Medicine, Faculty of
DSpace
UBCV
Qiu, Wansu
Doctor of Philosophy - PhD
University of British Columbia
Perinatal depression (PND) is a heterogenous disease where risk factors, symptoms, severity, treatment efficacy, and effects on children can differ depending on timing of onset. The majority of new cases occur with depression onset in the postpartum (postpartum depression; PPD). Very few people with PPD are adequately treated and even in these individuals only ~50% will reach remission with current front-line treatments of selective serotonin reuptake inhibitors (SSRIs). Inflammatory signalling has been related to antidepressant efficacy outside of the postpartum, and I investigated inflammatory mechanisms that may modulate the efficacy of fluoxetine (FLX) in the postpartum. In children, maternal SSRI treatment alters neurodevelopment, including an increased risk for autism spectrum disorder. In Chapter 4, I investigate oxytocin (OT) as a therapeutic in preadolescent offspring to ameliorate maternal treatment effects of CORT and FLX. In Chapter 2, postpartum FLX did not reduce passive coping behaviour in the dams and was commensurate with FLX induced elevations in hippocampal IL-1β levels, peripheral CXCL1 levels, and reduced plasma tryptophan concentrations. In Chapter 3, postpartum CORT treatment increased putative neurotoxic and neuroprotective metabolites within the tryptophan-metabolic pathway in dams. Maternal postpartum FLX did not reverse any postpartum CORT effects, and together with CORT, shifted the tryptophan metabolism towards neurotoxicity. In Chapter 4, maternal FLX reduced neurogenesis and IL-10, IL-13, IFN-γ expression in the hippocampus of offspring. Maternal CORT increased hippocampal neurogenesis and hippocampal IL-6:IL-10 ratio in both sexes, while periadolescent OT treatment reversed these and increased social behaviour in offspring of maternal CORT-treated dams. OT increased neurogenesis in the hippocampus of adult male offspring but not adult female offspring. Collectively, these data suggest the inefficacy of FLX long-term during the postpartum may be due to increased proinflammatory signalling in the hippocampus and reduced tryptophan, and future studies should investigate these pathways to improve long-term efficacy of SSRIs. Moreover, PPD and postpartum antidepressant treatments can have long-term consequences in offspring, which may be ameliorated with OT treatment prior to adolescence. Together, these data can facilitate a better understanding of the consequences of untreated and treated maternal PPD for better outcomes in mothers and children.
PERINATAL DEPRESSION AND TREATMENT IMPLICATIONS FOR MOTHER AND OFFSPRING: LESSONS FROM AN ANIMAL MODEL by Wansu Qiu B.Sc. Hons, University of Guelph, 2016 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Neuroscience) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2021 © Wansu Qiu, 2021 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Perinatal depression and treatment implications for mother and offspring: Lessons from an animal model submitted by Wansu Qiu in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience Examining Committee: Liisa A.M. Galea, Psychology, UBC Supervisor Joanne Weinberg, Cellular & Physiological Sciences, UBC Supervisory Committee Member Tim Oberlander, Pediatrics, UBC Supervisory Committee Member Angela Devlin, Pediatrics, UBC University Examiner Clare Beasley, Psychiatry, UBC University Examiner Additional Supervisory Committee Members: Joelle LeMoult, Psychology, UBC Supervisory Committee Member Jehannine Austin, Medical Genetics, UBC Supervisory Committee Member iii Abstract Perinatal depression (PND) is a heterogenous disease where risk factors, symptoms, severity, treatment efficacy, and effects on children can differ depending on timing of onset. The majority of new cases occur with depression onset in the postpartum (postpartum depression; PPD). Very few people with PPD are adequately treated and even in these individuals only ~50% will reach remission with current front-line treatments of selective serotonin reuptake inhibitors (SSRIs). Inflammatory signalling has been related to antidepressant efficacy outside of the postpartum, and I investigated inflammatory mechanisms that may modulate the efficacy of fluoxetine (FLX) in the postpartum. In children, maternal SSRI treatment alters neurodevelopment, including an increased risk for autism spectrum disorder. In Chapter 4, I investigate oxytocin (OT) as a therapeutic in preadolescent offspring to ameliorate maternal treatment effects of CORT and FLX. In Chapter 2, postpartum FLX did not reduce passive coping behaviour in the dams and was commensurate with FLX induced elevations in hippocampal IL-1β levels, peripheral CXCL1 levels, and reduced plasma tryptophan concentrations. In Chapter 3, postpartum CORT treatment increased putative neurotoxic and neuroprotective metabolites within the tryptophan-metabolic pathway in dams. Maternal postpartum FLX did not reverse any postpartum CORT effects, and together with CORT, shifted the tryptophan metabolism towards neurotoxicity. In Chapter 4, maternal FLX reduced neurogenesis and IL-10, IL-13, IFN-γ expression in the hippocampus of offspring. Maternal CORT increased hippocampal neurogenesis and hippocampal IL-6:IL-10 ratio in both sexes, while periadolescent OT treatment reversed these and increased social behaviour in offspring of maternal CORT-treated dams. OT increased neurogenesis in the hippocampus of adult male offspring but not adult female offspring. iv Collectively, these data suggest the inefficacy of FLX long-term during the postpartum may be due to increased proinflammatory signalling in the hippocampus and reduced tryptophan, and future studies should investigate these pathways to improve long-term efficacy of SSRIs. Moreover, PPD and postpartum antidepressant treatments can have long-term consequences in offspring, which may be ameliorated with OT treatment prior to adolescence. Together, these data can facilitate a better understanding of the consequences of untreated and treated maternal PPD for better outcomes in mothers and children. v Lay Summary Perinatal depression (PND) affects 15% of mothers, and untreated PND can have long-term consequences in children. However, there is a lack of efficacy of antidepressants in the mother and the safety of these drugs for children is questioned. Using a model of PND, we explored biological markers associated with the compromised efficacy of a common antidepressant fluoxetine (Prozac). I found that several biomarkers were associated with reduced effects of fluoxetine in the postpartum including, increased inflammation and reduced tryptophan. Maternal fluoxetine treatment blunted the immune system in adult offspring. Oxytocin, prior to adolescence, showed the potential to reverse some effects in offspring. Together, these data indicate that the inefficacy of antidepressants may be due to alterations in inflammation and that oxytocin may be an effective treatment in children exposed to PND and SSRIs. These findings provide future pathways to improve the symptoms of PND for both mother and children. vi Preface A distinct portion of Chapter 1 has been published as: Qiu, W., Hodges, T.E., Clark, E.L., Blankers, S.A., Galea, L.A.M. (2020). Perinatal depression: Heterogeneity of disease and in animal models. Frontiers in Neuroendocrinology, 59:100854. This review was conceived, planned, and written by all authors except S.A. Blankers. The majority of this publication were split evenly between W. Qiu and Dr. T.E. Hodges with the exceptions of section 1.4.3, which was written by E.L. Clark. The review was written under the supervision of Dr. L.A.M Galea. S.A. Blankers provided feedback and suggested edits prior to manuscript submission. A version of Chapter 2 has been published as: Qiu, W., Duarte-Guterman, P., Eid, R.S., Go, K.A., Lamers, Y., Galea, L.A.M. (2020). Postpartum fluoxetine increased maternal inflammatory signalling and decreased tryptophan metabolism: Clues for efficacy. Neuropharmacology, 175:108174. This experiment was conceived and designed by W. Qiu and Dr. L.A.M. Galea. W. Qiu executed all animal work and collected data with the assistance of K.A. Go. Dr. P. Duarte-Guterman and Dr. R.S. Eid assisted with data analyses. The laboratory of Dr. Y. Lamers were collaborated with for data analyses. W. Qiu performed all statistical analyses and wrote the manuscript with supervision and feedback from Dr. L.A.M. Galea. All other authors provided feedback and suggested edits prior to manuscript submission. A version of Chapter 3 has been published as: Postpartum corticosterone and fluoxetine shift the tryptophan-kynurenine pathway in dams (2021). Psychoneuroendocrinology, 130: 105273. This experiment was conceived and designed by W. Qiu and Dr. L.A.M. Galea. W. Qiu executed all vii animal work and collected data with the assistance of K.A. Go. The laboratory of Dr. Y. Lamers were collaborated with for data analyses. W. Qiu performed all statistical analyses and wrote the manuscript with supervision and feedback from Dr. L.A.M. Galea. All other authors provided feedback and suggested edits prior to manuscript submission. A version of Chapter 4 has been published as: Qiu, W., Go, K.A., Wen, Y., Duarte-Guterman, P., Eid, R.S., Galea, L.A.M. (2021). Maternal fluoxetine reduces hippocampal inflammation and neurogenesis in adult offspring with sex-specific effects of periadolescent oxytocin. Brain Behaviour and Immunity, in press. This experiment was conceived and designed by W. Qiu and Dr. L.A.M. Galea. W. Qiu executed all animal work and collected data with the assistance of K.A. Go. Y. Wen, Dr. P. Duarte-Guterman and Dr. R.S. Eid assisted with data analyses. W. Qiu performed all statistical analyses and wrote the manuscript with supervision and feedback from Dr. L.A.M. Galea. All other authors provided feedback and suggested edits prior to manuscript submission. All animal studies presented in this thesis were conducted in accordance with ethical guidelines set by the Canadian Council on Animal Care and were approved by the Animal Care Committee at the University of British Columbia (certificates A20-0218 and A20-0219). viii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables ............................................................................................................................. xvii List of Figures ........................................................................................................................... xviii List of Symbols .............................................................................................................................xx List of Abbreviations ................................................................................................................. xxi Glossary .................................................................................................................................... xxiii Acknowledgements .................................................................................................................. xxiv Dedication ...................................................................................................................................xxv Chapter 1: Introduction ................................................................................................................1 1.1 Major depressive disorder versus perinatal depression .................................................. 1 1.2 Heterogeneity of perinatal depression ............................................................................ 4 1.3 Considerations for pharmacological antidepressants and non-pharmacological treatments .................................................................................................................................... 8 1.3.1 Antidepressant efficacy: Antepartum and postpartum ............................................ 8 1.3.2 Changes in maternal SSRI pharmacokinetics ....................................................... 10 1.4 Physiology of motherhood and depression: A perfect storm ........................................ 11 1.4.1 Stress and stress hormones during pregnancy and postpartum ............................. 12 1.4.2 Maternal immune changes during pregnancy and the postpartum ....................... 14 ix 1.4.3 Serotonergic tone and metabolism during pregnancy and the postpartum ........... 16 1.4.4 Neuroplasticity during pregnancy and postpartum ............................................... 19 1.5 Rodent models of postpartum depression ..................................................................... 21 1.5.1 Gestational stress .................................................................................................. 22 1.5.2 Postpartum stress manipulations ........................................................................... 24 1.5.3 Postpartum corticosterone model .......................................................................... 25 1.5.4 Interventions in animal models ............................................................................. 27 1.6 Untreated maternal perinatal depression on offspring .................................................. 29 1.6.1 Evidence from preclinical animal models of gestational stress or postpartum CORT on offspring development.......................................................................................... 30 1.7 The impact of treating maternal perinatal depression with pharmacological antidepressants on children ....................................................................................................... 31 1.7.1 Evidence from preclinical studies on effects of maternal SSRI exposure on offspring behavioural outcomes ............................................................................................ 33 1.7.2 Evidence from preclinical studies on effects of maternal SSRI exposure on offspring physiological outcomes ......................................................................................... 35 1.8 Oxytocin as a potential remedy in offspring ................................................................. 36 1.9 Thesis overview and objectives .................................................................................... 38 Chapter 2: Postpartum fluoxetine increased maternal inflammatory signalling and decreased tryptophan metabolism: Clues for efficacy .............................................................40 2.1 Introduction ................................................................................................................... 40 2.2 Methods......................................................................................................................... 44 2.2.1 Animals ................................................................................................................. 44 x 2.2.2 Breeding and culling ............................................................................................. 45 2.2.3 Drug preparations and treatments ......................................................................... 46 2.2.4 Maternal care behaviour observations .................................................................. 46 2.2.5 Forced-swim test ................................................................................................... 47 2.2.6 Estrous cycle cytology .......................................................................................... 48 2.2.7 Blood and tissue collection ................................................................................... 48 2.2.8 Tissue homogenization ......................................................................................... 48 2.2.9 Cytokine and protein quantification...................................................................... 48 2.2.10 Plasma pyridoxic acid, pyridoxal, and tryptophan analyses ................................. 50 2.2.11 Statistical analyses ................................................................................................ 50 2.3 Results ........................................................................................................................... 51 2.3.1 Maternal CORT treatment significantly lowered adrenal mass ............................ 51 2.3.2 Maternal CORT treatment reduced nursing and increased time off the nest and these effects were restored by FLX treatment ...................................................................... 51 2.3.3 Maternal CORT treatment increased time spent immobile in FST2 .................... 52 2.3.4 Maternal FLX treatment increased IL-1 levels in the hippocampus and CXCL1 levels in plasma. Maternal CORT treatment reduced TNF-, and IFN- levels in the hippocampus and TNF- levels in plasma ........................................................................... 54 2.3.5 Maternal fluoxetine treatment significantly reduced plasma 4’-pyridoxic acid, pyridoxal, and tryptophan concentrations, regardless of maternal corticosterone. Maternal corticosterone increased plasma pyridoxal and pyridoxal 5’-phosphate concentrations ...... 59 2.3.6 Principal component analyses on brain cytokine and chemokine levels .............. 60 2.4 Discussion ..................................................................................................................... 65 xi 2.4.1 Corticosterone treatment reduced maternal care and increased passive coping behaviour65 2.4.2 Postpartum corticosterone treatment reduced two proinflammatory cytokines in the hippocampus and in plasma without affecting cytokine or chemokine levels in the frontal cortex ......................................................................................................................... 66 2.4.3 Fluoxetine treatment showed limited efficacy to reduce passive coping ............. 68 2.4.4 Fluoxetine treatment increased IL-1 in the hippocampus and plasma CXCL1, decreased plasma tryptophan, and altered vitamin B6 metabolism, which may be related to the inefficacy of fluoxetine in the postpartum ...................................................................... 69 2.5 Conclusions ................................................................................................................... 71 Chapter 3: Postpartum corticosterone and fluoxetine shift the tryptophan-kynurenine pathway in dams ..........................................................................................................................72 3.1 Introduction ................................................................................................................... 72 3.2 Methods......................................................................................................................... 74 3.2.1 Animals ................................................................................................................. 74 3.2.2 Drug treatments ..................................................................................................... 75 3.2.3 Blood collection .................................................................................................... 75 3.2.4 Tryptophan-kynurenine metabolite analyses ........................................................ 76 3.2.5 Statistical analyses ................................................................................................ 76 3.3 Results ........................................................................................................................... 77 3.3.1 Maternal fluoxetine decreased xanthurenic acid concentrations .......................... 77 xii 3.3.2 Maternal corticosterone increased metabolite concentrations and fluoxetine failed to correct this shift. Maternal corticosterone with fluoxetine shifted the neuroprotective-to-neurotoxic balance towards neurotoxicity ............................................................................ 77 3.3.3 Maternal corticosterone increased a coenzyme associated with the putative neurotoxic branch and fluoxetine failed to correct this shift. Maternal corticosterone with fluoxetine increased a cofactor associated with the putative neurotoxic branch .................. 80 3.3.4 Metabolite concentrations significantly correlated maternal depressive-like behaviour, maternal hippocampal TNF- concentratins, or relative adrenal mass .............. 80 3.4 Discussion ..................................................................................................................... 84 3.4.1 Maternal corticosterone increased more putative neurotoxic metabolites and putative neurotoxic branch coenzyme/cofactor .................................................................... 84 3.4.2 Maternal fluoxetine failed to reverse maternal corticosterone effects, and contributed towards a shift to neurotoxicity ......................................................................... 86 3.5 Conclusion .................................................................................................................... 87 Chapter 4: Maternal postpartum fluoxetine reduces hippocampal inflammation and neurogenesis in adult offspring with sex-specific effects of periadolescent oxytocin .............89 4.1 Introduction ................................................................................................................... 89 4.2 Methods......................................................................................................................... 94 4.2.1 Animals ................................................................................................................. 94 4.2.2 Dam breeding and offspring culling ..................................................................... 94 4.2.3 Maternal drug preparations and treatments........................................................... 95 4.2.4 Offspring weaning ................................................................................................ 96 4.2.5 Offspring drug preparations and treatments ......................................................... 96 xiii 4.2.6 Offspring puberty analysis and anogenital distance ............................................. 98 4.2.7 Behavioural assessment ........................................................................................ 98 4.2.7.1 3-chambered social investigation task .............................................................. 98 4.2.7.2 Elevated plus maze ........................................................................................... 99 4.2.8 Tissue Collection and tissue processing ............................................................... 99 4.2.9 Vaginal cytology for estrous cycle assessment ................................................... 100 4.2.10 Doublecortin Immunohistochemistry ................................................................. 100 4.2.11 Microscopy ......................................................................................................... 101 4.2.12 Tissue homogenization ....................................................................................... 101 4.2.13 Multiplex cytokine electrochemiluminescence immunoassays and protein quantification ...................................................................................................................... 101 4.2.14 Data Analyses ..................................................................................................... 102 4.3 Results ......................................................................................................................... 103 4.3.1 Oxytocin with TriozanTM increased sedation-like behaviour and prolonged recovery from sedation-like behaviour compared to oxytocin alone in both sexes ............ 103 4.3.2 Maternal corticosterone treatment accelerated pubertal onset in female offspring 104 4.3.3 Maternal fluoxetine treatment increased body mass whereas oxytocin treatment reduced body mass in both sexes during periadolescence and in adult males .................... 106 4.3.4 Both maternal corticosterone and fluoxetine treatment alone decreased anxiety-like behaviour in offspring .................................................................................................. 107 4.3.5 Periadolescent oxytocin treatment increased social investigation in both sexes 107 xiv 4.3.6 Oxytocin treatment increased neurogenesis in males but not in females. Maternal fluoxetine treatment reduced neurogenesis. Maternal corticosterone increased neurogenesis, whereas oxytocin, and oxytocin with TriozanTM reduced this elevation in neurogenesis by maternal corticosterone ....................................................................................................... 109 4.3.7 Maternal fluoxetine reduced proliferative DCX-positive cells in males and post-mitotic cells in both sexes in the ventral dentate gyrus. OT increased post-mitotic DCX-positive cells only in males ................................................................................................. 110 4.3.8 In both sexes, maternal fluoxetine decreased interleukin-10, interleukin-13, and interferon-γ, in the hippocampus ........................................................................................ 113 4.3.9 Maternal corticosterone shifted the offspring hippocampal cytokine profile towards a more proinflammatory profile via interleukin-6-to-interleukin-10 ratio; and offspring periadolescent oxytocin and oxytocin with TriozanTM treatment normalized this shift 114 4.3.10 Maternal fluoxetine treatment reduced overall immune signalling .................... 116 4.3.11 Neurogenesis was correlated with social behaviour in females while neurogenesis was correlated with anxiety like behaviour in male ............................................................ 117 4.4 Discussion ................................................................................................................... 118 4.4.1 Maternal postpartum fluoxetine reduced offspring neuroinflammation and neurogenesis in the hippocampus as well as anxiety-like behaviour in both sexes in adulthood119 4.4.2 Maternal corticosterone treatment increased offspring neuroinflammation and neurogenesis, and reduced anxiety-like behaviour in both sexes ....................................... 122 xv 4.4.3 Periadolescent oxytocin treatment reversed the effects of maternal fluoxetine or corticosterone treatment on various measures, and sex differences emerged with oxytocin treatment. ............................................................................................................................ 124 4.4.4 Possible explanations on differences between oxytocin and oxytocin with TriozanTM effects ................................................................................................................. 125 4.5 Conclusion .................................................................................................................. 126 Chapter 5: General discussions ................................................................................................127 5.1 Summary of experimental findings ............................................................................. 127 5.2 Postpartum corticosterone model of de novo depression ............................................ 130 5.2.1 Pulsatile corticosterone release is modelled by injections ............................ 133132 5.3 Limited efficacy of fluoxetine in the postpartum ................................................. 134133 5.4 Inflammatory profiles influence efficacy .................................................................... 136 5.4.1 Animal models of perinatal depression and inflammation ........................... 137136 5.5 Fluoxetine’s efficacy is associated with the tryptophan kynurenine pathway ............ 138 5.5.1 Indole synthesis pathway of tryptophan metabolism by the gut microbiota ...... 143 5.6 Treatments in dams lead to variability in offspring anxiety-like behaviour ............... 143 5.7 Periadolescent effects of oxytocin .............................................................................. 145 5.7.1 Outcomes in the immune system ........................................................................ 146 5.8 Future directions and clinical implications ................................................................. 147 5.9 Conclusions ................................................................................................................. 151 Bibliography ...............................................................................................................................153 Appendices ..................................................................................................................................196 Appendix A Supplemental figures for Chapter 4 ................................................................... 196 xvi A.1 Figure S1 ............................................................................................................. 196 A.2 Figure S2 ............................................................................................................. 197 A.3 Figure S3 ............................................................................................................. 198 A.4 Figure S4 ............................................................................................................. 200 A.5 Figure S5 ............................................................................................................. 202 A.6 Figure S6 ............................................................................................................. 204 A.7 Figure S7 ............................................................................................................. 205 A.8 Figure S8 ............................................................................................................. 207 A.9 Figure S9 ............................................................................................................. 208 Appendix B Supplemental table for Chapter 4 ....................................................................... 210 B.1 Table S1 .............................................................................................................. 210 xvii List of Tables Table 2.1. Number of rats in each estrous stage on FST2. ........................................................... 52 Table 2.2. Two-way ANOVA results of hippocampal (HPC) cytokines and chemokine data. ... 56 Table 2.3. Two-way ANOVA results of frontal cortex cytokines and chemokine data. .............. 56 Table 2.4. Two-way ANOVA results of plasma cytokines and chemokine data. ........................ 57 Table 2.5. Component loadings for the hippocampal cytokine variance of the first two components identified using principal component analysis (PCA). ............................................. 62 Table 2.6. Component loadings for the frontal cortex cytokine variance of the first two components identified using principal component analysis (PCA). ............................................. 62 Table 2.7. Component loadings for the plasma cytokine variance of the first two components identified using principal component analysis (PCA). ................................................................. 62 Table 4.1. Component loadings for the hippocampal cytokine variance of the first two components identified using principal component analysis (PCA). ........................................... 117 Table 5.1. Postpartum corticosterone model of de novo postpartum depression.. ..................... 128 Table 5.2. Periadolescent oxytocin treatment in offspring in the postpartum corticosterone model of de novo postpartum depression and maternal antidepressant treatments. .............................. 129 xviii List of Figures Figure 1.1. Comparison between biological changes during the perinatal period and during depression. ...................................................................................................................................... 7 Figure 1.2. Schematic comparison of brain and hippocampal development across the perinatal period between humans and rats. .................................................................................................. 22 Figure 2.1. Timeline of experiment in chapter 2. ......................................................................... 45 Figure 2.2. Effects of maternal corticosterone (CORT) and fluoxetine (FLX) treatment on relative adrenal mass, maternal care behaviour (total nursing, total off-nest behaviour), and immobility behaviour during forced swim test (FST). ................................................................. 53 Figure 2.3. Effects of maternal corticosterone (CORT) and fluoxetine (FLX) treatment on hippocampal and plasma cytokine levels. ..................................................................................... 58 Figure 2.4. Effects of maternal corticosterone (CORT) and fluoxetine (FLX) treatment on plasma tryptophan levels, 4’-pyridoxic acid, pyridoxal levels, and pryidoxal 5’-phosphate. ................... 60 Figure 2.5. Effects of maternal corticosterone (CORT) and fluoxetine (FLX) treatment on hippocampal , frontal cortex, and plasma cytokine principal component analysis (PCA) sample scores onto the first two components. ........................................................................................... 64 Figure 3.1. Schematic overview of the tryptophan-kynurenine pathway. .................................... 74 Figure 3.2. Effects of maternal corticosterone (CORT) and fluoxetine (FLX) on plasma tryptophan-kynurenine pathway metabolites. ............................................................................... 79 Figure 3.3. Effects of maternal corticosterone (CORT) and fluoxetine (FLX) treatment on tryptophan-kynurenine pathway coenzyme and cofactor. ............................................................ 81 xix Figure 3.4. Correlations between tryptophan-kynurenine metabolites and maternal depressive-like behaviour, maternal hippocampal cytokine levels, or maternal relative adrenal mass. ......... 83 Figure 4.1. Timeline of experiment in chapter 4. Briefly, dams were bred within the animal facility as previously reported (Qiu et al., 2020a). ....................................................................... 95 Figure 4.2. Sex differences in changes in puberty development during periadolescence (postnatal day 25-34). .................................................................................................................................. 105 Figure 4.3. Effects of maternal postpartum fluoxetine (FLX) treatment or corticosterone (CORT) treatment on offspring anxiety-like behaviour and social behaviour in adulthood. ................... 108 Figure 4.4. Effects of maternal postpartum fluoxetine (FLX) treatment or corticosterone (CORT) treatment and oxytocin (OT) and oxytocin with TriozanTM (OT+T) on offspring doublecortin (DCX)-positive cells in the dentate gyrus. .................................................................................. 113 Figure 4.5. Effects of maternal postpartum fluoxetine (FLX) treatment or corticosterone (CORT) treatment, and periadolescent oxytocin (OT) and oxytocin with TriozanTM (OT+T) treatment on offspring neuroinflammation. ..................................................................................................... 116 Figure 5.1. Schematic diagrams of various metabolic pathways investigated and reported in Chapter 2 and 3. .................................................................................................................... 142141 Figure 5.2. An infographic summary of proposed future directions for dams and offspring................................................................................................................................................ 