Hypoactivation of CRF receptors, predominantly type 2, in the medial-posterior BNST is vital for adequate maternal behavior in lactating rats

Abstract

Maternal behavior ensures the proper development of the offspring. In lactating mammals, maternal behavior is impaired by stress, the physiological consequence of central corticotropin-releasing factor receptor (CRF-R) activation. However, which CRF-R subtype in which specific brain area(s) mediates this effect is unknown. Here we confirmed that an intracerebroventricularly injected nonselective CRF-R antagonist enhances, whereas an agonist impairs, maternal care. The agonist also prolonged the stress-induced decrease in nursing, reduced maternal aggression and increased anxiety-related behavior. Focusing on the bed nucleus of the stria terminalis (BNST), CRF-R1 and CRF-R2 mRNA expression did not differ in virgin versus lactating rats. However, CRF-R2 mRNA was more abundant in the posterior than in the medial BNST. Pharmacological manipulations within the medial-posterior BNST showed that both CRF-R1 and CRF-R2 agonists reduced arched back nursing (ABN) rapidly and after a delay, respectively. After stress, both antagonists prevented the stress-induced decrease in nursing, with the CRF-R2 antagonist actually increasing ABN. During the maternal defense test, maternal aggression was abolished by the CRF-R2, but not the CRF-R1, agonist. Anxiety-related behavior was increased by the CRF-R1 agonist and reduced by both antagonists. Both antagonists were also effective in virgin females but not in males, revealing a sexual dimorphism in the regulation of anxiety within the medial-posterior BNST. In conclusion, the detrimental effects of increased CRF-R activation on maternal behavior are mediated via CRF-R2 and, to a lesser extent, via CRF-R1 in the medial-posterior BNST in lactating rats. Moreover, both CRF-R1 and CRF-R2 regulate anxiety in females independently of their reproductive status.

Figure 1.

Effect of nonspecific intracerebroventricular CRF-R1/2 manipulation on maternal care under nonstress conditions on LD1 (A) and stress conditions on LD5 (B), on maternal aggression during the maternal defense test on LD5 (C), and on anxiety-related behavior of lactating rats on LD3 (D). ABN (A, B, top) and sum of nursing (A, B, bottom) were scored for 60 min before and for 90 min after infusion (A) or 60 min after maternal defense (B). Maternal aggression was scored as number of attacks (C, top) and attack latency (C, bottom) by the resident. Anxiety-related behavior was measured as the percentage of time spent on the open arms (D, top) and the percentage of entries into the open arms (D, bottom) on the EPM. Dams received an acute intracerebroventricular infusion of VEH (5 μl of sterile Ringer's solution, pH 7.4), CRF-R1/2 agonist Ucn 1 (CRF-R1/2 ago; 1 μg/5 μl), or CRF-R1/2 antagonist D-Phe (CRF-R1/2 ant; 10 μg/5 μl). Data are presented as mean + SEM. n = 6–7 per group. **p ≤ 0.01, *p ≤ 0.05, (*) p = 0.06 versus VEH; ++p ≤ 0.01, (+)p = 0.07 versus previous time point (two-way ANOVA for repeated measures; factors: time × treatment).

Figure 2.

CRF-R1 (top) and CRF-R2 (bottom) mRNA expression in the medial and posterior part of the BNST comparing virgin and lactating rats. Data are presented as mean grain area + SEM. n = 4–7 per group. ** p ≤ 0.01 versus medial part (two-way ANOVA; factors: reproductive status × brain site). Representative photomicrographs from a lactating rat are shown on the right side (4× objective) Scale bar, 500 μm. Hybridization is evident as localized clumps of silver grains. ac, Anterior commissure.

Figure 3.

Histological localization of infusion cannula within the mpBNST. A, Cannula placement sites for subsequent drug infusion are shown as black dots within the mpBNST on schematic plates from the Paxinos and Watson (1998) stereotaxic atlas. B, Extent of substance spreading after ink infusion (black dot) in three rats is shown schematically as gray circles within the mpBNST. ac, Anterior commissure; f, fornix; ic, internal capsule; lv, lateral ventricle; sm, stria medullaris of the thalamus.

Figure 4.

Effect of intra-mpBNST CRF-R1 or CRF-R2 specific agonist (ago) or antagonist (ant) treatment on maternal care of lactating dams under nonstress conditions on LD1 (A) and stress conditions on LD5 (B). ABN (top) and sum of nursing (bottom) were scored for 60 min before and for 90 min after infusion (A) or 60 min after maternal defense (B). Under nonstress conditions, ABN and nursing were also observed for additional 60 min in the afternoon (A). Dams received an acute bilateral infusion of VEH (5 μl of sterile Ringer's solution; pH 7.4), CRF-R1 agonist human/rat CRF (CRF-R1 ago; 1 μg/0.5 μl), CRF-R1 antagonist CP-154,526 (CRF-R1 ant; 12 μg/0.5 μl), CRF-R2 agonist stresscopin (CRF-R2 ago; 3 μg/0.5 μl), or CRF-R2 antagonist Astressin-2B (CRF-R2 ant; 4 μg/0.5 μl) into the mpBNST. Data are presented as mean + SEM. n = 8–14 per group. **p ≤ 0.01, *p ≤ 0.05 versus VEH (two-way ANOVA for repeated measures; factors: time × treatment); +p ≤ 0.05, (+) p = 0.08 versus t −100 min (one-way ANOVA for repeated measures; factor: time).

Figure 5.

Effect of intra-mpBNST CRF-R1 or CRF-R2 specific agonist (ago) or antagonist (ant) treatment on maternal aggression of lactating rats measured in the maternal defense test. Maternal aggression against a virgin female intruder was scored during the 10 min trial. Number of attacks (left) and attack latency (right) by the resident is shown. For details on treatments, see legend to Figure 4. Data are presented as mean + SEM. n = 7–11 per group. **p ≤ 0.01, *p ≤ 0.05 versus VEH (one-way ANOVA; factor: treatment).

Figure 6.

Effect of intra-mpBNST CRF-R1 or CRF-R2 specific agonist (ago) or antagonist (ant) treatment on anxiety-related behavior of lactating and virgin female (A) and male rats (B) on the EPM or the LDB (males only). Virgin females were treated with antagonists only. The percentage of time spent on the open arms or the percentage of time spent in the light box during the 5 min tests is shown. For details on treatments, see legend to Figure 4. Data are presented as mean + SEM. n = 7–12 per group. **p < 0.01, *p ≤ 0.05 versus VEH (one-way ANOVA; factor: treatment).