Shank2-/- mice exhibit impaired pup retrieval and social attachment behavior but unaltered hormone levels (A) Experimental setup of the pup retrieval paradigm.Figure 3(A) Experimental setup of the pup retrieval paradigm. After 1-hour pup deprivation, pups were placed in three corners of the home cage that did not contain the nest. The mother retrieved the pups and crouches over them, engaging in maternal care responses (pup grooming, crouching, and nest building). (B) Shank2-/- mice showed significantly less pup retrieval both on postnatal day 1 and 2, two-way mixed ANOVA, effect of genotype: ***P<0.001, effect of day: P=0.258, day x genotype interaction: P=0.258, Shank2+/+ n = 12, Shank2-/- n = 9. Data information: All data are presented as mean ± SEM, NS: not significant. (C) Tracking path of a Shank2+/+ mother and a Shank2-/- mother during the pup retrieval assay. Left panel: Shank2+/+ mothers immediately retrieved the pups and started crouching over them in the nest location. Right panel: Shank2-/- mothers rarely retrieved the provided pups and failed to crouch over them in the nest location. In addition, Shank2-/- mothers showed no or impaired nest building. (D-G) Behavioral analysis of Shank2+/+ and Shank2-/- dams demonstrated a significant reduction in all major components of maternal behavior: (D) pup grooming, unpaired, two-tailed Student's t-test, ***P<0.001 (E) crouching, Mann-Whitney test, ***P<0.001 (F) nest building, Mann-Whitney test, ***P<0.001 and (G) Maternal interaction, unpaired, two-tailed Student's t-test, ***P<0.001. Shank2+/+ n = 12, Shank2-/- n = 9. Data information: All data are presented as mean ± SEM, NS: not significant. (H) Left panel: Example of a tracking trajectory of a Shank2-/- dams during the pup retrieval test. Shank2-/- dams investigated the provided pups (upper panel), which was further evident in the nose-tracking path of a Shank2-/- dam (lower panel). No significant difference was detected in the latency to approach the provided pups between Shank2+/+ and Shank2-/- mothers, Mann-Whitney test, P=0.943. Shank2+/+ n = 12, Shank2-/- n = 9. Data information: All data are presented as mean ± SEM, NS: not significant. (I) Levels of Oxytocin plasma and tissue concentration in Shank2+/+ and Shank2-/- mice. Left panel: No significant difference was detected between Oxytocin plasma concentration comparing Shank2+/+ with Shank2-/- mice, unpaired, two-tailed Student's t-test, P=0.276, Shank2+/+ n = 5, Shank2-/- n = 5. Right panel: Additionally, Shank2-/- mice displayed no significant differences in hypothalamic or pituitary Oxytocin concentrations, unpaired, two-tailed Student's t-test, P=0.536, Shank2+/+ n = 6, Shank2-/- n = 5; two-tailed Student's t-test, P=0.585, Shank2+/+ n = 5, Shank2-/- n = 5. Data information: All data are presented as mean ± SEM, NS: not significant. (J) Left panel: Electron micrographs of the posterior pituitary gland from Shank2+/+ and Shank2-/- mice in lower (left panel) and higher magnification. Scale bar (500 nm). Right diagram: No significant difference was detected in the number of vesicles in the posterior pituitary gland between Shank2+/+ and Shank2-/- mice, Mann-Whitney test, P=0.507, Shank2+/+ n = 3, Shank2-/- n = 3. (K-M) Additionally, Shank2-/- mice display no significant difference in: (K) Estradiol-, Mann-Whitney test, P=0.475, Shank2+/+ n = 6, Shank2-/- n = 7, (L) Progesterone, unpaired, two-tailed Student's t-test, P=0.570, Shank2+/+ n = 5, Shank2-/- n = 5 or (M) Prolactin- plasma concentrations, unpaired, two-tailed Student's t-test, P=0.980; Shank2+/+ n = 3, Shank2-/- n = 3. Data information: All data are presented as mean ± SEM, NS: not significant. E, endothelium; CL, capillary lumen; M, mitochondria; NG, neurosecretory granule; AE, axonal endings, HYP, Hypothalamus; PIT, Pituitary gland.