150148 xx List of Symbols : Alpha : Beta : Gamma xxi List of Abbreviations 3-HAA: 3-Hydroxyanthranilic acid 3-HK: 3-Hydroxykynurenine AA: Anthranilic acid ABN: Arched back nursing ANCOVA: Analysis of covariance ANOVA: Analysis of variance CORT: Corticosterone DCX: Doublecortin DSM-V: Diagnostic and Statistical Manual of Mental Disorders, 5th edition DXT: Dextrose EPDS: Edinburgh Postnatal Depression Scale EPM: Elevated plus maze FAD: Flavin adenine dinucleotide FLX: Fluoxetine FMN: Flavin mononucleotide FST: Forced swim test FST1: First forced swim test FST2: Second forced swim test GD: Gestational day HPA: Hypothalamic-pituitary-adrenal HPC: Hippocampus xxii ICD-10: International Statistical Classification of Diseases tenth edition IFN: Interferon IL: Interleukin KYN: Kynurenine KYNA: Kynurenic acid LPS: lipopolysaccharide MDD: Major depressive disorder OT: Oxytocin OT+T: Oxytocin with TriozanTM nanoformulation PBS: Phosphate buffered saline PC: Principal component PCA: Principal component analysis PD: Postnatal/Postpartum day PDSS: Postpartum Depression Screening Scale PND: Perinatal depression PPD: Postpartum depression SEM: Standard error of mean SSRI: Selective serotonin reuptake inhibitor TKP: Tryptophan kynurenine pathway TMC: N-trimethyl chitosan TNF: Tumor necrosis factor WHO: World Health Organization XA: Xanthurenic acid xxiii Glossary Antepartum: In discussing topics regarding effects in mothers/dams, refers to the gestational period before parturition. Neonatal/Postnatal: In discussing topics regarding effects in children/offspring, refers to the early period after birth. Periadolescence: In discussing topics regarding effects in children/offspring, refers to the developmental period immediately before, during, and immediately after adolescence. Peripartum: In discussing topics regarding effects in mothers/dams, refers to the period starting in gestation/pregnancy to after birth in the postpartum period, specifically in rodents before pup weaning. Perinatal: In discussing topics regarding effects in children/offspring, refers to the developmental period during gestation/pregnancy and after birth, specifically in rodents before pup weaning. Postpartum: In discussing topics regarding effects in mothers/dams, refers to the period after parturition, and specifically in rodents before pup weaning. xxiv Acknowledgements I would like to thank my supervisor, Dr. Liisa Galea, for her guidance and wonderful mentorship throughout this whole process. I have truly learned a lot, and enjoyed every moment of it (good or bad) during my stretch here in Dr. Galea’s lab. I would also like to thank my committee members, Dr. Jehannine Austin, Dr. Joelle LeMoult, Dr. Tim Oberlander, and Dr. Joanne Weinberg, for putting up with me all these years. I would also like to thank the University of British Columbia, and the Faculty of Medicine for their funding opportunities (Four Year Fellowship, IMH Marshall Scholarship). I would like to give gratitude to all my mentors (past and present as aforementioned), with a special shutout to Dr. Elena Choleris, my friends, and family. This would have not been possible without any of you. I would like to also acknowledge the hard work and long hours the numerous volunteers, RAs, and animal technicians have put in for this project to come to fruition. Thank you all for your endless support. xxv Dedication Peachfuzz and Jem(s) 1 Chapter 1: Introduction 1.1 Major depressive disorder versus perinatal depression The World Health Organization (WHO) identifies major depressive disorder (MDD) as the major leading cause of disability and a major contributor to the global burden of disease (Geneva World Health Organization, 2017). MDD affects more females than males across all age-groups, starting in adolescence (15-80+ years) (Seedat et al., 2009; Geneva World Health Organization, 2017). Aside from sex differences in disease prevalence, sex differences in symptom manifestation also exist (Silverstein, 2002; Marcus et al., 2008). For example, women diagnosed with MDD experience more ‘atypical’ symptoms such as hypersomnia and weight gain (Angst et al., 2002; Smith et al., 2008), and present with higher comorbidity for anxiety disorders (Lamers et al., 2011) than men diagnosed with MDD. The greater prevalence rate of depression in females compared to males is most salient during the reproductive years (i.e. 25-50 years of age) (Angst et al., 2002; Gutiérrez-Lobos et al., 2002). It is important to investigate unique experiences that occur during the reproductive years in females that may relate to greater susceptibility to depression. The perinatal period (during pregnancy and postpartum) is one time period that deserves investigation as the risk for de novo onset of any mental disorder is heightened during the postpartum (Munk-Olsen et al., 2006). In this thesis, the use of the terms “female” and “male” are used to describe the biological sex of individuals or rodents. Although the terms “women” and “men” are generally used to describe gender preferences of individuals, here it may also be used to describe those who are biologically female or male. This is due to often studies cited do not distinguish, or use the wrong term, between biological sex and gender preferences in individuals. Further, I recognize 2 that the pregnancy and postpartum experience are not exclusive to women, nor is the condition of PND. For the purpose of this thesis, I will be focusing only on females because we are modelling depression onset after the unique female experience of pregnancy and birth. Moreover, rats do not show gender preferences, thus we cannot model depression in those individuals who underwent pregnancy and postpartum but do not identify as women. Depression onset during pregnancy can be referred to as antepartum depression, whereas depression onset during the postpartum can be referred to as postpartum depression (PPD) (World Health Organization, 2003; American Psychiatric Association, 2013). The timing of depression onset likely has different implications for disease etiology, given the differences in physiological characteristics between antepartum and postpartum. Unfortunately, both the DSM-V and the International Statistical Classification of Diseases tenth edition (ICD-10) (World Health Organization, 2003) do not base their classification of perinatal depression (PND) on the recognition of the importance of the timing of onset of symptoms or of distinguishing PND as a de novo disorder. One major issue with the DSM-V definition is that, to be diagnosed with PND, there is a time restriction of occurrence between any time during pregnancy or up to the first four weeks postpartum. This generates an inability to distinguish between antepartum versus postpartum depression onset and negates any onset of depression after the first month postpartum (see section 1.2). Additionally, the diagnosis can only be applied to the current or most recent depressive episode (American Psychiatric Association, 2013), which impedes retrospective analyses in determining whether there is a history of depression (Uher et al., 2014). People presenting with depression in the perinatal period who have had a previous history of depression versus those presenting with de novo antepartum or postpartum depression likely have a different etiology and possibly treatment needs. Thus, it is important to understand whether triggers and 3 efficacy of treatment differ between de novo perinatal (antepartum or postpartum) onset versus a previous history of depression. Research indicates that there is an underdiagnosis of PND within the general population by as much as 50%, suggesting, in part, that this may be due to a failure to identify PND using current diagnostic tools and/or relying on self-report (Cox et al., 2016; Woody et al., 2017). This underdiagnosis may be due to similarities between some symptoms of depression and the common side-effects of pregnancy/postpartum, leading patients and practitioners to dismiss depression symptoms as natural perinatal discomforts (Jolley and Betrus, 2007). Symptoms of pregnancy including changes in appetite, increases in body mass/weight gain, sleep dysregulation, increased fatigue and irritability, and decreased concentration (Yonkers et al., 2009; American Psychiatric Association, 2013) are also seen in MDD. Two specific self-report screening tools have been developed for PND, the Edinburgh Postnatal Depression Scale (EPDS) and the Postpartum Depression Screening Scale (PDSS). Although both are reliable screening tools when compared to structured clinical interviews for DSM-V (Tandon et al., 2012; Vogeli et al., 2018), the sensitivity of both screen tools could be improved by reducing the cutoff scores, thus lowering the threshold for diagnoses, and including cultural appropriate measures (Chaudron et al., 2010; Tandon et al., 2012). For example, in low-income African American women, the sensitivity of major and minor depressive disorder screening tools was improved by reducing scale cutoff scores and thus lowering the threshold for diagnoses (Chaudron et al., 2010; Tandon et al., 2012). Overall, these data suggest a significant need for better screening and timeline inclusions for those diagnosed with PND. The ability to determine timing of depression onset is needed to gain an understanding of how timing of depression onset affects diagnosis, risk factors, and treatment barriers during these unique periods. 4 1.2 Heterogeneity of perinatal depression The greatest risk factor for PND is a previous history of depression (Flynn et al., 2006; Records and Rice, 2007; Bunevicius et al., 2009; Altemus et al., 2012; Tebeka et al., 2016; English et al., 2018). Interestingly, 43% of women with MDD relapsed during pregnancy, the majority of whom (68%) had discontinued their medication during pregnancy (reviewed in Cohen et al., 2006). Although many researchers have attempted to dissociate PND based on timing of onset and differences in symptom risk, many studies do not distinguish between participants with a previous history of depression from those with de novo PND (Fisher et al., 2016; McCall-Hosenfeld et al., 2016; Denckla et al., 2018; Fredriksen et al., 2017; Ahmed et al., 2019; Dekel et al., 2019; Wikman et al., 2019; reviewed in Baron et al., 2017; Santos et al., 2017). It is important to underscore that de novo onset of depression during the antepartum and postpartum have different implications than PND with a history of depression. For example, in those with a previous history of depression, PND symptoms may be a continuation of previous episodes (Kott and Brummelte, 2019). In addition, it is worthwhile noting that people who are currently taking antidepressants and/or have a current diagnosis of MDD and become pregnant would not necessarily be classified as having a PND subtype. Nonetheless, this may be an important population to consider (for more discussion on this see Kott and Brummelte, 2019). In this thesis, I concentrate on those with PND and I discuss differences in depressive symptom onset and manifestation in these different types of PND, those with de novo depression either antepartum or postpartum or those with a history of depression that reoccurs during pregnancy or the postpartum. Fewer women report with de novo antepartum symptoms onset (6-36% out of total participants) compared to women with de novo postpartum symptoms onset (56%-67% out of 5 total participants) (Fisher et al., 2016; Ahmed et al., 2019; Dekel et al., 2019; Wikman et al., 2019). Indeed, generally a larger subset of women that experience de novo depression have onset during the postpartum compared to onset during pregnancy (Postpartum Depression: Action Towards Causes and Treatment (PACT) Consortium, 2015; Fisher et al., 2016; Wikman et al., 2019.; reviewed in Shorey et al., 2018). Within the postpartum period looking at the percentage rate of symptoms onset in participants, de novo postpartum onset within the first 6 weeks post-parturition (67%) was higher than de novo postpartum onset after the first 6 months post-parturition (43%) (Wikman et al., 2019). Within the gestational period, Dekel and colleagues (2019) found that there were more cases of depression in the third trimester (10%) compared to the first trimester (4.9%), but these data did not exclude possible pre-existing conditions. Complicating matters, depression or anxiety during pregnancy are also significant risk factors for PPD (Robertson et al., 2004). All in all, these data suggest that there is heterogeneity in symptom onset with more de novo cases in the early postpartum compared to the antepartum period. Studies point to the postpartum as a time of greater susceptibility for first-time psychiatric disease, particularly in primiparous mothers compared to fathers (Munk-Olsen et al., 2006). Indeed, relative risk (per 1000 births) of first-time hospitalization for any psychiatric illnesses in primiparous mothers ranged from 1.68 to 7.31 for the first three months after giving birth, compared to 0.53 during pregnancy (Munk-Olsen et al., 2006). According to Banti and colleagues (2011), the cumulative incident rate for depression during postpartum is higher than during pregnancy (6.8% and 2.2% respectively) and first-time onset is also higher during the postpartum period than during pregnancy (5.7% and 1.6%, respectively). Global prevalence for first-time onset of depression during postpartum has been reported to be as high as 18% 6 (reviewed in Hahn-Holbrook et al., 2018; Shorey et al., 2018). Thus, data from several studies suggest that de novo PPD is more common than de novo antepartum depression. The manifestation of depression also differs depending on whether depression is de novo and the timing of the onset of depression (Postpartum Depression: Action Towards Causes and Treatment (PACT) Consortium, 2015; Fisher et al., 2016; Wikman et al., 2019). For example, women with depression onset during pregnancy have greater paranoia, lower obsessional/compulsive symptoms, and greater weight gain than do women with postpartum onset (Fisher et al., 2016). Furthermore, latent onset analyses indicated that more severe depression and greater anxiety was associated with depressive onset during pregnancy and a history of past depression (Postpartum Depression: Action Towards Causes and Treatment (PACT) Consortium, 2015). Fisher and colleagues (2016) found a higher risk of hypersomnia in women with pregnancy and postpartum depression onset compared to pre-pregnancy depression onset. Other distinguishing symptoms with onset have been noted with onset during pregnancy or the postpartum, but unfortunately these data were not separated for history of depression (Putnam et al., 2017). All in all, differing manifestations of symptoms in de novo antepartum versus postpartum depression provide more evidence that PND is not a homogeneous disorder. In Section 1.4, I turn to the biological mechanisms that may distinguish these two time periods and contribute to the changes in relative risk for depression (see Figure 1.1), but first I discuss treatment efficacy in reducing PND symptoms. 7 Figure 1.1. Comparison between biological changes during the perinatal period and during depression. Many of the changes that occur naturally during the perinatal period are similar to biological symptoms that occur during major depressive disorder (MDD). The similarity in biological outcomes with MDD may drive the increased the risk for depression during the perinatal period in susceptible people. HPA: Hypothalamic-pituitary-adrenal; ↑greater; ↓reduced. 8 1.3 Considerations for pharmacological antidepressants and non-pharmacological treatments A comprehensive review identified that only 13-16% of women receive treatment, of those diagnosed with PND and of these women, only half received adequate treatment, defined as six weeks of continued pharmacological or psychological treatment (Cox et al., 2016). Furthermore, perhaps most disturbingly, only about 3-5% of all PND cases reached remission (Cox et al., 2016). Interestingly, the authors also found lower treatment and remission rates for PPD compared to those with antepartum depression (Cox et al., 2016), which may be due to both biological and social factors. It should be noted that out of those who did receive adequate treatment approximately 50% of those women did reach remission suggesting, treatment if given properly it is effective for half of the population (Cox et al., 2016). For mothers, untreated or improperly treated depression can lead to maternal suicide or infanticide (Kimmel et al., 2019). In the postpartum period, suicide accounts for an astonishing 20% of maternal deaths (Lindahl et al., 2005). Indeed, the risk for suicide is low during pregnancy and high during the postpartum period (Lindahl et al., 2005; Pope et al., 2013; Esscher et al., 2016), highlighting the need for distinguishing between antepartum versus postpartum depression symptoms. Clearly, further efforts are warranted to improve treatment efficacy for PND. Here, I discuss the efficacy of selective serotonin reuptake inhibitors (SSRIs). 1.3.1 Antidepressant efficacy: Antepartum and postpartum SSRIs are a common class of antidepressants prescribed to treat PND (Latendresse et al., 2017; Sun et al., 2019), however, several studies have demonstrated that SSRIs are not entirely effective in reducing depressive scores in the perinatal period compared to placebo, suggesting that SSRIs have limited efficacy for PND (Appleby et al., 1997; Misri et al., 2004; Yonkers et 9 al., 2008; Bloch et al., 2012; Shankar et al., 2017; De Crescenzo et al., 2014; Molyneaux et al., 2014; Sharma, 2017). Treatment with the SSRI paroxetine did not improve depression scores compared to placebo at 8 weeks postpartum, although the overall remission rate was higher in the SSRI treatment group (Yonkers et al., 2008). Other studies comparing the effects of SSRI treatment with or without co-current psychotherapy on PPD found no differences between treatment groups, suggesting no additional benefit of SSRI treatment (Appleby et al., 1997; Misri et al., 2004; Bloch et al., 2012). Sertraline, another SSRI, reduced depression scores compared to placebo in women with de novo depression onset within the first 4 weeks postpartum, although in this study the treated group presented with lower depression scores at the onset (Hantsoo et al., 2014). In one study focusing on antepartum depression onset, SSRI treatment during gestation and/or the postpartum did not improve depression scores or predict recovery at 6 years postpartum (Shankar et al., 2017). All in all, these data suggest a low efficacy of SSRIs for reducing PND, but further studies are needed to determine relative efficacy dependent on timing onset and depression history. SSRI treatment during pregnancy or immediately after birth has been used to prevent PPD in women that have a history of depression with differing efficacy (Wisner et al., 2004; Suri et al., 2017; reviewed in Molyneaux et al., 2014). In one study, a 17-week trial of sertraline immediately after parturition resulted in a lower recurrence of depression in the postpartum (7%) compared to the recurrence of placebo-treated mothers (50%) (Wisner et al., 2004), but this study had a small number of participants. In contrast, when SSRIs were given during pregnancy, it did not prevent the onset of PPD (Suri et al., 2017). These data suggest that preventative antidepressant treatments in at-risk women may only be effective when given during the postpartum period. Overall, there is a paucity of data available, and we now consider differences 10 in maternal pharmacokinetics that could contribute to SSRI efficacy during the perinatal period (Sit et al., 2008, 2010). 1.3.2 Changes in maternal SSRI pharmacokinetics Pharmacokinetics refers to the absorption, metabolism, distribution, and elimination of a drug after administration. Many physiological changes occur during pregnancy, such as alterations in hepatic enzymes and gut motility, and during the postpartum, such as increased cardiac output and glomerular filtration (reviewed in Frederiksen, 2001). These physiological alterations can impact pharmacokinetics, including drug absorption, which contribute to antidepressant efficacy (reviewed in Frederiksen, 2001). Moreover, metabolism is decreased from mid-gestation to parturition, which returns to mid-gestation levels by 12 weeks postpartum depending on lactation (Butte et al., 1999; Sit et al., 2008; reviewed in Sit et al., 2006; Stuebe and Rich-Edwards, 2009). Thus, during pregnancy, changes in pharmacokinetics are marked by an increase in drug metabolism that is reduced at parturition and rebounds during the postpartum period. These data also suggest that SSRI dosage needs to be monitored carefully during pregnancy and the postpartum. Although studies are limited, for certain SSRIs, drug concentrations are lower during pregnancy, particularly in the third trimester, compared to baseline (Westin et al., 2017; Schoretsanitis et al., 2019). However, there is a caveat as analyses of metabolites between pregnancy and non-pregnancy indicate that the need for dose-readjustment can vary dramatically by type of SSRI. Some SSRIs (sertraline) metabolite analyses indicate a need to reduce dose, whereas other SSRIs (nortriptyline) metabolite analyses indicate a need to increase dose (Schoretsanitis et al., 2019). These changes highlight the significant differences in drug metabolism between pregnancy and the postpartum period, which may be indicative of changes in antidepressant efficacy between these two distinct periods. 11 Most SSRIs and other pharmacological antidepressants are catalyzed by the metabolic enzymes, CYP3A4 and CYP2D6 (reviewed in Deligiannidis et al., 2014). CYP3A4 and CYP2D6 enzymatic activity are increased during pregnancy and parturition but are reduced by 4 months postpartum (Tracy et al., 2005; Nylén et al., 2011; reviewed in Deligiannidis et al., 2014; Kim et al., 2018). The increases in CYP3A4 and CYP2D6 likely underlie the need to increase SSRI doses during pregnancy to treat PND (Hostetter et al., 2000). In a comprehensive study, pregnancy-like levels of 17β-estradiol and progesterone were found to regulate different isoforms of the phase 1 metabolism CYP450 family enzymes in human hepatocytes (Choi et al., 2013) and may underlie the changes in antidepressant metabolism during pregnancy. In addition, polymorphisms of CYP contribute to pharmacokinetics (Zobel and Maier, 2010; Fabbri et al., 2013; Singh et al., 2014; Koren and Ornoy, 2018), although the research to date has not yet considered the heterogeneity of PND (Wisner et al., 2010). As specified above, CYP3A4 and CYP2D6 enzymatic activity are reduced by 4 months postpartum relative to pregnancy, thus it should be expected that SSRI metabolism should be reduced, and lower doses are expected to be efficacious in the postpartum. Yet, in the clinical setting postpartum SSRI treatment still show little efficacy, and less women reach remission in the postpartum period than antepartum (Cox et al., 2016). In this thesis, I aim to understand why postpartum is a unique period of low SSRI efficacy and what are some possible underlying mechanisms modulating limited efficacy. 1.4 Physiology of motherhood and depression: A perfect storm During pregnancy and the postpartum, there are several physiological changes that occur that mirror the changes seen in depression. These changes may contribute to the greater 12 susceptibility to depression, creating the “perfect storm” for depression (see Galea and Frokjaer, 2019). I outline changes across pregnancy and the postpartum in stress hormones, immune signalling, serotonergic tone, and associated serotonergic metabolic factors (tryptophan-kynurenine pathway), and brain morphology, that may promote greater risk for PND (for more in-depth reviews on these topics see Hendrick et al., 1998; Malassiné et al., 2003; Hillerer et al., 2014; Napso et al., 2018; Noyola-Martínez et al., 2019). 1.4.1 Stress and stress hormones during pregnancy and postpartum The hypothalamic-pituitary-adrenal (HPA) axis undergoes significant change during pregnancy that may contribute to the increased susceptibility to psychiatric disorders during this period. High concentrations of basal cortisol are seen during pregnancy and the postpartum compared to non-postpartum period (de Weerth and Buitelaar, 2005). In contrast, lower levels of reactive cortisol are seen during the postpartum (Brunton et al., 2008), especially in women who are breastfeeding (Cox et al., 2015). These data show that HPA function is altered during pregnancy and in the postpartum, and lower stress reactivity in the postpartum may serve as a mechanism to protect postpartum women from high glucocorticoid exposure. Atypical diurnal and stress cortisol responses are linked to PPD (Bloch et al., 2005; Nierop et al., 2006; Taylor et al., 2009; Iliadis et al., 2015). For example, increased depressive symptoms in the postpartum were reported in women without the typical increase in bioavailable cortisol after waking (Taylor et al., 2009). Symptoms of antepartum and postpartum depression have been linked to high basal cortisol and high cortisol reactivity to stress at multiple timepoints: during pregnancy (Nierop et al., 2006), 6 weeks postpartum (Iliadis et al., 2015), as well as a year following birth (Bloch et al., 2005), although the direction of these effects can change with time and breastfeeding status. Only women with onset of depression in the 13 postpartum, and not women with onset of depression before or during pregnancy, had high levels of basal cortisol in the early postpartum (Iliadis et al., 2015). In addition, enhanced sensitivity of the HPA axis to corticotropin-releasing-hormone was found in women with a history of PPD one year or more after childbirth (Bloch et al., 2005). In women with a previous history of chronic depression, hypocortisolemia or low cortisol is seen up to one year postpartum (Groer and Morgan, 2007; reviewed in Seth et al., 2016). Moreover, Deligiannidis and colleagues (2016) found no difference in stress reactivity between pregnant women that scored low or high on a depressive symptom scale or that had a history of depression. In addition, although in a small sample, blunted HPA axis reactivity was found in women with the “blues” at 3 and 6 weeks postpartum, but not at 12 weeks postpartum (Magiakou et al., 1996). These latter findings may have been affected by breastfeeding as a blunted stress response is seen in breastfeeding multiparous mothers (Tu et al., 2006). Thus, the effect of PND on basal and stress reactive cortisol depends on when (pregnancy, postpartum) cortisol is measured as well as past depressive history, breastfeeding and parity, and whether the onset of depression occurred during pregnancy or in the postpartum. Different exposures to stressors during either pregnancy or postpartum can increase the risk for either antepartum or postpartum depression. For example, unsurprisingly, obstetric complications or unwanted pregnancy may be risk factors for antepartum depression, whereas birth complications or postpartum issues are a significant risk factor for PPD in the early postpartum period (Altemus et al., 2012; Yanikkerem et al., 2013; Postpartum Depression: Action Towards Causes and Treatment (PACT) Consortium, 2015; Rai et al., 2015; Dekel et al., 2019; Wikman et al., 2019). Postpartum issues such as infant colic, lack of partner support, and lack of breastfeeding were associated with early and late postpartum onset of depression 14 (Wikman et al., 2019) whereas parturition issues (interventions during delivery) were associated with early postpartum onset (Dekel et al., 2019; Wikman et al., 2019). Furthermore, unplanned pregnancies predicted depression during pregnancy and early postpartum onset (Wikman et al., 2019). These data suggest that depression symptom onset during the pregnancy, early postpartum, or in the late postpartum can have different biological and stressful event-related risk factors (Wikman et al., 2019). Overall, these data demonstrate that HPA function is dysregulated depending on a history of depression and HPA dysregulation may predict PND, particularly with an onset in the postpartum. 1.4.2 Maternal immune changes during pregnancy and the postpartum The maternal immune system undergoes significant changes during pregnancy and postpartum to maintain a healthy term pregnancy and delivery (Shimaoka et al., 2000; Hanna et al., 2006; Kraus et al., 2010; Christian and Porter, 2014; Simpson et al., 2016; Ziegler et al., 2018). Cytokines, the main signalling molecules of the immune system, in serum and the brain, fluctuate during pregnancy and the postpartum in humans and rodents (Shimaoka et al., 2000; Hanna et al., 2006; Orsi et al., 2006; Kraus et al., 2010; Christian and Porter, 2014; Simpson et al., 2016; Haim et al., 2017; Ziegler et al., 2018; Eid et al., 2019). Although there is considerable debate, the second trimester is regarded as a period of anti-inflammation (Shimaoka et al., 2000; Kraus et al., 2010), whereas the first and third trimester, parturition, and the postpartum are periods of proinflammation (Matthiesen et al., 1998; Simón et al., 1998; Keelan et al., 1999; Shimaoka et al., 2000; Hanna et al., 2006; Kraus et al., 2010; Christian and Porter, 2014; Simpson et al., 2016; Ziegler et al., 2018). During the first trimester, there is an influx of cytokines that suggests heightened immune activation in preparation for the successful implantation of the embryo (Simón et al., 1998). During the second trimester, the enhancement 15 of the maternal anti-inflammatory response system corresponds to the major fetal (Shimaoka et al., 2000) and placenta (Geirsson et al., 1985) development. During the third trimester, proinflammatory responses activate in preparation for the highly proinflammatory event, parturition (Keelan et al., 1999). In the human postpartum period, levels of proinflammatory cytokines either decrease, increase, or remain stable depending on the cytokine (Shimaoka et al., 2000; Kraus et al., 2010; Christian and Porter, 2014; Simpson et al., 2016). In a mouse study, Orsi and colleagues (2006) found a continued elevation of serum proinflammatory cytokines (interleukin (IL)-1beta(β), tumor necrosis factor-alpha (TNF-α), IL-12, IL-6, interferon-gamma (IFN-γ)) during gestation and the postpartum. In rats, the profile is different with serum levels of IL-10, IL-4, and TNF-α increasing during early postpartum compared to mid-gestation and IL-1β decreasing over this period (Eid et al., 2019). Both hippocampal levels of IL-6 and IL-10 were higher in early postpartum compared to virgin rats (Haim et al., 2017). Overall, maternal immune signalling undergoes significant change during pregnancy and the postpartum. In humans, elevated proinflammatory cytokines have been implicated in MDD and PND (Maes et al., 2000; Howren et al., 2009; Dowlati et al., 2010; Haapakoski et al., 2015; Simpson et al., 2016). Meta-analyses have found elevated levels of the cytokines, IL-6, TNF-α, and IL-1β are associated with MDD (reviewed in Howren et al., 2009; Dowlati et al., 2010; Haapakoski et al., 2015). TNF-α and IL-1β are mainly proinflammatory cytokines, whereas IL-6 is a pleiotropic cytokine with both pro- and anti-inflammatory properties (reviewed in Dinarello, 2000; Opal and DePalo, 2000). All three cytokines are also altered drastically in the perinatal period (Vassiliadis et al., 1998; Orsi et al., 2006; Kraus et al., 2010; Ziegler et al., 2018; Eid et al., 2019), and higher levels of all three are also associated with increased depression scores during the perinatal period (Maes et al., 2000; Corwin et al., 2008, 2015; Boufidou et al., 2009; Christian et al., 2009; 16 Cassidy-Bushrow et al., 2012; Simpson et al., 2016; Achtyes et al., 2019). Elevations in IL-6 and TNF-, also significantly increased the risk for PPD in the early postpartum period (Achtyes et al., 2019). In addition, IL-10, a predominantly anti-inflammatory cytokine, has also been associated with PND (Cassidy-Bushrow et al., 2012; Simpson et al., 2016; Bränn et al., 2017) with either an elevation during the second trimester (Cassidy-Bushrow et al., 2012) or a reduction in late pregnancy (Bränn et al., 2017). However, IFN-γ is reduced in PND in humans (Groer and Morgan, 2007). Furthermore, others have shown that IL-6 during pregnancy or at parturition can be a predictor of future PPD (Liu et al., 2016; Simpson et al., 2016). It should be noted that most of the evidence provided here, specifically in human studies, is based on circulating levels of cytokines, which may not reflect cytokine levels in brain (Gibb et al., 2011; Eid et al., 2020). However, there are suggestions of interaction between central and peripheral immune systems (reviewed by Louveau et al., 2015; Dantzer, 2018). Overall, data suggest a proinflammatory profile and fluctuating IL-10 levels throughout pregnancy are associated with PND. Certainly, the relationship between inflammation and the perinatal period is more complicated than we have described here (for a comprehensive review see Osborne and Monk (2013)). Nevertheless, there is evidence to suggest that inflammation plays a role in PND, but how it may impact antepartum versus postpartum depression onset remains to be determined. In Chapter 2, I investigate whether there are changes in peripheral inflammation and neuroinflammation in a model of de novo PPD. 1.4.3 Serotonergic tone and metabolism during pregnancy and the postpartum The serotonergic system is dynamically regulated throughout pregnancy and the postpartum period (reviewed in Lonstein, 2019; Pawluski et al., 2019). During rodent pregnancy and early postpartum, most studies find enhanced serotonin signalling, which is depending on the 17 brain region (Lonstein et al., 2003; Macbeth et al., 2008; Holschbach and Lonstein, 2017; see Lonstein, 2019; Pawluski et al 2019, for review). Study results can be mixed and this likely reflects different brain regions examined, different methods of measuring serotonin, and/or maternal behaviour, as lactation and/or exposure to pups can affect both brain and serum serotonin (Desan et al., 1988; Lonstein et al., 2003; Macbeth et al., 2008; Jury et al., 2015; Harding and Lonstein, 2016; Holschbach and Lonstein, 2017). Together, these studies indicate serotonin tone is influenced by several factors during pregnancy and in the postpartum. In humans, heightened tryptophan metabolism, the serotonin amino acid precursor, is seen in plasma and serum during pregnancy (Schröcksnadel et al., 2003; Lindsay et al., 2015), with a further elevation in the immediate postpartum period (1-3 days) up to 4-6 weeks postpartum, suggesting lower levels of serotonin in the perinatal period (Maes et al., 2002; Schröcksnadel et al., 2003; Kohl et al., 2005; Veen et al., 2016; Teshigawara et al., 2019). Intriguingly, only women with de novo PPD, but not antepartum depression or those with a history of MDD, had altered levels of tryptophan metabolites during pregnancy and the postpartum compared to healthy controls (Teshigawara et al., 2019). Lower peripheral concentrations of serotonin are observed in women with PPD (Aishwarya et al., 2013; Yildiz et al., 2017; Achtyes et al., 2019), and this reduction significantly increased the risk for PPD (Achtyes et al., 2019). These data suggest that women with de novo PPD may be particularly sensitive to heightened tryptophan metabolism during pregnancy and the postpartum (Kohl et al., 2005; Veen et al., 2016; Teshigawara et al., 2019). Overall, these changes in metabolism may lead to tryptophan depletion resulting in decreased serotonin synthesis, which may contribute towards the onset of PND symptoms (Maes et al., 2002; Kohl et al., 2005; Yildiz et al., 2017; Teshigawara et al., 2019). 18 Serotonergic tone in the brain and serum may be affected by levels of pregnancy-associated elevations in sex steroid hormones and decreases after parturition as there is a relationship between serotonergic signalling and sex hormones during the perinatal period (Pecins-Thompson et al., 1996; Berman et al., 2006; Frokjaer et al., 2015; Hu et al., 2019; reviewed in Barth et al., 2015). In euthymic women, the sudden withdrawal of sex steroid hormones may lead to a reduction in serotonin signalling in the brain (Pecins-Thompson et al., 1996; Berman et al., 2006; Frokjaer et al., 2015; Hu et al., 2019; reviewed in Barth et al., 2015). In addition, the genetic 5-HTTLPR polymorphisms in the serotonin transport gene (SLC6A4) may influence the effects of sex hormones and serotonin signalling on PND, as women with reduced estradiol and the SS 5-HTTLPR genotype had a higher risk for PPD than did women with the SL/LL 5-HTTLPR genotype (Hu et al., 2019). Various other factors influence serotonin signalling during the perinatal period, such as diet, metabolism, and the dynamic immune system during pregnancy and postpartum (reviewed by Murthi and Vaillancourt, 2020). Further research is needed to determine the direct role of the serotonin fluctuations on the risk for PND. It is possible, however, that reduced serotonergic tone in the dorsal raphe, the primary location for serotonin synthesis (Huang et al., 2019), in the postpartum (Holschbach and Lonstein, 2017) could contribute to lower efficacy of SSRIs in the postpartum in at risk populations (Navarre et al., 2010; Pawluski et al., 2012; Workman et al., 2016; Gobinath et al., 2018a). Together, data suggest serotonin signalling and precursor tryptophan metabolism are altered by pregnancy and postpartum and women with PND may be particularly sensitive to changes and fluctuating levels of serotonin and tryptophan metabolism. In Chapters 2 and 3, I investigate changes in tryptophan metabolism in postpartum dams in an animal model of de novo PPD. 19 1.4.4 Neuroplasticity during pregnancy and postpartum Many areas of the maternal brain undergo morphological changes during pregnancy and the postpartum period, most notably those brain areas involved in maternal behaviour undergo structural and functional changes during pregnancy and in the postpartum (Lorberbaum et al., 2002; Strathearn et al., 2009; Kim et al., 2011; reviewed by Hillerer et al., 2014). However, overall reductions in total brain volume (~6%) are also seen across pregnancy to childbirth that are restored by 6 months postpartum (Oatridge et al., 2002). In addition, specific regions of the brain (hippocampus, medial frontal cortices, prefrontal cortices, bilateral temporal cortices) show reduced gray matter volume during pregnancy that persists at least 2 years postpartum in first-time mothers compared to pre-conception (Hoekzema et al., 2017). Moreover, Hoekzema and colleagues (2017) showed that reductions in brain volume are highly predictive of increased maternal attachment to infants, indicating that these volume changes are related to the transition into motherhood and may function to mediate maternal care behaviours. Looking at a shorter time span, an increase in gray matter and cortical thickness is seen in several brain regions including the prefrontal cortex from parturition across the first 3 months postpartum (Kim et al., 2010; Kim et al., 2018). These increases are also related to positive thoughts on parenting. Perhaps not surprisingly, the pituitary increases in size during pregnancy and return to pre-pregnancy size after the first week postpartum in humans (Gonzalez et al., 1988; Elster et al., 1991; Dinç et al., 1998; Miki et al., 2005). These data show several changes in the brain from pregnancy to the postpartum. Notably, gray matter reductions in brain areas that are implicated in the neurobiology of depression, such as the hippocampus and prefrontal cortex, are seen across pregnancy to the early postpartum (reviewed in Lima-Ojeda et al., 2018) and are partially reversed in the later postpartum (Kim et al., 2010; Hoekzema et al., 2017; Kim et al., 2018). 