard laboratory chow (Harlan #2918) and water. Except for weekly cage changing rats were left undisturbed until entering the study in adulthood (PD70-77).      16 2.4 Adult drug administration For five consecutive days prior to the start of drug administration, rats were handled to habituate to experimenters and the body position utilized for injection, as well as administered pieces of Kellogg's® Froot Loops® (FL) to familiarize them to the palatable food used in the experimental behaviour task and avoid neophobic reactions. Starting on PD70-77 male and female rats from all three prenatal treatment groups were split into three drug administration groups (n=8-10/group). Rats were injected intraperitoneal (i.p.) with oxytocin (0.5 mg/kg or 1.0 mg/kg; OT.5 and OT1 respectively) or saline (SAL) for 10 consecutive days. One male and one female per litter, per drug treatment, were used in the study to control for litter effects. OT acetate salt (Bachem, CA, USA) was stored at -20°C until dissolved into 0.9% saline. All animals were injected with the same proportional volume (1ml/kg body weight) of solution; thus, OT solutions were made at different concentrations (OT.5 = 0.5 mg/ml, and OT1 = 1.0 mg/ml). The order in which animals received injections was randomized each day to decrease any effects of time of day (all injections occurred between 0800h and 1100h). The site of injection (left or right) was alternated each day.  These OT doses are consistent with previous literature and indicated to be high enough for some of the peptide to cross the blood brain barrier (Chakraborty, 2017). Furthermore, chronic administration was chosen because this pattern has previously shown alterations in HPA axis function, hippocampal neurogenesis, and anxiety-like behaviour. Through pilot testing it was determined that immediately following OT injection animals develop a specific and replicable set of short-term characteristics, which include: lethargy, sleepiness, immobility, piloerection, and mild hind-limb paresis. We will refer to this set of characteristics as sedative-like; these responses occur within 1-2 minutes following injection and then resolve, with the animals   17 displaying typical behaviour, within 45 ± 15 min. Therefore, animals were monitored following injection with the presence of any sedative-like effects denoted as a score of 1 and the absence of all sedative-like effects denoted as a score of 0. These scores were cumulated across the 10-day injection period. Due to these sedative-like effects all rats were given a 2 h recovery period following injection before any testing occurred.  2.5 Acute stress exposure & radioimmunoassay for corticosterone One hour after lights on (0800 h), and prior to injection on day 8, a basal blood sample (BAS) was collected from the tail vein of all animals. Basal sampling occurred within 3 minutes of initial cage movement. Immediately following basal sampling rats were injected as per usual and given a 2 h recovery period. This recovery period occurred to ensure that the sedative-like effects of OT had resolved and that animals returned to a basal level of corticosterone (CORT) following injection (analysis on a subset of SAL injected animals revealed that 2 h following injection both males and females return to basal levels of CORT, data not shown). Directly following this 2 h recovery period, rats were placed into restraint tubes for 30 min (restraint stress began for all animals between 1000 and 1100 h). Blood samples were collected from the tail vein of all animals immediately following restraint (response sample; RES) and 1 h later (recovery sample; REC).  Following collection, blood samples were kept at room temperature (RT) and centrifuged at 2400 rpm for 10 minutes. Serum was collected and stored at -20°C until analysis by radioimmunoassay. Total serum CORT was measured using a double antibody 125I radioimmunoassay kit (MP Biomedicals, Solon, OH, USA). Samples were run in duplicates and in accordance with the manufacturer’s instructions. Samples were randomized across assays with   18 an equal representation of sex, prenatal condition, and drug treatment in each assay. All three samples (BAS, RES, REC) from each animal were run within the same assay. The intra-assay coefficient of variation was less than 4.5%; inter-assay coefficient of variation less than 7.5%.  2.6 Behavioural test – novelty suppressed feeding (NSF) Prior to the novelty suppressed feeding task, and at 1700 h on day 8 of injection, all animals were food deprived overnight (18 h) in their home cages; ad libitum access to water continued. Food deprivation is the standard procedure used to motivate animals to feed. The NSF task is a test of anxiety-like behaviour that utilizes the animal’s tendency to exhibit neohypophagia, or the aversion to entering novel or open environments to feed, and forces rats into an anxiogenic conflict of remaining at the periphery or entering the open arena to consume familiar and rewarding food (Bodnoff et al., 1989; Campos et al., 2013). The main measure of anxiety-like behaviour is latency to feed. The NSF task was chosen because it has been shown to be sensitive to the administration of chronic anxiolytics and antidepressants, with reductions in latency to feed occurring along the same time course as drug effectiveness in humans (~2 weeks) (Bodnoff et al., 1989; Iijima et al., 2012). Further, ablation of adult hippocampal neurogenesis prevents the reductions in latency to feed following chronic antidepressants; in other words, the efficacy of antidepressant treatment is dependent on the presence of neurogenesis (Santarelli et al., 2003; David et al., 2009; Mateus-Pinheiro et al., 2013).  Following recovery from injection (2 h ± 5 min) on day 9, rats were placed individually in a novel open arena (60 × 60 × 50 cm), at a consistent corner of the arena, with one FL placed in the center. All testing occurred in a dimly lit room between 1000 and 1300 h. The NSF arena was thoroughly cleaned with 1% vinegar between animals. Immediately following NSF, rats were   19 placed singly into standard cages containing home cage bedding; latency to feed and amount of standard laboratory chow consumed within the first hour was measured to test for appetite differences.  The 10-min task was recorded and scored for additional behaviours. Previous findings indicate that both PAE and OT can alter locomotion (Uvnäs-Moberg et al., 1994; Hellemans et al., 2008; Brys et al., 2014; Leong et al., 2016); therefore, total distance traveled was calculated using the automated program Noldus Ethovision 3.1. It is also well known that PAE causes impulsivity or a lack of response inhibition (Olmstead et al., 2009; Muñoz-Villegas et al., 2017), therefore latency to investigate was scored and could then be compared to the main measure of latency to feed. Finally, PAE alters rearing behaviours (Gabriel et al., 2006; Hellemans et al., 2008), and OT alters self-grooming (Van Erp et al., 1993; Amico et al., 2004b). All of these additional behaviours (latency to investigate, rearing, and self-grooming) were hand scored by a single experimenter using Noldus Observer 5.0; the experimenter was blind to experimental groups and achieved an intra-rater reliability score of ~95%.  2.7 Tissue collection and processing Within approximately 2 h following injection on day 10, animals were anesthetized using isofluorane and perfused transcardially with 100-120 ml of phosphate buffered saline (PBS) followed by 175-210 ml of neutral buffered formalin (4% paraformaldehyde; PFA). Perfusions were accomplished using peristaltic perfusion pumps (flow rate of 25-30 ml/min). Brains were extracted and post-fixed in PFA overnight; fixed brains were transferred to 30% sucrose solution for cryoprotection until sectioning. Vaginal lavage samples were also collected to analyze estrous cycle phase at the time of termination.    20 Brains were sliced into 30 𝜇m coronal sections using a Leica SM2000R microtome (Richmond Hill, ON, Canada). Sections were collected in series of ten throughout the entire anterior-posterior extent of the hippocampus and stored in anti-freeze solution (comprised of 30% ethylene glycol, 20% glycerol, and 0.1M PBS) at -20°C until processing.  