Supplementary figures1_ Negr1 Deficiency Modulates Sex-Specific Neurobehavioral Adaptations to Social Isolation

1 Molecular and Behavioural effects of social isolation on Negr1-deficient mice. Arpana Reinsberg1, Katyayani Singh1, Mohan Jayaram1, Kaie Mikheim1, Mari-Anne Philips1, Eero Vasar1. qPCR results tables qPCR Tartgets Sex Prefrontal cortex Two-Way ANOVA Inhibitory neuronal markers Group housed Social isolation Factors Group Wt Mean ±SEM (transformed data) Group Negr1-/- Mean ±SEM (transformed data) SI Wt Mean ±SEM (transformed data) SI Negr1-/- Mean ±SEM (transformed data) X: Interaction #: Treatment $: Genotype F (transformed data) P (transformed data) Gad1 Males -6.135 ± 0.1100 -6.931± 0.08753 -6.314 ± 0.1371 -6571 ± 0.09341 Main effect: -$$ Interaction Treatment Genotype Post-hoc: - 1.082 0.2180 10.29 0.3066 0.6439 0.0032 Females -5.545 ± 0.08811 -5564 ± 0.4744 -5.584 ± 0.03948 -5.615 ± 0.06440 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.009287 0.4979 0.1562 0.9238 0.4852 0.6951 Gad2 Males -4.035 ± 0.07067 -4.101 ± 0.1117 -4.008 ± 0.09015 -3.893 ± 0.09879 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.9173 1.560 0.06734 0.3454 0.2207 0.7969 Females -3.582 ± 0.04560 -3.474 ± 0.08224 -3.560 ± 0.05225 -3.387 ± 0.03626 Main effect: -$ Interaction Treatment Genotype Post-hoc: - 0.3230 0.09224 6.122 0.05733 0.3433 0.0182 Pvalb Males -0.9851 ± 0.08887 -1.006 ± 0.06772 0.9479 ± 0.03572 -1.053 ± 0.06306 Main effect: - Interaction Treatment 0.3984 0.005404 0.5324 0.9419 2 Genotype Post-hoc: - 0.8918 0.3521 Females -1.313 ± 0.07495 -1.374 ± 0.04948 -1.327 ± 0.05317 -1.454 ± 0.04771 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.3412 0.6699 2.693 0.5628 0.4185 0.1095 Gad2 _SI_Unpaired t-test_Welch’s test Females -3.560 ± 0.05225 -3.387 ± 0.03656 Treatment 0.0152 3 qPCR Tartgets Sex Hippocampus Two-Way ANOVA Inhibitory neuronal markers Group housed Social isolation Factors Group Wt Mean ±SEM (transformed data) Group Negr1-/- Mean ±SEM (transformed data) SI Wt Mean ±SEM (transformed data) SI Negr1-/- Mean ±SEM (transformed data) X: Interaction #: Treatment $: Genotype F (transformed data) P (transformed data) Gad1 Males -4.509 ± 0.1851 -4.828 ± 0.1417 -4.474 ± 0.08811 -4.002 ± 0.1429 Main effect: -XX## Interaction Treatment Genotype Post-hoc: **Ctl Negr1-/-vs SI Negr1-/- 7.540 8.920 0.2825 0.0095 0.0051 0.5984 Females -4.799 ± 0.1587 -4.839 ± 0.1422 -4.631 ± 0.1899 -4.722 ± 0.09087 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.6139 0.05374 0.05374 0.4386 0.8180 0.8180 Gad2 Males -3.518 ± 0.08565 -3.686 ± 0.1017 -3.565 ± 0.06295 -3.634 ± 0.06796 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.3655 0.001450 2.012 0.5493 0.9698 0.1560 Females -3.589 ± 0.05239 -3.580 ± 0.05646 -3.492 ± 0.04706 -3.542 ± 0.04489 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.3537 1.763 0.1669 0.5559 0.1928 0.6854 Pvalb Males -2.575 ± 0.07649 -2.809 ± 0.05187 -2.641 ± 0.06595 -3.002 ± 0.08894 Main effect: -$$$ Interaction Treatment Genotype Post-hoc: **SI Wt vs SI Negr1-/- 0.7538 3.096 16.39 0.3915 0.0878 0.0003 Females -2.606 ± 0.04762 -2.706 ± 0.05406 -2.563 ± 0.09268 -2.809 ± 0.08078 Main effect: $ Interaction Treatment Genotype Post-hoc: - 1.053 0.1799 5.878 0.3117 0.6740 0.0205 4 qPCR Tartgets Sex Prefrontal cortex Two-Way ANOVA Neurotrophic-related Genes Group housed Social isolation Factors Group Wt Mean ±SEM (transformed data) Group Negr1-/- Mean ±SEM (transformed data) SI Wt Mean ±SEM (transformed data) SI Negr1-/- Mean ±SEM (transformed data) X: Interaction #: Treatment $: Genotype F (transformed data) P (transformed data) Fgfr2 Males -8.735 ± 0.1374 -8.793 ± 0.1880 -8.661 ± 0.2081 -8.570 ± 0.1743 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.1644 0.6567 0.007995 0.6878 0.4235 0.9293 Females -7.727 ± 0.1807 -8.018 ± 0.1119 -7.504 ± 0.1359 -7.904 ± 0.07371 Main effect: -$ Interaction Treatment Genotype Post-hoc: - 0.1707 1.618 6.748 0.6824 0.2128 0.0142 Bdnf Males -4.180 ± 0.09484 -4.266 ± 0.07222 -4.156 ± 0.05230 -4.-65 ± 0.02280 Main effect: - Interaction Treatment Genotype Post-hoc: - 1.942 3.093 0.001690 0.1737 0.0888 0.9675 Females -4.503 ± 0.08394 -4.449 ± 0.08081 -4.324 ± 0.08372 -4.552 ± 0.1219 Main effect: - Interaction Treatment Genotype Post-hoc: - 2.253 0.1601 0.8590 0.4121 0.6914 0.3601 Ntrk2(TrkB) Males -6.149 ± 0.1134 -6198 ± 0.08375 -6.004 ± 0.1182 -6.207 ± 0.04299 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.5500 0.4321 1.495 0.4637 0.5157 0.2304 Females -5.514 ± 0.1133 -5.749 ± 0.08784 -5.504 ± 0.08871 -5793 ± 0.08427 Main effect: -$$ Interaction Treatment Genotype 0.07990 0.03186 7.599 0.7791 0.8594 0.0092 5 Post-hoc: - Tcf4 Males -5.674 ± 0.1850 -5.815 ± 0.1477 -5.574 ± 0.1635 -5.658 ± 0.1946 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.02732 0.5524 0.4237 0.8697 0.4623 0.5193 Females -4.712 ± 0.2134 -5.348 ± 0.07288 -5.008 ± 0.2712 -5.430 ± 0.1239 Main effect: -$ Interaction Treatment Genotype Post-hoc: - 0.3026 0.9426 7.379 0.5859 0.3385 0.0103 6 qPCR Tartgets Sex Hippocampus Two-Way ANOVA Neurotrophic-related Genes Group housed Social isolation Factors Group Wt Mean ±SEM (transformed data) Group Negr1-/- Mean ±SEM (transformed data) SI Wt Mean ±SEM (transformed data) SI Negr1-/- Mean ±SEM (transformed data) X: Interaction #: Treatment $: Genotype F (transformed data) P (transformed data) Fgfr2 Males -6.153 ± 0.1410 -6.381 ± 0.09836 -6.109 ± 0.08745 -5.884 ± 0.06689 Main effect: -X# Interaction Treatment Genotype Post-hoc: *Ctl Negr1-/-vs SI Negr1-/- 4.759 6.756 0.0001530 0.0360 0.0136 0.9902 Females -6.321 ± 0.1505 -6.512 ± 0.2033 -6.218 ± 0.1296 -6.306 ± 0.11156 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.1193 1.047 0.8531 0.7318 0.3132 0.3620 Bdnf Males -3.537 ± 0.09984 -3.681 ± 0.05723 -4.047 ± 0.08875 -4.042 ± 0.1114 Main effect: #### Interaction Treatment Genotype Post-hoc: **Ctl Wt vs SI Wt 0.6795 23.15 0.5947 0.4154 <0.0001 0.4458 Females -3.999 ± 0.07033 -4.082 ± 0.04816 -4.063 ± 0.04676 -4.135 ± 0.07956 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.008121 0.8225 1.423 0.9287 0.3708 0.2412 Ntrk2(TrkB) Males -4.368 ± 0.2485 -4.885 ± 0.1305 -4.302 ± 0.09471 -3.775 ± 0.1247 Main effect: -XX## Interaction Treatment Genotype Post-hoc: ***Ctl Negr1-/-vs SI Negr1-/- 10.09 12.81 0.0008022 0.0032 0.0011 0.9776 Females -4.730 ± 0.2250 -4.741 ± 0.2075 -4.531 ± 0.2345 -4.583 ± 0.1211 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.01044 0.7796 0.02479 0.9192 0.3831 0.8758 7 Tcf4 Males -2.418 ± 0.2028 -2.768 ± 0.2733 -2.714 ± 0.1015 -2.094 ± 0.1248 Main effect: X Interaction Treatment Genotype Post-hoc: - 6.415 0.9797 0.4979 0.0160 0.3291 0.4851 Females -2.935 ± 0.2549 -2.925 ± 0.2394 -2.722 ±0.2521 -2.719 ± 0.09106 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.0003054 0.9043 0.0008342 0.9862 0.3480 0.9771 Tcf4_Negr1-/-_Unpaired _Welch’s test Males 0.1671 ± 0.02314 0.2410 ± 0.01907 Genotype 0.0250 8 qPCR Tartgets Sex Prefrontal cortex Two-Way ANOVA Synaptic adhesion Group housed Social isolation Factors Group Wt Mean ±SEM (transformed data) Group Negr1-/- Mean ±SEM (transformed data) SI Wt Mean ±SEM (transformed data) SI Negr1-/- Mean ±SEM (transformed data) X: Interaction #: Treatment $: Genotype F (transformed data) P (transformed data) Cntn1 Males -3.448 ± 0.09964 -3.679 ± 0.09330 -3.546 ± 0.08475 -3.639 ± 0.1167 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.4959 0.08564 2.700 0.4860 0.7715 0.1093 Females -2.982 ± 0.06758 -3.117 ± 0.06181 -3.074 ± 0.08346 -3.181 ± 0.1001 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.0311 0.9659 2.309 0.8609 0.3325 0.1373 Ntm1a Males -4.121 ± 0.1063 -4.326 ± 0.1007 -4.178 ± 0.1233 -4.335 ± 0.1671 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.03795 0.06947 2.096 0.08467 0.7937 0.1566 Females -3.546 ± 0.1123 -3.769 ± 0.09255 -3.556 ± 0.1069 -3.848 ± 0.1135 Main effect: $ Interaction Treatment Genotype Post-hoc: - 0.1085 0.1767 5.826 0.7438 0.6767 0.0210 Ntm1b Males -6.803 ± 0.09637 -6.756 ± 0.06743 -6.783 ± 0.1102 -6.484 ± 0.1896 Main effect: - Interaction Treatment Genotype Post-hoc: - 1.113 1.483 2.081 0.2994 0.2323 0.1588 Females -5.927 ± 0.08949 -6.108 ± 0.07492 -6.045 ± 0.1170 -6.165 ± 0.09386 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.1929 0.6034 2.659 0.66632 0.4425 0.1119 Lsamp1a Males -6.071 ± 0.07266 -6.130 ± 0.1241 -6.303 ± 0.1702 -6.287 ± 0.1334 Main effect: - 9 Interaction Treatment Genotype Post-hoc: - 0.080 2.154 0.025 0.7789 0.1514 0.8750 Females -5.384 ± 0.1504 -5.346 ± 0.07430 -5.293 ± 0.04744 -5.296 ± 0.09474 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.042 0.5374 0.033 0.8371 0.4684 0.8560 Lsamp1b Males -5.809 ± 0.02372 -6.089 ± 0.09130 -6.103 ± 0.1440 -6.003 ± 0.1399 Main effect: - Interaction Treatment Genotype Post-hoc: - 2.646 0.797 0.5956 0.1136 0.3787 0.4459 Females -5.272 ± 0.08106 -5.338 ± 0.06186 -5.329 ± 0.04886 -5.490 ± 0.1427 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.061 