An In Vivo Investigation into the Actions of the Hypothalamic Neuropeptide, QRFP A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health Christopher J Cook Faculty of Biology, Medicine and Health School of Medical Sciences 2017 Contents Abstract ........................................................................................................................................... 11 Declaration ........................................................................................................................................ 12 Copyright ........................................................................................................................................... 12 Acknowledgement ............................................................................................................................ 13 Chapter 1 Introduction ................................................................................................. 14 1.1 Energy homeostasis ................................................................................................................ 15 1.2 The control of food intake ...................................................................................................... 16 1.2.1 Peripheral signals regulating food intake .......................................................................... 17 1.2.2 Central aspects of food intake regulation ......................................................................... 19 1.2.3 Integration of peripheral and central signalling mechanisms ........................................... 21 1.2.4 Interconnectivity of hypothalamic nuclei in feeding regulation ....................................... 21 1.3 Homeostatic and hedonic control of feeding ......................................................................... 24 1.4 Control of energy expenditure ............................................................................................... 26 1.5 Central control of wakefulness/sleep ..................................................................................... 27 1.6 Nutrient sensing in the hypothalamus ................................................................................... 29 1.6.1 Glucose sensing ................................................................................................................. 30 1.6.2 Glucose-sensitive hypothalamic populations .................................................................... 30 1.6.3 Fatty acid sensing .............................................................................................................. 31 1.7 White Adipose Tissue ............................................................................................................. 32 1.7.1 Endocrine function of WAT ............................................................................................... 33 1.7.2 Neuronal control of WAT function .................................................................................... 34 1.7.3 Intracellular signalling in the maintenance of WAT depots .............................................. 34 1.8 RFamides................................................................................................................................. 37 1.8.1 Pyroglutamylated RFamide Peptide .................................................................................. 38 1.8.2 Gpr103 ............................................................................................................................... 39 1.8.3 QRFP and Gpr103 expression ............................................................................................ 40 1.8.4 Centrally-mediated effects of QRFP .................................................................................. 41 1.8.5 Action of QRFP in peripheral tissues ................................................................................. 42 Chapter 2 General Methods ......................................................................................... 44 2.1 In vivo methods ...................................................................................................................... 45 2.1.1 Animals .............................................................................................................................. 45 2.1.2 Reporter/cre mouse husbandry ........................................................................................ 45 2.1.3 Peptides ............................................................................................................................. 46 2.1.4 Stereotaxic surgery ............................................................................................................ 46 2.1.5 Feeding experiments ......................................................................................................... 48 2.1.6 Activity testing ................................................................................................................... 48 2.1.7 Oral glucose tolerance test (OGTT) ................................................................................... 49 2.1.8 Tissue dissection ................................................................................................................ 49 2.1.9 Transcardial perfusion for tissue fixation .......................................................................... 50 2.1.10 Operant response .............................................................................................................. 50 2.2 Ex vivo methods ...................................................................................................................... 52 2.2.1 Sectioning of frozen tissue ................................................................................................ 52 2.2.2 Immunohistochemistry ..................................................................................................... 53 2.2.3 Genotyping ........................................................................................................................ 55 2 2.2.4 Relative quantitative real time PCR (qPCR) ....................................................................... 57 2.2.5 End point reverse transcription PCR (RT-PCR) .................................................................. 61 2.3 Statistical Methods ................................................................................................................. 61 Chapter 3 The Location of QRFP in the Brain and Other Tissues ..................................... 62 3.1 Introduction ............................................................................................................................ 63 3.1.1 Objectives .......................................................................................................................... 64 3.2 Methods .................................................................................................................................. 65 3.2.1 Generation of transgenic Qrfp-cre mice ............................................................................ 65 3.2.2 QRFP peripheral expression .............................................................................................. 66 3.2.3 Immunohistochemical investigation of QRFP neurons ..................................................... 66 3.2.4 Efferent projection of QRFP neurons: cre-dependent tracing .......................................... 68 3.3 Results ..................................................................................................................................... 70 3.3.1 Qrfp expression ................................................................................................................. 70 3.3.2 Visualisation of QRFP in the brain ..................................................................................... 71 3.3.3 Visualisation of QRFP in the eye ........................................................................................ 75 3.3.4 Investigating QRFP co-localisation with other transmitters .............................................. 76 3.3.5 Efferent projection of QRFP neurons: cre-dependent tracing .......................................... 80 3.3.6 Anterograde tracing of QRFP neurons using AAVs ............................................................ 96 3.3.7 Retrograde tracing of QRFP projection targets ................................................................. 98 3.4 Discussion ............................................................................................................................... 99 3.4.1 Visualisation of QRFP in the brain ..................................................................................... 99 3.4.2 Investigation of co-expression of QRFP with other transmitters .................................... 100 3.4.3 Projections of QRFP neurons ........................................................................................... 102 3.4.4 Summary .......................................................................................................................... 104 Chapter 4 Metabolic Effects of QRFP .......................................................................... 105 4.1 Introduction .......................................................................................................................... 106 4.1.1 Objectives 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