2.8 Doublecortin immunohistochemistry Sections were rinsed in 0.1M PBS (5 × 10 min), treated with 0.6% hydrogen peroxide (30 min), rinsed again in 0.1M PBS (3 × 10 min), and incubated in primary antibody solution (RT for 1 h, 4°C for 23 h, on a rotator plate). Primary antibody solution contained: 0.001% goat anti-doublecortin (Santa Cruz Biotechnology [SC-8066], CA, USA), 0.04% Triton-X, and 3% normal rabbit serum in PBS. The following day, sections were rinsed in 0.1M PBS (5 × 10 min) and incubated in secondary antibody solution (4°C for 21 h 30 min, on a rotator plate). Secondary antibody solution contained: 0.05% biotinylated rabbit anti-goat (Vector Laboratories [lot# ZA0425] Burlington, ON, Canada) in PBS. The following day, sections were rinsed in 0.1M PBS (5 × 10 min) and incubated in AB complex (RT for 4 h, on a rotator plate). AB complex solution contained: 0.001% A and B stock (ABC Elite Kit, Vector) in PBS. Sections were then rinsed again in 0.1M PBS (3 × 10 min) and left at 4°C on a rotator plate overnight. The following day, sections were rinsed in 0.175M sodium acetate buffer (2 × 2 min), developed using diaminobenzidine (DAB Peroxidase Substrate Kit, Vector) in the presence of nickel (5 min 30 sec), and rinsed again in 0.175M sodium acetate buffer (2 × 2 min) and 0.1M PBS (3 × 10 min). Sections were mounted on slides, dried, and coverslipped with Permount (Fisher Scientific).     21 2.9 Microscopy All microscopy was accomplished blind to the experimental assignment of each animal. The number of DCX- expressing cells was quantified using a Nikon E600 brightfield microscope at 40X magnification. DCX is an endogenous microtubule-associated protein expressed in immature neurons (Brown et al., 2003). DCX is expressed for up to twenty-one days in adult rats. Due to the long-lasting expression of DCX it is a valuable measure of the effects of chronic treatments (such as OT) on neurogenesis. Further, because DCX is an endogenous marker, quantifying its expression does not require extra injections or discussion on the best timing of such injections, which is a constraint of exogenous methods (such as bromodeoxyuridine [BrdU]) (Brown et al., 2003).  Cell counts were evaluated in the dentate gyrus of five dorsal and five ventral sections from either the left or right side of the brain, with approximate Bregma coordinates of -3.0, -3.24, -3.48, -3.72, -4.08, -5.64, -5.76, -6.0, -6.12, and -6.24. Density calculations (number of cells per mm2) were determined following quantification of section areas using ImageJ32 (NIH, Bethesda, MN, USA). The dorsal and ventral dentate gyrus of the hippocampus were quantified separately because have been implicated in different functions, with the dorsal dentate gyrus involved in spatial memory and the ventral dentate gyrus involved in stress and anxiety-like responses (Fanselow and Dong, 2010).   2.10 Statistical analyses All data are expressed as mean ± SEM, and outliers (±2.5 SD>mean) were removed when appropriate (see supplementary figure 1 in Appendix A for list of outliers and missing data). Unless otherwise stated, data were analyzed using analyses of variance (ANOVA) with sex,   22 prenatal condition, and drug treatment as between-subjects factors. Due to the well-established sex differences in both basal and stress-response levels of CORT, male and female offspring were analyzed separately for this measure (Viau, 2002; Goel et al., 2014). ANOVA was followed by Fisher post hoc tests to examine significant main effects and interactions (IBM SPSS Statistics). Significant F statistics and p values are reported in the text; post hoc p values are reported in the figure legends. Differences were considered significant at p ≤ 0.05, and trends (p > 0.05 and < 0.09) were examined, as appropriate. In accordance with our hypothesis, five specific a priori comparisons were examined in males and females. A priori p-values were subjected to Bonferroni corrections were considered significant at p ≤ 0.01. There were no differences in the proportion of rats within each stage of the estrous cycle (proestrous, estrous, diestrous) by prenatal condition or drug treatment, and >90% of rats were in diestrous at the time of termination. Estrous cycling was included as a covariate but had no effect on any measure; thus, data was not stratified by estrous stage for subsequent analyses. Repeated measures ANOVAs were used to analyze the following: 1) maternal weight with GD (1, 7, 14, 21) or LD (1, 8, 15, 22) as the within-subjects factor, and prenatal condition as the between-subjects factor; 2) serum CORT levels with sample time (BAS, RES, REC) as within-subjects factors, and prenatal condition and drug treatment as between-subjects factors; 3) the density of DCX-expressing cells with brain region (dorsal, ventral) as within-subject factor, and sex, prenatal condition, and drug treatment as the between-subjects factor. For CORT data, in order to maintain animals within the repeated measures design outliers and missing samples were replaced with the respective group mean. Gestational weight and CORT analyses violated the assumption of sphericity (as examined by Mauchly’s test) and F-values were corrected using Greenhouse-Geisser estimates of sphericity.   23 As mentioned, animals were monitored following injection and the presence of any sedative-like effects were denoted and combined across the entire 10-day injections period. Thus, the levels of sedative-like effects for each individual animal ranged from 0-10, with 0 representing no sedative-like effect on any injection day and 10 representing sedative-like effects on every injection day. We expected that the sedative-like effects of OT would be differential across groups, therefore sedation data was analyzed using ANOVA with sex, prenatal condition, and drug treatment as between-subjects factors. This analysis focused on the specific effects of OT, hence SAL animals were removed; importantly, SAL animals showed zero incidences of sedative-like effects across the entire injection period. See figure 2 for an experimental timeline.   Figure 2 Experimental timeline Timeline not to scale. Abbreviations: C – control; PF – pair-fed; PAE – prenatal alcohol exposure; SAL – saline; OT.5 – oxytocin at 0.5 mg/kg; OT1 – oxytocin at 1.0 mg/kg; P – postnatal day; NSF – novelty suppressed feeding.          24 Chapter 3: Results 3.1 Alcohol-consuming dams showed reduced weight gain across gestation and early lactation As shown in Table 1, weight of pregnant females throughout gestation revealed that PAE and PF dams weighed less than C dams from GD 7 through GD 21, and PAE dams weighed less than PF dams on GD 21 (interaction of prenatal condition and GD [F(3.169,44.366)=25.149, p<0.0001]; this F value is corrected using Greenhouse-Geisser estimates of sphericity). Similarly, maternal weight throughout lactation revealed that PAE and PF dams weighed less than C dams on LD1 but caught up thereafter (interaction of prenatal condition and LD [F(6,84)=7.074, p<0.0001]). No differences were found for length of gestation, number of pups per litter, or number of pup deaths. Pregnancy Outcome Variables Prenatal Condition Groups  C PF PAE Number of pregnant dams 10 9 12 Length of gestation (d) 23.0 ± 0.0 23.0 ± 0.0 23.1 ± 0.08 Number of pups 15.5 ± 0.8 16.7 ± 0.6 15.5 ± 0.7 Number of pup deaths (PD1-22) 0 5 8 Dam Weight (g) GD1 263.1 ± 2.4 273.0 ± 4.8 269.6 ± 3.9 GD7 303.1 ± 3.1 283.8 ± 4.1# 282.8 ± 3.5## GD14 368.3 ± 4.9 337.6 ± 4.2### 332.1 ± 5.0### GD21 475.8 ± 6.2 444.3 ± 6.8# 416.8 ± 6.2###,✢ LD1 386.4 ± 6.5 358.0 ± 4.8# 343.8 ± 6.7### LD8 379.9 ± 5.3 366.3 ± 4.6 364.7 ± 6.6 LD15 381.0 ± 4.2 376.4 ± 5.1 371.2 ± 5.8 LD22 355.7 ± 4.9 354.9 ± 4.7 352.0 ± 4.4  Table 1 Pregnancy outcomes and maternal body weights throughout gestation and lactation Data are presented as mean ± SEM, pup deaths are presented as totals. Presence of a (#) indicates PAE and PF < C at #p<0.01, ##p<0.001, or ###p<0.0001; and (✢) indicates PAE < PF at ✢p<0.01.    