Figure 2. NL1 KO Mice Exhibit Minimal Social Behavior Abnormalities.

(A) NL1 WT and KO mice were allowed to interact sequentially with a novel inanimate target (i.e. an empty cage) and a social target (i.e. a caged adult conspecific mouse) in an open field. An initial 3-way mixed ANOVA found a significant main effect of genotype [3-way Mixed ANOVA; genotype (between-subjects factor): p<0.04; sex (between-subjects factor): p=0.97; target (within-subjects factor): p=0.76; genotype × sex interaction: p=.29; genotype × target interaction: p=0.38; sex × target interaction: p=0.38; genotype × sex × target interaction: p=0.59]. Further planned comparisons found that NL1 WT mice spent less time interacting with the social target than controls (left bars, *p<0.04, planned comparisons). Both NL1 WT and KO mice spent similar amounts of time interacting with the novel, inanimate target (right bars, p=0.13, planned comparisons). N = 23 littermate pairs. Legend in A applies to Panels A-F. (B) Time spent in interactions using the social vs. inanimate preference test. Mice were simultaneously exposed to a novel inanimate target (i.e. an empty cage) and a novel social target (i.e. a novel, caged adult conspecific mouse). The time spent interacting with inanimate and social targets was not different between NL1 KO mice and controls, and neither WT or NL1 KO mice preferred the social target over the inanimate target [n.s. = not significant; 3-way Mixed ANOVA; genotype (between-subjects factor): p=0.22; sex (between-subjects factor): p=0.16; target (within-subjects factor): p=0.08; genotype × sex interaction: p=0.27; genotype × target interaction: p=0.97; genotype × sex × target interaction: p=0.81]. N = 23 littermate pairs. (C) Time spent in interactions using the social novelty preference test. Mice were simultaneously exposed to a novel social target (i.e. a novel, caged adult conspecific mouse) and a familiar social target (i.e. a familiar, caged adult conspecific mouse). A 3-way mixed ANOVA found a main effect of target [3-way Mixed ANOVA; genotype (between-subjects factor): p=0.39; sex (between-subjects factor): p=0.50; target (within-subjects factor): p<0.003; genotype × sex interaction: p=0.31; genotype × target interaction: p=0.40; sex × target interaction: p=0.87; genotype × sex × target interaction: p=0.46]. However, planned comparisons within each genotype found that, unlike WT mice, NL1 KO mice did not exhibit a preference for the novel social target (**p<0.006, n.s. = not significant, p=0.10, planned comparisons). N = 23 littermate pairs (D) Time spent interacting with a freely moving juvenile mouse during a test of social learning [3-way Mixed ANOVA: genotype (between-subjects factor): p=0.39, sex (between-subjects factor): p<0.041; trial (within-subjects factor): p<0.0007; genotype × sex interaction: p=0.56; genotype × trial interaction: p=0.78; sex × trial interaction: p=0.07; genotype × sex × trial interaction: p=0.23]. Mice were initially allowed to interact with a juvenile target mouse for 2 minutes (Day 1), and NL1 KO mice showed normal direct social interaction with the juvenile. Seventy-two hours later (Day 4), mice were again allowed to interact with the same juvenile target mouse, and both WT and NL1 KO mice exhibited significant social learning (recognition memory), as indicated by a decrease in the amount of time spent interacting with the juvenile (*p<0.021 for WT and p<0.008 for KO, planned comparisons within each genotype). N = 23 littermate pairs. (E) The thickness (i.e. the height) of nests built from cotton nesting material was measured over a 90 minute observation period [3-way Mixed ANOVA: genotype (between-subjects factor): p<0.044, sex (between-subjects factor): p=0.35, time (within-subjects factor): p<0.000013, genotype × sex interaction: p=0.69, genotype × time interaction: p=0.15, sex × time interaction: p=0.18, genotype × sex × time interaction: p<0.032]. WT mice built thicker nests than NL1 KO mice (*p<0.021, planned comparison between genotypes at 90 minutes). N = 11 littermate pairs. Data represent means +/− SEM.