A University of Sussex DPhil thesis Available online via Sussex Research Online: http://sro.sussex.ac.uk/ This thesis is protected by copyright which belongs to the author. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Please visit Sussex Research Online for more information and further details ACOUSTIC SENSITIVITY OF THE VESTIBULAR SYSTEM AND MECHANICAL ANALYSIS OF THE TECTORIAL MEMBRANE IN MAMMALS Gareth Paul Jones Submitted for the degree of Doctor of Philosophy September 2011 2 DECLARATION I hereby declare that this thesis has not been, and will not be, submitted in whole or in part to another University for the award of any other degree. _______________ Gareth Paul Jones 3 ACKNOWLEDGEMENTS I thank my supervisors Professor Ian Russell and Dr. Andrei Lukashkin for their invaluable advice and guidance during my time as a DPhil student and in the preparation of this thesis. I also thank the BBSRC for providing the funding for my studentship. I thank my parents, David and Marylynn Jones, for the continual support and encouragement that they have provided throughout my prolonged stay in education. I thank my work colleagues (and friends) for creating an enjoyable and supportive working environment, particularly James Hartley for building almost every piece of equipment in our lab (and for knowing how to fix it), Thomas Weddell for his help and advice and Terri Roberts for her skilled assistance in creating sensitive pieces of equipment out of Falcon tubes and insulation tape. Finally I also thank a number of my friends for kindly contributing their time and efforts to proof reading my work, particularly Nicola Harman, Adele Carvil, Emma Haynes, and also my mother, Marylynn Jones. They have all unwittingly become fully accountable for any typos remaining in this documeant. 4 PREFACE The research presented in this thesis is the work of Gareth P. Jones under the supervision of Ian J. Russell and Andrei N. Lukashkin. This work covers two distinct topics related to mammalian hearing; the retained acoustic sensitivity of the mammalian vestibular system and the mechanical properties of the mammalian tectorial membrane (TM). This thesis contains 7 chapters and 1 appendix. Chapter 1 contains a general introduction to mammalian hearing and to the vestibular system and Chapter 2 describes the materials and methods used in both the vestibular and TM experiments. Chapters 3-6 are results chapters and include specific introductions to the experiments performed, results and discussion of results. Chapter 3 covers the topic of the acoustic sensitivity of the mammalian vestibular system and was previously published in the Journal of the Association for Research in Otolaryngology in Sept. 2010 (Jones et al., 2010). Chapter 3 is a reproduction of this paper, edited slightly to conform to the formatting of this document. The methods are included in the chapter, with additional methodological detail that was omitted from the published paper included in Chapter 2. The references have also been merged with the main REFERENCES section. For this paper the collection of DPOAE and CAP recordings to assess the hearing of the Nox3 mice was performed by Dr. Victoria A. Lukashkina. Chapters 4-6 investigate the material properties of the mammalian TM (Jones et al., 2011) and the methods for the experiments presented in these chapters are contained entirely in Chapter 2. Chapter 7 contains a general discussion of the data presented in this thesis and suggestions for future work in the two topic areas. Chapters 2 and 7 are split in to two sections relating to the vestibular and TM work respectively. All references cited in the text are listed alphabetically in the references section. APPENDIX 1 contains a video of the dissections performed to isolate the TM from the mouse cochlea. 5 UNIVERSITY OF SUSSEX Gareth Paul Jones Acoustic sensitivity of the vestibular system and mechanical analysis of the tectorial membrane in mammals SUMMARY This thesis cover two separate topics related to the function of the mammalian inner ear. Acoustic sensitivity of the vestibular system. Data are presented showing facilitation of the auditory startle response by tones outside the range of the mouse cochlea. The sensation of these low frequency tones is demonstrated to be mediated via the acoustically sensitive sacculus of the vestibular system by data collected from Nox3-/- mice. These mice lack the otoconia of the vestibular system and, unlike the wild-type mice, only show facilitation to tones within the range of the mouse cochlea, and not in response to tones <4 kHz. The mechanical properties of the tectorial membrane (TM). The mechanical properties of the TM are investigated using a laser interferometer-based method for tracking the longitudinal propagation of a radially shearing travelling wave in segments of TM isolated from the basal and apical regions of the wild-type cochlea. The properties of these travelling waves (wave propagation velocity and wave amplitude decay) are tracked over a range of stimulus frequencies (1-20 kHz). The viscoelastic properties, shear storage modulus (G’) and shear viscosity (η), are estimated over this frequency range and are found to be lower in the apical TM segments compared to the basal TM segments, indicating the apical region of the TM is less stiff than the basal region. These data are compared to data collected from TM segments isolated from the basal cochlear region of three mutant groups, each lacking expression of TM-specific proteins; α-tectorin (TectaY1870C/+), β-tectorin (Tectb-/-) and otoancorin (OtoaEGFP/EGFP), using the same laser interferometer-based method. The viscoelastic properties are estimated for each of the mutants and indicate varying degrees of loss of structural integrity in their respective TM segments. Reflective difference between the wild-types and mutants are also observed and compared. 6 CONTENTS DECLARATION ..................................................................................................................... 2 ACKNOWLEDGEMENTS ........................................................................................................ 3 PREFACE ............................................................................................................................. 4 SUMMARY .......................................................................................................................... 5 CONTENTS .......................................................................................................................... 6 LIST OF FIGURES ................................................................................................................ 12 ABBREVIATIONS ................................................................................................................ 16 VARIABLES ........................................................................................................................ 16 1 GENERAL INTRODUCTION .......................................................................................... 17 1.1 Anatomy of the mammalian ear ......................................................................... 17 1.1.1 The outer and middle ears ................................................................................... 17 1.1.2 The inner ear ........................................................................................................ 18 1.2 The vestibular system ......................................................................................... 20 1.2.1 Anatomy of the vestibular system ....................................................................... 20 1.2.2 Acoustic sensitivity of the vestibular system ....................................................... 22 1.2.2.1 Vestibular evoked myogenic potentials. ......................................................... 24 1.2.2.2 Frequency tuning of the sacculus ................................................................... 25 1.2.2.3 Neural projections ........................................................................................... 26 1.2.2.4 Relevance to mammalian hearing .................................................................. 26 1.2.2.5 Nox3 mice ....................................................................................................... 27 1.3 The cochlea ........................................................................................................ 29 1.3.1 Anatomy and passive mechanical properties ...................................................... 29 1.3.1.1 Organ of Corti .................................................................................................. 30 1.3.1.2 Supporting cells ............................................................................................... 31 1.3.1.3 Hair cells .......................................................................................................... 31 1.3.1.4 Basilar membrane ........................................................................................... 34 1.3.1.5 Tectorial membrane ........................................................................................ 35 1.3.1.5.1 Composition .............................................................................................