Sex differences in activated corticotropin-releasing factor neurons within stress-related neurocircuitry and hypothalamic–pituitary–adrenocortical axis hormones following restraint in rats
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
References (89)
- et al.
Sex differences in the behavioural response to persistent pain in rats
Neurosci Lett
(1994) - et al.
Sex-dependent effects of formalin and restraint on c-fos expression in the septum and hippocampus of the rat
Neuroscience
(1997) - et al.
Behavioural and hormonal effects of restraint stress and formalin test in male and female rats
Brain Res Bull
(1998) - et al.
Anxiety disorders: sex differences in prevalence, degree, and background, but gender-neutral treatment
Gend Med
(2007) - et al.
Sexual differentiation of human behavior: effects of prenatal and pubertal organizational hormones
Front Neuroendocrinol
(2011) - et al.
Expression of c-fos and BDNF mRNA in subregions of the prefrontal cortex of male and female rats after acute uncontrollable stress
Brain Res
(2005) - et al.
Corticotropin releasing hormone mRNA is elevated on the afternoon of proestrus in the parvocellular paraventricular nuclei of the female rat
Brain Res Mol Brain Res
(1990) - et al.
A detailed characterization of loud noise stress: intensity analysis of hypothalamo–pituitary–adrenocortical axis and brain activation
Brain Res
(2005) - et al.
Acute stress impairs spatial memory in male but not female rats: influence of estrous cycle
Pharmacol Biochem Behav
(2004) - et al.
The pattern of brain c-fos mRNA induced by a component of fox odor, 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), in rats, suggests both systemic and processive stress characteristics
Brain Res
(2004)
Sex differences in behavioral, neurochemical and neuroendocrine effects induced by the forced swim test in rats
Neuroscience
Differential forebrain c-fos mRNA induction by ether inhalation and novelty: evidence for distinctive stress pathways
Brain Res
Role of noradrenergic projections to the bed nucleus of the stria terminalis in the regulation of the hypothalamic–pituitary–adrenal axis
Brain Res Brain Res Rev
Exposure to bisphenol A during gestation and lactation causes loss of sex difference in corticotropin-releasing hormone-immunoreactive neurons in the bed nucleus of the stria terminalis of rats
Psychoneuroendocrinology
Gonadal steroid hormone receptors and sex differences in the hypothalamo–pituitary–adrenal axis
Horm Behav
Sex- and time-dependent changes in neurochemical and hormonal variables induced by predictable and unpredictable footshock
Physiol Behav
Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo–pituitary–adrenocortical responsiveness
Front Neuroendocrinol
Limbic system mechanisms of stress regulation: hypothalamo–pituitary–adrenocortical axis
Prog Neuropsychopharmacol Biol Psychiatry
Gender differences in corticotropin and corticosterone secretion and corticotropin-releasing factor mRNA expression in the paraventricular nucleus of the hypothalamus and the central nucleus of the amygdala in response to footshock stress or psychological stress in rats
Psychoneuroendocrinology
Corticosteroid–serotonin interactions in the neurobiological mechanisms of stress-related disorders
Neurosci Biobehav Rev
Gender, quality of life, and mental disorders in primary care: results from the PRIME-MD 1000 study
Am J Med
Plasma norepinephrine, epinephrine and corticosterone stress responses to restraint in individual male and female rats, and their correlations
Neurosci Lett
Neonatal sex hormones have ‘organizational’ effects on the hypothalamic–pituitary–adrenal axis of male rats
Dev Brain Res
Gender difference in hypothalamic–pituitary–adrenal axis response to alcohol in the rat: activational role of gonadal steroids
Brain Res
Serotonin–CRF interaction in the bed nucleus of the stria terminalis: a light microscopic double-label immunocytochemical analysis
Brain Res Bull
Estrous cycle influences on sexual diergism of HPA axis responses to cholinergic stimulation in rats
Brain Res Bull
Rat estrous cycle influences the sexual diergism of HPA axis stimulation by nicotine
Brain Res Bull
Gender differences in susceptibility to posttraumatic stress disorder
Behav Res Ther
Gender differences in depression in 23 European countries. Cross-national variation in the gender gap in depression
Soc Sci Med
Selective participation of the bed nucelus of the stria terminalis and CRF in sustained anxiety-like versus phasic fear-like responses
Prog Neuropsychopharmacol Biol Psychiatry
Gender differences in sympathoadrenal activity in rats at rest and in response to footshock stress
Int J Dev Neurosci
Effects of estrogen antagonists and agonists on the ACTH response to restraint stress in female rats
Neuropsychopharmacology
Female responses to acute and repeated restraint stress differ from those in males
Physiol Behav
Differential colocalization of estrogen receptor beta (ERbeta) with oxytocin and vasopressin in the paraventricular and supraoptic nuclei of the female rat brain: an immunocytochemical study
Proc Natl Acad Sci U S A
Circadian variation in basal plasma corticosterone and adrenocorticotropin in the rat: sexual dimorphism and changes across the estrous cycle
Endocrinology
Androgen and estrogen receptor-beta distribution within spinal-projecting and neurosecretory neurons in the paraventricular nucleus of the male rat
J Comp Neurol
Reproductive experience reduces circulating 17beta-estradiol and prolactin levels during proestrus and alters estrogen sensitivity in female rats
Endocrinology
Differential expression of oestrogen receptor α following reproductive experience in young and middle-aged female rats
J Neuroendocrinol
Neuroendocrine and behavioral responses and brain pattern of c-fos induction associated with audiogenic stress
J Neuroendocrinol
Connections of some auditory-responsive posterior thalamic nuclei putatively involved in activation of the hypothalamo–pituitary–adrenocortical axis in response to audiogenic stress in rats: an anterograde and retrograde tract tracing study combined with Fos expression
J Comp Neurol
Coexistence of glucorticoid receptor-like immunoreactivity with neuropeptides in the hypothalamic paraventricular nucleus
Exp Brain Res
Bed nucleus of the stria terminalis subregions differentially regulate hypothalamic–pituitary–adrenal axis activity: implications for the integration of limbic inputs
J Neurosci
The anteroventral bed nucleus of the stria terminalis differentially regulates hypothalamic–pituitary–adrenocortical axis responses to acute and chronic stress
Endocrinology
Risk factors predict post-traumatic stress disorder differently in men and women
Ann Gen Psychiatry
Cited by (102)
The sex-dependent and enduring impact of pubertal stress on health and disease
2023, Brain Research BulletinAcute exposure to microcystins affects hypothalamic-pituitary axes of male rats
2023, Environmental PollutionEffects of predator odor on anti-predation behavior and hypothalamic c-fos mRNA expression of Chinese mole shrew
2022, Applied Animal Behaviour Science