Corticotropin-releasing factor binds with high affinity to CRF receptor 1 (CRFR1) and is implicated in stress-related mood disorders such as anxiety and depression. Using a validated CRFR1-green fluorescent protein (GFP) reporter mouse, our laboratory recently discovered a nucleus of CRFR1 expressing cells that is prominent in the female rostral anteroventral periventricular nucleus (AVPV/PeN), but largely absent in males. This sex difference is present in the early postnatal period and remains dimorphic into adulthood. The present investigation sought to characterize the chemical composition and gonadal hormone regulation of these sexually dimorphic CRFR1 cells using immunohistochemical procedures. We report that CRFR1-GFP-ir cells within the female AVPV/PeN are largely distinct from other dimorphic cell populations (kisspeptin, tyrosine hydroxylase). However, CRFR1-GFP-ir cells within the AVPV/PeN highly co-express estrogen receptor alpha as well as glucocorticoid receptor. A single injection of testosterone propionate or estradiol benzoate on the day of birth completely eliminates the AVPV/PeN sex difference, whereas adult gonadectomy has no effect on CRFR1-GFP cell number. These results indicate that the AVPV/PeN CRFR1 is regulated by perinatal but not adult gonadal hormones. Finally, female AVPV/PeN CRFR1-GFP-ir cells are activated following an acute 30-min restraint stress, as assessed by co-localization of CRFR1-GFP cells with phosphorylated (p) CREB. CRFR1-GFP/pCREB cells were largely absent in the male AVPV/PeN. Together, these data indicate a stress and gonadal hormone responsive nucleus that is unique to females and may contribute to sex-specific stress responses.

Anxiety and depression are strikingly more prevalent in women compared with men. Dysregulation of corticotropin-releasing factor (CRF) binding to its cognate receptor (CRFR1) is thought to play a critical role in the etiology of these disorders. In the present study, we investigated whether there were sex differences in the effects of chronic variable stress (CVS) on CRFR1 cells using CRFR1-GFP reporter mice experiencing a 9-day CVS paradigm. Brains were collected from CVS and stress naïve female and male mice following exposure to the open field test. This CVS paradigm effectively increased anxiety-like behavior in female and male mice. In addition, we assessed changes in activation of CRFR1 cells (co-localization with c-Fos and phosphorylated CREB (pCREB)) in stress associated brain structures, including two sexually dimorphic CRFR1 cell groups in the anteroventral periventricular nucleus (AVPV/PeN; F>M) and paraventricular hypothalamus (PVN; M>F). CVS increased CRFR1-GFP cell number as well as the number of CRFR1/pCREB co-expressing cells in the female but not male AVPV/PeN. In the PVN, the number of CRFR1/pCREB co-expressing cells was overall greater in males regardless of treatment and CVS resulted in a male-specific reduction of CRFR1/c-Fos cells. In addition, CVS induced a female-specific reduction in CRFR1/c-Fos cells within the anteroventral bed nucleus of the stria terminalis and both sexes exhibited a reduction in CRFR1/c-Fos co-expressing cells following CVS within the ventral basolateral amygdala. Overall, these sex-specific effects of CVS on CRFR1 populations may have implications for sex differences in stress-induction of mood disorders.

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.

Sex differences in patterns of methamphetamine (MA) abuse have been reported with females (humans and rodents) showing an elevated addiction phenotype. Previous findings indicate MA-induced hypothalamic-pituitary-adrenal (HPA) axis activation is also sexually dimorphic with females exhibiting an elevated glucocorticoid release and differential neural activation patterns within HPA axis-associated brain regions. These effects may contribute to sex differences in abuse. To determine the role of gonadal hormones in mediating sex differences in MA-induced glucocorticoids, male and female C57BL/6J mice were gonadectomized or sham-operated, and following recovery, injected with MA (5mg/kg) and sacrificed 60min or 120min later. Blood was collected for corticosterone radioimmunoassay, and brains were used to assess c-Fos, and c-Fos co-localization with glucocorticoid receptor (GR). At 120min after MA injection, corticosterone levels were elevated in females compared to males and gonadectomy in males increased corticosterone to female levels. C-Fos was greater in females than males in the medial preoptic area, bed nucleus of the stria terminalis, basolateral amygdala, and central amygdala. Female gonadectomy had little effect on either corticosterone or c-Fos, while male gonadectomy elevated c-Fos in the central amygdala. Relative to sham males, gonadectomized males also showed decreased c-Fos/GR cell number in the CA3 hippocampal area compared to sham males, indicating a central site for attenuated negative feedback. Together, these findings indicate that androgens regulate MA-induced activation of the HPA axis, potentially by enhancing negative feedback. These sex and gonadal hormone effects on the HPA axis may contribute to sex differences in MA abuse patterns.

