CRF binding protein activity in the hypothalamic paraventricular nucleus is essential for stress adaptations and normal maternal behaviour in lactating rats.

To ensure the unrestricted expression of maternal behaviour peripartum, activity of the corticotropin-releasing factor (CRF) system needs to be minimised. CRF binding protein (CRF-BP) might be crucial for this adaptation, as its primary function is to sequester freely available CRF and urocortin1, thereby dampening CRF receptor (CRF-R) signalling. So far, the role of CRF-BP in the maternal brain has barely been studied, and a potential role in curtailing activation of the stress axis is unknown. We studied gene expression for CRF-BP and both CRF-R within the paraventricular nucleus (PVN) of the hypothalamus. In lactating rats, Crh-bp expression in the parvocellular PVN was significantly higher and Crh-r1 expression in the PVN significantly lower compared to virgin rats. Acute CRF-BP inhibition in the PVN with infusion of CRF(6-33) increased basal plasma corticosterone concentrations under unstressed conditions in dams. Furthermore, while acute intra-PVN infusion of CRF increased corticosterone secretion in virgin rats, it was ineffective in vehicle (VEH)-pre-treated lactating rats, probably due to a buffering effect of CRF-BP. Indeed, pre-treatment with CRF(6-33) reinstated a corticosterone response to CRF in lactating rats, highlighting the critical role of CRF-BP in maintaining attenuated stress reactivity in lactation. To our knowledge, this is the first study linking hypothalamic CRF-BP activity to hypothalamic-pituitary-adrenal axis regulation in lactation. In terms of behaviour, acute CRF-BP inhibition in the PVN under non-stress conditions reduced blanket nursing 60 min and licking/grooming 90 min after infusion compared to VEH-treated rats, while increasing maternal aggression towards an intruder. Lastly, chronic intra-PVN inhibition of CRF-BP strongly reduced maternal aggression, with modest effects on maternal motivation and care. Taken together, intact activity of the CRF-BP in the PVN during the postpartum period is essential for the dampened responsiveness of the stress axis, as well as for the full expression of appropriate maternal behaviour.

More than reproduction: Central gonadotropin-releasing hormone antagonism decreases maternal aggression in lactating rats.

Gonadotropin-releasing hormone (GnRH) is a major regulator and activator of the hypothalamic-pituitary-gonadal axis. Many studies have demonstrated the importance of GnRH in reproduction and sexual behaviour. However, to date, only a single study shows an involvement of GnRH in maternal behaviour where a 30% reduction of GnRH neurones abolishes a mother's motivation to retrieve pups. On this basis, we aimed to investigate the effects of acute central GnRH receptor blockade in lactating rats on maternal care under non-stress and stress conditions, maternal motivation in the pup retrieval test, maternal anxiety on the elevated plus maze, and maternal aggression in the maternal defence test. We found that acute central infusion of a GnRH antagonist ([d-Phe2,6 ,Pro3 ]-luteinising hormone-releasing hormone; 0.5 ng 5 µL-1 ) impaired a mother's attack behaviour against a female intruder rat during the maternal defence test compared to vehicle controls. However, in contrast to the previous study on reduced GnRH neurones, acute central GnRH antagonism did not affect pup retrieval, nor any other parameter of maternal behaviour or maternal anxiety. Taken together, GnRH receptor activation is mandatory for protection of the offspring. These findings shed new light on GnRH as a neuropeptide acting not exclusively on the reproductive axis but, additionally, on maternal behaviour including pup retrieval and maternal aggression.

Mom doesn't care: When increased brain CRF system activity leads to maternal neglect in rodents.

Mothers are the primary caregivers in mammals, ensuring their offspring's survival. This strongly depends on the adequate expression of maternal behavior, which is the result of a concerted action of "pro-maternal" versus "anti-maternal" neuromodulators such as the oxytocin and corticotropin-releasing factor (CRF) systems, respectively. When essential peripartum adaptations fail, the CRF system has negative physiological, emotional and behavioral consequences for both mother and offspring often resulting in maternal neglect. Here, we provide an elaborate and unprecedented review on the implications of the CRF system in the maternal brain. Studies in rodents have advanced our understanding of the specific roles of brain regions such as the limbic bed nucleus of the stria terminalis, medial preoptic area and lateral septum even in a CRF receptor subtype-specific manner. Furthermore, we discuss potential interactions of the CRF system with other neurotransmitters like oxytocin and noradrenaline, and present valuable translational aspects of the recent research.

