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Transgenic mice as a tool for depression research [Elektronische Ressource] : examples from the endocannabinoid and the corticotropin releasing hormone system / Michel-Alexander Steiner

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Transgenic mice as a tool for depression research: examples from the endocannabinoid and the corticotropin-releasing hormone system Dissertation zur Erlangung des Grades “Doktor der Naturwissenschaften“ im Promotionsfach Biochemie am Fachbereich Chemie, Pharmazie und Geowissenschaften der Johannes Gutenberg-Universität in Mainz Michel-Alexander Steiner geboren am 10.11.1976 in Wiesbaden Mainz, Oktober 2007 (Akakus, Libya, 2006) „Nur wer den Weg verliert, lernt ihn kennen.” (Afrikanisches Sprichwort) Table of contents Table of contents I Table of illustrations IV Summary IX Zusammenfassung X List of abbreviations XII CHAPTER 1 – GENERAL INTRODUCTION – THEORIES FOR AND ANIMAL MODELS OF DEPRESSION 1 1.1 The invention of genetically engineered mice 1 1.2 Pathophysiology of depression 2 1.3 Tests of depression- and anxiety-related behavior in rodents 3 1.3.1 Animal tests and models of depression 5 1.3.1.1 The Forced Swim Test (FST) 5 1.3.1.2 The Tail Suspension Test (TST) 7 1.3.1.3 Learned Helplessness 8 1.3.1.4 Chronic Mild Stress 8 1.3.1.5 Chronic Social Defeat 8 1.3.2 Animal tests of anxiety 9 1.4 The three major theories for the development of depression 10 1.4.1 The monoamine theory of depression 10 1.4.2 The neurotrophin theory of depression 11 1.4.3 The HPA axis theory of depression 13 1.5 Future directions of depression research 14 1.

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Published 01 January 2007
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Transgenic mice as a tool for
depression research:

examples from the
endocannabinoid and the corticotropin-releasing
hormone system


Dissertation
zur Erlangung des Grades

“Doktor der Naturwissenschaften“

im Promotionsfach Biochemie
am Fachbereich Chemie, Pharmazie und Geowissenschaften
der Johannes Gutenberg-Universität
in Mainz

Michel-Alexander Steiner
geboren am 10.11.1976 in Wiesbaden


Mainz, Oktober 2007










(Akakus, Libya, 2006)


„Nur wer den Weg verliert, lernt ihn kennen.”
(Afrikanisches Sprichwort)



Table of contents

Table of contents I
Table of illustrations IV
Summary IX
Zusammenfassung X
List of abbreviations XII

CHAPTER 1 – GENERAL INTRODUCTION – THEORIES FOR AND
ANIMAL MODELS OF DEPRESSION 1
1.1 The invention of genetically engineered mice 1
1.2 Pathophysiology of depression 2
1.3 Tests of depression- and anxiety-related behavior in rodents 3
1.3.1 Animal tests and models of depression 5
1.3.1.1 The Forced Swim Test (FST) 5
1.3.1.2 The Tail Suspension Test (TST) 7
1.3.1.3 Learned Helplessness 8
1.3.1.4 Chronic Mild Stress 8
1.3.1.5 Chronic Social Defeat 8
1.3.2 Animal tests of anxiety 9
1.4 The three major theories for the development of depression 10
1.4.1 The monoamine theory of depression 10
1.4.2 The neurotrophin theory of depression 11
1.4.3 The HPA axis theory of depression 13
1.5 Future directions of depression research 14
1.6 Objective 15

CHAPTER 2 – THE ENDOCANNABINOID SYSTEM AND DEPRESSION 17
2.1 Introduction to the role of endocannabinoid signaling in stress
processing 17
2.2 Cannabinoid type 1 receptors are required for the basal modulation of
the HPA axis 20
2.2.1 Summary 20
I 2.2.2 Introduction 22
2.2.3 Materials and Methods 24
2.2.4 Results 27
2.2.5 Discussion 34
2.3 Impaired cannabinoid receptor type 1 signaling interferes with stress
coping behavior in mice 38
2.3.1 Summary 38
2.3.2 Introduction 40
2.3.3 Materials and Methods 43
2.3.4 Results 46
2.3.5 Discussion 59
2.4 Antidepressant-like behavioral effects of impaired cannabinoid receptor
type 1 signaling coincide with exaggerated corticosterone secretion in
mice 64
2.4.1 Summary 64
2.4.2 Introduction 66
2.4.3 Materials and Methods 68
2.4.4 Results 72
2.4.5 Discussion 86
2.5 Endocannabinoid signaling influences forced swimming behavior in a
monoamine-independent manner 91
2.5.1 Summary 91
2.5.2 Introduction 93
2.5.3 Materials and Methods 96
2.5.4 Results 100
2.5.5 Discussion 109
2.6 Outlook and preliminary results from ongoing research on the role of
the endocannabinoid system in depression-like behaviors 114
2.6.1 Defining the site of action for and the neuronal subpopulation
involved in endocannabinoid-mediated HPA axis regulation 114
2.6.2 Investigating the role of endocannabinoid signaling for social
stress processing in mice 115
II 2.7 Generation of bacterial artificial chromosome-based transgenic mice
conditionally overexpressing a CB1 receptor – Venus fusion protein 118
2.7.1 Summary 118
2.7.2 Introduction 120
2.7.3 Materials and Methods 125
2.7.4 Results 133
2.7.5 Discussion 145

