Estrogen signaling in nociceptive neurons [Elektronische Ressource] / vorgelegt von Julia Annabelle Kuhn

Aa

Sujets

• Biologie

Informations

• Publié par: freie_universitat_berlin
• Publié le: 01 janvier 2010
• Nombre de lectures: 37
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

ESTROGEN SIGNALING IN NOCICEPTIVE
NEURONS
Dissertation

zur Erlangung des akademischen Grades des
Doktors der Naturwissenschaften (Dr. rer. nat.)

eingereicht im Fachbereich Biologie, Chemie, Pharmazie
der Freien Universität Berlin


vorgelegt von

Julia Annabelle Kuhn
aus Berlin

2010



















Die praktischen Arbeiten dieser Dissertation wurden vom 01.10.2006 bis 01.10.2009 unter der
Leitung von Dr. T. Hucho am Max-Planck-Institut für molekulare Genetik Berlin durchgeführt.

1. Gutachter: Frau Prof. Dr. Petra Knaus
2. Gutachter: Herr Prof. Dr. Christoph Stein

Disputation am 20. September 2010



Φύσις κρύπτεσθαι φιλεῖ.
Heraklit, Fragmente, B123


i
Table of contents
1 Summary................................................................................................................. 1
2 Zusammenfassung ................................................................................................. 3
3 Introduction............................................................................................................ 5
3.1 Primary sensory neurons ................................................................................................... 5
3.2 Estrogen in pain and nociception....................................................................................... 7
3.2.1 Sex differences in pain and nociception ...................................................................... 7
3.2.2 Pain modulation by estrogen ....................................................................................... 8
3.2.3 The steroid hormone estrogen ..................................................................................... 9
3.2.4 Classical estrogen signaling......................................................................................... 9
3.2.5 Non-classical estrogen signaling: Rapid effects and signaling via
membrane receptors................................................................................................... 10
3.2.6 The novel estrogen receptor GPR30.......................................................................... 10
3.2.7 Estrogen receptors in the nervous system.................................................................. 11
3.3 Signaling in nociceptive neurons..................................................................................... 12
3.3.1 Signaling towards sensitization ................................................................................. 12
3.3.2 The protein kinase C epsilon: An important second messenger in
pain signaling............................................................................................................. 14
3.3.3 The ion channel TRPV1 ............................................................................................ 16
4 Aim ........................................................................................................................ 20
5 Materials ............................................................................................................... 21
5.1 Antibodies and related compounds.................................................................................. 21
5.1.1 Primary antibodies..................................................................................................... 21
5.1.2 Secondary antibodies................................................................................................. 21
5.1.3 Fluorescent dyes and related compounds .................................................................. 22
5.2 Animals and cell lines...................................................................................................... 22
5.2.1 Animals...................................................................................................................... 22
5.2.2 Cell lines.................................................................................................................... 22
5.3 Vectors, constructs and proteins...................................................................................... 22
5.4 Primers and oligonucleotides........................................................................................... 23
5.5 Media, sera and supplements........................................................................................... 24 TABLE OF CONTENTS ii
5.6 Buffers ............................................................................................................................. 25
5.7 Drugs and chemicals........................................................................................................ 27
5.8 Kits and markers.............................................................................................................. 29
5.9 Laboratory equipment...................................................................................................... 29
5.10 Microscopes..................................................................................................................... 30
5.11 Software........................................................................................................................... 31
6 Methods................................................................................................................. 32
6.1 General methods.............................................................................................................. 32
6.1.1 RNA extraction and purification ............................................................................... 32
6.1.2 RT-PCR ..................................................................................................................... 32
6.1.3 Agarose gel electrophoresis....................................................................................... 34
6.2 Cell biological methods................................................................................................... 34
6.2.1 Primary rat DRG cultures.......................................................................................... 34
6.2.2 PKCε translocation assay .......................................................................................... 36
6.2.3 Calcium imaging........................................................................................................ 38
6.2.4 Combined assay: Calcium imaging and evaluation of PKCε
translocation in the same cell..................................................................................... 40
6.2.5 Primary mouse DRG cultures.................................................................................... 41
6.2.6 Live cell imaging of DRG cultures............................................................................ 42
6.2.7 F-11 cell culture......................................................................................................... 43
6.2.8 Transfection of F-11 cells.......................................................................................... 43
6.2.9 Analysis of in situ cytoskeleton of F-11 cells............................................................ 43
6.2.10 Live cell imaging of F-11 cells.................................................................................. 45
6.3 Biochemical and computational methods........................................................................ 46
6.3.1 Computational modeling ........................................................................................... 46
6.3.2 Tubulin binding assay................................................................................................ 47
6.4 Pain behavioral experiments............................................................................................ 49
6.4.1 Testing of the mechanical nociceptive threshold in the rat ....................................... 49
6.4.2 Testing of the mechanical nociceptive threshold in the mouse ................................. 52
6.5 Statistical analysis ........................................................................................................... 52 TABLE OF CONTENTS iii
7 Results ................................................................................................................... 53
7.1 GPR30 agonists induce mechanical hyperalgesia ........................................................... 53
7.1.1 Agonists of ERα and ERβ do not lead to rapid PKCε translocation
in DRG neurons......................................................................................................... 54
7.1.2 Inhibition of the adenylyl cyclase blocks estrogen-induced PKCε
translocation............................................................................................................... 58
7.1.3 GPR30 mRNA is expressed in DRGs ....................................................................... 59
7.1.4 The GPR30 agonist G-1 causes rapid PKCε translocation........................................ 59
7.1.5 The ERα/ ERβ inhibitor ICI 182,780 induces PKCε translocation ........................... 61
7.1.6 PKCε translocates in IB4-positive and TRPV1-expressing DRG
neurons....................................................................................................................... 62