Elsevier

Neuroscience

Volume 234, 27 March 2013, Pages 40-52
Neuroscience

Sex differences in activated corticotropin-releasing factor neurons within stress-related neurocircuitry and hypothalamic–pituitary–adrenocortical axis hormones following restraint in rats

https://doi.org/10.1016/j.neuroscience.2012.12.051Get rights and content

Abstract

Women may be more vulnerable to certain stress-related psychiatric illnesses than men due to differences in hypothalamic–pituitary–adrenocortical (HPA) axis function. To investigate potential sex differences in forebrain regions associated with HPA axis activation in rats, these experiments utilized acute exposure to a psychological stressor. Male and female rats in various stages of the estrous cycle were exposed to 30 min of restraint, producing a robust HPA axis hormonal response in all animals, the magnitude of which was significantly higher in female rats. Although both male and female animals displayed equivalent c-fos expression in many brain regions known to be involved in the detection of threatening stimuli, three regions had significantly higher expression in females: the paraventricular nucleus of the hypothalamus (PVN), the anteroventral division of the bed nucleus of the stria terminalis (BSTav), and the medial preoptic area (MPOA). Dual fluorescence in situ hybridization analysis of neurons containing c-fos and corticotropin-releasing factor (CRF) mRNA in these regions revealed significantly more c-fos and CRF single-labeled neurons, as well as significantly more double-labeled neurons in females. Surprisingly, there was no effect of the estrous cycle on any measure analyzed, and an additional experiment revealed no demonstrable effect of estradiol replacement following ovariectomy on HPA axis hormone induction following stress. Taken together, these data suggest sex differences in HPA axis activation in response to perceived threat may be influenced by specific populations of CRF neurons in key stress-related brain regions, the BSTav, MPOA, and PVN, which may be independent of circulating sex steroids.

Highlights

► Similar qualitative stress-induced neurocircuitry activation in male and female brain. ► Females have higher activation of PVN, BST, and MPOA after restraint stress. ► Females have higher stress-induced activation of CRF circuitry. ► Estradiol does not always augment stress-induced HPA axis activity.

Introduction

Stress can be an exacerbating or causal factor in the etiology of many diseases, including several psychological disorders. Some of these stress-influenced psychiatric illnesses are at least twice as prevalent in women than men, such as major depression (Linzer et al., 1996, Kessler et al., 2005, Van de Velde et al., 2010) and several anxiety disorders, such as posttraumatic stress and generalized anxiety disorders (Linzer et al., 1996, Stein et al., 2000, Holbrook et al., 2002, Tolin and Foa, 2006, Bekker and van Mens-Verhulst, 2007, Olff et al., 2007, Christiansen and Elklit, 2008, Vesga-López et al., 2008, Ditlevsen and Elklit, 2010). In humans these disorders are associated with dysregulation, either hypo- or hyper-activity, of the hypothalamic–pituitary–adrenocortical (HPA) axis and thus the HPA axis is currently a target of therapeutic treatments for these illnesses (Lanfumey et al., 2008, Lloyd and Nemeroff, 2011). In the brain, the paraventricular nucleus of the hypothalamus (PVN) controls activation of the HPA axis in response to either real or perceived threats, and release of the hormones adrenocorticotropic hormone (ACTH) and cortisol. If hyperactivity of the HPA axis truly underlies stress-related psychiatric illness in humans, female susceptibility to these illnesses could potentially be explained by differences in HPA axis activation following perceived, or psychological, threats or stressors.

In rats, there is a wealth of evidence that females can have a much larger magnitude of HPA axis activation to stress than males. Female rats reportedly release more ACTH and corticosterone (CORT) compared to male rats following a wide variety of acute stressful stimuli (Le Mevel et al., 1978, Livezey et al., 1985, Heinsbroek et al., 1991, Aloisi et al., 1994, Aloisi et al., 1998, Handa et al., 1994, Ogilvie and Rivier, 1997, Weinstock et al., 1998, Rivier, 1999, Drossopoulou et al., 2004, Seale et al., 2004, Viau et al., 2005, Larkin et al., 2010). In addition, activation of the PVN is significantly higher in females than males following various acute stressors, as indexed by either mRNA or protein products of the immediate early gene c-fos (Seale et al., 2004, Viau et al., 2005, Larkin et al., 2010). Presumably, gender-biased stress-induced activation of the PVN, and subsequent HPA hormone release are the result of corticotropin-releasing factor (CRF)-dependent differences, the primary PVN peptide controlling the release of ACTH from the pituitary (at least in rodents). Indeed, basal (Viau et al., 2005) and stress-induced CRF mRNA levels in the PVN have been reported to be higher in female compared to male rats (Aloisi et al., 1998, Iwasaki-Sekino et al., 2009). However, at least one group has reported the opposite effect after restraint stress (Sterrenburg et al., 2012), and Zavala and colleagues (Zavala et al., 2011) reported higher PVN c-fos (FOS) immunoreactivity in male compared to female rats after acute restraint. It remains unclear whether sex differences in PVN activation occurs specifically within CRF, or some other population, of neurons.

