The long-term effects of prenatal stress and/or antidepressant exposure in rats Pubblico

Abstract

Pregnancy expands a woman's health considerations beyond herself to include her unborn child. Approximately 10-20% of all pregnant women experience depression during pregnancy and pharmacological intervention may be indicated in a substantial proportion of these women. The purpose of this dissertation was to develop a model of clinically relevant prenatal exposure to an antidepressant and maternal depression during pregnancy with the ultimate goal of evaluating the long-term effects of these prenatal exposures on the offspring. Female Sprague-Dawley rats were implanted with osmotic minipumps that were found to deliver clinically relevant exposure to the antidepressant escitalopram compared to daily injections used in most of the extant literature. Subsequently, pregnant females were exposed on gestational days 10-20 to a chronic unpredictable mild stress paradigm that was verified to cause an increase in baseline corticosterone. Maternal behavior was continuously monitored over the first 10 days post parturition but no substantial difference in maternal care (nursing, licking and grooming, or no contact) was observed due to maternal exposure to stress and/or escitalopram. The adult male offspring were analyzed to determine the long-term effects of prenatal exposures. Baseline physiological measurements were largely unaltered by prenatal manipulations. Behavioral characterization of the male offspring, with or without pre-exposure to an acute restraint stressor prior to testing, did not reveal any group differences. Prenatal stress exposure resulted in a faster return of serum corticosterone towards baseline following the peak response to an acute restraint stressor, but not an airpuff startle stressor, in adulthood. Gene expression analysis of select brain regions through microarray and real time PCR revealed no significantly regulated transcripts due to prenatal exposures. This model of maternal depression and its treatment indicate that escitalopram use and/or stress during pregnancy produced no alterations in our measures of male adult behavior or the transcriptome, however prenatal stress exposure resulted in some evidence for increased glucocorticoid negative feedback following an acute restraint stress.The role of stressor and drug dosing or timing in extant studies suggests that study design should be carefully considered before implications for human health are ascribed to prenatal exposure to stress or antidepressant medication.

Table of Contents

Table of Contents

CHAPTER 1: INTRODUCTION AND BACKGROUND 1
Introduction 2
Historical Perspective 2
Defining The Clinical Problem 3
Routes of Fetal and Infant Exposure 7
Role of Serotonin in Development 11
Human Exposure Studies 13
Monoamine and Hormone Studies 13
Genome Association Studies 15
Behavioral Studies in Neonates and Children 17
Case Reports 21
Growth, Developmental, Gross Anatomical and Physiological Outcomes 22
Associations with Respiratory Distress and Pulmonary Hypertension 24
Associations with Congenital Malformations 26
Summary of Human Exposure Studies 28
Animal Exposure Studies 30
Translation of Development Between Animals and Humans 30
Tricyclic/Tetracyclic Antidepressant Animal Studies 31
Selective Serotonin Reuptake Inhibitor Antidepressant Animal Studies 37
Tables 47
Figures 51

CHAPTER 2: MODELING MATERNAL DEPRESSION AND ITS TREATMENT 57
Abstract 58
Introduction 60
Materials and Methods 63
Animals 63
Ethics Statement 63
Acute and Chronic Escitalopram Administration (Cohort One) 65
Jugular Catheter Implantation 66
Chronic Unpredictable Mild Stress Model (Cohort Two) 66
Chronic Restraint Stress Model (Cohort Three) 67
Corticosterone Assays 69
Statistical Analyses 69
Results 71
Fluoxetine Pharmacokinetics after Acute and Chronic Dosing 71
Comparison of Escitalopram Delivery and Detection Methods (Cohort One) 72
Chronic Unpredictable Mild Stress (Cohort Two) 73
Chronic Restraint Stress Model (Cohort Three) 74
Maternal Care Behavior 75
Maternal Endpoints After Chronic Unpredictable Mild Stress 76
Tables 84
Figures 88

CHAPTER 3: PRENATAL EXPOSURE TO ESCITALOPRAM AND/OR STRESS IN RATS: LIMITED EFFECTS ON ENDOCRINE OR BEHAVIORAL MEASURES IN ADULT MALE RATS 98
Abstract 99
Introduction 100
Materials and Methods 102
Animals 102
Ethics Statement 103
Escitalopram Administration and Chronic Unpredictable Mild Stress Model 103
Physiological Measures 104
Behavioral Testing 105
Jugular Catheter Studies 106
CRF Receptor Binding of the Anterior Pituitary 106
Statistical Analyses 107
Results 108
Offspring Endpoints after Chronic Unpredictable Mild Stress and/or Escitalopram Exposure 108
Behavioral Characterization of Male Adult Offspring 109
Serial Blood Sampling after Acute Stress 110
CRF Receptor Binding in the Anterior Pituitary 111
Discussion 112
Tables 118
Figures 119