When mothers neglect their offspring: an activated CRF system in the BNST is detrimental for maternal behavior.

Becoming a mother is an intense experience that not only changes a woman's life but is also paralleled by multiple central adaptations. These changes evolve before parturition and continue to persist into lactation, thereby ensuring the full commitment of the mother to care for the newborns. Most of our knowledge on these adaptations that drive the peripartum brain come from rodent animal models. On one side, it is known that maternal behavior is initiated and maternal mood is stabilized by an upregulation of the pro-maternal neuropeptide systems' activity of oxytocin and arginine-vasopressin. On the other side, signaling of the rather anti-maternal corticotropin-releasing factor system triggers maternal neglect and increases maternal anxiety. Here, we discuss how the corticotropin-releasing factor system based in the limbic bed nucleus of the stria terminalis negatively affects maternal behavior and maternal mood. Moreover, we apply microdialysis and acute pharmacological interventions to demonstrate how the corticotropin-releasing factor system potentially interacts with the pro-maternal oxytocin system in the posterior bed nucleus of the stria terminalis to trigger certain aspects of maternal behavior.

Maternal stress and the MPOA: Activation of CRF receptor 1 impairs maternal behavior and triggers local oxytocin release in lactating rats.

Maternal behavior and anxiety are potently modulated by the brain corticotropin-releasing factor (CRF) system postpartum. Downregulation of CRF in limbic brain regions is essential for appropriate maternal behavior and an adaptive anxiety response. Here, we focus our attention on arguably the most important brain region for maternal behavior, the hypothalamic medial preoptic area (MPOA). Within the MPOA, mRNA for CRF receptor subtype 1 (protein: CRFR1, gene: Crhr1) was more abundantly expressed than for subtype 2 (protein: CRFR2, gene: Crhr2), however expression of Crhr1, Crhr2 and CRF-binding protein (protein: CRFBP, gene: Crhbp) mRNA was similar between virgin and lactating rats. Subtype-specific activation of CRFR, predominantly CRFR1, in the MPOA decreased arched back nursing and total nursing under non-stress conditions. Following acute stressor exposure, only CRFR1 inhibition rescued the stress-induced reduction in arched back nursing while CRFR1 activation prolonged the decline in nursing. Furthermore, inhibition of CRFR1 strongly increased maternal aggression in the maternal defense test. CRFR1 activation had anxiogenic actions and reduced locomotion on the elevated plus-maze, however neither CRFR1 nor R2 manipulation affected maternal motivation. In addition, activation of CRFR1, either centrally or locally in the MPOA, increased local oxytocin release. Finally, inhibition of CRFBP (a potent regulator of CRFR activity) in the MPOA did not affect any of the maternal parameters investigated. In conclusion, activity of CRFR in the MPOA, particularly of subtype 1, needs to be dampened during lactation to ensure appropriate maternal behavior. Furthermore, oxytocin release in the MPOA may provide a regulatory mechanism to counteract the negative impact of CRFR activation on maternal behavior.

Brain CRF-binding protein modulates aspects of maternal behavior under stressful conditions and supports a hypo-anxious state in lactating rats.

Reduced corticotropin-releasing factor (CRF) receptor activation in the postpartum period is essential for adequate maternal behavior. One of the factors contributing to this hypo-activity might be the CRF-binding protein (CRF-BP), which likely reduces the availability of free extracellular CRF/urocortin 1. Here, we investigated behavioral effects of acute CRF-BP inhibition using 5µg of CRF(6-33) administered either centrally or locally within different parts of the bed nucleus of the stria terminalis (BNST) in lactating rats. Additionally, we assessed CRF-BP expression in the BNST comparing virgin and lactating rats. Central CRF-BP inhibition increased maternal aggression during maternal defense but did not affect maternal care or anxiety-related behavior. CRF-BP inhibition in the medial-posterior BNST had no effect on maternal care under non-stress conditions but impaired the reinstatement of maternal care following stressor exposure. Furthermore, maternal aggression, particularly threat behavior, and anxiety-related behavior were elevated by CRF-BP inhibition in the medial-posterior BNST. In the anterior-dorsal BNST, CRF-BP inhibition increased only non-maternal behaviors following stress. Finally, CRF-BP expression was higher in the anterior compared to the posterior BNST but was not different between virgin and lactating rats in either region. Our study demonstrates a key role of the CRF-BP, particularly within the BNST, in modulating CRF's impact on maternal behavior. The CRF-BP is important for the reinstatement of maternal care after stress, for modulating threat behavior during an aggressive encounter and for maintaining a hypo-anxious state during lactation. Thus, the CRF-BP likely contributes to the postpartum-associated down-regulation of the CRF system in a brain region-dependent manner.

