The social bonding defect is caused by the phenotype of Shank2-/- mice and cannot be attributed to general cognitive and olfactory deficits (A) Shank2-/- dams failed to nurture their pups.Figure 2(A) Shank2-/- dams failed to nurture their pups. Pups nurtured by Shank2+/+ mice display milk in their stomach (arrowheads, upper left panel), while milk was absent in the stomach of Shank2-/- pups (arrowhead, lower left panel). Pups of Shank2+/+ mice gradually gained weight (black circles) whereas no weight gain was observed in pups delivered by Shank2-/- mothers (blue circles), two-way mixed ANOVA, effect of genotype: ***P<0.001, effect of day: P=0.962, day x genotype interaction: ***P<0.001, Shank2+/+ n = 12, Shank2-/- n = 9. (B, C) Pups of Shank2-/- mice (genotype Shank2+/-) were cross-fostered by a Shank2+/+ female, while pups of the wild-type mouse (genotype Shank2+/+) were given to a Shank2-/- mothers. In contrast to pups (+/-) given to WT mothers (black circles), +/+ pups gradually lost weight if cross-fostered by Shank2-/- mothers (blue circles). Two-way mixed ANOVA effect of genotype: *P=0.041, effect of day: P=0.134, day x genotype interaction: ***P<0.001, Shank2+/+ n = 5, Shank2-/- n = 6. (D) Olfactory habituation/dishabituation ability was evaluated in female and male Shank2+/+ and Shank2-/- by the cumulative time spent sniffing a sequential series of non-social odors (water, almond, banana) and social odors (unfamiliar pup urine; unfamiliar male or female urine) delivered on cotton swabs. (E) Shank2-/- female mice showed a clear preference (dishabituation: banana # 3 vs. pup odor # 1) for pup odor in comparison to Shank2+/+ mice, two-way mixed ANOVA, effect of trial: ***P<0.001, effect of genotype: P=0.923, trial x genotype interaction: P=0.892. Additionally, Shank2-/- female mice displayed normal habituation response (pup odor #1-3) toward pup odor, two-way mixed ANOVA, effect of trial: **P<0.006, effect of genotype: P=0.742, trial x genotype interaction: P=0.907. Shank2+/+ n = 8, Shank2-/- n = 10. (F) Shank2-/- male mice showed a clear preference (dishabituation: banana # 3 vs pup odor # 1) for pup odor in comparison to Shank2+/+ mice, two-way mixed ANOVA, effect of trial: ***P<0.001, effect of genotype: P=0.787, trial x genotype interaction: P=0.769. Additionally, Shank2-/- male mice displayed normal habituation response (pup odor #1-3) toward pup odor, two-way mixed ANOVA, effect of trial: ***P<0.001, effect of genotype: P=0.553, trial x genotype interaction: P=0.813, Shank2+/+ n = 10, Shank2-/- n = 10. (G) Schematic illustration of the novel object recognition test. After a 30 min habituation phase, Shank2+/+ and Shank2-/- mice were allowed to investigate two identical objects in the open field arena (training session). 10 min later, one of the objects was replaced with a novel object (test session). Lower panels show a representative tracking path for a mouse in each test session. (H) Shank2+/+ and Shank2-/- mice displayed a significant preference for the novel object versus the familiar one in the test session, two-way mixed ANOVA, effect of object: ***P<0.001, effect of genotype: P=0.167, object x genotype interaction: P=0.667, Shank2+/+ n = 9, Shank2-/- n = 9. Data information: All graphs are presented as mean ± SEM, NS: not significant. (I) Additionally, no significant difference between Shank2+/+ and Shank2-/- was evident in the spontaneous alternation behavior during a Y Maze task, unpaired, two-tailed Student's t-test, P=0.146. Shank2+/+ n = 10, Shank2-/- n = 10. Data information: All graphs are presented as mean ± SEM, NS: not significant.