20 Reductions to neuroplasticity in the hippocampus during pregnancy and postpartum are seen in rodents (Shingo et al., 2003; Furuta and Bridges, 2005; Darnaudéry et al., 2007; Leuner et al., 2007; Pawluski and Galea, 2007; Rolls et al., 2008; Pawluski et al., 2010; Leuner and Sabihi, 2016; Eid et al., 2019). Primiparous rats show reduced survival of new neurons in the hippocampus from mid-gestation across the postpartum period (Eid et al., 2019; Pawluski and Galea, 2007). Reduced cell proliferation (the production of new cells) in the dentate gyrus of the hippocampus is seen in mid, but not in early, gestation and in early, but not late, postpartum period compared with nulliparous rats (Shingo et al., 2003; Furuta and Bridges, 2005; Darnaudéry et al., 2007; Leuner et al., 2007; Pawluski and Galea, 2007; Rolls et al., 2008; Pawluski et al., 2010; Leuner and Sabihi, 2016; Eid et al., 2019). The reduced levels of cell proliferation and neurogenesis across the postpartum are due to pregnancy alone (Pawluski and Galea, 2007) and are mediated by adrenal steroids (Leuner et al., 2007). Furthermore, withdrawal from a hormone simulated pregnancy reduced cell proliferation in the hippocampus (Green and Galea, 2008), suggesting that it is the hormonal fluctuations across parturition that initiate the reductions in neurogenesis in the early postpartum. Thelectomized females (inability to nurse) had increased neurogenesis compared to sham and “foster” moms (De Guzman et al., 2018), suggesting that nursing, perhaps via oxytocin, decreases neurogenesis in dams. Experience with mothering via “fostering” (nulliparous rats exposed to pups) showed increased survival of new neurons and cell proliferation in the hippocampus compared to primiparous or nulliparous rats (Pawluski and Galea, 2007). These data suggest that pregnancy and maternal experience differentially influence neurogenesis in the hippocampus. Other brain regions also show reduced plasticity as prolactin cells of the pituitary are reduced on postpartum day (PD) 7 (Haggi et al., 1986) and cell survival is reduced in the dorsal 21 raphe in the postpartum (Holschbach and Lonstein, 2017). Unlike the hippocampus, the subventricular zone, another site of continual neurogenesis in rodents, is associated with increased neurogenesis during pregnancy (Shingo et al., 2003). Overall, these data provide evidence that the brain undergoes several changes in response to pregnancy and the postpartum. Many of the brain regions affected by pregnancy and the postpartum also undergo substantial adaptations with depression that may underscore the greater susceptibility to develop psychiatric illness in the early postpartum (O’Hara and Swain, 1996; O’Hara and McCabe, 2013). 1.5 Rodent models of postpartum depression In both the human and rodent, specifically rats and mice, pregnancy, various hormones, such as glucocorticoids and ovarian hormones, follow a similar pattern of rising throughout pregnancy and drop in the immediate postpartum period (reviewed by Pawluski et al., 2009; Duarte-Guterman et al., 2019). However, there are differences between species, specifically regarding the regulation, production, and fluctuation of progesterone (reviewed in Bonney, 2013). These differences include the drop in progesterone prior to the onset of parturition in rats and mice that is not seen in humans (reviewed by Pawluski et al., 2009; Ratajczak et al., 2010), and difference sources (ovarian versus placental) of progesterone production between rodents and humans respectively (reviewed by Ratajczak et al., 2010). Additionally, placental structures differ between species, although differences are argued to be insufficient to negate using rodent models (Clark, 2014). Moreover, another major difference between human and rats/mice is that rodent pups are born “prematurely” relative to human infants, such that the first two weeks postpartum in rats and mice are developmentally equal to the third trimester of human fetal development (reviewed by Rice and Barone, 2000). Nevertheless, human and rat/mice 22 pregnancies are similar enough between species to allow comparisons between clinical and preclinical studies in dams, and in offspring, rodent models may even pose an advantage over human studies due to the early birth of offspring (discussed below in section 1.7). Figure 1.2. Schematic comparison of brain and hippocampal development across the perinatal period between humans and rats. 1Reviewed by Kilia et al., 2013; 2Reviewed by Klintsova et al., 2012; 3Reviewed by Bayer et al., 1993. Critical developmental window indicated by pink arrow. Period of perinatal brain growth spurt indicated by solid lines for both humans and rats. Perinatal hippocampal dentate development indicated by dashed lines, with arrow indicating continued development for both human and rats. Inset figures (neonatal pup, developing fetus in utero) provided by BioRender.com. 1.5.1 Gestational stress Data from a large European population epidemiological study determined that stress precedes approximately 90% of depression cases (Angst et al., 2002). To simulate this relationship between stress and depression in animal models, researchers have employed many 23 different methods of inducing stress to induce depressive-like behaviour in rodents (Wainwright and Galea, 2013). To model PND, different protocols have utilized psychosocial and/or physical stress during gestation. Gestational stress paradigms have used either restraint stress, overcrowding stress, or inescapable swim stress to induce depressive-like behaviour (Darnaudéry et al., 2004; Smith et al., 2004; O’Mahony et al., 2006; Hillerer et al., 2011; Pawluski et al., 2011; Jodi L. Pawluski et al., 2012; Haim et al., 2014, 2016; Leuner et al., 2014; Posillico and Schwarz, 2016). Typically, stress protocols are employed during mid (gestational day (GD) 11-17) or mid to late gestation (GD7-21), and these time differences have significant impacts on outcome variables. Furthermore, the length of the stressor varies per protocol with restraint stress typically lasting for 1-2.5 hours per day, whereas inescapable swim stress may be employed as short as 5-40 minutes per day (Leuner et al., 2014; Posillico and Schwarz, 2016). One limitation of daily repeated stress protocols is the potential for animals to habituate to the stressor (Grissom and Bhatnagar, 2009). Due, in part, to habituation concerns, Haim and colleagues (2016) used variable restraint stress, where the duration and number of sessions of restraint stress were randomized daily from GD 7 to 20. Longer paradigms (GD7-20) induce depressive-like behaviour (immobility in the forced swim test (FST)) in the early (Smith et al., 2004; Haim et al., 2014) and the late postpartum period prior to weaning (PD21-24; O’Mahony et al., 2006; Haim et al., 2014; Haim et al., 2016; Leuner et al., 2014). Shorter stress paradigms confined to either early (GD4-16), mid (GD11-17), or last week of gestation do not alter depressive-like behaviour in late gestation (Pawluski et al., 2011), early postpartum (Hillerer et al., 2011; Posillico and Schwarz, 2016), or late postpartum (Darnaudéry et al., 2004). Reduced maternal care is also found after repeated gestational stress (Leuner et al., 2014; Smith et al., 2004), demonstrating 24 another aspect of PND. Overall, longer daily gestational restraint stress and inescapable swim stress reliably increase depressive-like behaviour in the postpartum period. Gestational stress paradigms result in increased anxiety-like behaviour but few studies have seen changes in hippocampal plasticity in the postpartum (Hillerer et al., 2011; Darnaudéry et al., 2004; Pawluski et al., 2011; 2012). Increased anxiety-like behaviour is seen during early postpartum (PD8) (Hillerer et al., 2011) and in late postpartum (PD26) after gestational stress (Darnaudéry et al., 2004; but see Leuner et al., 2014). However, gestational stress paradigms do not reduce neurogenesis in the hippocampus (Pawluski et al., 2011; 2012) unlike what is seen in other chronic stress paradigms (Gobinath et al., 2015). Instead, Pawluski and colleagues (2011) found increased hippocampal cell proliferation in response to chronic restraint stress in mid-gestation (GD11-17). It is possible that a longer or different gestational stress paradigm may elicit different effects on hippocampal neurogenesis. Interestingly, reductions in frontal cortex spine density are evident due to gestational stress (Leuner et al., 2014; Haim et al., 2014; 2016), suggesting that the frontal cortex may be more sensitive to gestational stress than the hippocampus (for a more in-depth review of the effects of perinatal stress on the frontal cortex see Kolb et al., 2017). Overall, models of gestational stress effectively induce PND-like symptoms with co-morbid anxiety, reduced frontal cortex integrity, but not reduced hippocampal integrity, in rodent dams during both the early and late postpartum period. 1.5.2 Postpartum stress manipulations Stress exposure during the early-mid postpartum period is also used to induce depressive-like behaviour in rodent dams. Common stressors include maternal separation and limited bedding stress, but it is important to note that both the dam and the offspring are subjected to the stressor in these models (Brunson et al., 2005; Champagne and Meaney, 2006; Ivy et al., 2008; 25 reviewed in Alves et al., 2019). Indeed, models of maternal separation and limited bedding stress are often used as models of early-life adversity in offspring, and these models do reduce maternal care behaviour, which has significant implications for offspring development (Brunson et al., 2005; Champagne and Meaney, 2006; Ivy et al., 2008; reviewed in Alves et al., 2019). In addition, these models increase maternal depressive-like behaviour during the mid-postpartum (PD16) (Murgatroyd et al., 2015) and late postpartum (PD22-26) (Boccia et al., 2007; Maniam and Morris, 2010; Sung et al., 2010). Maternal separation stress, however, increases depressive-like behaviour only when dams are stressed during the early-to-mid postpartum period (PD1-14) (Boccia et al., 2007; Maniam and Morris, 2010; Sung et al., 2010) but not during the entire postpartum period (PD1-21) (Aguggia et al., 2013). Maternal separation also increases anxiety-like behaviour in dams tested in the late postpartum (PD21) (Aguggia et al., 2013; Maniam and Morris, 2010). In the hippocampus, cell proliferation was reduced in dams that underwent daily 6 h maternal separation from PD1-PD14 (Sung et al., 2010), but few studies have investigated this. Thus, several stressors can be used to induce depressive-like behaviour in the postpartum in rats, but the effects of these stressors may be dependent on what point of the postpartum period they are implemented. 1.5.3 Postpartum corticosterone model In the protocols described above, the stressors affect the offspring in addition to the dams. Although this is a reliable method of early-life adversity in offspring, it may confound the effects on the dams, as offspring vocalizations will change the elicited maternal care behaviours from the dams (Walker et al., 2003). Thus, our laboratory created another model of PND, in which rodent dams are exposed to daily corticosterone (CORT) injections during the entire postpartum period (Brummelte et al., 2006). This method results in elevated CORT levels in dams 26 (Brummelte et al., 2010; Kott et al., 2018), which mirrors elevated levels of cortisol seen in mothers with PPD (Iliadis et al., 2015; Osborne et al., 2018). Particularly, this model captures the elevated postpartum cortisol seen in those women with symptoms only in the postpartum period (Iliadis et al., 2015). Thus, with manipulations only in the postpartum, this method can model those individuals with de novo PPD, where symptoms onset is only in the postpartum period without directly affecting offspring. In the postpartum CORT model, we see increased depressive-like behaviours in the late postpartum period and disrupted maternal care behaviour early in the postpartum (Brummelte et al., 2006; Brummelte and Galea, 2010; Workman et al., 2016; Gobinath et al., 2018a). However, maternal postpartum CORT does not induce anxiety-like behaviour in rodent dams (Brummelte et al., 2006; Brummelte and Galea, 2010; Gobinath et al., 2018a; Overgaard et al., 2018). Unlike the gestational stress paradigms, neurogenesis in the hippocampus is reduced with postpartum CORT (Brummelte and Galea, 2010; Workman et al., 2016; Gobinath et al., 2018a), even below those seen in nulliparous animals given CORT (Workman et al., 2016), indicating the integrity of the hippocampus is affected by postpartum CORT. Interestingly, maternal CORT exposure throughout the perinatal period or during pregnancy alone elicited different phenotypes than when CORT was administered during the postpartum only (Brummelte and Galea, 2010). When high maternal CORT was given during both the last 10 days of gestation and throughout the postpartum period or during pregnancy alone, both these models disrupted maternal care behaviour and reduced hippocampal integrity. However, at neither time period did prolonged maternal CORT result in an increase in passive coping behaviour, unlike what was seen when CORT was administered in the postpartum period only (Brummelte and Galea, 2010). This suggests the possibility that maternal CORT may be most effective at inducing passive coping specifically during the postpartum period, and that 27 CORT exposure does not result in uniform changes to endophenotypes but depends on the time of exposure during the perinatal period. Overall, different models of postpartum manipulations can be used to model PND. 1.5.4 Interventions in animal models Studies have investigated the effects of SSRIs in rodent models of PND with variable effects on efficacy related to the paradigm (Pawluski et al., 2012; Gemmel et al., 2016; Haim et al., 2016a; Workman et al., 2016; Gobinath et al., 2018a; Overgaard et al., 2018). Briefly, gestational stress models using SSRIs have broader efficacy than postpartum manipulations. We will discuss these studies below. Chronic exposure to the SSRI citalopram during the postpartum reversed the increase in depressive-like behaviour and structural changes in the basolateral amygdala and nucleus accumbens following gestational stress (Haim et al., 2016). Furthermore, 4 weeks of the SSRI fluoxetine (GD15-PD21) reversed some gestational stress effects including disrupted maternal care behaviour, depressive-like behaviours, HPA hyperactivity, increased tryptophan metabolism, and synaptophysin in the maternal hippocampus (Gemmel et al., 2016; Kiryanova et al., 2016; Salari et al., 2016). Additionally, chronic fluoxetine treatment during the postpartum increased the number of immature neurons in the maternal hippocampus after gestational stress, however, gestational stress alone did not reduce neurogenesis in the hippocampus (Pawluski et al., 2012). Together these data indicate the efficacy of the SSRIs citalopram and fluoxetine on depressive-like behaviours is altered by gestational stress. In postpartum models of PND, the efficacy of SSRIs is limited. The SSRIs fluoxetine and paroxetine did not reduce depressive-like behaviour in the late postpartum or reverse reductions in maternal hippocampal neurogenesis due to maternal CORT treatment (Workman et al., 2016; 28 Gobinath et al., 2018a; Overgaard et al., 2018;). However, SSRI treatment reversed disrupted maternal care behaviour in the early postpartum period using the postpartum CORT model (Workman et al., 2016, Gobinath et al., 2018a). Intriguingly, fluoxetine is effective in increasing neurogenesis in females outside of the postpartum period (Mahmoud et al., 2016; Workman et al., 2016), and as noted earlier, fluoxetine in conjunction with maternal exercise can increase neurogenesis in the postpartum (Gobinath et al., 2018a). In Chapter 2 and 3, I will investigate factors that may relate to limited efficacy in the postpartum, which in short, may be associated with SSRI-induced changes in the maternal central and peripheral immune profiles, and the tryptophan-kynurenine metabolic pathway. However, it is important to note that in most animal studies a single dose of an SSRI is used, and it is possible that other doses may be more effective. Nevertheless, the typical dose used in all of these studies was higher than clinically relevant dosage (5mg/kg paroxetine or 10 mg/kg fluoxetine versus 1.33 mg/kg in humans; Latendresse et al., 2017). Thus, it is unlikely that higher dose contributes to the limited efficacy of SSRI in these studies. I am particularly interested in fluoxetine because it has remained one of the top three SSRIs prescribed to treat depression in the United States, at least during 1996-2015 (Luo et al., 2020) and can appear in breast milk at higher concentrations compared to other SSRIs (Berle and Spigset, 2011). The effects of maternal SSRI treatment on children are discussed below (section 1.7). Interestingly, in a novel animal model of previous depression and continuous antidepressant exposure from preconception to gestation, the SSRI, sertraline, was not effective at reducing depressive-like behaviour in female rats (Kott et al., 2018). This model recapitulates the subset of people who present with treated MDD preconception but choose to terminate SSRI treatment prior to or during pregnancy (Kott et al., 2018). Specifically, it was found that females 29 who were treated with CORT prior to gestation and who continued sertraline treatment throughout gestation displayed depressive-like endophenotypes (reduced maternal care behaviour, increased passive coping) compared to females treated with a vehicle as well as females that discontinued sertraline treatment mid-gestation (GD16) (Kott et al., 2018). Thus, in this model, continuous sertraline treatment through the perinatal period did not confer resilience and displayed even less efficacy to treat depressive-like endophenotypes in the postpartum. Overall, these data suggest that SSRIs on their own may not be as effective to alleviate depressive-like behaviours or neurogenesis in the hippocampus in the postpartum, which depends in part on the model of PND used (gestational stress or postpartum CORT). To understand some of the biological mechanism as to why SSRI show limited efficacy in the late postpartum, in Chapter 2 and 3, I examine inflammation and TKP in the late postpartum in dams. 1.6 Untreated maternal perinatal depression on offspring Untreated maternal depression can have severe short-term and long-term consequences in children that stem from infancy and persist into adulthood (reviewed by Goodman, 2019). Children of mothers with PND show impaired cognitive, social, and emotional development, which can result in higher risks for depression and anxiety disorders, conduct disorders, antisocial behaviour, and attention deficit hyperactive disorder (Goodman, 2019). These consequences on behaviour due to maternal depression are not moderated by duration and severity of maternal depression alone (reviewed by Goodman et al., 2011; Stein et al., 2014; Goodman, 2019; Goodman et al., 2020). The increased risk for behavioural changes due to PND in children can depend on genetic, epigenetic, HPA modulation, placental effects, or more specifically for PPD, reduced maternal sensitivity/attachment, parenting, or nutritional 30 status/transfer of stress hormones or other biomarkers via breast milk (Di Benedetto et al., 2020; Goodman et al., 2020). Maternal antidepressant treatment, coupled with maternal depression, can also modulate outcomes in children via effects on the mother or indirect effects in utero or via breastmilk (reviewed by Rotem‐Kohavi and Oberlander, 2017), these effects are discussed below. 1.6.1 Evidence from preclinical animal models of gestational stress or postpartum CORT on offspring development As discussed above in section 1.5.2, various animal models utilizing stress, such as maternal separation, in the postpartum, induce maternal depressive-like endophenotypes in the dam, can also directly affect the offspring (Champagne and Meaney, 2006; reviewed in Alves et al., 2019). Thus, postpartum stress, such as maternal separation, directly impact offspring and in fact are used as models of early-life adversity, and not solely PND. Therefore, I turn my focus to animal models of PND where only dams are manipulated. In order to model behavioural outcomes (higher risk for depression and anxiety disorders) in children, studies have examined depressive-like endophenotypes in offspring from animal models of PND. In the gestational stress model, variable effects have been reported for behaviour and inflammatory markers in adult offspring (Ehrlich and Rainnie, 2015; Salari et al., 2016; Zohar et al., 2016; Gatta et al., 2018; He et al., 2018; Gur et al., 2019; Liao et al., 2019; reviewed by Weinstock, 2017; Enayati et al., 2020; Creutzberg et al., 2021). Variable effects on depressive-like behaviour and inflammation depend on the protocol of gestational stress, strain/species of animal, and age of offspring when tested (reviewed by Weinstock, 2017; Enavati et al., 2020). However, in general, there are more studies showing increased anxiety-like behaviour in adult offspring, dysfunction in the HPA-axis (reviewed by Weinstock, 2017; 31 Creutberg et al., 2021), decreased social investigation (de Souza et al., 2013; Gur et al., 2019), and reduced neurogenesis in the hippocampus (Rayen et al., 2015) with gestational stress. In the postpartum CORT model of de novo PPD, there are also variable outcomes in anxiety-like behaviour and adult hippocampal neurogenesis, but consistent effects show HPA dysregulation in offspring (Gobinath et al., 2016, 2018b). Maternal postpartum CORT consistently increases HPA negative feedback in male and female adult offspring (Gobinath et al., 2016, 2018b). Moreover, in adulthood, studies find decreased dorsal hippocampal neurogenesis in females with maternal postpartum CORT, which was not seen in males (Gobinath et al., 2016, 2017), but see (Gobinath et al., 2018b). More variable effects are seen with maternal postpartum CORT on anxiety-like behaviour with one study showing an increase (Brummelte et al., 2006), but two studies showing no significant effects on anxiety-like behaviour (Gobinath et al., 2016, 2018b) in both male and female adult offspring. Together, different animal models of PND that focus on treatment to the dams only, can lead to long-term endocrine and neurological outcomes in adult offspring. In the postpartum CORT model, however, offspring social behaviour and inflammation have not been explored. In Chapter 4, using this model, offspring anxiety-like behaviour, social behaviour, hippocampal neurogenesis and neuroinflammation will be investigated in adulthood (also discussed in sections 1.7, 1.8). 1.7 The impact of treating maternal perinatal depression with pharmacological antidepressants on children Perinatal SSRI exposure has variable effects in children (reviewed by Rotem-Kohavi and Oberlander, 2017). Short-term behavioural effects in the postnatal period include poor neonatal 32 adaptation syndrome, which include respiratory distress, temperature instability, feeding difficulties, irritability, sleep problems, tremors (Olivier et al., 2015; reviewed by Rotem-Kohavi and Oberlander, 2017), and altered pain and stress responses (Oberlander et al., 2005, 2002). Behavioural effects can extend into later life, where poor neonatal adaption predict later life social difficulties (Klinger et al., 2011). It is important to note that perinatal SSRI exposure does not always have negative effects in offspring. Specifically, prenatal exposure to SSRIs is associated with accelerated language development (Weikum et al., 2012), increased readiness to interact (Weikum et al., 2013), and increased infant functional synchronicity and hyperconnectivity in auditory resting-state networks (Rotem-Kohavi et al., 2019). Clinical data suggests an increased risk for autism spectrum disorder (ASD) in children of PND mothers treated with SSRIs (Croen et al., 2011; Rai et al., 2013; Andalib et al., 2017). However, these findings are controversial as not all studies see this increased risk for ASD with SSRI exposure (reviewed by Rotem-Kohavi and Oberlander, 2017; Rommel et al., 2020). Thus, the literature on perinatal exposure to SSRIs suggests both positive and negative effects on child development. It can be difficult to disentangle the effects of maternal depression and exposure to SSRIs in children (Oberlander and Zwaigenbaum, 2017) as it is unethical to investigate the independent effects of perinatal SSRI exposure without the onset of maternal depression, nor is it ethical to withhold PND treatment. Animal models give a substantial advantage over human studies because it does allow researchers to independently manipulate variables. Moreover, the early postnatal period in rodents is roughly equivalent to the human third trimester, specifically in terms of the development of the serotonergic system (Suri et al., 2015), as well as the development of the neuroendocrine and other neural systems (Matthews, 2002) see Figure 1.2. By investigating in rodent animal models ex utero, it eliminates modulatory effects of the 33 placenta on offspring neurobehavioural development (Hsiao and Patterson, 2012), while still investigating the critical developmental period (third trimester in humans/early postnatal in rodents; Figure 1.2). Thus, in this thesis, in Chapter 4, I investigate the effects of postnatal SSRI treatment to the dams on offspring. 1.7.1 Evidence from preclinical studies on effects of maternal SSRI exposure on offspring behavioural outcomes Given that there is clinical evidence suggesting an increased risk for ASD due to perinatal SSRIs (Croen et al., 2011; Rai et al., 2013; Andalib et al., 2017), researchers have investigated the effects of SSRI exposure on social behaviour, a core phenotype of ASD (American Psychiatric Association, 2013), and anxiety-like behaviour, due to its high comorbidity rate in individuals with ASD (van Steensel et al., 2011). Here, again mirroring the clinical literature (reviewed by Rotem-Kohavi and Oberlander, 2017; Rommel et al., 2020), there is variability in outcomes in animal models. In animal models of gestational stress, SSRI treatment to dams during the perinatal period has variable effects on depressive-like, anxiety-like, and social behaviour in adult offspring depending on sex (Bourke et al., 2013; Ehrlich et al., 2015; Boulle et al., 2016a, 2016b; Kiryanova et al., 2016, 2017; reviewed by Ramsteijn et al., 2020). The variability in the gestational stress model may be due to the timing of exposure to SSRIs in offspring (in utero, ex utero, via breast milk), as studies are not consistent as to when SSRIs treatments are given to dams (gestation, postpartum) (Bourke et al., 2013; Ehrlich et al., 2015; Boulle et al., 2016a, 2016b; Kiryanova et al., 2016, 2017; reviewed by Ramsteijn et al., 2020). In the postpartum CORT model, SSRI treatment is only given to the dams in the postpartum period (Workman et al., 2016; Gobinath et al., 2018a; Qiu et al., 2020a, Chapter 2). In this model, maternal 34 postpartum FLX increased anxiety-like behaviour in males only (Gobinath et al., 2016) or has no significant effect on anxiety-like behaviour (Gobinath et al., 2018b). In these studies, external factors such as test order and number of multiple behavioural tests, leading to overall environmental chaos could have contributed towards variable effects in offspring (Gobinath et al., 2016, 2018b), similar to clinical literature (Dhaliwal et al., 2017, 2020). Indeed, in SSRI-exposed children, executive function abilities differ by chaotic environment defined as high noise, high commotion, low regular routine (Dhaliwal et al., 2017, 2020). In the postpartum CORT model, in experimental designs with fewer tests in offspring, there are no significant effects of maternal SSRIs on adult offspring anxiety-like behaviour (Gobinath et al., 2018b). However, in designs where a multitude of various tests in offspring were conducted, maternal SSRIs led to an increase in anxiety-like behaviour in adult male offspring, indicating a chaotic/stressful environment may contribute to this phenotype (Gobinath et al., 2016). Collectively, regardless of animal model of PND, maternal SSRI treatment can alter outcomes in offspring behaviour. Interestingly, perinatal FLX may normalize maternal gestational stress effects in male offspring in outcomes such as depressive-like and anxiety-like behaviour (Boulle et al., 2016a; Salari et al., 2016). These effects of maternal postpartum FLX are not seen following postpartum CORT (Gobinath et al., 2016, 2018b). In Salari et al., (2016), maternal FLX treatment was given during both gestation and postpartum, whereas in Gobinath et al., (2016, 2018b), FLX were given in the postpartum period only. Thus, differences may lie within the route of exposure of SSRI to the offspring either in utero exposure versus ex utero via breast milk. Together, data suggest that although some benefits of maternal SSRI treatment has been found in offspring, these effects are inconsistent across models and may have to do with the timing of exposure. 35 1.7.2 Evidence from preclinical studies on effects of maternal SSRI exposure on offspring physiological outcomes Serotonin is a significant modulator of neurogenesis (reviewed by Alenina and Klempin, 2015), such that chronic FLX treatment increases hippocampal neurogenesis in naïve adult males (Malberg et al., 2000; Santarelli et al., 2003). Given this relationship, adult hippocampal neurogenesis outcomes in offspring due to maternal SSRI treatment is also of particular interest in animal models. In a model of gestational stress, postpartum FLX with gestational stress decreased adult neurogenesis compared to postpartum FLX alone in both sexes (Rayen et al., 2015). In the postpartum CORT model, adult hippocampal neurogenesis is decreased in both males and females due to postpartum FLX (Gobinath et al., 2018b), although this can be more variable in males (Gobinath et al., 2016). Together, perinatal SSRI exposure can lead to sex-specific effects on adult hippocampal neurogenesis depending on timing of exposure (gestation-postpartum, postpartum alone) and animal model of PND. Individuals with ASD show neuroinflammation (reviewed Matta et al., 2019; Liao et al., 2020), reduced hippocampal connectivity during memory tasks (Cooper et al., 2017), and hippocampal cell size but increased cell density (Bauman and Kemper, 2005). Although, hippocampal volume data have been mixed, nevertheless, changes in the hippocampus have been identified as a possible biomarker for ASD (reviewed by Pagnozzi et al., 2018; Li et al., 2019). Due to these associations, I explore the outcomes of maternal postpartum SSRI treatment in the postpartum CORT model on offspring hippocampal neurogenesis, hippocampal neuroinflammation, social behaviour, and anxiety-like behaviour in adulthood. In addition, therapeutics should be investigated not only in mothers to improve outcomes, but also in children to reverse maternal effects. In chapter 4, I investigate periadolescent oxytocin as one such 36 possible therapeutic to reverse maternal treatment effects in both male and female offspring to further understand sex differences in offspring due to maternal SSRI treatment in an animal model of de novo PPD. It should be noted that here, I have only discussed specific outcomes in adulthood as I am interested primarily in the long-term effects on adult offspring from maternal treatments. But it is important to understand that there are maternal treatments that may affect neonatal or juvenile offspring differently (for review see Gemmel et al., 2018; Hutchison et al., 2020). 1.8 Oxytocin as a potential remedy in offspring Oxytocin (OT) is a large neuropeptide that is released from the magnocellular cells in the hypothalamus and is linked to attachment behaviours (Insel, 1992). OT functions to modulate a variety of effects, notably in mothers, OT release aids in uterine contractions in preparation for parturition (Uvnäs-Moberg et al., 2019). During the early postpartum period, OT also functions to increase maternal-to-infant prosocial interactions in humans (Szymanska et al., 2017), and other related maternal care behaviours in rodents (Okabe et al., 2017). Further, in rodents, OT modulates adult hippocampal neurogenesis (Leuner et al., 2012; Duarte-Guterman et al., 2020), inflammation (Yuan et al., 2016; An et al., 2019), anxiety-like behaviour (Yoshida et al., 2009; reviewed by Neumann and Slattery, 2016), and social behaviour (reviewed by Neumann, 2008). OT has also been considered as a potential candidate as a therapeutic for maternal depression (Kim et al., 2014). In neonates, the early expression of OT receptors may indicate that early life OT signalling and circuitry has organization effects on the brain during early development (Miller and Caldwell, 2015). Additionally, mutations in OT peptide structure and receptor 37 polymorphisms have been associated with the developmental disorder, ASD (Jacob et al., 2007). These findings, together with the prosocial effects of OT, suggest OT may be a good candidate as a possible remedy for individuals with ASD (Huang et al., 2021). Indeed, based on one recent meta-analysis, intranasal OT improves social functioning in individuals with ASD (Huang et al., 2021). However, it is noted that the effects from clinical studies are variable based on differences in dose, a high percentage of male participants suggesting a sex bias in the effects of OT in humans, and the heterogeneity of the disorder (Huang et al., 2021). Sex differences are seen in the effects of OT in rat as peripheral OT treatment increases social behaviour and OT-positive expressing cells in adult males but not females (Duarte-Guterman et al., 2020). Further, a single OT administration in the posterior bed nucleus of the stria terminalis increases social behaviour in adult males and not females (Dumais et al., 2016). Unfortunately, there are minimal comprehensive analyses of sex differences in OT mediated outcomes (Dumais and Veenema, 2016), thus it is important to do a thorough investigation of the effect of OT by sex. Interestingly, in animal models of gestational stress, maternal stress reduces hippocampal OT gene expression and OT-positive magnocellular hypothalamic cells in adult male offspring depending on region (de Souza et al., 2013; Gatta et al., 2018), and in the hypothalamus in adult female vole offspring (He et al., 2018). Moreover, gestational stress alters OT receptor gene expression dependent on brain region, with increased expression in adult male hippocampus (Gatta et al., 2018) and decreased expression in cortex (Gur et al., 2019). Adolescent stress also decreases OT receptor expression in the paraventricular nucleus of the hypothalamus in adult males, but not in adult females (Asgari and McCormick, in press). Currently, there are no studies investigating the effects of postpartum CORT nor perinatal SSRI exposure in animal models of 38 PND on offspring OT circuitry. Given that maternal CORT and FLX influences many of the same outcomes that OT influences (hippocampal neurogenesis, anxiety-like behaviours), we were curious if this could be a possible therapeutic to offspring that had been exposed to maternal postpartum CORT and/or FLX. OT however is a large hydrophilic neuropeptide that when given peripherally, does not readily cross the blood-brain-barrier (Mens et al., 1983). Additionally, OT has a short half-life of 3-9 min in blood (Churchland and Winkielman, 2012). Here, we introduced a novel nanomedicine delivery platform, TiozanTM (Ovensa Inc) to prolong OT half-life and facilitate it across the blood-brain-barrier for central delivery (Ovensa Inc., 2020). Thus, Chapter 4 investigates the effects of OT versus OT with TriozanTM, as a possible remedy in offspring to negate the effects of maternal treatment. 1.9 Thesis overview and objectives The overarching goal of this thesis is to understand possible mechanism(s) underlying the efficacy of FLX in dams and safety of maternal postpartum FLX treatment in the offspring, as well as a possible remedy in offspring to negate any maternal treatment effects in an animal model of de novo PPD. The following experimental chapters outline studies investigating the effects of maternal postpartum antidepressant (FLX) treatment in an animal model of de novo PPD (postpartum CORT) on behaviour, immune signalling, and neuroplasticity in dams and female and male offspring. Chapter 2: Are there changes in neuroimmune profile and tryptophan metabolism in an animal model of de novo PPD and do they coincide with SSRI efficacy? I hypothesize that maternal postpartum CORT treatment will induce depressive-like endophenotypes (reduced 39 maternal care behaviour in the early postpartum, increased passive coping behaviour in the late postpartum). Moreover, postpartum FLX will have limited efficacy to reverse CORT effects on passive coping behaviour in the late postpartum, which will be associated with changes in maternal hippocampal, frontal cortex, and circulating inflammatory markers and vitamin B6-dependent tryptophan metabolism. Chapter 3: Are changes in the tryptophan-kynurenine pathway in an animal model of de novo PPD and do they coincide with SSRI efficacy? I hypothesize maternal postpartum CORT will induce a depressive-like phenotypes in the TKP including elevations in putative neurotoxic metabolites and putative neurotoxicity in the tryptophan-kynurenine metabolic pathway and postpartum FLX will have limited efficacy to reverse CORT-induced effects. Chapter 4: Do maternal postpartum CORT and/or FLX influence neuroimmune profile, neurogenesis, and behaviour in adult offspring and can offspring periadolescent OT offset changes due to maternal treatment? I hypothesize maternal postpartum CORT and FLX treatment will alter offspring puberty development, adult social and anxiety-like behaviours, hippocampal neurogenesis and inflammation in adult male and female offspring. Moreover, periadolescent OT treatment will show potential to reverse at least some of the maternal treatment effects from CORT or FLX in offspring, in a sex-dependent manner. 