Antagonism of V1b receptors promotes maternal motivation to retrieve pups in the MPOA and impairs pup-directed behavior during maternal defense in the mpBNST of lactating rats.

Recent studies using V1b receptor (V1bR) knockout mice or central pharmacological manipulations in lactating rats highlighted the influence of this receptor for maternal behavior. However, its role in specific brain sites known to be important for maternal behavior has not been investigated to date. In the present study, we reveal that V1bR mRNA (qPCR) and protein levels (Western blot) within either the medial preoptic area (MPOA) or the medial-posterior part of the bed nucleus of the stria terminalis (mpBNST) did not differ between virgin and lactating rats. Furthermore, we characterized the effects of V1bR blockade via bilateral injections of the receptor subtype-specific antagonist SSR149415 within the MPOA or the mpBNST on maternal behavior (maternal care under non-stress and stress conditions, maternal motivation to retrieve pups in a novel environment, maternal aggression) and anxiety-related behavior in lactating rats. Blocking V1bR within the MPOA increased pup retrieval, whereas within the mpBNST it decreased pup-directed behavior, specifically licking/grooming the pups, during the maternal defense test. In addition, immediately after termination of the maternal defense test, V1bR antagonism in both brain regions reduced nursing, particularly arched back nursing. Anxiety-related behavior was not affected by V1bR antagonism in either brain region. In conclusion our data indicate that V1bR antagonism significantly modulates different aspects of maternal behavior in a brain region-dependent manner.

CRF-R1 activation in the anterior-dorsal BNST induces maternal neglect in lactating rats via an HPA axis-independent central mechanism.

Adequate maternal behavior in rats requires minimal corticotropin-releasing factor receptor (CRF-R) activation in the medial-posterior bed nucleus of the stria terminalis (mpBNST). Based on the architectural heterogeneity of the BNST and its distinct inter-neural connectivity, we tested whether CRF-R manipulation in another functional part, the anterior-dorsal BNST (adBNST), differentially modulates maternal behavior. We demonstrate that in the adBNST, activation of CRF-R1 reduced arched back nursing (ABN) and nursing, whereas activation of CRF-R2 resulted in an initial reduction in nursing but significantly increased the incidence of ABN 5h after the treatment. Following stressor exposure, which is detrimental to maternal care, ABN tended to be protected by CRF-R1 blockade. Maternal motivation, maternal aggression, and anxiety were unaffected by any manipulation. Furthermore, under basal and stress conditions, activation of adBNST CRF-R1 increased plasma ACTH and corticosterone concentrations, whereas stimulation of adBNST CRF-R2 increased basal plasma ACTH and corticosterone concentrations, but blocked the stress-induced increase in plasma corticosterone secretion. Moreover, both the CRF-R1 and -R2 antagonists prevented the stress-induced increase in plasma corticosterone secretion. Importantly, elevated levels of circulating corticosterone induced by intra-adBNST administration of CRF-R1 or -R2 agonist did not impact maternal care. Finally, Crf mRNA expression in the adBNST was increased during lactation; however, Crfr1 mRNA expression was similar between lactating and virgin rats. In conclusion, maternal care is impaired by adBNST CRF-R1 activation, and this appears to be the result of a central action, rather than an effect of elevated circulating levels of CORT. These data provide new insights into potential causes of disturbed maternal behavior postpartum.

Salivary oxytocin concentrations in response to running, sexual self-stimulation, breastfeeding and the TSST: The Regensburg Oxytocin Challenge (ROC) study.