CHAPTER 4: PRENATAL EXPOSURE TO ESCITALOPRAM AND/OR STRESS IN RATS: NO EFFECTS ON GENE EXPRESSION MEASURES IN ADULT MALE RATS 123
Abstract 124
Introduction 125
Materials and Methods 128
Animals 128
Ethics Statement 129
Escitalopram Administration and Chronic Unpredictable Mild Stress Model 129
Gene Expression Studies 130
Statistical Analyses 132
Results 133
Microarray Analysis of the Amygdala, Hippocampus, and Hypothalamus 133
Real-Time PCR Analysis of the Hippocampus 134
Discussion 135
Tables 138
Figures 140

CHAPTER 5: GENERAL DISCUSSION AND CONCLUSION 148
Discussion 149
Future Directions 156

APPENDIX A: HYPOTHALAMIC AND HIPPOCAMPAL GENE EXPRESSION IN NEONATAL AND ADULT RATS AFTER PRENATAL ESCITALOPRAM EXPOSURE 159
Abstract 160
Introduction 161
Materials and Methods 163
Animals 163
Ethics Statement 163
Escitalopram Administration 164
Animals 164
Drug Treatment 164
Real Time PCR 165
Statistical Analyses 166
Results 167
Experiment 1: Weight Gain of Pregnant Dams 167
Hippocampal Gene Expression in Neonatal Rats Prenatally-Exposed to Escitalopram Via Continuous Exposure or Daily Injections 167
Experiment 2: Hypothalamic Gene Expression in Adult Male Rats Prenatally-Exposed to Escitalopram 168
Tables 169
Figures 172

APPENDIX B: HIPPOCAMPAL EPIGENETIC MODIFICATIONS IN ADULT RATS AFTER PRENATAL STRESS AND/OR ESCITALOPRAM EXPOSURE 179
Abstract 180
Introduction 181
Materials and Methods 183
Chromatin Immunoprecipitation Studies 183
Genomic DNA Isolation for 5-hydroxymethylcytosine Sequencing Studies 184
Sequencing of 5-hmC-Enriched and Input Genomic DNA 185
Sequence Alignment, Binning, and Peak Identification 186
Results 188
Optimization of Cross-Linking and Sonication Times for Hypothalamic Tissue 188
Elucidation of the H3K9-Ac and H3K9-Me3 Binding Sites in Genomic Regions of Target Genes 188
H3K9-Ac and H3K9-Me3 Histone Modifications Due to Prenatal Exposures in Genomic Regions of Target Genes 188
ChIP-Sequencing of Adult Hippocampal Tissue After Prenatal Stress 189
Tables 191
Figures 193
References 201
 
List of Tables
Table 1.1. Impact of Motherisk counseling on perception of teratogenic risk. 47
Table 1.2. Summary of endpoints after prenatal tricyclic/tetracyclic antidepressant exposure in animals 48
Table 1.3. Summary of endpoints after prenatal SSRI exposure in animals 49
Table 2.1. Summary of the chronic unpredictable mild stress model. 84
Table 2.2 Behavioral and corticosterone endpoints in pregnant rats exposed to the chronic restraint stress model. 85
Table 2.3. Behavioral and corticosterone endpoints in male and female offspring. 86
Table 2.4. Maternal and litter endpoints. 87
Table 3.1. Physiologic measures in adulthood. 118
Table 4.1. Primer sequences for real time PCR. 138
Table 4.2. Primer abbreviations and optimal primer concentrations for real time PCR. 139
Table A.A.1. Litter weights and male-female ratio after prenatal exposure to saline or escitalopram delivered through a daily injection or minipump administration. 169
Table A.A.2. Abbreviations and primer sequences for monoamine receptor, transporter, and neuropeptide targets. 170
Table A.A.3. Abbreviations and primer sequences for neurotrophic factor and transcription factor targets. 171
Table A.B.1. Chromatin immunoprecipitation primer sequences and concentrations. 191
Table A.B.2. Read summary of ChIP-Seq experiment 192

