Fig. 5. Differential regulation of social behavior by the canonical and non-canonical NLGN3 signaling.
a–d NRXN1β- and PTPδA3B–-induced postsynaptic differentiation (a and b) and NLGN3 accumulation (c and d) of cortical neurons from Nlgn3hse (a and c) and Nlgn3mf (b and d) mice. Shank2, gephyrin, and NLGN3 immunostaining signals were quantified on the right (n = 54 and 66 NRXN1β-beads and 51 and 77 PTPδA3B–-beads for wild-type and Nlgn3hse neurons, respectively for (a). n = 51 and 43 NRXN1β-beads and 57 and 60 PTPδA3B–-beads for wild-type and Nlgn3mf neurons, respectively for (b). n = 35 and 33 NRXN1β-beads, 32 and 36 PTPδA3B–-beads, and 30 and 34 Fc-beads for wild-type and Nlgn3hse neurons, respectively for (c). n = 28 and 23 NRXN1β-beads, 29 and 34 PTPδA3B–-beads, and 22 and 26 Fc-beads for wild-type and Nlgn3mf neurons, respectively for (d). Scale bars, 5 μm. e Coimmunoprecipitation of NLGN3-PTPδ complex from striatal synaptosomal fraction of Nlgn3hse and Nlgn3mf mutant mice. Representative images (left) and co-IP efficiency of PTPδ normalized to NLGN3 for immunoprecipitated samples (right) are shown (n = 4 experiments each for mutants and controls). f–i Three-chamber sociability test of Nlgn3hse mutant mice (f and g) (N = 19 and 18 for mutants and controls, respectively) and Nlgn3mf mutant mice (h and i) (N = 19 and 17 for mutants and controls, respectively). Time spent in each chamber (f and h) and preference index (((time spent around stranger cage/(Time spent around stranger cage + Time spent around empty cage)) × 100) – 50) (g and i) are presented. j–n Reciprocal social interaction test of Nlgn3hse mice (j and k) (N = 13 and 14 pairs for mutant vs. mutant and control vs. control, respectively) and Nlgn3mf mice (l and m) (N = 14 and 13 pairs for mutant vs. mutant and control vs. control, respectively). Total duration of proximity events (j and l) and numbers of elevated head-head contact (k and m) during 30 min test are presented. n Elevated head-head contact observed in Nlgn3mf mice. o and p Rotation speed to fall off in each trial of Nlgn3hse mice (N = 17 and 16 for mutants and controls, respectively) (o) and Nlgn3mf mice (N = 37 and 35 for mutants and controls, respectively) (p) in accelerating rotarod. Data in (a–d) are presented as box plots. The horizontal line in each box shows the median, the box shows the IQR and the whiskers are 1.5 × IQR. Data in (e–m, o, and p) represent mean ± s.e.m. *p < 0.05, **p < 0.01 and ***p < 0.001, Two-sided Student’s t-test in (a, b, e, f, j, l, m and p), Mann–Whitney U-test in (g), Tukey’s test in (c and d), and two-sided Bonferroni post-test in (f and h) (comparisons with dotted line). See Supplementary Table 4 for additional statistics and exact p values.