BEHAVIOURAL NEUROPHARMACOLOGY OF FEAR AND ANXIETY INVOLVING CHOLECYSTOKININ2 RECEPTORS JUSTIN MOHAMED FAROOK B.Sc., University of Kerala, India. (1996) A THESIS SUBMITTED IN PARTIAL FULFILLMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2003 ABSTRACT PVG hooded and Sprague-Dawley (SD) rats differ in a variety of anxiety and fear related behavioral measures. PVG hooded rats are more sensitive to the effects of cholecystokinin-2 (CCK2) receptor specific agonist and antagonist drugs as indicated by behavioral and molecular assays (Farook et al, 2001). The studies from this thesis showed that molecular differences (Farook et al, 2001; Wang et al, 2003) between PVG hooded and SD rats parallel their differences on intraperitoneal administration (I.P.) of CCK2 receptor specific agonist (CCK-4) and CCK2 receptor specific antagonist drugs (LY 225910, LY288513, and CR2945) on a variety of anxiety and fear related animal models. I.P administration of a cholecystokinin-1 (CCK1) antagonist, lorglumide (CR1409) failed to show any significant response in PVG hooded rats but showed reliable significance in SD rats. cDNA microarray and reverse transcriptase-polymerase chain reaction (RTPCR) experiments were then carried out in collaboration to determine the CCK2 receptor expression among these strains of rats. The results showed that CCK2 receptors were expressed to a greater extent in the PVG hooded rats. Anxiety habituation studies using a selective CCK2 receptor agonist (BC264) on day-8 resulted in reversal of freezing behavior in PVG hooded rats which was not observed in SD rats. These results strongly suggest that CCK2 receptors mediate the freezing behavior and the differential expression of these receptors underlie the strain difference in such behavior. Furthermore, this study also brings additional evidence for the possible existence of CCK2 receptor subsites, CCK2A and CCK2B probably corresponding to different coupling sites of the CCK2 receptor. ii PUBLICATIONS 1. Farook JM, McLachlan CS, Zhu YZ, Lee L, Moochhala SM, Wong PTH (2004). The CCK(2) agonist BC264 reverses freezing behavior habituation in PVG hooded rats on repeated exposures to a cat. Neuroscience Letters, January 30; 355(3): 205-8. 2. Farook JM, Wang Q, Moochhala SM, Zhu ZY, Lee L, Wong PTH (2004). Distinct regions of periaqueductal gray (PAG) are involved in freezing behavior in hooded PVG rats on the cat-freezing test apparatus. Neuroscience Letters, January 9; 354(2): 139-42. 3. Farook JM, Zhu YZ, Wang H, Moochhala S, Lee L, Wong PTH (2001). Strain differences in freezing behavior of PVG hooded and Sprague-Dawley rats: differential cortical expression of cholecystokinin2 receptors. Neuroreport, August 28; 12(12):2717-20 4. Farook JM, Zhu YZ, Wang Q, Moochhala SM, Lee L, Wong PTH (2004). Analysis of strain difference in behavior to Cholecystokinin (CCK) receptor mediated drugs in PVG hooded and Sprague-Dawley rats using elevated plusmaze test apparatus. Neuroscience Letters, April 1; 358(3):215-9. 5. Wang H, Zhu YZ, Farook JM, Moochhala S, Teo AL, Lee LK, Wong PTH (2003). Genetic variations in CCK2 receptor in PVG hooded and Sprague-Dawley rats and its mRNA expression on cat exposure. Behavioral Neuroscience, April; 117(2):385-90 6. Wang H, Zhu YZ, Wong PTH, Farook JM, Teo AL, Lee LK, Moochhala S (2003). cDNA microarray analysis of gene expression in anxious PVG and SD rats after cat-freezing test. Experimental Brain Research, April; 149(4):413-21 7. Zhu YZ, S Moochhala, Wang H, Farook JM, Greengrass C J, Ting WL, Lee HS, Lee EJD and Wong PTH (2000). Up-regulation of gene expression in rats that showed high anxiety on the elevated plus-maze. Singapore Medical Journal, (41): 25-27. iii CONFERENCE ABSTRACTS 1. Farook JM, Spiess S, Zhu YZ, Moochalla SM, and Wong PTH. Investigation of Behavioral Responses Induced by Selective Stimulation of CCK2 Receptor Mediated Drugs on Primates using threat/confrontation Model 3rd FAONS Congress. September 28th to October 1st, 2002. Seoul, Korea. 2. Farook JM, Zhu YZ, Moochhala S, Hong Wang and Wong PTH, A genetic and behavioral analysis of anxiety-related behaviors in SD and PVG rats. (2000): 56. Federation of Asian-Oceanian Neurosciences Societies (FAONS) Symposium 2000 & The 20th Scientific Meeting of the Hong Kong Society of Neurosciences, 7-10 December 2000, The University of Hong Kong, China. 