40 Chapter 2: Postpartum fluoxetine increased maternal inflammatory signalling and decreased tryptophan metabolism: Clues for efficacy 2.1 Introduction Perinatal depression (PND) affects approximately 15% of women within the general population (O’hara and Swain, 1996; Shorey et al., 2018), and depression onset can occur de novo during pregnancy or in the postpartum. Untreated PND can have severe and long-lasting effects on the mother and her children (Beck, 1999; Grace et al., 2003; Weissman et al., 2006). However, the efficacy and safety of maternal antidepressant use during pregnancy and/or postpartum has been challenged in both humans and animal models (Oberlander et al., 2009; Sharp et al., 2010; De Crescenzo et al., 2014; Workman et al., 2016; Suri et al., 2017; Gobinath et al., 2018a). Thus, it is important to determine physiological factors during the perinatal period that may contribute to reduced drug efficacy during the perinatal period. One of the strongest predictors of PND is stressful life events (Robertson et al., 2004). Additionally, women with PND show dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis (Jolley et al., 2007; Glynn et al., 2013; Osborne et al., 2018) including higher evening salivary cortisol, the main stress hormone, when compared to healthy controls (Iliadis et al., 2015; Osborne et al., 2018). Animal models of depression often utilize stress as an inducer of depressive-like endophenotypes (Krishnan and Nestler, 2008) and models of PND use either gestational stress (Hillerer et al., 2011; Pawluski et al., 2011; Haim et al., 2014) or postpartum corticosterone (CORT) (Brummelte et al., 2006; Brummelte and Galea, 2010; Workman et al., 2013). Both models of PND recapitulate different etiologies and ontogeny timelines of 41 depressive-like endophenotypes, which mirrors the heterogeneity of PND (Postpartum Depression: Action Towards Causes and Treatment (PACT) Consortium, 2015; Galea and Frokjaer, 2019). Gestational stress models depression that would occur de novo during pregnancy, whereas postpartum corticosterone models depression that occurs de novo in the postpartum only. Although depression during pregnancy is one of the greatest predictors of postpartum depression, at least 40% of women with PND will develop depression for the first time during the postpartum period (Munk-Olsen et al., 2006; Wisner et al., 2013). Intriguingly, in rodent models, the effects of gestational stress on depressive-like endophenotypes can be partially restored with pharmacological antidepressants, such as selective serotonin reuptake inhibitors (SSRIs) (Gemmel et al., 2016; Haim et al., 2016), while, there is less efficacy of SSRIs during the postpartum using the CORT-induced model (Workman et al., 2016; Gobinath et al., 2018a). In the CORT-induced model of postpartum depression, SSRI treatment is effective at rescuing disrupted maternal care in the short term (days 2-8 after parturition) but is ineffective in alleviating passive coping or reduced hippocampal neurogenesis in the long-term (days 23-24 after parturition) (Workman et al., 2016; Gobinath et al., 2018a). Based on these data, it is important to understand the possible mechanisms that may be related to the lack of efficacy of SSRIs during the late postpartum period, and we chose to examine two potential factors: inflammation and plasma metabolites of the vitamin B6 dependent tryptophan-kynurenine pathway. Inflammation is associated with major depressive disorder (MDD) as patients diagnosed with MDD show higher circulating proinflammatory cytokines, including tumour necrosis factor (TNF)-, interleukin (IL)-6, and IL-1 (Howren et al., 2009; Dowlati et al., 2010; Haapakoski et al., 2015). In addition, pregnancy and the postpartum also induce changes in the immune system 42 in the hippocampus (Haim et al., 2017; Sherer et al., 2017) and in blood (Eid et al., 2019; reviewed by Duarte-Guterman et al., 2019). Elevated levels of circulating proinflammatory immune markers, particularly the cytokines IL-1, interferon-gamma (IFN-) and IL-6, are reported in women with PND (Groer and Morgan, 2007; Corwin et al., 2008) or associated with depression scores in the perinatal period (Maes et al., 2000), implicating the immune system in PND. These changes in the immune system during the perinatal period may offer clues as to the lack of SSRI efficacy during this period. Indeed, SSRI treatment in non-responsive patients either show no reduction in cytokine levels or continued elevation of cytokine levels (Syed et al., 2018), while depressed patients in remission show reductions in circulating cytokine levels in response to SSRI treatment (Hannestad et al., 2011; Syed et al., 2018). Moreover, Haroon and colleagues (2018) found an association between higher levels of circulating cytokines TNF and IL-6, and the number of failed antidepressant treatment trials (Haroon et al., 2018). These findings suggest that inflammation signalling may give us clues as to the lack of SSRI efficacy in the postpartum period. Another possible mechanism associated with antidepressant efficacy is tryptophan metabolism and the vitamin B6 dependent tryptophan-kynurenine pathway. Elevated tryptophan metabolism as indicated by lower tryptophan concentrations (Hughes et al., 2012; Myint et al., 2013; Ogawa et al., 2014; Doolin et al., 2018; Pu et al., 2020) and higher metabolite concentrations are associated with MDD (Myint et al., 2007; Savitz, 2017; Ogyu et al., 2018; Pu et al., 2020). Furthermore, inflammation can increase tryptophan metabolism (Myint et al., 2013), and thus influence serotonin synthesis, and this relationship is one proposed mechanism through which inflammation can lead to depression onset (Myint et al., 2013). Importantly, MDD individuals who are non-responders to SSRI treatment had higher baseline metabolite 43 markers of tryptophan metabolism (Sun et al., 2020). Interestingly, the tryptophan-kynurenine pathway regulates hippocampal neurogenesis via IL-1 (Zunszain et al., 2012), and neurogenesis is decreased in the CORT-induced model of postpartum depression (Brummelte and Galea, 2010; Workman et al., 2016; Gobinath et al., 2018a). Thus, levels of tryptophan metabolism may be a modulator of SSRI efficacy. Two brain regions, the hippocampus and the frontal cortex, are both highly associated with depression (Koenigs and Grafman, 2009; Fanselow and Dong, 2010). For example, a meta-analysis indicates that people diagnosed with MDD for at least two years present with smaller hippocampal volume (McKinnon et al., 2009). In a separate meta-analysis of resting-state functional connectivity studies, MDD was found to be characterized by hypoconnectivity in frontoparietal regions important for regulation of attention and emotion (Kaiser et al., 2016). Interestingly, the postpartum period is associated with reduced gray matter in both the hippocampus and various regions in the prefrontal cortex (Hoekzema et al., 2017). Models of PND result in decreased spine density in dams in the prelimbic medial frontal cortex (Haim et al., 2014; Leuner et al., 2014; Haim et al., 2016) and reduced neurogenesis in the hippocampus (Pawluski and Galea, 2007; Workman et al., 2016; Gobinath et al., 2018a). Thus, it is clear that the pathoetiology of depression involves disruptions to both the frontal cortex and hippocampus, two areas that are examined in this chapter. Here, I sought to characterize markers of inflammation in the maternal hippocampus, frontal cortex, and plasma, and plasma tryptophan metabolism markers in a CORT-induced model of postpartum depression and SSRI treatment. I hypothesized that CORT treatment would induce depressive-like endophenotypes, including disruptions in maternal care behaviour and increased passive coping behaviour. Furthermore, I expected that the SSRI, fluoxetine (FLX), 44 will show limited efficacy at reducing CORT-induce depressive-like endophenotypes. I also hypothesized that FLX treatment would alter markers of inflammation and tryptophan metabolism, which could be related to the limited efficacy seen by FLX in the postpartum. 2.2 Methods 2.2.1 Animals Forty adult female Sprague-Dawley rats and six male rats (2.5 months old) were purchased from Charles River (Montreal, QC, Canada), however, only thirty-seven females were included in the final study due to pregnancy and birth-related complications. All animals were initially housed in same-sex pairs upon arrival in polycarbonate cages (24 x 16 x 46 cm) with aspen chip bedding. Males and females were housed in separate colony rooms. All animals were maintained on a 12 h: 12 h light/dark cycle (lights on at 07:00) and given standard rat chow (Jamieson's Pet Food Distributors Ltd, Delta, BC, Canada) and autoclaved tap water ad libitum. All animals were allowed to acclimatize to the facilities for seven days and handled for five days prior to the onset of the experiment. Females were randomly assigned to one of four experimental groups (described below), and males were utilized only as breeders. All protocols were in accordance with ethical guidelines set by the Canadian Council for Animal Care and were approved by the University of British Columbia Animal Care Committee. For an overview of experimental procedures, refer to Figure 2.1. 45 Figure 2.1. Timeline of experiment in chapter 2. Briefly, animals were bred within the animal facility and kept undisturbed throughout gestation. Date of birth was designated postpartum day (PD) 0. On PD2 animals received daily injections of either corticosterone (CORT) or oil vehicle and fluoxetine (FLX) or dextrose vehicle for 21 days. Maternal care observations occurred during PD2-7. Forced-swim test (FST) was conducted on PD22 and PD23. Pups remained with the dams until experimental endpoint. 2.2.2 Breeding and culling Breeding and culling protocols were followed as previously described (Workman et al., 2013). Briefly, males were single-housed for 24 hrs prior to breeding. Upon breeding, two females were co-housed with a single male overnight. Dams were divided into 4 cohorts of 8-12 dams per cohort, no more than 2-months apart between the first and last cohort. Vaginal lavage samples were collected the following morning and females were deemed pregnant if sperm was detected (considered gestational date (GD)1). Females were single-housed on GD1 and left undisturbed for the duration of gestation in a separate colony room, except for weekly cage-changing and weighing consistent with past studies (Workman et al., 2016). Day of birth is regarded as postpartum day (PD) 0. All litters were culled randomly on PD1 (the following day after birth of the pups) to 5 females and 5 males per litter. A total of two dams received cross-fostered pups from aged-matched groups because original litters did not 46 meet the requirement of 5 females and 5 males. The first week postpartum, PD1-7 is regarded as “early postpartum” and the last week postpartum PD17-PD23, is regarded as “late postpartum”. 2.2.3 Drug preparations and treatments All treatments were administered as previously described (Gobinath et al., 2018a). All animals received daily injections beginning from PD2 to PD23 between 08:00-10:00 towards the beginning of the inactive phase for our animals, mirroring high evening cortisol in humans with PPD (Iliadis et al., 2015; Osborne et al., 2018). Dams received corticosterone (CORT; 40mg/kg, s.c.; Sigma-Aldrich, St. Louis, MO, USA) or its vehicle (OIL; sesame oil + 10% EtOH, 1ml/kg, s.c.) and either fluoxetine (FLX; 10mg/kg, s.c.; Sequoia Research Products, Pangbourne, UK) or its vehicle, dextrose (DXT; 5% dextrose in sterile water, 1ml/kg, s.c.). Thus, all dams were randomly assigned to one out of four treatment groups (OIL+DXT, n = 9; OIL+FLX, n = 10; CORT+DXT, n = 9; and CORT+FLX, n = 9). FLX and an emulsion of CORT were prepared every three days. This dose of CORT has reliably produced a depressive-like phenotype in dams with reduced maternal care, reduced neurogenesis in the hippocampus, and increased immobility in the forced swim test (Brummelte et al., 2006; Gobinath et al., 2018a). The FLX dose was chosen as it has previously shown to reverse maternal care deficits seen with maternal CORT (Workman et al., 2016; Gobinath et al., 2018a) and to increase brain derived neurotrophic factor and hippocampal cell proliferation after 21 days of injections in both sexes (Hodes et al., 2010), and is higher than clinically relevant dose (Sriraman et al., 2015; Latendresse et al., 2017). 2.2.4 Maternal care behaviour observations Maternal care behaviour observations were conducted twice per day (at 11:30-12:30 and 16:30-17:30) for six days during PD2-7 in the home cage by trained observers. Observations occurred at least 2 hours after injections and after the previous observation. Each observation per 47 animal lasted for 20 minutes per day (10 minutes per morning and afternoon session). The following maternal care behaviours were scored: (a) nursing, which included arched-back nursing (arched-back, actively nursing pups), blanket nursing (in nursing position but resting on-top of pups), and passive nursing (lying on the side and nursing pups); (b) licking (licking pups while not nursing any other pups), (c) self-grooming (not engaging with pups, and self-grooming), (d) nest building (actively building nest), and (e) time spent off-nest (off-nest and not engaging with pups). 2.2.5 Forced-swim test Dams were tested in the forced-swim test (FST) on PD22 (FST1) for 15min and PD23 (FST2) for 5min to assess the effects of CORT and/or FLX on passive coping behaviour as previously described (Workman et al., 2016; Gobinath et al., 2018a). FST is a reliable measure of antidepressant efficacy (Lucki, 1997; Cryan et al., 2002). All behavioural testing occurred at least 2 hours after injections in a separate behavioural room. Briefly, all dams were placed into a water tank containing warm, clean water (24-26°C) in dim light. Behaviours were videotaped and scored by a blinded scorer using BEST Collection Software (Educational Consulting, Inc, Hobe Sound, FL, USA). During FST1, pups were left undisturbed outside of behavioural room and weaned during FST2. Three specific behaviours were coded for: (1) swimming (animals were actively moving their limbs), (2) immobility (immobile with only movements necessary to stay floating), and (3) climbing along tank walls (animals were actively moving limbs along the tank walls, attempting to climb). Data for 4 samples is missing due to a recording malfunction. 48 2.2.6 Estrous cycle cytology Vaginal lavage samples were taken prior to FST2 and were counterstained using cresyl-violet solution to be assessed later for estrous cycle stages based on McLean et al., (2012) and Cora et al., (2015). 2.2.7 Blood and tissue collection Immediately after FST2, all animals were euthanized by rapid decapitation, and trunk blood was collected within three minutes of touching the cage. Plasma was collected into cold EDTA coated tubes and centrifuged 4h later for 10min at 4°C. Data for 5 samples were lost. Brains and adrenal glands were extracted on a cold surface. Hippocampus (0.60mm above bregma to -7.20mm below bregma) and frontal cortex (6.12mm to 0.60mm above bregma) regions were microdissected over ice and flash-frozen immediately after extraction on dry ice and stored at -80°C until assayed for protein and cytokine levels. Adrenal glands were collected and weighed. 2.2.8 Tissue homogenization Following previous protocols (Bodnar et al., 2017; Eid et al., 2019), briefly, all tissues were homogenized in 200 ul cold lysis buffer (150 mM NaCl, 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X, containing a cocktail of protease and phosphatase inhibitors) using the Omni Bead Ruptor 24 (Omni International. Kennesaw, GA, USA). Immediately after homogenization, all tissue samples were centrifuged at 4°C at 13.2 rpm for 20min. Supernatant was then collected and stored in -80°C for protein and cytokine analysis. 2.2.9 Cytokine and protein quantification Cytokine concentrations were measured as previously described (Galea et al., 2018; Eid et al., 2019) using a multiplex electrochemiluminescence immunoassay kit (V-PLEX 49 Proinflammatory Panel 2, Rat) from Meso Scale Discovery (Rockville, MD, USA) used according to the manufacturer’s instructions. Samples were run in duplicates to quantify in multiplex in each sample: interferon-gamma (IFN-), interleukin-1-beta (IL-1), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-) and chemokine (C-X-C motif) ligand 1 (CXCL1). Plates were read using a Sector Imager 2400 (Meso Scale Discovery), and data analyses were conducted using the Discovery Workbench 4.0 software (Meso Scale Discovery). Lower limits of detection (LLOD) were as follows (pg/mL) for HPC: IFN-: 0.568, IL-1: 2.31, IL-4: 0.399, IL-5: 9.35, IL-6: 5.78, IL-10: 2.53, IL-13: 1.08, TNF-: 1.36, and CXCL1: 0.357; for frontal cortex: INF-: 0.179, IL-1: 2.79, IL-4: 0.143, IL-5: 4.08, IL-6: 8.67, IL-10: 0.603, IL-13: 0.429, TNF-: 0.331, and CXCL1: 0.467; for plasma INF-: 0.227, IL-1: 1.39, IL-4: 0.0858, IL-5: 4.77, IL-6: 22.1, IL-10: 1.85, IL-13: 0.544, TNF-: 0.250, and CXCL1: 0.232. No values were below LLOD for the HPC and frontal cortex. the number of samples that fell below the LLOD were IFN-  (), IL-1: 27 (84.4%), IL-4: 5 (15.6%), IL-5: 4 (12.5%), IL-6: 12 (37.5%), IL-10: 4 (12.5%), IL-13: 21 (63.6%), CXCL1: 0 (0%), and TNF-: 0 (0%). Plasma cytokines with at least 80% of the data in the detectable range were included in the data analyses (IL-4, IL-5, IL-10, CXCL1, and TNF-), ANOVAs were calculated. Cytokine samples were eliminated if duplicates had high intra-assay coefficients (>20%) of variations (n = 7). Total protein levels were quantified in tissue homogenates using the Pierce Microplate BCA Protein Assay Kit in triplicates following the manufacturer’s protocol (Thermo Scientific Pierce Protein Biology, Thermo Fisher Scientific, Waltham, MA, USA). Cytokine levels in the 50 HPC and frontal cortex were normalized to total protein levels and reported as pg cytokine/mg of protein. Plasma cytokines are reported as pg/ml. 2.2.10 Plasma pyridoxic acid, pyridoxal, and tryptophan analyses We quantified vitamin B6 and related metabolites because vitamin B6 in its coenzyme form pyridoxal 5’-phosphate is a critical coenzyme in tryptophan metabolism (Bender et al., 1990). Plasma 4’-pyridoxic acid, pyridoxal, pyridoxal 5’-phosphate, and tryptophan concentrations were quantified using isotope dilution liquid chromatography coupled with tandem mass spectrometry (ABSciex API4000; AB SCIEX, Framingham, MA, U.S.A.) based on the method developed by Midttun et al., (2005, 2009) with modifications. The intra- and interassay CVs for these assays were all ≥7%. 2.2.11 Statistical analyses All data (maternal behaviours, relative adrenal mass, FST behaviours, tryptophan metabolites, and cytokines) were analyzed separately using two-way analysis of variance (ANOVA) with CORT (CORT or OIL) and FLX (FLX or DXT) as between-subject factors unless otherwise specified. Relative adrenal mass was calculated as adrenal mass/body mass. Effect sizes were reported for significant effects as Ƞp2 or Cohen’s d. Post hoc comparisons used Newman-Keuls. A priori comparisons were subjected to Bonferroni correction. As an exploratory approach, principal component analyses (PCA) were conducted to determine networks (components) that best explain cytokines level variances within a specific brain region (HPC or PFC). Principal component scores for individual sample data for the first two components generated were then analyzed using ANOVA models to derive information regarding the amount of variance accounted for by cytokine profiles within circulating and neural tissue (Eid et al., 2019). All ANOVA and correlation data analyses were analyzed using 51 Statistica (v. 9, StatSoft, Inc., Tulsa, OK, USA). PCA data analyses were conducted using R (3.4.3) statistical analysis software with the FactoMineR package. Effects were considered statistically significant if p ≤ 0.05 and trends are discussed if p ≤ 0.10. A priori comparisons were based on previous literature showing that postpartum CORT reduces nursing and increases time spent off the nest, while postpartum FLX restores these deficits (Workman et al., 2016; Gobinath et al., 2018a). Pearson product-moment correlations were conducted between cytokines within circulating and neural tissue. Outliers were eliminated if two standard deviations away from the mean (n = 1-3 for maternal behaviour). 2.3 Results 2.3.1 Maternal CORT treatment significantly lowered adrenal mass CORT treatment significantly reduced relative adrenal mass compared to oil vehicle-treated animals (main effect of CORT; F(1, 33) = 103.120, p < 0.001, Ƞp2 = 0.758; Figure 2.2A). No other significant effects or interactions were found (all p’s ≥ 0.403). 2.3.2 Maternal CORT treatment reduced nursing and increased time off the nest and these effects were restored by FLX treatment CORT+DXT showed significantly lower total time nursing compared to all other groups and postpartum FLX restored the CORT-induced decrease in nursing relative to OIL+DXT (a priori: p = 0.028, Cohen’s d = 1.148), OIL+FLX (p = 0.012, Cohen’s d = 1.330), and CORT+FLX (p = 0.012, Cohen’s d = 1.436); main effect of FLX: F(1, 31) = 4.713, p = 0.038, Ƞp2 = 0.132, all other main or interaction effects were not significant (p’s > 0.09), Figure 2.2B). CORT+DXT group showed significantly higher total time spent off-nest compared to OIL+DXT (a priori: p = 0.010, Cohen’s d = 1.756); OIL+FLX (p = 0.009, Cohen’s d = 1.249); 52 CORT+FLX (p = 0.042, Cohen’s d =1.469); main effect of CORT, F(1, 28) = 6.207, p = 0.019, Ƞp2 = 0.181; all other main or interaction effects were not significant (p’s > = 0.11 (Figure 2.2C)). No other significant effects on other maternal care behaviours were found (all p’s ≥ 0.126). 2.3.3 Maternal CORT treatment increased time spent immobile in FST2 Regardless of FLX, CORT treatment, significantly increased time spent immobile during FST2 compared to oil vehicle-treated animals (main effect of CORT, F(1, 29) = 4.464, p = 0.043, Ƞp2 = 0.133 (Figure 2.2D)). No other significant effects of treatment on time spent immobile were found (all p’s ≥ 0.167). The number of animals in each estrous cycle stage is shown in Table 2.1. Using estrous cycle stage as a covariate did not alter the findings, nor was there a significant effect of the covariate (p = 0.437). Furthermore, there was no significant difference between groups on estrous cycle phase (χ2 = 7.87, p = 0.547). There was a trend towards significance for CORT treatment to reduce time spent swimming compared to oil vehicle-treated animals (main effect of CORT, p = 0.086) but no other significant effects on time spent climbing were found (all p’s ≥ 0.379; data not shown). Time spent climbing was not affected by any of the treatments (all p’s ≥ 0.168). Estrous cycle stage n Percentage Proestrus 8 21.62% Estrus 6 16.22% Metestrus 8 21.62% Diestrus 15 40.54% Table 2.1. Number of rats in each estrous stage on FST2. 53 Figure 2.2. Effects of maternal corticosterone (CORT) and fluoxetine (FLX) treatment on relative adrenal mass, maternal care behaviour (total nursing, total off-nest behaviour), and immobility behaviour during forced swim test (FST). A. CORT treatment significantly reduced relative adrenal mass. *Indicate significance at p < 0.05. B. CORT + dextrose vehicle (DXT)-treated group showed significantly lower total nursing time compared to all other groups. *Indicate significance at p < 0.05. C. CORT+DXT treated groups showed a significantly higher total time off-nest when compared to all other groups. *Indicate significance at p < 0.05. D. CORT treatment significantly increased the time spent immobile in FST2. *Indicate significance at p < 0.05 Data represented in means + standard error of the mean (SEM), overlaid with individual data points. n’s = 8-10 per group. 54 2.3.4 Maternal FLX treatment increased IL-1 levels in the hippocampus and CXCL1 levels in plasma. Maternal CORT treatment reduced TNF-, and IFN- levels in the hippocampus and TNF- levels in plasma FLX treatment significantly increased levels of IL-1 in the hippocampus compared to vehicle-treated animals (main effect of FLX, F(1, 30) = 4.906, p = 0.034, Ƞp2 = 0.140 (Figure 2.3A). FLX treatment did not significantly affect any other cytokines, chemokine, IL-6:IL-10 ratio, or IFN-:IL-10 ratio in the maternal hippocampus (all p ≥ 0.194; Table 2.2). CORT treatment significantly reduced TNF- and IFN- levels in the hippocampus compared to vehicle treatment (main effect of CORT: F(1, 30) = 10.748, p = 0.003, Ƞp2 = 0.264; F(1, 31) = 5.108, p = 0.031, Ƞp2 = 0.141, Figure 2.3 B and C, respectively). There were trends towards significance for an effect of CORT to reduce levels of IL-1, IL-6, and IL-10 in the hippocampus compared to vehicle treatment, (p = 0.086, p = 0.067, p = 0.082 respectively). CORT treatment did not significantly affect any other cytokines, chemokine, IL-6:IL-10 ratio, or IFN-:IL-10 in the maternal hippocampus (all p’s ≥ 0.161; see Table 2.2). No other significant interaction effects between CORT and FLX treatment were observed in the maternal hippocampus (all p ≥ 0.339; Table 2). Further, there were no significant effects of CORT or FLX treatment, nor of their interaction, on cytokines, chemokines, IL6:IL-10 ratio, or IFN-:IL-10 levels in the maternal frontal cortex (all p’s ≥ 0.102; see Table 2.3). FLX treatment significantly increased plasma CXCL1 levels (main effect of FLX: F(1, 28) = 4.581, p = 0.041, Ƞp2 = 0.141; Figure 2.3D). CORT treatment significantly reduced plasma TNF- levels compared to vehicle treatment (main effect of CORT: F(1, 28) = 17.971, p < 0.001, Ƞp2 = 0.391; Figure 2.3E). FLX treatment tended to increase plasma TNF- levels (p = 55 0.102). There were no significant effects for any other cytokine or ratios in plasma (all p’s ≥ 0.102; see Table 2.4). HPC Variable F-value p-value Ƞp2 IFN- CORT 5.108 *0.031 0.141 FLX 0.010 0.921 < 0.001 CORT x FLX 0.001 0.971 < 0.001 IL-10 CORT 3.230 †0.082 0.097 FLX 0.143 0.708 0.005 CORT x FLX 0.220 0.643 0.007 IL-13 CORT 2.480 0.126 0.079 FLX 0.074 0.787 0.003 CORT x FLX 0.299 0.588 0.010 IL-1 CORT 3.159 †0.086 0.095 FLX 4.906 *0.034 0.140 CORT x FLX 0.017 0.896 < 0.001 IL-4 CORT 1.298 0.263 0.039 FLX 0.137 0.714 0.004 CORT x FLX 0.013 0.911 < 0.001 IL-5 CORT 0.011 0.916 < 0.001 FLX 1.122 0.298 0.036 CORT x FLX 0.159 0.692 0.005 IL-6 CORT 3.744 †0.062 0.102 FLX 0.840 0.366 0.025 CORT x FLX 0.307 0.583 0.009 CXCL1 CORT 0.002 0.965 < 0.001 FLX 0.408 0.527 0.012 CORT x FLX 0.234 0.632 0.007 TNF- CORT 10.748 *0.003 0.264 FLX 0.580 0.452 0.019 CORT x FLX 0.115 0.737 0.004 IL-6:IL-10 CORT 0.496 0.487 0.016 FLX 0.365 0.550 0.012 CORT x FLX 0.052 0.821 0.002 IFN-:IL-10 CORT 0.018 0.894 < 0.001 FLX < 0.001 0.989 < 0.001 CORT x FLX 0.033 0.858 0.001 56 Table 2.2. Two-way ANOVA results of hippocampal (HPC) cytokines and chemokine data. *Indicate significance at p < 0.05. †Indicate trend at p < 0.10. n’s = 7-10 per group. Frontal cortex Variable F-value p-value Ƞp2 IFN- CORT 0.148 0.703 0.005 FLX 0.143 0.707 0.005 CORT x FLX 0.420 0.522 0.013 IL-10 CORT < 0.001 0.978 < 0.001 FLX 0.305 0.585 0.010 CORT x FLX 0.246 0.623 0.008 IL-13 CORT 0.435 0.514 0.013 FLX 0.015 0.904 < 0.001 CORT x FLX 0.559 0.460 0.017 IL-1 CORT 0.066 0.799 0.002 FLX 0.180 0.674 0.006 CORT x FLX 0.124 0.727 0.004 IL-4 CORT 0.006 0.938 < 0.001 FLX 0.276 0.603 0.008 CORT x FLX 0.378 0.543 0.011 IL-5 CORT 0.034 0.855 0.001 FLX 0.288 0.585 0.009 CORT x FLX 0.402 0.530 0.012 IL-6 CORT 0.384 0.540 0.011 FLX < 0.001 0.987 < 0.001 CORT x FLX 1.139 0.294 0.033 CXCL1 CORT 2.845 0.102 0.089 FLX 1.712 0.201 0.056 CORT x FLX 0.004 0.952 < 0.001 TNF- CORT 1.220 0.278 0.037 FLX 0.969 0.332 0.029 CORT x FLX 0.012 0.912 < 0.001 IL-6:IL-10 CORT 0.057 0.812 0.002 FLX 0.123 0.728 0.004 CORT x FLX 0.299 0.588 0.009 IFN-:IL-10 CORT 0.043 0.837 0.001 FLX 0.004 0.949 < 0.001 CORT x FLX 0.345 0.562 0.012 Table 2.3. Two-way ANOVA results of frontal cortex cytokines and chemokine data. n’s = 7-10 per group. 57 Plasma Variable F-value p-value Ƞp2 IL-10 CORT 0.158 0.694 0.006 FLX 1.760 0.195 0.059 CORT x FLX 0.105 0.749 0.003 IL-4 CORT 1.835 0.187 0.064 FLX 0.492 0.489 0.018 CORT x FLX 0.009 0.925 < 0.001 IL-5 CORT 0.100 0.754 0.004 FLX 0.130 0.721 0.005 CORT x FLX 0.866 0.360 0.030 CXCL1 CORT 0.747 0.395 0.026 FLX 4.581 *0.041 0.141 CORT x FLX 0.026 0.872 0.001 TNF- CORT 17.970 *< 0.001 0.391 FLX 2.855 0.102 0.092 CORT x FLX 0.254 0.618 0.009 Table 2.4. Two-way ANOVA results of plasma cytokines and chemokine data. *Indicate significance at p < 0.05. n’s = 7-10 per group. Three cytokines positively correlated between hippocampal levels and frontal cortex levels, IL-10, IL-4, and IL-5 (r = 0.5090, p = 0.002; r = 0.4303, p = 0.008; r = 0.4000, p = 0.019). Although only the correlations with IL-10 remained significant after correcting for multiple comparisons. However, none of the plasma cytokines significantly correlated with hippocampal or frontal cortex cytokines, although there were trends for TNF-a (frontal: r = 0.356, p = 0.051; hippocampus: r = 0.351, p = 0.073), but they are not significant after correcting for multiple comparisons. 58 Figure 2.3. Effects of maternal corticosterone (CORT) and fluoxetine (FLX) treatment on hippocampal and plasma cytokine levels. A. FLX treatment significantly increased levels of hippocampal interleukin (IL)-1. B. CORT treatment significantly reduced hippocampal tumour necrosis factor(TNF)- levels. C. CORT treatment significantly reduced hippocampal interferon-gamma (IFN-) levels. D. FLX increased chemokine (C-X-C motif) ligand 1 (CXCL1) levels in plasma. E. CORT treatment decreased TNF- levels in plasma. Data represented in means + standard error of the mean (SEM), overlaid with individual data points. *Indicate significance at p < 0.05. n’s = 7-10 per group. 59 2.3.5 Maternal fluoxetine treatment significantly reduced plasma 4’-pyridoxic acid, pyridoxal, and tryptophan concentrations, regardless of maternal corticosterone. Maternal corticosterone increased plasma pyridoxal and pyridoxal 5’-phosphate concentrations Maternal FLX treatment significantly decreased plasma concentrations of tryptophan (Figure 2.4A), 4’-pyridoxic acid, and pyridoxal, (Figure 2.4B-C) compared to DXT-treated animals (main effect of FLX: F(1, 29) = 4.925, p = 0.034, Ƞp2 = 0.145; F(1, 29) = 4.399, p = 0.044, Ƞp2 = 0.132; F(1, 29) = 7.995, p = 0.008, Ƞp2 = 0.216; respectively) but not pyridoxal 5’-phosphate (p = 0.546; Figure 4D). A priori analysis found that the OIL+FLX group showed significantly lower levels of plasma pyridoxal compared to all other groups (all p’s ≤ 0.006, all Cohen’s d ≥ 1.266; Figure 2.4C). Maternal CORT significantly increased plasma pyridoxal (main effect of CORT, F(1, 29) = 9.549, p = 0.004, Ƞp2 = 0.248; Figure 2.4C), pyridoxal 5’-phosphate levels (main effect of CORT, F(1, 29) = 19.392, p ≤ 0.001, Ƞp2 = 0.40; Figure 2.4D), and there was a trend for maternal CORT to increase plasma pyridoxic acid levels (p = 0.079; Figure 2.4B). There were no significant interaction effects for all metabolites (all p’s > 0.128). 60 Figure 2.4. Effects of maternal corticosterone (CORT) and fluoxetine (FLX) treatment on plasma tryptophan levels, 4’-pyridoxic acid, pyridoxal levels, and pryidoxal 5’-phosphate. A. FLX treatment significantly reduced plasma tryptophan levels. B. FLX treatment significantly reduced plasma 4’-pyridoxic acid levels. C. OIL+FLX treatment significantly reduced pyridoxal levels compared to all other groups. D. CORT treatment significantly increased pyridoxal 5’-phosphate levels. *Indicate significance at p < 0.05. Data represented in means + standard error of the mean (SEM), overlaid with individual data points. n’s = 7-10 per group. 2.3.6 Principal component analyses on brain cytokine and chemokine levels Within the hippocampus, the PCA method found the first two principal components explained 63.70% of the variation (component 1 explaining 52.40% and component 2, 11.30%). All nine cytokines and chemokines showed significant loadings onto component 1 (all p’s < 61 0.001); IL-10 showed a significant negative loading onto component 2 (p < 0.001), whereas CXCL1 (p = 0.004) and IL-1 (p < 0.001) showed a significant positive loading onto component 2 (Table 2.5). Within the frontal cortex, the PCA method found the first two principal components explained 72.39% of the variation (component 1 explaining 59.40% and component 2, 12.99%). All cytokines and chemokines except IL-6 showed significant loadings on component 1 (all p’s < 0.001), IL-6 (p < 0.001), and CXCL1 (p < 0.001) showed a significant positive loadings on component 2, and IL-10 (p < 0.001) showed a significant negative loading on component 2 (Table 2.6). Within plasma, the PCA method found the first two principal components explaining 73.62% of the variation (component 1 explaining 49.97% and component 2, 23.65%). All cytokines and chemokines but TNF- showed significant loadings on component 1 (all p’s < 0.001), IL-5 (p < 0.001), and TNF- (p < 0.001) showed significant positive loadings on component 2, whereas IL-4 (p < 0.001), showed a significant negative loading on component 2 (Table 2.7). Variables Component 1 Component 2 IFN- *0.897 0.153 IL-10 *0.705 *-0.550 IL-13 *0.809 -0.268 IL-1 *0.660 *0.572 IL-4 *0.780 -0.122 IL-5 *0.642 -0.159 IL-6 *0.576 -0.141 CXCL1 *0.555 *0.462 TNF- *0.813 0.138 62 Table 2.5. Component loadings for the hippocampal cytokine variance of the first two components identified using principal component analysis (PCA). Networks are interpreted using significant loadings set by variables denoted by *. All denoted values are determined by statistically significant contributions of variables towards the specific component (network). *Indicate significance at p = 0.001. n’s = 9-10 per group. Variables Component 1 Component 2 IFN- *0.862 0.262 IL-10 *0.893 *-0.353 IL-13 *0.634 0.199 IL-1 *0.835 0.174 IL-4 *0.893 -0.303 IL-5 *0.922 -0.188 IL-6 0.277 *0.806 CXCL1 *0.473 *0.333 TNF- *0.870 -0.136 Table 2.6. Component loadings for the frontal cortex cytokine variance of the first two components identified using principal component analysis (PCA). Networks are interpreted using significant loadings set by variables denoted by *. All denoted values are determined by statistically significant contributions of variables towards the specific component (network). *Indicate significance at p = 0.001, n’s = 9-10 per group. Variables Component 1 Component 2 IL-10 *0.872 -0.080 IL-4 *0.891 *-0.370 IL-5 *0.543 *0.367 CXCL1 *0.803 0.182 TNF- 0.058 *0.933 Table 2.7. Component loadings for the plasma cytokine variance of the first two components identified using principal component analysis (PCA). Networks are interpreted using significant loadings set by variables denoted by *. All denoted values are determined by statistically significant contributions of variables towards the specific component (network). *Indicate significance at p = 0.001. n’s = 9-10 per group. 63 We analyzed sample scores onto principal component 1 using ANOVA, CORT treatment significantly decreased the sample scores onto principal component 1 within the HPC cytokine data set (main effect of CORT, F(1, 33) = 4.568, p = 0.040, Ƞp2 = 0.122 (Figure 2.5A)) but not the frontal cortex (p = 0.234; Figure 2.5B). No other significant main or interaction effects between CORT and FLX treatment were found when analyzing component 1 sample scores within the HPC, frontal cortex, or plasma cytokine data sets (all p’s ≥ 0.343; Figure 2.5A-C). We analyzed sample scores onto principal component 2 using ANOVA. CORT treatment significantly decreased the sample scores onto principal component 2 within the plasma cytokine data set (main effect of CORT, F(1, 33) = 12.827, p = 0.001, Ƞp2 = 0.280 (Figure 2.5F)). No other significant main or interaction effects between CORT and FLX treatment were found when analyzing component 2 sample scores within the HPC, frontal cortex, or plasma data sets (all p’s ≥ 0.209; Figure 2.5D-F). 64 Figure 2.5. Effects of maternal corticosterone (CORT) and fluoxetine (FLX) treatment on hippocampal , frontal cortex, and plasma cytokine principal component analysis (PCA) sample scores onto the first two components. A. CORT treatment significantly lowered hippocampal cytokine sample scores onto PCA component 1. B. Frontal cortex cytokine sample scores onto PCA component 1. No statistically significant effects were found. C. Plasma cytokine sample scores onto PCA component 1. No statistically significant effects of CORT and FLX were found. D. Hippocampal cytokine sample scores onto PCA component 2. No statistically significant effects were found. E. Frontal cortex cytokine sample scores onto PCA component 2. No statistically significant effects were found. F. CORT treatment significantly lowered plasma cytokine sample scores onto PCA component 2. *Indicate significance at p < 0.05. Data represented in means + standard error of the mean (SEM), overlaid with individual data points. n’s = 9-10 per group. 65 2.4 Discussion Postpartum FLX treatment increased hippocampal IL-1 levels, plasma CXCL1 levels, and reduced plasma tryptophan, 4’-pyridoxic acid, and pyridoxal concentrations regardless of CORT treatment. Postpartum CORT treatment decreased two proinflammatory cytokines (TNF- and IFN-) in the maternal hippocampus and plasma (TNF-), and increased plasma pyridoxal 5’-phosphate concentrations. Consistent with previous reports, postpartum CORT treatment decreased maternal care behaviour, increased passive coping behaviour, and reduced adrenal mass, and while postpartum FLX restored maternal behaviours, it did not rescue passive coping behaviours (Brummelte et al., 2006; Brummelte and Galea, 2010; Workman et al., 2016; Gobinath et al., 2018a). Our findings point to the possibility that the lack of efficacy of FLX to restore passive coping is via its effects on IL-1 CXCL1, and tryptophan metabolism. 2.4.1 Corticosterone treatment reduced maternal care and increased passive coping behaviour Consistent with our previous research, postpartum CORT treatment significantly decreased total time spent nursing and increased total time spent off-nest (Brummelte et al., 2006; Brummelte and Galea, 2010; Workman et al., 2016; Gobinath et al., 2018a). This finding is corroborated by other models of postpartum stress (Brunson et al., 2005; Ivy et al., 2008; Kurata et al., 2009; Nephew and Bridges, 2011; Murgatroyd et al., 2015; Moussaoui et al., 2016). As maternal care behaviour is thought to be highly rewarding (Mattson et al., 2001, 2003; Ferris et al., 2005), the reduction in maternal care may be an expression of reduced reward-seeking or anhedonia-like behaviour, which may be a representation of the clinical anhedonia phenotype of depression (Murgatroyd et al., 2015; reviewed by Post and Leuner, 2019). 