25 3.2 Oxytocin reduced weight gain in males, but not females Not surprisingly, body weight analyses revealed that females weighed less initially and had a lower percent weight gain than males (main effect of sex on initial weight [F(1,158)=1068.001, p<0.0001] and percent weight gain [F(1,157)=9.616, p=0.002]). Additionally, as shown in Table 2, in males, OT.5 and OT1-treated groups had a lower percent weight gain than SAL-treated group (interaction of sex and drug treatment on percent weight gain [F(2,157)=4.510, p=0.012]). No other significant main effects or interactions were found.   Prenatal Condition and Drug Treatment Groups   Weight Variables C PF PAE SAL OT.5 OT1 SAL OT.5 OT1 SAL OT.5 OT1  Males  Initial weight (g) 502.0±14.0 493.5±9.8 498.1±9.9 487.4±18.2 480.3±14.3 509.0±21.8 495.0±24.7 480.8±16.9 499.5±11.1  % weight gain (d 1-10) 4.8±0.4 2.1±0.8✻✻ 0.9±0.6✻✻^ 5.4±0.6 3.9±0.8✻✻ 2.6±1.1✻✻ 5.0±0.5 2.1±0.7✻✻ 1.2±0.6✻✻^  Females  Initial weight (g)✭✭✭ 291.2±9.2 304.4±12.2 292.0±7.3 289.0±7.8 303.3±7.1 289.8±6.4 296.0±8.4 302.0±9.7 275.8±11.3  % weight gain (d 1-10)✭ 3.0±0.8 2.1 ± 1.2 1.2±1.0 2.8±1.0 0.6±0.9 1.8±0.6 1.7±0.8 1.3±0.7 2.6±1.0  Table 2 Adult weights throughout drug treatment Data are presented as mean ± SEM. Presence of a (✭) indicates F < M at ✭p<0.01 or ✭✭✭p<0.0001. Presence of a (✻) indicates male OT.5 and OT1 < male SAL at ✻✻p<0.001. Further, (^) indicates an a priori comparison; in males, the OT-dependent decrease in percent weight gain remains significant in C and PAE animals exposed to higher OT dose: C OT1 < C SAL and PAE OT1 < PAE SAL at ^p<0.01.   3.3 Oxytocin administration resulted in short-term sedative-like effects For all 10 days of injections the OT-treated groups (OT.5, n=57; OT1, n=60) had 909 incidences of sedative-like effects (OT.5 = 75.8% and OT1 = 79.5%), whereas the SAL-treated group (n=58) had zero incidence of sedative-like effects. Accordingly, we were interested in the specific sedative-like effects of the different OT doses across our prenatal condition groups. As shown in Figure 3A, analysis of sedation data revealed that females exhibited fewer sedative-like effects than males (main effect of sex [F(2,117)=5.535, p=0.02]). Additionally, PAE and PF   26 animals exhibited fewer sedative-like effects than C animals (main effect of prenatal condition [F(1,117)=5.138, p<0.01]). No other significant main effects or interactions were found.  3.4 Oxytocin administration resulted in long-term decreases in locomotor activity; prenatal alcohol exposure resulted in hyperactivity As illustrated in Figure 3B, analysis of total distance traveled revealed that females traveled a greater distance than males (main effect of sex [F(1,156)=52.525, p<0.0001]. Interestingly, PAE males traveled a greater distance than C and PF males, while PAE and C females traveled a greater distance than PF females (trend for interaction of sex and prenatal condition [F(2,156)=2.872, p=0.06]). Finally, OT.5 and OT1-treated groups traveled a shorter distance than the SAL-treated group (main effect of drug treatment [F(2,156)=23.366, p<0.0001]). No other significant main effects or interactions were found.   27  Figure 3 Short-term sedative-like effects of oxytocin and long-term locomotor activity Data are presented as mean ± SEM. A. Sedative-like effects of oxytocin: Measured immediately following an injection. Presence of a (✭) indicates F < M at ✭p<0.05, and (#) indicates PAE and PF < C at #p<0.05. B. Locomotor activity: Measured during the NSF task. Presence of a (✭) indicates F > M at ✭✭✭p<0.0001. Presence of a (#) indicates male PAE > male C and PF, as well as female PAE > female PF at ###p<0.0001; further, (✢) indicates female PF < female C at ✢p<0.01. Lastly, (*) indicates OT.5 and OT1 < Sal at ***p<0.0001, with (^) indicating an a priori comparison; in both males and females, the OT-dependent decrease in activity remains significant in C animals: C OT.5 < C Sal and C OT1 < C Sal at ^p<0.01.      OT.5OT1OT.5OT1OT.5OT10246810Level of Sedative-like EffectsMales: Sedative-like EffectsControl PAEPF#SALOT.5OT1SALOT.5OT1SALOT.5OT1010002000300040005000Distance (cm)Males: Total Distance TraveledControl PAEPF^ ^*** ******###OT.5OT1OT.5OT1OT.5OT10246810Level of Sedative-like EffectsFemales: Sedative-like EffectsControl PAEPF«#SALOT.5OT1SALOT.5OT1SALOT.5OT1010002000300040005000Distance (cm)Females: Total Distance TraveledControl PAEPF*^********###^«««A.B.✢   28 3.5 Prenatal alcohol exposure altered latency to feed, but not to investigate, in both males and females the novelty suppressed feeding task As illustrated in Figure 4A, analyses of latency to feed revealed that females showed reduced latency compared to males (main effect of sex [F(1,154)=11.34, p=0.001]), and PAE showed reduced latency compared to C and PF animals (main effect of prenatal condition [F(1,154)=8.479, p=0.0003]. As shown in Figure 4B, analyses of appetite differences revealed that females consumed more than males in the home cage (main effect of sex [F(1,156)=4.789, p=0.03]). Importantly, no other group differences were found for appetite, and there were no significant differences found for latency to investigate (Figure 4C).   29  Figure 4 Feeding behaviour in the novelty suppressed feeding task Data are presented as mean ± SEM. A. Latency to Feed: Measured during the NSF task. Presence of a (★) indicates F < M at ★★p=0.001, and (#) indicates PAE > C and PF at #p<0.01 or ###p<0.0001. B. Latency to Investigate: Measured during the NSF task. No significant group differences were found. C. Appetite Differences: Measured in the home cage following NSF testing. Presence of a (★) indicates F > M at ★p=0.03. No other group differences were found. SALOT.5OT1SALOT.5OT1SALOT.5OT1050100150200250300350Latency (s)Males: Latency to FeedControl PAEPF####SALOT.5OT1SALOT.5OT1SALOT.5OT105101520Amount / BW (g/kg)Males: Appetite DifferencesControl PAEPF SALOT.5OT1SALOT.5OT1SALOT.5OT1050100150200250300350Latency (s)Females: Latency to FeedControl PAEPF####««SALOT.5OT1SALOT.5OT1SALOT.5OT105101520Amount / BW (g/kg)Females: Appetite DifferencesControl PAEPF«SALOT.5OT1SALOT.5OT1SALOT.5OT1050100150200250300350Latency (s)Males: Latency to InvestigateControl PAEPFSALOT.5OT1SALOT.5OT1SALOT.5OT1050100150200250300350Latency (s)Females: Latency to InvestigateControl PAEPFA.B.C.  30 3.6 Prenatal alcohol exposure reduced rearing in both sexes, whereas, oxytocin and prenatal alcohol exposure differentially increased self-grooming in males and females  As shown in Figure 5A, analyses of rearing behaviour revealed that PAE animals reared less than C and PF animals (main effect of prenatal condition [F(2, 156)=37.667, p<0.0001]). Alternatively, as illustrated in Figure 5B, analyses of self-grooming behaviour revealed that C and PF females self-groomed less than their respective male counterparts, a sex difference that did not exist in PAE animals; additionally, PAE females self-groomed more than C females (interaction of sex and prenatal condition [F(2, 153)=3.411, p=0.036]). Further, females in the OT.5 and OT1-treated groups self-groomed less than their respective male counterparts, a sex difference that did not exist in the SAL-treated group; additionally, males in the OT.5 and OT1-treated groups self-groomed more than males in the SAL-treated group (interaction of sex and drug treatment [F(2, 153)=4.008, p=0.02]). These behaviours were recorded during the NSF task. No other significant main effects or interactions were found.   31  Figure 5 Rearing and self-grooming in the novelty suppressed feeding task Data are presented as mean ± SEM. A. Rearing: Presence of a (#) indicates PAE > C and PF at ###p<0.0001, with (^) indicating an a priori comparison; in both males and females, this PAE-dependent increase in rearing remains significant in SAL animals: PAE SAL < C SAL at ^p<0.01. B. Self-Grooming: Presence of a (★) indicates F < M at ★p<0.05 (sex difference present in C and PF groups, as well as OT.5 and OT1 groups). Presence of a (#) indicates female PAE > female C at #p<0.05. Lastly, presence of (*) indicates OT.5 and OT1 males > SAL males at *p<0.05, with (^) indicating an a priori comparison; in males, the OT-dependent increase in self-grooming remains significant in C animals: C OT1 > C SAL at ^p<0.01.     