The function of anxiety fear related genes in animal models

THE FUNCTION OF ANXIETY/FEAR-RELATED GENES IN ANIMAL MODELS ---THE ROLE OF CCK, 5-HT AND CRF SYSTEMS IN THE ELEVATED PLUS MAZE, CAT EXPOSURE TEST AND FEAR CONDITIONING WANG HONG MMed,

THE FUNCTION OF ANXIETY/FEAR-RELATED GENES

IN ANIMAL MODELS -THE ROLE OF CCK, 5-HT AND CRF SYSTEMS IN THE ELEVATED PLUS

MAZE, CAT EXPOSURE TEST AND FEAR CONDITIONING

WANG HONG (MMed, BSc)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE

2003

Acknowledgements i

ACKNOWLEDGEMENTS

Firstly, I wish to express my deepest appreciation and thanks to my

supervisor, Dr Zhu Yi Zhun, Department of Pharmacology, NUS, for his excellent

guidance and immense support throughout the course of this study

Secondly, I would like to thank my co-supervisor, Associate Professor Shabbir M Moochhala, Defence Medical Research Institute, Singapore, for his

proficient guidance and constant encouragement throughout the course of this study

Thirdly, I would also like to thank my co-supervisor, Professor Joachim Spiess, Department of Molecular Neuroendocrinology, Max Planck Institute for

Experimental Medicine, Germany, for his expert direction and stimulating discussion throughout the course of this study

Associate Professor Wong Tsun Hon, Peter and Associate Professor Tan Chay Hoon, Department of Pharmacology, NUS, are highly appreciated for their

invaluable help in the research work and the preparation of paper publication

I am very grateful to Mr Cedomir Todorovic, Max Planck Institute for Experimental Medicine, Germany, for his patient instruction and great help in the animal behavior study in Germany Mr Justin Mohamed Farook, Department of Pharmacology, NUS, is acknowledged for the behavior work in Singapore

I would also like to thank Dr Thomas Zeyda, Dr Jelena Radulovic and Ms Christina Schrick, Max Planck Institute for Experimental Medicine, Germany, for their helpful suggestion, valuable discussion and technical support

I am greatly thankful to my parents and brother for their complete support throughout my career

Acknowledgements ii

Last, I would like to express my intense appreciation and thanks to my beloved husband, Chen Hui, for his full support and continual encouragement in the past 10 years

This work was supported by grants from the National University of Singapore (R-184-000-033-214), Defense Medical Research Institute (P46-1-1/99), Singapore, and Max Planck Institute for Experimental Medicine, Germany

Publications iii

PUBLICATIONS

1 Wang H, Zhu YZ, Moochhala S, Farook JM, Lee LK, Wong PT Gene

expression of CCK2 and serotonin receptors in SD rats exhibiting high and low anxiety in the elevated plus maze 2003, submitted

2 Wang H, Zhu YZ, Farook JM, Moochhala S, Teo AL, Lee LK, Wong PT Genetic variations in CCK2 receptor in PVG hooded and Sprague-Dawley

rats and its mRNA expression on cat exposure Behav Neurosci 2003, 117(2):

385-90

3 Wang H, Zhu YZ, Wong PT, Farook JM, Teo AL, Lee LK, Moochhala S cDNA microarray analysis of gene expression in anxious PVG and SD rats

after cat-freezing test Exp Brain Res 2003, 149(4): 413-21

4 Wang H, Zhu YZ, Tang YK, Lau G, Moochhala S, Tan CH Gene expression

of serotonin receptor subtypes and cholecystokinin-B receptor in suicide

patients J Neurochem 2001, 77 (Supp 1): 53

5 Farook JM, Zhu YZ, Wang H, Moochhala S, Lee L, Wong PT Strain

differences in freezing behavior of PVG hooded and Sprague-Dawley rats:

differential cortical expression of cholecystokinin2 receptors Neuroreport

2001, 12(12): 2717-20

6 Zhu YZ, Moochhala S, Wang H, Farook JM, Greengrass CJ, Teo WL, Lee

HS, Lee EJD, Wong PT-H Up-regulation of gene expression in rats that

showed high anxiety on the elevated plus-maze Singapore Medical Journal

2000, 41(3): 25-27

Table of Contents iv

TABLE OF CONTENTS

ACKNOWLEDGEMENTS

PUBLICATIONS

TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION

1 Anxiety and fear

1.1 D EFINITION

1.2 Anxiety disorders

2 Cholecystokinin (CCK) and its receptors

2.1 CCK: a neurotransmitter

2.1.1 Discovery of CCK and its molecular forms

2.1.2 Distribution of CCK-related peptides in the central nervous

system

2.2 CCK receptors

2.2.1 Characterization of two CCK receptor subtypes: CCK1 and

CCK2

2.2.2 Distribution of CCK receptors

2.2.3 The molecular biology of the CCK1 and CCK2 receptors

i iii

iv viii

x

1

1

1

2

3

3

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6

8

Table of Contents v

2.3 Function of CCK-related peptides and CCK receptors

2.3.1 Role in anxiety

2.3.2 Role in memory process

3 5-HT and its receptors

3.1 Introduction

3.2 5-HT and anxiety

3.3 5-HT receptor subtypes

3.3.1 5-HT1 receptors

3.3.2 5-HT2 receptors

3.3.3 5-HT3 receptors

3.3.4 Other 5-HT receptors

4 CRF and CRF receptors

4.1 Structure and distribution of CRF-like peptides

4.2 CRF receptor subtypes

4.2.1 Characterization of two CRF receptor subtypes: CRF1 and

CRF2

4.2.2 CRF receptor expression in the mammalian CNS and

periphery

4.2.3 Pharmacology of CRF1 and CRF2 receptors

4.3 CRF-binding protein

4.4 Role of the CRF system in anxiety

4.4.1 CRF and anxiety

4.4.2 Role of CRF1 receptors in anxiety

4.4.3 Role of CRF2 receptors in anxiety

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Table of Contents vi

4.5 Role of the CRF system in memory and learning

5 Animal Models

5.1 Introduction

5.2 Validity criteria of an animal model

5.3 Elevated plus maze test

5.4 Cat exposure test

5.5 Fear conditioning

6 The hypothesis of this study

rats exhibiting high and low anxiety in the elevated plus maze

CHAPTER 3: Differences in behavior profile and gene expression of

CHAPTER 4: cDNA microarray analysis of gene expression profile in

anxious PVG and SD rats after cat exposure

and antagonist LY225910 in PVG hooded and SD rats on cat exposure

SD rats

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Table of Contents vii

CHAPTER 7: The crosstalk between CCK and CRF systems in anxiety

and fear conditioning

REFERENCES

LIST OF TABLES/FIGURES

TABLES

FIGURES

107

121

127

161

164

171

Abbreviations viii

ABBREVIATIONS

AD Alzheimer's disease

Arc rat activity-regulated cytoskeleton-associated gene

BZ benzodiazepine

CCK cholecystokinin

CNS central nervous system

CRF corticotropin-releasing factor

CS conditioned stimulus

CSF cerebrospinal fluid

DA dopamine

EPM elevated plus-maze

GAD generalized anxiety disorder

Abbreviations ix

LC locus coeruleus

MAP microtubule-associated protein

NA noradrenaline

PET positron emission tomography

SD Sprague-Dawley

SVG sauvagine

UCN urocortin

URO urotensin

Summary x

SUMMARY

The significance of anxiety or fear in health and disease is well recognized today, but the underlying cell biological and molecular mechanisms are not clear due

to the observation that various neurotransmitters and neuromodulators affect anxiety Our study tested the involvement of CCK, 5-HT and CRF systems in anxiety and fear processes and their crosstalk in the central nervous system

Elevated plus maze is a well-validated animal model of anxiety Two groups

of rat: high- and low-anxiety, were chosen from 88 SD rats according to the percentage of time spent in the open arms After RT-PCR amplification of CCK2 and 5-HT receptor subtypes, CCK2 receptor mRNA expression was found to be significantly higher in the cortex and cerebellum of the high-anxiety group as opposed to the low-anxiety group and 5-HT receptor subtypes were expressed in the altered expression pattern in the various brain regions

As exposure to the elevated plus maze is a relatively mild stimulus for rats, another animal model/the cat exposure test, and two rat strains/PVG hooded and SD rats, were employed in the following study SD rats were in a low-anxious state, while PVG hooded exhibited highly anxious behavior when exposed to the cat RT-PCR results showed that the expression of CCK2 receptor mRNA tended to be higher

in PVG rats compared with SD rats and significantly so in the cortex and hippocampus A cDNA microarray consisting of 5,931 genes was employed to analyze the gene expression profiles between the cortex of anxious PVG hooded and

SD rats after cat exposure We detected 16 up-regulated and 38 down-regulated genes between PVG hooded and SD rats Some of these genes have not yet been

Summary xi

associated with anxiety (e.g fibroblast growth factor, FGF), while other genes were recently found to be expressed in an anxious state (e.g rat nerve growth factor-induced [NGFI-A] gene) The expression of three genes was confirmed by RT-PCR

The treatment of a CCK2 agonist (CCK4) or antagonist (LY225910) showed

different effects in two rat strains on cat exposure However, there was no significant alteration of CCK2 receptor gene expression in the cortex and hippocampus of control, agonist- and antagonist-treated groups In addition, four variations were detected in the coding region of CCK2 receptor gene in PVG hooded and SD rats

We shifted the behavior study from rats to mice in the last part of this project,

as this will allow us to compare the difference between wild type and knockout mice CCK4 was found to induce an anxiogenic effect in C57BL/6J mice on the elevated plus maze after intracerebroventricular (i.c.v.) or i.h., but not i.s injection This effect could be blocked by pretreatment with the CCK2 receptor antagonist CR2945

or the CRF receptor antagonist (Glu11,16)-astressin I.c.v injection of CCK4 before training, but not after training, reduced the freezing behavior of mice on both context- and tone-dependent fear conditioning This impairment could be blocked by the pretreatment with (Glu11,16)-astressin 15 minutes before the application of CCK4, while injection of (Glu11,16)-astressin alone did not exhibit any significant effect The finding that (Glu11,16)-astressin could block the CCK4 effects suggested that CRF receptors mediate CCK4 actions at least in part

In summary, our experiments demonstrated that the CCK system is involved

in anxiety and fear memory 5-HT, like CCK, is also anxiety-related and might be involved in the physiological functions of the CCK system Moreover, the crosstalk

Summary xii

between the CCK and CRF systems may play an important role in anxiety and fear conditioning