66 Postpartum CORT treatment increased passive coping behaviour in the FST, consistent with previous reports (Brummelte et al., 2006; Brummelte and Galea, 2010; Workman et al., 2016; Gobinath et al., 2018a). Passive coping behaviour, measured via immobility in the second FST, has been interpreted as behavioural despair (reviewed in Krishnan and Nestler, 2011), which may be representative of the depressed mood and hopelessness within a clinical phenotype of depression. Overall, postpartum CORT induces behavioural endophenotypes of depression in rodent dams in our model of de novo postpartum depression. Although PND can refer to depression during pregnancy or the postpartum, many more women suffer from de novo depression in the postpartum compared to during pregnancy (Munk-Olsen et al., 2006; Shorey et al., 2018; Wikman et al., 2019). Here, using postpartum CORT, we specially model de novo postpartum depression that occurs in the majority of first-time depression in the perinatal period (see Galea and Frokjaer, 2019). 2.4.2 Postpartum corticosterone treatment reduced two proinflammatory cytokines in the hippocampus and in plasma without affecting cytokine or chemokine levels in the frontal cortex Patients with MDD (Howren et al., 2009; Dowlati et al., 2010; Haapakoski et al., 2015) and PND (Groer and Morgan, 2007; Osborne and Monk, 2013) show elevated levels of circulating proinflammatory cytokines, particularly TNF-, IL-6, and IL-1. Based on these clinical phenotypes, one might predict postpartum CORT treatment, which induces depressive-like endophenotypes (Brummelte et al., 2006; Brummelte and Galea, 2010; Workman et al., 2016; Gobinath et al., 2018a) would elevate proinflammatory cytokines in our model. However, postpartum depression is associated with lowered circulating IFN- levels compared to controls 67 (Groer and Morgan, 2007), indicating the proinflammatory phenotype of MDD is not seen in postpartum depression. This effect is consistent with our findings that postpartum CORT treatment reduced two proinflammatory cytokines (TNF- and IFN-) in the maternal hippocampus and plasma TNF-, without significantly affecting other cytokines or chemokine measured in the hippocampus or the frontal cortex. Moreover, Groer and Morgan (2007) found significant changes in circulating cytokine ratios (IFN- versus IL-10), suggesting possible changes in cytokine profiles. This effect is similar to what we found in our study, while we did not see changes in IFN- versus IL-10 ratio, we did see significant changes in cytokine profiles due to CORT treatment indicated by the PCA method. Interestingly, in a model of sub-chronic gestational stress (during gestational days 16-22), rat dams showed a decrease in IL-1 gene expression in the frontal cortex (Posillico and Schwarz, 2016). Although in the present study, we did not see any significant changes in the frontal cortex, we did see a non-significant reduction in hippocampal IL-1 protein with postpartum CORT. Although circulating levels of cytokines may differ from central levels (Gibb et al., 2011), we see similarities in the effects of CORT on TNF- levels between hippocampus and plasma, suggesting some interaction between brain and plasma immune systems, which is corroborated by previous literature (reviewed by Louveau et al., 2015; Dantzer, 2018). In addition, circulating IFN- was reduced by postpartum CORT treatment in our rodent model and in humans with PND (Groer and Morgan, 2007), suggesting translational relevance. Interestingly, we did find positive correlations between hippocampal and frontal cortex cytokine levels for three cytokines (IL-10, IL-14, and IL-5), while only IL-10 remained significant after correcting for multiple comparisons, and results should be interpreted 68 with cautions, nevertheless, these results suggest some interaction between different brain regional immune profiles. Altogether, our findings with maternal CORT are consistent with the clinical data indicating lowered circulating IFN-γ in postpartum depression (Corwin et al., 2008, 2015; Skalkidou et al., 2009; Okun et al., 2011; Buglione-Corbett et al., 2018; Miller et al., 2019) and are likely related to the anti-inflammatory effect of glucocorticoids (reviewed by Sorrells and Sapolsky, 2007). These findings suggest that with the altered immune profile during the perinatal period, the changes in the inflammatory system in PND may not be representative of, or similar to, those found in MDD. 2.4.3 Fluoxetine treatment showed limited efficacy to reduce passive coping We found that FLX treatment rescued disrupted maternal care behaviour early in the postpartum (PD 2-7) but not passive coping behaviour later in the postpartum (PD23), in line with previous research (Brummelte et al., 2006, Brummelte and Galea, 2010, Workman et al., 2016, Gobinath et al., 2018a). Interestingly, FLX treatment with voluntary exercise reduces passive coping behaviour in this model of postpartum depression (Gobinath et al., 2018a). Other studies have also noted a lack of efficacy in the postpartum with pharmacological antidepressants (SSRIs) using passive coping behaviour (Craft et al., 2010; Overgaard et al., 2018). The limited efficacy of SSRIs in the postpartum period is also seen in humans (Sharma and Sommerdyk, 2013; Sharma, 2017). Here, in the model of de novo postpartum depression, I recapitulate similar findings in the limited efficacy of SSRIs as reported in the clinical field to treat PND (Yonkers et al., 2008; Sharp et al., 2010) and specifically in the postpartum period (Sharma and Sommerdyk, 2013; Sharma, 2017). It is interesting to note that when utilizing gestational stress to model depression onset during the antenatal period, the SSRI citalopram, was able to reduce passive 69 coping behaviour (Haim et al., 2016). Thus, there appears to be a significant pathophysiological difference in SSRI efficacy and response to treatment depending on the etiology of PND in rodent models (gestational stress versus postpartum CORT exposure). 2.4.4 Fluoxetine treatment increased IL-1 in the hippocampus and plasma CXCL1, decreased plasma tryptophan, and altered vitamin B6 metabolism, which may be related to the inefficacy of fluoxetine in the postpartum FLX treatment increased levels of hippocampal IL-1 and plasma CXCL1 levels, and decreased plasma tryptophan metabolism. Our results are consistent with another study that showed FLX-treated (non-partum) female mice had increased IL-1 gene expression in the hippocampus coupled with limited efficacy to reduce passive coping behaviour and anhedonia in the sucrose preference test (Duda et al., 2017). Overall, these data, coupled with our own, suggest an association between the lack of efficacy of FLX and increased IL-1 inflammation in the hippocampus of female rodents, which may be associated with elevated circulating CXCL1 levels. Furthermore, SSRI treatment-resistant patients with MDD show elevated circulating IL-1 levels after SSRI treatment (Syed et al., 2018), suggesting an association between elevated IL-1 levels and reduced treatment efficacy in both clinical studies and preclinical models. Future studies should determine if elevated IL-1 or the chemokine, CXCL1 are causal and crucial for SSRI efficacy. We also observed a reduction in plasma tryptophan concentrations in our rodent dams due to FLX treatment. This reduction may contribute to the limited behavioural efficacy of maternal FLX in postpartum rats. Lower circulating concentration of tryptophan, a serotonin precursor, is observed in patients with MDD (Hughes et al., 2012; Myint et al., 2013; Ogawa et 70 al., 2014; Doolin et al., 2018; Pu et al., 2020) and a reduced rate of serotonin synthesis may lead to the onset of depressive symptom. Although, lower concentrations of peripheral tryptophan are seen in the postpartum, regardless of depressive symptoms, lower concentrations of the tryptophan metabolite, kynurenine are seen only in postpartum women with depression (Veen et al., 2016). Moreover, only women with de novo postpartum depression had altered levels of tryptophan metabolites during the peripartum compared to healthy controls, and not those with antepartum depression or a history of depression (Teshigawara et al., 2019). These effects suggest that although parity itself can lower tryptophan concentrations, those with PND, particularly de novo postpartum depression, show altered tryptophan-to-kynurenine metabolism compared to healthy non-partum and postpartum controls and this is consistent with the present data. It should also be noted that drug metabolism increases in the perinatal period and that doses of SSRIs may need to be increased (Sit et al., 2010). Here, I show despite our relatively high dose of FLX compared to clinical doses (Sriraman et al., 2015; Latendresse et al., 2017) that tryptophan concentration is still reduced. Additionally, we found significant decreases in vitamin B6 metabolism due to FLX treatment). Thus, it is possible for vitamin B6 to play a significant modulatory role in depression onset. Here, the changes in vitamin B6 metabolites due to FLX treatment may be a representation of disrupted vitamin B6 metabolism. These effects may, in part, lower the efficacy of FLX to reduce depressive-like endophenotypes in the postpartum. Interestingly, in human hippocampal progenitor cell cultures, the ability for the cytokine IL-1 to decrease neurogenesis is highly dependent on the tryptophan metabolic pathway (Zunszain et al., 2012). In addition to this association, vitamin B6 can inhibit cytokine (IL-1 IL-6, and TNF-) gene expression in cultured macrophages (Shan et al., 2020) and lower 71 pyridoxal 5’-phosphate concentrations correlate with high inflammation in rheumatoid arthritis (Chiang et al., 2005). Derivatives of vitamin B6 can also increase neurogenesis in the hippocampus in adult male mice (Yoo et al., 2011). Overall, these data suggest the immune system, tryptophan metabolism, vitamin B6, and hippocampal integrity are highly correlated with each other and may act together to influence depression etiology and SSRI efficacy. 2.5 Conclusions In conclusion, while maternal CORT treatment-induced passive coping behaviour, maternal FLX treatment failed to reverse this effect. We identified two possible mechanisms that may contribute towards SSRI inefficacy in the postpartum as maternal FLX: (1) increased hippocampal IL-1 and plasma CXCL1 levels, and (2) reduced plasma levels of tryptophan and vitamin B6 metabolism. Ultimately, our study contributes to the body of literature to suggest a possible association between an altered cytokine profile and the pathophysiology of PND. Future studies are needed to determine if altered immune profiles, tryptophan and vitamin B6 metabolism can act as biomarkers and improve pharmacological treatments for PND. 72 Chapter 3: Postpartum corticosterone and fluoxetine shift the tryptophan-kynurenine pathway in dams 3.1 Introduction Perinatal depression (PND) is a heterogeneous disease, in which symptoms and remission vary depending on depression onset timing and depression history (reviewed in Galea and Frokjaer, 2019; Qiu et al., 2020b). The first three months postpartum is a period of high susceptibility for first time psychiatric disease in females but not in males (Munk-Olsen et al., 2006). Postpartum depression (PPD) affects 15% of females with approximately 40% of these cases being individuals experiencing depressive symptoms for the first time (Wisner et al., 2013). Maternal postpartum corticosterone (CORT) is used to model de novo PPD in rodents (reviewed in Qiu et al., 2020b). This model mimics elevated cortisol levels seen in those women with depression symptoms postpartum (Iliadis et al., 2015). Maternal postpartum CORT consistently induces various depressive-like endophenotypes, such as reduced maternal care behaviour, increased passive coping behaviour, and reduced hippocampal integrity (Workman et al., 2016; Gobinath et al., 2018; Qiu et al., 2020a, Chapter 2). Inflammation and altered metabolism in the tryptophan-kynurenine pathway (TKP) are associated with major depressive disorder (MDD) and PND (Corwin et al., 2008; Maes et al., 2011; Haapakoski et al., 2015). The immune system and TKP are related as proinflammatory cytokines, such as interferon (IFN)- and interleukin (IL)-1, can increase enzymatic conversion of tryptophan into its downstream metabolite, kynurenine (reviewed by Maes et al., 2011). Higher expression of proinflammatory cytokines such as IL-6, tumor necrosis factor (TNF)-, 73 IFN-, and IL-1 are associated with both MDD and PND (Corwin et al., 2008; Haapakoski et al., 2015). TKP can be divided into either a putative “neurotoxic” pathway or a “neuroprotective” pathway and higher activation in the putative neurotoxic branch is seen in MDD and PND (see Figure 3.1; reviewed in Maes et al., 2011). Curiously, PND is associated with higher kynurenic acid (KYNA) (Teshigawara et al., 2019), but MDD is associated with reduced KYNA (reviewed by Maes et al., 2011). Previously, we found reduced cytokine (TNF- and IFN-) expression and elevated concentrations of a TKP enzyme cofactor (vitamin B6) due to postpartum CORT (Qiu et al., 2020a), indicating an altered immune system and TKP in this model of de novo PPD. Selective serotonin reuptake inhibitors (SSRIs) are the first line treatment for PND although efficacy of SSRIs varies with timing of depression onset as those with postpartum onset have lower efficacy than those with antenatal onset (Cox et al., 2016). Both inflammation and TKP are implicated in antidepressant efficacy both in humans (Syed et al., 2018; Sun et al., 2020) and in rodents (Qiu et al., 2020a). Individuals with MDD who are non-responsive to SSRI treatment show either no change or an elevation of circulating cytokine levels (Syed et al., 2018) and display higher baseline tryptophan metabolism defined by kynurenine to tryptophan ratio (Sun et al., 2020). Previously, limited efficacy of the SSRI, fluoxetine (FLX), to decrease depressive-like behaviour, was commensurate with increased levels of hippocampal IL-1 and decreased circulating tryptophan concentrations (Qiu et al., 2020a). Thus, tryptophan catabolism via TKP may be disrupted by CORT and could be a potential biomarker of SSRI efficacy, and the present study sought to understand this relationship. Here, using a model of de novo PPD, we investigated the effects of maternal postpartum CORT, to induce depressive-like endophenotypes, and concurrent FLX to understand how these treatments may affect metabolites 74 in the TKP. We hypothesized that maternal postpartum CORT treatment will alter TKP metabolite concentrations and that postpartum FLX will have limited efficacy to inhibit these effects. Figure 3.1. Schematic overview of the tryptophan-kynurenine pathway. Proinflammatory cytokines such as interleukin (IL)-1, tumor necrosis factor (TNF)-, and interferon (IFN)-, can act on the enzyme indoleamine 2,3-dioxygenase and glucocorticoids such as corticosterone can act on the enzyme tryptophan 2,3-dioxygenase to increase the enzymatic metabolism of tryptophan to kynurenine. Proinflammatory cytokines also act on the kynurenine 3-monooxygenase metabolizing kynurenine into 3-hydroxykynurenine in the neurotoxic branch of the tryptophan-kynurenine pathway (reviewed in Maes et al., 2011l; Savitz, 2020; Troubat et al., 2020). 3.2 Methods 3.2.1 Animals Forty adult female Sprague-Dawley rats and six male rats (2.5 months old) were purchased from Charles River (Montreal, QC, Canada). Males were used as breeders, and co- 75 housed with two females until pregnancy (determined via sperm in daily vaginal lavage samples). All pregnant rats were undisturbed until parturition. Thirty-seven females were included in the study. Protocols were in accordance with ethical guidelines set by the Canadian Council for Animal Care and were approved by the University of British Columbia Animal Care Committee. 3.2.2 Drug treatments All methods are previously described in Qiu et al. (2020a). Briefly, all dams received daily injections postpartum, starting from two days after birth, for 22 days. All injections were given subcutaneously (s.c.) and occurred between 08:00-10:00. Dams received CORT (40 mg/kg, s.c.; Sigma-Aldrich, St. Louis, MO, USA) or its vehicle (OIL; sesame oil + 10% EtOH, 1ml/kg, s.c.) and either FLX (10mg/kg, s.c.; Sequoia Research Products, Pangbourne, UK) or its vehicle, dextrose (DXT; 5% dextrose in sterile water, 1ml/kg, s.c.). Dams were randomly assigned to one out of four treatment groups (OIL+DXT, n = 9; OIL+FLX, n = 10; CORT+DXT, n = 9; and CORT+FLX, n = 9). All animals underwent maternal observations from postnatal day (PD) 2-8 and then testing in the forced swim test (FST) on PD21-22 and these data were published previously (Qiu et al., 2020a). We found postpartum CORT reduced total nursing and increased time off the nest and postpartum FLX reversed these effects in the early postpartum. However, on PD23, we found in these rats that postpartum CORT increased immobility in the FST that was not rescued by FLX, an effect that was consistent with past research (Workman et al., 2016; Gobinath et al., 2018a). 3.2.3 Blood collection At least 2h after the last injection and immediately after the FST, all animals were euthanized by rapid decapitation, brains were extracted and trunk blood was collected within 76 three minutes of touching the cage. Plasma was collected in cold EDTA (made in house) coated microcentrifuge tubes, immediately placed on ice to help preserve contents in plasma, and centrifuged 4h later for 10min at 4°C. Data for 5 samples were not collected due to human error. We also examined inflammation in the periphery, hippocampus and frontal cortex and these results have been published elsewhere (Qiu et al., 2020a). Briefly, we found that postpartum FLX elevated hippocampal IL-1 and circulating CXCL1 and postpartum CORT reduced IFN- and TNF-. Here we examined plasma TKP metabolites. 3.2.4 Tryptophan-kynurenine metabolite analyses We quantified kynurenine (KYN), kynurenic acid (KYNA), xanthurenic acid (XA), anthranilic acid (AA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), riboflavin (vitamin B2) and its cofactor, flavin adenine dinucleotide (FAD), using isotope dilution liquid chromatography coupled with tandem mass spectrometry (ABSciex API4000; AB SCIEX, Framingham, MA, U.S.A.) based on a modified method by Midttun et al. (2005). The intra- and inter-assay CVs for the quantitation of these analytes were all < 7%. Using a novel exploratory approach, a neuroprotective-to-neurotoxic z score ratio was calculated to account for the balance between multiple putative neuroprotective metabolites or neurotoxic metabolites/cofactors: (zKYNA + zAA) / (z3-HK + z3-HAA + zB2). 3.2.5 Statistical analyses All metabolites and metabolite ratio data were analyzed using two-way general linear model of analysis of covariance (ANCOVA) with CORT (CORT or OIL) and FLX (FLX or DXT) as between-subject factors and estrous cycle stage as covariate, unless otherwise specified. Effect sizes were reported as Ƞp2 or Cohen’s d. Post hoc comparisons used Newman-Keuls. A priori comparisons were subjected to Bonferroni correction. Pearson’s product-moment 77 correlations were conducted between behaviour, and cytokine data, adrenal mass, and TKP metabolites. All statistical analyses were analyzed using Statistica software (v. 9, StatSoft, Inc., Tulsa, OK, USA). The covariate of estrous cycle stage and body mass did not significantly affect any analyses (all p’s ≥ 0.097). Outliers were removed when two standard deviations away from the mean, and this happened twice in the neuroprotective-to-neurotoxic ratio. Assumptions of normal distributions and homogeneity of variance were meet and verified with tests for normality and Barlett’s test for all dependent variables. 3.3 Results 3.3.1 Maternal fluoxetine decreased xanthurenic acid concentrations Maternal FLX treatment decreased plasma XA compared to vehicle DXT treatment (main effect of FLX: F(1, 29) = 6.009, p = 0.020, Ƞp2 = 0.172; Figure 3.2A), with no other significant main or interaction effects (all p’s ≥ 0.296). 3.3.2 Maternal corticosterone increased metabolite concentrations and fluoxetine failed to correct this shift. Maternal corticosterone with fluoxetine shifted the neuroprotective-to-neurotoxic balance towards neurotoxicity Maternal CORT increased plasma AA (main effect of CORT: F(1, 29) = 44.506, p < 0.001, Ƞp2 = 0.605), 3-HK (F(1, 29) = 9.282, p = 0.005, Ƞp2 = 0.242), and 3-HAA (F(1, 29) = 33.693, p < 0.001, Ƞp2 = 0.537) concentrations compared to vehicle-treated animals (Figure 3.2B-D), with no other significant main or interaction effects (all p’s ≥ 0.253). Maternal CORT also increased plasma KYNA (F(1, 29) = 17.731, p < 0.001; Ƞp2 = 0.379; Figure 3.2E) with no other significant effects (all p’s ≥ 0.747). CORT+FLX significantly lowered the neuroprotective-to-neurotoxic z score ratio compared to all other treatment groups, indicating a shift towards the 78 neurotoxic pathway (a priori all p’s ≤ 0.007; Cohen’s d = 1.785 compared to OIL+DXT, Cohen’s d = 1.372 compared to OIL+FLX, Cohen’s d = 1.963 compared to CORT+DXT; interaction between CORT and FLX, F(1, 27) = 3.906, p = 0.058, Ƞp2 = 0.126; main effect of CORT, F(1, 27) = 4.265, p = 0.049, Ƞp2 Ƞp2 = 0.136; main effect of FLX, F(1, 27) = 10.055, p = 0.004, Ƞp2 = 0.271, Figure 3.2F). There were no other significant effects on KYN or other metabolite concentrations (p = 0.410) 79 Figure 3.2. Effects of maternal corticosterone (CORT) and fluoxetine (FLX) on plasma tryptophan-kynurenine pathway metabolites. A. FLX significantly decreased plasma xanthurenic acid concentrations. B.-E. CORT treatment significantly increased plasma anthranilic acid concentrations (B.), plasma 3-hydroxykynurenine concentrations (C.), plasma 3-hydroxyanthranilic acid concentrations (D.), and plasma kynurenic acid concentrations (E.). F. CORT+FLX significantly decreased the neuroprotive-to-neurotoxic z score ratio compared to all other groups. *p < 0.05. Data represented in means + standard error of the mean, overlaid with individual data points, n’s = 7-10 per group. 80 3.3.3 Maternal corticosterone increased a coenzyme associated with the putative neurotoxic branch and fluoxetine failed to correct this shift. Maternal corticosterone with fluoxetine increased a cofactor associated with the putative neurotoxic branch Vitamin B2 and its cofactor, FAD, are critical for the enzymatic conversion of KYN to the neurotoxic metabolite 3-HK, playing an important role in the neurotoxic pathway of TKP. Maternal CORT increased plasma vitamin B2 (main effect of CORT: F(1, 29) = 5.070, p = 0.032, Ƞp2 = 0.149), with no other significant effects (all p’s ≥ 0.254, Figure 3.3A). CORT+FLX had significantly higher FAD than all other groups (all p’s ≤ 0.021; Cohen’s d = 1.718 compared to OIL+DXT, Cohen’s d = 2.166 compared to OIL+FLX, Cohen’s d = 1.325 compared to CORT+DXT; interaction between CORT and FLX, F(1, 29) = 3.888, p = 0.050, Ƞp2 = 0.118; main effect of CORT, F(1, 29) = 13.432, p = 0.001, Ƞp2 = 0.316; no significant main effect of FLX, p = 0.151, Figure 3.3B). No signifiant main or interaction effects were found on FMN concentrations, (all p’s ≥ 0.211). 3.3.4 Metabolite concentrations significantly correlated maternal depressive-like behaviour, maternal hippocampal TNF- concentratins, or relative adrenal mass Plasma flavin mononucleotide positively correlated with time spent immobile in the forced swim test (r(29) = 0.381, p = 0.038) (Figure 3.4A). Hippocampal levels of the proinflammatory cytokine, TNF- negatively correlated with both 3-HAA (r(29) = -0.405, p = 0.026) and AA (r(29) = -0.414, p = 0.023), Figure 3.4B-C. Relative adrenal mass negatively correlated with KYNA, (r(32) = -0.482, p = 0.004), 3-HK (r(32) = -0.565, p = 0.001), 3-HAA (r(32) = -0.663, p < 0.001), AA (r(32) = -0.682, p < 0.001), and FAD (r(32) = -0.506, p = 0.003), Figure 3.4D-H. There were no other significant correlations (all p’s ≥ 0.064). 81 Figure 3.3. Effects of maternal corticosterone (CORT) and fluoxetine (FLX) treatment on tryptophan-kynurenine pathway coenzyme and cofactor. A. CORT significantly increased plasma vitamin B2 concentrations. B. CORT+FLX significantly increased plasma flavin adenine dinucleotide (FAD) concentrations compared to all other groups. *p < 0.05. Data represented in means + standard error of the mean, overlaid with individual data points, n’s = 7-10 per group. 82 83 Figure 3.4. Correlations between tryptophan-kynurenine metabolites and maternal depressive-like behaviour, maternal hippocampal cytokine levels, or maternal relative adrenal mass. A. Plasma flavin mononucleotide positively correlated time spent immobile in the forced swim test. B. Plasma 3- hydroxyanthranilic acid (3-HAA) negatively correlated with hippocampal tumor-necrosis factor (TNF)- levels. C. Plasma anthranilic acid (AA) negatively correlated with hippocampal TNF- levels. D. Plasma kynurenic acid (KYNA) negatively correlated with relative adrenal mass. E. Plasma 3-hydroxykynurenine (3-HK) negatively correlated with relative adrenal mass. F. Plasma 3-HAA negatively correlated with relative adrenal mass. G. Plasma AA negatively correlated with relative adrenal mass. H. Plasma flavin adenine dinucleotide (FAD) negatively correlated with relative adrenal mass. 84 3.4 Discussion Maternal postpartum CORT increased two neurotoxic TKP metabolites, 3-HK and 3-HAA, and one coenzyme of the neurotoxic pathway, vitamin B2. However, postpartum CORT also increased two neuroprotective metabolites, KYNA and AA, without significantly affecting KYN concentrations. Further, maternal postpartum CORT with postpartum FLX shifted the neuroprotective-to-neurotoxic balance towards neurotoxicity and increased FAD, a coenzyme to drive conversion to the neurotoxic pathway. Maternal postpartum FLX treatment alone decreased plasma XA concentration. Together, these data indicate maternal postpartum CORT upregulated TKP towards the neurotoxic pathway, and this effect together with FLX increased neurotoxic signalling in the TKP. 3.4.1 Maternal corticosterone increased more putative neurotoxic metabolites and putative neurotoxic branch coenzyme/cofactor Maternal postpartum CORT increased plasma concentrations of neurotoxic metabolites, 3-HK and 3-HAA, which is consistent with findings using inflammation-induced depression models (Parrott et al., 2016; Tao et al., 2020). Inflammation-induced depression, via lipopolysaccharide, results in higher concentrations of 3-HK in the frontal cortex and hippocampus of male mice (Tao et al., 2020). Lipopolysaccharide-induced depressive endophenotypes are dependent on the activation of the neurotoxic pathway of TKP, via 3-HK, at least in male mice (Parrot et al., 2016). Maternal CORT also increased the neuroprotective metabolite AA, which is consistent with findings showing elevated peripheral concentrations in both male and female individuals with high risk for MDD and in an animal model of depression (Sakurai et al., 2020). Together these results suggest that maternal postpartum CORT activates the neurotoxic pathway of TKP, which shares similarities to other models of depression as well 85 as in individuals with MDD, and therefore may prove to be an effective therapeutic target in the future. KYNA concentrations are reduced in MDD (reviewed by Maes et al., 2011), however a meta-analysis revealed that lower KYNA concentrations with MDD were found when there was a higher percentage of male patients (Ogyu et al., 2018), indicating a possible sex difference in this effect. Indeed, higher gestational KYNA concentrations were found in women with the onset of postpartum depressive symptoms (Teshigawara et al., 2019). This latter finding is consistent with our findings that maternal postpartum CORT increased depressive-like endophenotypes (Qiu et al., 2020a) and in the present study increased plasma KYNA. It is possible the increases in both neuroprotective and neurotoxic metabolites by maternal postpartum CORT reflects higher TKP activation, which is upregulated by glucocorticoids in both rodents and humans (reviewed in Maes et al., 2011). Specifically, CORT can upregulate tryptophan 2,3-dioxygenase activity, increasing the breakdown of tryptophan to kynurenine (reviewed by Maes et al., 2011). This effect occurs as a first step in the TKP, prior to the division of metabolites into either the neuroprotective or neurotoxic pathway. Regardless, postpartum CORT increased activation of the neurotoxic pathway via elevated concentrations of two neurotoxic metabolites (3-HK and 3-HAA), and the neurotoxic branch coenzyme vitamin B2, and its cofactor FAD. Thus, higher activation of the neurotoxic branch may have contributed towards the CORT-induced depressive-like endophenotypes reported previously in these same animals (Qiu et al., 2020a). In this regard it is notable that flavin mononucleotide, a precursor to FAD, was positively correlated with passive coping in the FST. Future studies could use this as therapeutic target and/or increase the efficacy of FLX by targeting this pathway. 86 3.4.2 Maternal fluoxetine failed to reverse maternal corticosterone effects, and contributed towards a shift to neurotoxicity Previously, we reported in these same animals, a significant decrease in plasma tryptophan concentration and higher levels of the proinflammatory cytokine, IL-1 in the maternal hippocampus due to FLX treatment in the same animals (Qiu et al., 2020a). Higher levels of IL-1 can also upregulate TKP (reviewed in Maes et al., 2011), so this may have contributed to the current findings. Maternal postpartum FLX decreased plasma XA concentration, which is consistent with data in individuals with MDD (Colle et al., 2020). XA has not been specifically described as either neuroprotective or neurotoxic, but its downstream effects on the glutaminergic system have been implicated in psychiatric diseases (reviewed by Fazio et al., 2018). Moreover, maternal postpartum FLX with CORT increased FAD, a cofactor in the neurotoxic branch, and shifted the balance of the neuroprotective-to-neurotoxic ratio towards the neurotoxic branch. Previously, in these same animals, postpartum FLX failed to reverse depressive-like endophenotypes (Qiu et al., 2020a). Further, we found significant negative correlations between hippocampal TNF- levels and plasma 3-HAA and AA concentrations. Although this finding is partially inconsistent with a study in MDD individuals where TNF was positively associated with plasma TKP (Haroon et al., 2020), this other study used sex as covariate and did not consider treatment effects in their analyses. The present findings coupled with the results of higher IL-1 and reduced tryptophan concentrations with maternal postpartum FLX (Qiu et al., 2020a) may have contributed towards the limited efficacy of FLX previously reported in this model of de novo PPD. It is important to note that in this study we examined peripheral levels of TKP metabolites and given in the past we have seen some differences between circulating versus 87 neural levels of cytokines (Qiu et al., 2020a), it is very possible that brain levels of TKP metabolites may be different. However, Haroon and colleagues (2020) compared peripheral plasma TKP activation and metabolites were positively correlated with cerebrospinal fluid TKP activation and metabolites, indicating some consistency. Using postpartum CORT, we show a number of phenotypes that overlap with the human presentation of de novo postpartum depression (reviewed in Qiu et al., 2020b). As stated earlier, the efficacy for antidepressants is lower for those with postpartum depression compared to antepartum (Cox et al., 2016). Further, women with PPD, show early remission with SSRI treatment, which disappears with longer treatment (Sharp et al., 2010), suggesting short-term but not long-term efficacy of SSRIs. Our own findings mirror the human data as we have found that SSRIs reverse the maternal care deficits seen in the short term (PD 2-8) but do not reverse the effects on passive coping or on hippocampal integrity in the long term (PD 23) seen with postpartum CORT (Workman et al., 2016; Gobinath et al., 2018a; and Qiu et al., 2020a; reviewed by Qiu et al., 2020b). As perinatal depression is most common in women with a previous history of depression it would be important to determine if other PND models of antenatal depression similar effects on the TKP. 3.5 Conclusion Overall, high maternal postpartum CORT, which models women an onset of depression during the postpartum that show elevated cortisol levels, upregulated metabolites in the neurotoxic pathway of the TKP, consistent with the profiles of TKP in MDD. Maternal postpartum CORT also led to higher concentrations of neuroprotective metabolites, KYNA and AA, both of which have been associated with either MDD or PPD. This suggests that the association between TKP metabolites and depression may be an important therapeutic target. 88 Maternal postpartum FLX commensurate with maternal CORT increased TKP activation by increasing FAD concentrations and shifting the neuroprotection-to-neurotoxic balance towards the neurotoxic pathway of TKP. These results indicate that higher activation of metabolites of the neurotoxic TKP pathway may be a biomarker of depression and targeting this pathway may lead to novel therapeutic targets for PND and MDD. 89 Chapter 4: Maternal postpartum fluoxetine reduces hippocampal inflammation and neurogenesis in adult offspring with sex-specific effects of periadolescent oxytocin 4.1 Introduction The postpartum is a time of high susceptibility to de novo psychiatric disorders (Munk-Olsen et al., 2006). Perinatal depression (PND) is a highly heterogeneous disorder in which symptoms and remission can be varied dependent on the time of depression onset and whether or not there was a preexisting history of depression (Galea and Frokjaer, 2019; Qiu et al., 2020b). The most prominent risk factor for PND is a previous history of depression and in these cases, it is not advised to stop the use of antidepressants as this can also pose a risk to mother and child (Angelotta and Wisner, 2017). However, approximately 60% of those with depression onset during the postpartum period, or postpartum depression (PPD), are experiencing depression for the first time (Munk-Olsen et al., 2006; Wisner et al., 2013). PPD affects approximately 15% of mothers (Shorey et al., 2018). Thus, the majority of mothers with PPD present with de novo depression and unfortunately are untreated or inadequately treated, and even in those that are treated, only a small (3-5%) percentage reach full remission (Cox et al., 2016). Both PPD and its treatment have consequences for child development. Female children of mothers with PND are at higher risk for depression themselves (Weissman et al., 2006), and male children are at higher risk for antisocial behaviour disorders (Beck, 1999; Hay et al., 2010). It can be difficult to disentangle the effects of maternal depression and antidepressant treatment on child development 90 (Oberlander et al., 2009; Olivier et al., 2015). Thus, animal models offer an advantage as one can independently manipulate antidepressant treatment in an animal model of depression. Although selective serotonin reuptake inhibitors (SSRIs) are a common class of antidepressants prescribed to treat PPD (Latendresse et al., 2017), the safety and efficacy of SSRIs have been questioned in both the clinical and preclinical setting (Sharp et al., 2010; Workman et al., 2016; Gobinath et al., 2018a). SSRI treatments during the perinatal period have been linked to various neurodevelopmental outcomes in the exposed offspring, including altered brain development, changes in infant behaviour, and reduced executive function, but these effects often depend on a variety of factors, such as household stress (reviewed in Oberlander et al., 2009; Rotem‐Kohavi and Oberlander, 2017; Dhaliwal et al., 2017, 2020). Indeed, some effects of SSRI exposure in utero may be beneficial, as studies have shown accelerated language development (Weikum et al., 2012) and increased infant functional synchronicity and hyperconnectivity in auditory resting-state networks (Rotem-Kohavi et al., 2019). Although there is conflicting evidence, prenatal SSRIs have been associated with increased risk for autism spectrum disorder (ASD) (Croen et al., 2011; Gentile, 2015; Man et al., 2015; Fatima et al., 2018; Morales et al., 2018; but see Clements et al., 2015; Kobayashi et al., 2016). ASD is a developmental disorder characterized by persistent deficits in social abilities and anxiety (American Psychiatric Association, 2013) and is associated with enlarged volume in the hippocampus (Schumann et al., 2004; Dager et al., 2007; Groen et al., 2010; Chaddad et al., 2017; Reinhardt et al., 2020). Direct postnatal administration of SSRIs to preweaning offspring resulted in social deficits in adolescence and adulthood (reviewed by Gemmel et al., 2018). However, direct exposure of offspring to SSRIs does not mirror the human condition where 91 antidepressant treatment is administered to mothers. As such animal models of peripartum SSRI treatment of the dams in a model of PND would better imitate the human condition. PND is typically modelled using gestational stress to model antepartum depression, or postpartum stress/corticosterone (CORT) to model de novo PPD (for review see Qiu et al., 2020b). Gestational stress models depression onset during the antepartum and often use maternal perinatal (gestation and postpartum) SSRI administration (Gemmel et al., 2016; Haim et al., 2016). Maternal SSRIs (gestational and postpartum) with gestational stress enhance social investigation in females and increase play in males, coupled with reduced hippocampal plasticity in both sexes (Rayen et al., 2011; Gemmel et al., 2017, 2019). As noted above, de novo depression in the perinatal period is most common in the postpartum (Munk-Olsen et al., 2006). Thus, it is also important to understand the effects of maternal SSRI use in the postpartum in an animal model of de novo PPD in offspring. Maternal postpartum SSRI exposure reduced adult hippocampal neurogenesis (Gobinath et al., 2018b) and increased anxiety-like behaviour in male but not female adult offspring (Gobinath et al., 2016) with or without postpartum CORT. These studies suggest different phenotypes depending on the duration and timing of maternal SSRI use, sex, and whether depressive-like behaviours are induced during gestation or postpartum. Inflammation is associated with depression as individuals with either major depressive disorder or PND show elevated proinflammatory cytokines such as IL-6, IL-1, TNF- compared to healthy controls (Corwin et al., 2008; Howren et al., 2009; Dowlati et al., 2010; Haapakoski et al., 2015). Intriguingly, changes in levels of circulating proinflammatory cytokines can reflect treatment response in major depressive disorder (MDD), as responders to treatment show decreased or stabilized proinflammatory cytokine levels, but non-responders to treatment with MDD can show increased cytokines levels (Syed et al., 2018). We observed a 92 similar pattern in an animal model of de novo PPD, as we found that postpartum exposure to the SSRI, fluoxetine (FLX), increased IL-1β in the maternal hippocampus, coincident with limited efficacy to treat depressive-like endophenotypes (Qiu et al., 2020a). However, whether this has consequences on neuroinflammation in the offspring is not known. In studies of gestational stress, adult male offspring showed elevations in the proinflammatory cytokines IL-1 TNF-, and IL-6 in the hippocampus (Diz-Chaves et al., 2013; Zhang et al., 2016), but to our knowledge, no study has investigated the link between maternal perinatal SSRI exposure and neuroinflammation in the offspring. However, maternal immune activation influences neuroinflammation in the offspring with either increases or decreases in proinflammatory cytokines depending on timing and the paradigm used (reviewed in Gumusoglu and Stevens, 2019). Oxytocin (OT) is a neuropeptide that has been used in clinical trials for ASD (Guastella et al., 2010, 2015; Anagnostou et al., 2012; Domes et al., 2013; Tachibana et al., 2013). Although two previous meta-analyses found no significant effects of OT treatment on ASD symptoms (Ooi et al., 2017; Wang et al., 2019), a recent meta-analysis found intranasal OT significantly improved social functioning in individuals with ASD (Huang et al., 2021) (Huang et al., 2021). These discrepancies may be due to timing of OT administration relative to assessment, wide age range of the participants, and sex differences in the effects of OT. Although the mechanisms underlying potential effects of OT in treating ASD symptoms are not known, OT modulates anxiety (Yoshida et al., 2009; reviewed in Neumann and Slattery, 2016) and social behaviour (reviewed by Neumann, 2008), and regulates inflammation (Yuan et al., 2016; An et al., 2019) and adult hippocampal neurogenesis (Leuner et al., 2012; Duarte-Guterman et al., 2020). However, it is not known if these same relationships are seen in females. 