Figure 1: Basic behaviour of Tcf4tg mice upon IR and EE. A) Body weight was higher in EE mice (p<0.0001). B–C)Light-dark preference B) Latency to enter dark box was longer in IR than EE mice (p<0.0001). C)Dark preference.D–H)Open field.D)Time in the centre was reduced by EE (p<0.0001) and Tcf4tg (p=0.0064), particularly in EETcf4tg (p<0.01, Bonferroni). E) Rearing was affected by IR (p<0.0001) andTcf4tg (p=0.0458). F–H)Time moving, distance and speed were increased by IR (p<0.0001 for all).I)In Water Maze IR mice swam faster (p<0.0001). J–K)Hole board.J)Nose pokes.K)Exploration time was increased by EE (p=0.0044) but reduced in EETcf4tg mice (p<0.05, Bonferroni, genotype effect p=0.0428). Bar graphs show means with SEM. Tests were analysed with Two-way ANOVA, unless stated differently.
Figure 2: Basic behaviour ofTcf4tg mice upon IR and EE. A–D)Y-maze. A)Total arm exploration is increased upon IR (p<0.0001).B)Total number of alternations is reduced by IR (p=0.0003).C)IR mice explore arms of the maze more than EE in first (p<0.0001) and second interval (p<0.0001).D)IR mice make fewer alternations in first (p=0.0145) and second interval (p=0.0259). E)Prepulse inhibition is reduced by IR at 75 dB (p=0.0134) and 80 dB (p=0.0057). F)Startle response to 120 dB pulse is affected by housing (p<0.0001) and genotype (p=0.0013). In EE group it is reduced inTcf4tg (p<0.01, Bonferroni).G)In tail suspension test IR mice fight less (p<0.0001) implying reduced motivation.H–I)Social interaction. Neither sociability nor social memory are alterations were observed. J)Hot plate. Thermal pain sensitivity was increased by IR (p<0.0001). K)Pain threshold for electric shocks is increased upon IR (p<0.0001).L)Water Maze, visible platform: EE mice reach the platform faster than IR mice (p<0.0001, RM ANOVA). Bar graphs represent mean with SEM. All tests were analysed using Two-way ANOVA, unless stated differently.
Figure 3: Basic behaviour of agedTcf4tg mice upon IR and EE. A)Body weight was increased in IR mice from the age of 8 months andTcf4tg mice were slightly heavier that wt in IR group.B–C)Light-dark preference.B)IR mice entered the dark compartment later than EE mice (p<0.0001).C)IR mice displayed higher dark preference (p<0.0001).D–G)Open field.D)IR mice spent less time in the centre (p=0.002).
E)Rearing was reduced upon IR (p=0.0133) and particularly inTcf4tg mice (p=0.0344, t-test), but without G×E. F)Time moving (p<0.0001),G)covered distance (p<0.0001) andH)running speed (p<0.0001) were increased upon IR.I)In Water Maze mean swim speed (all phases) was affected by housing (p<0.0001) and G×E (p=0.0352). In IR groupTcf4tg mice swam faster than wt (p<0.05, Bonferroni).J–K)Hole board.
J)Number of hole nose pokes was reduced in IR (p=0.002), similarly toK)exploration time (p=0.0072). Bar graphs represent mean with SEM. All tests were analysed using Two-way ANOVA, unless stated differently.
Figure 4: Basic behaviour of agedTcf4tg mice upon IR and EE. A)EPM. IR mice spent less time in open (p<0.001) and more time in closed arms (p=0.0332). B–E)Y-maze.B)IR animals explored more arms in total than EE (p<0.0001). C)IR mice made in total fewer alternations than EE mice (p=0.0045). D)In both intervals IR mice made more arm choices than EE mice (both p<0.0001). E)IR mice made fewer alternations in first interval (p=0.0119). In the second interval effects of housing and genotype were not significant; however, in EE groupTcf4tg mice made more alternations than wt mice (p<0.05, Bonferroni).