CHAPTER 3 – THE CORTICOTROPIN-RELEASING HORMONE SYSTEM
153 AND DEPRESSION
3.1 Introduction to the corticotropin-releasing hormone system and
depression 153
3.2 Conditionally overexpressing mouse mutants highlight paradoxical
antidepressant-like effects of corticotropin-releasing hormone 155
3.2.1 Summary 155
3.2.2 Introduction 157
3.2.3 Materials and Methods 159
3.2.4 Results 166
3.2.5 Discussion 186

CHAPTER 4 – FINAL DISCUSSION 192

5 LIST OF REFERENCES 197

6 APPENDIX 220
6.1 Generated vectors and oligonucleotide sequences 220
6.2 Acknowledgements 225
6.3 Epilogue 227
6.4 Curriculum vitae 228
6.5 Kapitel 1 und 2 “Das Endocannabinoid-System – Physiologie und
230 klinische Bedeutung“


III Table of illustrations

Figures Page
Figure 1.3.1.1 The Forced Swim Test (FST)……………………………...… 6
+/+Figure 2.2.1 Plasma corticosterone and ACTH levels in CB1 and
-/-CB1 mice……………...……………………………………... 27
Figure 2.2.2 ACTH secretion in primary pituitary cell cultures from
+/+ -/-CB1 and CB1 mice……………………………..………… 29
Figure 2.2.3 Levels of CRH mRNA expression in the PVN of female
+/+ -/-CB1 and CB1 mice……………...………...……………… 30
Figure 2.2.4 Co-localization of CB1 receptor and CRH mRNA in several
extra-hypothalamic brain regions………………….…………. 31
Figure 2.2.5 Levels of MR and GR mRNA expression in the PVN and
+/+ -/-hippocampus of CB1 and CB1 mice……………………. 33
+/+ -/-Figure 2.3.1 Behavioral response of CB1 and CB1 mice in the
forced swim test…………………….………………………… 47
Figure 2.3.2 Effects of the CB1 receptor antagonist SR141716 on
forced swimming behavior in C57BL/6N mice…………….. 49
Figure 2.3.3 Effects of the CB1 receptor antagonist SR141716 on
+/+ -/-forced swimming behavior in CB1 and CB1 mice…….. 51
Figure 2.3.4 Effects of desipramine on forced swimming behavior in
+/+ -/-CB1 and CB1 mice……………………………………….. 52
Figure 2.3.5 Effects of paroxetine on forced swimming behavior in
+/+ -/-CB1 and CB1 mice……………………………………….. 54
+/+ -/-Figure 2.3.6 VGLUT1 mRNA expression levels in CB1 and CB1
mice…………………………………………………………..… 57
IV +/+ -/-Figure 2.3.7 BDNF mRNA expression levels in CB1 and CB1
mice……...…………………………………………………….. 58
Figure 2.4.1 Acute pharmacological blockade of CB1 receptors dose-
dependently increases injecton stress- and forced swim
test (FST) stress-induced corticosterone secretion….…… 72
Figure 2.4.2 Acute pharmacological blockade of CB1 receptors
increases forced swim test (FST) stress-induced
+/+ -/-corticosterone secretion in CB1 but not in CB1
mice……………………………………………………………. 73
Figure 2.4.3 Genetic deletion of CB1 receptors enhances basal and
forced swim test (FST) stress-induced corticosterone
secretion in a time-dependent manner……………………... 75
Figure 2.4.4 Subchronic pharmacological blockade of CB1 receptors
enhances injection stress- and forced swim test (FST)
stress-induced corticosterone secretion in a time-
dependent manner…………..……………………………….. 77
Figure 2.4.5 Behavioral and neuroendocrine responses to the forced
swim test (FST) in CB1 receptor-deficient mice following
treatment with desipramine.................................................. 79
Figure 2.4.6 Lack of interaction between desipramine and SR141716
treatment in C57BL/6N mice………………………………… 81
Figure 2.4.7 Chronic blockade of CB1 receptors by SR141716 leads to
reduced responsiveness to corticosterone stimulating
effects of an acute SR141716 challenge…………………… 84
Figure 2.4.8 Chronic blockade of CB1 receptors by SR141716 alters
behavioral and neuroendocrine responses to the forced
swim test (FST)……………………………………………….. 85
Figure 2.5.1 Validation of EthoVision video-tracking for the detection of
immobility behavior in the mouse FST……………………… 101
V Figure 2.5.2 The pharmacological blockade of CB1 receptor signaling
induces antidepressant-like effects in the mouse FST……. 103
Figure 2.5.3 Pharmacological enhancement of endocannabinoid
signaling does not influence immobility behavior in the