93 In the present study, we sought to determine whether OT during pre-adolescence would influence adult neurogenesis, inflammation and behaviour and could be considered a viable candidate to offset any effects of maternal CORT and FLX treatments. As discussed above, both untreated maternal depression and perinatal SSRI exposure can have long-term consequences on offspring behaviour and neurogenesis in a sex-dependent manner, but the effects on offspring neuroinflammation are not known. In this study, we sought to examine the effects of maternal SSRI treatment in the postpartum on adult male and female offspring anxiety-like behaviour, social investigation, hippocampal neurogenesis, and neuroinflammation, in an animal model of de novo maternal PPD. Hippocampal neurogenesis is also modulated by inflammation (Zunszain et al., 2012), and has been linked to social and anxiety-like behaviours (Lagace et al., 2010; Mohammad et al., 2018), suggesting these outcomes are interrelated. However, most studies have used only male rodents. Methods that increase neuroinflammation (lipopolysaccharide treatment, high-fat diet, chronic ethanol) also increase anxiety-like behaviour and decrease hippocampal neurogenesis in male rodent (Pascual et al., 2015; Noronha et al., 2019; Gomes et al., 2020; Zheng et al., 2021). Given the effects of PND and SSRI treatment on offspring social development, we also tested the effects of periadolescent OT to offset outcomes in the offspring following maternal treatments and whether this treatment affects pubertal timing. Thus, in the present study, we examined the long-term effects of maternal CORT treatment, as a model of de novo maternal PPD, and concurrent SSRI treatment on offspring anxiety-like and social behaviour, adult hippocampal neurogenesis, and neuroinflammation. We expected that maternal CORT or FLX treatments, given their ability to alter neuroinflammation in the dams (Qiu et al., 2020a) would alter neuroinflammation in the 94 offspring. Moreover, we hypothesized that OT would be a viable therapeutic to offset any changes we saw with maternal treatments in the male and female offspring. 4.2 Methods 4.2.1 Animals Forty adult female Sprague-Dawley rats and six male rats (2.5 months old) were purchased from Charles River (QC, Canada) and used for breeding. However, only thirty-seven females were included in the final study due to pregnancy and birth-related complications (reabsorption/failure to maintain pregnancy or stillbirth/infanticide). All animals were initially housed in same sex pairs upon arrival in polycarbonate cages (24 x 16 x 46 cm) with aspen chip bedding. All males and females were housed in separate colony rooms. All animals were maintained on a 12 h: 12 h light/dark cycle (lights on at 7:00 am) and given standard rat chow (Jamieson's Pet Food Distributors Ltd, Delta, BC, Canada) and autoclaved tap water ad libitum. Dams were randomly assigned to one out of four experimental groups (described below), and males were utilized only as breeders. All protocols were in accordance with ethical guidelines set by Canada Council for Animal Care and were approved by the University of British Columbia Animal Care Committee. For an overview of experimental procedures, refer to Figure 4.1. 4.2.2 Dam breeding and offspring culling All parental breeding and culling protocols followed as previously described (Workman et al., 2016; and Qiu et al., 2020a). All maternal data were reported in Qiu et al., (2020a). Briefly, females were paired with breeder males overnight, and vaginal lavage samples were obtained in the morning. Females were deemed pregnant if sperms were present in lavage sample (considered gestational day (GD)1). All females were single-housed on GD1 and remained 95 undisturbed until birth except for weekly cage changes and weighing. Day of birth was considered postnatal day (PD) 0. All litters were culled on PD1 (the following day after birth), by randomly selecting 5 female and 5 male offspring per litter. Figure 4.1. Timeline of experiment in chapter 4. Briefly, dams were bred within the animal facility as previously reported (Qiu et al., 2020a). Maternal treatment of either oil vehicle or corticosterone (CORT) and/or dextrose vehicle or fluoxetine (FLX) were given daily (s.c.) to dams from postnatal day (PD) 2 to 23. Offspring were weaned from dams on PD 23. Offspring received daily injections (i.p.) of phosphate-buffered saline (PBS), oxytocin (OT), or oxytocin+TriozanTM (OT+T) for 10 days from PD 25-34. Offspring underwent behavioural testing in adulthood between PD 71-73. 4.2.3 Maternal drug preparations and treatments All treatments and protocols were administered as previously described in Qiu et al., (2020a). Briefly, dams received either corticosterone (CORT; Sigma-Aldrich, St. Louis, MO, USA; 40mg/kg, s.c.) or vehicle (OIL; sesame oil + 10% EtOH; 1ml/kg, s.c.) and either fluoxetine (FLX; Sequoia Research Products, Pangbourne, UK; 10mg/kg, s.c.) or vehicle (DXT; 5% dextrose in sterile H2O; 1ml/kg, s.c.). Thus, dams were randomly assigned into 1 out of 4 treatment groups (OIL-DXT, OIL-FLX, CORT-DXT, and CORT-FLX; n = 9-10/group). Dams 96 received the treatments from PD2 to PD23 (for 22 days) between 8:00-10:00am. All dams were separated from their litters for no more than 5min per day for treatment administrations. FLX and an emulsion of CORT were prepared every 3 days. The dose of CORT has reliably produced a depressive-like phenotype and reduced maternal care behaviour in dams (Brummelte et al., 2006; Brummelte and Galea, 2010; Workman et al., 2016; Gobinath et al., 2018a; Qiu et al., 2020a). CORT and FLX treatment were given to the dams in the postpartum period, to better recapitulate the human condition (SSRIs given to mothers not infants). Maternal CORT and FLX can influence offspring directly as they can be detected in breastmilk (Weissman et al., 2004; Brummelte et al., 2010) or indirectly via changes in maternal behaviour. 4.2.4 Offspring weaning Briefly, all offspring were weaned on PD23 and same-sex siblings were pair-housed and separated into different colony rooms by sex. Offspring were assigned to one of three treatment conditions upon weaning (Table S1): phosphate buffered saline (PBS), oxytocin (OT), or oxytocin in adjuvant with TriozanTM (OT+T; prepared by PO-Laboratories, Dorval, QC, Canada). Three pups per litter per sex were used in the experiment with 1 of each sex given vehicle, OT or OT+T treatment (described below). OT treatment or OT+T treated animals were paired with either a PBS-treated control or an untouched control. 4.2.5 Offspring drug preparations and treatments OT is a large neuropeptide that when peripherally-administered leads to a 0.002-0.003% penetration of the blood-brain-barrier (BBB) (Mens et al., 1983), which may be due to its relatively short half-life (approximately 3-9min) (Churchland and Winkielman, 2012) or metabolism by the gut (Ferrie et al., 2013) leading to reduced levels available to cross the BBB. Thus, OT may require a drug delivery system to lengthen its half-life for therapeutic use and 97 better its permeability across the BBB. Hence, we used a novel nanotechnology, TriozanTM, as the delivery system to facilitate OT across the BBB. TriozanTM is a nanomedicine delivery platform based on a high value-added chitosan derivative, N-trimethyl chitosan (TMC), commercialized by Ovensa Inc. TriozanTM is a form of TMC, which encapsulates and binds molecules such as OT to protect OT from degradation. It allows for the accumulation of therapeutics in targeted tissues with minimal loss. This allows for a more controlled release of OT, via effective penetration of the BBB. For offspring treatment, OT (0.5mg/kg, i.p.; Bachem AG, Bubendorf, Switzerland) were prepared by dissolving in sterilized 0.1M PBS. TriozanTM nanomedicine delivery platform was determined to have encapsulated free OT at 45-55% efficacy by high performance liquid chromatography (PO-Laboratories, Dorval, QC, Canada) with total concentration of 250.8g/ml corrected to 0.5mg/kg dosage (i.p.) using sterile PBS as previously described in Duarte-Guterman et al., (2020). Sterilized 0.1M PBS were prepared in lab and utilized as vehicle controls. All treatments were administered as daily injections (i.p.) for 10 days during the periadolescent period (PD 25-34) between 9:30-10:30am. As oxytocin injections can cause sedation-like effects (Uvnäs-Moberg et al., 1994; Hess et al., 2016; Duarte-Guterman et al., 2020), all offspring were monitored for 30min after injections for sedation-like effects using a 3- point scale: 0 = no sedation, 1 = mild sedation-like, 2 = complete sedation-like, at three different time points (5min, 10m, and 30min post-injection). OT has effects on muscles to both constrict and produce relaxation depending on the types of muscle and muscle tone (Altura and Altura, 1984; Wooldridge et al., 2002), and it is possible that the sedation-like effects seen after OT (Uvnäs-Moberg et al., 1994; Hess et al., 2016; Duarte-Guterman et al., 2020) are attributable to OT’s effects on muscles. 98 4.2.6 Offspring puberty analysis and anogenital distance ASD is a developmental disorder in which symptoms can manifest at an early age (Ozonoff et al., 2008), and given we were administering OT during periadolescence, we assessed various developmental measures (puberty, anogenital distance, and body mass development) throughout peri-adolescence and into adulthood in offspring. Pubertal development was assessed throughout the peri-adolescence period. Different puberty development observations were conducted in females versus males. In females, vaginal opening was observed and in males, testicle descension was observed. Observations were rated on a scale of 0-2. For females, 0 was rated as not open, and 2 was rated as fully exposed vaginal openings, thus reaching puberty criteria. For males, 0 indicated no visible testicles, and 2 indicated testicles fully descended into scrotum. Observations were taken throughout offspring treatment, daily for 10 days, and results were reported using this numerical scale. Additionally, anogenital distance was measured using a digital caliper throughout offspring treatment, daily for 10 days as a secondary measure of pubertal development. 4.2.7 Behavioural assessment All behavioural analysis occurred during adulthood (PD71-72). All behavioural tests were videotaped and later coded by blind experimenter using the BEST analysis software (Educational Consulting, Inc, Hobe Sound, FL, USA). Males and females were tested separately, and all chambers were cleaned with 70% isopropanol between animals. 4.2.7.1 3-chambered social investigation task Briefly, the apparatus consisted of three chambered arenas (100 cm × 40 cm × 40 cm; see schematic in Figure 4.3C). First, experimental animals were individually allowed to freely investigate the entire arena for 10 minutes prior to testing. The experimental animal was then 99 briefly restricted to the center chamber while a novel same sex conspecific animal (i.e. the stimulus rat) was placed under a wire cage on one of the side chambers (stimulus side; Figure 4.3C). An empty cage was placed on the opposite side (object side; Figure 4.3C). The experimental animal was then allowed 10 minutes of investigation time throughout the whole apparatus. Social investigation behaviour was quantified as the amount of time spent on the side of the chamber with the stimulus animal (in seconds). 4.2.7.2 Elevated plus maze Briefly, the apparatus consists of two open arms bisected by two closed arms enclosed by 40 cm opaque walls (each arm was 50 × 10 cm, and the center area was 10 × 10 cm), elevated 40 cm above ground. All animals were individually placed into the center of the apparatus facing an open arm and allowed to freely explore the apparatus undisturbed for 5 minutes. The time spent in open arm (in seconds) was quantified, and less time spent in the open arms was used as a measure of anxiety-like behaviour as previously described (Gobinath et al., 2016). 4.2.8 Tissue Collection and tissue processing On PD 74, all animals were decapitated, and trunk blood was collected within 3 minutes of entering the room. Brains were extracted and left hemispheres were dissected on a cold surface to obtain hippocampi (HPC). All left hemisphere HPC tissue were flash frozen immediately on dry ice after extraction and stored at -80°C until assayed for protein and cytokine levels. The right hemispheres of the brain were fixed in cold 4% paraformaldehyde overnight at 4ºC and then transferred to 30% sucrose in phosphate buffer and stored at 4ºC until sectioning. All right hemispheres were rapidly frozen and the HPC sectioned at 30 µm in a series of 10 using a freezing microtome (Leica, Richmond Hill, ON, Canada). Sections were stored in antifreeze 100 (30 % ethylene glycol/ 20% glycerol; Sigma) at -20ºC until processing. Adrenal glands were also extracted immediately after decapitation and weighed. 4.2.9 Vaginal cytology for estrous cycle assessment Vaginal cells were collected by lavage taken prior to decapitation. Cells were placed on glass slides, counterstained with cresyl-violet, and then examined to assess for estrous cycle stages based on McLean et al. (2012) and Cora et al. (2015). 4.2.10 Doublecortin Immunohistochemistry Immunohistochemistry for doublecortin (DCX) was conducted as previously described (Gobinath et al., 2016, 2017). DCX is an endogenous microtubule-associated protein (Nacher et al., 2001), that is expressed in immature neurons between 4 hours to 21 days after production in rats (Brown et al., 2003; Yagi et al., 2020). The morphology of DCX-expressing cells is used to determine the maturity stages (proliferative, intermediate, and post-mitotic) of new neurons (Plümpe et al., 2006). This gives an advantage over other methods of investigating neurogenesis because it allows for inference of the maturity of the cell based on morphology. Briefly, right hemisphere HPC sections were rinsed with 0.1M phosphate-buffered saline, then treated with 0.3% hydrogen peroxide (30 min). Tissue was rinsed then incubated in primary DCX antibody solution (1:1000, goat anti-DCX, Santa Crux Biotechnology, Santa Cruz, CA, USA) at 4˚C for approximately 24hrs. Rinsed tissue sections were then incubated in secondary antibody solution (1:500, rabbit anti-goat, Vector Laboratories, Burlington, ON, Canada) at 4˚C for approximately 24 h, then incubated in Avidin Biotin complex (ABC elite kit, 1:1000, Vector) for 4 h. Sections were developed using 3,3’-diaminobenzidine + nickel (DAB Peroxidase substrate kit, Vector), mounted on superfrost slides, dehydrated with increasing concentrations of ethanol, cleared with Xylene, and coverslipped with Permount (Fisher Scientific) for microscopy. DCX is expressed in 101 immature neurons (Nacher et al., 2001) and thus is used as a quantification for adult hippocampal neurogenesis. 4.2.11 Microscopy HPC sections (1/10th) were counted for DCX-expressing cells using the 40x objective of an Olympus C×22LED brightfield microscope. For DCX-expression, hippocampal regions were split into dorsal and ventral region because the dorsal and ventral regions have different functions (Fanselow and Dong, 2010; Kheirbek and Hen, 2011). Two hundred cells (n = 100 within the dorsal region; n = 100 within the ventral region) were randomly selected and classified based on morphological attributes for cell maturity analyses (Plümpe et al., 2006). Percentage of proliferative cells and postmitotic cells were analyzed. Two sections per region were randomly selected and counted by experimenters, blind to the treatment conditions. 4.2.12 Tissue homogenization Following previous protocols (Bodnar et al., 2017), the left hemisphere HPC was homogenized in cold lysis buffer using the Omni Bead Ruptor 24 (Omni International. Kennesaw, GA, USA). Immediately after homogenization, all samples were centrifuged at 4°C at 13.2 rpm for 20min. The supernatant was then collected and stored in -80 ˚C for future protein and cytokine analyses. 4.2.13 Multiplex cytokine electrochemiluminescence immunoassays and protein quantification Cytokine levels were quantified using a multiplex electrochemiluminescence immunoassay kit (V-PLEX Proinflammatory Panel 2, Rat) from Meso Scale Discovery (Rockville, MD, USA), which was used according to manufacturer’s instructions (described in Bodnar et al., 2017). Samples were run in duplicates, and the following 8 cytokines and 1 102 chemokine were concurrently quantified in each sample: interferon gamma (IFN-), interleukin (IL)-1-beta, IL-4, IL-5, IL-6, IL-10, IL-13, tumor necrosis factor alpha (TNF-) and the chemokine, CXCL1. Plates were read using a Sector Imager 2400 (Meso Scale Discovery), and data analyses were conducted using the Discovery Workbench 4.0 software (Meso Scale Discovery). Lower limits of detection were as follows (pg/mL) reported by individual plates: INF-: 0.885, 0.290, 0.265, 0.157, 0.218; IL-1: 4.23, 2.20, 4.38, 1.73, 1.83; IL-4: 0.573, 0.366, 0.217, 0.154. 0.145; IL-5: 36.0, 14.4, 16.9, 13.4, 11.6; IL-6: 6.27, 7.97, 5.78, 4.23, 3.83; IL-10: 3.02, 0.825, 0.850, 0.719, 1.130; IL-13: 0.880, 0.731, 0.384, 0.423, 0.405; TNF-: 0.707, 0.403, 0.229, 0.192, 0.181; and CXCL1: 6.12, 5.49, 1.02, 1.37, 0.433. Total protein levels were quantified in tissue homogenates in triplicates using the Pierce Microplate BCA Protein Assay Kit (Thermo Scientific Pierce Protein Biology, Thermo Fisher Scientific, Waltham, MA, USA), which was used as a covariate in all analyses. 4.2.14 Data Analyses All data were analyzed using four-way analysis of variance (ANOVA) unless otherwise specified, with maternal CORT (OIL, CORT), maternal FLX (DXT, FLX), offspring treatment (PBS, OT, and OT+T), and offspring sex (male, female) as between-subject factors. Total sedation-like scores were calculated (summation of all 10 injection day scores) and analyzed repeated measures ANOVA with the same between-subjects factors described above and post-injection time (5min, 10min, and 30min) as within-subject factors. Adrenal mass was analyzed relative to final body mass (adrenal mass / final body mass). Number of DCX-expressing cells and hippocampal volume were analyzed using repeated measures ANOVA with the same between-subjects factors as described above and region (dorsal, ventral) as the within-subjects factor. Cytokine analyses were analyzed using a four-way analysis of covariance (ANCOVA) 103 with maternal CORT (OIL, CORT), maternal FLX (DXT, FLX), offspring treatment (PBS, OT, and OT+T), offspring sex (male, female) as between-subject factors, and hippocampal protein concentrations as a covariate factor. As an exploratory approach, principal component analyses (PCA) were conducted to determine networks (components) that could explain cytokines level variances. Principal component scores for individual sample data for the first two components generated were then analyzed using ANCOVA models to derive information regarding the amount of variance accounted for by cytokine profiles within hippocampal tissue, modified from Eid et al. (2019). Effect sizes (partial eta squared (Ƞp2) and Cohen’s d) are reported for significant effects. Post-hoc comparisons used Newman-Keuls. A priori comparisons were subjected to Bonferroni correction. Pearson’s product-moment correlations were conducted between behaviour and adult hippocampal neurogenesis. All data ANOVA, ANCOVA, and correlation analyses were analyzed using Statistica software (v. 9, StatSoft, Inc., Tulsa, OK, USA). PCA data analyses were conducted using R (3.4.3) statistical analysis software with the FactoMineR package. All effects were considered statistically significant if p ≤ 0.05, trends are discussed if p ≤ 0.10. Outliers that fell more than 2 standard deviations away from the mean, were eliminated from the analyzes. 4.3 Results 4.3.1 Oxytocin with TriozanTM increased sedation-like behaviour and prolonged recovery from sedation-like behaviour compared to oxytocin alone in both sexes Offspring that received OT+T treatment showed higher sedation-like scores (p = 0.004, Cohen’s d = 0.434), and took longer to recover from sedation-like effects 10 and 30 min after injection (p’s ≤ 0.019, Cohen’s d’s ≥ 0.305) than offspring receiving OT treatment alone, 104 regardless of sexes (main effect of treatment, F(2, 145) = 934.761, p < 0.001, Ƞp2 = 0.928; significant interaction between time after injection and treatment, F(4, 290) = 97.212, p < 0.001, Ƞp2 = 0.573; Figure S1A-B). No other main or interaction effects were found (p’s ≥ 0.063). 4.3.2 Maternal corticosterone treatment accelerated pubertal onset in female offspring Females showed more indications of puberty development earlier than their male littermates, F(1, 152) = 34.922, p < 0.001, Ƞp2 = 0.187, and males showed higher percent change in anogenital distance, F(1, 148) = 9.576, p = 0.002, Ƞp2 = 0.061 (Figure 4.2A-B), regardless of maternal treatment. Due to the clear sex differences in the timing and physical puberty development between male and females (reviewed by Spear, 2000; Bell, 2018), we used different puberty development measurements between males and females, thus analyses were conducted separately by sexes. Female offspring of CORT-treated dams exhibited more indications of pubertal development earlier than females of OIL-treated dams (main effect of CORT, F(7, 74) = 9.213, p = 0.003, Ƞp2 = 0.111; Figure 4.2C, Figure S1C), however this effect was not seen in the male offspring (p = 0.732; Figure 4.2D, Figure S1D). No other main effects or interaction effects were reported in either sex on puberty development (p’s ≤ 0.178), nor were there any significant main effects or interactions in percent change of anogenital distance (p’s ≤ 0.096). 105 Figure 4.2. Sex differences in changes in puberty development during periadolescence (postnatal day 25-34). A. Females show more indications of puberty than males between PD25-34. B. Males had higher percent change in anogenital distance than females between PD25-34. C. Female offspring of dams treated with maternal postpartum corticosterone (CORT) showed more signs of puberty than females of dams treated with oil. D. No effect of maternal postpartum CORT in male offspring. *Indicates significance at p < 0.05. Data represented in means + standard error of the mean (SEM), overlaid with individual data points. 106 4.3.3 Maternal fluoxetine treatment increased body mass whereas oxytocin treatment reduced body mass in both sexes during periadolescence and in adult males Offspring of dams exposed to maternal FLX treatment alone (OIL-FLX) had higher percent change in body mass between PD25 and PD34 than all other groups (OIL-DXT, p = 0.025, Cohen’s d = 0.400; CORT-DXT, p = 0.030, Cohen’s d = 0.327; CORT-FLX, p = 0.005, Cohen’s d = 0.505; interaction effect: maternal CORT and FLX treatments, F(1, 149) = 6.345, p = 0.013, Ƞp2 = 0.041; Figure S2A). OT and OT+T treatment significantly lowered percent change in body mass during periadolescence from PD25 to PD34, compared to PBS treated offspring regardless of sex (main effect of treatment, F(2, 149) = 13.998, p < 0.001, Ƞp2 = 0.158; OT: p = 0.029, Cohen’s d = 0.308; OTT: p < 0.001, Cohen’s d = 0.713; Figure S2B). OT+T treatment reduced percent change in body mass from PD25 to PD34 compared to OT treatment, (p = 0.002, Cohen’s d = 0.444; Figure S2B). As expected, male offspring had higher percent change in body mass during periadolescence from PD25 to PD34 than female offspring (main effect of sex, F(1, 149) = 14.000, p < 0.001, Ƞp2 = 0.459; Figure S2C). There was a significant main effect of maternal CORT (F(1, 149) = 3.82, p = 0.05, Ƞp2 = 0.025) but no other significant main or interaction effects were found (p’s ≥ 0.123). As expected, males weighed significantly more than females on the final day of the experiment (main effect of sex, p < 0.001, F(1, 165) = 1638.960, Ƞp2 = 0.908; Figure S2D), OT and OT+T treatment significantly lowered final body mass in adulthood only in male offspring compared to PBS treated offspring (p = 0.014, Cohen’s d = 0.539; p = 0.002, Cohen’s d = 0.743, respectively; treatment by sex interaction, p = 0.032, F(2, 165) = 3.526, Ƞp2 = 0.041; Figure S2E-F). Females had significantly higher relative adrenal mass than males (main effect of sex, F(1, 107 164) = 9.020, p = 0.003, Ƞp2 = 0.052; Figure S2G). There were no other significant main effects or interaction effects (p’s ≥ 0.069). 4.3.4 Both maternal corticosterone and fluoxetine treatment alone decreased anxiety-like behaviour in offspring Maternal CORT-DXT or maternal OIL-FLX treatment but not CORT-FLX increased time spent in open arms, indicating less anxiety-like behaviour compared to the vehicle treatment group regardless of sex (p = 0.033, Cohen’s d = 0.578; and p = 0.012, Cohen’s d = 0.613, respectively; interaction effect between maternal CORT by FLX treatment, F(1, 167) = 11.044, p = 0.001, Ƞp2 = 0.062, Figure 4.3A, Figure S3A-B). Females showed less anxiety-like behaviour than males, regardless of maternal or offspring treatment group (main effect of sex, F(1, 167) = 7.992, p = 0.005, Ƞp2 0.046, Figure 4.3B). No other significant main effects or interaction effects were found (p’s ≥ 0.332). 4.3.5 Periadolescent oxytocin treatment increased social investigation in both sexes OT treatment increased social investigation compared to control treatment only in groups from dams that received maternal CORT treatment regardless of sex (compared to the OIL-PBS p = 0.042, Cohen’s d = 0.513; and compared to CORT-PBS p = 0.030, Cohen’s d = 0.844; significant interaction effect of maternal CORT treatment and offspring oxytocin treatment, F(2, 151) = 3.411, p = 0.036, Ƞp2 = 0.043; Figure 4.3D, Figure S3C-D). There was also an interaction between maternal CORT, maternal FLX treatment and offspring sex (F(1, 151) = 3.412, p = 0.067, Ƞp2 = 0.022), however no significant post hoc comparisons were found. There was a main effect of offspring OT treatment, F(2, 151) = 2.612, , p = 0.077, Ƞp2 = 0.033), but no other significant main effects or interaction effects (p’s ≥ 0.115). 108 Figure 4.3. Effects of maternal postpartum fluoxetine (FLX) treatment or corticosterone (CORT) treatment on offspring anxiety-like behaviour and social behaviour in adulthood. A. Maternal postpartum FLX alone and CORT alone increased time spent in open arms of the elevated plus maze, indicating lower anxiety-like behaviour in both sexes. B. Females spent more time in open arms of the elevated plus maze, indicating lower anxiety-like behaviour. C. Figure representation of the 3-chambered social investigation task. Social investigation is measured by the amount of time spent with stimulus animal. D. Maternal CORT treatment with offspring OT increased time spent with stimulus animal as a proxy for social investigation in the 3-chambered social investigation task in both sexes. *Indicate significance at p < 0.05 Data represented in means + standard error of the mean (SEM), overlaid with individual data points. 109 Fluctuating levels in gonadal hormones due to the estrous cycle have been suggested to influence anxiety-like behaviour and social behaviour (Frye et al., 2000), thus vaginal cytology to identify estrous cycle stage was determined. Estrous cycle stage was not affected by maternal or offspring treatment nor did it influence any behaviours measured. No main effect or interaction effects were found (p’s ≤ 0.127). 4.3.6 Oxytocin treatment increased neurogenesis in males but not in females. Maternal fluoxetine treatment reduced neurogenesis. Maternal corticosterone increased neurogenesis, whereas oxytocin, and oxytocin with TriozanTM reduced this elevation in neurogenesis by maternal corticosterone Regardless of hippocampal region, males treated with OT had significantly more DCX-expressing cells than males treated with PBS or OT+T (PBS: p = 0.030, one-tailed, Cohen’s d = 0.474; OT+T: p = 0.006, Cohen’s d = 0.810), but there was no significant effect of OT in females (p’s ≥ 0.473; OT treatment by sex interaction, F(2, 161) = 4.323, p = 0.014, Ƞp2 = 0.051), Figure 4.4E-F, Figure S4I-J. Maternal CORT increased DCX-expressing cells, regardless of region or sex, only in vehicle-treated groups (p = 0.007, Cohen’s d = 0.554; maternal CORT by OT treatment, F(2, 161) = 3.817, p = 0.024, Ƞp2 = 0.045), as this not seen in groups that were also treated with either of the formulations of OT (p’s ≥ 0.492; Figure 4.4G; Figure S4I-J). Maternal FLX reduced DCX-expressing cells only in the ventral dentate gyrus in the oil treated groups (compared to OIL-DXT, p < 0.001, Cohen’s d = 0.812; CORT-DXT, p < 0.001, Cohen’s d = 0.811; CORT-FLX, p < 0.001, Cohen’s d = 0.741; interaction between maternal FLX, maternal CORT and region, F(1, 161) = 9.614, p = 0.002, Ƞp2 = 0.056; Figure 4.4H; Figure S5I-J). There were also significant 110 interactions and main effects (interaction between maternal FLX treatment and region, F(1, 161) = 10.820, p = 0.001, Ƞp2 = 0.063; maternal CORT by region F(1, 161) = 6.114, p = 0.0145, Ƞp2 = 0.036. Significant main effects of sex, F(1, 161) = 17.010, p < 0.001, Ƞp2 = 0.095; maternal FLX exposure, F(1, 161) = 7.367, p = 0.007, Ƞp2 = 0.0434; maternal CORT exposure, F(1, 161) = 5.457, p = 0.021, Ƞp2 = 0.033 and dentate gyrus region, F(1, 161) = 646.353, p < 0.001, Ƞp2 = 0.801) but no other significant main effects or interaction effects were found (p’s ≥ 0.128). 4.3.7 Maternal fluoxetine reduced proliferative DCX-positive cells in males and post-mitotic cells in both sexes in the ventral dentate gyrus. OT increased post-mitotic DCX-positive cells only in males Maternal FLX reduced proliferative DCX-expressing cells only in the ventral dentate gyrus in males (compared to DXT, p = 0.001, Cohen’s d = 0.441; interaction between maternal FLX, sex and region, F(1, 159) = 6.638, p = 0.011, Ƞp2 = 0.040; Figure 4.4I-J, Figure S6A-B) and not females (p ≥ 0.415). There were also significant main effects of sex (F(1, 159) = 5.537, p = 0.020, Ƞp2 = 0.034 and dentate gyrus region, F(1, 159) = 294.373, p < 0.001, Ƞp2 = 0.649) but no other significant main effects or interaction effects were found (p’s ≥ 0.065). Regardless of hippocampal region, males treated with OT has significantly more post-mitotic DCX-expressing cells than males treated with PBS or OT+T (PBS: p = 0.025, Cohen’s d = 0.454; OT+T: p = 0.011, Cohen’s d = 0.723; interaction between treatment and sex, F(2, 159) = 4.252, p = 0.016, Ƞp2 = 0.051) but there were no significant effects of OT in females (p ≥ 0.557), Figure 4.4K-L. Maternal FLX reduced post-mitotic DCX-expressing cells only in the ventral dentate gyrus in the oil treated group, regardless of sex (compared to OIL-DXT, p < 0.001, Cohen’s d = 0.898; CORT-DXT, p < 0.001, Cohen’s d = 0.949; CORT-FLX, p < 0.001, Cohen’s d = 0.829; interaction between maternal FLX, maternal CORT and region F(1, 159) = 111 8.476, p = 0.004, Ƞp2 = 0.051, Figure 4.4M; Figure S6C-D). There were also other significant interactions and main effects (interaction between maternal FLX and region, F(1, 159) = 10.999, p = 0.001, Ƞp2 = 0.065; maternal CORT by region, F(1, 159) = 5.775, p = 0.017, Ƞp2 = 0.035; maternal CORT by FLX, F(1, 159) = 3.888, p = 0.050, Ƞp2 = 0.024; main effects of sex, F(1, 159) = 10.315, p = 0.002, Ƞp2 = 0.061; maternal FLX, F(1, 159) = 8.183, p = 0.005, Ƞp2 = 0.049; maternal CORT, F(1, 159) = 4.948, p = 0.027, Ƞp2 = 0.031 and dentate gyrus region, F(1, 159) = 423.614, p < 0.001, Ƞp2 = 0.727) but no other significant effects were found (p’s ≥ 0.101). 112 113 Figure 4.4. Effects of maternal postpartum fluoxetine (FLX) treatment or corticosterone (CORT) treatment and oxytocin (OT) and oxytocin with TriozanTM (OT+T) on offspring doublecortin (DCX)-positive cells in the dentate gyrus. A. Representative photomicrograph of a proliferative DCX-positive cell in the granule cell layer of the dentate gyrus in a female (black arrowhead). B. Representative photomicrograph of a proliferative DCX-positive cell in the granule cell layer of the dentate gyrus in a male (black arrowhead). C. Representative photomicrograph of post-mitotic DCX-positive cells in the granule cell later of the dentate gyrus in a female. D. Representative photomicrograph of post-mitotic DCX-positive cells in the granule cell later of the dentate gyrus in a male. E. Offspring OT and OT+T treatment had no effect on female offspring DCX-positive cells throughout the dentate gyrus. F. Offspring OT treatment increased DCX-positive cells throughout the dentate gyrus in male offspring. G. Maternal CORT treatment increased DCX-positive cells in the dentate gyrus in both sexes. OT and OT+T treatment normalized this effect. H. Maternal FLX treatment alone decreased DCX-positive cells in the ventral dentate gyrus in both males and females. I. Maternal FLX treatment had no significant effect on female offspring proliferative DCX-positive cells in the ventral dentate gyrus. J. Maternal FLX treatment decreased proliferative DCX-positive cells in the ventral dentate gyrus in male offspring. K. OT and OT+T treatment had no effect on female offspring post-mitotic DCX-positive cells across the dentate gyrus. L. OT treatment increased post-mitotic DCX-positive cells across the dentate gyrus in male offspring. M. Maternal FLX treatment alone decreased post-mitotic DCX-positive cells in the ventral dentate gyrus in both sexes. *Indicate significance at p < 0.05. Data represented in means + standard error of the mean (SEM), overlaid with individual data points. 4.3.8 In both sexes, maternal fluoxetine decreased interleukin-10, interleukin-13, and interferon-γ, in the hippocampus Maternal FLX treatment decreased IL-10, IL-13, and IFN- levels in the hippocampus of offspring in both sexes (main effect of FLX, IL-10: F(1, 146) = 5.509, p = 0.020, Ƞp2 = 0.036; IL-13: (1, 150) = 5.407, p = 0.021, Ƞp2 = 0.035; IFN-: F(1, 153) = 3.750, p = 0.05, Ƞp2 = 0.024), Figure 4.5A-C, Figures S7A-C, Figure S7G-I. Females had lower hippocampal TNF- levels than males (main effect of sex, F(1, 151) = 5.531, p = 0.020, Ƞp2 = 0.035; Figure 4.5D). No other 114 significant main or interaction effects were found on adult offspring IL-10, IL-13, IFN-, and TNF- levels (p’s ≥ 0.106). OT+T treatment lowered hippocampal IL-5 levels compared to PBS and OT treatment, (p = 0.031, Cohen’s d = 0.454, p = 0.046, Cohen’s d = 0.466, respectively; main effect of offspring OT treatment, F(2, 151) = 2.949, p = 0.050, Ƞp2 = 0.038; Figure 4.5E, Figure S7D, Figure S7J), but there were no other significant main or interaction effects were found on adult offspring IL-5 levels (p’s ≥ 0.163). No significant main or interaction effects were found on adult offspring IL-1, IL-4, IL-6, and CXCL1 levels. 4.3.9 Maternal corticosterone shifted the offspring hippocampal cytokine profile towards a more proinflammatory profile via interleukin-6-to-interleukin-10 ratio; and offspring periadolescent oxytocin and oxytocin with TriozanTM treatment normalized this shift To examine further changes in immune profile, IL-6:IL-10 cytokine ratios were determined as a proxy for the proinflammatory-to-anti-inflammatory balance (Wood et al., 2015; de Brito et al., 2016; Eid et al., 2020). Maternal CORT treatment increased IL-6:IL-10 ratio in the offspring hippocampus, which was reduced with both formulations of OT, regardless of sex (interaction between maternal CORT treatment and offspring OT treatment, F(2, 145) = 5.735, p = 0.004; Ƞp2 = 0.073; p’s ≤ 0.010; Cohen’s d = 0.487-0.837; Figure 4.5F; Figure S7E, Figure S7K). There are no other significant main or interaction effects (p’s ≥ 0.889). 