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Final Copy 2019 01 23 Smith This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Smith, Phil Title: A systems biology approach using electrophysiology and modelling to determine the membrane clock General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. A systems biology approach using electrophysiology and modelling to determine the membrane clock By Philip Smith “A dissertation submitted to the University of Bristol in accordance with the requirements for award of the degree of Doctor of Philosophy in the Faculty of Life Sciences.” School of Physiology, Pharmacology, and Neuroscience University of Bristol September 2018 Word Count: 33,310 Abstract Circadian rhythms (endogenous 24-hour rhythms of physiology and behaviour) are vital to health and wellbeing; disruption of these rhythms has been linked with health problems such as depression, diabetes, and cancer. Modern times have seen an increase in the use of artificial lighting at night, which has been associated with growing circadian disruption in the general population. This means that understanding of the circadian machinery is increasingly important. Previous studies have shown that clock neurons in the fruit fly Drosophila melanogaster called the lateral neurons ventral (LNvs) are an important part of the circadian system and display electrical properties that vary with the time of day, particularly in action potential firing rate. However, the mechanism of how these neurons produce this change is not yet fully understood. To address this, we investigated the role of voltage-gated potassium channels in generating different activity profiles during the day and night. We utilised electrophysiological techniques, computational modelling, and behavioural assays to show that changes in the current of certain ion channels, namely Shaw (Kv3) current being highest at dawn and Shal (Kv4) current being highest at dusk, support a shift between the more active morning state and the less active evening state of LNv neuronal activity. These ion channels are key in the generation of functional circadian rhythms, where disruption is detrimental to sleep patterns, and in supporting good overall health of the fly. Computational modelling of these channels led to a model of the LNv neuronal activity that allowed the first implementation of an electrophysiological and modelling technique called dynamic clamp in Drosophila. The model was subsequently applied to investigation of human essential tremor and the associated channel Kv9 showing increased neuronal hyperexcitation. These results enhance the understanding of how the LNvs function and potentially offer more insight into their physiological importance. i ii Dedication and acknowledgments There are numerous people who have supported this project and generously given their time and advice. First of all, thanks to my two supervisors, Dr James Hodge and Prof Krasimira Tsaneva-Atanasova who have lent invaluable help over the course of this project. Also, Prof Neil Marrion and Prof Elek Molnar, whose feedback and insight helped to shape the project. Many thanks also go to the lab technicians as well as current and previous members of the Hodge lab: Jack Curran, Kiah Tasman, Hannah Julienne, Amanda Deakin, Simon Lowe, James Higham, and especially Edgar Buhl for help in setting up the electrophysiology and behaviour experiments. Additional thanks go to Prof Lorraine Clark and members of her lab for the opportunity to collaborate on the essential tremor project and all the hard- work that went into making that happen. iii iv Author’s declaration I declare that the work in this dissertation was carried out in accordance with the requirements of the University's Regulations and Code of Practice for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate's own work. Work done in collaboration with, or with the assistance of, others, is indicated as such. Any views expressed in the dissertation are those of the author. SIGNED: ............................................................. DATE:.......................... v vi Table of contents Abstract .................................................................................................................................... i Dedication and acknowledgments ..................................................................................... iii Author’s declaration .............................................................................................................. v Table of contents ................................................................................................................. vii List of figures ......................................................................................................................... xi List of tables ........................................................................................................................ xiii List of abbreviations ............................................................................................................ xv Chapter 1 – Introduction ....................................................................................................... 1 1.1 Circadian rhythms ....................................................................................................... 1 1.2 Molecular regulation of circadian rhythms in Drosophila and mammals ............ 2 1.3 Neuronal regulation of rhythms in Drosophila and mammals .............................. 3 1.4 Voltage-gated potassium channels and other ion channels ................................ 6 1.5 Drosophila as a model organism for circadian research ...................................... 9 1.6 Aims of the thesis ....................................................................................................... 9 Chapter 2 - Materials & methods ...................................................................................... 11 2.1 Flies ............................................................................................................................ 11 2.1.1 Fly husbandry .................................................................................................... 11 2.1.2 Fly stocks ............................................................................................................ 12 2.2 Electrophysiology ...................................................................................................... 14 2.2.1 Brain dissection ................................................................................................. 14 2.2.2 Whole cell patch clamp..................................................................................... 15 2.2.3 Voltage protocol ................................................................................................. 16 2.2.4 Recording solutions .......................................................................................... 17 2.2.5 Pharmacology .................................................................................................... 17 2.2.6 Dynamic clamp .................................................................................................. 20 2.3 Behavioural assays .................................................................................................. 21 2.3.1 Longevity assay ................................................................................................. 21 2.3.2 Drosophila activity monitor (DAM) .................................................................. 21 2.4 Computational modelling ......................................................................................... 25 2.4.1 Channel models ................................................................................................. 25 2.4.2 Whole cell model ............................................................................................... 28 2.5 Statistics ..................................................................................................................... 28 vii Chapter 3 – Electrophysiology .......................................................................................... 30 3.1 Electrophysiology theory ........................................................................................
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