3. Farook JM, Zhu YZ, Moochalla SM, Hong W and Wong PTH, Influence of age-related changes on freezing behavior in the cat - freezing test apparatus. 2001. Cognitive Neroscience Society 8th Annual Meeting, 23-27 March 2001, New York, USA. 4. Farook JM, Zhu YZ, Moochhala S, Wang H, Teo M, Lee EJD and Wong PTH. Strain differences in fear-motivated behaviour of rats. 2001. Combined Annual Scientific Meeting, 8-9 September 2001, National University of Singapore, Singapore. iv ACKNOWLEDGEMENTS My deepest thanks are to my supervisor(s) Prof: Peter Wong and Prof Shabbir Moochhala at the National University of Singapore for giving me the opportunity to undertake the research for this degree. I am thankful to Mrs. Ting Wee Lee for her excellent technical assistance during the entire course of the work as well as to Mr. Ishak Bin Ismail and Mr. Haridass for their assistance in constructing the various apparatus that were required for the behavioral experiments. I am grateful to the scholarship awarded by the National University of Singapore without which this work would not have been possible. v TABLE OF CONTENTS Abstract…………………………………………………………………………….…….….……ii Publications………………………………………………………………………….…… ….iii Conference Abstracts……………………………………………………………….….…………iv Acknowledgements………………………………………………………………….……………v Table of Contents…………………………………………………………………… … ……vi List of Figures……………………………………………………………………….…… …… ix List of Tables……………………………………………………………………….…… … .xiii List of Abbreviations…………………………………………………………………… .… …xiv Chapter 1: Introduction…………………………………………………………………………1 1.1 Cholecystokinin (CCK)…… …… ….… 1.1.1 Cholecystokinin as a Neurotransmitter ………………………… …………………3 1.1.2 Cholecystokinin Receptors ………………………………………………… ….6 1.1.3 Distribution of Cholecystokinin in the brain and CCK receptors in the brain…… .8 1.1.4 Interactions of Cholecystokinin…………………………………………………….12 1.2 Anxiety…………………………………………………………………………… ….…13 1.2.1 Cholecystokinin (CCK) and Anxiety………………………………………… .… 14 1.3 Neurophysiology of Fear……………………… ………………………….….…… ….17 1.4 Defensive Behavior………………………………….……… ………… ….…… ……20 1.4.1 Cholcystokinin (CCK) and Defensive Behavior……………………………………21 1.5 The Role of Rat Strains in Experimental Psychopharmacology…………………………22 vi Chapter 2: Materials and Methods…………………………………………………… .….…26 2.1 Subjects………………………………………………………………………… ………26 2.2 Drugs………………………………………………………………………… …………26 2.3 Behavioural Testing Protocols………………………………………………… ….……28 2.3.1 Cat-freezing test…………………………………………………………… ….……29 2.3.2 Cat-odor test………………………………………………………………… .……30 2.2.3 Eat-drink test………………………………………………………………… ……30 2.3.4 Runway test………………………………………………………………………… 32 2.3.5 Vocalization test………………………………………………………… …….……32 2.3.6 Vibrissae stimulation test…………………………………………………………….33 2.3.7 Light-dark test……………………………………………………………… ………33 2.3.8 Elevated-plus maze test………………………………………………………….… .34 2.4 Periaqueductal gray (PAG) lesioning………………………………………….…… .…36 2.5 Anxiety habituation…………………………………………………….……….… .… .38 2.6 cDNA microarray……………………………………………………………….……… 40 2.7 Reverse transcription-polymerase chain reaction (RT-PCR)…………….….……….….41 2.8 Elevated-plus maze test (repeated experiment)………… ………………………………41 2.9 Statistical Analysis………………………………………………………….…… …42 Chapter 3: Results……………………………………………………………………….….….43 3.1.1 Behavioral Measures in the cat-freezing test……………………………….… …….43 3.1.2 Behavioral Measures in the Cat-odor test…………….…………………….……….…48 3.1.3 Behavioral Measures in the Eat-drink test……………………………….….…………56 3.1.4 Behavioral Measure in the Runway test……………………………………………….66 3.1.5 Behavioral Measures in the Vocalization test…………………………….…….…… .67 3.1.6 Behavioral Measures in the Vibrissae stimulation test………………………… ….…68 3.1.7 Behavioral Measures in the Light-dark test……………………………………………79 vii 3.1.8 Behavioral Measures in the Elevated-plus maze test………………………………… 87 3.2 Periaqueductal gray (PAG) lesioning………………………………………………….… .97 3.3 Anxiety habituation………………………………………………………………… .100 3.4 cDNA microarray and RT-PCR……………………………………………………….….104 3.5 Elevated-plus maze test (repeated experiment)………………….…………………… ….106 Chapter 4: Discussion…………………………………………………………………………111 References………………………………………………………………………………… ….132 viii LIST OF FIGURES Figure Page Number 1. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910(0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on freezing behaviour on the cat-freezing test…………………………… …… .