Role of Voltage-gated Sodium Channel Isoforms in Electrophysiological Properties of Neurons Innervating the Viscera in Mice Andelain Kristiseter Erickson School of Medicine Discipline of Medicine The University of Adelaide Thesis submitted for the degree of Doctor of Philosophy 17th May 2019 Table of Contents Citation listing ........................................................................................................................................ vi Abstract .................................................................................................................................................. vii Declaration of originality ..................................................................................................................... viii Acknowledgments .................................................................................................................................. ix Abbreviations .......................................................................................................................................... x Chapter 1 Visceral Pain .......................................................................................................................... 2 1.1 Abstract ......................................................................................................................................................... 2 1.2 Introduction .................................................................................................................................................... 2 1.3 Clinical relevance .......................................................................................................................................... 3 1.4 Visceral comorbidities .................................................................................................................................... 3 1.5 Extrinsic sensory afferent innervation of the colon and bladder .................................................................... 4 1.6 Afferent subtypes innervating the colon and bladder .................................................................................... 5 1.7 Conclusions ................................................................................................................................................. 14 1.8 Disclosure statement ................................................................................................................................... 14 1.9 Acknowledgements ..................................................................................................................................... 15 1.10 Literature cited ........................................................................................................................................... 15 Chapter 2 Voltage-gated sodium channels: (Nav)igating the field to determine their contribution to visceral nociception ......................................................................................................................... 24 2.1 Abstract ....................................................................................................................................................... 24 2.2 Introduction .................................................................................................................................................. 25 2.3 Nav1.1 .......................................................................................................................................................... 29 2.4 Nav1.2 .......................................................................................................................................................... 29 2.5 Nav1.3 .......................................................................................................................................................... 29 2.6 Nav1.4 .......................................................................................................................................................... 30 2.7 Nav1.5 .......................................................................................................................................................... 30 2.8 Nav1.6 .......................................................................................................................................................... 30 2.9 Nav1.7 .......................................................................................................................................................... 31 2.10 Nav1.8 ........................................................................................................................................................ 33 2.11 Nav1.9 ........................................................................................................................................................ 34 2.12 Conclusion ................................................................................................................................................. 38 2.13 Additional information ................................................................................................................................ 40 2.14 Author contributions ................................................................................................................................... 40 2.15 Funding ...................................................................................................................................................... 40 2.16 Literature cited ........................................................................................................................................... 40 Chapter 3 Domain organization, gating mechanisms, and neurotoxin binding sites of mammalian voltage-gated sodium channels ........................................................................................................... 48 3.1 Pore-forming α-subunit and auxiliary β-subunits ......................................................................................... 48 3.2 Mechanism of voltage-dependent activation ............................................................................................... 48 ii ` 3.3 Mechanism of fast inactivation ..................................................................................................................... 49 3.4 Nav channel neurotoxin binding sites ........................................................................................................... 49 3.5 Literature cited ............................................................................................................................................. 50 Aims ........................................................................................................................................................ 52 Chapter 4 Methods ................................................................................................................................ 53 4.1 Animals and chronic visceral hypersensitivity model ................................................................................... 53 4.2 Retrograde labeling of colon-innervating neurons and dissociated dorsal root ganglia cell culture ............ 53 4.3 RNA extraction and reverse-transcription quantitative polymerase chain reaction of whole dorsal root ganglia ........................................................................................................................................................................... 54 4.4 Single-cell quantitative polymerase chain reaction of colon-innervating dorsal root ganglia neurons ......... 55 4.5 Whole-cell current-clamp electrophysiology of colon-innervating dorsal root ganglia neurons ................... 55 4.6 Whole-cell voltage-clamp electrophysiology of colon-innervating dorsal root ganglia neurons ................... 56 4.7 Literature cited ............................................................................................................................................. 57 Chapter 5 Active electrophysiological properties of colon-innervating dorsal root ganglia neurons from healthy mice and TNBS-treated mice .......................................................................... 58 5.1 Thoracolumbar and lumbosacral dorsal root ganglia from healthy mice and TNBS-treated mice have similar expression levels of Nav1.1-Nav1.9 (scn1a-scn11a) and β-subunits β1-β4 (scn1b-scn4b) ............................... 58 5.2 Thoracolumbar colon-innervating dorsal root ganglia from healthy mice and TNBS-treated mice have comparable expression of Nav1.1-Nav1.9 (scn1a-scn11a) at the single-cell level ............................................. 60 5.3 Thoracolumbar and lumbosacral colon-innervating dorsal root ganglia from healthy mice express β-subunits β1-β4 (scn1b-scn4b) at the single-cell level ...................................................................................................... 61 5.4 Baseline rheobase in colon-innervating dorsal root ganglia neurons is affected by spinal origin, number of days in culture, soma diameter, and resting membrane potential, but not TNBS treatment .............................. 62 5.5 Colon-innervating dorsal root ganglia neurons from TNBS-treated mice do not display a higher number of action potentials at 2-times rheobase compared to neurons from healthy mice ................................................ 63 5.6 Baseline peak sodium current density in colon-innervating dorsal root ganglia