Maternal experience can promote a long-lasting increase in maternal motivation. This maintenance of caregiving behaviors, rather than avoidant or agnostic responses towards young, is advantageous for the survival of subsequent offspring. We have previously reported that maternal motivation is associated with differential immediate early gene expression in central motivation circuits and aversion circuits. Here we ask how these circuits come to differentially respond to infant cues. We used Targeted Recombination in Active Populations (TRAP) to identify cells that respond to pups in maternally hesitant TRAP2;Ai14 virgin female mice. Following an initial 60 min exposure to foster pups, virgin TRAP2;Ai14 mice were injected with 4-hydroxytamoxifen to induce recombination in c-Fos expressing cells and subsequent permanent expression of a red fluorescent reporter. We then examined whether the same cells that encode pup cues are reactivated during maternal memory retrieval two weeks later using c-Fos immunohistochemistry. Whereas initial pup exposure induced c-Fos activation exclusively in the medial preoptic area (MPOA), following repeated experience, c-Fos expression was significantly higher than baseline in multiple regions of maternal and central aversion circuits (e.g., ventral bed nucleus of the stria terminalis, nucleus accumbens, basolateral amygdala, prefrontal cortex, medial amygdala, and ventromedial nucleus of the hypothalamus). Further, cells in many of these sites were significantly reactivated during maternal memory retrieval. These data suggest that cells across both maternal motivation and central aversion circuits are stably responsive to pups and thus may form the cellular representation of maternal memory.

Gonadal hormone actions through androgen receptor (AR) and estrogen receptor alpha (ERα) regulate sex differences in hypothalamic-pituitary-adrenal (HPA) axis responsivity and stress-related behaviors. Here we tested whether corticotropin releasing factor (CRF) expressing neurons, which are widely known to regulate neuroendocrine and behavioral stress responses, co-express AR and ERα as a potential mechanism for gonadal hormone regulation of these responses. Using Crh-IRES-Cre::Ai9 reporter mice we report high co-localization of AR in CRF neurons within the medial preoptic area (MPOA), bed nucleus of the stria terminalis (BST), medial amygdala (MeA), and ventromedial hypothalamus (VMH), moderate levels within the central amygdala (CeA) and low levels in the paraventricular hypothalamus (PVN). Sex differences in CRF/AR co-expression were found in the principal nucleus of the BST (BSTmpl), CeA, MeA, and VMH (males>females). CRF co-localization with ERα was generally lower relative to AR co-localization. However, high co-expression was found within the MPOA, AVPV, and VMH, with moderate co-expression in the arcuate nucleus (ARC), BST, and MeA and low levels in the PVN and CeA. Sex differences in CRF/ERα co-localization were found in the BSTmpl and PVN (males>females). Finally, we assessed neural activation of CRF neurons in restraint-stressed mice and found greater CRF/c-Fos co-expression in females in the BSTmpl and periaqueductal gray, while co-expression was higher in males within the ARC and dorsal CA1. Given the known role of CRF in regulating behavioral stress responses and the HPA axis, AR/ERα co-expression and sex-specific activation of CRF cell groups indicate potential mechanisms for modulating sex differences in these functions.

Corticotropin-releasing factor (CRF) signaling through CRF receptor 1 (CRFR1) regulates autonomic, endocrine, and behavioral responses to stress, as well as behavioral changes during the maternal period. Previous work in our lab reported higher levels of CRFR1 in female, compared to male, mice within the rostral anteroventral periventricular nucleus (AVPV/PeN), a brain region involved in maternal behaviors. In this study, we used CRFR1-GFP reporter mice to investigate whether the reproductive status (postpartum vs. nulliparous) of acutely stressed females affects levels of CRFR1 in the AVPV/PeN and other regions involved in maternal functions. Compared to nulliparous, postpartum day 14 females showed increased AVPV/PeN CRFR1-GFP immunoreactivity and an elevated number of restraint stress-activated AVPV/PeN CRFR1 cells as assessed by immunohistochemical co-localization of CRFR1-GFP and phosphorylated CREB (pCREB). The medial preoptic area (MPOA) and paraventricular hypothalamus (PVN) of postpartum mice showed modest decreases in CRFR1-GFP immunoreactivity, while increased CRFR1-GFP/pCREB co-expressing cells were found in the PVN following restraint stress relative to nulliparous mice. Tyrosine hydroxylase (TH) and CRFR1-GFP co-localization was also assessed in the AVPV/PeN and other regions and revealed a decrease in co-localized neurons in the AVPV/PeN and ventral tegmental area of postpartum mice. Corticosterone analysis of restrained mice revealed blunted peak, but elevated recovery, levels in postpartum compared to nulliparous mice. Finally, we investigated projection patterns of AVPV/PeN CRFR1 neurons using female CRFR1-Cre mice and revealed dense efferent projections to several preoptic, hypothalamic, and hindbrain regions known to control stress-associated and maternal functions. Together, these findings contribute to our understanding of the neurobiology that might underlie changes in stress-related functions during the postpartum period.