Behavioural neuropharmacology of fear and anxiety involving cholecystokinin2 receptors

BEHAVIOURAL NEUROPHARMACOLOGY OF FEAR
AND ANXIETY INVOLVING CHOLECYSTOKININ
2
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



ii
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 (CCK
2
) 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 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 models.
I.P administration of a cholecystokinin-1 (CCK
1
) 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 (RT-
PCR) experiments were then carried out in collaboration to determine the CCK
2
receptor
expression among these 2 strains of rats. The results showed that CCK
2
receptors were
expressed to a greater extent in the PVG hooded rats. Anxiety habituation studies using a
selective CCK
2
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 CCK
2
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 CCK
2
receptor subsites,
CCK
2A
and CCK
2B

probably corresponding to different coupling sites of the CCK
2

receptor.











iii
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 cholecystokinin
2
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 plus-
maze 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 CCK
2
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.

iv
CONFERENCE ABSTRACTS


1. Farook JM
, Spiess S, Zhu YZ, Moochalla SM, and Wong PTH. Investigation of
Behavioral Responses Induced by Selective Stimulation of CCK
2
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.



















v
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.

vi
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

1.1.1 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



vii
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


viii
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

ix
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

x
(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

xi
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 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 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


xii
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

xiii
LIST OF TABLES

Table Page Number

1. Neurotransmitters co-localized with CCK…………………………………….….…….5
2. Selective CCK
2
receptor agonists……………… ………………………….……… …9
3. Selective CCK
2
receptor antagonists…………………………………………… ……10
4. Commonly used animal models of anxiety…………………………………………….18

xiv
LIST OF ABBREVIATIONS

A/DTB anxiety/defence test battery
ANOVA analysis of variance
ADHD attention deficit hyperactivity disorder
BZP benzodiazepines
BLA basolateral complex of amygdala
CCK cholecystokinin
CCK-LI cholecystokinin-like immunoreactivity
CE central nucleus of amygdala
CGRP calcitonin gene-related peptide
cm centimetre
CNS central nervous system
CRF corticotropin-releasing factor
CSF cerebrospinal fluid
DA dopamine
dPAG dorsal periaqueductal gray
EPM elevated plus-maze
F/DTB fear/defence test battery
g gram
GABA γ-aminobutyric acid
5-HT 5-hydroxytryptamine
h hour
ICLAS international council for laboratory animal science
i.p intraperitoneal
IUPHAR international union of pharmacology
kg kilogram
LH lateral hypothalamus
min minute
MET methionine

NAc nucleus accumbens
NTS nucleus tractus solitarius
OFT open field test
PPI prepulse inhibition
RT-PCR reverse-transcriptase polymerase chain reaction
SN subthalamic nucleus
sec second
VTA ventral tegmental area
VIP vasoactive intestinal peptide
vPAG ventral periaqueductal gray
µg microgram