Intranasal oxytocin (OXT) application is emerging as a potential treatment for socio-emotional disorders associated with abnormalities in OXT system (re-) activity. The crucial identification of patients with such abnormalities could be streamlined by the assessment of basal and stimulus-induced OXT concentrations in saliva, using a simple, stress-free sampling procedure (i.e. an OXT challenge test). We therefore established the Regensburg Oxytocin Challenge (ROC) test to further validate salivary OXT concentrations as a practical, reliable and sensitive biomarker. OXT concentrations were quantified by radioimmunoassay in samples collected at home by healthy adult male and female volunteers before and after running ("Run") or sexual self-stimulation ("Sex"). In lactating women, salivary OXT concentrations were quantified before, during and after breastfeeding. Salivary OXT along with salivary cortisol and heart rate were monitored in healthy adult participants undergoing the Trier Social Stress Test (TSST). The home-based "Run" and "Sex" challenges as well as the laboratory-based TSST caused quantifiable, rapid, and consistent increases in salivary OXT (approximately 2.5-fold after 10-15min), which were similar for men and women. Breastfeeding did not result in measurably increased salivary OXT levels, probably because the short pulses of OXT release characteristic for lactation were missed. Taken together, ROC tests reliably assess the responsiveness of the OXT system (i.e., the increase in salivary OXT concentrations as compared to basal levels) to challenges such as "Run" and "Sex" at home or psychosocial stress (TSST) in the laboratory. Further studies with larger sample numbers are essentially needed in order to reveal individual differences in ROC test outcomes depending on, for example, genetic or environmental factors.

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

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.

The interplay of conditional NCAM-knockout and chronic unpredictable stress leads to increased aggression in mice.

The neural cell adhesion molecule (NCAM) is a key regulator of brain plasticity. Substantial evidence indicates that NCAM is down-regulated by exposure to sustained stress and chronic stress seems to lead to increased aggression. In addition, constitutional NCAM deletion in mice has been shown to lead to increased intermale aggression and altered emotionality Forebrain-specific postnatal NCAM knockout was previously shown to impair cognitive function, particularly when animals were exposed to subchronic stress, but the effects on emotional and social behavior remain unclear. In this study, we investigated the potential interplay of a forebrain-specific postnatal NCAM deletion and exposure to different lengths of repeated stress (i.e. subchronic: 14 days; chronic: 29 days) on aggressive and emotional behavior. Our results show that postnatal deletion of NCAM in the forebrain leads to increased aggression and altered emotionality depending on the duration of stress, whereas conditional NCAM knockout has no basal impact on these behaviors. These findings support the involvement of NCAM in the regulation of emotional and aggressive behaviors, suggesting that diminished NCAM expression might be a critical vulnerability factor for the development of these behavioral alterations under repeated exposure to stress.

Reduced brain corticotropin-releasing factor receptor activation is required for adequate maternal care and maternal aggression in lactating rats.

The brain corticotropin-releasing factor (CRF) system triggers a variety of neuroendocrine and behavioural responses to stress. Whether maternal behaviour and emotionality in lactation are modulated by CRF has rarely been investigated. In the present study, we measured CRF mRNA expression within the parvocellular part of the paraventricular nucleus in virgin and lactating Wistar rats bred for high (HAB) and low (LAB) anxiety-related behaviour or non-selected for anxiety (NAB). Further, we intracerebroventricularly infused synthetic CRF or the CRF receptor (CRF-R) antagonist D-Phe to manipulate CRF-R1/2 non-specifically in lactating HAB, LAB, and NAB dams, and monitored maternal care, maternal motivation, maternal aggression, and anxiety. The CRF mRNA expression in the parvocellular part of the paraventricular nucleus was higher in HAB vs. LAB rats independent of reproductive status. The lactation-specific decrease of CRF mRNA was confirmed in LAB and NAB dams but was absent in HAB dams. Intracerebroventricular CRF decreased maternal care under basal conditions in the home cage in all breeding lines and reduced attack behaviour in HAB and LAB dams during maternal defence. In contrast, D-Phe rescued maternal care after exposure to maternal defence in the home cage without influencing maternal aggression. Furthermore, D-Phe decreased and CRF tended to increase anxiety in HAB/NAB and LAB dams, respectively, suggesting an anxiogenic effect of CRF in lactating females. In conclusion, low CRF-R activation during lactation is an essential prerequisite for the adequate occurrence of maternal behaviour.