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Glial Cell Mechanisms Regulate Alcohol Sedation in Drosophila Melanogaster

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Glial Cell Mechanisms Regulate Alcohol Sedation in Drosophila Melanogaster Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2019 Glial cell mechanisms regulate alcohol sedation in Drosophila melanogaster Kristen M. Lee Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Genetics Commons, and the Molecular and Cellular Neuroscience Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/5780 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. © Kristen Mary Lee 2019 All Rights Reserved Glial cell mechanisms regulate alcohol sedation in Drosophila melanogaster A dissertation submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. by Kristen Mary Lee Bachelor of Science, Wagner College, 2014 Director: Mike Grotewiel, PhD Assistant Graduate Dean Associate Professor, Department of Human & Molecular Genetics Virginia Commonwealth University Richmond, Virginia March, 2019 ii ACKNOWLEDGEMENT First and foremost, I would like to thank my mentor, Dr. Mike Grotewiel, who has been extremely supportive of my research endeavors. When I walked into your office five years ago, I never would have imaged that a small screen would have blossomed into a dissertation project. Thank you for not only letting me pursue this project, but for being just as excited about it as I am. I feel extremely grateful to have you as a mentor, and I truly appreciate all of your advice, it has shaped me into a better scientist. I would also like to thank Dr. John Bigbee. Thank you for being a haven of support and guidance throughout my time at VCU. And thank you for being the advisor, and so supportive, of the NeuroNerds. This organization has allowed me to form so many new friendships and has given me a sense of community outside of the lab. I would like to thank my committee members, Drs. Dupree, Fuss, Davies and Lister who have been extremely enthusiastic at all my committee meetings. Thank you for providing helpful critiques and experimental ideas, and for always having an open door when I needed any additional guidance. I have appreciated all your support. I would like to thank my F31 co-sponsor, Dr. Knapp, for being so present and responsive. Thank you for coming to my committee meetings and providing advice on my fellowship application. You guidance was vital for my fellowship’s success. iii A huge thank you also goes out to (Silly) Rebecca Schmitt, who has managed to be in the seat next to me almost every day these last five years. I could say a lot of weird stuff here, but to put it simply – thank you for turning a very small office into a home. I also thank all the Grotewiel lab members pass and present – Ian, Jena, Laura, and especially Brandon – you have all been awesome people that I have looked forward to spending each day with. I am extremely grateful to have Christopher Dean in my life. Thank you for filling my free time with adventure, good food, and cute animals. I appreciate your endless encouragement. Last but certainly not least, I would like to thank my family. My parents, Tom and Kathy, have been a constant source of support throughout my life. Thank you for being present and active in all my endeavors, I wouldn’t be here today without you both. My brother and sister, Matt and Monica, are two of the best and funniest people I know. Thank you for always being a phone call away, and for knowing how to make me smile and laugh. I thank my family for coming to visit me in Richmond so frequently, and for making New Jersey such a hard place to leave every time I visit home. Your support has meant the world to me. iv Table of Contents Acknowledgment ..............................................................................................................ii List of Tables ..................................................................................................................vii List of Figures ................................................................................................................viii List of Abbreviations ........................................................................................................xi Abstract ..........................................................................................................................xv Chapter 1: Introduction ....................................................................................................1 A. Alcohol abuse and alcohol use disorder …………………………………………1 B. Drosophila melanogaster as a model to study alcohol-related behaviors …....2 C. Drosophila melanogaster as a model to study adult