Additional file 4: of Enlarged dendritic spines and pronounced neophobia in mice lacking the PSD protein RICH2

Figure S4. Behavioral analysis of RICH2−/− mice II. RICH2+/− and RICH2−/− mice were analyzed and compared to wild type littermates. Using several paradigms, ASD-like behavior (a-g), and learning and memory (h-m) were assessed. a) Nesting behavior: Histogram of average nest score taken 24 h after a nestlet was introduced in the home cage. No significant difference among RICH2+/+, RICH2+/− and RICH2−/− mice was present in nest quality scores (Kruskal-Wallis analysis, chi-square: 1.133, df = 2, p = 0.567). b-g) Sociability and preference for social novelty scores in the automated three chambered social approach task. b) Normal sociability was found in all genotypes (RICH2+/+, RICH2+/− and RICH2−/−). All genotypes spend significantly more time sniffing the wire cage containing a stranger mouse vs. the empty wire cage (two-way mixed ANOVA, genotype by stimulus interaction F2.28 = 1.063, p = 0.359; stimulus main effect F1.28 = 130.248, p < 0.001; main effect of the genotype F2.28 = 1.151, p = 0.331; Post-tests of stimulus effect in all three genotypes: RICH2+/+ p = 0.001; RICH2+/− p = 0.001; RICH2−/− p = 0.002). c) Similarly, all genotypes demonstrated a significant preference for spending time in the side containing the stranger mouse, versus time in the chamber with the empty wire cage (two-way mixed ANOVA, genotype by stimulus interaction F2.28 = 0.349, p = 0.708; stimulus main effect F1.28 = 19.186, p < 0.001; main effect of the genotype F2.28 = 0.459, p = 0.636; Post-tests of stimulus effect in all three genotypes: RICH2+/+ p = 0.05; RICH2+/− p = 0.027; RICH2−/− p = 0.016). d) In the second part of the test however, while in the social novelty task, all genotypes still show a significant preference for stranger 2, indicated as time spent sniffing the wire cage (two-way mixed ANOVA, genotype by stimulus interaction F2.28 = 0.557, p = 0.579; stimulus main effect F1.28 = 28.307, p < 0.001; main effect of the genotype F2.28 = 0.835, p = 0.444; Post-tests of stimulus effect in all three genotypes: RICH2+/+ p = 0.029; RICH2+/− p = 0.01; RICH2−/− p = 0.018). e) There was no significant preference for the time spent in the chamber containing the stranger 2 in RICH2−/− mice (two-way mixed ANOVA, genotype by stimulus interaction F2.28 = 0.163, p = 0.850; stimulus main effect F1.28 = 21.557, p < 0.001; main effect of the genotype F2.28 = 3.010, p = 0.065). f, g) No significant differences were detected in the number of transitions between genotypes during the sociability (f) sociability (two-way mixed ANOVA, genotype by stimulus interaction F2.28 = 1.715, p = 0.198; stimulus main effect F1.28 = 0.072, p = 0.790; main effect of the genotype F2.28 = 0.847, p = 0.439) and social novelty task (g) (two-way mixed ANOVA, genotype by stimulus interaction F2.28 = 1.185, p = 0.320; stimulus main effect F1.28 = 5.864, p = 0.022; main effect of the genotype F2.28 = 3.395, p = 0.048; Post-tests of stimulus effect in all three genotypes: RICH2+/+ p = 0.470; RICH2+/− p = 0.432; RICH2−/− p = 0.081; main effect of genotype RICH2+/− vs RICH2−/− p = 0.044). h, i) Spontaneous alternation behavior in the Y-maze labyrinth, a hippocampus dependent task of spatial working memory. h) No significant difference between genotypes in the percentage of alternation during the 5 min test session in the Y maze labyrinth (Kruskal-Wallis ANOVA; chi-square: 2.717, df = 2, p = 0.257). i) Additionally, no significant difference in the number of entries between genotype (F2.28 = 2.053, p = 0.147; one way ANOVA) was detected. j-m) Learning and memory testing using the Morris Water Maze. j, k) Training trials for the visible as well as invisible platform. For each day of training, data were averaged across the four daily trials and in all two - way mixed ANOVAs, “day” was treated as the repeated measurement. j) During the acquisition training of the visible platform test, all three genotypes showed similar learning curves over 3 days (two-way mixed ANOVA, genotype by trial interaction F4.56 = 1.946, p = 0.147; main effect of the trial F2.56 = 111.035, p < 0.001; main effect of the genotype F2.28 = 0.591, p = 0.560; Post-tests of the trial effect: RICH2+/+ p < 0.001; RICH2+/− p < 0.001; RICH2−/− p < 0.001). k) During the acquisition training of the invisible platform test, all three genotypes showed similar learning curves over 6 days (two-way mixed ANOVA, genotype by trial interaction F10.140 = 0.437, p = 0.901; main effect of the trial F5.140 = 11.357, p < 0.001; main effect of the genotype F2.28 = 0.919, p = 0.411; Post-tests of the trial effect: RICH2+/+ p < 0.001; RICH2+/− p = 0.002; RICH2−/− p = 0.005). l) Track length was similar across genotypes (two-way mixed ANOVA, genotype by trial interaction F10.140 = 0.292, p = 0.982; main effect of the trial F5.140 = 9.179, p < 0.001; main effect of the genotype F2.28 = 0.648, p = 0.531; Post-tests of the trial effect: RICH2+/+ p = 0.03; RICH2+/− p = 0.009; RICH2−/− p = 0.007). m) In the probe trial, all genotypes spent more time in the training quadrant (T: Target, AL: Adjacent left, O: Opposite), however not significant (RICH2+/+: (F3.60 = 0.391, p = 0.76); RICH2+/−: (F3.30 = 0.808, p = 0.500); RICH2−/−: (F3.57 = 0.513, p = 0.675), within group repeated measures ANOVA). a-m) All analyses and statistics were performed with n = 10 RICH2+/+, n = 12 RICH2+/−, n = 9 RICH2−/− mice. Data are shown as mean, ± SEM. (TIF 1485 kb)