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Characterization of the Ion Transporter NKCC1 in the Field of Chemosensation

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Characterization of the Ion Transporter NKCC1 in the Field of Chemosensation Characterization of the Ion Transporter NKCC1 in the Field of Chemosensation Dissertation to obtain the degree Doctor Rerum Naturalium (Dr.rer.nat.) at the Faculty of Biology and Biotechnology Ruhr-University Bochum International Graduate School of Biosciences Ruhr-University Bochum (Department of Cellphysiology) submitted by Claudia Haering from Dortmund, Germany Bochum (April, 2015) First Referee: Prof. Dr. Dr. Dr. Hatt Second Referee: Prof. Dr. Wiese Charakterisierung des Ionentransporters NKCC1 in der Chemosensorik Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät Biologie und Biotechnologie an der Internationalen Graduiertenschule Biowissenschaften der Ruhr-Universität Bochum angefertigt im Lehrstuhl für Zellphysiologie vorgelegt von Claudia Haering aus Dortmund, Deutschland Bochum (April, 2015) Referent: Prof. Dr. Dr. Dr. Hatt Korreferent: Prof. Dr. Wiese ERKLÄRUNG Hiermit erkläre ich, dass ich die Arbeit selbständig verfasst und bei keiner anderen Fakultät eingereicht und dass ich keine anderen als die angegebenen Hilfsmittel verwendet habe. Es handelt sich bei der heute von mir eingereichten Dissertation um sechs in Wort und Bild völlig übereinstimmende Exemplare. Weiterhin erkläre ich, dass digitale Abbildungen nur die originalen Daten enthalten und in keinem Fall inhaltsverändernde Bildbearbeitung vorgenommen wurde. Bochum, den (Claudia Haering) Table of contents 1. Introduction __________________________________________________________ 1 1.1 General introduction - Olfaction_________________________________________ 1 1.1.1 The olfactory system ______________________________________________ 1 1.1.2 Cellular composition of the main olfactory epithelium ____________________ 2 1.1.3 The olfactory receptor _____________________________________________ 4 1.1.4 The olfactory signal transduction pathway _____________________________ 6 1.1.5 Neurogenesis of the adult olfactory epithelium __________________________ 9 1.1.6 Cloride concentration of OSN ______________________________________ 11 1.2 Assumed chloride transporter of OSNs __________________________________ 11 1.2.1 The cation-chloride cotranporter NKCC1 _____________________________ 12 1.2.2 Involvement of NKCC1 in plasticity of the brain _______________________ 14 1.2.3 Involvement of NKCC1 in neurogenesis______________________________ 16 1.2.4 Regulation of NKCC1 ____________________________________________ 17 1.2.5 NKCC1 Knock-out mice __________________________________________ 19 2. Objectives ___________________________________________________________ 20 3. Materials and Methods ________________________________________________ 21 3.1 Materials __________________________________________________________ 21 3.1.1 Equipment _____________________________________________________ 21 3.1.2 Chemicals _____________________________________________________ 22 3.1.3 Buffer and solutions _____________________________________________ 23 3.1.4 Consumables ___________________________________________________ 26 3.1.5 Kits __________________________________________________________ 27 3.1.6 Antibodies _____________________________________________________ 27 3.1.7 Oligonucleotides ________________________________________________ 28 3.2 Methods __________________________________________________________ 31 3.2.1 Animals _______________________________________________________ 31 3.1.2 Preparation of olfactory epithelium __________________________________ 31 3.1.3 RNA isolation __________________________________________________ 32 3.1.4 Complementary DNA (cDNA) sythesis ______________________________ 32 3.1.5 RNA sequencing (RNA-Seq) analysis _______________________________ 32 3.1.6 Reverse transcription polymerase chain reaction (RT-PCR) ______________ 33 3.1.7 Agarose gel electrophoresis ________________________________________ 34 3.1.8 SDS-Polyacrylamide gel eletrophoresis (PAGE) _______________________ 35 3.1.9 Western blotting ________________________________________________ 35 3.1.10 Electro-olfactogram (air-phase) ____________________________________ 35 3.1.11 Submerged electro-olfactogram ___________________________________ 36 3.1.12 Cilia preparation (calcium shock method)____________________________ 37 3.1.13 Preparation of olfactory epithelium cryosections ______________________ 37 3.1.14 Immunohistochemistry __________________________________________ 37 3.1.15 Hematoxylin-/Eosin-staining ______________________________________ 38 3.1.16 Measurement of neuronal layer thickness and Cell counting _____________ 38 3.1.17 Microscopy ___________________________________________________ 39 3.1.18 Statistical data analysis __________________________________________ 