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Download Thesis This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ The Development and Function of Cerebellar Nucleo-Olivary Neurons Prekop, Hong-Ting Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. END USER LICENCE AGREEMENT Unless another licence is stated on the immediately following page this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ You are free to copy, distribute and transmit the work Under the following conditions: Attribution: You must attribute the work in the manner specified by the author (but not in any way that suggests that they endorse you or your use of the work). Non Commercial: You may not use this work for commercial purposes. No Derivative Works - You may not alter, transform, or build upon this work. Any of these conditions can be waived if you receive permission from the author. Your fair dealings and other rights are in no way affected by the above. Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 10. Oct. 2021 Hong-Ting Prekop Centre for Developmental Neurobiology King’s College London Thesis submitted for the degree of Doctor of Philosophy (PhD) September 2017 Abstract The neuronal circuitry between the cerebellum and inferior olive is of crucial importance in motor function. While the climbing fibres that send olivary signals to the cerebellum have been shown to play a significant role in modulating cerebellar output, little is known of the origins or function of the nucleo-olivary neurons of the cerebellar nuclei that send reciprocal feedback to the inferior olive. In this thesis, the Sox14 gene is identified as a novel genetic developmental marker for nucleo-olivary neurons of the lateral and interposed, but not medial, cerebellar nuclei. Using Sox14-GFP and Sox14-Cre knock-in and Sox14 knock-out mouse lines, in combination with birth dating, marker analysis and tract tracing techniques, I characterised the projections and development of nucleo-olivary neurons. These experiments established that Sox14 is expressed in early born GABAergic nuclear projection neurons that exclusively target the inferior olive. A separate population of Sox14+ cells in Nucleus Y target the oculomotor nucleus. Sox14 expression is observed from E12.5 directly ventral to the nuclear transitory zone, where glutamatergic nuclear cells are known to reside during development. Injection of Cre-dependent AAV-mCherry-flex-dtA was used to drive expression of diphtheria toxin A subunit in Sox14 expressing cells. Ablation of Sox14+ nucleo- olivary neurons leads to some deficits in motor performance and motor learning. However, no deficits in associative motor learning were observed, suggesting that models of associative learning that invoke a key role for the nucleo-olivary feedback may be incorrect or incomplete. 2 This work establishes that nucleo-olivary neurons of the lateral and interposed cerebellar nuclei comprise a homogeneous and genetically distinct population and sheds light on the function of this projection in cerebellar function. Furthermore, this thesis establishes Sox14 transgenic mice as a unique tool in cerebellar research that will provide an important window on the function of the cerebellum in future studies. 3 Acknowledgments / funding With gratitude, this thesis represents independent research funded by the National Institute for Health Research (NIHR) Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King’s College London. The views expressed are my own and not necessarily those of the NHS, the NIHR, or the Department of Health. I would like to thank Prof. Thomas Jessell and Dr. Laskaro Zagoraiou for the generation of the Sox14-Gfp and Sox14-Cre mouse lines. The mice are central to the research, so thank you for the generosity in making the mice available. Thank you to Stephanie and Reinko at Noldus, who arranged the trial period for use of the Erasmus ladder. They were so kind in arranging to come to help me set up and provided technical assistance in the analysis of the data. I owe much of this work to the dedicated supervision from Alessio Delogu and Richard Wingate. Alessio, I can’t think of a more competent and knowledgeable mentor that I could have had. The range of skills that you have acquired throughout your career and was able to impart onto me (as best as you could!) is inspiring. I have learnt a lot of technical skills and even though not all of them worked out, you encouraged me to troubleshoot and supported new ideas. Richard, our discussions helped me enormously to think through the data and put them into