SALOT.5OT1SALOT.5OT1SALOT.5OT10123Duration (s)Males: Bouts of RearingControl PAEPF###^SALOT.5OT1SALOT.5OT1SALOT.5OT10123Duration (s)Females: Bouts of RearingControl PAEPF^###SALOT.5OT1SALOT.5OT1SALOT.5OT10246810Duration (s)Males: Bouts of Self-Grooming***^Control PAEPFSALOT.5OT1SALOT.5OT1SALOT.5OT10246810Duration (s)Females: Bouts of Self-GroomingControl PAEPF#« « «« «A.B.  32 3.7 Prenatally alcohol exposed males showed differential corticosterone levels across time, whereas low dose oxytocin reduced corticosterone response to acute stress in males Due to the inherent large sex differences in basal and response CORT levels in rodents (Goel et al., 2014) that may obscure statistical findings, serum CORT levels were analyzed separately in each sex. Not surprisingly, analyses of CORT concentrations revealed that the response levels were significantly higher than basal and recovery levels for males in all prenatal conditions. However, an inverse relationship was found between basal and recovery levels for PF and PAE males, such that recovery was significantly lower than basal for PF males but significantly higher than basal for PAE males (interaction of time and prenatal condition [F(2.322,88.229)=3.315, p<0.034]; this F value is corrected using Greenhouse-Geisser estimates of sphericity). Additionally, as shown in Figure 6A, PAE males showed differential CORT levels across time: PAE males displayed lower basal levels than C and PF males, lower response levels than PF males, and higher recovery levels than PF and C males (trending higher than C males [p=0.058]). Finally, as shown in Figure 6A, OT.5-treated males showed reduced CORT response levels compared to SAL-treated males (trend for interaction of time and OT treatment [F(2.322,88.229)=2.397, p=0.089]; this F value is corrected using Greenhouse-Geisser estimates of sphericity). No other significant main effects or interactions were found in males. In females, analyses of CORT similarly revealed that the response levels were significantly higher than basal and recovery levels; additionally, recovery levels were significantly lower than basal levels (main effect of time [F(1.513,121.032)=235.229, p<0.0001]; this F value is corrected using Greenhouse-Geisser estimates of sphericity). No other group differences were found in females (see Figure 6B).    33   Figure 6 Corticosterone profile before and after acute restraint stress Data are presented as mean ± SEM. For clarity, prenatal conditions are separated along the x-axis. Sexes were analyzed separately. A. Males: All animals responded to stress, with RES > BAS and REC at p<0.0001. However, recovery and basal levels differed for PF and PAE animals, such that PF REC < PF BAS and PAE REC > PAE BAS at p<0.05. Presence of a (#) indicates PAE is different than C and/or PF at #p<0.05. More specifically, PAE BAS < C and PF BAS; PAE RES < PF RES; and PAE REC > C and PF REC. Graphical insert more clearly shows PAE REC > C and PF REC. Presence of (*) indicates OT.5 < SAL at *p<0.05. B. Females: All animals responded to stress, with RES > BAS and REC at p<0.0001. All animals also returned to a lower recovery level than basal, with REC < BAS at p<0.0001. No significant groups differences were found.   BASRESRECBASRESRECBASRESREC020406080Corticosterone (µg/dL)Males: Basal, Response, and Recovery CORT## #* * *BASRESRECBASRESRECBASRESREC050100150Corticosterone (µg/dL)Females: Basal, Response, and Recovery CORTA.B.COT1PF PAEOT.5SALC PF PAESALOT.5OT1SALOT.5OT1SALOT.5OT105101520Corticosterone (µg/dL)Males: Recovery CORTControl PAEPF#  34 3.8 Both males and females prenatally exposed to alcohol showed elevated doublecortin expression in the dorsal hippocampus Within the dorsal dentate gyrus, both PAE and PF animals had higher density of DCX-expressing cells than C animals (see Figure 7A); by contrast, within the ventral dentate gyrus only PF animals had higher density than C animals (see Figure 7B) (interaction of region and prenatal condition [F(2,156)=6.640, p=0.002]). No other significant main effects or interactions were found.   35  Figure 7 Density of doublecortin expressing cells in the dorsal and ventral dentate gyrus of the hippocampus  A. Representative photomicrographs of the granule cell layer (GCL) in the dorsal and ventral dentate gyrus showing DCX-immunoreactive cells. Images generated by Aperio ScanScope CS slide scanner (Leica Biosystems, Concord, ON) and zoomed in at 20X magnification using Aperio ImageScope (Leica Biosystems, Buffalo Grove, IL). A single representative slide was scanned by Wax It Histology Services Inc. B. Density of DCX-expressing cells in the dorsal and ventral dentate gyrus. Data are presented as mean ± SEM. Presence of a (★) indicates Ventral < Dorsal at ★p<0.05 for all prenatal conditions; and (#) indicates PAE and PF > C in dorsal dentate gyrus, and PF > C in ventral dentate gyrus at ##p<0.001 or #p<0.05.     36 Chapter 4: Discussion Our results demonstrate that PAE and OT administration induce compelling neurogenic, endocrine, and neurobehavioural alterations. PAE males and females exhibited increased density of immature neurons in the dorsal dentate gyrus. In addition, PAE males and females displayed an inappropriate behavioural response in the NSF task as they consumed the food more quickly compared to controls. Further, both PAE males and females also demonstrated altered endocrine and behavioural responses that may be linked to stress-coping. In particular, PAE males and females exhibited increased locomotor activity levels (increased distance traveled) but decreased exploration (decreased rearing behaviour) in the NSF task. PAE females demonstrated greater behavioural changes showing increased self-grooming, whereas PAE males exhibited endocrine changes showing attenuated CORT recovery following acute restraint stress.  Interestingly, OT-treated animals exhibited sedative-like effects immediately following injection, as well as reduced distance traveled in the NSF task. PAE and OT differentially altered locomotor activity, with PAE resulting in increased locomotor activity and OT resulting in decreased locomotor activity. The decreased locomotor activity following OT may preferentially impact hyperactive PAE animals. Alternatively, utilizing the methods in this study OT was not able to mitigate the effects of PAE on hippocampal neurogenesis or CORT recovery. However, low dose OT reduced CORT response to acute restraint stress.  Together our results demonstrate a possible role for OT in attenuating locomotor hyperactivity following PAE. Our findings are an important extension of previous work on altered neurogenic, endocrine, behavioural responses in PAE animals. Further, our results support and extend the literature on the use of OT as a therapeutic intervention. The ultimate novelty of this study lies in the utilization of OT-administration as an intervention for PAE.    37 4.1 Body weight As expected, ethanol-consuming and pair-fed consuming dams had reduced body weight throughout gestation and into early lactation compared to control dams, which parallels many previous findings (Weinberg, 1988, 1992; Osborn et al., 1996; Sliwowska et al., 2010; Uban et al., 2010, 2013). However, catch-up growth occurred and in adulthood, offspring of ethanol and pair-fed consuming dams weighed the same as control offspring, which again corroborates previous literature (Hellemans et al., 2008). OT-treated males, across prenatal groups, had a lower percent weight gain from day 1 to 10 of treatment than SAL-treated males, but this was not seen in females. Previous studies show altered weight gain following central administration of OT in male and female rats (Arletti et al., 1989, 1990; Bowen et al., 2011; Spetter and Hallschmid, 2017). Specifically, food intake is acutely reduced following both central and peripheral OT administration, which is associated with reduced weight gain in males (Arletti et al., 1989, 1990; Spetter