F)In TST IR mice showed reduced fighting time (p<0.0001).G–H)Social interaction.G)Sociability was not altered in any of the groups. H)Social memory was increased in IR mice (p=0.0203). I)In hot plate test pain threshold was increased upon IR (p<0.0001). J–K)MWM.J)In visible platform phase IR mice learned slower than EE mice (p<0.0001, RM ANOVA).K–L)Perseveration (% distance in previous target quadrant) was not altered in reversal learning nor in delayed matching to place. Bar graph represent mean with SEM. All tests were analysed using Two-way ANOVA, unless stated differently.
Figure 5: Basic behaviour ofTcf4C mice. A)Body weight was reduced in Tcf4C mice at the age of 4 weeks (p=0.0472, Mann Whitney test,Tcf4C n=23, wt n=16) but not at 14 weeks (Tcf4C n=16, wt n=14).
B–C)Light-dark preference. No significant changes were detected.D–H)Open field.D)Tcf4C mice spent less time in the centre, which suggest mildly increased anxiety.E–H)Neither rearing nor locomotor activity was altered in any of the parameters. I)In Morris Water Maze swimming speed was reduced inTcf4C mice (p=0.02229, Mann Whitney test).J–K)Hole board exploration was not changed inTcf4C. Bar graph represent mean with SEM.
Figure 6: Basic behaviour ofTcf4C mice. A)Elevated plus maze.B–E)Y-maze.B)Total arm exploration was not altered inTcf4C mice.C)Total alternation number was mildly reduced inTcf4C animals (p=0.014, t-test). D) Tcf4C mice explored the arms less than wt in first (p=0.0469, t-test), but not in the second interval. E) Number of alternations was reduced in Tcf4C mice only in the second interval (p=0.0475, t-test). G) Sociability and social memory were normal. H) Pain sensitivity was unaffected in Tcf4C mice.I–J)Prepulse inhibition was normal, but startle response to 120 dB pulse was reduced inTcf4C mice (p=0.0311, Two-way RM ANOVA), particularly in post-test (p<0.05, Bonferroni). K)In MWM visible platformTcf4C mice showed no impairments. Bar graphs represent mean with SEM.
Table1:CognitioninTcf4tgandTcf4Cmice:domains,traitsandmeasures.DatafromtheYoungTcf4tgIR–EEandSD–ctrlcohortsaswellasfromTcf4C cohorthousedinIR.PG—globalp-value. SuperdomainDomainTraitMeasurePGEEenvironmenttg/wtIRenvironmenttg/wtSDenvironmenttg/wtIRenvironmentko/wtG×EINTERACTION EffectStatisticPEffectStatisticPEffectStatisticPEffectStatisticPPtg/wt×IR/EEPtg/wt×SD/ctrl COGNITIVESYMPTOMCLASSmultivariate*-0.127t80=-0.4380.663-1.811t86=-2.890.005-1.997t82=-4.372<0.001-1.632t86=-3.723<0.0010.0190.315 SpatialLearningmultivariate*0.015t76=0.040.969-4.103t82=-2.3970.019-3.899t85=-6.578<0.001-4.112t82=-6.670<0.0010.0220.039 Memoryrecallmultivariate*0.645t50=1.210.2320.002t53=0.0040.997-4.321t56=-6.698<0.001-1.456t26=-1.6820.105ns0.047 