Fig. 5
From: Enlarged dendritic spines and pronounced neophobia in mice lacking the PSD protein RICH2
Repetitive behavior, fear of novel objects, and impaired motor learning in RICH2−/− mice. a–d Novel object recognition task for short and long term memory. a) Percent time of mice spent exploring during different stages of the novel object recognition task for short-term memory. Mice were exposed to two identical objects during the training session (AA). 10 min later during the test session, the animals were exposed to a familiar object (A) and a novel object (B). All genotypes showed a significant preference for the novel object after 10 min (two-way mixed ANOVA, genotype by target interaction F2.21 = 1.252, p = 0.306; target main effect F1.21 = 37.531, p < 0.001; Post-tests of target effect in all three genotypes: RICH2+/+ p = 0.006 (n = 9); RICH2+/− p = 0.007 (n = 11); RICH2−/− p = 0.034 (n = 4)). b Percent time of mice spent exploring during different stages of the novel object recognition task for long term memory. Long term memory was assessed 24 h after the training session (10 min exposure to the familiar object (A)). Wild type animals as well as RICH2+/− showed a significant preference for the novel object (B) after 24 h (two-way mixed ANOVA, genotype by target interaction F1.15 = 0.412, p = 0.531; target main effect F1.15 = 28.305, p < 0.001; Post-tests of target effect in genotypes: RICH2+/+ p = 0.016 (n = 7); RICH2+/− p = 0.003 (n = 11)). Analysis of RICH2−/−could not be performed since mice which explored objects less than 10 s were excluded from the analysis. Only two RICH2−/− met the criteria for inclusion and thus no conclusion for long term memory can be drawn based on the remaining two RICH2−/− animals for statistical analysis. c) Percent time of mice spent exploring during different stages of the novel location task for spatial working memory. Mice were exposed to two identical objects, during the training session. 10 min later, one of the familiar object was moved to a novel location. A significant preference for the “moved” object during the test session for each genotype was measured (two-way mixed ANOVA, genotype by target interaction F2.17 = 0.575, p = 0.236; target main effect F1.17 = 90.745, p < 0.001; Post-tests of target effect in genotypes: RICH2+/+ p = 0.001 (n = 7); RICH2+/− p = 0.001 (n = 8); RICH2−/− p = 0.028 (n = 5)). d Tracking path of mice before and after the presentation of novel objects during the novel object recognition task as well as novel location task. RICH2−/−mice actively avoid spending time in the proximity of the novel object and show reduced locomotor response towards novel objects. e The open field arena was separated in a non object and object zone during the novel object recognition test. There was a significant difference between genotypes regarding preference for a zone after introduction of objects (two-way mixed ANOVA, genotype and test session interaction F4.56 = 2.557, p = 0.049; main effect of the genotype F2.28 = 10.613, p < 0.001; main effect of the test session F2.56 = 13.607, p = 0.001). No side preference was present in the habituation phase of the novel object recognition test (open field arena without objects) (two-way mixed ANOVA, genotype and zone interaction F2.28 = 2.544, p = 0.097; main effect of the zone F1.28 = 1.044, p = 0.316: main effect of the genotype F2.28 = 0.718, p = 0.497). RICH2−/− mice showed a significant preference for the non object zone in comparison to the object zone in both ‘identical objects test’ (two-way mixed ANOVA, genotype and zone interaction F2.28 = 8.608, p = 0.001; main effect of the zone F1.28 = 16.567, p = 0.372: main effect of the genotype F2.28 = 0.016, p = 0.984) (RICH2−/− p = 0.01) and ‘novel object test’ (two-way mixed ANOVA, genotype and zone interaction F2.28 = 8.744, p = 0.001; main effect of the zone F1.28 = 10.795, p = 0.003: main effect of the genotype F2.28 = 0.131, p = 0.878) (RICH2−/− p = 0.011). f A significant difference among genotypes was detected regarding freezing behavior after introduction of novel objects (two-way mixed ANOVA, genotype by test session interaction F4.56 = 3.280, p = 0.04; main effect of test session F2.56 = 0.478, p = 0.623, main effect of the genotype F2.28 = 5.452, p = 0.01). No significant difference in freezing behavior was found during the habituation phase of the novel object recognition test. However, significant genotype differences were found during identical object presentation (Post-test RICH2+/+ vs RICH2−/− p = 0.019; RICH2+/− vs RICH2−/− p = 0.011). Additionally, during novel object presentation significant differences among genotypes (Post-test RICH2+/+ vs RICH2−/− p = 0.012; RICH2+/− vs RICH2−/− p = 0.017) were detected. g Further, a significant genotype difference was found measuring track length (two-way mixed ANOVA, genotype by test session interaction F4.56 = 1.739, p = 0.194; main effect of test session F2.56 = 8.612, p = 0.003, main effect of the genotype F2.28 = 7.399, p = 0.003). No significant difference in track length was detected during the habituation phase of the novel object recognition test. However, significant genotype differences were found during identical object presentation (Post-test RICH2+/+ vs RICH2−/− p = 0.002, RICH2+/− vs RICH2−/− p = 0.023). Additionally, during novel object presentation, a significant difference among genotypes (Post-test RICH2+/+ vs RICH2−/− p = 0.002, RICH2+/− vs RICH2−/− p = 0.01) was detected. h Number of rearings in novel environment. No significant difference of RICH2−/− mice compared to wild type animals was detected in general exploratory behavior (F2.26 = 0.083, p = 0.920; one way ANOVA). i For the analysis of ASD-like behavior, repetitive self-grooming was measured over a period of 10 min. RICH2−/− mice display a significant increase in time spent self grooming in comparison to wild type mice. A One Way Analysis revealed a significant difference among genotypes (F2.28 = 4.791, p = 0.016). Bonferroni post hoc analysis revealed a significant difference between RICH2+/+ and RICH2−/− mice (t = 4.47, p = 0.043). j Significant genotype differences were found during rotarod test (two-way mixed ANOVA, genotype by trial interaction F14.196 = 2.202, p = 0.019; main effect of trial F7.14 = 16.845, p < 0.001, main effect of the genotype F2.28 = 3.025, p = 0.065). Post hoc analysis indicated a significant difference between RICH2+/+ and RICH2 −/− (p = 0.02) mice in the latency to fall of the rotarod. e–j Analysis and statistics were performed with n = 10 RICH2+/+, n = 12 RICH2+/−, n = 9 RICH2−/− mice. Data are shown as mean, + SEM