University of Cincinnati

U UNIVERSITY OF CINCINNATI Date: I, , hereby submit this original work as part of the requirements for the degree of: in It is entitled: Student Signature: This work and its defense approved by: Committee Chair: Approval of the electronic document: I have reviewed the Thesis/Dissertation in its final electronic format and certify that it is an accurate copy of the document reviewed and approved by the committee. Committee Chair signature: The role of serotonin in brain development and 3,4- methylenedioxymethamphetamine-induced cognitive deficits A Dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in the Graduate program in Molecular and Developmental Biology of the College of Medicine May 2009 by Tori Lynn Schaefer B.S., College of Mount St. Joseph 2004 Committee Chair: Michael T. Williams, Ph.D. Charles V. Vorhees, Ph.D. Kenneth Campbell, Ph.D. Gary A. Gudelsky, Ph.D. Steve Danzer, Ph.D. ABSTRACT Serotonin (5-hydroxytryptamine, 5-HT) is thought to be important during brain development and is one of the first neurotransmitters to appear. It appears to act as a neurotrophic factor supporting the growth and maturation of both serotonergic and non- serotonergic cells during the pre and early postnatal periods prior to its role as a neurotransmitter. Disruption of 5-HT functioning during human development is thought to be associated with autism and schizophrenia. Early developmental exposure to stress or drugs of abuse disrupt 5-HT development and produces altered cognitive ability. To better understand the relationship between the developing serotonergic system and long- term cognitive function we employed the use of a genetic model in which 5-HT levels are depleted ~80% throughout life and the use of a model of 3,4- methylenedioxymethamphetamine (MDMA) exposure in which administration from postnatal day (P)11-20 is used; a period analogous to the second half of human gestation. Pet-1 is a transcription factor that is restricted in the brain to 5-HT neurons and has been shown to be important for their development and function. A loss of Pet-1 results in an 80% reduction of the number of 5-HT neurons as well as 5-HT tissue content. Pet-1 knockouts were tested as adults in both the Cincinnati and Morris water mazes that assess path integration and spatial learning, respectively. A reduction in cognitive ability was not observed in Pet-1 knockout mice, although they displayed decreased locomotor activity, increased marble burying, and increased startle reactivity. To better assess the role 5-HT plays during development for circuits involved in memory formation, we used a model of MDMA exposure that has previously been reported to produce protracted path integration and spatial learning deficits that last late into adulthood. In adults, MDMA i produces an initial release of 5-HT followed by dramatic depletions, and these depletions can last for weeks. It was previously shown that neonatal MDMA administration on P11, the first day of the exposure period known to result in memory impairment (P11-20), also produced significant depletions in 5-HT. We further characterized the degree of 5-HT depletion that was experienced by rats exposed to P11 MDMA exposure and compared this to 5-HT reductions induced by other substituted amphetamines as well as determining the length and degree of depletion following P11-20 MDMA exposure. Substantial 5-HT depletions existed during all ten days of MDMA exposure, however, these depletions were not observed on P30. Citalopram (CIT), a highly selective SSRI, was used in combination with P11-20 MDMA exposure to attenuate the 5-HT depletions. Thereafter, cognitive ability was assessed in MDMA-treated animals with attenuated 5- HT depletions. The combination of CIT and MDMA did not improve learning ability; however CIT treatment alone produced deficits in path integration learning. These data suggest that 5-HT alterations during vulnerable critical periods can affect later cognitive ability, and they suggest that depletions in 5-HT alone may not account for cognitive dysfunction in neonatal rats treated with MDMA. Examination of specific 5-HT receptors is likely the next step to understand the mechanism involved in 5-HT disruption during development and later cognitive ability. ii ACKNOWLEDGEMENTS I would like to thank my thesis advisor, Dr. Michael Williams, for his patience, support, and guidance throughout my graduate career. I am grateful to have the opportunity to work with Michael and am thankful to have him as an advisor. I would also like to thank Dr. Charles Vorhees for his additional guidance throughout my graduate career. It has been an incredible experience to work with these excellent scientists who I can comfortably call friends as well as colleagues. I would also be remiss if not thanking current and former my lab mates: Matt Skelton, Devon Graham, Curt Grace, Amanda Braun, and Mary Moran; I have been truly blessed to work with such wonderful and intelligent people. I would also like to thank my dissertation committee