MOLECULAR UNDERPINNINGS OF ANXIETY

REGULATION: NOVEL INSIGHTS INTO THE ROLE OF THE PURINERGIC AND OXYTOCINERGIC SYSTEMS WITHIN THE PARAVENTRICULAR

NUCLEUS OF THE HYPOTHALAMUS

DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER

NATURWISSENSCHAFTEN (DR. RER. NAT.) DER FAKULTÄT FÜR

BIOLOGIE UND VORKLINISCHE MEDIZIN DER UNIVERSITÄT

REGENSBURG

vorgelegt von

Stefanie Martinetz

aus

München

im Jahr

2014

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Das Promotionsgesuch wurde eingereicht am:

27. Mai 2014

Die Arbeit wurde angeleitet von:

Prof. Dr. rer. nat. Inga D. Neumann

Unterschrift:

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Content Overview

Introduction .............................................................................................................................. 11

Materials & Methods ............................................................................................................... 37

Results ...................................................................................................................................... 57

Discussion ................................................................................................................................. 90

Conclusions and perspectives ................................................................................................ 109

Summary ................................................................................................................................ 112

Deutsche Zusammenfassung ................................................................................................. 115

List of abbreviations ............................................................................................................... 118

Bibliography ............................................................................................................................ 123

CV and list of publications ...................................................................................................... 146

Danksagung ............................................................................................................................ 148

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Content

Introduction .............................................................................................................................. 11

1. Positive and negative aspects of anxiety ...................................................................... 11

2. Neurobiology of anxiety ................................................................................................ 12

3. Drug treatment of anxiety disorders ............................................................................. 14

4. In vivo models of anxiety ............................................................................................... 16

5. Regulation of anxiety by neuropeptides ....................................................................... 18

5.1. The neuropeptide oxytocin .................................................................................... 18

5.2. Molecular mechanism of oxytocin’s effect on anxiety .......................................... 19

5.3. Further involvement of neuropeptides in anxiety ................................................. 20

6. ATP as a neurotransmitter ............................................................................................ 22

6.1. P2X4 receptor ........................................................................................................ 25

7. Protein synthesis ........................................................................................................... 26

8. MicroRNAs and the regulation of gene expression ...................................................... 29

8.1. General information............................................................................................... 29

8.2. MicroRNAs in brain and behaviour ........................................................................ 30

8.3. Regulation of the microRNA pathway ................................................................... 32

9. Aims and outline of the present thesis ......................................................................... 33

9.1. Aim 1: Identification of a novel anxiolytic factor within the PVN of rats .............. 34

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9.2. Aim 2: Elucidation of the role of de novo protein synthesis in the mediation of the

(long-term) anxiolytic effect of oxytocin .......................................................................... 34

9.3. Aim 3: Determination of the control of microRNA expression levels by OT in the

hypothalamus ................................................................................................................... 35

Materials & Methods ............................................................................................................... 37

1. Animals .......................................................................................................................... 37

2. Surgical Procedures ....................................................................................................... 37

3. Cells ............................................................................................................................... 38

3.1. H32 cells ................................................................................................................. 38

3.2. Be(2)-M17 cells ...................................................................................................... 39

3.3. Primary hypothalamic neurons .............................................................................. 39

3.4. Cell stimulation ...................................................................................................... 40

4. Behavioural studies ....................................................................................................... 40

5. RNA studies ................................................................................................................... 42

5.1. RNA extraction ....................................................................................................... 42

5.2. Microarray .............................................................................................................. 42

5.3. Deep Sequencing ................................................................................................... 43

5.4. PCR and qPCR ......................................................................................................... 44

5.5. Northern Blot ......................................................................................................... 47

6. Protein studies............................................................................................................... 48

6.1. Protein extraction .................................................................................................. 48

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6.2. SDS-PAGE and Western Blot analysis .................................................................... 50

6.3. Labelling, tagging and affinity purification of newly synthesized proteins ........... 52

6.4. Dot Blot .................................................................................................................. 54

6.5. Immunohistochemistry .......................................................................................... 54

7. Statistics ........................................................................................................................ 55

Results ...................................................................................................................................... 57

Part I: P2X4R as a regulator of anxiety in the PVN of Wistar rats ........................................ 57

Experiment 1: Validation of a microarray study showing differences in gene expression

profile in the PVN of female virgin HAB and LAB rats ...................................................... 57

Experiment 2: Immunostaining of the ATP-receptor P2X4R in the hypothalamus of rats

.......................................................................................................................................... 59

Experiment 3: Effects of the regulation of P2X4R within the PVN on anxiety-like

behaviour in Wistar rats ................................................................................................... 61

Experiment 4: Effects of the regulation of P2X4R within the PVN on anxiety-like

behaviour in HAB and LAB rats ........................................................................................ 65

Experiment 5: Elucidation of possible signalling cascades in hypothalamic cells following

P2X4R activation ............................................................................................................... 66

Part II: Oxytocin activates protein synthesis in the rat hypothalamus ................................ 72

Experiment 1: Effect of OT on key modulators of protein synthesis ...........................