JONATHAN C BEAN Genetic Labeling Reveals Novel Cellular
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JONATHAN C BEAN Genetic Labeling Reveals Novel Cellular Targets of Schizophrenia Susceptibility Gene ErbB4 and Neuregulin-1 – ErbB4 Signaling In Monoamine Neurons (Under the direction of LIN MEI) Neuregulin 1 (NRG1) and its receptor ErbB4 are schizophrenia risk genes. NRG1-ErbB4 signaling plays a critical role in neural development and regulates neurotransmission and synaptic plasticity. Nevertheless, its cellular targets remain controversial. ErbB4 was thought to be expressed in excitatory neurons although recent studies have disputed this view. Utilizing mice that express a fluorescent protein under the promoter of the ErbB4 gene, I determined in what cells ErbB4 is expressed and their identity. ErbB4 was widely expressed in the mouse brain, being highest in amygdala and cortex. Almost all ErbB4-positive cells were GABAergic in cortex, hippocampus, basal ganglia, and most of amygdala in neonatal and adult mice, suggesting GABAergic transmission as a major target of NRG1-ErbB4 signaling in these regions. Non-GABAergic, ErbB4- positive cells were present in thalamus, hypothalamus, midbrain and hindbrain. In particular, ErbB4 was expressed in both dopamine neurons in the substantia nigra and ventral tegmental area and in serotoninergic neurons of raphe nuclei, but not in norepinephrinergic neurons of the locus coeruleus. In hypothalamus, ErbB4 was present in neurons that express oxytocin. ErbB4 was expressed in a group of cells in the subcortical areas that are positive for S100β. These results identify novel cellular targets of NRG1-ErbB4 signaling. Finally, perfusion of NRG1 into the medial prefrontal cortex enhanced both dopamine and serotonin release but with differing time courses. INDEX WORDS: Neuregulin-1, ErbB4, Serotonin, Oxytocin, S100β, GABA, Schizophrenia GENETIC LABELING REVEALS NOVEL CELLULAR TARGETS OF SCHIZOPHRENIA SUSCEPTIBILITY GENE ERBB4 AND NEUREGULIN-1 – ERBB4 SIGNALING IN MONOAMINE NEURONS by JONATHAN C BEAN DISSERTATION Submitted to the Faculty of the College of Graduate Studies of Georgia Regents University Augusta Georgia In Partial Fulfillment of the Requirement For the Degree of DOCTOR OF PHILOSOPHY April 2015 ProQuest Number: 3729648 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. ProQuest 3729648 Published by ProQuest LLC (2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106 - 1346 TABLE OF CONTENTS Page ACKNOWLEDGEMENTS …………………………………………………………….vii LIST OF FIGURES ………………………………………………………………….…ix LIST OF TABLES …………………………………………………………………..….xii 1. INTRODUCTION ……………………………………………………………………1 1.1 Statement of a problem .……………………………………………………….1 1.2 Statement of specific aims….……………………………………………….…3 1.3 Review of literature ...…………….…………………………………………….5 1.3.1 Schizophrenia ……….…………………………………………………..5 1.3.1.1 History ……………….…………………………………………..5 1.3.1.2 Symptomology ….……………………………………………... 6 1.3.1.3 Epidemiology and Etiology ………….……………………… 14 1.3.2 Neuregulin-1 and ErbB4 …….…………………………………………27 1.3.2.1 History of Growth Factors and Their Receptors……….….. 27 1.3.2.2 Structure and Function…….………………………………… 30 1.3.2.3 Function in the Brain …………….……………………………38 1.3.2.4 Neuregulin-1 and ErbB4 Association with Schizophrenia ..44 1.3.2.5 Controversies in Expression Profile …….………………….51 2. MATERIAL AND METHODS.……………………………………………..………53 2.1 Generation of ErbB4-Reporter and ErbB4-Reporter; GAD67::GFP Mice..53 iv v 2.2 Neuron Culture………………………………………………………………….56 2.3 Analysis of ErbB4-Positive Cells……………………………………………...59 2.4 Immunofluorescent Labeling……………………………...…………………..60 2.5 Microdialysis…………………………………………………………………….61 2.6 High Performance Liquid Chromatography (HPLC)………………………..62 2.7 Statistical Analysis……………………………………………………………..63 3. RESULTS …………………………………………………………………………...65 3.1 Generation and Validation of ErbB4-Reporter Mice………………………..65 3.2 Density of ErbB4-Positive Cells Varies Across Brain Regions……………68 3.3 Olfactory System……………………………………………………………….72 3.4 Cortex……………………………………………………………………………78 3.5 Hippocampus…………………………………………………………………...87 3.6 Basal Ganglia…………………………………………………………………...92 3.7 Amygdala………………………………………………………………………..98 3.8 Thalamus………………………………………………………………………102 3.9 Hypothalamus…………………………………………………………………108 3.10 Midbrain………………………………………………………………………114 3.11 Hindbrain and Cerebellum………………………………………………….124 3.12 White Matter Areas and Choroid Plexus……………………………….....130 3.13 Neuregulin-1 Enhances Dopamine and Serotonin Release……………134 4. DISCUSSION ……………………………………………………………………..139 4.1 ErbB4-positive