Circadian regulation of the vertebrate habenula A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2019 Adriana Basnakova School of Biological Sciences, Division of Molecular and Cellular Function Table of Contents Abbreviations .............................................................................................................................. 4 Abstract ........................................................................................................................................ 6 Declaration ................................................................................................................................... 7 Copyright statement ................................................................................................................... 7 Acknowledgements .................................................................................................................... 8 Chapter 1 Introduction .............................................................................................................. 10 Circadian rhythms ................................................................................................................... 10 Circadian clock at molecular level ........................................................................................... 10 Organisation of the circadian system in vertebrates .............................................................. 12 Zebrafish circadian oscillators ............................................................................................. 13 Master circadian pacemaker in mammals .......................................................................... 16 Habenula ................................................................................................................................. 22 Suborganisation of habenula in mammals ......................................................................... 22 Suborganisation of habenula in zebrafish .......................................................................... 25 Neurotransmitter profile of mammalian habenula ............................................................ 26 Neurotransmitter profile of zebrafish habenula ................................................................. 28 Lateralisation....................................................................................................................... 29 Role of habenula in regulation of behaviour ...................................................................... 29 Habenula as a component of circadian system .................................................................. 32 Thesis aims .............................................................................................................................. 35 Chapter 2 Circadian rhythms in the habenula of zebrafish .................................................. 37 Introduction ............................................................................................................................ 37 Calcium and phosphorylated-ERK rhythms in brain ........................................................... 37 Calcium and phosphorylated-ERK monitoring in zebrafish ................................................ 38 Aims..................................................................................................................................... 40 Part I: Monitoring circadian variations in the habenula using bioluminescence ................... 41 Methods .............................................................................................................................. 41 Results ................................................................................................................................. 43 Discussion ............................................................................................................................ 46 Part II: Monitoring circadian variations in the habenula using immunohistochemistry of pERK ................................................................................................................................................ 47 Methods .............................................................................................................................. 47 Results ................................................................................................................................. 49 Discussion ............................................................................................................................ 55 Part III: Monitoring circadian variation in the habenula using calcium imaging .................... 58 Methods .............................................................................................................................. 58 Results ................................................................................................................................. 60 2 Discussion ............................................................................................................................ 64 Summary and evaluation of the techniques ........................................................................... 66 Chapter 3 Blocking the clock in the zebrafish habenula ...................................................... 68 Introduction ........................................................................................................................ 68 Aims..................................................................................................................................... 69 Methods .............................................................................................................................. 70 Results ................................................................................................................................. 73 Discussion ............................................................................................................................ 81 Chapter 4 Circadian rhythms and influence of modulators in the mouse habenula ......... 85 Introduction ........................................................................................................................ 85 Aims..................................................................................................................................... 88 Methods .............................................................................................................................. 89 Results ................................................................................................................................. 93 Discussion .......................................................................................................................... 114 Chapter 5 General discussion ............................................................................................... 118 The habenula oscillator and its electrical output ................................................................. 118 Daily changes in habenula activity in vertebrates ................................................................ 118 Potential role of rhythmic habenula ..................................................................................... 119 Role of AVP and OT neuromodulators in regulation of the vertebrate habenula ................ 120 Role of habenula oscillator in circadian regulation of brain neurotransmission and behaviour .............................................................................................................................................. 121 Future work ........................................................................................................................... 122 Chapter 2 ........................................................................................................................... 122 Chapter 3 ........................................................................................................................... 122 Chapter 4 ........................................................................................................................... 124 Evaluation of mouse and zebrafish animal models for investigation of brain oscillators .... 125 Implications of habenula research ........................................................................................ 127 References ............................................................................................................................... 128 Final word count: 48 928 3 Abbreviations AANAT Aralkylamine N-acetyltransferase ACh Acetylcholine aCSF Artificial cerebrospinal fluid AP Action potential ARC Arcuate nucleus of the hypothalamus AVP Arginine vasopressin BMAL Brain and muscle arnt-like cAMP Cyclic adenosine monophosphate CCGs Clock-controlled genes CLZN Coelenterazine CNS Central nervous system CREB Cyclic adenosine monophosphate response element-binding CRY Cryptochrome CT Circadian time DA Dopamine DD Constant darkness dpf days post-fertilisation DRN Dorsal raphe nucleus EGFP Enhanced green fluorescent protein ERK Extracellular signal-regulated kinase FR Fasciculus retroflexus GA GFP-aequorin GA-opt GFP-aequorin codon optimised GABA Gamma-aminobutyric acid GECI Genetically encoded calcium indicator GFP Green fluorescent protein HCN Hyperpolarisation-activated cyclic nucleotide-gated hpf hours post-fertilisation Hz Hertz IHC Immunohistochemistry IPN