The function of anxiety fear related genes in animal models 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

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

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

plus-maze 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

Tables 164

Table 1-1 Some animal (behavior) models of anxiety/fear

Conditioned responses Unconditioned responses

Geller-Seifter conflict

Vogel conflict

Conflict tests: pigeons, primates

Four-plate test

Conditioned emotional response (CER)

Conditioned taste aversion (CTA)

Fear potentiated startle

Defensive burying

Active/passive avoidance

Learned helplessness

Electrical brain stimulation (dPAG)

Elevated plus-maze (and zero-maze)

Open field Light/dark exploration Free exploration Holeboard Staircase test Social interaction Social competition Ultrasonic vocalization (pain or separation)

Human threat (primates) 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

Table 2-1 The primer sequence, expected length of PCR product and corresponding GenBank accession number of β-actin, CCK2 and 5-HT receptors

length (bp)

GenBank accession number β-actin se

* se: sense primer as: antisense primer bp: basepair

Tables 166

Table 3-1 Freezing behavior and locomotor activity of PVG hooded and SD rats on

cat exposure test

Locomotor activity Rat strain Freezing behavior on cat

exposure (% session time) Control Cat-exposed PVG hooded

Sprague-Dawley

87±3 9±3

811±132 1007±153

170±9 548±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

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

* se: sense primer as: antisense primer bp: basepair

length (bp)

GenBank accession number Arc se

Arc as 5’-ATACCAGTCTTGGGTGCCAG-3’ 5’-AGTGTCTGGTACAGGTCCCG-3’ 357 U19866 NGFI-A se

NGFI-A as 5’-GCAACAATTTGTGGCCTGAA-3’ 5’-GAGTTGGGACTGGTAGGTGT-3’ 512 M18416 5HT3R se

5HT3R as

5’- TTATGTGTATGTGCACCATCAAGGTGAA-3’

5’-ACAGCAGCGTGTCCAGCACATATCCCACC-3’

956 U59672 β-actin se

β-actin as

5’-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3’

Tables 168

Table 4-2 The 16 up-regulated genes in PVG hooded rats compared to SD rats

ratio gene name

2.1 Rat fibroblast growth factor putative isoform 2 (FGF) mRNA, complete cds, alternatively spliced 2.0 Mouse FK506 binding protein 51 mRNA, complete cds

1.8 Messenger RNA for rat preproalbumin

1.7 Rat gastric intrinsic factor mRNA, complete cds

1.6 Rat MHC class I antigen (RT1.EC3) gene, complete cds

1.6 Mouse S3-12 mRNA, complete cds

1.6 Rat caveolin-3 mRNA, complete cds

1.5 Rat mRNA for pre-alpha-inhibitor, heavy chain 3

1.5 Rat synaptotagmin VI mRNA, complete cds

1.5 Mouse ERCC2 gene, genomic sequence

1.5 Mouse major histocompatibility locus class II region

1.5 Rat mRNA (rls2var1) for leuserpin-2

1.5 Rat Wistar transforming growth factor beta-3 mRNA, complete cds

1.5 Rat mRNA for fibronectin

1.5 Mouse mRNA for type II 57 kd keratin

1.5 Mouse protein phosphatase 1 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)

Table 4-3 The 38 up-regulated genes in SD rats compared to PVG hooded rats

ratio gene name

-2.0 Rat activity-regulated cytoskeleton-associated gene (Arc) mRNA, complete cds

-1.9 Rat carbonic anhydrase III (CA3) mRNA, complete cds

-1.8 Mouse (Mus musculus domesticus) transcription factor IID (Tbp) mRNA, complete cds -1.8 Rat partial mRNA for MHC class Ia A2h antigen (RT1-A2h gene)

-1.7 Mouse myelin proteolipid protein mRNA, complete cds

-1.7 Rat SC1 protein mRNA, complete cds

-1.7 Rat prealbumin (transthyretin) mRNA, complete cds

-1.7 Rat nerve growth factor-induced (NGFI-A) gene, complete cds

-1.7 Rat prostaglandin F2a receptor mRNA, complete cds

-1.6 rat myelin basic protein (mbp) gene mrna

-1.6 Rat mRNA for neurodegeneration associated protein 1, complete cds

-1.6 Rat (clone pCNPII) 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPII) mRNA, complete cds -1.6 Rat microtubule-associated protein 1A MAP1A (Mtap-1) mRNA, complete cds

-1.6 Rat mRNA for microtubule associated protein IB

-1.6 Rat partial GR gene for glucocorticoid receptor, promoter region

-1.6 63 kDa calmodulin-stimulated phosphodiesterase

-1.6 Rat mRNA for cytoplasmic dynein 74 kD intermediate chain

-1.6 Rat neural membrane protein 35 mRNA, complete cds

-1.6 Rat non-processed neurexin I-beta mRNA, complete cds

-1.6 microtubule-associated protein, MAP-115

-1.6 Rat rab GDI alpha mRNA

-1.6 Rat Ca2+-dependent activator protein (CAPS) mRNA, complete cds

-1.5 Mouse epidermal keratin type I intermediate filament gene, exon 1

-1.5 Rat mRNA for calcineurin A

-1.5 Rat Myo5a mRNA for myosin-Va, complete cds

-1.5 Mouse mRNA for neuroserpin

-1.5 Mouse GABA-A receptor gamma-2 subunit mRNA, complete cds

-1.5 Rat kinesin-like protein KIF1B (KIF1B) mRNA, partial cds

-1.5 Mouse zinc finger protein (Peg3) mRNA, complete cds

-1.5 Mouse mRNA for SmN protein

-1.5 Rat GABA-A receptor alpha-1 subunit gene, complete cds

-1.5 Mouse ubiquitinating enzyme E2-20K mRNA, complete cds

-1.5 Rat mRNA for SNERG-1 protein

-1.5 Rat mRNA for cathepsin L (EC 3.4.22.15)