and Hallschmid, 2017). Conversely, both increases and decreases in food intake and body weight have been reported in females (Björkstrand and Uvnäs-Moberg, 1996). Differential results in males compared to females is not surprising given the sex differences in OT system development and function (Neumann, 2008; Tamborski et al., 2016). OT acts on many systems throughout the body, including gastrointestinal tone and motility (Llewellyn-Smith et al., 2012; Welch et al., 2014); OT modulation of weight may suggest altered hypothalamic control of digestion, metabolism, or hunger. Importantly, despite differences in weight gain, OT-treated animals in this study did not show altered food consumption in the home cage following NSF.    38 4.2 Sedative-like effects of oxytocin This study adds to previous literature on the sedative-like effects of OT, including lethargy, sleepiness, immobility, piloerection, and mild hind-limb paresis, with the additional systematic examination of OT effects across multiple days. OT acts on many systems throughout the body controlled by central autonomic and somatic responses (reviewed in Gimpl and Fahrenholz, 2009). OT acts to increase neuronal excitability in GABAergic (i.e. inhibitory) transmission in many brain regions (Huber et al., 2005). Similar to classic sedatives, such as benzodiazepines, OT increases GABAergic transmission in the amygdala (Viviani et al., 2010), which project to the hypothalamus and brainstem to trigger autonomic responses (Huber et al., 2005).  Human and animal studies indicate that OT is expressed throughout the periphery (Ohlsson et al., 2006; Greenwood and Hammock, 2017; reviewed in Gimpl and Fahrenholz, 2001) with effects on cardiovascular regulation (Higa et al., 2002), pain relief (Caldwell et al., 1987), gastrointestinal motility (Llewellyn-Smith et al., 2012; Welch et al., 2014), muscular contraction and maintenance (Elabd et al., 2014), renal control of mineral and osmoregulation (reviewed in Gimpl and Fahrenholz, 2001), and thermoregulation (Lipton and Glyn, 1980). We illustrate that the sedative-like effects of OT occur within minutes of administration and subside within 1 hr, which mirrors the timeline of peak OT content in plasma and brain following peripheral administration (Neumann et al., 2013). We suggest that the sedative-like effects of OT occur through increased central autonomic processes impacting one or many of the following: increased parasympathetic cardiac control (reduced heart rate, respiration, and blood pressure), increased analgesia, decreased gastric tone and motility, reduced stress response, and/or altered muscle contraction. Interestingly, OT-treated females exhibited fewer sedative-like effects than males. As mentioned, there are well-known sex differences in OT system development and   39 function (Neumann, 2008; Tamborski et al., 2016). Specifically, initial expression of oxytocin mRNA in development occurs earlier in females (Tamborski et al., 2016), and adult females have higher oxytocin expression (Carter, 2007). Therefore, females may be less sensitive to the doses utilized in this study.  Importantly, the sedative-like effects of OT following injection resolve within 1 hour. Interestingly, these sedative-like effects have not been reported following central or nasal administration and may be specific to peripheral injection as this technique involves higher dosage than central or nasal administration. During peripheral injection the high levels of exogenous OT may affect central processes and may also immediately act on peripheral OT receptors. Nasal administration of OT in humans has shown no robust or reliable side-effects (MacDonald et al., 2011).  4.2.1 Prenatal alcohol exposure alters sedative-like response to oxytocin Interestingly, in this study PAE animals showed fewer sedative-like effects than controls following OT administration. PAE animals may be less sensitive to OT administration at the doses used due to altered OT system development and functioning; indeed, PAE male and female rodents have been shown to have altered OT receptor binding in the amygdala in adulthood (Kelly et al., 2009). Altered OT receptor binding following PAE is not entirely surprising given the reduced number of OT neurons and OT mRNA levels in adult male and female rodents following prenatal stress (Lee et al., 2007; De Souza et al., 2013; He et al., 2018). Furthermore, given the widespread effects of ethanol on developing systems it is not uncommon for individuals with FASD to respond differently to the same drug dose utilized in unexposed individuals (Stratton et al., 1996). PF animals also showed reduced sedative-like effects   40 following OT administration; this is not surprising given the aforementioned alterations in the OT system following prenatal stress. Although pair-feeding is included as a control for the reduced food intake that occurs in alcohol-consuming animals, this condition encompasses similarities to prenatal stress (see section 4.6 for detailed discussion of pair-feeding).  4.3 Locomotor activity 4.3.1 Hyperactivity following prenatal alcohol exposure  Here we illustrate locomotor hyperactivity in PAE animals, which is possibly the most common behavioural outcome following PAE, and has been previously illustrated in both male and female rats (Gilbertson and Barron, 2005; Hellemans et al., 2008; Brys et al., 2014), as well as in primates, mice, and guinea pigs (Becker and Randall, 1989; Gibson et al., 2000; Schneider et al., 2001). Hyperactivity is also a common symptom of FASD in humans. Indeed, in humans it has been suggested that prenatal exposure to alcohol is likely the leading cause of ADHD, with the rates as high as 85% in the FASD population (Burd, 2016).  4.3.2 Reduced locomotor activity following oxytocin Previous literature has shown that OT administration decreases locomotor activity (Uvnäs-Moberg et al., 1994; Maejima et al., 2015; Angioni et al., 2016; Leong et al., 2016). However, the majority of these measures occur immediately or within 30 min following injection. Here, we illustrate reduced locomotor activity approximately 2 hr following OT injection. In previous studies the effects of OT on locomotor activity may be confounded by the immediate sedative-like effect; alternatively, the reduced locomotion in this study reflects long-term effects of OT at   41 a time when the sedative-like effects have subsided. This distinction may be important as the mechanisms underlying immediate sedative-like effects and persistent hypoactivity may differ.  4.4 Novelty suppressed feeding  4.4.1 Altered anxiety-like and stress-coping behaviour following prenatal alcohol exposure We demonstrate that PAE animals show decreased latency to feed, which classically suggests less anxiety-like behaviour. Reduced anxiety-like behaviour has been previously shown following PAE in the elevated plus maze (Carneiro et al., 2005; Brolese et al., 2014). Reductions in anxiety-like behaviour are not necessarily an adaptive response and may represent a lack of appropriate risk-assessment or increased hyperactivity (Campos et al., 2013). Interestingly, our data do not illustrate differences in latency to investigate the food. In other words, all animals investigate the food at a similar time but only PAE animals are quicker to consume the food. The equivalent latency to investigate between PAE and C animals suggests that levels of anxiety-like behaviour do not differ. Instead, PAE animals may be quicker to consume the food due to the aforementioned hyperactivity. In particular, in this context hyperactivity may be related to impulsivity or a lack of appropriate risk-assessment. PAE animals have been shown to exhibit increased impulsivity and risk-taking (Olmstead et al., 2009; Muñoz-Villegas et al., 2017). Moreover, individuals with FASD experience deficits in executive functioning, including lack of response inhibition, impulsivity, and risk-taking (Paolozza et al., 2014). Importantly, appetite does not play a role in the latency to feed measures in this study as no prenatal group differences were found in home cage food consumption. However, females were quicker to feed in both the   42 NSF task and the home cage. This is not surprising, given that female rats have been shown to overeat chow during recovery from food restriction (Lenglos et al., 2013).  