mouse FST………….……………………………………….… 105
Figure 2.5.4 Effects of PCPA or AMPT pre-treatment on SR141716-
induced behavioral and hormonal responses to forced
swim stress………………………………………………...….. 106
Figure 2.5.5 Forced swim stress-induced alterations in brain
endocannabinoid levels…………………………….………… 108
Figure 2.7.1 Overview of the CB1-EGFP fusion construct of plasmid
pMS1……………………………………………………………. 133
Figure 2.7.2 Expression of the CB1-EGFP fusion protein in HEK293
cells……………………………………………………………… 134
Figure 2.7.3 CB1-EGFP receptor trafficking in response to
agonist/antagonist stimulation………………………………… 135
Figure 2.7.4 Agonist-stimulated CB1-EGFP-mediated CRE-luciferase
reporter gene expression……………………………………… 136
Figure 2.7.5 Agonist-stimulated CB1-EGFP-mediated MAPK
phosphorylation………………………………………………… 137
Figure 2.7.6 Overview of the genomic organization of the CB1 receptor
gene…………………………………………………………….. 139
Figure 2.7.7 Overview of the CB1-Venus-Easy recombination
cassette…………………………………………………………. 139
Figure 2.7.8 Overview of the CB1-Venus-Soph recombination
cassette……………………………………………………….… 140
Figure 2.7.9 Screening of lox2272/Cre-mediated recombination of CB1-
Venus-Soph……………………………………………………. 141
VI Figure 2.7.10 PCR-Screening of putative CB1-Venus-Soph-BACs after
homologous recombination………………………………….. 142
Figure 2.7.11 Restriction digest of CB1-Venus-Soph-BAC……………….. 143
Figure 2.7.12 Genomic DNA screening of putative CB1-Venus-Soph-
BAC transgenic mice via PCR……………………………….. 144
Figure 3.2.1 Generation of CNS-restricted CRH overexpressing mice…. 167
Figure 3.2.2 Verification of the CNS-restricted overexpression of CRH in
CRH-COE-Nes mice…………………………………………. 168
Figure 3.2.3 CRH overexpression from the R26 locus results in a gene
dosage dependent increase of CRH protein content in the
brain……………………………………………………………. 169
Figure 3.2.4 Stress induced HPA axis hyperactivity in CRH-COE-Nes
171 mice is sex dependent………………………………………..
Figure 3.2.5 CRH overexpression leads to increased explorative
172 behavior………………………………………………………....
Figure 3.2.6 CRH overexpression leads to increased active stress
coping behavior in antidepressant screening paradigms….. 174
Figure 3.2.7 Reduced floating in the FST is not mediated via the CRH-
R2 system……………………………………………………… 176
Figure 3.2.8 Forebrain restricted overexpression of CRH does not affect
antidepressant-like behavior in the forced swim test………. 177
hom
Figure 3.2.9 Antidepressant-like behavior of CRH-COE -Nes mice is
partly mediated by increased catecholaminergic
neurotransmission, which originates from CRH-mediated
hyperactivation of the locus coeruleus……………….……… 181
Figure 3.2.10 Transgenic CRH overexpression does not influence
hippocampal monoamine efflux during forced swimming… 184


VII Tables Page
Table 2.2.1 Co-expression of CB1 receptor and CRH mRNA in extra-
32 hypothalamic brain regions……………………………….…...
Table 2.3.1 Enzymatic activity of monoamine oxidase (MAO) A and B
+/+ -/-in various brain areas of female CB1 and CB1 mice
under basal conditions…………………….………………...… 55
Table 2.3.2 Monoamine and metabolite concentrations in hippocampus
tissue of subchronically vehicle- or SR141716-treated
+/+ -/-female CB1 and CB1 mice 10 min after FST on day 2 56
Table 3.2.1 Monoamine and metabolite concentrations in hippocampal
tissue of vehicle and PCPA pre-treated male CRH-COE-
Nes animals…………………………………………………..… 179
Table 3.2.2 Monoamine and metabolite concentrations in hippocampal
tissue of vehicle and AMPT pre-treated male CRH-COE-
Nes animals………………………………………………..…… 180
Table 3.2.3 Monoamine and metabolite concentrations in hippocampal
baseline dialysates of CRH-COE-Nes animals (averaged
over 2 h of baseline sampling)………………………………... 183
Supplementary
Table 6.1.1 List of plasmids…………………………………………………. 220
Supplementary
Table 6.1.2 List of oligonucleotides………………………………………… 222


VIII