CHARACTERISATION OF INPUT AND OUTPUT MECHANISMS IN THE ZEBRA FINCH CIRCADIAN SYSTEM by CATHERINE LINDA JONES A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Biosciences College of Life and Environmental Sciences The University of Birmingham September 2011 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Circadian rhythms are biochemical, physiological, or behavioural over 24 hours. The avian circadian system is complex, involving numerous oscillators in the brain. I characterised two hypothalamic input mechanism (melatonin receptors and light) and one output mechanism (vasotocin) in the zebra finch. Melatonin receptors were cloned and expression levels investigated in the brain and in peripheral tissues. Receptors were found in all tissues, with some pronounced rhythmic mRNA expression. Tissue-specific differences in temporal distribution, peak expression and amplitude suggests melatonin have varied roles in different tissues and different receptors control/influence these roles. Effect of light in the hypothalamus was investigated by exposing light into the dark phase of an LD cycle and studying the difference in C-FOS expression. C-FOS was found in hypothalamic nuclei associated with photic transduction. C-FOS-IR cells were also found in the two known avian hypothalamic oscillators, the LHN and SCN. Arginine-Vasotocin is a neuropeptide involved in numerous bodily and nervous tissue functions, secreted within the hypothalamus and pituitary gland. Immunofluorescent experiments showed marked differences in expression, as different zeitgeber times and between species. This study has improved our understanding of avian circadian systems, providing new insights into the hypothalamic oscillator of a complex circadian organisation. ii Dedication This thesis is dedicated to my mother, father, brother (Caroline, Roger and David) and Grandma (Betty) who have shown me continued love and support in whatever I choose to do, and for that I am truly grateful. Zebra Finches from the breeding aviary. iii Acknowledgements I am very grateful to my supervisor Roland Brandstaetter for wonderful opportunity of doing this thesis, all your help, advice and direction he gave me throughout the course of this study. This project would not exist without your invaluable patience, encouragement and support throughout the past four years. Thanks to Dr. Gisela Helfer; for introducing me to the wonderful world molecular biology. Your help, advice and encouragement along the way were greatly appreciated and will not be forgotten. I am thankful to my colleagues at the University of Birmingham; particularly Laine Wallace and Anthony Jones for their advice and help in the genomics laboratory. Thanks go to Pete Jones and Phil Archer in the BMSU; for taking care of the birds for my experiments. I would also like to thank my PhD colleagues from the school of bioscience for keeping me sane and supporting me throughout my PhD, the laughs and drinks along the way were greatly appreciated. My deepest thanks go to my family Caroline, Roger, David, Linda and Betty and the Burgess‟s; your love and support is always there when I need it most and is always greatly received. To Richard Tudor, thanks go to you for your love and support throughout my PhD, writing of this thesis and molecular advice along the way. For keeping me smiling and going, it is all very much appreciated. And to all my friends who have been there for me when I needed them most and kept me going. Finally, thanks are due to the School of Biosciences, University of Birmingham and the Biotechnology and Biological Sciences Research Council (BBSRC) for the funding of this project. iv Contents Page CHAPTER 1 – GENERAL INTRODUCTION 1.1 Circadian system 1 1.1.1 Introduction 1 1.1.2 The molecular clockwork 4 1.1.3 Hypothalamus 9 1.1.4 Photic information 12 1.2 Avian circadian system 17 1.2.1 Hypothalamus 19 1.2.2 Pineal gland 25 1.2.3 Retina 26 1.3 Neurochemistry of circadian systems 27 1.3.1 Hormones 27 1.3.1.1 Melatonin 30 1.3.1.2 Arginine-vasotocin (AVT) 33 1.3.1.3 Somatostatin (SS) 34 1.3.2 Other neurotransmitter and neuropeptides 35 1.3.2.1 Serotonin (5-hydroxytryptamine, 5-HT) 37 1.3.2.2 Neuropeptide Y (NPY) 37 1.3.2.3 Vasoactive intestinal polypeptide (VIP) 39 1.3.2.4 Gamma-aminobutyric acid (GABA) 40 1.4 Summary 41 1.5 Thesis Aims and Objectives 43 1.6 Species of interest: Zebra finch (Taeniopygia guttata) 45 CHAPTER 2 - MATERIALS AND METHODS 2.1 Animals and husbandry 48 2.1.1 Animals 