List of tables/figures 161 LIST OF TABLES/FIGURES Table 1-1 Some animal (behavior) models of anxiety/fear Table 2-1 The primer sequence, expected length of PCR product and corresponding GenBank accession number of β-actin, CCK2 and 5-HT receptors Table 3-1 Freezing behavior and locomotor activity of PVG hooded and SD rats on cat exposure test Table 4-1 The primer sequence, expected length of PCR product and corresponding GenBank accession number of Arc, NGFI-A, 5-HT3R and β-actin Table 4-2 The 16 up-regulated genes in PVG hooded rats compared to SD rats Table 4-3 The 38 up-regulated genes in SD rats compared to PVG hooded rats Table 4-4 Receptors of some neurotransmitters which were differentially expressed and their abundance ratios between PVG hooded and SD rats Figure 1-1 Predicted structure of human preprocholecystokinin Figure 1-2 Schematic representation of the rat CCK1 receptor Figure 1-3 Schematic representation of the rat CCK2 receptor Figure 1-4 Alignment of members of the corticotropin-releasing factor (CRF) peptide family Figure 1-5 Dendrogram showing the relatedness of corticotropin-releasing factor receptors (CRF-Rs) from different species Figure 1-6 Interactions between mammalian corticotropin releasing factor (CRF)like peptides, their receptors and the CRF binding protein (CRF-BP) Figure 2-1 The illustration of the elevated plus maze for SD rats List of tables/figures 162 Figure 2-2 Variations in anxious level in SD rats on the elevated plus-maze Figure 2-3 Gene expression of CCK2 receptor between the high and low anxiety rats screened by the elevated-plus maze Figure 2-4 Gene expression of 5-HT1A receptor between the high and low anxiety rats screened by the elevated-plus maze Figure 2-5 Gene expression of 5-HT1B receptor between the high and low anxiety rats screened by the elevated-plus maze Figure 2-6 Gene expression of 5-HT2A receptor between the high and low anxiety rats screened by the elevated-plus maze Figure 2-7 Gene expression of 5-HT3 receptor between the high and low anxiety rats screened by the elevated-plus maze Figure 3-1 CCK2 receptor gene expression in six brain regions of PVG hooded and SD rats after cat exposure Figure 4-1 Representative images of cDNA microarray results comparing PVG hooded and SD rats Figure 4-2 The expression of mRNA for Arc, NGFI-A, 5-HT3 receptor (5HT3R) and β-actin in PVG hooded and SD rats by reverse transcription-polymerase chain reaction (RT-PCR) Figure 5-1 Effects of intraperitoneal administration (0.1 mg/kg) of LY225910 and CCK-4 in PVG hooded and SD rats on freezing behavior and locomotor activity Figure 5-2 CCK2 receptor gene expression in cortex and hippocampus of PVG hooded and SD rats among the control, CCK-4 and LY225910 groups Figure 6-1 Genetic variants in the coding region of the CCK2 receptor gene Figure 7-1 The stereotaxic apparatus List of tables/figures 163 Figure 7-2 Anatomical localization of the injection sites of i.c.v., i.h and i.s Figure 7-3 The elevated plus maze apparatus Figure 7-4 Fear conditioning apparatus Figure 7-5 Dose-dependent anxiogenic action of CCK4 in C57BL/6J mice in the elevated plus-maze Figure 7-6 The anxiogenic effect of CCK4 in the elevated plus-maze was blocked by CCK2 receptor antagonist CR2945 Figure 7-7 The anxiogenic effect of CCK4 in the elevated plus-maze was blocked by h/rCRF receptor antagonist acetic astressin Figure 7-8 The effect of CCK4 on context- and tone-dependent fear conditioning Figure 7-9 h/rCRF receptor antagonist acetic astressin blocked the effect of CCK4 on context- and tone-dependent fear conditioning Figure 7-10 The anxiogenic effect of CCK4 after i.h injection in the elevated plusmaze was blocked by h/rCRF receptor antagonist acetic astressin Figure 7-11 The effect of CCK4 on context- and tone-dependent fear conditioning after i.h injection Figure 7-12 The i.s injection of CCK4 did not produce anxiogenic effect in C57BL/6J mice in the elevated plus-maze 164 Tables Table 1-1 Some animal (behavior) models of anxiety/fear Conditioned responses Unconditioned