WM-recallmultivariate*1.344t50=1.7210.0910.139t54=0.1930.848-2.024t56=-2.3110.025nsns WMtprobe1*1.242t24=1.7620.0910.182t26=0.2570.799-2.25t27=-2.3630.026nsns WMdprobe1*1.446t24=1.8740.0730.095t26=0.1310.897-1.797t27=-2.2520.033nsns WM-remoterecallmultivariate*-0.055t50=-0.0780.938-0.079t52=-0.1070.915-6.619t56=-5.944<0.001-1.456t54=-1.6550.104ns<0.001 WMtprobe2*-0.044t24=-0.0720.9430.0002t25<0.0011.000-8.099t27=-7.002<0.001-1.333t26=-1.9340.064ns0.001 WMdprobe2*-0.066t24=-0.0840.934-0.157t25=-0.1940.848-5.139t27=-6.644<0.001-1.579t26=-1.5090.143ns0.001 Perseverationmultivariate*-0.621t50=-0.8490.400-7.877t54=-2.9190.005-4.698t56=-6.020<0.001-6.862t54=-4.632<0.0010.0140.046 WMtrevsum*-0.283t24=-0.4730.641-8.016t26=-2.5370.018-4.289t27=-6.172<0.001-6.469t26=-4.555<0.0010.0200.042 WMdrevsum*-0.959t24=-1.0700.295-7.738t26=-2.6980.012-5.107t27=-5.727<0.001-7.255t26=-3.874<0.0010.0280.040 SpatiallearningWMinitiallearningmultivariate*-0.093t50=-0.1280.899-2.598t54=-1.4690.148-3.035t56=-3.3780.001-4.834t54=-4.815<0.001nsns WMthpsum*0.230t24=0.3150.755-2.294t26=-1.1980.242-3.253t27=-3.3040.003-7.645t26=-5.572<0.001nsns WMdhpsum*-0.415t24=-0.5410.593-2.902t26=-1.5560.132-2.816t27=-3.2520.003-2.024t26=-2.3930.024nsns BaselineWMvisibleplatformmultivariate*0.63t50=0.8140.4191.653t54=0.9290.357-0.791t54=-1.0260.309ns controlWMtvpsum*0.468t24=0.5650.5772.273t26=1.1730.252-0.783t26=-0.9880.332ns WMdvpsum*0.792t24=1.1980.2421.033t26=0.5550.584-0.799t26=-1.0360.310ns Fearmemorymultivariate*-0.413t54=-0.9390.352-0.807t58=-2.0560.0440.068t48=0.1790.8580.806t53=1.4330.158nsns Contextmemorymultivariate*-0.417t52=-0.830.410-0.066t58=-0.1440.886-0.318t23=-1.1290.2710.763t46=1.5120.137nsns ContextmemoryFCcontextns0.097t26=0.1490.8820.093t28=0.150.882-0.318t23=-1.1290.2711.126t23=1.7720.090nsns RemotecontextFCrcontext*-1.034t24=-1.5930.124-0.225t28=-0.3090.7600.383t21=0.6510.522ns Cuememorymultivariate*-0.467t52=-0.7290.469-1.548t58=-3.4370.0010.453t23=0.8010.4310.935t54=1.5850.119nsns CuememoryFCcue*-0.175t26=-0.2340.817-1.628t28=-2.5690.0160.453t23=0.8010.4310.967t24=1.4470.161nsns RemotecuememoryFCrcue*-0.689t24=-0.9810.336-1.468t28=-2.1770.0381.150t28=1.6770.105ns Socialfearmemorymultivariatens1.153t56=2.0990.040 SocialfearmemorySIsocavoidancens0.883t28=1.5940.122 RemoresocialfearmemorySIrsocavoidancens1.255t26=1.3930.175 WorkingmemoryYmazealternations*0.104t26=0.1750.8630.439t28=0.5810.566-0.218t28=-0.3000.767-2.032t28=-2.6660.013nsns
able2:EnvironmentaleffectsintheG×Ecohorts:Tcf4tgYoungIR–EEandTcf4tgSD–Control.PG—globalp-value.IReffectonsuperdomainanxietyissignificantforthesumscore(univariatetest,p=0.013).