115 116 Figure 4.5. Effects of maternal postpartum fluoxetine (FLX) treatment or corticosterone (CORT) treatment, and periadolescent oxytocin (OT) and oxytocin with TriozanTM (OT+T) treatment on offspring neuroinflammation. A. Maternal FLX treatment decreased offspring hippocampal IL-10 levels in both sexes. B. Maternal FLX treatment decreased offspring hippocampal IL-13 levels in both sexes. C. Maternal FLX treatment decreased offspring hippocampal interferon (IFN)- levels in both sexes. D. Females had lower tumor necrosis factor (TNF)- levels than males. E. OT+T treatment decreased offspring hippocampal interleukin (IL)-5 levels in both sexes. F. Maternal CORT treatment increased the IL-6:IL-10 ratio as an approximation of the proinflammation-to-anti-inflammation index, indicating a shift towards neuroinflammation. G. Maternal FLX treatment reduced overall sample loadings onto the first principal component found using the exploratory principal component analysis method. *Indicates significance at p < 0.05 Data represented in means + standard error of the mean (SEM), overlaid with individual data points. 4.3.10 Maternal fluoxetine treatment reduced overall immune signalling An exploratory approach was used to determine immune signalling profiles across all 9 cytokines/chemokine (Bodnar et al., 2017; Eid et al., 2020; Qiu et al., 2020a). Within the offspring hippocampus, the PCA method found the first two principal components explained 65.05% of the variation (component 1 explaining 45.32% and component 2, 19.73%). All nine cytokines and chemokines showed significant positive loadings onto component 1 (p’s < 0.001). IFN-, IL-1, and IL-6 showed significant positive loadings onto component 2 (p’s < 0.001); IL-13, IL-10, IL-5, and TNF-, showed significant negative loadings onto component 2 (p’s < 0.001) (Table 4.1). We analyzed sample scores onto principal component 1 and found that maternal FLX treatment significantly decreased the sample scores onto principal component 1 (main effect of FLX, F(1, 157) = 6.145, p = 0.014, Ƞp2 = 0.038; Figure 4.5G, Figure S7F, Figure 117 S7L). There were no other significant effects were found for principal component 1 (p’s ≥ 0.184) or principal component 2 (p’s ≥ 0.239). Variables Component 1 Component 2 IFN- *0.628 *0.706 IL-10 *0.815 *-0.333 IL-13 *0.839 *-0.283 IL-1 *0.527 *0.670 IL-4 *0.867 -0.002 IL-5 *0.772 *-0.374 IL-6 *0.528 *0.507 CXCL1 *0.251 0.081 TNF- *0.589 *-0.483 Table 4.1. Component loadings for the hippocampal cytokine variance of the first two components identified using principal component analysis (PCA). Networks are interpreted using significant loadings set by variables denoted by *. All denoted values are determined by statistically significant contributions of variables towards the specific component (network). *Indicate significance at p = 0.001. 4.3.11 Neurogenesis was correlated with social behaviour in females while neurogenesis was correlated with anxiety like behaviour in male Females had significant negative correlations of hippocampal neurogenesis with social behaviour across groups. Time spent with social stimulus negatively correlated with DCX-positive cells in the ventral (r(8)= -0.729, p = 0.026, PBS-OIL-DXT) and dorsal dentate gyrus (r(7) = -0.715, p = 0.046, PBS-CORT-FLX) in females (Figure S8A-B). There were no other significant correlations (all p’s ≥ 0.057). Interestingly, most of the negative and positive correlations of hippocampal neurogenesis with behaviour in males were with anxiety-like behaviour. In males, time spent in open arms in 118 EPM negatively correlated with DCX-positive cells in the ventral region (r(8) = -0.695, p = 0.038, PBS-CORT-DXT); r(8) = -0.671, p = 0.048, OT-CORT-DXT), Figure S8C-D. In OT-treated males, time spent in open arms in EPM positively correlated with number of proliferative DCX-positive cells in the dorsal dentate gyrus (r(8) = 0.670, p = 0.048, OIL-DXT), Figure S8E. In OT-treated males, time spent with social stimulus positively correlated with proliferative DCX-positive cells in the ventral dentate gyrus (r(9) = 0.884, p = 0.001, OIL-FLX), Figure S8F. There were no other significant correlations (all p’s ≥ 0.067). 4.4 Discussion Maternal postpartum CORT and/or FLX influenced neuroinflammation, neuroplasticity and behaviour of adult offspring and offspring treatment with oxytocin during periadolescence offset some of these alterations. Maternal postpartum FLX reduced anxiety-like behaviour, hippocampal neurogenesis, and hippocampal cytokine levels in adult offspring of both sexes. Similarly, maternal postpartum CORT reduced anxiety-like behaviour but unlike maternal postpartum FLX, it increased neurogenesis and the IL-6:IL-10 ratio in the hippocampus in the adult offspring of both sexes. Periadolescent OT elicited sex differences as OT treatments increased neurogenesis in adult males but not females, whereas OT treatments rescued the maternal FLX reduction in neurogenesis in adult females but not in adult male offspring. Periadolescent OT treatment interacted with maternal CORT to normalize the increased hippocampal IL-6:IL-10 ratio and to increase social investigation in both sexes. Lastly, the nanoparticle TriozanTM altered the pattern of findings from OT alone for behaviour, hippocampal IL-5 levels and neurogenesis. Together, we found that maternal postpartum CORT or FLX treatment affected various neurobehavioural outcomes in adult offspring, and periadolescence 119 OT treatment in these offspring showed some efficacy to ameliorate maternal treatment effects on these outcomes. 4.4.1 Maternal postpartum fluoxetine reduced offspring neuroinflammation and neurogenesis in the hippocampus as well as anxiety-like behaviour in both sexes in adulthood In our study, maternal FLX treatment reduced hippocampal neuroinflammatory signalling. Indeed, maternal postpartum FLX was associated with reduced PC1 scores, suggesting that 45% of the variability in neuroinflammation in the offspring was due to maternal postpartum FLX. Individually, maternal FLX treatment reduced three cytokines in the hippocampus of both sexes: the anti-inflammatory cytokine, IL-10, the proinflammatory cytokine, IFN- and the pleiotropic cytokine, IL-13. Together this suggests that maternal postpartum FLX treatment may be suppressing overall cytokine expression in adult male and female offspring. These results are interesting as the dams had higher hippocampal IL-1 and circulating CXCL1 levels due to FLX treatment (Qiu et al., 2020a), and taken together, the increased maternal inflammation corresponded with dampened inflammation in adult offspring. Intriguingly, somewhat consistent with our findings, peripartum immune challenges in dams can lead to a lower proinflammatory response in adult offspring after the offspring themselves have been exposed to a second lipopolysaccharide (LPS) immune challenge in offspring adulthood (Clark et al., 2019) or in periadolescence (Lasala and Zhou, 2007; Surriga et al., 2009). In our study, the behavioural battery may have acted as enough of a challenge to reduce inflammation in the offspring, akin to those findings. However, maternal immune challenges with LPS or polyinosinic:polycytidylic acid, without second immune challenges in offspring, often result in elevations in cytokine levels (Basta-Kaim et al., 2012; Garay et al., 2013; Purves-Tyson et al., 120 2019). To our knowledge, no other studies have examined the effects of maternal SSRIs treatment on offspring neuroinflammation. Maternal postpartum FLX treatment decreased adult hippocampal neurogenesis in male and female offspring in the ventral dentate gyrus. These findings are consistent with other studies indicating that postpartum maternal FLX reduced hippocampal neurogenesis in adulthood (Gobinath et al., 2016, 2018b; Rayen et al., 2015). However, maternal postpartum FLX does not always result in reduced neurogenesis and these effects may depend on age, sex, region of the hippocampus, whether or not gestational stress was present, and/or how many behavioural tasks preceded perfusions (Rayen et al., 2011, 2015; Gobinath et al., 2016, 2017, 2018b). Different outcomes in response to maternal FLX may also depend on the differences in the developmental trajectory of neurogenesis in the hippocampus in response to maternal FLX (Gobinath et al., 2017). Collectively, data indicate that maternal postpartum FLX treatment decreases neurogenesis in the hippocampus of adult male and female offspring, replicating previous studies (Gobinath et al., 2016, 2018b; Rayen et al., 2015). In the present study, maternal FLX reduced anxiety-like behaviour in both sexes in adulthood, partially consistent with previous results (Boulle et al., 2016a; Kiryanova et al., 2016). However, there are equivocal effects of postpartum FLX in the literature on anxiety-like behaviour that depend on age, sex, testing procedures or gestational stress, with studies showing increased anxiety-like behaviour in adult males (Gobinath et al., 2016), decreased in adult males (Boulle et al., 2016a; Kiryanova et al., 2016), no change in adult females (Boulle et al., 2016b; Kiryanova et al., 2017), or no change in adolescent males and females (Rayen et al., 2011). The variable effects of FLX during development is also seen in human populations that depend on various environmental factors, including maternal stress, genetic variations, and mother-child 121 interactions (for review, see Hutchison et al., 2020). The observed variability in anxiety-like outcomes may also lie in the order and number of behaviour tasks conducted as higher anxiety-like phenotypes appear to be more robust when tested first (Gobinath et al., 2016, 2018b). Furthermore, it is possible that the addition of 10-day injections in the periadolescent period in the current study may have contributed to the differences in anxiety-like phenotype between the current and previous studies (Gobinath et al., 2016, 2018b). In our previous studies, we have seen that increased anxiety-like behaviour with maternal FLX in male offspring only manifests when multiple (four) behavioural tests are done consecutively over multiple days (Gobinath et al., 2016) but not when only one test is done (Gobinath et al., 2018b). Intriguingly, studies in children have found something similar as overall household chaos affected the relationship between maternal SSRI exposure and executive function in the exposed children (Dhaliwal et al., 2020). It is possible the differences between this study and previous studies (Gobinath et al., 2016; 2018b) could be due to the number of behavioural tasks animals were tested in, which might have contributed towards overall “chaos” the animals experienced during the experiment. More research is needed to understand this relationship. Interestingly, neurogenesis parameters were coupled with behaviour in sex-specific ways, with females showing associations with social investigation but males showing associations with anxiety-like behaviour. This suggests that neurogenesis may be more tied to different behaviours between the sexes, as anxiety-like behaviour has been associated with neurogenesis in males in past research (Pascual et al., 2015; Mohammad et al., 2018; Noronha et al., 2019; Gomes et al., 2020; Zheng et al., 2021) but few studies have examined females. However, due to the number of correlations conducted, the results should be interpreted with caution. Together, our data with 122 previous studies, suggest that variability in anxiety-like and social behaviour may depend on age, sex, and previous experience. 4.4.2 Maternal corticosterone treatment increased offspring neuroinflammation and neurogenesis, and reduced anxiety-like behaviour in both sexes Maternal CORT increased adult offspring neuroinflammation, increasing the hippocampal IL-6:IL-10 ratio, indicating a shift towards the proinflammatory profile in both adult male and female offspring. This is similar to previous research in animal models of gestational stress, where maternal gestational stress increased proinflammatory cytokines, IL-1 IL-6, and TNF- mRNA expression in the hippocampus of adult male offspring (Diz-Chaves et al., 2013; Zhang et al., 2016). Interestingly, maternal CORT treatment decreased hippocampal TNF- and IFN-, two proinflammatory cytokines, in the dam as well as peripheral TNF- (Qiu et al., 2020a), but increased the proinflammatory ratio in the offspring hippocampus. Together, these data indicate that maternal peripartum stress or CORT can increase neuroinflammation in adult offspring. In the present study, maternal CORT increased hippocampal neurogenesis in both sexes, consistent with other studies using gestational stress or postpartum CORT (Rayen et al., 2015; Gobinath et al., 2016). However, gestational stress effects on hippocampal neurogenesis in offspring depend on the duration/intensity of gestational stress (Fujioka et al., 2006), where shorter durations of stress (3 days mid-gestation) increased neurogenesis in adult offspring, but longer gestational stress protocols reduced neurogenesis in adult offspring (Lemaire et al., 2000; Fujioka et al., 2006; Madhyastha et al., 2013; Liao et al., 2019). Similarly, maternal postpartum CORT can reduce neurogenesis in offspring that vary by age (Gobinath et al., 2017) and behavioural testing (Gobinath et al., 2018b). Collectively, short-term maternal stress or maternal 123 CORT treatment, depending on experience, can increase adult hippocampal neurogenesis in offspring. Female offspring of dams exposed to postpartum CORT treatment showed accelerated indications of puberty development compared to controls. This may share some similarities to findings as exposure to maternal depression and family stress were associated with early puberty maturation in adolescent girls (Rudolph and Troop-Gordon, 2010). Maternal postpartum CORT treatment decreased anxiety-like behaviour in both adult sexes. This contrasts with previous findings from our laboratory with no changes in anxiety-like behaviour due to maternal CORT (Gobinath et al., 2016; 2018b), and other studies using gestational stress that find increased anxiety-like behaviour in adult male offspring (Lordi et al., 2000; Patin et al., 2005; Boulle et al., 2016a, 2016b; Liao et al., 2019). In one study, decreased anxiety-like behaviour was seen after gestational stress, however, only in juvenile male, but not female, offspring (Baker et al., 2008). It is unclear why we saw decreased anxiety-like behaviour in this study, but it may have had to do with the periadolescent injections over ten days. This injection stress during periadolescence may have inoculated the offspring to further stress in adulthood, providing improved resilience to perturbations in environment. Indeed, early life stress can act as stress inoculation training that buffers stress response later in life (Parker et al., 2006; reviewed in Lyons et al., 2010). Nevertheless, the influence of stress and CORT in the perinatal period can have dramatically variable effects on anxiety-like behaviours that likely result from the environmental conditions during the testing period and during development. 124 4.4.3 Periadolescent oxytocin treatment reversed the effects of maternal fluoxetine or corticosterone treatment on various measures, and sex differences emerged with oxytocin treatment. OT and OT with TriozanTM normalized the shift towards neuroinflammation (IL-6:IL-10 ratio) induced by maternal CORT treatment in both male and female offspring hippocampus, consistent with the anti-inflammatory effects of OT (Yuan et al., 2016; An et al., 2019). This finding is also consistent with another study which showed that a single infusion of OT in adulthood reduced hippocampal proinflammatory gene expression in adult male mice offspring after maternal separation stress (Amini-Khoei et al., 2017). Periadolescent OT treatment increased neurogenesis in the hippocampus only in adult male offspring, suggesting that OT can have sex-specific effects to modulate neurogenesis in the hippocampus. This result is in partial agreement with previous literature, indicating OT treatment in adulthood increased neurogenesis in the ventral hippocampus of adult male rats (Leuner et al., 2012). Interestingly, OT and OT with TriozanTM normalized the increase in neurogenesis due to maternal CORT treatment in both male and females. OT alone increased social investigation in both male and female offspring but only when dams were treated with postpartum CORT. This finding is consistent with previous literature, indicating that central OT infusion increased social interaction only in those animals of mothers exposed to prenatal stress (Lee et al., 2007). In the human literature, OT has greater stress-buffering effects in women with early life adversity than placebo (Riem et al., 2020), suggesting again, that OT can work to influence behaviour in a context-specific manner. Together with our data, these effects suggest that oxytocin can boost social and stress interactions more so in the presence of early life adversity. 125 Furthermore, in addition to the sex differences found in neurogenesis due to OT treatment, we also found that only adult male offspring treated with OT and OT with TriozanTM weighed less than control-treated males. The reduced body mass gain may be due to the anorexigenic effects of OT (reviewed by Leslie et al., 2018). Interestingly, OT treatment elicited sex differences that were not present without periadolescent treatment. Sex differences are seen in OT mediated behaviours (Dumais et al., 2016; Duarte-Guterman et al., 2020; reviewed by Bredewold and Veenema, 2018; Caldwell, 2018) and in central OT and OT receptor expression (reviewed Dumais and Veenema, 2016; Duarte-Guterman et al., 2020). Thus, it is perhaps not surprising that we observed sex differences that were revealed by periadolescent OT treatment. 4.4.4 Possible explanations on differences between oxytocin and oxytocin with TriozanTM effects The nanoparticle, coupled with OT, did elicit some differences compared to OT alone. OT with TriozanTM induced changes in hippocampal IL-5, social behaviour, and sedation-like behaviour in both sexes. Coupled with TriozanTM, OT showed more robust effects in reducing hippocampal IL-5 in adult offspring and increasing sedation-like behaviour in both sexes than OT alone. However, the effects of OT on social behaviour and increasing hippocampal neurogenesis were only found with OT alone, which may be due to the interaction between the gut microbiome and OT (Erdman and Poutahidis, 2016). The gut microbiome is involved in regulating social behaviour (Stilling et al., 2018; reviewed by Sarkar et al., 2020) and adult hippocampal neurogenesis (Ogbonnaya et al., 2015; Möhle et al., 2016). Thus, it is possible that the effects of OT alone without TriozanTM may have been due to possible interactions between the gut microbiome and the brain. Further research is required to determine the role of the gut microbiome in the effects of OT peripheral injections. 126 4.5 Conclusion Overall, we show maternal postpartum CORT and/or FLX treatments had short- and long-term consequences on the offspring. We found that maternal treatments influenced female pubertal development and periadolescent body mass in the short-term and in the long-term we found that maternal treatments influenced body mass, anxiety-like and social behaviour, as well as hippocampal neurogenesis and neuroinflammation. Offspring periadolescence OT treatment showed efficacy to offset maternal treatment effects on social behaviour and adult hippocampal neurogenesis but not on puberty development or anxiety-like behaviour, and with limited effects on neuroinflammation (IL-6:IL-10 ratio). We found significant sex differences in the effects of periadolescence OT treatment on puberty in females and in adulthood in males on body mass and neurogenesis in the hippocampus. Intriguingly, maternal treatments that resulted in increased neuroinflammation in the dam were associated with dampened inflammation in the offspring and vice versa, suggesting that maternal neuroinflammation via maternal treatments has programming effects on the offspring neuroimmune system. Untreated PPD can result in cognitive and emotional disturbances in children that have long-lasting effects into adulthood. Therefore, it is important to determine safe and efficacious treatments for the mother while also improving child development outcomes. 127 Chapter 5: General discussions 5.1 Summary of experimental findings Throughout this dissertation I used an animal model of de novo PPD to investigate the effects of maternal CORT and antidepressant treatment (FLX) on the dam and offspring. The central goal was to identify potential mechanisms of FLX inefficacy to ameliorate the depressive-like endophenotypes that develop in the long-term. Specifically, I studied key inflammatory signalling markers and pathways that have been related to antidepressant inefficacy in othercontexts (Syed et al., 2019; Zhang et al., 2019; Benedetti et al., 2021). In Chapter 2 and 3, I report how maternal CORT induces depressive-like endophenotypes and concurrent maternal FLX treatment affects the maternal immune system, and the maternal tryptophan-kynurenine metabolic pathway. Chapter 4 investigates the effects of maternal treatments and periadolescent OT treatment on offspring outcomes in adulthood on social and anxiety-like behaviours, hippocampal neurogenesis and neuroinflammation. The major findings were: 1) postpartum FLX increased maternal hippocampal IL-1 plasma CXCL1 levels, and decreased plasma tryptophan concentrations, which coincided with limited efficacy to reduce depressive-like endophenotypes in the late postpartum (Chapter 2, Qiu et al., 2020a); 2) postpartum CORT increased more putative neurotoxic metabolites and coenzymes, and postpartum FLX with CORT shifted the tryptophan-kynurenine pathway neuroprotective-to-neurotoxic balance towards neurotoxicity (Chapter 3, Qiu et al., 2021a); 3) maternal FLX treatment decreased offspring adult hippocampal neurogenesis and hippocampal immune profile regardless of sex (Chapter 4, Qiu et al., 2021b); 4) periadolescent offspring oxytocin treatment ameliorated maternal CORT effects on adult offspring regardless of sex and 128 increased adult hippocampal neurogenesis in males only (Chapter 4, Qiu et al., 2021b). In summary, these data indicate maternal postpartum CORT can induce various depressive-like endophenotypes in dams similar to various clinical biomarkers of depression, and postpartum SSRI treatment shows limited efficacy in the long-term, which mirrors findings in humans with PPD (reviewed by De Crescenzo et al., 2014; Molyneaux et al., 2018; Sharma, 2017). Furthermore, maternal treatments have short- and long-term effects on offspring, and periadolescent OT treatment showed potential to negate many of the maternal CORT treatment effects. Collectively, using a preclinical animal model of de novo PPD, these findings suggest multiple pathways could be exploited to improve the efficacy of maternal SSRI treatments in the postpartum and one possible therapeutic to offset offspring outcomes following maternal postpartum depression. Corticosterone effects Antidepressant (fluoxetine) effects Depressive-like (passive coping) behaviour ↑ Was not efficacious Maternal care behaviour ↓ Was efficacious Hippocampal plasticity ↓ Was not efficacious Immune profile ↓ HPC IFN-, TNF-* ↑ HPC IL-1, plasma CXCL1* Vitamin B6-dependent tryptophan-kynurenine pathway* ↑ neurotoxic branch metabolites* ↓ Tryptophan, vitamin B6 cofactors, ↑ neurotoxicity* Table 5.1. Postpartum corticosterone model of de novo postpartum depression. Summary of findings in rat dams due to postpartum corticosterone or fluoxetine effects of studies presented in Chapter 2-3 as well as Workman et al., 2013, 2015; Gobinath et al., 2018. *Indicates novel findings based on work from Qiu et al., 2020, 2021b. 129 Maternal corticosterone effects Maternal antidepressant (fluoxetine) effects Offspring oxytocin effects Preadolescence (during 10-day injection period) Puberty scores ↑ in females only - - Anogenital distance - - - Sedation-like scores - - ↑ OT with TriozanTM than OT alone Change in body mass - ↑ ↓ Adulthood Final body mass - - ↓ Adrenal mass - - - Anxiety behaviour ↓ ↓ - Social behaviour - - ↑ OT with maternal corticosterone Neurogenesis ↑ ↓; ↓ proliferative immature neurons in males only ↑ in males only; Normalized maternal corticosterone effects Neuroimmune profile ↑ HPC IL-6:IL-10 ratio ↓ HPC IFN-, IL-10, IL-13 Normalized maternal corticosterone effects; ↓ HPC IL-5 OT with TriozanTM Table 5.2. Periadolescent oxytocin treatment in offspring in the postpartum corticosterone model of de novo postpartum depression and maternal antidepressant treatments. Summary of finding in offspring due to maternal treatments or offspring treatments presented in Chapter 4 (Qiu et al., 2021a). 130 5.2 Postpartum corticosterone model of de novo depression It is crucial to note that animal models do not fully recapitulate the human condition. However, it is equally important to note that both MDD and PND are heterogenous conditions (Goldberg, 2011; Galea and Frojaker, 2019) and that there is still much to understand about these conditions. In terms of translation from animal models to human disease, more research is needed to better understand both the models and the disease itself. Animal models can show face (similar symptoms to the human condition), construct (it should relate to underlying etiology of the human condition and biological markers), and predictive (treatment efficacy) validity. Indeed, I show the postpartum CORT model used in this thesis has face, construct, and predictive validity as there are several similarities to clinical de novo postpartum depression. First, this model consistently shows face validity as high CORT postpartum increases depressive-like endophenotypes that mimic various symptoms seen in the human condition, such as reduced maternal care behaviour and increased passive coping behaviour (Brummelte et al., 2006; Brummelte and Galea, 2010; Brummelte et al., 2010; Workman et al., 2016; Gobinath et al., 2016; Qiu et al., 2020a). Passive coping behavior as measured by immobility in the FST is not without some controversy. It has been interpreted as learned helplessness or behavioural despair (reviewed in Krishnan and Nestler, 2011), which may be representative of the depressed mood and hopelessness within a clinical phenotype of depression. However, it is important to acknowledge that passive-coping itself may be an adaptive behavioural response to preserve energy for survival (de Kloet and Molendijk, 2016). Much has been written about the interpretations of immobility in the FST and the reader is directed to excellent reviews on the subject (Krishnan and Nestler, 2011, Slattery and Cryan, 2012). What is accepted is that immobility in the FST has been used as an early (acute) screen for antidepressant efficacy (Cryan 131 et al., 2002; Salttery and Cryan, 2012) outside of any animal model of depression. In addition, all animal models of depression (CUS, CORT, withdrawal from hormone simulated pregnancy, bulbectomy) all result in increased immobility in the FST (Stoffel and Craft, 2004; Brummelte et al., 2006; Tasset et al., 2008; Wainwright et al., 2016; Mahmoud et al., 2016; Hu et al., 2017; Eid et al., 2020), an effect that is more prominent in rats compared to mice. Furthermore, on the interpretation of behavioural despair sickness behaviour, which is an adaptive response to conserve energy for healing, has been proposed to be depression-inducing, which supports the inflammatory hypothesis of depression (Dantzer, 2016). Together these findings suggest that although a behaviour (passive-coping) may be adaptive, the exacerbation of this adaptive behaviour may still represent an aspect of depressive-like behaviour. These findings suggest, whether or not immobility in the FST is called passive coping or behavioural despair, it has consistently with respect to increased levels in rat models of depression. Furthermore, in terms of construct validity (or similarities to biomarkers and physiological mechanisms), high CORT postpartum is associated with elevated cortisol levels seen in PPD (Iliadis et al., 2015) and reduces hippocampal integrity (Workman et al., 2013; Workman et al., 2016; Gobinath et al., 2016), which is also seen in MDD (Boldrini et al., 2019). The results from Chapter 2 and 3 add to construct validity for this model as I found increased neurotoxic tryptophan-kynurenine pathway metabolites, and reduced IFN- with postpartum CORT (Qiu et al., 2020a; Qiu et al., 2021a), both of which are seen in PPD (Corwin et al., 2015; Veen et al., 2016). Finally, our postpartum CORT model shows predictive validity, as SSRIs are effective in the short term by rescuing maternal care deficits, and not efficacious in the long-term (Workman et al., 2016; Gobinath et al., 2016; Qiu et al., 2020a), which reflects what is seen in the PND 132 literature (reviewed by De Crescenzo et al., 2014; Molyneaux et al., 2014; Sharma, 2017). Together, these studies highlight the major strengths of the postpartum high CORT model with good face, construct and predictive validity to model de novo PPD. It must be noted that some of my findings from Chapters 2 and 3 do not mimic the human condition (decreased TNF-, no effect on IL-6, and increased KYNA, a putative neuroprotective metabolite of the tryptophan-kynurenine pathway), as these biomarkers are related to PND (Boufidou et al., 2009; Wang et al., 2018). However, it is also important to know that there is a lack of literature on the biomarkers associated with de novo PPD. As such null or opposing results, from the literature in PND or MDD, should not be taken as deterrents, but as an understanding that conditions are complex. These findings can pave the way for future experiments to understand why these factors were not as predicted and whether is it the animal model or is it that de novo PPD may be expressed differently than PND. A strength of this model is that both the depression model (postpartum CORT) and FLX treatment are given to the dams directly, which reflects the clinical reality where treatments are given solely to the mothers. Moreover, any effects on the offspring are not directly due to manipulations in offspring but via the mothers, which again mimics the human condition. This may cause more variation in offspring results (Gobinath et al., 2016; Gobinath et al., 2018; Qiu et al., 2021b, Chapter 4) than direct manipulations in offspring as this tends to yield more consistent results (reviewed by Shah et al., 2018). It is important to understand that animal models should recapitulate the disease and, in this respect, it is worthwhile mentioning that there is variability in the effects of PND on children (reviewed by Grace et al., 2003; Santos et al., 2017; Farías-Antúnez et al., 2018; Slomian et al., 2019). In short, the postpartum CORT model 133 of de novo PPD has strengths that encapsulate the human PPD condition in symptoms, biomarkers, treatment efficacy, and the indirect exposure in offspring. 5.2.1 Pulsatile corticosterone release is modelled by injections Natural CORT release occurs in two patterns, ultradian and circadian in both humans and rodents (reviewed by Lightman and Conway-Campbell, 2010; Russell and Lightman, 2019; Spiga et al., 2015). Daily glucocorticoid release follows a diurnal circadian release pattern, where typically CORT release peaks in the morning of humans in preparation for waking, or prior to the active evening phase in rodents. In addition, glucocorticoids are also released in a daily ultradian pulsatile pattern. Due to this nature of CORT release, to better model the natural condition, ideally exogenous CORT treatments should follow pulsatile patterns. A single injection of CORT will result in an immediate increase in CORT and subsequent decline in plasma levels of CORT (Conway-Campbell et al., 2010; reviewed in Spencer and Deak, 2017), thus fulfilling a pulsatile daily pattern. Furthermore, daily injections of a single high dose of 40mg/kg CORT were found to be more effective in producing increased passive coping and sustained elevated morning serum CORT levels throughout the treatment period in non-partum female rats compared to either CORT in drinking water or CORT pellet implantations (Kott et al., 2016). Although silastic pellets are popular as a method of hormone treatment, they have non-uniform variable release in both females and males (Kott et al., 2016; Robinson et al., 2016). Thus, the data suggest daily CORT injections are a valid method to model elevated pulsatile CORT release, which possibly follow a pulsatile circadian pattern similar to what is naturally observed in naïve animals. Importantly, likely due to its normal pulsatile nature, CORT is often only effective, when given in a pulsatile fashion (Kalafatakis et al., 2018; reviewed by Lightman et al., 2020). 134 Pulsatile CORT can mediate emotional recognition better than continuous CORT (Kalafatakis et al., 2018). The effects of FLX to increase neurogenesis in the hippocampus in adult male rats is also dependent on pulsatile CORT (injections) as this effect is not seen when CORT levels are at high sustained levels (pellet CORT) (Huang and Herbert, 2006; AlAhmed and Herbert, 2008). Together data suggest pulsatile CORT is required for optimal treatment effects and daily CORT injections lead to better pulsatile release than other methods of administration. 5.3 Limited efficacy of fluoxetine in the postpartum Tachyphylaxis is defined broadly as previously effective pharmacological treatments that lose their efficacy after prolonged use (Cohen et al., 1968). Pharmacological antidepressants are prone to tachyphylaxis (Lieb and Balter, 1984). Specifically, loss of efficacy occurs as early as 6-weeks after initiation of treatment, leading to increased risk for depression recurrence and relapse in up to 57% of individuals (reviewed by Kinrys et al., 2019). In PND individuals, the remission rates are even lower, at only 3-5%, with slightly worse remission in those with postpartum onset (~3%) compared to those with antepartum onset (~5%) (Cox et al., 2016). Using the postpartum CORT model of de novo PPD, we find that the SSRI, FLX, reversed short-term deficits in maternal care but not the longer-term effects on passive coping or hippocampal integrity, indicating tachyphylaxis-like event in the postpartum in our model (Chapter 2; Workman et al., 2016; Gobinath et al., 2018a; Qiu et al., 2020a). In the late postpartum, neither FLX nor paroxetine are efficacious in reducing passive coping behaviour (Workman et al., 2016; Gobinath et al., 2018a; Overgaard et al., 2018; Qiu et al., 2020a, Chapter 2) or increasing the reduced hippocampal integrity (Workman et al., 2013, 2016; Gobinath et al., 2018a). FLX was however efficacious at rescuing maternal care behaviour 135 in the short-term in the early postpartum period (Workman et al., 2016; Gobinath et al., 2018a; Qiu et al., 2020a; Chapter 2). The same regimen of FLX is efficacious in adult male rats treated with high CORT (Huang and Herbert 2006) and in middle-age females after chronic unpredictable stress to increase hippocampal neurogenesis (Mahmoud et al., 2016). Collectively these results show there is a loss of efficacy with longer term use of SSRIs in the postpartum, suggesting that there is something unique about the postpartum that requires more attention to boost efficacy of SSRIs. The success of FLX to rescue reduced maternal care behaviour in the early postpartum (Qiu et al., 2020a; Chapter 2) is consistent with previous studies (Workman et al., 2016; Gobinath et al., 2018a). Peripartum (antepartum and postpartum) FLX treatment (>5mg/kg dose in drinking water), reversed gestational stress induced high passive coping behaviour and low sucrose preference in the early postpartum in dams (Salari et al., 2016; Amani et al., 2021; Bashiri et al., 2021). Together data suggest that SSRIs are indeed efficacious in the early postpartum, but similar to the clinical data (reviewed by De Crescenzo et al., 2014; Molyneaux et al., 2018; Sharma, 2017), it loses its efficacy in the long-term. One possible mechanism for the differences in efficacy may be due to hormone levels (estrogens and progesterone) in the early postpartum and late postpartum. Hormonal levels rise during the pregnancy and drop during the immediate to early postpartum (reviewed in Pawluski et al., 2009), and fluctuate in the lactation period depending on ovarian tissue or blood (Taya and Greenwald, 1982). Indeed, hormonal status modulates SSRI efficacy in humans and in rodents at least for some dependent variables (Zanardi et al., 2007; Mahmoud et al., 2016). However, we did not measure hormonal levels during the early or late postpartum period therefore it is uncertain if ovarian hormone status influences SSRI efficacy in this model of de novo PPD and should be considered in future work. 136 In Chapter 2 and 3, I found evidence to suggest inflammation and tryptophan metabolism may be associated with efficacy, which is discussed below. Interestingly, the limited efficacy in the late postpartum is only observed in the postpartum CORT model of de novo PPD. Using a gestational stress model of PND, postpartum citalopram or FLX treatment reduced depressive-like endophenotypes (passive coping behaviour, structural changes in the nucleus accumbens, and medial frontal cortex, elevated serotonin turnover rate in the frontal cortex) in late postpartum dams (Haim et al., 2016; Gemmel et al., 2016). Other studies have shown perinatal (gestational and postpartum) FLX treatment reversed gestational stress induced depressive-like behaviours in the late gestation and early postpartum as well as HPA hyperactivity in the late gestation (Salari et al., 2016). Together, data suggest SSRIs are more efficacious in the gestational stress model of PND. One explanation to why SSRIs efficacy differ, may be the differences in inflammatory profile between the two models (discussed in section 5.4.1) and/or hormonal status as noted earlier. 