responses Geller-Seifter conflict Elevated plus-maze (and zero-maze) Vogel conflict Open field Conflict tests: pigeons, primates Light/dark exploration Four-plate test Free exploration Conditioned emotional response (CER) Holeboard Conditioned taste aversion (CTA) Staircase test Fear potentiated startle Social interaction Defensive burying Social competition Active/passive avoidance Ultrasonic vocalization (pain or separation) Learned helplessness Human threat (primates) Electrical brain stimulation (dPAG) Fear/anxiety-defence test batteries *Procedures may be different on the basis of whether or not they involve conditioning (adapted from Rodgers and Dalvi, 1997) dPAG: dorsal periaqueductal grey matter 165 Tables Table 2-1 The primer sequence, expected length of PCR product and corresponding GenBank accession number of β-actin, CCK2 and 5-HT receptors Gene Primer Expected length (bp) GenBank accession number β-actin se β-actin as CCK2 se CCK2 as 5-HT1A se 5-HT1A as 5-HT1B se 5-HT1B as 5-HT2A se 5-HT2A as 5-HT3 se 5-HT3 as 5’-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3’ 5’-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3’ 5’-CGCCTAAGAACGGTCACCAACG-3’ 5’-AACAATCACTAGCAGCATTCGCACC-3’ 5’-AGCATCTCCGACGTGACCTTCAGCTACCA-3’ 5’-GCTCCCTTCTTTTCCACCTTCCTGACAGT-3’ 5’-GCCGACGACTACATTTACCAGGACTCCAT-3’ 5’-TGCCGGTCTTGTTGGGTGTCTGTTTCA-3’ 5’-CATGCCTCTCCATTCTTCATCTCCAGGAA-3’ 5’-CAAGGTGGCTTCTTTCTGAAGTGACTTGA-3’ 5’-GAGACCATCTTCATTGTGCAGCTGGTGCA-3’ 5’-ACAGCAGCGTGTCCAGCACATATCCCACC-3’ 838 BC014401 783 M99418.1 635 NM_012585.1 643 NM_022225.1 611 NM_017254.1 956 U59672 * se: sense primer as: antisense primer bp: basepair 166 Tables Table 3-1 Freezing behavior and locomotor activity of PVG hooded and SD rats on cat exposure test Rat strain Freezing behavior on cat Locomotor activity exposure (% session time) Control Cat-exposed PVG hooded 87±3 811±132 170±9 Sprague-Dawley 9±3 1007±153 548±51 * Freezing behavior was expressed as the percentage of session time (20 min) Controls were exposed to only an empty cat compartment Locomotor activity was expressed as total number of beam interruptions during the test period Freezing was significantly different between the two strains, t = 25.449, p< 0.001 One-way ANOVA for locomotor activity: F(3,12) = 11.996, p< 0.001 Post hoc Tukey: significantly different between control and cat-exposed for both PVG (p< 0.005) and SD (p < 0.05) rats 167 Tables Table 4-1 The primer sequence, expected length of PCR product and corresponding GenBank accession number of Arc, NGFI-A, 5-HT3R and β-actin Gene Primer Expected length (bp) GenBank accession number Arc se Arc as NGFI-A se NGFI-A as 5HT3R se 5HT3R as β-actin se β-actin as 5’-ATACCAGTCTTGGGTGCCAG-3’ 5’-AGTGTCTGGTACAGGTCCCG-3’ 5’-GCAACAATTTGTGGCCTGAA-3’ 5’-GAGTTGGGACTGGTAGGTGT-3’ 5’- TTATGTGTATGTGCACCATCAAGGTGAA-3’ 5’-ACAGCAGCGTGTCCAGCACATATCCCACC-3’ 5’-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3’ 5’-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3’ 357 U19866 512 M18416 956 U59672 838 BC014401 * se: sense primer as: antisense primer bp: basepair Tables 168 Table 4-2 The 16 up-regulated genes in PVG hooded rats compared to SD rats ratio 2.1 2.0 1.8 1.7 1.6 1.6 1.6 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 gene name Rat fibroblast growth factor putative isoform (FGF) mRNA, complete cds, alternatively spliced Mouse FK506 binding protein 51 mRNA, complete cds Messenger RNA for rat preproalbumin Rat gastric intrinsic factor mRNA, complete cds Rat MHC class I antigen (RT1.EC3) gene, complete cds Mouse S3-12 mRNA, complete cds Rat caveolin-3 mRNA, complete cds Rat mRNA for pre-alpha-inhibitor, heavy chain Rat synaptotagmin VI mRNA, complete cds Mouse ERCC2 gene, genomic sequence Mouse major histocompatibility locus class II region Rat mRNA (rls2var1) for leuserpin-2 Rat Wistar transforming growth factor beta-3 mRNA, complete cds Rat mRNA for fibronectin Mouse mRNA for type II 57 kd keratin Mouse protein phosphatase binding protein PTG mRNA, complete cds * Ratio is the balanced differential expression