SuperdomainDomainMeasurePGIRcomparedtoEESDcomparedtoCTRLEffectStatisticPEffectStatisticP COGNITIVESYMPTOMCLASSmultivariate*-2.745t166=-4.703<0.001-3.702t160=-7.314<0.001SpatialLearningmultivariate*-5.384t158=-4.421<0.001-8.061t161=-13.08<0.001Memoryrecallmultivariate*-0.961t103=-1.740.085-7.948t106=-11.53<0.001Perseverationmultivariate*-10.502t104=-4.143<0.001-10.466t106=-12.75<0.001Spatiallearningmultivariate*-6.38t104=-4.185<0.001-5.904t106=-7.289<0.001Baselinecontrolmultivariate*-10.15t104=-7.378<0.001Fearmemorymultivariate*-0.329t112=-0.6940.489-0.050t126=-0.1510.880Contextmemorymultivariate*1.821t110=4.125<0.001-0.451t46=-1.4330.159Cuememorymultivariate*-2.479t110=-4.16<0.0010.352t46=0.7030.486SocialfearmemorymultivariatensWorkingmemoryWorkingmemoryYmazealternations*-1.69t54=-2.2480.0290.193t55=0.2030.840NEGATIVESYMPTOMCLASSmultivariate*-2.367t231=-4.861<0.001-0.235t173=-0.890.375PainsensitivityPainsensitivityHPpain*-6.658t54=-3.0470.004AnxietyAnxietymultivariate*0.756t112=2.3030.023*-1.899t172=-3.609<0.001CuriosityCuriositymultivariate*3.33t112=3.4760.001-1.087t113=-1.5960.113MotivationMotivationmultivariate*-5.245t113=-4.597<0.0011.446t114=4.358<0.001POSITIVESYMPTOMCLASSmultivariate*-5.51t114=-7.32<0.001-0.261t114=-0.7780.438HyperactivityAmbulationmultivariate*-7.232t229=-10.63<0.0010.149t231=0.2150.830Speedmultivariate*-4.431t107=-4.397<0.001-0.608t109=-1.440.153
Proteome analysis of 4 weeks oldTcf4tg and wt male mice (line TMEBBL6).
(a) CaMKIIalpha, syn-aptosomes
(b) HOMER1, synapto-somes
(c) VAMP1, cytoplas-matic fraction
(d) VAMP2, syn-aptosomes and cytoplasmatic franction Figure 7: Validation of proteomics candidates by western blotting(whole blots). Proteins from synapto-somes and cytosolic fractions of prefrontal cortices of 4 weeks old TMEBBl6 male mice. For details see Results section 4.1.5 on page 45
Figure 8: Serumβ-endorphin levels in mice housed in IR and EE. No difference was detected between the two groups of animals. The result has to be interpreted with caution due to very low measured concentra-tions and high standard deviaconcentra-tions.
Figure 9: Pain sensitivity in 7.5 weeks old EE, IR and SD mice. To avoid the influence of prolonged isolation after the end of the stress period, we assessed whether differences in pain sensitivity apparent after 3.5 weeks of IR (n=13), SD (n=6) and EE (n=13). Hot plate test revealed no significant differences;
however the median of IR latencies is higher than of EE and SD mice. Presumably, hypoalgesia in IR animals develops over time, starts to emerge already after 3 weeks of IR and would be more pronounced after longer time. The result suggests that hypoalgesia may be specific to IR and not to SD.
The cut-off value in this experiment was 90 s. Animals that failed to jump or lick their back paws within this time were assigned the value of 90 s.A)Bar graphs represent medians with interquartile range. B)Dot plots show the same data for individual animals.
Figure 10: RT-qPCR in PFC and hypothalamus of wt EE, IR and SD mice. Genes associated with pain sensitivity or psychotic disorders were chosen. A–E) PFC, cDNA in dilution 1:25. F–K) PFC, dilution 1:40. L–O)Hypothalamus, dilution 1:100. A)Pomcwas upregulated in IR compared to EE in PFC (p=0.0222, Kruskal-Wallis test). C–O)None of the other tested genes showed significant differences between the groups. Plots show single animals (dots) and group means (relative to Actb). AverageCt ranges are shown in boxes. Genes: Actbβ-actin,Pomcproopiomelanocortin,Pdynprodynorphin,Penk proenkephalin,Cnr1cannabinoid receptor 1,Drd1adopamine receptor 1a,Disc1disrupted in schizophrenia, Comt1catechol-O-methyltransferase,Nrg1-IIIneuregulin 1 type III,Prlvbparvalbumin,Gad1andGad2 glutamate decarboxylase 1 and 2,Oprk1opioid kappa receptor 1. Other genes included in the screen are not shown due to low expression or high standard deviations.
