Peripheral Mechanisms of Touch and Pain
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Peripheral Mechanisms of Touch and Pain Jane Elizabeth Sexton A thesis submitted for the degree of Doctor of Philosophy to University College London Wolfson Institute for Biomedical Research University College London 2016 Declaration I, Jane Elizabeth Sexton 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. 1 Abstract This thesis uses transgenic approaches to alter expression of candidate mechanosensors in physiological and pathological conditions to determine their contribution to the sensations of touch and pain. Transient receptor potential (TRP) ion channels have highly conserved roles in sensory function and a great deal of evidence implicates them in the process of mechanotransduction. Their propensity to form heteromeric complexes as well as the functional redundancy they exhibit makes them difficult to study. We used animals with global deletion of multiple canonical TRP (TRPC) channels to minimise the effects of these features and found that TRPC1, TRPC3, TRPC5 and TRPC6 have a modality specific and combinatorial role in innocuous mechanosensation. Next, we looked at the role of TRPC channels and TRPA1 and TRPV1 in mechanical hypersensitivity in the monosodium-iodoacetate (MIA) model of Osteoarthritis (OA). The results show TRPC3, TRPC6 and TRPV1 do not contribute to mechanical hypersensitivity in OA. However, TRPA1 appears to be necessary for the full manifestation of OA induced mechanical hypersensitivity. We also investigated the role of Annexin A6 in mechanosensation and OA. The Annexin A6 protein binds to NMB-1, a blocker of mechanically activated currents. We found that it negatively regulates mechanosensation and that by overexpressing the protein using a gene therapy approach we are able to partially attenuate mechanical hypersensitivity in the MIA model of OA. Finally, we investigated the role of ASIC4 in pain and mechanosensation through generation and behavioural characterisation of a global knockout mouse. The function of ASIC4 is largely unknown but in vitro it regulates expression and function of other ASIC subunits, some of which have roles in noxious and mechanosensory processes. We found a role for ASIC4 in formalin induced pain and in visceral inflammation which is the first report of a somatosensory function for this poorly understood protein. 2 Funding I would like to thank the following for funding this project: Publications Sexton, JE, Desmonds, T, Quick, K, Taylor, R, Abramowitz, J, Forge, A, Kros, CJ, Birnbaumer, L and Wood, JN. (2015). The contribution of TRPC1, TRPC3, TRPC5 and TRPC6 to touch and hearing. Neuroscience letters 610:36-42. Sexton, JE, Vernon, J and Wood, JN (2014) TRPs and Pain. In: Nilius, B. and Flockerzi, V. (eds.) Mammalian Transient Receptor Potential (TRP) Cation Channels, Volume II. Handbook of Experimental Pharmacology Volume 223. Springer International Publishing. pp 873-897 3 Contents 1 Introduction .............................................................................................................. 18 1.1 Introduction to Neurobiology ........................................................................... 18 1.2 Mechanosensation ............................................................................................ 22 1.2.1 Primary afferent neurons and mechanosensory end organs ...................... 22 1.2.2 The electrophysiological properties of mechanosensitive fibres .............. 25 1.2.3 Potential mechanisms of mechanotransduction ........................................ 27 1.2.4 Mechanotransduction in the inner ear ....................................................... 29 1.2.5 Mechanotransduction candidates .............................................................. 31 1.3 Pain and nociception ........................................................................................ 33 1.3.1 Inflammatory and neuropathic pain .......................................................... 36 1.3.2 Central pain pathways ............................................................................... 43 1.4 Transient Receptor Potential (TRP) channels .................................................. 45 1.4.1 TRP channel expression in sensory neurons ............................................. 47 1.4.2 TRP channel structure and heteromer formation ...................................... 49 1.4.3 TRP channels in Mechanosensation.......................................................... 51 1.4.4 TRP channels and pain .............................................................................. 54 1.5 Acid sensing ion channels ................................................................................ 58 1.5.1 ASIC expression ....................................................................................... 59 1.5.2 Biophysical properties of ASICs ............................................................... 61 1.5.3 ASICs in mechanosensation ...................................................................... 65 1.5.4 ASICs in pain ............................................................................................ 67 1.6 Annexin A6 ...................................................................................................... 72 1.7 Voltage gated sodium channels ........................................................................ 74 1.8 Targeted gene deletion strategies in transgenic mice ....................................... 77 1.8.1 Cre/loxP system ........................................................................................ 77 1.8.2 Flp/FRT system ......................................................................................... 77 4 2 Aims of this project.................................................................................................. 78 3 Materials and Methods............................................................................................. 80 3.1 Molecular Biology ............................................................................................ 80 3.1.1 DNA extraction for genotyping ................................................................ 80 3.1.2 Polymerase chain reaction for genotyping ................................................ 81 3.1.3 Gel electrophoresis .................................................................................... 81 3.1.4 RNA extraction ......................................................................................... 81 3.1.5 cDNA synthesis ......................................................................................... 82 3.1.6 Agarose gel extraction and DNA sequence analysis ................................ 82 3.1.7 Reverse transcription polymerase chain reaction (RT-PCR) .................... 83 3.1.8 Quantitative RT-PCR ................................................................................ 83 3.2 Immunohistochemistry ..................................................................................... 84 3.2.1 Tissue collection........................................................................................ 84 3.2.2 Sectioning .................................................................................................. 84 3.2.3 Immunostaining ........................................................................................ 84 3.2.4 Analysis ..................................................................................................... 84 3.3 Western blotting ............................................................................................... 85 3.3.1 Tissue preparation ..................................................................................... 85 3.3.2 Protein quantification ................................................................................ 85 3.3.3 SDS-PAGE ................................................................................................ 85 3.3.4 Wet transfer ............................................................................................... 85 3.3.5 Blocking and antibody incubation ............................................................ 85 3.4 Antibodies ........................................................................................................ 87 3.5 Behavioural testing ........................................................................................... 88 3.5.1 Von Frey ................................................................................................... 88 3.5.2 Cotton Swab test ....................................................................................... 88 3.5.3 Randall Selitto ........................................................................................... 88 5 3.5.4 Hargreaves................................................................................................. 89 3.5.5 Hot Plate .................................................................................................... 89 3.5.6 Thermal Place Preference ......................................................................... 89 3.5.7 Rotarod ...................................................................................................... 90 3.5.8 Swim Test ................................................................................................. 90 3.5.9 Trunk Curl Test ......................................................................................... 90 3.5.10 Auditory testing ........................................................................................