Following the perception of stressful stimuli, the hypothalamic-pituitary-adrenal (HPA) axis becomes activated to restore internal homeostasis and promote survival. Known to be tightly regulated, exposure to a stressor triggers the HPA axis to produce the neuropeptides corticotropin releasing factor (CRF) and arginine vasopressin (AVP) which ultimately results in the release of glucocorticoids such as cortisol (primarily in humans) or corticosterone (primarily in rodents) (Herman et al., 2016, Herman et al., 2020; Vale et al., 1981). These glucocorticoids form a negative feedback loop where cortisol feeds back to the hypothalamus to result in the cessation of the stress response (Herman et al., 2016, Herman et al., 2020; Vale et al., 1981), however overactivation of the HPA axis often results in dysregulation and the inability to effectively mitigate the stress response (Herman et al., 2016, Herman et al., 2020). Constant high level cortisol secretion and HPA axis hyperactivity results in a greater likelihood of developing stress-related diseases such as anxiety and depression (Frimodt-Møller et al., 2019; Karin et al., 2020; Keller et al., 2017).

Women are at twice the risk of being diagnosed with stress-related disorders such as anxiety and depression when compared to men (Altemus et al., 2014; Gater et al., 1998; Kessler et al., 1994; Weissman et al., 1996), and gonadal steroid hormones have been suggested to contribute to this sex difference. Androgens such as testosterone (T) are known to inhibit the HPA axis and behavioral stress responses (Burgess and Handa, 1992; Handa et al., 1994a; Lund et al., 2004; Suzuki et al., 2001; Viau and Meaney, 1996). Androgen treatment in humans and rodents has been shown to decrease the release of stress hormones adrenocorticotropic hormone (ACTH) from the pituitary and cortisol/corticosterone from the adrenal cortex (Handa et al., 1994b; Lund et al., 2004, Lund et al., 2005, Lund et al., 2006; Kalil et al., 2013; Michopoulos et al., 2012; Mitsushima et al., 2006; Rubinow et al., 2005; Seale et al., 2004; Williamson and Viau, 2008). Androgen administration has also been demonstrated to decrease indices of anxiety and depression in humans and rodents (Aikey et al., 2002; Amore et al., 2009; Aydogan et al., 2012; Fernández-Guasti and Martínez-Mota, 2005; Wang et al., 1996). Highlighting a critical role of androgen receptor (AR) in the regulation of stress-related behaviors, individuals with Complete Androgen Insensitivity Syndrome (CAIS), who have dysfunctional androgen receptor (AR), show high rates of depression and anxiety disorders (Engberg et al., 2017; Fliegner et al., 2014). Furthermore, rodents with a similar condition termed testicular feminization mutation (Tfm), which results in loss of AR function, show elevated stress-induced corticosterone levels and anxiety-like behavior when compared to rodents lacking the mutation (Hamson et al., 2014; Zuloaga et al., 2008a, Zuloaga et al., 2008b, Zuloaga et al., 2011).

Estrogens however function to work oppositely to androgens, where they enhance HPA axis activity resulting in increased ACTH and corticosterone release (Burgess and Handa, 1992; Handa et al., 1994a; Lund et al., 2004; Suzuki et al., 2001; Viau and Meaney, 1996). However, effects of estrogens on anxiety-like behaviors and the HPA axis depend on whether estrogen receptor alpha (ERα) or beta (ERβ) signaling pathways are preferentially activated (Handa and Weiser, 2014; Lund et al., 2005; Weiser and Handa, 2009). Selective ERα binding results in an increase in HPA axis activity as well as anxiety-like behavior (Handa and Weiser, 2014; Hrabovszky et al., 2004; Lund et al., 2005, Lund et al., 2006; Weiser and Handa, 2009) and overexpression of ERα in specific brain regions increases anxiogenic responses (Spiteri et al., 2012). However, activation of ERβ appears to have a similar function to AR, where it decreases HPA axis activity (Handa et al., 1994b; Lund et al., 2004, Lund et al., 2005, Lund et al., 2006) and both depressive- and anxiety-like behavior (Imwalle et al., 2004; Lund et al., 2005; Rocha et al., 2005).

Although the role of gonadal hormones and their receptors in regulating behavioral and neuroendocrine stress responses are well established, the specific cell types and brain regions they act on to produce these effects are largely unknown (Handa et al., 2009; Zuloaga et al., 2020). One cell phenotype that has been suggested to regulate effects of gonadal steroid hormones on stress responses is CRF expressing neurons. CRF neurons in various brain regions have been shown to play critical roles in regulating both behavioral and neuroendocrine responses to stress (Binder and Nemeroff, 2010; Subbannayya et al., 2013). For example, PVN CRF-expressing neurons are widely known to regulate HPA axis function as well as stress-related behavioral responses (Daviu et al., 2020; Füzesi et al., 2016; Hodges and Felling, 1970). Furthermore, CRF neurons in regions of the extended amygdala critically regulate anxiety-related behaviors and fear learning (Asok et al., 2018; Kim et al., 2017; McCall et al., 2015; Pliota et al., 2018; Pomrenze et al., 2015, Pomrenze et al., 2019; Sahuque et al., 2006; Sanford et al., 2017). What remains unclear is whether androgen actions at AR and estrogen actions at ERα are capable of altering CRF expression and activation of CRF expressing neurons. In this study, we aimed to determine whether CRF neurons in various regions of the mouse brain co-localize AR and ERα, and if this differed by sex, as a potential mechanism for gonadal hormone regulation of stress-related functions associated with CRF. Since there are robust sex differences in neuroendocrine and behavioral stress responses (Zuloaga et al., 2020; Handa et al., 2022; Sheng et al., 2021), and these functions are known to be regulated by CRF neurons (Binder and Nemeroff, 2010; Subbannayya et al., 2013), we also explored whether stress-induced activation of various CRF neuron populations differed between male and female mice. This information may serve to identify specific cell populations that contribute to sex differences in the regulation of stress responses.