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The Effects of the General Anaesthetic Propofol on Drosophila Larvae Drew Min Su Cylinder Bachelor of Science

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The Effects of the General Anaesthetic Propofol on Drosophila Larvae Drew Min Su Cylinder Bachelor of Science The effects of the general anaesthetic propofol on Drosophila larvae Drew Min Su Cylinder Bachelor of Science A thesis submitted for the degree of Master of Philosophy at The University of Queensland in 2019 Queensland Brain Institute Abstract Although general anaesthetics have been in use since the mid-19th century, the mechanism by which these drugs induce reversible loss of consciousness is still poorly understood. Previous research has indicated that general anaesthetics activate endogenous sleep pathways by potentiating GABAA receptors in wake-promoting neurons. However, more recent studies have demonstrated that general anaesthetics also inhibit synaptic release through interactions with the SNARE complex, an integral part of presynaptic neurotransmitter release machinery in all neurons. The presynaptic and postsynaptic mechanisms may thus be linked in a two-step process: at low doses, general anaesthetics activate sleep-promoting circuits, thereby producing unconsciousness, while at the higher doses necessary for surgery, general anaesthetics inhibit presynaptic release machinery brain-wide, thereby causing a total loss of behavioural responsiveness. While this hypothesis remains speculative, it is testable in animal models. This study develops larval Drosophila melanogaster as an animal model to test this hypothesis in the context of a common intravenous GABA-acting general anaesthetic, propofol. Although presynaptic effects of general anaesthetics have been studied in larval neuromuscular junction preparations, there is not much data for how these drugs affect larval behaviour or brain activity. General anaesthesia is easily addressed in animal models because it can be described as a state of decreased responsiveness which can be assessed using diverse behavioural endpoints. In this study, a series of behavioural assays were designed and tested to assess the effect of GABA-acting general anaesthetics and sedative drugs on Drosophila larvae. These assays were then used to explore key parts of the pre- and postsynaptic mechanisms thought to underlie general anaesthesia. It was found that knockdown of the RDL subunit, the main anaesthetic target in the GABA receptor, did not confer resistance to propofol. However, deletion of the N-terminus of the H3 domain in syntaxin1A, a key component of the SNARE complex, resulted in resistance to propofol and isoflurane. These results suggest that propofol and isoflurane act through similar mechanisms and that this mechanism involves a presynaptic component that transcends life stage or brain size. Larval Drosophila thus provide a valuable counterpart to adult flies for investigating presynaptic mechanisms of anaesthesia. i Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. ii Publications included in this thesis No publications included. iii Submitted manuscripts included in this thesis No manuscripts submitted for publication. Other publications during candidature No other publications. Contributions by others to the thesis Chapter 2: Dr Jessica Waanders helped design and perform the high performance liquid chromatography experiments. Chapter 3: Dr Oressia Zalucki constructed UAS-HA syntaxin227. Deniz Ertekin helped perform the immunohistochemistry experiments and helped write the associated methods. Chapter 4: Dr Lucy Heap performed the watershed segmentation of the two-photon microscopy data mentioned in this chapter. iv Statement of parts of the thesis submitted to qualify for the award of another degree No works submitted towards another degree have been included in this thesis Research Involving Human or Animal Subjects No animal or human subjects were involved in this research v Acknowledgements I am first and foremost most grateful to Dr Bruno van Swinderen who has been an extraordinarily patient and understanding supervisor. I am also thankful to my co- supervisor, Dr Victor Anggono, with whom I completed my first lab rotation. Thank you to my committee members: Dr Nela Durisic, Dr Kai-Hsiang Chuang, and Dr Helen Cooper who always kept me on my toes with insightful questions and gave me plenty of advice that helped shape this project. Thank you to all the members of the van Swinderen Lab: Dr Leonie Kirszenblat, Dr Martyna Grabowska, Dr Lucy Heap, Dr Kai Feng, Dr Michael Troup, Adam Hines, Rhiannon Jeans, and Deniz Ertekin. Your enthusiasm and quirky sense of humour made work enjoyable, and your expertise made this thesis possible. In particular, thank you to Dr Michael Troup for your guidance on this project. Thank you also to Dr Kunle Bademosi who taught me how to perfectly fillet a maggot, and Dr Lucy Heap whose MATLAB wizardry made chapter 4 possible. I would also like to give special thanks to Deniz Ertekin who gamely answered all of my questions no matter how stupid or pedantic. This thesis would also not have been half as enjoyable without my MPhil cohort: Sam Armstrong, Julius Alpay, Clare Harris, Chai Chee Ng, and Maleeha Waqar. You have been an inspiring group to go on this journey with, and I’m excited to see your future successes. In particular Chai Chee, Maleeha, and Dr Consuelo Santamaria Ferrada have been an immeasurable support and have kept me relatively sane throughout this project. I could have not asked for a better home-away-from-home. Finally, I would of course like to thank my parents and sister. California is a long way away but, even from there, you were still able to offer sage advice. Your constant encouragement and support is the reason I was able to get where I am now. vi Financial support This research was supported by the University of Queensland Research Training Tuition Fee Offset Scholarship. Keywords General anaesthesia, Drosophila, larva, behaviour, chemotaxis, anaesthetic, sedative, propofol, isoflurane, gaboxadol vii Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 110903, Central Nervous System, 40% ANZSRC code: 110301, Anaesthesiology, 30% ANZSRC code: 060801, Animal Behaviour, 30% Fields of Research (FoR) Classification FoR code: 1109, Neurosciences, 100% viii Table of Contents Abstract ................................................................................................................................. i Declaration by author ........................................................................................................... ii Publications included in this thesis ..................................................................................... iii Submitted manuscripts included in this thesis .................................................................... iv Other publications during candidature ................................................................................ iv Contributions by others to the thesis ................................................................................... iv Statement of parts of the thesis submitted to qualify for the award of another degree ........ v Research Involving Human or Animal Subjects ................................................................... v Acknowledgements ............................................................................................................. vi Financial support ............................................................................................................... vii Keywords ........................................................................................................................... vii Australian and New Zealand Standard Research Classifications (ANZSRC) ................... viii Fields of Research (FoR) Classification ............................................................................ viii Table of Contents ............................................................................................................... ix List of Figures ...................................................................................................................
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