-1.5 Rat GluT and GluT-R glutamate transporter mRNA, complete cds

-1.5 Rat gamma-adducin mRNA, complete cds

-1.5 Rat D-binding protein mRNA, complete cds

-1.5 Rat metabotropic glutamate receptor 2 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)

Tables 170

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 1 through 9

-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)

Figure 1-1 Predicted structure of human preprocholecystokinin The signal peptide

consists of residues 20 to 1 The amino terminal flanking peptide consists of residues 1 to 25 The largest characterized form from brain and intestine, CCK-58, consists of residues 26 to 83 Other active molecular forms are derived from this precursor, such as CCK-39, CCK-33, CCK-22, CCK-7, and CCK-5 (Nobel et al., 1999)

Figures 172

Figure 1-2 Schematic representation of the rat CCK1 receptor showing the

postulated transmembrane topology, sites for putative NH2-linked glycosylation (tridents), serine and threonine phosphorylation by PKC and protein kinase A (PO3), and conserved cysteines in the first and second ECLs, possibly forming a disulfide bridge, and a possible palmitoylated conserved cysteine in the cytoplasmic tail (Nobel et al., 1999) NH2 -, N terminus; COOH -, C terminus

Figure 1-3 Schematic representation of the rat CCK2 receptor showing the

postulated transmembrane topology, sites for putative NH2-linked glycosylation (tridents), serine and threonine phosphorylation by PKC and protein kinase A (PO3), and conserved cysteines in the first and second ECLs, possibly forming a disulfide bridge, and a possible palmitoylated conserved cysteine in the cytoplasmic tail (Nobel et al., 1999) NH2 -, N terminus; COOH -, C terminus

Figures 174

Figure 1-4 Alignment of members of the corticotropin-releasing factor (CRF)

peptide family The amino acids that are homologous between the CRF peptides are boxed Human stresscopin-related peptide (SRP) and human urocortin II (UCNII) are identical Human stresscopin (SCP) and human UCNIII also have identical amino acid sequences (Dautzenberg and Hauger, 2002) Abbreviations: h, human; m, mouse; o, ovine; SVG, sauvagine; URO, urotensin I

Figure 1-5 Dendrogram showing the relatedness of corticotropin-releasing factor

receptors (CRF-Rs) from different species (Spiess et al., 1998) cCRF-R1, chicken CRF-R1; hCRF-R, human CRF-R; mCRF-R, mouse CRF-R; rCRF-R, rat CRF-R;

xCRF-R, Xenopus CRF-R

Figures 176

Figure 1-6 Interactions between mammalian corticotropin releasing factor

(CRF)-like peptides, their receptors and the CRF binding protein (CRF-BP) Binding to the CRF-BP or activation of CRF1 or CRF2 receptors stimulates or decreases a wide range of physiological responses (Dautzenberg and Hauger, 2002) Abbreviations: SCP, stresscopin; UCN, urocortin

Figure 2-1 The illustration of the elevated plus maze for SD rats The plus-maze

was elevated by 50 cm above floor level and comprised of 2 (opposite) closed and 2 open arms, each 50 cm by 10 cm The closed arms had 40 cm high side and end walls This apparatus was set up in a test room with a video camera mounted to the maze Each rat was tested on the elevated plus-maze apparatus for a stipulated period

of 300 seconds

Figures 178

Figure 2-2 Variations in anxious level in SD rats on the elevated plus-maze The

time spent on the open arms was expressed as percentage of the total session time (300 s) Five rats with the longest open arm times were used as the low anxiety group and 5 rats with the shortest open arm times were used as the high anxiety group

Figure 2-3 Gene expression of CCK2 receptor between the high (solid bars) and low (open bars) anxiety rats screened by the elevated-plus maze PCR products were quantified based on the electrophoresis of the 1% agarose gel using NIH image-processing software Ratios of the corresponding density (CCK2 /β-actin) were calculated for each sample and used for quantitative calculations and comparisons The PCR bands that were statistically significant were shown above the

corresponding bars The top band in each group was β-actin and the bottom was the target product * p<0.05

Figures 180

Figure 2-4 Gene expression of 5-HT1A receptor between the high (solid bars) and low (open bars) anxiety rats screened by the elevated-plus maze PCR products were quantified based on the electrophoresis of the 1% agarose gel using NIH image-processing software Ratios of the corresponding density (5-HT1A /β-actin) were calculated for each sample and used for quantitative calculations and comparisons The PCR bands that were statistically significant were shown above the

corresponding bars The top band in each group was β-actin and the bottom was the target product * p<0.05