Corticotropin-releasing factor infusion in the bed nucleus of the stria terminalis of lactating mice alters maternal care and induces behavioural phenotypes in offspring

Abstract

The peripartum period is accompanied by numerous physiological and behavioural adaptations organised by the maternal brain. These changes are essential for adequate expression of maternal behaviour, thereby ensuring proper development of the offspring. The corticotropin-releasing factor (CRF) plays a key role in a variety of behaviours accompanying stress, anxiety, and depression. There is also evidence that CRF contributes to maladaptations during the peripartum period. We investigated the effects of CRF in the bed nucleus of the stria terminalis (BNST) of lactating mice during maternal care and analysed locomotor activity and anxiety-like behaviour in the offspring. The BNST has been implicated in anxiety behaviour and regulation of the stress response. The effects of intra-BNST CRF administration were compared with those induced by the limited bedding (LB) procedure, a model that produces altered maternal behaviour. BALB/cJ dams were exposed to five infusions of CRF or saline into the BNST in the first weeks after birth while the LB dams were exposed to limited nesting material from postnatal days (P) 2-9. Maternal behaviour was recorded in intercalated days, from P1-9. Offspring anxiety-like behaviour was assessed during adulthood using the open-field, elevated plus-maze, and light/dark tests. Both intra-BNST CRF and LB exposure produced altered maternal care, represented by decreased arched-back nursing and increased frequency of exits from the nest. These changes in maternal care resulted in robust sex-based differences in the offspring's behavioural responses during adulthood. Females raised by CRF-infused dams exhibited increased anxiety-like behaviour, whereas males presented a significant decrease in anxiety. On the other hand, both males and females raised by dams exposed to LB showed higher locomotor activity. Our study demonstrates that maternal care is impaired by intra-BNST CRF administrations, and these maladaptations are similar to exposure to adverse early environments. These procedures, however, produce distinct phenotypes in mice during young adulthood and suggest sex-based differences in the susceptibility to poor maternal care.

Figure 1

Experimental design (created with BioRender.com).

Figure 2

Cannula placement. (a) Representative image after staining (haematoxylin and eosin). (b) Placement of the cannula in a coronal section of the mouse brain; circles for saline-infused (SAL) dams and diamonds for corticotropin-releasing factor-infused (CRF) dams.

Figure 3

Maternal behaviour: Arched-back nursing. (a) Frequency of ABN in SAL and CRF dams per block. (b) Frequency of ABN in CT and LB dams per block. Data are presented as means SEM of all days from each block. **p < 0.01, repeated-measures ANOVA, treatment effect. n = 5–7 dams per group. SAL, saline-infused dams; CRF, corticotropin-releasing factor-infused dams; CT, control dams; LB, stressed dams; ABN, arched-back nursing.

Figure 4

Maternal behaviour: Exits from the nest. (a) Frequency of exits from the nest in SAL and CRF dams per block. (b) Frequency of exits from the nest in CT and LB dams per block. Data are presented as means SEM of all days from each block. **p < 0.01 repeated-measures ANOVA, treatment effect. n = 5–7 dams per group. SAL, saline-infused dams; CRF, corticotropin-releasing factor-infused dams; CT, control dams; LB, stressed dams.

Figure 5

Body weight. (a) Body weight (in grams) of SAL and CRF offspring on P9. (b) Body weight (in grams) of SAL and CRF offspring on P21. (c) Body weight (in grams) of SAL and CRF offspring on P58. (d) Body weight (in grams) of CT and LB offspring on P9. (e) Body weight (in grams) of CT and LB offspring on P21. (f) Body weight (in grams) of CT and LB offspring on P58. Results are presented as means SEM. *p < 0.05, t-test; ***p < 0.001, two-way ANOVA, sex effect. n = 5–7 families per group (P9 and P21); n = 10–12 animals per group (P58). SAL, offspring of saline-infused dams; CRF, offspring of corticotropin-releasing factor-infused dams; CT, offspring of control dams; LB, offspring of stressed dams.

Figure 6

Parameters of the open field test. (a) Locomotor activity (in meters) of SAL and CRF offspring. (b) Time spent (in seconds) in the centre zone by SAL and CRF offspring. (c) Number of rearing behaviours by SAL and CRF offspring. (d) Locomotor activity (in meters) of CT and LB offspring. (e) Time spent (in seconds) in the centre zone by CT and LB offspring. (f) Number of rearing behaviours by CT and LB offspring. Results are presented as means SEM. * p < 0.05, two-way ANOVA, treatment-effect. n = 10–12 animals per group. SAL, offspring of saline-infused dams; CRF, offspring of corticotropin-releasing factor-infused dams; CT, offspring of control dams; LB, offspring of stressed dams.

Figure 7

Parameters of the elevated plus-maze. (a) Time spent (in seconds) in the open arms by SAL and CRF offspring. (b) Number of entries into the open arms by SAL and CRF offspring. (c) Number of stretching behaviour by SAL and CRF offspring. (d) Time spent (in seconds) in the open arms by CT and LB offspring. (e) Number of entries into the open arms by CT and LB offspring. (f) Number of stretching behaviour by CT and LB offspring. Results are presented as means SEM. Two-way ANOVA, interaction-effect, where CRF males increased their time while CRF females decreased. *p < 0.05, 2-way ANOVA, treatment-effect. n = 10–12 animals per group. SAL, offspring of saline-infused dams; CRF, offspring of corticotropin-releasing factor-infused dams; CT, offspring of control dams; LB, offspring of stressed dams.

Figure 8

Parameters of the light/dark test. (a) Latency (in seconds) to enter the dark zone of SAL and CRF offspring. (b) Number of head pokes by SAL and CRF offspring. (c) Latency (in seconds) to enter the dark zone of CT and LB offspring. (d) Number of head pokes by CT and LB offspring. Results are presented as means SEM. *p < 0.05, **p < 0.01, two-way ANOVA, treatment-effect. n = 10–12 animals per group. SAL, offspring of saline-infused dams; CRF, offspring of corticotropin-releasing factor-infused dams; CT, offspring of control dams; LB, offspring of stressed dams.