Pathway-specific expression of calcitonin receptors in hypothalamic and brain stem nuclei regulating food intake and transcriptomic changes in hypothalamic orexin neurons after fasting [Elektronische Ressource] / vorgelegt von Ailing Ji

Yanzhang Li

Sujets

• Gesundheit

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2005
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Aus dem Institut für Anatomie und Zellbiologie
der Philipps-Universität Marburg
Abteilung Molekulare Neurowissenschaften
Direktor: Professor Dr. med. E. Weihe





Pathway-specific expression of calcitonin receptors in
hypothalamic and brain stem nuclei regulating food
intake and transcriptomic changes in hypothalamic
orexin neurons after fasting



Inaugural Dissertation
zur Erlangung des Doktorgrades der Humanbiologie
(Dr. rer. physiol.)


dem Fachbereich Humanmedizin
der Philipps-Universität Marburg
vorgelegt


von
Ailing Ji
aus Henan, V. R. China


Marburg 2005

Angenommen vom Fachbereich Humanmedizin
der Philipps-Universität Marburg am 22. 07. 2005
Gedruckt mit Genehmigung des Fachbereichs
Dekan: Prof. Dr. med. Bernhard Maisch
Referent: Prof. Dr. med. Eberhard Weihe
Korreferent: Prof. Dr. Karlheinz Voigt

Contents

CONTENTS
1. INTRODUCTION ......................................................................................1
1.1 The control of food intake by the central nervous system........................................................1
1.1.1 Feeding-related nuclei of the rodent hypothalamus ..........................................................3
1.1.2 Feeding-related regions of the brain stem.........................................................................5
1.2 Neuropeptides in the control of food intake and energy balance.............................................6
1.2.1 Orexigenic and anorexigenic peptides................................................................................6
1.2.2 The calcitonin peptide superfamily .....................................................................................7
1.3 The calcitonin receptors ..............................................................................................................9
1.4 Receptor-activity-modifying proteins ......................................................................................10
1.5 Distribution of rat calcitonin receptor isoforms......................................................................12
1.6 Aims ............................................................................................................................................13
2 MATERIALS AND METHODS ...............................................................15
2.1 Materials......15
2.1.1 Equipment .........................................................................................................................15
2.1.2 Chemicals and reagents ....................................................................................................15
2.1.3 Buffers and solutions.........................................................................................................18
2.1.4 Animals .............................................................................................................................23
2.1.5 Radioactive nucleotides23
2.1.6 Kits ....................................................................................................................................23
2.1.7 Enzymes23
2.1.8 Oligonucleotides ...............................................................................................................24
2.1.9 DNA and RNA size markers ..............................................................................................25
2.1.10 Other supplies..................................................................................................................25
2.2 Methods.......26
2.2.1 Animals26
2.2.2 Tissue preparation ............................................................................................................26
2.2.2.1 Coating of glass slides ...............................................................................................26
2.2.2.2 Paraffin sections.........................................................................................................26
2.2.2.3 Frozen sections27
2.2.3 Immunocytochemistry .......................................................................................................27
2.2.3.1 Double sequential immunostaining............................................................................27
2.2.3.2 Double immunofluorescence .....................................................................................28
2.2.4 Laser capture microdissection (LCM) ..............................................................................28
2.2.4.1 Microdissection of orexin neurons.............................................................................28
2.2.4.2 Microdissection of the area postrema (AP) and of the nucleus of the
solitary tract (NTS)...............................................................................................................29
2.2.5 RNA isolation ....................................................................................................................29
2.2.6 Reverse transcriptase polymerase chain reaction (RT-PCR)............................................30
2.2.6.1 Complementary DNA (cDNA) synthesis...................................................................30
2.2.6.2 Polymerase chain reaction (PCR)30
2.2.7 Agarose gel electrophoresis..............................................................................................31
2.2.8 Cloning..............................................................................................................................31
2.2.9 Probes ...............................................................................................................................33
2.2.9.1 Complementary RNA probes.....................................................................................33
2.2.9.2 Oligonucleotide probes33
2.2.10 In situ hybridization (ISH)35
I Contents

2.2.10.1 Prehybridization.........................................................................................................35
2.2.10.2 Hybridization .............................................................................................................35
2.2.10.3 Posthybridization .......................................................................................................35
2.2.10.4 Detection....................................................................................................................36
2.2.10.5 Double in situ hybridization.......................................................................................36
2.2.11 Grain counting analysis .................................................................................................37
2.2.12 Microarray analysis........................................................................................................38
2.2.12.1 Target preparation......................................................................................................38
2.2.12.2 Eukaryotic Target Hybridization ...............................................................................41
2.2.12.3 Washing, Staining and Scanning42
2.2.12.4 Microarray analysis....................................................................................................43
3 RESULTS...............................................................................................44
3.1 Gene expression analysis of calcitonin receptor isoforms in rat brain..................................44
3.1.1 RT-PCR analysis of CT and CT expression in rat brain.............................................44 (a) (b)
3.1.2 Cellular expression patterns of calcitonin receptor mRNAs in rat brain
revealed by in situ hybridization ......................................................................................45
3.2 RT-PCR analysis of CT and CT transcripts in the RNA extracts of (a) (b)
microdissected area postrema (AP) and nucleus of the solitary tract (NTS)........................54
3.3 Expression of calcitonin receptor isoforms in phenotype-identified neurons in
hypothalamus .............................................................................................................................55
3.3.1 Arcuate nucleus (Arc) .......................................................................................................55
3.3.2 Paraventricular nucleus (PVN).........................................................................................57
3.3.3 Lateral hypothalamic area (LHA).....................................................................................58
3.4 Expression of calcitonin receptor isoforms in phenotype-identified neurons
in brain stem...............................................................................................................................60
3.5 Identification of CT receptor isoforms in orexin neurons......................................................61
3.5.1 ISH with isoform-specific oligonucleotides.......................................................................61
3.5.2 Identification of CT receptor isoforms in microdissected orexin-immunoreactive
neurons by RT-PCR ..........................................................................................................62
3.6 Receptor-activity-modifying proteins (RAMPs) expression in orexin neurons....................63
3.7