1.1 TCF4 and neurodevelopment . . . 4
1.2 Structure of humanTCF4 . . . 5
2.1 Breeding strategy of theTcf4knockout mouse lines . . . 17
3.1 Environmental paradigms: IR, EE and SD . . . 18
3.2 Fear conditioning paradigm . . . 21
3.3 Morris water maze paradigm . . . 22
3.4 Creating behavioural profiles of mice . . . 25
3.5 Genotyping strategy of theTcf4knockout mouse lines . . . 27
3.6 Genotyping PCR electrophoresis: representative gel pictures . . . 29
4.1 LTP and LTD in hippocampal CA1 ofTcf4tg mice . . . 38
4.2 Spine analysis inTcf4tg mice . . . 39
4.3 Electron microscopy: analysed parameters . . . 40
4.4 Synapse morphology inTcf4tg mice . . . 41
4.5 RT-qPCR with RNAseq candidates inTcf4tg mice . . . 45
4.6 Validation of proteomics candidates by western blotting . . . 50
4.7 Tcf4expression inTcf4C knockout mice . . . 51
4.8 Tcf4C mouse morphometrics . . . 51
4.9 Behavioural profiles of wildtype IR and SD mice . . . 53
4.10 Stress hormones are increased in blood after acute SD . . . 54
4.11 Cognition inTcf4tg mice upon IR and EE . . . 55
4.12 Cognition in agedTcf4tg mice upon IR and EE . . . 55
4.13 Environmental effects in the G×E cohorts . . . 56
4.14 Cognition inTcf4C mice . . . 57
4.15 Behavioural profiles ofTcf4tg andTcf4C mice . . . 59
4.16 IR-induced hypoalgesia: validation of RNAseq candidates by RT-qPCR . . . 66
5.1 Comparison of PTHS mutations with the mutation inTcf4C mice . . . 70
5.2 Bell-shaped relationship betweenTcf4expression and cognition . . . 71
5.3 Isolation rearing-induced changes in DRGs and hypothalamus . . . 76
1 Basic behaviour ofTcf4tg mice upon IR and EE . . . 99
2 Basic behaviour ofTcf4tg mice upon IR and EE . . . 100
3 Basic behaviour of agedTcf4tg mice upon IR and EE . . . 101
4 Basic behaviour of agedTcf4tg mice upon IR and EE . . . 102
5 Basic behaviour ofTcf4C mice . . . 103
6 Basic behaviour ofTcf4C mice . . . 104
7 Validation of proteomics candidates by western blotting (whole blots) . . . 107
8 Serumβ-endorphin levels in mice housed in IR and EE . . . 108 9 Pain sensitivity in 7.5 weeks old EE, IR and SD mice . . . 108 10 RT-qPCR in PFC and hypothalamus of wt EE, IR and SD mice . . . 109
2.1 Chemicals and reagents. . . 13
2.2 Laboratory supplies. . . 14
2.3 Laboratory equipment and software. . . 14
3.1 Directionality and dimension reduction . . . 26
3.2 Genotyping primers . . . 27
3.3 PCR master-mixes . . . 28
3.4 Standard PCR programs . . . 28
3.5 Standard RT-qPCR . . . 31
3.6 Primers used for RT-qPCR . . . 32
4.1 RNAseq: Genes deregulated in PFC ofTcf4tg mice . . . 42
4.2 RNAseq: Genes deregulated in hippocampus ofTcf4tg mice . . . 43
4.3 Proteomics in cytosol . . . 46
4.4 Proteomics in synaptosomes . . . 47
4.5 Genetic main effects inTcf4tg andTcf4C mice in different environments . . . 60
4.6 Genes downregulated in hypothalamus of wt mice upon isolation rearing . . . 62
4.7 Genes regulated in DRGs of wt mice upon isolation rearing . . . 62
4.8 Gene sets upregulated in hypothalamus upon isolation rearing . . . 64
4.9 Gene sets downregulated in hypothalamus upon isolation rearing . . . 64
4.10 Gene sets upregulated in DRGs upon isolation rearing . . . 64
4.11 Gene sets downregulated in DRGs upon isolation rearing . . . 65
1 Cognition inTcf4tg andTcf4C mice: domains, traits and measures. . . 105
2 Environmental effects in G×E cohorts . . . 106