TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Entwicklungsgenetik Genetically dissecting the role of CRHR2 and UCN2 in the control of emotional behavior Adam Kolarz Vollständiger Ausdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. S. Scherer Prüfer der Dissertation: 1. Univ.-Prof. Dr. W. Wurst 2. Priv.-Doz. Dr. C. Wotjak (Ludwig-Maximilians-Universität München) Die Dissertation wurde am 20.02.2015 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 05.06.2015 angenommen. Table of content Table of content Table of content ............................................................................................. 3 Table of figures .............................................................................................. 8 List of abbreviations ...................................................................................... 11 Abstract ........................................................................................................ 18 Zusammenfassung ........................................................................................ 20 The Three Oddest Words ..................................................................................................................................... 22 1 Introduction ............................................................................................ 23 1.1 Depression and anxiety ........................................................................................................................ 23 1.1.1 Neurobiology of depression and anxiety disorders. ....................................................................... 23 1.1.2 Serotonergic dysfunction in anxiety and mood disorders .............................................................. 24 1.2 Stress as an environmental factor contributes to development of psychiatric disorders. ................... 25 1.2.1 Stress response ............................................................................................................................... 25 1.2.2 Regulation of the HPA axis .............................................................................................................. 27 1.2.2.1 Afferent projections to the PVN – initiation and integration of the stress signal .................. 27 1.2.2.2 Extinction of the stress response ........................................................................................... 29 1.3 Beyond the monoaminergic theory of depression ............................................................................... 30 1.3.1 The CRH/UCN system in the brain................................................................................................... 30 1.3.1.1 CRH and its role in anxiety and mood disorders .................................................................... 32 1.3.2 CRH receptors.................................................................................................................................. 33 1.3.2.1 Corticotropin-releasing hormone receptor type 1 (CRHR1) ................................................... 33 1.3.2.2 Corticotropin-releasing hormone receptor type 2 (CRHR2) ................................................... 35 1.3.2.2.1 CRHR2 and its role in stress response regulation.............................................................. 36 1.3.2.2.2 Stress recovery - CRHR2 in the bed nucleus of the stria terminalis. ................................. 38 1.3.2.2.3 In search of the anxiogenic role of CRHR2 - the lateral septum ....................................... 39 1.3.2.2.4 Linking stress and regulation of serotonergic system – CRHR2 in the dorsal raphe nucleus ........................................................................................................................................... 40 1.3.3 CRH-related peptides with affinity toward CRHR2 – Urocortins ..................................................... 42 1.3.3.1 Urocortin 1 (UCN1) ................................................................................................................. 42 1.3.3.2 Urocortin 2 (UCN2) ................................................................................................................. 43 1.3.3.3 Urocortin 3 (UCN3) ................................................................................................................. 44 1.4 Transgenic mouse models ................................................................................................................... 45 1.4.1 Conventional vs. conditional mouse models ................................................................................... 46 2 Aims of the thesis .................................................................................... 48 3 Materials and Methods ........................................................................... 49 3 Table of content 3.1 Materials.............................................................................................................................................. 49 3.1.1 Buffers and solutions ....................................................................................................................... 49 3.1.1.1 Buffers for agarose gel electrophoresis .................................................................................. 49 3.1.1.2 Solutions for in situ hybridization (ISH) .................................................................................. 49 3.1.1.3 Immunohistochemistry solutions ........................................................................................... 52 3.1.1.4 LacZ staining solutions ............................................................................................................ 53 3.1.1.5 Solutions for western blotting ................................................................................................ 54 3.1.1.6 HPLC Solutions ........................................................................................................................ 55 3.1.2 Oligonucleotides for genotyping ..................................................................................................... 56 3.1.3 Oligonucleotides for qPCR ............................................................................................................... 57 3.1.4 mRNA probes for ISH ....................................................................................................................... 57 3.1.5 Antibodies ....................................................................................................................................... 57 3.1.6 Animals ............................................................................................................................................ 58 3.2 Methods ............................................................................................................................................... 60 3.2.1 Preparation and analysis of nucleic acids ........................................................................................ 60 3.2.1.1 Polymerase chain reaction (PCR) ............................................................................................ 60 3.2.1.2 RNA isolation .......................................................................................................................... 61 3.2.1.3 Reverse transcription (RT) PCR ............................................................................................... 62 3.2.1.4 Quantitative real-time PCR (qPCR) ......................................................................................... 62 3.2.2 Plasma corticosterone analysis ....................................................................................................... 63 3.2.3 Tissue preparation ........................................................................................................................... 64 3.2.4 In situ hybridization (ISH) ................................................................................................................ 64 3.2.4.1 Radioactive labeling of probes ............................................................................................... 64 3.2.4.2 Purification of the riboprobes ................................................................................................ 66 3.2.4.3 Pre-treatment of cryo-slides................................................................................................... 66 3.2.4.4 Hybridization .......................................................................................................................... 67 3.2.4.5 Washing .................................................................................................................................. 67 3.2.4.6 Autoradiography ..................................................................................................................... 68 3.2.4.7 Quantification of relative expression ..................................................................................... 68 3.2.5 Immunohistochemistry ................................................................................................................... 68 3.2.6 Nissl staining ...................................................................................................................................