“I Am Among Those Who Think That Science Has Great Beauty. A
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“I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.” Marie Curie “The most exciting phrase to hear in science, the one that heralds new discoveries, is not Eureka! (I found it!) but rather, "hmm.... that's funny...."” Isaac Asimov “Science is a wonderful thing if one does not have to earn one's living at it.” Albert Einstein University of Alberta The effects of neurosteroids and neuropeptides on anxiety-related behavior by Elif Engin A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Psychology ©Elif Engin Fall 2009 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission. Examining Committee Dallas Treit, Psychology Frederick Colbourne, Psychology Clayton Dickson, Psychology Peter Hurd, Psychology Kathryn Todd, Psychiatry Anantha Shekhar, Psychiatry, Indiana University For Nur and Marc You are the light in my mind, the warmth in my heart. Abstract Anxiety disorders are the most prevalent of all psychiatric conditions. However, current pharmacological treatments for anxiety disorders are characterized by one or more of the following deficiencies: 1) unwanted side effects, 2) partial efficacy, 3) addictive potential, and 4) delayed onset of therapeutic effects. These therapeutic liabilities motivate the search for better pharmacological treatments. This research effort has been concentrated in three broad, neuropharmacological domains: 1) Sub-unit specific GABAA receptor agonists, 2) Neurosteroids, and 3) Neuropeptides. The general purpose of this thesis was to advance our understanding of the putative anxiolytic potential of neurosteroids and neuropeptides, and their neural mechanisms of action, as revealed by intracerebral infusion studies in animal models of anxiety. Chapter 1 of this thesis will provide a systematic review of what is now known about the behavioral effects of intra-cerebrally infused agonists and antagonists of anxiolytic compounds in animal models of anxiety. A theoretical context in which to view the empirical work is also outlined. Chapter 2 will provide a brief introduction to neurosteroids and neuropeptides, and their potential as anxiolytic drugs as suggested by the current literature. In Chapter 3, the anxiolytic-like effects of the neurosteroid allopregnanolone were examined in the amygdala, the hippocampus or the medial prefrontal cortex. Allopregnanolone had site- and test-specific anxiolytic effects, causing anxiolysis following infusion into the amygdala and the medial prefrontal cortex. In Chapter 4, the anxiety-related effects of two receptor antagonists of the neuropeptide arginine vasopressin were investigated in the hippocampus. Anxiolytic effects were specific to both receptor sub-type and by infusion site. In chapter 5, the putative anxiolytic and antidepressant effects of the neuropeptide somatostatin were investigated. Intracerebroventricular microinfusion of somatostatin produced anxiolytic- like and antidepressant-like signatures in distinct domains. In chapter 6, selective agonists for each of the 5 G-protein coupled somatostatin receptors were administered to rats. Intracerebroventricular administration of an sst2 agonist produced anxiolytic-like effects, whereas an antidepressant-like effect was observed following the administration of both sst2 and sst3 agonists. In summary, the present thesis provides important clues to the neurochemical correlates of anxiety, and its potential treatment with alternative compounds such as neuropeptides. Acknowledgements I am grateful beyond measure to my supervisor Dr. Dallas Treit, who accepted me as a graduate student when I knew next to nothing about neuroscience, guided me patiently through my first steps in the field, listened to all my ideas no matter how naïve they might have been, calmed me down when things appeared overwhelming, always believed in me and supported me throughout my early development as a scientist. It has been a great honor working with you, and I can only hope that one day I may become a mentor half as good as you. I would also like to thank Dr. Clayton Dickson for his support and all my committee members for all their helpful and constructive comments that improved the material in this thesis significantly. Several short-term and long-term members of the Treit lab have been a great source of both scientific and emotional support during my long hours of lab work. I would like to thank my fellow graduate student Kris McEown, and our laboratory assistants Tim Collins, Brittany Dingley, Andrea Kokotilo, Andrew Patrick, Connie Sobsey, Jenn Stellbrink for all their contributions. Problems became resolvable, hardships became bearable, pessimism became avoidable, just because of friends and family. Alisha, Eric, Robin, Connie, Jenn, Murat, Volkan, Serkan, and everyone else who were always there for me: Thank you for making my life in Edmonton so rich and happy. Peter, you have changed my life at a time when change was most welcome, in a most welcome way. Thank you for being so amazing. Nur and Marc, I don’t even know where I would be now without you. This thesis, and many other things in my life wouldn’t have been possible without you. Saying thank you will never be enough, and I will be searching for the right words of gratefulness for the rest of my life. And finally, my dear parents, for whom I will always be a little child. Here I am, standing with a PhD in my hand, yet I know you are still wondering if I put my scarf on this morning and whether I am getting enough vitamin C. Thank you for keeping the child in me always alive, and thank you for worrying about everything, so that there is not much left for me to worry about. I know the compromises you have made for us and I will be forever grateful. Table of Contents CHAPTER 1 GENERAL INTRODUCTION: THE EFFECTS OF INTRA- CEREBRAL DRUG INFUSIONS ON ANIMALS' UNCONDITIONED FEAR REACTIONS .........................................................................................1 1. General Introduction ..................................................................................... 2 2. Intra-Cerebral Infusion Studies and Brain Anxiety Circuits..................... 4 2.1. Animal Models of Unconditioned Anxiety Reactions ........................ 5 2.2. Effects of Intra-Cerebally Infused Compounds in Animal Models of Anxiety.............................................................................................................. 7 2.2.1. Periaqueductal gray (PAG)...........................................................7 2.2.2. Raphe nuclei and locus coeruleus...............................................11 2.2.3. Hypothalamus.............................................................................13 2.2.4. Amygdala ...................................................................................15 2.2.5. Septum........................................................................................19 2.2.6. Hippocampus..............................................................................22 2.2.7. Medial prefrontal cortex.............................................................26 2.3. Conclusions............................................................................................. 28 References.......................................................................................................... 33 CHAPTER 2 NEUROSTEROIDS AND NEUROPEPTIDES IN ANXIETY RESEARCH ....................................................................................................64 1. New Directions in Anxiety Research......................................................... 65 2. Neurosteroids ............................................................................................. 65 3. Neuropeptides ............................................................................................ 66 FIGURES AND TABLES ............................................................................... 71 References.......................................................................................................... 73 CHAPTER 3 THE ANXIOLYTIC-LIKE EFFECTS OF ALLOPREGNANOLONE MICROINFUSED INTO THE AMYGDALA, MEDIAL PREFRONTAL CORTEX, OR HIPPOCAMPUS .........................76 1. Introduction........................................................................................................ ............................................................................................................................. 77 2. General Methods........................................................................................... 80 2.1. Subjects.................................................................................................... 80 2.2. Surgery....................................................................................................