PAE-induced hyperactivity is represented by an increase in total distance traveled. However, PAE animals also exhibit decreased rearing behaviour, which represents less exploration. Previous studies have suggested that changes in exploration can occur independently of alterations in locomotor activity (Abel, 1995). Specifically, decreased exploration, as measured by rearing in this study, may represent increased anxiety or stress-coping behaviour or may represent less motor activity. PAE animals often show differential behavioural responsiveness to stress. The presence of chronic injection stress may drive the decrease in rearing following PAE. In fact, exposure to chronic mild stress or CRH reduces both locomotor hyperactivity and rearing in PAE animals (Gabriel et al., 2006; Hellemans et al., 2008). Both single and chronic SAL injections elevate plasma CORT and ACTH levels, similar to chronic mild stress (Kiss and Aguilera, 1993; Freiman, 2016). Thus, chronic injection stress may similarly act to reduce rearing in PAE animals. Locomotion remains elevated in this study, suggesting that exploratory behaviour in PAE animals may be more sensitive to prior stress. PAE females in this study show increased levels of self-grooming. Self-grooming occurs most frequently in novel environments in rodents as a stress-coping or de-arousal behaviour, but can also represent cleaning, thermoregulation, and social communication (Fernández-Teruel and Estanislau, 2016; Kalueff et al., 2016). Interestingly, rodent models of anxiety, ADHD, and ASD all display high locomotor activity and increased self-grooming (Kalueff et al., 2016). This is important because animal models of PAE, as well as the symptomology of FASD in humans, display significant behavioural overlap with all three of these neuropsychiatric and neurodevelopmental disorders. The increased self-grooming in PAE females may be induced by   43 an amplified response to the novelty of the task or may be exacerbated by heightened stress levels following chronic injections. A previous study has shown that females are more vulnerable to chronic mild stress, and respond differently than males to the combination of chronic mild stress and an additional novel task (Dalla et al., 2005).  Taken together, PAE animals do not show more anxiety-like behaviour as assessed by the classic measurement of latency to feed. However, the overall behavioural profile does reflect distinctive behavioural responses to prior stress and novel conditions. PAE animals show hyperactivity, less vertical exploration, and possible impulsivity or inappropriate risk-assessment. Females show more enhanced behavioural alterations, with elevated hyperactivity, heightened impulsivity (quicker latency to feed), and more self-grooming compared to males.  4.4.2 Oxytocin increased self-grooming in adult males but not females Increases in self-grooming following both central and peripheral administration of OT have been previously shown in male and female rodents (Drago et al., 1986; Van Erp et al., 1993; Amico et al., 2004b). However, the exact mechanism and functional significance of increased grooming following OT remains unclear. The increase in self-grooming of PAE females in this study may partially explain the lack of increased self-grooming in OT-treated females, with the effects of PAE on female self-grooming masking any effects of OT.     44 4.5 Basal and stress-induced corticosterone levels 4.5.1 Prenatal alcohol exposure dysregulates corticosterone levels in adult males but not females PAE and the HPA activity have a complex and reciprocal relationship; PAE results in HPA dysregulation throughout life, and exposure to stress exacerbates many neurobehavioural deficits in PAE. In this study, three samples were collected to measure CORT under basal, response, and recovery conditions, and we observed effects of PAE on HPA activity in males but not females; sexually-dimorphic effects of PAE on HPA activity are commonly detected (reviewed in Weinberg et al., 2008).  We illustrate differential CORT levels across time in PAE males. Firstly, PAE males show lower basal CORT levels compared to C and PF. Most previous studies have shown no differences in basal CORT in PAE animals (Weinberg et al., 1996; Glavas et al., 2007; for review see Weinberg et al., 2008; Hellemans et al., 2010). However, the effects of PAE on HPA activity is heavily dependent on experimental context, which could result in varying outcomes. Under the condition of chronic injection stress, it is possible that PAE males exhibit lower basal CORT due to altered diurnal rhythm or reduced central regulation. Secondly, we do not observe hyperresponsivity to stress in PAE males. PAE commonly results in HPA hyperresponsiveness to stressors in males and females (Weinberg et al., 2008; Hellemans et al., 2010); however, other studies show no changes in stress response to acute restraint in PAE males (Weinberg, 1988), or show hyperresponsiveness only to prolonged or intense restraint stress (Weinberg, 1992; Hellemans et al., 2010). Acute restraint stress in this study may not have been intense enough to elicit hyperresponsiveness in PAE males. Finally, PAE males show higher recovery CORT compared to PF and C. This replicates previous studies in PAE animals showing deficits in   45 recovery after restraint stress (Weinberg, 1988; Weinberg et al., 1996), and deficits in HPA negative feedback (Osborn et al., 1996). In our study, PF males show elevated response CORT compared to PAE animals; this is not surprising given the stressful nature of this control condition (see section 4.6 for detailed discussion of pair-feeding). In females, no group differences were found across time, but basal levels were significantly higher than response levels. It is likely that any group differences in CORT levels were abolished by the heightened basal CORT levels in this study. A previous study from our lab has shown adult female basal CORT levels following 10-days of CMS as high as 5.7 ± 2.1 µg/dL (Uban et al., 2013). In comparison, the average female basal level of CORT in this study was 36.2 ± 2.7 µg/dL. Unlike CMS, chronic injection stress is predictable, and may have resulted in higher basal CORT levels through anticipation of injection upon morning entry of the investigator into the colony room. In fact, similar values of ~40 µg/dL have been shown 30 min following i.p. injections of saline, with no habituation occurring across 14 days of injections (Kiss and Aguilera, 1993). Further, heightened basal CORT levels in females supports our suggestion that the enhanced behavioural alterations in females in this study may represent distinctive responses to prior stress.  