39 4. Results ______________________________________________________________ 40 4.1 Genotyping of NMRI background mice __________________________________ 40 4.2 Localization of NKCC1 in the murine olfactory epithelium __________________ 41 4.3 Localization of olfactory signal transduction proteins in the murine olfactory epithelium ________________________________________________________ 42 4.4 Transcriptome analysis of the olfactory epithelium _________________________ 43 4.4.1 RNA-Sequencing analysis of olfactory epithelium of NKCC1+/+ and NKCC1-/- mice __________________________________________________________ 44 4.4.2 RNA-Seq: Expression analysis of chloride-associated cotransporters _______ 45 4.4.3 RNA-Seq: Expression analysis of olfactory signal transduction proteins and adaptation-related proteins _________________________________________ 49 4.4.4 RNA-Seq: Expression analysis of olfactory receptors ___________________ 50 4.4.5 RNA-Seq: Expression analysis of significantly regulated genes ___________ 53 4.4.6 RT-PCR: Expression of chloride-associated cotransporters _______________ 55 4.4.7 RT-PCR: Expression of signal transduction proteins and adaptation-related proteins ________________________________________________________ 56 4.4.8 RT-PCR: Expression of olfactory receptors ___________________________ 57 4.5 Protein expression analysis of the olfactory epithelium of NKCC1+/+ and NKCC1-/- mice _____________________________________________________________ 58 4.5.1 Protein expression analysis of signal transduction proteins and adaptation- related proteins __________________________________________________ 58 4.5.2 Protein expression analysis of a ciliary protein and development- related proteins ________________________________________________________ 59 4.6 Doublecortin expression in the murine olfactory epithelium of a DCX-promo-EGFP transgenic mouse __________________________________________________ 60 4.7 Electro-olfactogram recordings ________________________________________ 61 4.7.1 Air-phase EOG recordings of NKCC1+/+ and NKCC1-/- mice: Odorant concentration dependency _________________________________________ 62 4.7.2 Air-phase EOG recording of NKCC1+/+, NKCC1+/- and NKCC1-/-mice _____ 62 4.7.3 Air-phase EOG recording of NKCC1+/+ and NKCC1-/- mice: Henkel100 versus 10 odorants mixture ______________________________________________ 64 4.7.5 Air-phase electro-olfactogram of NKCC1+/+ and NKCC1-/- mice: Repetitive stimulation with Henkel100 ________________________________________ 64 4.7.6 Submerged EOG recordings of NKCC1+/+ and NKCC1-/- mice: Calcium- activated chloride channel inhibition with niflumic acid and tannic acid _____ 65 4.7.7 Submerged electro-olfactogram of NKCC1+/+ and NKCC1-/- mice: Calcium- activated chloride channel inhibition with niflumic acid __________________ 66 4.8 Morphological changes of the olfactory epithelium in NKCC1-/- mice __________ 67 4.8.1 Measurements of turbinate lengths of NKCC1+/+ and NKCC1-/- mice _______ 68 4.8.2 Measurements of the olfactory neuronal layer thickness of NKCC1+/+, NKCC1+/- and NKCC1-/- mice ______________________________________________ 68 4.8.3 Differences of the cell number in the olfactory epithelium of NKCC1+/+, NKCC1+/- and NKCC1-/- mice ______________________________________ 69 5. Discussion ___________________________________________________________ 71 5.1 Localization of NKCC1 in the murine olfactory epithelium __________________ 71 5.2 Localization of olfactory signal transduction proteins in the murine olfactory epthelium ________________________________________________________ 72 5.3 Transcriptome analysis of the olfactory epithelium of NKCC1+/+ and NKCC1-/- mice ________________________________________________________________ 72 5.3.1 Expression analysis of chloride-associated cotransporters ________________ 73 5.3.2 Expression analysis of olfactory signal transduction proteins and adaptation- related proteins __________________________________________________ 76 5.3.3 Expression analysis of olfactory receptors ____________________________ 76 5.3.4 Expression analysis of significantly regulated genes ____________________ 77 5.4 Western blot analysis of the olfactory epithelium of NKCC1+/+ and NKCC1-/- mice ________________________________________________________________ 78 5.5 EOG recordings ____________________________________________________ 80 5.6 Morphological changes of the olfactory epithelium of NKCC1+/+, NKCC1+/- and NKCC1-/- mice ____________________________________________________ 82 5.7 The impact of NKCC1 in chloride accumulation of OSNs or how NKCC1 is a modulator of neurogenesis ___________________________________________ 83 6. Conclusion ___________________________________________________________ 86 6.1 Summary __________________________________________________________ 86 6.2 Zusammenfassung
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