CNS glial cells ………….5 C.1 A functional overview of mammalian astrocytes ……………………………….7 C.2 A functional overview of Drosophila melanogaster cortex glia and astrocytes …………………………………………………………………………………………...14 D. Glial responses to alcohol administration and their role in alcohol-related behaviors ………………………………………………………………………………19 D.1 Mammalian astrocytes and alcohol …………………………………………….19 D.2 Mammalian microglia and alcohol ……………………………………………...21 D.3 Mammalian oligodendrocytes and alcohol …………………………………….22 D.4 Drosophila melanogaster surface glia and alcohol …………………………...23 E. Significance ………………………………………………………………………...24 v Chapter 2: Materials & Methods ....................................................................................26 A. Universal Methods ………………………………………………………………..26 B. Chapter Specific Methods ………………………………………………………..29 Chapter 3: Characterization of a CNS-glia specific GeneSwitch fly stock .....................38 A. Introduction ………………………………………………………………………...38 B. Results ……………………………………………………………………………..39 B.1 Identifying GeneSwitch reagents that induce LacZ transgene expression during adulthood ………………………………………………………………………39 B.2 The 59929 GeneSwitch transgene induces UAS-transgene expression in CNS glia ………………………………………………………………………………..44 B.3 The RU486 feeding regiment does not alter alcohol sedation sensitivity in control flies …………………………………………………………………………….46 Chapter 4: Cysteine proteinase 1 regulates Drosophila alcohol sedation by functioning in adult cortex glia ..........................................................................................................48 A. Introduction ………………………………………………………………………...48 B. Results ……………………………………………………………………………..51 B.1 Identifying glial genes that influence alcohol-related behavior in Drosophila ………………………………………………………………………………………...…51 B.2 Cp1 knockdown in all CNS glia alters alcohol sedation without influencing internal alcohol levels …………………………………………………………………63 B.3 Expression of Drosophila pseudoobscura Cp1 rescues alcohol sedation sensitivity due to knockdown of endogenous Drosophila melanogaster Cp1 in glia …………………………………………………………………………………………...72 B.4 Cp1 expression specifically in cortex glia regulates alcohol sedation ……...83 B.5 Cp1 in rapid tolerance development ……………………………………………88 B.6 Cp1 knockdown in CNS glia during adulthood alters alcohol sedation …….91 vi C. Discussion ………………………………………………………………………….95 Chapter 5: Tyramine synthesis, vesicular packaging and the SNARE complex function coordinately in astrocytes to regulate Drosophila alcohol sedation .............................105 A. Introduction ……………………………………………………………………….105 B. Results ……………………………………………………………………………107 B.1 Tdc2 functions in CNS glia, specifically astrocytes, to regulate alcohol sedation sensitivity …………………………………………………………………..107 B.2 Tdc2 regulates alcohol sedation sensitivity in CNS glia during adulthood through a pharmacodynamic mechanism ………………………………………...117 B.3 Manipulation of tyramine synthesis in astrocytes impacts alcohol sedation ………………………………………………………………………………………….124 B.4 Alcohol sedation is influenced by vesicular packaging and release machinery in glia ………………………………………………………………………………….132 B.5 The tyramine receptor is important for alcohol sedation ……………………147 C. Discussion ………………………………………………………………………...152 Chapter 6: Discussion .................................................................................................155 A. Summary and future directions ………………………………………………...155 B. Translatability to mammals ……………………………………………………..157 References …………………………………………………………………………………...161 Appendix ……………………………………………………………………………………...187 Vita …………………………………………………………………………………………….204 vii List of Tables Table 4.1. Screen results: expressing RNAi against genes known to be expressed in glia and measuring alcohol sedation sensitivity ……………………………………………55 Table 4.2. Expression of RNAi against Cp1, axo, Jhl-21, nemy and Ent2 in glia does not alter internal alcohol concentrations ………………………………………………………...59 Table 4.3. Expression of RNAi against Cp1, axo, Jhl-21, nemy and Ent2 in neurons does not alter alcohol sedation sensitivity ………………………………………………….60 Table 4.4. Expression of Cp1 RNAi in each glial cell subtype individually ……………..87 Table 5.1. Identifying glial genes that regulate alcohol sedation ……………………….110 Table 5.2. Manipulated expression of Tdc2 in each glial cell subtype individually …...112 Table 5.3. Lethality associated with UAS-Tdc2 expression in glia ……………………..142 Table 5.4. Expressing RNAi against individual components of the vesicular release machinery in adult glia does not alter alcohol sedation …………………………………145 Table 5.5. Pan-neuronal expression
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