… .44 2. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on locomotor activity on the cat-freezing test…………………………………………… 46 3. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on freezing behavior on cat-odor test .50 4. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on flat back behavior on cat-odor test…………………………………………………… 52 5. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on curve back behavior on cat-odor test……………………………………………… .54 6. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on eat frequency behaviour on eat-drink test………………………………………………60 7. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on eat duration behaviour on eat-drink test……………………………………… .………62 8. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on drink frequency behavior on eat-drink test…………………………………… ………64 9. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 ix (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on avoidance number………………………………………………………………… 69 10. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on startle behaviour…………………………………………………………………… 71 11. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on jump reaction behavior………………………………………………………………73 12. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on defensive biting behavior………………………………………………………….…75 13. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on Jump attack behavior…………………………………………… …………77 14. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats for time spent in the light chamber……………………………………………… ………81 15. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats for time spent in the dark chamber………………………………………………….……83 16. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on transitions…………………………………………………………………………… .85 17. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on the time spent in open arm………………………………………………………… .89 18. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats x on the time spent in closed arm……………………………………………………… 91 19. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on the number of entries in open arm…………………………………………………….93 20. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on the number of entries in the closed arm………………………………………….……95 21. Composite of lesion electrodes locations aimed at the dlPAG and vlPAG with the reference to the Paxinos and Watson atlas (1999). Some sites overlapped……………………………………………………………………….……… 98 22. Freezing behavior expressed as a percentage of the total session time (20 min) for vlPAG and dlPAG lesioned rats. Locomotor activity was measured as the total number of beam interruptions for the total session time (20 minutes)……………………………………………………………………… 99 23. Freezing behavior and locomotor activity expressed as the total session time (20 min) during the presence or absence of electrical lesioning of dlPAG or vlPAG lesioned rats on daily exposure to a cat for days 1-7…………….… 101 24. Effects of daily habituation of PVG hooded and Sprague-Dawley rats for days 1-7……………………………………………………………………………….102 25. Effects of BC264 (0.3µg/kg) on PVG hooded and Sprague-Dawley rats on day-8 after daily exposure to a cat for days…………………………………….… .103 26. CCK receptor expression in PVG hooded and SD rats: cDNA microarray and RT-PCR………………………………………………………………………………105 27. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), in PVG hooded and SD rats on the time spent in open arm…………………………………………………………………………… .107 28. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), in PVG hooded and SD rats on the time spent in closed arm………………………………………………………………………… .108 29. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg) in PVG hooded and SD rats on the number of entries in open arm .109 xi 30. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg) in PVG hooded and SD rats on the number of entries in closed arm .110 xii LIST OF TABLES Table Page Number 1. Neurotransmitters co-localized with CCK…………………………………….….…….5 2. Selective CCK2 receptor agonists……………… ………………………….