Impaired experience-dependent maternal care in presynaptic active zone protein CAST-deficient dams

Abstract

Although sociological studies affirm the importance of parental care in the survival of offspring, maltreatment-including child neglect-remains prevalent in many countries. While child neglect is well known to affect child development, the causes of maternal neglect are poorly understood. Here, we found that female mice with a deletion mutation of CAST (a presynaptic release-machinery protein) showed significantly reduced weaning rate when primiparous and a recovered rate when multiparous. Indeed, when nurturing, primiparous and nulliparous CAST knock out (KO) mice exhibited less crouching time than control mice and moved greater distances. Contrary to expectations, plasma oxytocin (OXT) was not significantly reduced in CAST KO mice even though terminals of magnocellular neurons in the posterior pituitary expressed CAST. We further found that compared with control mice, CAST KO mice drank significantly less water when nurturing and had a greater preference for sucrose during pregnancy. We suggest that deficiency in presynaptic release-machinery protein impairs the facilitation of some maternal behaviours, which can be compensated for by experience and learning.

Figure 1

Reproductive performance was impaired in primiparous CAST KO mothers. (A) The weaning rate was significantly lower in CAST KO dams than in CAST heterozygous (HT) or other genotype-deficient dams at the first reproductive experience (primiparity); **p < 0.01 by one-way ANOVA, Tukey’s test. (B) HE stained sections of the mammary gland. (C) Nest-building scores were significantly lower in CAST KO mice at primiparity than in WT mice (WT: 3.20; KO: 1.89; *p < 0.05 by t-test). 5-point nest scoring: 0, untouched nest material; 1, moved nest material; 2, flat nest; 3, <half cupped dome; 4, half-height dome; 5, > half-height or full dome. (bottom) Typical nests of score 3 made by WT mice and score 1 by KO mice. (D) Nurturing analysis. Monitoring of WT and KO dams while they took care of newborn pups (P0–3 days). After the 20 min initial observation, pups were deprived of house cage for 1 h. Subsequently, they were placed in each corner of the home cage, except for the nest corner (dotted circle), and then WT or KO dams were returned to the nest and monitored for 20 min (nurturing analysis). (E–G) From the video record, the number of pups retrieved (G), latency to retrieve each pup (F), and crouching time for all three pups (E,G) were measured. If the number of retrieved pups was less than three, crouching time was 0. The KO mice missed retrieving one of three pups (red arrow in D). Crouching time at the initial observation and during nurturing was significantly lower in primiparous KO mice (n = 20) than in WTs (n = 13) (H–J) In multiparous KO mice, initial crouching time was similar to that seen in WTs (H). While the WT mice retrieved all three pups within 1 min, the KO mice took longer, but the number or pups and the time spent crouching did not differ significantly (I,J). WT (3), KO (9); mean ± SEM, *p < 0.05, **p < 0.01 (Student’s t-test).

Figure 2

Increased mobility in nurturing CAST KO dams. (A,B) Nurturing dams in the home cage were tracked using video capture. The WT mice stayed mostly in the nest for breastfeeding and to keep the pups warm. In contrast, the KO mice moved around the cage. The distance travelled was significantly higher in the KO mice, and correlated with the reduction in crouching time. WT (5), KO (7); mean ± SEM, *p < 0.05 (Student’s t-test). (C,D) To measure exploratory activity, open field activity was measured with male and virgin female WT and KO mice. The distance travelled by KO mice did not differ significantly from WT males or females. Furthermore, WT and KO mice exhibited a similar trend for traveling mostly in the peripheral zone (outer) compare with the centre zone (inner). Male WT (6), KO (12), Female WT (3), KO (4); mean ± SEM. (E–H) WT and KO mice performed an olfactory habituation/discrimination test via a cotton-tip presentation-based task. Six-to-ten-week old mice were pretrained with mineral oil-soaked cotton swabs, then exposed to two different odorants (100 µM octanol and benzaldehyde), with 3 trials per odorant. Sniffing duration indicated that both WT and KO mice were able to detect and discriminate between the distinct odorants (E,F). On a separate day, the mice performed a similar task using male and female urine after undergoing pretraining on distilled water. Habituation and discrimination of urine samples was comparable between KO and WT mice (G,H). Female WT (7), KO (6); mean ± SEM.

Figure 3

Maternal care in virgin females. (A–D) Virgin adult female WT and KO mice were exposed to pups for two consecutive days. The first contact time (A), number of retrieved pups (B), latency to retrieve pups (C), and crouching time over three pups (D) were measured. WT (8), KO (9), mean ± SEM, *p < 0.05 (Student’s t-test).

Figure 4

Contribution of OXT release from the pituitary gland. (A) Immunohistochemistry showed that CAST was distributed in the posterior pituitary, suggesting the CAST in magnocellular neurons of the hypothalamus might regulate the release of OXT. In KO mice, the expression of family protein ELKS seemed complementarily enhanced in the posterior region. (B) Ratio of fluorescent intensity of anti-ELKS antibody between posterior and anterior pituitary indicated the enhancement of ELKS expression in the posterior region of KO mice. WT (4), KO (5), mean ± SEM, ***p < 0.005 (Student’s t-test). (C) Immunohistochemistry of hormones including OXT, growth hormone, prolactin, and thyroid-stimulating hormone (TH) indicated their distribution in the pituitary gland. Unlike other hormones that were released from the anterior pituitary, OXT was only distributed in the posterior region. (D) Concentration of plasma OXT in male WT and KO mice. OXT release to plasma did not differ significantly between WT and KO mice. WT (3), KO (5), mean ± SEM.

Figure 5

CAST KO mice exhibited less water intake during nurturing and higher sucrose preference during primiparous pregnancy. (A–C) The sucrose preference test was performed with WT and KO females before and after their first pregnancies. Total water intake increased during nurturing in both WT and KO mice, however the amount was significantly lower in KO mice (A). While both WT and KO mice preferred to drink the sucrose water (B), and sucrose preference was significantly higher in KO mice during their primiparous pregnancy (C). (D–F) The same test was performed on multiparous females. Although sucrose preference was higher in the KO mice during pregnancy, the difference was not significant (F). WT (10), KO (9), mean ± SEM, *p < 0.05 (Student’s t-test).