members, Drs. Michael Williams, Chip Vorhees, Gary Gudelsky, Steve Danzer, and Kenny Campbell for their support, guidance and interest in my research. I am honored to be a graduate of the Molecular and Developmental Biology Graduate Program. I would like to thank the staff of the Division of Developmental Biology and the Division of Neurology, both past and present. Finally, I would like to thank my family, especially my wonderful husband Jonathan. His support, patience and encouragement have made this possible. I would like to thank my mother, Kim, for supporting whatever I decided to do with my life. iv TABLE OF CONTENTS List of figures vii Chapter 1 Introduction 1 The serotonergic system 1 Serotonin depletion 5 Pet-1 7 MDMA 9 o History and prevalence 9 MDMA-induced 5-HT alterations 10 Other MDMA-induced alterations 12 o Behavior 14 o Learning and memory 14 Dissertation synopsis 19 References 20 Figures 40 Hypothesis and Specific Aims 41 Chapter 2: Mouse Pet-1 knock-out induced 5-HT disruption results in a lack of cognitive deficits and an anxiety phenotype complicated by hypoactivity and defensiveness Title page 42 Abstract 43 Introduction 44 Material and Methods 46 Results 52 Discussion 56 References 63 Figures 71 Chapter 3: A comparison of monoamine and corticosterone levels 24 hours following (+)methamphetamine, (±)3,4-methylenedioxymethamphetamine, cocaine, (+)fenfluramine, or (±)methylphenidate administration in the neonatal rat Title page 78 Abstract 89 Introduction 80 Materials and Methods 82 Results 86 Discussion 88 References 97 Figures 106 v Chapter 4: Short- and long-term effects of (+)-methamphetamine and (±)-3,4- methylenedioxymethamphetamine on monoamine and corticosterone levels in the neonatal rat following multiple days of treatment Title page 113 Abstract 114 Introduction 115 Material and Methods 118 Results 122 Discussion 127 References 132 Figures 143 Chapter 5: Alterations to learning and memory following neonatal exposure to 5-HT altering drugs: individual and combined effects of MDMA and citalopram Title page 153 Abstract 154 Introduction 156 Material and Methods 159 Results 168 Discussion 178 References 187 Figures 199 Chapter 6: Discussion Conclusions 208 Discussion 208 Critical period of 5-HT development 210 Alternative 5-HT hypothesis 212 Future studies 214 References 219 Figures 227 vi LIST OF FIGURES Chapter 1 Figure 1. Simplified schematic of serotonergic signaling 40 Chapter 2 Figure 1. Elevated zero maze 71 Figure 2. Locomotor activity 72 Figure 3. Marble burying 73 Figure 4. Light-Dark box exploration 73 Figure 5. MWM hidden platform trials 74 Figure 6. MWM shift phase initial heading error 75 Figure 7. Cincinnati water maze 76 Figure 8. Locomotor activity with MA challenge 77 Chapter 3 Table 1. Body weights 106 Figure 1. Corticosterone concentrations in plasma 107 Figure 2. Serotonergic markers in the neostriatum 108 Figure 3. Dopaminergic markers in the neostriatum 109 Figure 4. Serotonergic markers in the hippocampus 110 Figure 5. Comparison of MWM effects 111 Chapter 4 Figure 1. Body weights 143 vii Figure 2. Corticosterone concentrations in plasma 144 Figure 3. Serotonergic markers in the neostriatum after P11-15 exposure 145 Figure 4. Dopaminergic markers in the neostriatum after P11-15 146 exposure Figure 5. Serotonergic markers in the neostriatum after P11-20 exposure 147 Figure 6. Dopaminergic markers in the neostriatum after P11-20 148 exposure Figure 7. Serotonergic markers in the hippocampus after P11-15 149 exposure Figure 8. Serotonergic markers in the hippocampus after P11-20 151 exposure Figure 9. Time course of CORT and 5-HT changes 152 Chapter 5 Table 1. 5-HIAA, 5-HIAA/5-HT ratio, DA, DOPAC, DOPAC/DA ratio, 199 NGF, BDNF, and CORT on P12, 16, and 21 Table 2. Light-Dark test, Elevated zero maze, and Straight channel 201 Figure 1. Hippocampus 5-HT: P11-20 exposure 202 Figure 2. P12, P16, and P21 5-HT levels 203 Figure 3. Body weights 204 Figure 4. Locomotor activity 205 Figure 5. Cincinnati water maze 206 Figure 6. Morris water maze 207 viii Chapter 6 Table 1. Summary of hippocampal 5-HT, CORT, and CWM learning 227 Figure 1. Schematic of 5-HT neuron 228 ix CHAPTER 1: INTRODUCTION The monoamine 5-hydroxytryptamine (5-HT) was first isolated from serum and was named “serotonin” because of its vasoconstrictive effects (Rapport et al. 1948). Serotonin was later detected in the mammalian brain and had a wide distribution throughout the central nervous system. Many aspects of mammalian physiology are influenced by the effects of 5-HT, including cardiovascular regulation, respiration, gastrointestinal function, and more centrally controlled functions including circadian rhythm, appetite, aggression, sensorimotor activity, sexual behavior, mood, cognition, and learning and memory. Although many therapeutic drugs with serotonergic activity can alleviate some of the symptoms associated with psychological and cognitive dysfunction we still do not have a good understanding of the disease etiologies.

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