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 i Acknowledgements 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 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 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 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 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 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 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 i PUBLICATIONS iii TABLE OF CONTENTS iv ABBREVIATIONS viii SUMMARY x CHAPTER 1: INTRODUCTION 1 Anxiety and fear 1.1 DEFINITION 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 3 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 Table of Contents 2.3 Function of CCK-related peptides and CCK receptors v 2.3.1 Role in anxiety 10 2.3.2 Role in memory process 13 5-HT and its receptors 14 3.1 Introduction 14 3.2 5-HT and anxiety 15 3.3 5-HT receptor subtypes 16 3.3.1 5-HT1 receptors 17 3.3.2 5-HT2 receptors 20 3.3.3 5-HT3 receptors 22 3.3.4 Other 5-HT receptors 24 CRF and CRF receptors 25 4.1 Structure and distribution of CRF-like peptides 25 4.2 CRF receptor subtypes 27 4.2.1 Characterization of two CRF receptor subtypes: CRF1 and CRF2 27 4.2.2 CRF receptor expression in the mammalian CNS and periphery 4.2.3 Pharmacology of CRF1 and CRF2 receptors 28 29 4.3 CRF-binding protein 30 4.4 Role of the CRF system in anxiety 31 4.4.1 CRF and anxiety 31 4.4.2 Role of CRF1 receptors in anxiety 33 4.4.3 Role of CRF2 receptors in anxiety 35 Table of Contents 4.5 Role of the CRF system in memory and learning Animal Models vi 37 39 5.1 Introduction 39 5.2 Validity criteria of an animal model 40 5.3 Elevated plus maze test 42 5.4 Cat exposure test 44 5.5 Fear conditioning 46 The hypothesis of this study 48 CHAPTER 2: Gene expression of CCK2 and serotonin receptors in SD rats exhibiting high and low anxiety in the elevated plus maze 50 CHAPTER 3: Differences in behavior profile and gene expression of CCK2 receptor in PVG hooded and SD rats in cat exposure 64 CHAPTER 4: cDNA microarray analysis of gene expression profile in anxious PVG and SD rats after cat exposure 74 CHAPTER 5: Differences in the effect of CCK2 receptor agonist CCK4 and antagonist LY225910 in PVG hooded and SD rats on cat exposure 89 CHAPTER 6: Genetic variations of CCK2 receptor in PVG hooded and SD rats 97 Table of Contents vii CHAPTER 7: The crosstalk between CCK and CRF systems in anxiety and fear conditioning 107 CHAPTER 8: CONCLUSIONS AND FUTURE WORKS 121 REFERENCES 127 LIST OF TABLES/FIGURES 161 TABLES 164 FIGURES 171 Abbreviations viii ABBREVIATIONS 5-HT serotonin, 5-hydroxytryptamine ACTH adrenocorticotropic hormone AD Alzheimer's disease ahCRF alpha-helical CRF AMV-RT avian myeloblastosis virus reverse transcriptase anti-Svg-30 anti-sauvagine-30 Arc rat activity-regulated cytoskeleton-associated gene BZ benzodiazepine cAMP cyclic adenosine monophosphate CCK cholecystokinin CNS central nervous system CORT corticosterone CRF corticotropin-releasing factor CRF-BP CRF-binding protein CS conditioned stimulus CSF cerebrospinal fluid DA dopamine DEPC diethylpyrocarbonate DMSO dimethyl sulphoxide dNTP deoxyribonucleoside triphosphate EPM elevated plus-maze ESTs expressed sequence tags F/A DTB Fear/anxiety Defence Test Battery FGF fibroblast growth factor GABA γ-aminobutyric acid GAD generalized anxiety disorder GPCRs G-protein coupled receptors h/rCRF human and rat CRF HPA hypothalamic–pituitary–adrenal Abbreviations ix i.c.v intracerebroventriculus i.h the dorsal hippocampus i.s the lateral intermediate septal area IEGs immediate-early genes LC locus coeruleus MAP microtubule-associated protein NA noradrenaline NGFI-A rat nerve growth factor-induced gene A oCRF ovine CRF PET positron emission tomography PTSD post-traumatic stress disorder RT-PCR reverse transcription-polymerase chain reaction SD Sprague-Dawley SVG sauvagine UCN urocortin URO urotensin x Summary 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 RTPCR 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 factorinduced [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  ... Pharmacology of CRF1 and CRF2 receptors 28 29 4.3 CRF-binding protein 30 4.4 Role of the CRF system in anxiety 31 4.4.1 CRF and anxiety 31 4.4.2 Role of CRF1 receptors in anxiety 33 4.4.3 Role of CRF2... Role of CRF2 receptors in anxiety 35 Table of Contents 4.5 Role of the CRF system in memory and learning Animal Models vi 37 39 5.1 Introduction 39 5.2 Validity criteria of an animal model 40 5.3... exposure test 44 5.5 Fear conditioning 46 The hypothesis of this study 48 CHAPTER 2: Gene expression of CCK2 and serotonin receptors in SD rats exhibiting high and low anxiety in the elevated plus