……… …9 3. Selective CCK2 receptor antagonists…………………………………………… ……10 4. Commonly used animal models of anxiety…………………………………………….18 xiii LIST OF ABBREVIATIONS A/DTB ANOVA ADHD BZP BLA CCK CCK-LI CE CGRP cm CNS CRF CSF DA dPAG EPM F/DTB g GABA 5-HT h ICLAS i.p IUPHAR kg LH MET NAc NTS OFT PPI RT-PCR SN sec VTA VIP vPAG µg anxiety/defence test battery analysis of variance attention deficit hyperactivity disorder benzodiazepines basolateral complex of amygdala cholecystokinin cholecystokinin-like immunoreactivity central nucleus of amygdala calcitonin gene-related peptide centimetre central nervous system corticotropin-releasing factor cerebrospinal fluid dopamine dorsal periaqueductal gray elevated plus-maze fear/defence test battery gram γ-aminobutyric acid 5-hydroxytryptamine hour international council for laboratory animal science intraperitoneal international union of pharmacology kilogram lateral hypothalamus minute methionine nucleus accumbens nucleus tractus solitarius open field test prepulse inhibition reverse-transcriptase polymerase chain reaction subthalamic nucleus second ventral tegmental area vasoactive intestinal peptide ventral periaqueductal gray microgram xiv [...]... 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on the number of entries in the closed arm………………………………………….……95 21 Composite of lesion electrodes locations aimed at the dlPAG and vlPAG with the reference to the Paxinos and Watson atlas (1999) Some sites overlapped……………………………………………………………………….……… 98 22 Freezing behavior expressed as a percentage of the total session time (20 min) for vlPAG and. .. Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), in PVG hooded and SD rats on the time spent in closed arm………………………………………………………………………… .108 29 Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg) in PVG hooded and SD rats on the number of entries in open arm .109 xi 30 Effects of intraperitoneal... as the total number of beam interruptions for the total session time (20 minutes)……………………………………………………………………… 99 23 Freezing behavior and locomotor activity expressed as the total session time (20 min) during the presence or absence of electrical lesioning of dlPAG or vlPAG lesioned rats on daily exposure to a cat for days 1-7…………….… 101 24 Effects of daily habituation of PVG hooded and Sprague-Dawley... 1-7……………………………………………………………………………….102 25 Effects of BC264 (0.3µg/kg) on PVG hooded and Sprague-Dawley rats on day-8 after daily exposure to a cat for 7 days…………………………………….… 103 26 CCK receptor expression in PVG hooded and SD rats: cDNA microarray and RT-PCR………………………………………………………………………………105 27 Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), in PVG hooded and SD rats on the... in closed arm……………………………………………………… 91 19 Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg), LY 288513 (3, 5, 10mg/kg), CR 2945 (0.1, 1, 3mg/kg), and Lorglumide (3mg/kg) in PVG hooded and SD rats on the number of entries in open arm…………………………………………………….93 20 Effects of intraperitoneal administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05,... administration of CCK-4 (0.05, 0.1, 0.2mg/kg), LY 225910 (0.05, 0.1, 0.2mg/kg) in PVG hooded and SD rats on the number of entries in closed arm .110 xii LIST OF TABLES Table Page Number 1 Neurotransmitters co-localized with CCK…………………………………….….…….5 2 Selective CCK2 receptor agonists……………… ………………………….……… …9 3 Selective CCK2 receptor antagonists…………………………………………… ……10 4 Commonly used animal models of anxiety ………………………………………….18... LIST OF ABBREVIATIONS A/DTB ANOVA ADHD BZP BLA CCK CCK-LI CE CGRP cm CNS CRF CSF DA dPAG EPM F/DTB g GABA 5-HT h ICLAS i.p IUPHAR kg LH min MET NAc NTS OFT PPI RT-PCR SN sec VTA VIP vPAG µg anxiety/ defence test battery analysis of variance attention deficit hyperactivity disorder benzodiazepines basolateral complex of amygdala cholecystokinin cholecystokinin-like immunoreactivity central nucleus of amygdala... nervous system corticotropin-releasing factor cerebrospinal fluid dopamine dorsal periaqueductal gray elevated plus-maze fear/ defence test battery gram γ-aminobutyric acid 5-hydroxytryptamine hour international council for laboratory animal science intraperitoneal international union of pharmacology kilogram lateral hypothalamus minute methionine nucleus accumbens nucleus tractus solitarius open field . BEHAVIOURAL NEUROPHARMACOLOGY OF FEAR AND ANXIETY INVOLVING CHOLECYSTOKININ 2 RECEPTORS JUSTIN MOHAMED FAROOK B.Sc., University of Kerala, India DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2003 ii ABSTRACT PVG hooded and Sprague-Dawley (SD) rats differ in a variety of anxiety and. administration (I.P.) of CCK 2 receptor specific agonist (CCK-4) and CCK 2 receptor specific antagonist drugs (LY 225910, LY288513, and CR2945) on a variety of anxiety and fear related animal