Corticotropin-releasing factor (CRF) and its primary receptor (CRFR1) are critical regulators of behavioral and neuroendocrine stress responses. CRFR1 has also been associated with stress-related behavioral changes in postpartum mice. Our previous studies indicate dynamic changes in CRFR1 levels and coupling of CRFR1 with tyrosine hydroxylase (TH) and oxytocin (OT) neurons in postpartum mice. In this study, we aimed to determine the time course of these changes during the postpartum period.

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.

Sex differences in neural structures are generally believed to underlie sex differences reported in anxiety, depression, and the hypothalamic-pituitary-adrenal axis, although the specific circuitry involved is largely unclear. Using a corticotropin-releasing factor receptor 1 (CRFR1) reporter mouse line, we report a sexually dimorphic distribution of CRFR1 expressing cells within the paraventricular hypothalamus (PVN; males > females). Relative to adult levels, PVN CRFR1-expressing cells are sparse and not sexually dimorphic at postnatal days 0, 4, or 21. This suggests that PVN cells might recruit CRFR1 during puberty or early adulthood in a sex-specific manner. The adult sex difference in PVN CRFR1 persists in old mice (20-24 months). Adult gonadectomy (6 weeks) resulted in a significant decrease in CRFR1-immunoreactive cells in the male but not female PVN. CRFR1 cells show moderate co-expression with estrogen receptor alpha (ERα) and high co-expression with androgen receptor, indicating potential mechanisms through which circulating gonadal hormones might regulate CRFR1 expression and function. Finally, we demonstrate that a psychological stressor, restraint stress, induces a sexually dimorphic pattern of neural activation in PVN CRFR1 cells (males >females) as assessed by co-localization with the transcription/neural activation marker phosphorylated CREB. Given the known role of CRFR1 in regulating stress-associated behaviors and hormonal responses, this CRFR1 PVN sex difference might contribute to sex differences in these functions.

Stress-related mood disorders like anxiety and depression are more prevalent in women than men and are often associated with hypothalamic-pituitary-adrenal (HPA) axis dysregulation. Androgen actions through androgen receptors (ARs) decrease HPA axis responses and stress-associated behaviors. Corticotropin releasing factor (CRF) and its binding to CRF receptor 1 (CRFR1) is also critical for regulation of the HPA axis, anxiety, and depression. We first determined CRFR1/AR co-localization patterns in male and female CRFR1-GFP mice. High co-localization was found within the paraventricular nucleus (PVN), dorsolateral and anteroventral subdivisions of the bed nucleus of the stria terminalis (BSTdl and BSTav), medial preoptic area (MPOA), and posterodorsal medial amygdala (MePD). We next determined whether the non-aromatizable androgen dihydrotestosterone (DHT) regulates CRFR1 expression and stress-induced activation of CRFR1-expressing cells. In the PVN, CRFR1-GFP cell number decreased following gonadectomy (GDX), but DHT treatment reversed this effect. GDX-DHT treated mice also had a decreased CRFR1-GFP cell number within the BSTdl compared to gonad intact and GDX-untreated groups. Following restraint stress GDX-blank mice showed fewer c-Fos/CRFR1 co-localized neurons in the MePD compared to gonad intact and GDX-DHT groups indicating decreased stress-induced activation of CRFR1 neurons following GDX. Higher plasma corticosterone (CORT) was found in GDX males compared to GDX-DHT and sham males following restraint stress, with a negative correlation between PVN CRFR1+ neurons and corticosterone levels 30- and 90-min following restraint. Together these findings show androgens can directly alter CRFR1 levels in the brain which may have implications for sex differences in regulation of the HPA axis and stress-related behaviors.

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.