5.4 Inflammatory profiles influence efficacy Although the direct mechanism of how SSRIs reduce symptoms of depression are unknown, one proposed mechanism is via the anti-inflammatory properties of SSRIs (Dionisie et al., 2021). Moreover, differences in baseline immune profile may predict response to antidepressants (Zhang et al., 2019; Benedetti et al., 2021). In individuals with MDD, higher baseline IL-1, TNF- and C-reactive protein significantly predicted treatment non-response to antidepressant treatments (SSRI or serotonin-norepinephrine reuptake inhibitors) (Zhang et al., 2019; Benedetti et al., 2021). Interestingly, in another study, proinflammatory cytokines such as IL-1, TNF, IFN-, and IL-6 showed a continual increase in those who are non-responders to 137 SSRI treatment compared to a steady level or decline in responders (Syed et al., 2019). Together, these data suggest significantly different immune profiles at baseline and/or in response to treatment can predict or influence SSRI efficacy. Although, these data are in MDD cases, not PND, the data suggest crucial interactions between SSRIs and the immune system on treatment efficacy and future research should examine these potential interactions in PND. 5.4.1 Animal models of perinatal depression and inflammation In Chapter 2, using the postpartum CORT model of de novo PPD, I found significant reductions in two proinflammatory cytokines, TNF- and IFN- in the maternal hippocampus, and a reduction of TNF- in plasma (Qiu et al., 2020a). While depression is often associated with increases in proinflammatory cytokines, there are instances of reductions in cytokine expression as well. Interestingly our data match what has been show in individuals with PPD, as reductions in peripheral IFN- have also been reported (Corwin et al., 2015). Additionally, using a gestational stress model of PND, frontal cortex IL-1 and IFN- were increased in the late gestation but not early postpartum (Lenz et al., 2019). Together data suggests that both MDD and PND or model of MDD, PND can lead to potentially different cytokine signatures. Moreover, pregnancy and the postpartum have different immune signatures (Orsi et al., 2006; Eid et al., 2019), thus depending on timing of treatment, cytokine expressions are different, possibly modulated by differences in gonadal hormonal levels (Arakawa et al., 2014). One study has found unpredictable mild stress or high daily CORT treatment both induced depressive-like behaviour, increased serum TNF-, IL-1, and IL-6 levels compared to controls in adult male rats, and in the brain proinflammatory effects depended on the brain region (Zhao et al., 2019). In the postpartum CORT model, I report no significant changes in any 138 cytokines in the maternal frontal cortex (Chapter 2, Qiu et al., 2020a). High CORT is well known to dampen inflammatory responses (reviewed by Chakraborty et al., 2021), but the effects are not all encompassing. Despite postpartum CORT dampening inflammation based on2 cytokines in the hippocampus and one cytokine in plasma, it did not affect frontal cortex immune profile, illustrating the lack of a complete dampening. In future, examining network interactions across multiple regions may be a better approach than examining individual cytokines. Currently, to my knowledge, there are no published papers reporting the effects of SSRIs on cytokine milieu in the gestational stress model. Whereas in the postpartum CORT model, as reported in Chapter 2 (Qiu et al., 2020a), FLX increased hippocampal IL-1 and plasma CXCL1. Therefore, it is difficult to compare whether differences in the immune profile are underlying the differences in SSRI efficacy between animal models of PND and future studies are encouraged to investigate these effects. Future studies can test whether FLX induced elevated IL-1 is directly correlated with the limited FLX efficacy in the late postpartum. 5.5 Fluoxetine’s efficacy is associated with the tryptophan kynurenine pathway Tryptophan, a precursor for serotonin, is metabolized into downstream catabolites via the tryptophan-kynurenine pathway, and this pathway is stimulated by cytokines such as IL-1, TNF-, and IFN- (reviewed by Savitz, 2017). It is unclear in my studies, if higher activations of the tryptophan-kynurenine pathway and reduced tryptophan concentrations led to reduced serotonin availability and synthesis. In my studies, postpartum FLX reduced tryptophan concentrations (Chapter 2, Qiu et al., 2020a; Figure 5.1). Moreover, investigating other factors within the tryptophan-kynurenine pathway, postpartum FLX reduced two forms of vitamin B6, pyridoxal and 4’-pyridoxic acid, and XA levels (Chapter 3, Qiu et al., 2021a; Figure 5.1). Both 139 reductions in XA and low vitamin B6 status have both been associated with depression in humans (Hvas et al., 2004; Nanri et al., 2013; Colle et al., 2020; Kafeshani et al., 2020; Ryan et al., 2020; Arteaga-Henriquez et al., 2021). Additionally, I found significant elevations in putative neurotoxic (3-HK, 3-HAA) tryptophan-kynurenine pathway metabolites due to postpartum CORT treatment (Chapter 3, Qiu et al., 2021a; Figure 5.1). Higher putative neurotoxic metabolites are associated with depression (Savitz et al., 2015; Meier et al., 2016). Together, data suggest changes in tryptophan-kynurenine pathway seen in the postpartum CORT model are similar to endophenotypes seen in the human condition, and possibly contributed to depressive-like endophenotypes in the late postpartum (Chapter 2, Qiu et al., 2020a; Chapter 3, Qiu et al., 2021b). I also found maternal postpartum CORT elevated putative neuroprotective (KYNA, AA) metabolites (Chapter 3, Qiu et al., 2021a; Figure 5.1). It is crucial to note that although both KYNA (reviewed in Stone et al., 2012; Lovelace et al., 2017; Barone, 2019) and AA (Kanchanatawan et al., 2018) have been reported to be neuroprotective, there is evidence to suggest higher concentrations of these metabolites are associated with neuropsychiatric disease. Elevated levels of AA have been correlated with higher depression scores in female individuals, and not males (Steiner et al., 2021). Elevated KYNA concentrations have also been associated with other neuropsychiatric disease such as schizophrenia, multiple sclerosis, Parkinson’s disease, and bipolar disorder with psychotic episodes (Hartai et al., 2005a, 2005b; Erhardt et al., 2017; Plitman et al., 2017; Sellgren et al., 2019; Marx et al., 2020). It is possible, that the elevation in KYNA in CORT-treated animals is also a compensatory putative neuroprotection effect against all other putative neurotoxic effects or reflects the possibility that it is not neuroprotective in females as much of the research has not examined sex differences. 140 A major limitation of Chapter 3 is the lack of analysis of other downstream metabolites. Particularly, within the tryptophan-kynurenine pathway, the metabolite quinolinic acid has been cited to have putative neurotoxic properties (reviewed in Stone et al., 2012; Lovelace et al., 2017; Barone, 2019). Thus, we cannot fully conclude if postpartum FLX or CORT are completely shifting the tryptophan-kynurenine pathway metabolism towards putative neurotoxicity. It should also be noted that these neurotoxic or neuroprotective properties of tryptophan-kynurenine pathway metabolites are controversial, and are therefore described as “putative” and results should be interpreted with caution. Moreover, we also did not measure 5-hydroxytryptophan, serotonin, or any of its downstream metabolite concentrations via the serotonin synthesis stream (Figure 5.1) in Chapter 2 or 3. Interestingly, it should be noted that SSRIs increase expression of tryptophan hydroxylase, an enzyme in the serotonin synthesis pathway, in adult male rats (Kim et al., 2002; Shishkina et al., 2007; Klomp et al., 2014). Future studies are encouraged to investigate further in the serotonin synthesis pathway of tryptophan metabolism and the tryptophan-kynurenine pathway in this model of PPD and postpartum SSRI treatment to further strengthen these results. 141 142 Figure 5.1. Schematic diagrams of various metabolic pathways investigated and reported in Chapter 2 and 3. A. Schematic diagram of the tryptophan-kynurenine metabolic pathway and the serotonin synthesis pathway with a summary of maternal postpartum corticosterone or fluoxetine treatment effects reported in Chapter 2 and 3 (Qiu et al., 2020a; Qiu et al., 2021a). B. Schematic diagram of the vitamin B6, a coenzyme of the tryptophan-kynurenine pathway, with a summary of maternal postpartum corticosterone or fluoxetine treatment effects reported in Chapter 2 (Qiu et al., 2020a). C. Schematic diagram of the vitamin B2 metabolic pathway, a coenzyme of the tryptophan-kynurenine pathway, with a summary of maternal postpartum corticosterone or fluoxetine treatment effects reported in Chapter 3 (Qiu et al., 2021a). 143 5.5.1 Indole synthesis pathway of tryptophan metabolism by the gut microbiota The majority of systemic tryptophan is metabolized in the gut via the tryptophan-kynurenine pathway or the indole synthesis pathway (reviewed by Agus et al., 2018). Overall metabolism by the gut microbiome, by either pathway, can result in reduced availability of systemic tryptophan to be produced into serotonin (Gheorghe et al., 2019). Although the indole synthesis pathway has not gained as much attention as the tryptophan-kynurenine pathway, there is some evidence to suggest the indole synthesis pathway is highly involved in the pathoetiology of inflammatory conditions such inflammatory bowel disease (reviewed by Agus et a., 2018; Gheorghe et al., 2019) and obesity (Gheorghe et al., 2019). Inflammatory bowel disease and obesity are both highly correlated with depression (Preiss et al., 2013; Fond et al., 2014; Martin-Rodriguez et al., 2015; Choi et al., 2019). Overall, the gut microbiome has gained considerable attention in the field of neuropsychiatry and warrants further investigation in both depression pathoetiology and antidepressant efficacy. 5.6 Treatments in dams lead to variability in offspring anxiety-like behaviour Serotonin is a crucial neurotrophic factor during early development (Di Pino et al., 2004; Kepser and Homberg, 2015; Shah et al., 2018). Direct manipulations of the postnatal serotonin system using SSRIs in neonatal rodent offspring can have detrimental effects that lead to increased anxiety-like behaviour, depressive-like behaviour, and reduced social behaviours in adulthood (Shah et al., 2018). In Chapter 4, maternal FLX exposure reduced anxiety-like behaviour, in contrast to other papers where maternal postpartum FLX increased anxiety-like behaviour in adult male offspring (Gobinath et al., 2016) or had no effect (Gobinath et al., 2018b). Maternal perinatal SSRI treatment in the gestational stress model also has variable 144 effects on anxiety-like behaviour, as studies have shown either decreased anxiety-like behaviour (Boulle et al., 2016a; Kiryanova et al., 2016) or no change in anxiety-like behaviour in adulthood (Bourke et al., 2013; Ehrlich et al., 2015). These variability in outcomes following maternal SSRI treatments in animal models of PND mirror variability seen in human data (reviewed by Hutchison et al., 2020). Thus, regardless of whether literature is in human or animal models, maternal SSRI treatment can lead to variable effects in child and offspring anxiety-like behaviour. In humans, levels of household stress/chaos can moderate the effects of maternal perinatal depression and SSRI treatment on children (reviewed in Oberlander et al., 2009; Rotem‐Kohavi and Oberlander, 2017; Dhaliwal et al., 2017, 2020). Particularly, when household chaos was low, regardless of maternal SSRI treatment, maternal prenatal mood disturbances led to lower executive function in children compared to children of mothers without prenatal mood disturbances. However, when household chaos was high, maternal SSRI treatment led to lower executive function in children compared to children non-SSRI treated mothers (Dhaliwal et al., 2017, 2020). Although it is unclear why I saw decreased anxiety-like behaviour in Chapter 4 (Qiu et al., 2021b), it is possible periadolescent injections of oxytocin, are a stress inducing factor and led to stress inoculation and later resilience in those animals. Stress inoculation during adolescence, particularly when it is a repeated stress (Grissom and Bhatnagar, 2009), reduces passive coping behaviour and anxiety-like behaviour in adulthood in male rodents (Kendig et al., 2011; Suo et al., 2013; Brockhurst et al., 2015). In Chapter 4, all offspring animal were subjected to repeated 10 days of injection stress, which may have led to stress adaptation that resulted in reduced anxiety-like behaviour in adulthood. Collectively, data suggest that various “household” or colony disturbing chaos and stress, such as periadolescent injections and/or periadolescent 145 testing, could have functioned similar to household chaos may lead to different interactions between PND and SSRI on offspring. 5.7 Periadolescent effects of oxytocin OT is a large neuropeptide is released from the magnocellular cells in the hypothalamus and functions to modulate a variety of effects and sex-dependent outcomes (Dumais et al., 2016; Duarte-Guterman et al., 2020). A single OT infusion into the central nucleus of the amygdala increased social behaviours under the context of maternal gestational stress in males (Lee et al., 2007). In an early-gestational stress model in pigs, a single intranasal OT treatment reversed gestational stress induced reduction in passive coping behaviour, but had no effect without gestational stress in females (Rault et al., 2013). Similarly in Chapter 4, I found OT only increased social behaviour under the context of maternal postpartum CORT treatment. These are collectively intriguing results as they suggest that OT can have effects depending on the context, findings that mirror data in humans (Bartz et al., 2011; Rilling et al., 2012). I also report some sex-specific effects of periadolescent OT treatment, where OT increased neurogenesis in adult males and not females (Qiu et al., 2021b, Chapter 4). Additionally, preliminary work investigating the gut microbiome in the same offspring animals reported in Chapter 4, found sex-specific effects of OT on the gut microbiome (Qiu et al., in prep; Figure S9). In brief, oxytocin treatment decreased the abundance of Clostridium in adult female offspring but increased in adult male offspring (Qiu et al., in prep; Figure S9). Moreover, OT with TriozanTM decreased the abundance of Firmicutes in adult female offspring but had no effect in males (Qiu et al., in prep; Figure S9). The gut microbiome has also been suggested to regulate social behaviour (Stilling et al., 2018; reviewed by Sarkar et al., 2020) and adult 146 hippocampal neurogenesis (Ogbonnaya et al., 2015; Möhle et al., 2016). Thus, it is possible the sex-specific effect of OT on the gut microbiome is another mechanism that modulates social behaviour in females, whereas in males it may also depend on adult hippocampal neurogenesis in addition to the gut microbiome. Nevertheless, to confirm this hypothesis, further research is required. Together, these data suggest OT increases social behaviour, the mechanism through which those effects are achieved may depend on sex-specific system (neurogenesis, gut microbiome). Thus, further emphasising the importance of incorporating sex differences research in the preclinical field. 5.7.1 Outcomes in the immune system In offspring, IL-6:IL-10 ratio was used as a proxy for inflammatory-to-anti-inflammatory profile, indicating a shift towards the proinflammatory profile in both adult male and female offspring. Although IL-6 is a pleotropic cytokine, under the context of depression, neurogenesis, and the neuroendocrine system, it has been associated with proinflammation and can be reduced by anti-inflammatory agents (Abbasi et al., 2012, Kim et al., 2016). In this thesis, the term neuroinflammation have been used to indicate changes in inflammation in the brain, not always with a shift toward a proinflammatory profile. It is acknowledged that our assay also included various anti-inflammatory and pleotropic cytokines. Thus, depending on the signaling proteins uncovered our data is not exclusively indicative of neuro-proinflammation but changes in the neuroimmune signalling profile and cytokine/chemokine expression. 147 5.8 Future directions and clinical implications The serotonin system is implicated in depression (Oxenkrug, 2010; Fakhoury, 2016) and is altered by pregnancy and motherhood (reviewed by Lonstein, 2019; Pawluski et al., 2019). SSRIs function to prevent serotonin reuptake from the presynaptic synaptic cleft for degradation (Vaswani et al., 2003). However, given the low efficacy of SSRIs especially for postpartum depression (reviewed by De Crescenzo et al., 2014; Molyneaux et al., 2018; Cox et al., 2016; Sharma, 2017), the use of adjuvant treatments with SSRIs and other possible treatments should be studied. Here, in this thesis I found evidence that the inadequate efficacy of SSRIs to treat de novo PPD is associated with reduced tryptophan concentrations, higher inflammation, and higher putative neurotoxicity in the tryptophan-kynurenine pathway. However, as specified previously, it is not known if changes in tryptophan-kynurenine pathway and reduced tryptophan contribute towards low serotonin synthesis. Thus, future research should investigate the serotonin system and synthesis pathway in addition to tryptophan-kynurenine pathway and the indole synthesis pathway in this animal model of de novo PPD. Based on the increased maternal hippocampal IL-1 due to postpartum FLX (Chapter 2, Qiu et al., 2020a), and the association of SSRI efficacy and increased IL-1 observed in humans (Syed et al., 2019), future studies can investigate whether blocking inflammation can lead to increased SSRI efficacy. Specifically, using an IL-1 receptor antagonist such as anakinra, future studies can block IL-1 receptor mediated inflammation signalling to determine if FLX efficacy can be improved. As specified in section 5.5.1 previously, the gut microbiome is highly involved in tryptophan metabolism. A key future direction would be to understand how the gut microbiome affects serotonin synthesis, depression, and SSRI efficacy. There are suggestions that pregnancy 148 and postpartum can alter the human gut microbiome, although effects may depend on body mass, diet, or gestational weight gain (Koren et al., 2012; Edwards et al., 2017; Aatsinki et al., 2018). Nevertheless, investigation of natural changes in gut microbiome due to pregnancy and postpartum and how this may contribute to the onset of PPD can help both the understanding of PPD pathoetiology and the discovery of new treatments. Another future direction would be to investigate the effects of diet (calorie-dense, enriched versus regular) or dietary supplements (omega-3 fatty acid, vitamins, probiotics/prebiotics) on depressive-like endophenotypes and SSRI efficacy in the postpartum. Additionally, downstream metabolites of the tryptophan-kynurenine pathway can affect the glutaminergic system, which is also implicated in depression onset (reviewed by Savitz, 2020). Future studies are highly encouraged to consider the glutaminergic system in any studies using this animal model of de novo PPD. See Figure 5.2 for a summary of future directions. In offspring, future directions can include exploring the effects of maternal postpartum FLX on neonatal serotonin synthesis, metabolism, and transmission and how changes in this neurotrophic factor in early life affect long-term effects on offspring. Moreover, both the tryptophan-kynurenine pathway and the glutaminergic system is associated with ASD (Lim et al., 2016). Future studies can also incorporate analyses of the glutaminergic system in offspring. See Figure 5.3 for a summary of future directions. Increased risk for attention deficit hyperactive disorder has also been associated with perinatal SSRI exposure (Clements et al., 2015). Various animal tasks (Russell, 2011; Wickens et al., 2011) have been suggested to be reliable tests for attention deficit hyperactive disorder associated endophenotypes. Future directions can thus include those behavioural tasks when investigating offspring. 149 Given that findings on offspring anxiety with maternal SSRIs can vary dramatically it may be informative to test for anxiety-like behaviour under different parameters. Unpredictable events are more likely to induce sustained anxiety than predictable events (Grillon et al., 2004), especially if tasks are paired with an aversive event (reviewed in Ennaceur, 2014). Thus, modifying the EPM or using the light/dark box paired with an aversive unpredictable mild foot shock (Dias et al., 2013) may lead to a more reliable measurement of offspring anxiety-like behaviours. Furthermore, the autonomic system has been implicated in both anxiety (Hyde et al., 2019) and ASD (Kushki et al., 2013). Anxiety is also suggested to mediate the relationship between the autonomic nervous system dysfunction and ASD (Taylor et al., 2021), possibly with interactions of the gut microbiome (Kong et al., 2021). Thus, these relationships should be explored further in future studies. 150 Figure 5.2. An infographic summary of proposed future directions for dams and offspring. 151 5.9 Conclusions Overall, although animal models come with inherent limitations, the animal model of de novo PPD and postpartum antidepressant treatments also offer many advantages and strengths. The postpartum CORT model used in this thesis shows face (induces depressive-like behaviour), construct (changes in IFN-, and tryptophan-kynurenine pathway metabolites), and predictive validity (limited efficacy of SSRIs) (Chapter 2, 3; Qiu et al., 2020a, 2021a). I found evidence to suggest inflammation, tryptophan availability, and tryptophan-kynurenine pathway metabolism are associated with the limited efficacy of SSRIs (Chapter 2, 3; Qiu et al., 2020a, 2021a), all of which have been implicated in SSRI efficacy in a clinical setting (Syed et al., 2019; Zhang et al., 2019; Sun et al., 2020; Benedetti et al., 2021). Moreover, the method of administering both postpartum CORT, to induce depressive-like endophenotypes, and postpartum SSRIs to dams more closely mimics the human condition (Chapter 4; Qiu et al., 2021b). I also report the significance of using OT as a possible remedy to negate maternal CORT treatment effects in offspring, while having limited efficacy to reverse maternal FLX effects (Chapter 4; Qiu et al., 2021b). One notable caveat is the reduced anxiety-like behaviour seen in male and female offspring due to maternal CORT or FLX treatment (Chapter 4; Qiu et al., 2021b), which contradicted previous reports (Gobinath et al., 2016; Gobinath et al., 2018b). However, one possible explanation for this effect is adolescent stress inoculation. This, then opens up the possibility of investigating whether the behavioural effects of maternal treatments may be reversed by adolescent mild stress exposure for future experiments. In the clinical setting, both untreated maternal PND and perinatal SSRI treatment can have severe consequences in the children. Both the efficacy of SSRIs in mothers and the safety 152 of maternal SSRI treatment leading to exposure in children has been questioned. In this thesis, my data emphasizes that a) human maternal PPD can be modelled in rodents; b) there are possible mechanisms that may affect SSRI efficacy in an animal model of PPD (inflammation, tryptophan availability, tryptophan-kynurenine pathway metabolism); and c) there are possible remedies in offspring to ameliorate maternal treatment effects (OT, possibly adolescent stress inoculation). Collectively data presented here can serve as a foundation for future research in maternal PPD and SSRI treatment for better outcomes in both the mothers and her children. 153 Bibliography Aatsinki, A.-K., Uusitupa, H.-M., Munukka, E., Pesonen, H., Rintala, A., Pietilä, S., Lahti, L., Eerola, E., Karlsson, L., Karlsson, H., 2018. Gut Microbiota Composition in Mid-Pregnancy Is Associated with Gestational Weight Gain but Not Prepregnancy Body Mass Index. 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Brain, Behaviour, and Immunity 91, 505–518. https://doi.org/10.1016/j.bbi.2020.11.007 Ziegler, S.M., Feldmann, C.N., Hagen, S.H., Richert, L., Barkhausen, T., Goletzke, J., Jazbutyte, V., Martrus, G., Salzberger, W., Renné, T., Hecher, K., Diemert, A., Arck, P.C., Altfeld, M., 2018. Innate immune responses to toll-like receptor stimulation are altered during the course of pregnancy. Journal of Reproductive Immunology 128, 30–37. https://doi.org/10.1016/j.jri.2018.05.009 Zobel, A., Maier, W., 2010. Pharmacogenetics of antidepressive treatment. Eur Arch Psychiatry Clin Neurosci 260, 407–417. https://doi.org/10.1007/s00406-009-0091-4 Zohar, I., Shoham, S., Weinstock, M., 2016. Perinatal citalopram does not prevent the effect of prenatal stress on anxiety, depressive-like behaviour and serotonergic transmission in adult rat offspring. European Journal of Neuroscience 43, 590–600. https://doi.org/10.1111/ejn.13150 Zunszain, P.A., Anacker, C., Cattaneo, A., Choudhury, S., Musaelyan, K., Myint, A.M., Thuret, S., Price, J., Pariante, C.M., 2012. Interleukin-1β: A New Regulator of the Kynurenine Pathway Affecting Human Hippocampal Neurogenesis. Neuropsychopharmacology 37, 939–949. https://doi.org/10.1038/npp.2011.277 196 Appendices Appendix A Supplemental figures for Chapter 4 A.1 Figure S1 Figure S1. Sedation-like effects of oxytocin (OT) and oxytocin with TriozanTM (OT+T) after injections during the periadolescence period (postnatal day 25-34). A. OT+T treatment led to higher sedation-like effects overall compared to OT treatment. B. Animals treated with OT+T had slower recovery from sedation-like effects, indicated by higher sedation scores 10min and 30min post-injection compared to OT treatment. C. Female offspring of dams treated with maternal postpartum corticosterone (CORT) showed more signs of puberty than females of dams treated 197 with oil. D. No effect of maternal postpartum CORT in male offspring. *Indicates significance at p < 0.05. Data represented in means + standard error of the mean (SEM), overlaid with individual data points. A.2 Figure S2 This is Appendix A, Section 2. Figure S2. Effects of maternal postpartum fluoxetine (FLX) treatment on percent change in body mass during periadolescence (postnatal day 25-34), and sex differences in offspring oxytocin (OT) and oxytocin with TriozanTM (OT+T) on percent change in body mass during periadolescence, final body mass and adrenal mass. A. Maternal postpartum FLX treatment increased percent change in body mass in both sexes during postnatal day (PD) 25-34. B. OT and OT+T treatment reduced percent change in percent change in body mass in both sexes 198 between PD25-34. Offspring of both sexes treated with OT+T showed less percent change in body mass than OT treated offspring. C. Male offspring had higher percent change in percent change in body mass between PD25-34 than females. D. Male offspring had higher final body mass. E. Oxytocin treatment had no effect on final body mass in females. F. OT and OT+T treatment reduced final body mass in males. G. Females had higher relative adrenal mass than males. *Indicate significance at p < 0.05. Data represented in means + standard error of the mean (SEM), overlaid with individual data points. A.3 Figure S3 199 Figure S3. Effects of maternal postpartum fluoxetine (FLX) treatment or corticosterone (CORT) treatment on offspring anxiety-like behaviour and social behaviour in adulthood. A. Maternal postpartum FLX alone and CORT alone increased time spent in open arms of the elevated plus maze, indicating lower anxiety-like behaviour in females. B. Maternal postpartum FLX alone and CORT alone increased time spent in open arms of the elevated plus maze, indicating lower anxiety-like behaviour in males. C. Maternal CORT treatment with offspring OT increased time spent with stimulus animal as a proxy for social investigation in the 3-chambered social investigation task in females. D. Maternal CORT treatment with offspring OT increased time spent with stimulus animal as a proxy for social investigation in the 3-chambered social investigation task in males. *Indicate significance at p < 0.05. Data represented in means + standard error of the mean (SEM), overlaid with individual data points. 200 A.4 Figure S4 201 Figure S4. Effects of maternal postpartum corticosterone (CORT) treatment and oxytocin (OT) and oxytocin with TriozanTM (OT+T) on offspring doublecortin (DCX)-positive cells in the dentate gyrus. A. Representative photomicrograph of DCX-positive cells in dorsal dentate gyrus in a female control animal. B. Representative photomicrograph of DCX-positive cells in dorsal dentate gyrus in a male treated with phosphate-buffered saline (PBS) vehicle. C. Representative photomicrograph of DCX-positive cells in dorsal dentate gyrus in a male treated with OT. D. Representative photomicrograph of DCX-positive cells in dorsal dentate gyrus in a male treated with OT+T. E. Representative photomicrograph of DCX-positive cells in ventral dentate gyrus in a female control animal. F. Representative photomicrograph of DCX-positive cells in ventral dentate gyrus in a male treated with PBS vehicle. G. Representative photomicrograph of DCX-positive cells in ventral dentate gyrus in a male treated with OT. H. Representative photomicrograph of DCX-positive cells in ventral dentate gyrus in a male treated with OT+T. I. Maternal CORT treatment increased DCX-positive cells in both the dorsal and ventral dentate gyrus in female offspring. OT and OT+T treatment reversed this effect. J. Maternal CORT treatment increased DCX-positive cells in both the dorsal and ventral dentate gyrus in male offspring. OT and OT+T treatment reversed this effect. Offspring OT treatment increased DCX-positive cells grouping the dorsal and ventral dentate gyrus together in male offspring (no effect in females). *Indicated significant maternal CORT by offspring OT treatment interaction; #indicate significant OT treatmeny by sex interaction, significance at p < 0.05. K. Representative photomicrograph of DCX-positive cells in dorsal dentate gyrus in a PBS-treated female offspring of a dam treated with maternal postpartum oil vehicle. L. Representative photomicrograph of DCX-positive cells in dorsal dentate gyrus in a PBS-treated male offspring of a dam treated with maternal postpartum oil vehicle. M. Representative photomicrograph of DCX-positive cells in dorsal dentate gyrus in a PBS-treated female offspring of a dam treated with maternal postpartum CORT. N. Representative photomicrograph of DCX-positive cells in dorsal dentate gyrus in a PBS-treated male offspring of a dam treated with maternal postpartum CORT. O. Representative photomicrograph of DCX-positive cells in ventral dentate gyrus in a PBS-treated female offspring of a dam treated with maternal postpartum oil vehicle. P. Representative photomicrograph of DCX-positive cells in ventral dentate gyrus in a PBS-treated male offspring of a dam treated with maternal postpartum oil vehicle. Q. Representative photomicrograph of DCX-positive cells in ventral dentate gyrus in a PBS-treated female offspring of a dam treated with maternal postpartum CORT. R. Representative photomicrograph of DCX-positive cells in ventral dentate gyrus in a PBS-treated male offspring of a dam treated with 202 maternal postpartum CORT. *Indicate significance at p < 0.05. Data represented in means + standard error of the mean (SEM), overlaid with individual data points. A.5 Figure S5 Figure S5. Effects of maternal postpartum corticosterone (CORT) and maternal postpartum fluoxetine (FLX) treatment on offspring doublecortin (DCX)-positive cells in the dentate gyrus. A. Representative photomicrograph of DCX-positive cells in ventral dentate gyrus in a female offspring of a dam treated with maternal postpartum oil vehicle and maternal postpartum dextrose (DXT) vehicle. B. Representative photomicrograph of 203 DCX-positive cells in ventral dentate gyrus in a female offspring of a dam treated with maternal postpartum CORT and maternal postpartum DXT vehicle. C. Representative photomicrograph of DCX-positive cells in ventral dentate gyrus in a female offspring of a dam treated with maternal postpartum oil vehicle and maternal postpartum FLX. D. Representative photomicrograph of DCX-positive cells in ventral dentate gyrus in a female offspring of a dam treated with maternal postpartum CORT and maternal postpartum FLX. E. Representative photomicrograph of DCX-positive cells in ventral dentate gyrus in a male offspring of a dam treated with maternal postpartum oil vehicle and maternal postpartum dextrose (DXT) vehicle. F. Representative photomicrograph of DCX-positive cells in ventral dentate gyrus in a male offspring of a dam treated with maternal postpartum CORT and maternal postpartum DXT vehicle. G. Representative photomicrograph of DCX-positive cells in ventral dentate gyrus in a male offspring of a dam treated with maternal postpartum oil vehicle and maternal postpartum FLX. H. Representative photomicrograph of DCX-positive cells in ventral dentate gyrus in a male offspring of a dam treated with maternal postpartum CORT and maternal postpartum FLX. I. Maternal FLX treatment without maternal CORT decreased DCX-positive cells in the ventral dentate gyrus in female offspring. J. Maternal FLX treatment without maternal CORT decreased DCX-positive cells in the ventral dentate gyrus in male offspring. Representative figures are taken in male offspring unless otherwise specified. *Indicate significance at p < 0.05. Data represented in means + standard error of the mean (SEM), overlaid with individual data points. 204 A.6 Figure S6 Figure S6. Effects of maternal postpartum fluoxetine (FLX) treatment on offspring proliferative and post-mitotic doublecortin (DCX)-positive cells in the ventral dentate gyrus. A. Maternal FLX treatment had no effect on female offspring proliferative DCX-positive cells in the ventral dentate gyrus. B. Maternal FLX treatment decreased proliferative DCX-positive cells in the ventral dentate gyrus in male offspring. C. Maternal FLX treatment alone decreased post-mitotic DCX-positive cells in the ventral dentate gyrus in female offspring. D. Maternal FLX treatment alone decreased post-mitotic DCX-positive cells in the ventral dentate gyrus in male offspring. *Indicate significance at p < 0.05. Data represented in means + standard error of the mean (SEM), overlaid with individual data points. 205 A.7 Figure S7 206 Figure S7. Effects of maternal postpartum fluoxetine (FLX) treatment or corticosterone (CORT) treatment, and oxytocin (OT) and oxytocin with TriozanTM (OT+T) on offspring neuroinflammation. A. Females had lower tumor necrosis factor (TNF)- levels than males. B. Maternal FLX treatment decreased offspring hippocampal (interleukin) IL-10 levels in females. C. Maternal FLX treatment decreased offspring hippocampal IL-13 levels in females. D. Maternal FLX treatment decreased offspring hippocampal interferon (IFN)- levels in females. E. OT+T treatment decreased offspring hippocampal IL-5 levels in females. F. Maternal CORT treatment increased the IL-6:IL-10 ratio as an approximation of the proinflammation-to-anti-inflammation index, indicating a shift towards neuroinflammation in females. G. Maternal FLX treatment reduced overall sample loadings onto the first principal component found using the exploratory principal component analysis method in females. H. Maternal FLX treatment decreased offspring hippocampal (interleukin) IL-10 levels in males. I. Maternal FLX treatment decreased offspring hippocampal IL-13 levels in males. J. Maternal FLX treatment decreased offspring hippocampal interferon (IFN)- levels in males. K. OT+T treatment decreased offspring hippocampal IL-5 levels in males. L. Maternal CORT treatment increased the IL-6:IL-10 ratio as an approximation of the proinflammation-to-anti-inflammation index, indicating a shift towards neuroinflammation in males. M. Maternal FLX treatment reduced overall sample loadings onto the first principal component found using the exploratory principal component analysis method in males. *Indicates significance at p < 0.05. Data represented in means + standard error of the mean (SEM), overlaid with individual data points. 207 A.8 Figure S8 208 Figure S8. Correlations between neurogenesis and behaviour. A. Time spent with social stimulus negatively correlated with doublecortin (DCX)-positive cells in the ventral dentate gyrus in female offspring of the PBS-OIL-DXT group. B. Time spent with social stimulus negatively correlated with DCX-positive cells in the ventral dentate gyrus in female offspring of the PBS-CORT-FLX group. C. Time spent in open arms of the elevated plus maze (EPM) negatively correlated with DCX-positive cells in the ventral dentate gyrus in male offspring of the PBS-CORT-DXT group. D. Time spent in open arms of the EPM negatively correlated with DCX-positive cells in the ventral dentate gyrus in male offspring of the OT-CORT-DXT group. E. Time spent in open arms of the EPM positively correlated with proliferative DCX-positive cells in the dorsal dentate gyrus in male offspring of the OT-OIL-DXT group. F. Time spent with social stimulus positively correlated with proliferative DCX-positive cells in the ventral dentate gyrus in male offspring of the OT-OIL-FLX group. A.9 Figure S9 Figure S9. Effects of, offspring periadolescent oxytocin (OT) and oxytocin with TriozanTM (OT+T) on offspring neuroinflammation. A. Periadolescent OT+T decreased the abundance of Firmicutes, a phylum of 209 bacteria associated with inflammation in female offspring. B. No effect of OT nor OT+T treatment on Firmicutes abundance in male offspring. C. OT and OT+T decreased the abundance of Clostridium, a genus of bacteria associated with inflammation in female offspring. D. OT treatment increased Clostridium abundance in male offspring. 210 Appendix B Supplemental table for Chapter 4 B.1 Table S1 Maternal treatments Offspring treatment Female Male OIL-DXT PBS PBS PBS PBS 9 9 OIL-FLX 9 8 CORT-DXT 8 9 CORT-FLX 9 8 OIL-DXT OT OT OT OT 9 9 OIL-FLX 9 10 CORT-DXT 8 9 CORT-FLX 9 9 OIL-DXT OT+T OT+T OT+T OT+T 6 6 OIL-FLX 7 8 CORT-DXT 6 6 CORT-FLX 6 6 Table S1. Number of animals per treatment group.
Thesis/Dissertation
eng
Neuroscience
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Galea, Liisa A. M.
Perinatal depression and treatment implications for mother and offspring : lessons from an animal model
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