Using Electrophysiology to Explore Retinal Function in Autosomal Dominant Optic Atrophy A thesis submitted to Cardiff University for the degree of Doctor of Philosophy By Enyam Komla Amewuho Morny School of Optometry and Vision Sciences Cardiff University October 2016 Supervisors Marcela Votruba, Tom Margrain and Alison Binns DECLARATION This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed ………………………… Date: 10/10/2016 STATEMENT 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of PhD. Signed ………………………… Date: 10/10/2016 STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated, and the thesis has not been edited by a third party beyond what is permitted by Cardiff University’s Policy on the Use of Third Party Editors by Research Degree Students. Other sources are acknowledged by explicit references. The views expressed are my own. Signed ………………………… Date: 10/10/2016 STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed ………………………….. Date: 10/10/2016 STATEMENT 4: BAR ON ACCESS APPROVED I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loans after expiry of a bar on access previously approved by the Academic Standards and Quality Committee. Signed ………………………….. Date: 10/10/2016 i SUMMARY Autosomal dominant optic atrophy (ADOA) is an inherited optic neuropathy due to mutation in the OPA1 gene. Patients present with bilateral optic nerve head pallor and loss of visual function. Patients also show a reduction in the P50:N95 ratio of the pattern electroretinogram (PERG) and thinning of the retinal nerve fibre layer (RNFL) and macula. In a mouse model of ADOA, previously generated in this laboratory, the defect first manifested as a dendritic pruning of RGCs, which appeared to be ON- centre specific. Electrophysiological evidence in both humans and the mutant mice showed a reduction in the photopic negative component (PhNR) of the brief flash electroretinogram (ERG). The separation of the photopic ERG into ON- and OFF-pathway components using long-duration monochromatic (red) flash on a rod suppressing blue background, provided an opportunity to assess ON- and OFF-RGC function in ADOA, which had not been previously investigated in humans. This study therefore aimed to assess the effect of ADOA on the red-on blue PhNR-ON and PhNR-OFF components to determine whether the PhNR-ON was a selective marker for ADOA. In this thesis, a protocol was developed for recording long duration red-on-blue PhNRs from the macula (focal) and entire retina (full-field). A comparison of the retinotopic characteristics of the PhNRs (brief and long-duration) with the N95 of the PERG and the distribution of RGCs in the retina showed that the PhNR was capable of assessing RGC function. Retinal function was then probed in twelve participants with ADOA and sixteen controls using focal and full-field long duration ERG, full-field brief flash ERG and PERG. Retinal structure was also assessed using optical coherence tomography. In conclusion, this thesis found that the PhNR-ON and PhNR-OFF amplitudes were equally affected with no evidence of a preferential ON-pathway loss. ii ACKNOWLEDGEMENTS Praise to the LORD, the Almighty, the God of all creation; Who now as then reveals in man His Glory. This PhD journey for me has been altogether exciting, not because it was smooth sailing, but because it was full of numerous twists and turns as well as high and low points. Its successful completion has been by the amazing and indispensable grace of God, and by the immeasurable support from so many people. I am particularly grateful to my supervisors Marcela, Tom and Alison. They have been my wise and gracious mentors and dependable guides through this journey. I couldn’t have asked for a better deal. Acknowledgements also go to Mr Kishan Patel, who collected some of the electrophysiology data for the study in Chapter 4 under the Cardiff Undergraduate Research Opportunities Programme (CUROP) 2015. A tonne of gratitude to the Fantastic Flors, my “sister-in-arms” in the “Land of Strangers”. #WWIDWY? Big-ups go to my colleagues in 2.11, Chris, Seine, Kathy, Sharon, Caroline, Katie, Jimmy, Bert, Rupal and Laura for being good company and loyal subjects for my long experiments. Thanks also to Ashley, Tony and Katie Mortlock, my helpful sounding boards and to Sue Hobbs, the repository of all wisdom and knowledge of the school. And to Atsufui and Xoese, for not giving up on me and bearing with me the many times I said I’d finish and I didn’t. I love you. iii TABLE OF CONTENTS DECLARATION ................................................................................................... i SUMMARY.......................................................................................................... ii ACKNOWLEDGEMENTS .................................................................................. iii TABLE OF CONTENTS ..................................................................................... iv LIST OF FIGURES ............................................................................................. x LIST OF TABLES .............................................................................................. xv LIST OF ABBREVIATIONS ............................................................................ xviii CHAPTER ONE – INTRODUCTION ......................................................... 19 1.1 Introduction to the Thesis ..................................................................... 19 1.2 Overview of the Retinal Architecture .................................................... 20 1.2.1 The Photoreceptors ....................................................................... 22 1.2.2 The Bipolar Cells ........................................................................... 23 1.2.3 The Retinal Ganglion Cells ............................................................ 23 1.2.4 ON and OFF Pathways of the Retina ............................................ 27 1.3 Autosomal Dominant Optic Atrophy ..................................................... 29 1.3.1 Prevalence and Epidemiology ....................................................... 30 1.3.2 Clinical Features............................................................................ 30 1.3.3 Syndromic dominant optic atrophy ................................................ 37 1.3.4 The OPA1 Gene and its Functions ................................................ 39 1.3.5 Pathogenesis and Molecular Genetics of ADOA ........................... 41 1.3.6 Peculiar Susceptibility of RGCs in ADOA ...................................... 42 1.3.7 Other Loci Associated with Hereditary Optic Atrophy .................... 43 1.3.8 Current Management .................................................................... 45 1.3.9 Mouse Models: What they tell us about OPA1 in ADOA ............... 47 1.4 Ocular Electrophysiology ..................................................................... 52 1.4.1 The Electroretinogram ................................................................... 52 1.4.2 The Flash Electroretinogram ......................................................... 53 1.4.3 The Characteristics of the PhNR ................................................... 64 1.4.4 The Pattern ERG (PERG) ............................................................. 77 1.4.5 Electrophysiological Findings in ADOA ......................................... 80 1.5 Rationale of the Study.......................................................................... 81 1.6 Hypothesis ........................................................................................... 82 1.7 Aims of the Study ................................................................................. 83 iv CHAPTER TWO – ELECTROPHYSIOLOGICAL PROTOCOL DEVELOPMENT ............................................................................................... 84 2.1 Introduction .......................................................................................... 84 2.2 General Methods ................................................................................. 84 2.2.1 Participants ................................................................................... 84 2.2.2 Light Stimulator ............................................................................. 85 2.2.3 Calibration of equipment ............................................................... 88 2.2.4 Electrodes ..................................................................................... 89 2.2.5 Electronic recording system .......................................................... 89 2.2.6 Procedures .................................................................................... 90 2.2.7 Signal Analysis .............................................................................. 90 2.3 Experiment 1: Determining Stimulus Duration ..................................... 91 2.3.1 Introduction ................................................................................... 91 2.3.2 Methods .......................................................................................