List of Figures
Figure 1.1. Use of antidepressants during pregnancy: 1996-2005. 51
Figure 1.2. Kaplan-Meier curves illustrating the time to relapse after discontinuation of antidepressants during pregnancy. 53
Figure 1.3. Percent of pregnant women taking prescription drugs arranged by the Food and Drug Administration labeling categories between 1996-2000 in the United States. 54
Figure 1.4. Representative autoradiographs of in utero exposure to SRIs. 55
Figure 1.5. Comparison of human and rat pharmacokinetics of the antidepressant escitalopram during pregnancy. 56
Figure 2.1. Determination of fluoxetine pharmacokinetics after acute subcutaneous dosing in non-pregnant females. 88
Figure 2.2. Dose-finding study to determine optimal dose of fluoxetine in pregnant rats. 89
Figure 2.3. Pharmacokinetic study to determine optimal vehicle and detection method for escitalopram in pregnant rats. 90
Figure 2.4. Serum drug concentrations following escitalopram administration. 91
Figure 2.5. Serum escitalopram concentrations compared to weight throughout pregnancy. 92
Figure 2.6. Corticosterone measurements during the chronic unpredictable mild stress model. 93
Figure 2.7. Maternal contact behavior following chronic unpredictable mild stress and escitalopram administration during pregnancy. 94
Figure 2.8. Maternal licking and grooming behavior and nursing behavior. 96
Figure 3.1. Timeline of chronic unpredictable mild stress and/or escitalopram administration during pregnancy and measurements in adulthood. 119
Figure 3.2. Behavioral characterization of adult male offspring prenatally exposed to stress and/or escitalopram. 120
Figure 3.3. HPA axis endocrine response following acute stress in adult male offspring prenatally exposed to stress and/or escitalopram. 121
Figure 3.4. CRF receptor binding in the anterior pituitary. 122
Figure 4.1. Average signal intensity across chip number in microarray data. 140
Figure 4.2. Heat map and hierarchical clustering of microarray results clustered by brain region. 141
Figure 4.3. Representative SAM analysis Q-Q plot. 142
Figure 4.4. Power analysis from SAM. 143
Figure 4.5. Hierarchical clustering of microarray results by specific brain region. 144
Figure 4.6. Real time PCR of hippocampal tissue from adult males. 145
Figure 4.7. Real time PCR of hippocampal tissue from adult male offspring with or without subchronic stress in adulthood. 146
Figure 5.1. Teratological relevance of animal studies versus clinical relevance of human studies. 158
Figure A.A.1. Weight gain during pregnancy of animals exposed to minipump delivery or daily injections. 172
Figure A.A.2. Hippocampal gene expression of serotonergic targets in neonatal rats. 173
Figure A.A.3. Hippocampal gene expression of neuropeptidergic targets and Bdnf in neonatal rats. 174
Figure A.A.4. Monoamine receptor and transporter gene expression in the hypothalamus of adult male animals prenatally exposed to escitalopram. 175
Figure A.A.5. Neuropeptide target gene expression in the hypothalamus of adult male animals prenatally exposed to escitalopram. 176
Figure A.A.6. Neurotrophic factor and associated target gene expression in the hypothalamus of adult male animals prenatally exposed to escitalopram. 177
Figure A.A.7. Transcription factor gene expression in the hypothalamus of adult male animals prenatally exposed to escitalopram. 178
Figure A.B.1. Optimization of cross-linking and sonication times for H3K9-Ac hypothalamic tissue. 193
Figure A.B.2. Approximate binding sites of acetylated and trimethylated H3K9 histone. 194
Figure A.B.3. Histone modifications in the Crh promoter region. 195
Figure A.B.4. Histone modifications in the Nr3c1 promoter region. 196
Figure A.B.5. Histone modifications in the Slc6a4 promoter region. 197
Figure A.B.6. Genomic features of 5-hydroxymethylcytosine DNA in adult male hippocampal tissue. 198
Figure A.B.7. Chromosome-wide distribution of 5-hydroxymethylcytosine sites. 199
Figure A.B.8. MACS peaks analysis of the adult male hippocampus from controls or animals prenatally exposed to stress. 200

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience Health Sciences, Pharmacology
Parola chiave	HPA neuropsychopharmacology epigenetics hypothalamic-pituitary-adrenal axis animal pharmacokinetics glucocorticoids animal models of depression prenatal stress prenatal antidepressant prenatal SSRI
Committee Chair / Thesis Advisor	Nemeroff, Charles B, University of Miami Owens, Michael J, Emory University
Committee Members	Miller, Gary W, Emory University Weinshenker, David, Emory University Howell, Leonard, Emory University

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