In mammals, the postpartum period is a time of great behavioral and biological dynamism. Most of these changes are understood to occur to increase offspring survival and ensure developmental success, while the role of other postpartum neurobiological modifications remains unknown. During pregnancy and the early postpartum period, adaptations related to stress include a complex sequence of changes to alter hypothalamic-pituitary-adrenal (HPA) axis activity. In general, following the perception of a stressor, a signaling cascade is initiated by the hypothalamic release of corticotropin-releasing factor (CRF) to signal the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary which then signals the release of glucocorticoids from the adrenal cortex. Corticotropin-releasing factor receptor 1 (CRFR1) is a receptor involved with initiating the physiological stress response and is important for both stress- and maternal-related behaviors (Gammie et al., 2007; Smith et al., 1998). Previous work in our lab has demonstrated postpartum alterations to the expression of CRFR1 in stress/maternal behavior-regulating brain regions including the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus. In the PVN, there is a decrease in CRFR1 that reaches the lowest expression at postpartum day (PD) 7 (De Guzman et al., 2023).

This decrease in CRFR1 coincides with the decrease in overall CRF signaling and increased CRF binding protein—which sequesters free CRF—and both of these adaptations have been experimentally linked in rats to enable typical maternal care (Sanson et al., 2024). Intra-PVN inhibition of CRF binding protein or infusion of CRF, which preferentially binds to CRFR1 (Gammie et al., 2007) both lead to interruptions to maternal care behaviors like hovering, licking and grooming, and pup retrieval. It may be necessary for CRFR1 to be downregulated in this brain area to allow for the display of typical maternal behaviors. In the SON, we have observed increases in CRFR1 expression during the mid-late postpartum period, such that no CRFR1 is present before pregnancy, and expression of this receptor is detectable starting around PD14 (De Guzman et al., 2023).

Relatedly, oxytocin (OT) is a nonapeptide secreted by cells of the PVN and SON and plays a crucial role in maternal care. Alongside prolactin, OT enables nursing as the primary trigger for milk release during lactation and is commonly implicated in maternal stress buffering to facilitate continued pup care despite stressful conditions (Ng et al., 2023). Recent work has uncovered a phenomenon unique to the postpartum period in mice in which OT neurons in the hypothalamus begin to express CRFR1 for the first time following parturition (Ugartemendia et al., 2022; De Guzman et al., 2023). OT neurons and CRFR1-expressing neurons are typically distinct cell populations outside of the postpartum period (De Guzman et al., 2023; Ramot et al., 2017; Ugartemendia et al., 2022). The functional significance of this phenomenon is unclear, but we have characterized a pattern of increase across the postpartum period in both the number of co-localized CRFR1/OT neurons and the percent of OT neurons expressing CRFR1 such that peak co-expression occurs at approximately PD21, coinciding with the time of typical rodent weaning (Curley et al., 2009; De Guzman et al., 2023). Thus, it seems that there may be an increase in these co-expressing neurons as a factor of greater maternal experience.

Given this apparent relationship between maternal experience and co-localization of CRFR1 in OT neurons, it is possible that the appearance and subsequent increase of CRFR1/OT neurons may relate to the appearance of one or more maternal behaviors. In rodents, maternal behaviors are a suite of programmed social behaviors demonstrated following parturition and include licking and grooming of pups, nest building, hovering over the nest, pup retrieval, aggression towards intruders, and nursing (Champagne et al., 2001). Studies in rodents (primarily rats) demonstrate that many of these behaviors improve with greater maternal experience, as multiparous dams—those who have had multiple births—display improved maternal care and greater resilience to challenges (Aguggia et al., 2019; Nephew et al., 2009).

Previous literature has demonstrated the general improvements in maternal behaviors and even stress susceptibility in rodent dams that have had multiple pregnancies as compared with primiparous dams who have only given birth once (Künkele and Kenagy, 1997). Multiparous rats have been shown to be more resilient to the stress of pup separation as multiparous dams undergoing chronic daily pup separation did not display more anxiety-like behavior and were less negatively impacted in a spatial memory task, unlike their primiparous counterparts (Aguggia et al., 2019). Aggression towards intruder males–a phenomenon primarily observed in postpartum rodents to defend their pups from potential infanticide–may also be increased in multiparous rats (Nephew et al., 2009). Additionally, multiparity may confer a benefit of faster pup retrieval and increased hypothalamic oxytocin compared to first-time mothers in mice (Lopatina et al., 2011). We aimed to investigate whether the expression of CRFR1/OT co-localization is altered in postpartum dams that have had multiple successive pregnancies and births (i.e., multiparous mice) to determine whether maternal behavioral changes in multiparous rodents might be transiently associated with differences in expression of CRFR1/OT neurons.

In addition to multiparity's influence on CRFR1/OT neuron expression, there may also be changes in other brain areas relevant to maternal adaptations. Our lab has previously found that in the medial preoptic area (MPOA), a region implicated in a number of maternal behaviors, there is a decrease in CRFR1+ neurons across the postpartum period between PD1 and PD28 when compared to reproductively naïve (nulliparous) females (De Guzman et al., 2021, De Guzman et al., 2023). CRFR1 may have a specific role in the MPOA during the postpartum period in that its downregulation may be adaptive to enable pup-care behavior. In rats, selective activation of CRFR1 neurons in the MPOA caused disruptions to maternal care, suggesting relative inactivity or perhaps decreased expression of CRFR1 may be important for the display of typical maternal behavior (Klampfl et al., 2018). Given the changes to maternal behavior in multiparous rodents, it's possible that these may coincide with changes to CRFR1 expression in the MPOA of more experienced dams, which has not yet been established.

Previous work from our lab has also found an effect of increased CRFR1 cells in the anteroventral periventricular nucleus (AVPV) of postpartum mice compared with nulliparous females (De Guzman et al., 2021). Outside of reproductive experience, our lab demonstrated a sex difference in CRFR1 neurons in the AVPV in that females have substantially more CRFR1 than males; in adulthood, males have almost no CRFR1 in this region (Rosinger et al., 2017, Rosinger et al., 2019). High levels of activation of CRFR1 neurons are seen following restraint stress in females but not males, suggesting this region may be important for stress regulation in females. The further increase in postpartum mice may point to an adaptation important for stress regulation during the postpartum period, but whether this effect is present in multiparous dams is unknown. As multiparous rodents may have more resilience to stress (Aguggia et al., 2019), this transient increase in CRFR1 in primiparous dams may be attenuated during subsequent postpartum experiences.

There are several aspects of the rodent maternal period that could trigger or be triggered by changes in CRFR1 as well as OT neuron expression of CRFR1. Exposure to offspring and experience-based enhancements to maternal care are the postpartum experiences emphasized in the present study. Postpartum day 7 was selected as an assessment point to examine potential differences in CRFR1 or CRFR1/OT due to consistently low hypothalamic expression at PD7 in previous primiparous experiments (De Guzman et al., 2021, De Guzman et al., 2023), making it suitable to detect receptor level changes. PD6–7 were also optimal for assaying maternal behavior concurrent to posited CRFR1/OT expression changes due to pup retrieval, maternal aggression, and high pup care demands being most prominent during the first postpartum week. We hypothesized that relative to primiparous dams, more experienced dams who are rearing their third litters would display early-postpartum alterations in CRFR1 and CRFR1/OT co-expression and that these changes may coincide with enhanced maternal behavior as measured by pup retrieval and maternal defense tests.