48 2.1.1.1 Zebra Finch 48 2.1.1.2 Chicken and Japanese quail 48 v 2.1.2 Synchronising aviaries 49 2.1.3 Lighting conditions 49 2.2 Molecular procedures 52 2.2.1 Tissue sampling 52 2.2.2 RNA extraction 54 2.2.3 RNA analysis 54 2.2.4 cDNA synthesis 55 2.2.5 Gene cloning of Mel-1A, Mel-1B and Mel-1C receptors 55 2.2.5.1 Ligation 56 2.2.5.2 Transformation of cells with vector ligated cells 58 2.2.5.3 FastPlasmid Miniprep 60 2.2.5.4 EcORI digestion 60 2.2.5.5 Ethanol precipitation 60 2.2.6 DNA sequencing and identification of zebra finch Mel-1A, Mel-1B and Mel -1C receptors cDNA 61 2.2.7 Reverse transcription polymerase chain reaction (RT-PCR) 62 2.2.7.1 Primer design 63 2.2.8 Optimising RT-PCR cycling conditions for Mel-1A, Mel-1B and Mel-1C primers 65 2.2.9 Semi-quantitative analysis of RT-PCR for Mel-1A, Mel-1B and Mel-1C 69 2.2.10 Gel electrophoresis 69 2.2.10.1 Agarose gel electrophoresis 69 2.2.10.1.1 RNA gels 70 2.2.10.2 Polyacrylamide gel electrophoresis 70 2.2.11 Visualisation and analysis 71 2.3 Immunofluorescent procedures 73 2.3.1 Tissue sampling, perfusion and processing of the brains 73 2.3.2 Sectioning tissues 76 2.3.3 Immunofluorescent protocol 78 2.3.4 Immunofluorescent stains 82 2.3.4.1 Melatonin receptor Mel-1A, Mel-1B and Mel-1C 82 2.3.4.2 C-FOS 82 2.3.4.3 Arginine-vasotocin (AVT) 83 2.3.4.3.1 Zebra finch 83 2.3.4.3.2 Chicken and Japanese quail 84 vi 2.3.4.4 Hoechst staining (Bisbenzimide H 33258) 84 2.3.5 Microscopy and analysis 85 CHAPTER 3 - MOLECULAR METHODOLOGY VALIDATION 3.1 Introduction 86 3.2 Results 95 3.2.1 RNA isolation and analysis 95 3.2.1.1 Brain tissues 95 3.2.1.2 Peripheral tissues 96 3.2.2 Cloning and DNA sequence analysis 98 3.2.2.1 Phylogenetic analysis 104 3.2.3 Optimising RT-PCR conditions for melatonin receptor analysis 107 3.2.3.1 Optimising PCR magnesium concentration and annealing temperatures 107 3.2.3.2 Optimising PCR cycle number 108 3.2.3.3 Testing cDNA concentrations 109 3.3 Discussion 112 CHAPTER 4 - MELATONIN RECEPTOR EXPRESSION IN THE ZEBRA FINCH BRAIN AND PERIPHERAL TISSUES 4.1 Introduction 115 4.2 Results 118 2.2.1 Zebra finch expression of melatonin receptors in the brain tissues 122 2.2.2 Zebra finch expression of melatonin receptors in the peripheral tissues 135 4.3 Discussion 142 CHAPTER 5 – MELATONIN MEMBRANE RECEPTOR LOCALISATION IN TWO AVIAN CIRCADIAN OSCILLATORY REGIONS 5.1 Introduction 148 5.2 Results 154 5.2.1 Antibodies and trials 154 5.2.2 Melatonin receptor expression in the hypothalamus at ZT6 and ZT18 157 vii 5.2.3 Melatonin receptor expression in the retina at ZT6 and ZT18 169 5.3 Discussion 180 CHAPTER 6 – LIGHT INDUCED c-fos EXPRESSION IN THE ZEBRA FINCH HYPOTHALAMUS 6.1 Introduction 184 6.2 Results 189 6.2.1 C-FOS expression in the zebra finch hypothalamus after one hour light exposure in the early dark period 191 6.2.2 C-FOS expression in the zebra finch hypothalamus after one hour light exposure in the late dark period 197 6.2.3 Comparing the effect that a one hour light exposure has in the early dark period to one hour light exposure in the late dark period, in the zebra finch hypothalamus 203 6.3 Discussion 208 CHAPTER 7 - ARGININE-VASOTOCIN (AVT) EXPRESSION IN THE ZEBRA FINCH HYPOTHALAMUS 7.1 Introduction 211 7.2 Results 217 7.2.1 AVTergic cell expression in the hypothalamus under LD conditions 217 7.2.1.1 Immunofluorescent image analysis 217 7.2.1.2 Immunofluorescent cell counts analysis 223 7.2.2 AVTergic cell expression in the hypothalamus under dimLL conditions 226 7.2.2.1 Immunofluorescent images analysis 226 7.2.2.2 Immunofluorescent cell count analysis 230 7.2.3 Comparing AVTergic cell expression under LD and dimLL conditions in the hypothalamus 233 7.3 Discussion 247 viii CHAPTER 8 - SPECIES COMPARISON OF AVTergic CELLS IN THE HYPOTHALAMUS AT ZT1 8.1 Introduction 250 8.2 Results 254 8.2.1 Immunofluorescent image analysis 254 8.2.2 Comparative AVTergic cell profiles in different species at ZT1 257 8.2.3 Comparative AVTergic cell count analysis at ZT1 260 8.3 Discussion 269 CHAPTER 9 - GENERAL DISCUSSION 9.1 General Discussion 272 9.2 Future work 280 REFERENCES 286 APPENDIX I Supplementary Material 1.1 Supplementary material for materials and methods, Chapter Two 1.1.1 Solutions and Mediums 320 1.2 Supplementary material for results Chapters Three-Eight 1.2.1 Sequence alignments for the phylogenetic tree analysis 323 1.2.2 Statistical analysis for the melatonin receptor mRNA expression in the zebra finch the brain tissues, in Chapter Four