Very little research thus far has focused on sex differences in the activation of brain regions associated with PVN relative activity following processive stressors, defined as psychological stimuli that activate stress response systems regardless of whether or not the threat is real. However, uncovering how sex might influence these particular pathways may be especially important for understanding sex- and stress-influenced psychiatric disorders in humans. We have previously identified regions that are associated with HPA axis activation to processive stress in male rats using audiogenic stress, including the ventrolateral septum, the anteroventral division of the bed nucleus of the stria terminalis (BSTav), the subiculum, and the medial preoptic area (MPOA), and c-fos mRNA expression in these regions was found to be highly correlated with PVN activity and HPA axis hormone release (Burow et al., 2005). Others have implicated such regions as the medial prefrontal cortex and medial nucleus of the amygdala as limbic structures capable of affecting HPA axis responses to perceived threats (Emmert and Herman, 1999, Herman et al., 2003, Herman et al., 2005, Day et al., 2004). Importantly, several studies have shown sex differences in some of these regions. For example, sex differences in activation have been observed after either formalin injection or restraint stress in the septum (Aloisi et al., 1997) and frontal cortex (Figueiredo et al., 2002). Females show less activity in the medial prefrontal cortex after inescapable tailshock than males, despite females having greater HPA axis hormone release following this stressor than males (Bland et al., 2005). Of particular interest however, are potential sex differences in the BSTav and the MPOA that could affect HPA axis activity. Both of these regions have CRF-producing neurons, and they both contain dense numbers of both androgen and estrogen receptors (Simerly et al., 1990). Specifically, CRF-containing neurons in the fusiform nucleus of the BST send direct projections to the PVN (Dong et al., 2001). In addition, a sexually dimorphic population of CRF neurons exists in the MPOA (McDonald et al., 1994, Funabashi et al., 2004), which is a morphologically and functionally sexually differentiated region involved in the control of reproductive behavior containing dense amounts of steroid hormone receptors (Tobet and Hanna, 1997). Furthermore, this region has been found to be the site of inhibitory action of androgens on HPA axis activity in male rats (Viau and Meaney, 1996, Williamson et al., 2010). Indeed, several researchers have named these particular regions as likely candidates for sex-specific influences on stress-induced HPA axis function (Viau, 2002, Herman et al., 2003, Herman et al., 2005). However to date, no research has focused on stress-induced sex differences in these areas simultaneously.

Therefore to investigate possible sex differences in stress-induced neurocircuitry following acute stress, we exposed male and female rats to 30 min of restraint. Because many studies have demonstrated estrous cycle influences on both basal (Atkinson and Waddell, 1997) and stress-induced (Viau and Meaney, 1991, Rhodes et al., 2002, Rhodes et al., 2004, Conrad et al., 2004, Iwasaki-Sekino et al., 2009, Larkin et al., 2010) ACTH and CORT release, we included females in three stages of the estrous cycle: diestrus, proestrus, and estrus. In contrast to our previous studies in male rats using noise stress, we used restraint stress in this study due to the large volume of literature examining the effect of sex on responses to this stressor in particular. We first utilized the immediate early gene c-fos as a general marker of neuronal activation, in order to measure stress-induced brain activity across a wide selection of regions with dissimilar neuroanatomical characteristics. We then investigated brain regions that were found to have a sex-specific activation to determine colocalization of CRF with c-fos using dual fluorescence in situ hybridization (FISH). Finally, we manipulated sex steroid levels in females and compared acute stress-induced HPA axis hormone activation in females with prolonged exposure to silastic capsules containing estradiol or vehicle, compared to intact male and female animals.

Section snippets

Experiments 1 and 2: animals

Young adult (2–3-month-old) Sprague–Dawley rats (Harlan Laboratories, Indianapolis, IN, USA) were allowed to acclimate in the colony for at least 1 week without manipulation upon arrival. All animals were originally group-housed but were singly housed in the same room just prior to stress manipulation (Experiment 1), or following surgery (Experiment 2), and were maintained on a 12:12 h light:dark cycle (lights on at 6 am) under constant temperature and humidity conditions, and were provided access

Experiment 1: HPA axis hormones

Fig. 1 displays HPA axis hormone plasma concentrations immediately prior to and immediately following, 30 min of restraint. A repeated measures ANOVA revealed that restraint significantly increased ACTH levels in both males and females as reflected by a main effect of stress (F1,27 = 46.54, p < 0.001; Fig 1A). In addition, females had higher ACTH levels compared to males, as revealed by a significant main effect of sex (F1,27 = 13.59, p = 0.001). There was also a significant stress by sex interaction (F

Discussion

The fact that HPA axis activation in response to a variety of stressful stimuli can be affected by sex and sex steroids is well established; the exact mechanism by, and level at, which this can occur remains obscure. The present study focuses on the forebrain neural circuit associated with stress-induced HPA axis activation, whether sex differences in stress-induced HPA axis hormone release are accompanied by differences in the regulation of processive stress, and the extent to which HPA axis

Conclusion

In summary, these results demonstrate that HPA axis responses to acute restraint stress are affected by sex in a complex way, and that regions outside of the PVN should be considered when exploring sex differences in brain activation following acute stress. Following restraint, similar stress responsive neurocircuitry is activated in the female compared to male brain, although the magnitude of this activation in certain brain regions is gender specific, despite displaying similar activation of

Acknowledgements

This work was supported by NIH R01 MH077152 awarded to S. Campeau. The authors would like to thank Dr. Robert Spencer (University of Colorado at Boulder, Department of Psychology and Neuroscience) for generously providing the authors with the restraint tubes used in this experiment and for his invaluable support of this work. The authors would also like to thank Jon Roberts (University of Colorado at Boulder, Department of Psychology and Neuroscience, Staff member) for providing technical

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