4.5.2 Low dose oxytocin reduces corticosterone response to acute stress in males Consistent with previous literature, OT reduced CORT levels in response to restraint stress (Windle et al., 1997, 2004; Cohen et al., 2010). However, only administration at the low OT dose resulted in a reduced CORT response, suggesting a dose-dependent effect. Many different pharmacological agents, including anxiolytics, elicit dose-dependent effects on endocrine, neurogenic, and behavioural outcomes (Guimarães et al., 1990; Wilson et al., 2004; Mahmoud et   46 al., 2016). Furthermore, the anxiolytic and social recognition effects of OT have been shown to follow a similar U-shaped dose response curve, with only low doses facilitating such behaviours (Popik et al., 1992; Uvnäs-Moberg et al., 1994). Previous effects of OT on CORT response levels are robustly and consistently shown after central administration in both males and females (Windle et al., 1997, 2004; Cohen et al., 2010). Research on CORT response following peripheral OT administration is limited. It is possible that the lower OT dose administered peripherally in this study more closely represents the low doses used in central administration. Due to the sex differences in OT system development and function (Neumann, 2008; Tamborski et al., 2016), males and females may possess different dose-response curves, with females less sensitive to the effects of peripheral OT at the doses utilized in this study. Further, as previously mentioned, it is possible that any group differences in females were abolished by the heightened basal CORT levels in this study. Interestingly, visual inspection of the graph indicates that the effect of low OT dose on the CORT response in males is driven by the C and PF groups. The low OT dose does not seem to reduce CORT responses in PAE animals. As previously mentioned, it is not uncommon for individuals with FASD to respond differently from unexposed individuals to the same drug dose (Stratton et al., 1996). Moreover, we utilized a ~2 h recovery period to prevent the confounding sedative-like effects of OT on endocrine and behavioural measures; however, as mentioned, the peak levels of OT content following peripheral injection are within 30-min in the brain (Neumann et al., 2013). Therefore, it is also possible that peripheral OT may more robustly reduce CORT response to stress when administered roughly 30-min before stress exposure. Nevertheless, these findings broaden our understanding of OT effects on stress regulation.    47 4.6 Hippocampal neurogenesis 4.6.1 Prenatal alcohol exposure increased doublecortin-density in dorsal hippocampus in adult males and females Previous literature on the effects of PAE on hippocampal neurogenesis is comprised of a range of age groups, varying alcohol exposure models, and different neurogenesis markers. As previously mentioned, our liquid ethanol diet is given across the entire gestation period, which is roughly equivalent to the first 2 trimesters in humans. Studies utilizing this 2-trimester model have shown that PAE reduces cell proliferation and neuronal survival in adult males (Redila et al., 2006; Sliwowska et al., 2010), and reduces the proportion of new neurons and glia cells in adult females (Uban et al., 2010). Furthermore, PAE reduces the number of immature neurons in aged females (Gil-Mohapel et al., 2014). As far as we know, the effects of PAE, utilizing this 2-trimester model, on immature neurons in adulthood has not been measured. Our study revealed enhanced DCX-expression or increased density of immature neurons in the dorsal dentate gyrus following PAE in both males and females. The extension of results to include a measure of immature neurons may help create a more complete picture of neurogenic changes across time following PAE. In males, chronic exposure to ethanol in utero results in reduced cell proliferation, which may trigger compensatory mechanisms that lead to increases in neuronal differentiation or levels of immature neurons. However, in the long-term this increased density of immature neurons does not result in increased density of mature neurons, suggesting subsequent periods of cell death. In females, similar compensatory mechanisms leading to higher levels of immature neurons and subsequent cell death likely occurs, but PAE females do not show the initial reduced cell proliferation (Redila et al., 2006; Uban et al., 2010). This is consistent with previous studies showing no effect of chronic stress on cell proliferation in   48 females (Westenbroek et al., 2004). Possible compensatory mechanisms include altered cell cycle length and neuronal maturation rates (Gil-Mohapel et al., 2010). In fact, ethanol exposure early in development results in increased cell cycle length, slowed cell proliferation, and delayed neuronal maturation (Jacobs and Miller, 2001; Singh et al., 2009). It is likely that these altered mechanisms persist long-term and may underlie the decreased cell proliferation but increased immature neurons shown in adulthood following PAE. Hippocampus development, specifically growth and maturation of the dentate gyrus, occurs during the early postnatal period in the rat; thus, 2-trimester models do not measure the direct effects of alcohol exposure on the major period of hippocampal development. Models of 3-trimester alcohol exposure often show enhanced, and sometimes distinctive, deficits in hippocampal cell proliferation and neurogenesis (reviewed in Gil-Mohapel et al., 2010). Utilizing a 3-trimester model, Gil-Mohapel et al. (2011) demonstrate enhanced NeuroD expression following PAE in adolescence, which is an immature neuronal marker with the same expression profile as DCX. This study shows similar enhanced immature neuronal levels in PAE adults following a 2-trimester model. Importantly, exposure to ethanol just during the 3rd trimester equivalent does not alter DCX expression in late adolescence (Helfer et al., 2009), suggesting the effects of PAE on immature neurons requires chronic alcohol exposure. Data from the present study support and extend previous literature on altered neurogenic capability following PAE. However, further research is needed to elucidate the temporal and mechanistic effects of PAE on hippocampal neurogenesis.  Lastly, our findings are specific to the dorsal dentate gyrus, which has been implicated in cognition, learning and memory, visuospatial navigation, and locomotion (Epp et al., 2007; Fanselow and Dong, 2010). As mentioned, individuals with FASD have deficits in learning and   49 memory, display hyperactivity, show impulsivity, and lack response inhibition (Astley, 2000; Mattson et al., 2001; Paolozza et al., 2014). Importantly, the behavioural profile of PAE animals in this study include hyperactivity and altered latency to feed, which likely reflect impulsivity and reduced response inhibition. Therefore, altered neurogenesis in the dorsal hippocampus may contribute to such behaviour in PAE animals, as well as deficits in humans with FASD. Interestingly, PF animals showed enhanced DCX density in the dorsal and ventral dentate gyrus. The effects of pair-feeding on neurogenesis are not surprising given that this condition likely involves some levels of prenatal stress for the offspring due to maternal hunger and increased arousal (see section 4.6 for a detailed discussion of pair-feeding).  4.6.2 Oxytocin did not increase doublecortin-density in the hippocampus A previous study by Leuner et al. (2012) showed increased cell proliferation following acute administration of both central and peripheral OT, as well as enhanced neuronal survival following chronic peripheral OT administration. OT did not alter neurogenic measures in this study. However, Leuner’s study and ours have several methodological differences that likely explain the dissimilar results. The study by Leuner et al. measured the number of BrdU-labeled cells three weeks following cessation of OT injections. Upon injection, BrdU labels cells in the dentate gyrus that are within the S-phase; three weeks later the majority of these new cells express mature neuronal markers. Therefore, Leuner et al. (2012) demonstrate that chronic OT treatment results in enhanced neuronal survival three weeks after the end of treatment. In comparison, DCX-expression was utilized in this study and reflects the immediate effects of chronic OT treatment on proliferating cells and immature neurons. Although the effects of chronic peripheral OT injection on cell proliferation are not known, acute peripheral OT   50 injection rapidly increases cell proliferation (Leuner et al., 2012). However, during subsequent periods of cell death these additional cells are lost. Taken together, it is possible that chronic OT injection does not alter cell proliferation or neuronal differentiation (number of immature neurons) but provides a protective mechanism around mature neurons, preventing cell death and ultimately leading to increased neuronal survival in the long-term.  4.7 The effects of pair-feeding Here, we show reduced sedative-like effects in PF animals, decreased locomotor activity in PF females, altered CORT levels across time in PF males, and enhanced DCX-density in the dorsal and ventral dentate gyrus in PF males and females. As previously