context. Your enthusiasm for the subject and positivity in general is infectious and was crucial in keeping me on track, particularly over the writing up period. I also have many to thank and acknowledge in both the labs that I was a part of. Thanks to Polona and Olivier for the collaborative efforts in the production of AAVs and for general lab assistance. Without you both, my weekends would have been filled with long journeys for short tasks in the lab! Thanks to Marcela, Tristan, Micha, Margarita, Flo and Leigh for all the help provided for my work at the CDN, the pub trips, coffee breaks, constant snacks and for increased happiness in general! I would like to acknowledge assistance with some of the data presented in this thesis. I had the opportunity to supervise Niyoti for her master’s research project and Daniel for his Erasmus project, who both helped with the production of the Cre-expressing 4 HEK293 cell line presented in Chapter 3. I am very grateful to Anna, an Erasmus student, who I only knew for a short while before I left the lab. She helped with the Gad1 ISH for the ablation analysis presented in Chapter 5 and repeating some of the BrdU data presented in Chapter 4. Special thanks to the other labs at the Wohl and IoP, especially to Cathy Fernandes for lending me equipment for mouse behavioural tests and helping me to perform the pilot testing in Appendix D. Thank you to all those in Deepak Srivastava’s lab for help in primary cell culture work and the antibodies “borrowed”. Thank you to all in the Wohl for the lunch times and the socials. You all made it such a pleasant and friendly work environment to be part of. During the relentless hours spent in the animal facility, I would like to thank Ted, John and Louis for their dedication and assistance in caring for the mice and for me in times of panic. This research project has been a steadfast pursuit during four years in which my life has radically changed. I must thank all those outside my “science world” for their support and affection throughout. To my various housemates, especially Esme and Hannah, thank you for bearing with my crazy use of the fridge. Christchurch Kensington is the sole reason I chose to complete a PhD in London. Thank you to all in this adopted family for your love, prayers and friendship over the years. Thank you to my real family for loving me regardless of what path I choose in life. Mum and dad, you are my role models, with your hard work ethic and inexhaustible love for others. You have shown me what it looks like to prioritise people and give time generously. Chris, thank you for dating, proposing to and marrying a PhD student! Your support has been unconditional and constant. Thank you for the emotional support, the endless cups of tea and the proof-reading. You have made all the transitions over the past years effortless with your loving compromises. Lastly, I would like to thank God for his work in my life. Soli Deo gloria. 5 The highest reward for a person's toil is not what they get for it, but what they become by it. -John Ruskin 6 Table of Contents Abstract 2 Acknowledgments / funding 4 Table of Contents 7 Table of Figures 15 Table of Tables 17 1 Introduction 18 1.1 What are Cerebellar nuclei? 18 1.2 Structure of the cerebellum 20 1.3 Nuclear cells as components of models of cerebellar function 24 1.3.1 Circuitry is linked to function 24 1.3.2 Convergent Networks within the cerebellar nuclei 26 1.3.3 Cerebellar role in Motor Learning 29 1.3.4 Cerebellar role in Motor timing 31 1.3.5 Non-motor functions of the cerebellum 32 1.4 Nucleus anatomy and Cellular diversity 33 1.4.1 Glutamatergic Projection Neurons 34 1.4.2 Nucleo-Olivary Projection Neurons 35 1.4.3 Glycinergic Projection Neurons 35 1.4.4 Interneurons 36 1.5 Development of the Cerebellum 38 1.6 Development of Cerebellar nuclei 40 1.6.1 Origin of glutamatergic neurons 41 1.6.2 Origin of GABAergic projection neurons 43 1.6.3 Origin of Other nuclear neurons 44 1.6.4 Nucleogenesis and cell migration 45 1.6.5 Evolution and the diversification of cerebellar nuclei 47 7 1.7 Cerebellar nuclei and disease 48 1.8 Sox14 and the Cerebellar nuclei 53 2 Characterisation 54 2.1 Introduction 54 2.1.1 The Sox14 gene 54 2.1.2 Identifying the cells of the cerebellar nuclei 56 2.1.2.1 GABA and GAD 58 2.1.2.2 Calcium binding proteins 59 2.1.3 Aims of the Chapter 60 2.2 Results 61 2.2.1 Sox14+ cells are non-uniformly distributed across the cerebellar nuclei 61 2.2.2 Identification of Sox14+ cells by immunohistochemistry 69 2.2.2.1
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