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ATP-Activated Currents and Calcium Signalling in Adult Murine Supporting Cells

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ATP-Activated Currents and Calcium Signalling in Adult Murine Supporting Cells ATP-activated currents and calcium signalling in adult murine supporting cells Piotr Sirko University College London Thesis submitted for the degree of Doctor of Philosophy 1 I, Piotr Sirko, 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 Abstract Purinergic P2 receptors are present in the adult organ of Corti however their role and cellular effects of their stimulation are unclear. To date studies have been hampered by the relative difficulty of accessing intact adult organ of Corti tissue. In this study a novel dissection technique which enabled easy access to the apical turn of the adult mouse organ of Corti was used. The goal of the current study was to determine the distribution of P2 receptors and determine the effect of ATP on membrane conductances in inner sulcus (IS) supporting cells and supporting cells surrounding the IHC afferent synapse in the adult mouse organ of Corti. Purinergic P2 receptors were localized immunohistochemically and functionally to multiple supporting cells of the organ of Corti. In patch clamp electrophysiology recordings application of extracellular ATP to gap junction coupled IS cells, elicited inward currents and transiently increased resting membrane resistance while making the resting zero current potential more positive. Transmitted brightfield imaging showed that the electrophysiological changes were accompanied by a decrease in cytoplasmic Brownian movement. In IS cells decoupled with gap junction blockers stimulation with ATP elicited monophasically declining membrane currents. In contrast in decoupled border cells surrounding the IHC afferent synapse ATP activated biphasic currents. The initial component of the currents elicited in border cells desensitized during sustained stimulation and showed high inward rectification, characteristic of some P2X receptor subtypes. In contrast the delayed component was identified as a Ca2+-activated Cl- channel. It is argued that these conductances could help decrease the potassium concentration around the IHCs during periods of sustained IHC stimulation and thus affect the membrane potential of these sensory cells. In addition, actively propagating, gap junction-mediated, extracellular ATP-independent Ca2+ waves were observed to propagate along the organ of Corti in the IS and Deiters’ cell regions. These increases in intracellular Ca2+ levels were periodic. Currently the role of the waves is unclear, however increases in intracellular Ca2+ of a similar frequency and periodicity have been linked to the regulation of gene expression. Thus the observed waves could be a new mechanism regulating gene expression in the organ of Corti. 3 Acknowledgements Acknowledgements I would like to thank my supervisor Prof. Jonathan Ashmore for giving me the opportunity to become an independent and impartial scientist. I would also like to thank Dr. Jonathan Gale for discussion and support when designing and conducting experiments. I would like to thank Miriam for providing invaluable help in the laboratory numerous times, post-docs: Jime, Paromita, Roberta and Zoe as well as fellow students: Assel, Claudia, Elena, Lorcan, Lucile, Sara, and Stephanie for interesting discussions and inspiration. Finally, I would like to thank Paulina for giving me the energy and confidence necessary to continue throughout the years and my parents for their continued support. 4 Table of Contents Table of Contents LIST OF FIGURES ...................................................................................................... 10 LIST OF TABLES ........................................................................................................ 14 LIST OF ACRONYMS ................................................................................................ 15 1 INTRODUCTION ................................................................................................. 17 1.1 STRUCTURE OF THE COCHLEA .......................................................................... 17 1.1.1 Inner ear anatomy ....................................................................................... 17 1.1.2 Anatomy of the organ of Corti .................................................................... 17 1.1.3 The development of the organ of Corti ....................................................... 21 1.1.4 Tectorial membrane, structural role and morphology, contact with hair cells 21 1.1.5 Hearing ....................................................................................................... 21 1.2 IMPORTANCE OF COCHLEAR SUPPORTING CELLS AND GAP JUNCTIONS IN THE ORGAN OF CORTI .......................................................................................................... 22 1.2.1 Gap junctions in the organ of Corti ............................................................ 22 1.2.2 Role of supporting cells and gap junctions in the organ of Corti ............... 24 1.2.3 Role of supporting cells at the afferent synapse ......................................... 26 1.2.4 Purinergic receptors ................................................................................... 27 1.2.5 Purinergic signalling in the cochlea ........................................................... 30 1.3 IMPORTANCE OF ATP SIGNALLING IN THE COCHLEA ........................................ 31 1.3.1 Protective role of cochlear ATP signalling ................................................. 31 1.3.2 Purinergic cotransmission in the cochlea ................................................... 32 1.3.3 Effects of extracellular ATP-dependent intracellular Ca2+ regulation on cochlear supporting cells ........................................................................................ 32 1.3.4 Mechanism of propagation of ATP-mediated Ca2+ waves .......................... 33 1.3.5 Significance of spontaneous ATP-mediated Ca2+ waves in the neonatal cochlea .................................................................................................................... 34 2 MATERIALS AND METHODS ......................................................................... 37 2.1 GENERAL REAGENTS ........................................................................................ 37 2.2 EX VIVO PREPARATION ..................................................................................... 37 2.2.1 Obtaining and dissecting adult mouse cochlear tissue ............................... 37 5 Table of Contents 2.3 ELECTROPHYSIOLOGY ...................................................................................... 37 2.3.1 Solutions ...................................................................................................... 37 2.3.2 Recording electrodes and puff pipettes ....................................................... 37 2.3.3 Recording equipment .................................................................................. 38 2.3.4 Cell identification ........................................................................................ 38 2.3.5 Application of P2 agonists .......................................................................... 39 2.3.6 Blocking gap junctional coupling ............................................................... 39 2.3.7 Electrophysiological patch clamp recordings ............................................ 39 2.3.8 Correction of liquid junction potentials ...................................................... 40 2.3.9 Recording protocols used ............................................................................ 40 2.3.10 Data analysis ........................................................................................... 42 2.4 IMMUNOHISTOCHEMISTRY ............................................................................... 42 2.4.1 Fixation ....................................................................................................... 42 2.4.2 Immuno-labelling ........................................................................................ 43 2.4.3 Confocal imaging ........................................................................................ 44 2.5 BRIGHTFIELD IMAGING..................................................................................... 44 2.5.1 Brightfield imaging and analysis ................................................................ 44 2.6 CALCIUM IMAGING .......................................................................................... 45 2.6.1 Calcium indicator loading and intracellular Ca2+ imaging ....................... 45 2.6.2 Measuring the duration of intracellular Ca2+ increase .............................. 45 2.6.3 Measuring the percentage change of Ca2+ indicator signal ....................... 46 2.6.4 Subtracting background fluorescence ......................................................... 46 2.6.5 Comparing the intracellular Ca2+ increase evoked by different P2 receptor agonists ................................................................................................................... 46 2.6.6 Treatment with P2 receptor antagonists and apyrase ................................ 47 2.6.7 Laser ablation of hair cells ......................................................................... 48 2.6.8 Measurement of Ca2+ wave properties ....................................................... 48 2.6.9 Measurement of Ca2+ wave parameters from kymographs ........................ 49 2.7 DATA PRESENTATION
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