between two samples If the ratio is positive, the gene in PVG channel is up regulated; the negative ratio means the gene in SD channel is up regulated (cds: coding DNA sequence) Tables 169 Table 4-3 The 38 up-regulated genes in SD rats compared to PVG hooded rats ratio -2.0 -1.9 -1.8 -1.8 -1.7 -1.7 -1.7 -1.7 -1.7 -1.6 -1.6 -1.6 -1.6 -1.6 -1.6 -1.6 -1.6 -1.6 -1.6 -1.6 -1.6 -1.6 -1.5 -1.5 -1.5 -1.5 -1.5 -1.5 -1.5 -1.5 -1.5 -1.5 -1.5 -1.5 -1.5 -1.5 -1.5 -1.5 gene name Rat activity-regulated cytoskeleton-associated gene (Arc) mRNA, complete cds Rat carbonic anhydrase III (CA3) mRNA, complete cds Mouse (Mus musculus domesticus) transcription factor IID (Tbp) mRNA, complete cds Rat partial mRNA for MHC class Ia A2h antigen (RT1-A2h gene) Mouse myelin proteolipid protein mRNA, complete cds Rat SC1 protein mRNA, complete cds Rat prealbumin (transthyretin) mRNA, complete cds Rat nerve growth factor-induced (NGFI-A) gene, complete cds Rat prostaglandin F2a receptor mRNA, complete cds rat myelin basic protein (mbp) gene mrna Rat mRNA for neurodegeneration associated protein 1, complete cds Rat (clone pCNPII) 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPII) mRNA, complete cds Rat microtubule-associated protein 1A MAP1A (Mtap-1) mRNA, complete cds Rat mRNA for microtubule associated protein IB Rat partial GR gene for glucocorticoid receptor, promoter region 63 kDa calmodulin-stimulated phosphodiesterase Rat mRNA for cytoplasmic dynein 74 kD intermediate chain Rat neural membrane protein 35 mRNA, complete cds Rat non-processed neurexin I-beta mRNA, complete cds microtubule-associated protein, MAP-115 Rat rab GDI alpha mRNA Rat Ca2+-dependent activator protein (CAPS) mRNA, complete cds Mouse epidermal keratin type I intermediate filament gene, exon Rat mRNA for calcineurin A Rat Myo5a mRNA for myosin-Va, complete cds Mouse mRNA for neuroserpin Mouse GABA-A receptor gamma-2 subunit mRNA, complete cds Rat kinesin-like protein KIF1B (KIF1B) mRNA, partial cds Mouse zinc finger protein (Peg3) mRNA, complete cds Mouse mRNA for SmN protein Rat GABA-A receptor alpha-1 subunit gene, complete cds Mouse ubiquitinating enzyme E2-20K mRNA, complete cds Rat mRNA for SNERG-1 protein Rat mRNA for cathepsin L (EC 3.4.22.15) Rat GluT and GluT-R glutamate transporter mRNA, complete cds Rat gamma-adducin mRNA, complete cds Rat D-binding protein mRNA, complete cds Rat metabotropic glutamate receptor mRNA, primary transcript * Ratio is the balanced differential expression between two samples If the ratio is positive, the gene in PVG channel is up regulated; the negative ratio means the gene in SD channel is up regulated (cds: coding DNA sequence) 170 Tables Table 4-4 Receptors of some neurotransmitters which were differentially expressed and their abundance ratios between PVG hooded and SD rats ratio gene type gene name -1.5 GABA receptor Rat GABA-A receptor alpha-1 subunit gene, complete cds -1.3 Rat mRNA for GABA-BR1b receptor -1.3 Rat GABAB1 receptor (GABABR1) gene, exons through -1.3 Rat GABA-A receptor alpha-6 subunit gene, complete cds -1.2 Rat GABA-A receptor alpha-4 subunit gene, complete cds -1.2 Rat GABA-A receptor delta-subunit gene, complete cds 1.2 Rat mRNA for GABA(A) receptor gamma-1 subunit -1.2 Rat GABA-A receptor alpha-5 subunit gene, complete cds -1.2 Rat GABA-A receptor alpha-6 subunit cDNA 1.3 CCK receptor Rat brain cholecystokinin receptor mRNA, complete cds -1.2 Rat gene for cholecystokinin type-A receptor (CCKAR), complete cds 1.2 5-HT3 receptor Rat 5HT3 receptor mRNA, complete cds * Ratio is the balanced differential expression between two samples If the ratio is positive, the gene in PVG channel is up regulated; the negative ratio means the gene in SD channel is up regulated (cds: coding DNA sequence) Figures 187 Figure 5-1 Effects of intraperitoneal administration (0.1 mg/kg) of LY225910 and CCK4 in PVG hooded and SD rats on freezing behavior (top panel) and locomotor activity (bottom panel) Freezing was expressed as percentage of session time (20 min) One-way ANOVA: F(5,20) = 478.23, p