Repeated pup presentation does not induce maternal behavior in Shank2-/- mice that display a circuit specific disruption of social attachment behavior.Figure 5(A) Maternal behavior can be induced in female mice, which have never been exposed to pups (naive females) by repeated social exposure to pups (trial 1-5). Virgin female mice start to retrieve pups and engage in maternal care responses (pup grooming, crouching, nest building), thereby becoming attached to the nest location as indicated by the tracking path. (B) No significant difference was detected for latency to approach the provided pups between Shank2+/+ and Shank2-/- naive females, unpaired, two-tailed Student's t-test, P=0.677. Shank2+/+ n = 9, Shank2-/- n = 9. Data information: All data are presented as mean ± SEM, NS: not significant. (C) Shank2-/- naive females fail to become maternal after repeated pup exposure, as demonstrated by the significant reduction of pup retrieval during trial 1-4 and the test session on day 5 compared to Shank2+/+ induced females, two-way mixed ANOVA, effect of genotype: ***P<0.001, effect of trial: P=0.282, trial x genotype interaction: P=0.168, Shank2+/+ n = 9, Shank2-/- n = 9. Data information: All data are presented as mean ± SEM, NS: not significant. (D) Upper Panel: Tracking path of Shank2+/+ and Shank2-/- mice during social exposure to pups in the 20 min test session (trial 5). Shank2+/+ female mice become attracted to the nest location engaging in maternal care responses, Shank2-/- mice, however, display no preference for the pups, or participate in nest building (lower panel). (E-H) Repeated pup exposure does not induce major components of social attachment behavior in Shank2-/- females: (E) pup grooming, unpaired, two-tailed Student's t-test, ***P<0.001 (F) crouching, Mann-Whitney test, ***P<0.001 and (G) nest building, Mann-Whitney test, ***P<0.001, (H) maternal interaction, unpaired, two-tailed Student's t-test, ***P<0.001 (trial 5), Shank2+/+ n = 9, Shank2-/- n = 9. Data information: All data are presented as mean ± SEM, NS: not significant. (I, Examination of the neuronal activation pattern after pup presentation in Shank2+/+ and Shank2-/- mice using c-Fos immunocytochemistry. Schematic map of the neuronal pathway regulating social attachment behavior in mice. J) Activation of the circuit starts via olfactory input (OB-AON-Amygdala) or tactile input from the pups to the MPOA of the hypothalamus and proceeds via the VTA-NA-VP axis to the output region of the circuit inducing attraction toward infants. After non-exposure to pups and social exposure to pups (6 h) coronal sections (40 μm) of Shank2+/+ and Shank2-/- mice were prepared and the number of c-Fos positive cells in this neuronal pathway analyzed. Shank2-/- mice displayed major impairment in the neuronal activation pattern of the circuit regulating social attachment behavior in comparison to Shank2+/+ mice. AON, two-way ANOVA, effect of genotype: P=0.118, effect of pup exposure: **P=0.003, pup exposure x genotype interaction: P=0.561, AMY, two-way ANOVA, effect of genotype: P=0.227, effect of pup exposure: ***P<0.001, genotype x pup exposure interaction: P=0.073, LS, two-way ANOVA, effect of genotype: ***P<0.001, effect of pup exposure: ***P<0.001, genotype x pup exposure interaction: ***P<0.001, BNST, two-way ANOVA, effect of genotype: P=0.201, effect of pup exposure: ***P<0.001, genotype x pup exposure interaction: P=0.205, MPOA, two-way ANOVA, effect of genotype: *P=0.013, effect of pup exposure: **P=0.003, genotype x pup exposure interaction: *P=0.01. VTA, two-way ANOVA, effect of genotype: **P=0.008, effect of pup exposure: **P=0.002, genotype x pup exposure interaction: **P=0.007. NAcc, two-way ANOVA, effect of genotype: **P=0.003, effect of pup exposure: **P=0.002, pup exposure x genotype interaction: **P=0.014. Shank2+/+ and Shank2-/- without pup contact n = 3, Shank2+/+ and Shank2-/- with pup contact n = 5. Data information: All data are presented as mean ± SEM, NS: not significant. (K) Representative coronal brain section (left panel) and example of c-Fos immunoreactivity in the MPOA (right panel) of Shank2+/+ and Shank2-/- pup exposed females (scale bar: 200 µm). The dotted line outlines the third ventricle (3V). Arrowheads indicate c-Fos positive cells. (L) Levels of synaptic proteins in the crude synaptosomes fraction (P2) from the hypothalamus of Shank2+/+ and Shank2-/- mice. Shank2+/+ n = 5 (black bars), Shank2-/- n = 5 (blue bars). Unpaired, two-tailed Student's t-test.