mentioned, pair-feeding accounts for the reduced food intake associated with alcohol consumption. However, pair-feeding is a confounded “control” group. Alcohol consumption is associated with decreased food intake, although animals eat ad libitum; however, unlike alcohol-consuming dams, PF dams are not fed ad libitum but rather receive a reduced ration of food, equivalent to that consumed by a PAE partner. As a result, PF dams commonly consume their entire ration of food within a few hours after it is presented, which effectively results in food deprivation until the next feeding (Gallo and Weinberg, 1981). This abnormal feeding pattern coupled with increased maternal hunger and arousal, likely results in some level of prenatal stress for the pair-fed fetuses. Prenatal stress, in itself, may have serious long-term impacts on the development and functioning of the limbic-HPA axis in offspring. In addition to this possible confound, pair-feeding also does not control for the nutritional consequences of alcohol, such as altered absorption and nutrient utilization (Weinberg, 1984). Previous research has shown that reduced food consumption often accompanies chronic alcohol-intake in humans (reviewed in Yeomans et al., 2003). In humans,   51 the overall effects associated with chronic alcohol consumption cannot be disentangled from reduced food intake; further, it is highly likely that the results of one may intensify or diminish the effects of the other. The PF group was included in the initial animal model studies of PAE to control for the reduced food intake and confirm that effects of PAE were not simply due to altered nutrition. However, in view of the fact that alcohol is now firmly established as a known teratogen, and our understanding of the confounds that occur with a pair-feeding regimen, the utility of the PF group may be questioned and the inclusion of this group should ultimately depend on the experimental question. In this study we are interested in the effects of in utero alcohol consumption on neurogenic, endocrine, and behavioural domains, as well as investigating possible modulation by OT administration. The unique effects of PF in this study (decreased locomotor activity in females, increased CORT response to acute restraint stress in males, and enhanced DCX-density in the ventral dentate gyrus of males and females) are similar in many ways to those seen in animals following prenatal stress (Vallée et al., 1997; Mirescu et al., 2004; O’Connor et al., 2005; Halligan et al., 2007; Moriceau et al., 2009; Raineki et al., 2012). Furthermore, a previous study in our laboratory has shown enhanced neuronal survival in PF animals (Uban et al., 2010). Alternatively, the similar effects of PF and PAE in this study (reduced sedative-like effects following OT treatment and enhanced DCX-density in the dorsal dentate gyrus) likely reflects similar outcomes that may occur through different mechanisms.  4.8 Oxytocin as a possible intervention following prenatal alcohol exposure: limitations and future directions Our data supports and extends our understanding of the effects of PAE on neurogenic, endocrine, and behavioural outcomes, as well as extending the discussion on administration of   52 peripheral OT. OT was not able to mitigate the effects of PAE on hippocampal neurogenesis or CORT recovery following acute stress utilizing the methods in this study; however, PAE animals differentially responded to the sedative-like effects of OT. Therefore, it is possible that PAE animals possess altered OT sensitivity compared to control animals, and that the doses utilized in this study may not have been optimized for modulation of outcomes following PAE. Nevertheless, PAE and OT differentially altered locomotor activity, and the decreased locomotor activity following OT may preferentially impact hyperactive PAE animals. As previously mentioned, hyperactivity is arguably the most common behavioural phenotype following PAE in animal models, as well as reflects a common symptom of FASD in humans. Attenuation of the hyperactivity following PAE may support reductions in learning and memory deficits, attention problems, impulsivity, and risk-taking. Future studies examining whether OT can mitigate the effects of PAE on tasks specific to these cognitive domains are required.  Consistent with previous literature, low dose OT reduced CORT response to acute restraint stress, but this effect was driven by the C and PF groups. The results of this study do not exclude the ability of OT to modulate other aspects of the HPA axis, including receptor expression, CRH and ACTH levels, and negative feedback. HPA dysregulation is a common finding following PAE, and thus OT may modulate the CORT response in PAE animals following other stressors, which vary in duration and intensity from the acute restraint stress utilized in this study. The modulation of HPA axis activity following OT in PAE represents a potential avenue for future studies. Moreover, the 2 h recovery period following injection may have masked any robust effects of OT on modulation of the HPA axis in this study. Likewise, our data do not exclude the possibility that OT modulates other neurogenic measures (such as cell proliferation, long-term neuronal survival, and cell death) or other factors associated with hippocampal functioning (such   53 as synaptic plasticity, neurotransmitter modulation, or gliogenesis). The effects of OT on hippocampal neurogenesis likely depend on the timing and type of measurement utilized; future research on the temporal profile and mechanism(s) underlying the effects of OT on hippocampal neurogenesis are required. Finally, this study does not show the well-known anxiogenic effects of PAE or anxiolytic effects of OT. However, the quick latency to feed across all animals within the study suggests a very mild effect of novelty or stress in this task. It is likely that OT could not have further reduced the latency to feed in this task as the animals had already reached the lower limit. Further, the hyperactivity of PAE animals confounded the interpretation of anxiety-like behaviour in this study. The effects of OT as an anxiolytic would likely be more pronounced in tasks that involve greater novelty or stress. Future research on the anxiolytic effects of OT could examine the combination of PAE and chronic stress, which often exacerbates anxiety-like behaviour in PAE animals (Hellemans et al., 2008).  Lastly, the ability of OT to modulate the effects of PAE on alternative measures, such as bonding with a caregiver, social behaviour, and/or sexual functioning, were not examined and remain a target for future research. Such future research could begin to examine the role of OT treatment across the lifespan, such as increasing PAE offspring bonding with caregiver during the pre-weaning period and attenuating social behaviour deficits in adolescence. Finally, nasal administration of OT is currently being explored in clinical trials for ASD and has shown no robust or reliable side-effects, such administration may also be a viable option for individuals with FASD. 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Curr Opin Clin Nutr Metab Care 6:639–644.     73 Appendices Appendix A: Supplementary figures Measurement Outliers Missing Data Maternal Weight 0 0 Adult Weight 0 0 Sedative-like Effects 0 0 Distance Traveled 0 2 Latency to Feed 3 1 Latency to Investigate 2 2 Appetite Differences 0 2 Rearing 0 2 Self-Grooming 3 2 Basal Corticosterone 2* 6 (3*) Response Corticosterone 0 3 Recovery Corticosterone 4* 9 (6*) Doublecortin Density 2 2  Supplementary Figure 1 Outliers and missing data Table indicates outliers (±2.5 SD>mean) removed from analysis and missing data. Missing data includes collection, sampling, and/or processing errors. Presence of a (*) indicates outliers or missing data that were replaced with the group mean in order to retain samples within a repeated measurement analysis. No more than two animals from the same group were removed from or replaced within any one analysis.     

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