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Alex James Clark The use of microfluidic chambers to study action potential propagation and stimulus transduction in sensory neurons in vitro Alex James Clark SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY 1 I, Alex James Clark, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis 2 Abstract Primary afferent sensory neurons can be incredibly long single cellular structures, often traversing distances of over one metre in the human. Cutaneous sensory stimuli are transduced in the periphery by specialised end-organs or free nerve endings which enable the coding of the stimulus into electrical action potentials that propagate towards the central nervous system. Despite significant advances in our knowledge of sensory neuron physiology and ion channel expression, many commonly used techniques fail to accurately model the primary afferent neuron in its entirety. In vitro experiments often focus on the cell somata and neglect the fundamental processes of peripheral stimulus transduction and action potential propagation. Despite this, these experiments are frequently used as a model for cellular investigations of the receptive terminals. Crucially, somal responses may not represent the functional expression of ion channels in the axon and end terminals. The aim of this thesis was to develop a system using compartmentalised culture chambers and ratiometric calcium imaging to directly and accurately compare the sensitivity and functional protein expression of isolated neuronal regions in vitro. Using this preparation I demonstrate that the nerve terminals of cultured DRG neurons can be depolarised to induce action potential propagation, which has both a TTX-resistant and TTX-sensitive component. Furthermore, I show that there is a differential regulation of proton sensitivity between the sensory terminals and somata in cultured sensory neurons. I also go on to show that capsaicin sensitivity is highly dependent on embryonic dissection age. This novel approach enables a comprehensive method to study the excitability characteristics and regional sensitivity differences of cultured sensory neurons on a single cell level. Examination of the sensory terminals is crucial to further understand the properties and diversity of DRG sensory neurons. 3 Acknowledgements First and foremost I would like to thank my supervisor Martin, for his help and guidance throughout my PhD. I greatly appreciate the skills and knowledge he has passed on to me during my time in his lab, and I’m sure these will help hugely during the course of my scientific career. To the other members of the Koltzenburg lab, both past and present, thank you for everything you have all done to help me on my way. I have learnt so much from your help in the lab to the conversations over many cups of coffee! I gratefully acknowledge Mona, who has been a constant source of good advice over the years. It has been a pleasure to work with all of you during my PhD. I would like to particularly thank Guillermo and Gipi for helping me to establish the microfluidic chambers in Martin’s lab. Thank you for taking your time to answer my questions and showing me the ropes. Your help was pivotal in getting me up and running in the lab! I am very grateful to Niral and Erik, for their helpful insight into using radioactivity. To ‘the boys’, thanks for always being there for beers and darts - the ‘Cittie’ has become a home away from home! Your enthusiasm for celebrations has made for some very memorable occasions! To my parents, you are a constant source of love and encouragement and for that I am forever grateful. You have always believed in me and endlessly supported me. You have taught me to work hard and take pride in all I do. I would not be where I am today without the support from both of you. To my best friend and fiancée Carol, you have always been there for me, to bear the brunt of the frustrations and share in the joy of success. Thank you for your editing help (!), your culinary delights and for always being able to cheer me up and keep a smile on my face. Finally, we can begin to plan our big day! Finally, I dedicate the work in this thesis to my Grandad, Czes Mitmanski. You were such a warm and friendly person, constantly interested in all I do and always ready to listen over a glass of Rioja! Your stories, smiles and hugs will never be forgotten. I hope I have made you proud. 4 Contents page Abstract………………………………………………………………………………………………………………………………………….3 Acknowledgements ............................................................................................................................. 4 List of tables ........................................................................................................................................ 9 Abbreviations .................................................................................................................................... 10 Chapter 1 .............................................................................................................................................. 15 Introduction ......................................................................................................................................... 15 1.1. Sensory neurons .................................................................................................................. 16 1.1.1. Sensory afferent subtypes .............................................................................................. 16 1.1.2. DRG neurogenesis .......................................................................................................... 20 1.1.3. Neurotrophin dependence and role of the Runx transcription factors ......................... 21 1.1.4. The role of the low affinity p75 neurotrophin receptor ................................................. 23 1.2. Sensory transduction .......................................................................................................... 25 1.2.1. TRP channel architecture and activation properties ...................................................... 26 1.2.2. TRP channel chemo- and thermoreception ................................................................... 26 1.2.3. Embryonic emergence of TRP channel expression......................................................... 29 1.2.4. Detection of protons ...................................................................................................... 30 1.2.5. Mechanoreception ......................................................................................................... 33 1.3. Action potential propagation – Voltage Gated Sodium Channels ....................................... 35 1.3.1. Nav1.1 ............................................................................................................................. 36 1.3.2. Nav1.3 ............................................................................................................................. 37 1.3.3. Nav1.6 ............................................................................................................................. 37 1.3.4. Nav1.7 ............................................................................................................................. 37 1.3.5. Nav1.8 ............................................................................................................................. 38 1.3.6. Nav1.9 ............................................................................................................................. 39 1.4. Axonal Transport ................................................................................................................. 40 1.4.1. Retrograde neurotrophin signalling ............................................................................... 40 1.5. Methods to investigate the properties of DRG neurons ..................................................... 43 1.5.1. Compartmentalised chambers ....................................................................................... 45 5 1.6. Aims ..................................................................................................................................... 49 Chapter 2 .............................................................................................................................................. 51 Materials and Methods ........................................................................................................................ 51 2.1. Fabrication of master moulds by soft lithography .............................................................. 52 2.2. Fabrication of epoxy templates ........................................................................................... 55 2.3. PDMS replication ................................................................................................................. 56 2.4. Irreversible binding using a plasma cleaner ........................................................................ 57 2.5. Quantifying the flow across the microgroove array ............................................................ 57 2.6. Preparation for culture ........................................................................................................ 62 2.7. DRG neuron culture ............................................................................................................. 62 2.8. Medium changes and chemo-attraction protocol .............................................................
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