Cells In the Olfactory Bulb And Deficits In Olfaction Seen In Schizophrenia……………………………………………………………………..140 vi 4.2 ErbB4-positive Cells Are Enriched In the Intercalated Nucleus Of the Amygdala and Blunted Affect Seen In Schizophrenia………………………...141 4.3 ErbB4 in GABA or Glutamatergic Neurons………………………………...144 4.4 ErbB4 In the Hypothalamus And Neuregulin-1 – ErbB4 Signaling’s Possible Role In Metabolism……………………………………………………..147 4.5 Glia Cells Positive For ErbB4 In the Thalamus……………………………148 4.6 ErbB4 Is Selectively Expressed In Monoamine Neurons Implicated In Psychiatric Disorders……………………………………………………………..149 4.7 Limitations of ErbB4-reporter Mice………………………………………….150 4.8 Neuregulin-1 Enhanced Monoamine Release………………………….....153 4.9 Translations Implications For Altered Neuregulin-1 – ErbB4 Signaling...156 5. SUMMARY ……………………………………………………………………… 158 6. LITERATURE CITED …………………………………………………………… 160 ACKNOWLEDGEMENTS The path to completion of my Ph.D. has been a long tortuous journey fraught with many challenges. Along the way I have gotten support from many people whom I am immensely appreciative of. To my Ph.D. committee: Drs. Darrell Brann, Alvin Terry, Lynnette McCluskey, and Almira Vazdarjanova, and to my reader Dr. Albert Pan, you have been critical of my science and pushed me to do my best work and for that I am thankful. Your critiques and suggestions have improved the final product of my dissertation and of my published work. To all the past and present members of Drs. Mei and Xiong’s labs, you have been supportive during my struggles, critical when I needed it most, and were there to celebrate with me during my successes. Special thanks to Drs. Dongmin Yin and Chengyong Shen for their mentorship and support. To Dr. Xiong, you have given me much needed critical feedback and many good suggestions on my project over the years. To my mentor Dr. Lin Mei, you have always been supportive of me, never given up on me, and always pushed me to do more. I could not have hoped for a better Ph.D. mentor and I will forever be proud to say I trained with Lin Mei. I cannot thank you enough for what you have done for me and I cannot imagine being better positioned to move forward into a successful scientific career under anyone else’s direction. Thank you. vii viii Finally, to my mother Katherine Bean, my father Jerry Bean, and my daughter Raven Bean, pursuing a Ph.D. and a career in science has been a dream of mine but has come with many sacrifices. I realize that most of these of come at the expense of my family life and to you. For all the sacrifices you have made for me to allow me to follow this dream I will forever be grateful. For this, I dedicate the work herein to you. Thank you. LIST OF FIGURES Figure Page 1. Monoaminergic Systems and Their Receptors ………………………………….12 2. Cholinergic System and GABA and Glutamatergic Circuity …………………..17 3. Recombination and Meiosis ………………………….…………………………..25 4. Neuregulin-1 Isoforms, ErbBs and ErbB4 Isoforms …………………………….31 5. Neuregulin-1 – ErbB4 Signaling …………………………………………………36 6. Neuregulin-1 – ErbB4 Signaling in the Brain ………………………………..….40 7. Genotyping results for ErbB4-reporter and GAD67::GFP mice ……………….55 8. Control Data for Microdialysis and HPLC ………………………………………..64 9. tdTomato Labels ErbB4-expressing Neurons …………………………………..66 10. Unique Distribution of ErbB4-positive Cells In Brain Regions ………………69 11. ErbB4-positive Cells Are Enriched In the Glomerulus and Mitral Cell Layers of Olfactory Bulb ……………...……………………………………….…………………74 12. ErbB4-positive Cells In Cortex Are Enriched In Layer 2/3, Are GABAergic Interneurons and Positive For PV …………………………………………….……...80 13. ErbB4-positive Cells In Cortex and Hippocampus of Neonatal Mice Are GABAergic Interneurons ………………………………………………………………85 14. ErbB4-positive Cells In Hippocampus Are Concentrated In Stratum Pyramidal, Are GABAergic Interneurons and Positive For PV ………….………..88 ix x 15. ErbB4-positive Cells In the Basal Ganglia Show Unique Distribution and Are GABAergic Interneurons ………………………………………………………………94 16. ErbB4-positive Cells In the Amygdala Are Concentrated In the Intercalated Nucleus ……………………………..…………………………………………………..99 17. ErbB4-positive Cells Are Enriched In Medial Habenula ……………………..103 18. S100β Was Detected In ErbB4-positive Cells In Thalamus, Hypothalamus, Midbrain and Hindbrain …………………………………………………………...…106 19. ErbB4-positive Cells Enriched In the Paraventricular and Dorsomedial Hypothalamic Nuclei …………………………………………………………………110 20. ErbB4-positive Cells Co-localize With Oxytocin But Not Vasopressin In the Paraventricular Hypothalamic Nucleus