0.347 0.505 0.8060 0.5591 0.4817 Negr1 Males -5797 ± 0.2579 -6.131 ± 0.2517 Genotype 0.3664 Females -5.300 ± 0.1658 -5.307 ± 0.1657 Genotype 0.9790 16 Bdnf_Negr1-/- _Unpaired_Welch’s test -3.871 ± 0.05976 -4.091 ± 0.06628 Genotype 0.0187 Ntrk2(TrkB)_Negr1-/- _Unpaired_Welch’s test -4.809 ± 0.1233 -4200 ± 0.1272 Genotype 0.0015 qPCR Tartgets Both sexes combined Prefrontal cortex Two-Way ANOVA Synaptic adhesion Group housed Social isolation Factors Group Wt Mean ±SEM (transformed data) Group Negr1-/- Mean ±SEM (transformed data) SI Wt Mean ±SEM (transformed data) SI Negr1-/- Mean ±SEM (transformed data) X: Interaction #: Treatment $: Genotype F (transformed data) P (transformed data) Cntn1 -3.165 ± 0.06545 -3.328 ± 0.08302 -3.292 ± 0.06215 -3.398 ± 0.09169 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.1392 1.639 3.078 0.7102 0.2045 0.0836 Ntm1a -3834 ± 0.1001 -4.047 ± 0.09233 -3.867 ± 0.1068 -4.079 ± 0.1120 Main effect: $ Interaction Treatment Genotype Post-hoc: - 9.272e-005 0.09962 4.275 0.9923 0.7532 0.0421 Ntm1b -6.270 ± 0.1239 -6.340 ± 0.1016 -6.311 ± 0.1181 -6.352 ± 0.09842 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.01725 0.05597 0.2520 0.8959 0.8137 0.6174 Lsamp1a -5.802 ± 0.1331 -5.738 ± 0.1142 -5.798 ± 0.1443 -5.765 ± 0.1404 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.01372 0.007523 0.1337 0.9071 0.9311 0.7157 Lsamp1b -5.600 ± 0.08593 -5.694 ± 0.1028 -5.716 ± 0.1155 -5.686 ± 0.1061 Main effect: - Interaction Treatment Genotype 0.3626 0.2766 0.09582 0.5489 0.6005 0.7578 17 Post-hoc: - Negr1_Unpaired_Welch’ s test -5.536 ± 0.1570 -5.719 ± 0.1745 Genotype 0.4396 qPCR Tartgets Both sexes combined Hippocampus Two-Way ANOVA Synaptic adhesion Group housed Social isolation Factors Group Wt Mean ±SEM (transformed data) Group Negr1-/- Mean ±SEM (transformed data) SI Wt Mean ±SEM (transformed data) SI Negr1-/- Mean ±SEM (transformed data) X: Interaction #: Treatment $: Genotype F (transformed data) P (transformed data) Cntn1 -2.343 ± 0.06799 -2.438 ± 0.06486 -2.374 ± 0.06194 -2.276 ± 0.05240 Main effect: - Interaction Treatment Genotype Post-hoc: - 2.368 1.103 0.0006099 0.1282 0.2970 0.9804 Ntm1a -4.162 ± 0.08780 -4.198 ± 0.08156 -4.140 ± 0.07082 -4.020 ± 0.06680 Main effect: - Interaction Treatment Genotype Post-hoc: - 0.9984 1.668 0.2974 0.3209 0.2006 0.5871 Ntm1b -5.701 ± 0.1056 -5.826 ± 0.08951 -5.682 ± 0.09063 -5.516 ± 0.06606 Main effect: - Interaction Treatment Genotype Post-hoc: - 2.635 3.386 0.05335 0.1087 0.0697 0.8180 Lsamp1a -4.105 ± 0.09293 -4.243 ± 0.07704 -4.064 ± 0.06735 -3.869 ± 0.06862 Main effect: X## Interaction Treatment Genotype Post-hoc: **Ctl Negr1-/- vs SI Negr1-/- 4.692 7.285 0.1371 0.0336 0.0086 0.7122 Lsamp1b -4.526 ± 0.08712 -4.672 ± 0.07602 -4.539 ± 0.06738 -4.411 ± 0.06323 Main effect: - Interaction Treatment Genotype 3.407 2.764 0.01518 0.0689 0.1006 0.9023 18 Post-hoc: - Negr1_Unpaired_Welch’s test -4.521 ± 0.1314 -4.509 ± 0.1243 Genotype 0.9492