Stress and the reproductive axis.

There exists a reciprocal relationship between the hypothalamic-pituitary-adrenal (HPA) and the hypothalamic-pituitary-gonadal (HPG) axes, wherein the activation of one affects the function of the other and vice versa. For example, both testosterone and oestrogen modulate the response of the HPA axis, whereas activation of the stress axis, especially activation that is repeating or chronic, has an inhibitory effect upon oestrogen and testosterone secretion. Alterations in maternal care can produce significant effects on both HPG and HPA physiology, as well as behaviour in the offspring at adulthood. For example, changes in reproductive behaviour induced by altered maternal care may alter the expression of sex hormone receptors such as oestrogen receptor (ER)α that govern sexual behaviour, and may be particularly important in determining the sexual strategies utilised by females. Stress in adulthood continues to mediate HPG activity in females through activation of a sympathetic neural pathway originating in the hypothalamus and releasing norepinephrine into the ovary, which produces a noncyclic anovulatory ovary that develops cysts. In the opposite direction, sex differences and sex steroid hormones regulate the HPA axis. For example, although serotonin (5-HT) has a stimulatory effect on the HPA axis in humans and rodents that is mediated by the 5-HT1A receptor, only male rodents respond to 5-HT1A antagonism to show increased corticosterone responses to stress. Furthermore, oestrogen appears to decrease 5-HT1A receptor function at presynaptic sites, yet increases 5-HT1A receptor expression at postsynaptic sites. These mechanisms could explain the heightened stress HPA axis responses in females compared to males. Studies on female rhesus macaques show that chronic stress in socially subordinate female monkeys produces a distinct behavioural phenotype that is largely unaffected by oestrogen, a hyporesponsive HPA axis that is hypersensitive to the modulating effects of oestrogen, and changes in 5-HT1A receptor binding in the hippocampus and hypothalamus of social subordinate female monkeys that are restored or inverted by oestrogen replacement. This review summarises all of these studies, emphasising the profound effect that the interaction of the reproductive and stress axes may have on human reproductive health and emotional wellbeing.

A comparison of two repeated restraint stress paradigms on hypothalamic-pituitary-adrenal axis habituation, gonadal status and central neuropeptide expression in adult male rats.

The available evidence continues to illustrate an inhibitory influence of male gonadal activity on the hypothalamic-pituitary-adrenal (HPA) axis under acute stress. However, far less is known about how these systems interact during repeated stress. Because HPA output consistently declines across studies examining repeated restraint, the potential mechanisms mediating this habituation are often inferred as being equivalent, even though these studies use a spectrum of restraint durations and exposures. To test this generalisation, as well as to emphasise a potential influence of the male gonadal axis on the process of HPA habituation, we compared the effects of two commonly used paradigms of repeated restraint in the rodent: ten daily episodes of 0.5 h of restraint and five daily episodes of 3 h of restraint. Both paradigms produced comparable declines in adrenocorticotrophic hormone and corticosterone between the first and last day of testing. However, marked differences in testosterone levels, as well as corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) expression, occurred between the two stress groups. Plasma testosterone levels remained relatively higher in animals exposed to 0.5 h of restraint compared to 3 h of restraint, whereas forebrain gonadotrophin-releasing hormone (GnRH) cell counts increased in both groups. AVP mRNA was increased after 3 h, but not after 0.5 h of repeated restraint, in the medial parvicellular paraventricular nucleus and in the posterior bed nucleus of the stria terminalis (BST), and increased with 0.5 h of repeated restraint in the medial amygdala. CRH mRNA was increased after 3 h, but not after 0.5 h of repeated restraint, in the central amygdala and anterior BST. The data obtained illustrate that, despite comparable declines in HPA responses, the pathways recruited for stress adaptation appear to be distinct between restraint groups. Given the extreme sensitivity of limbic AVP to testosterone, and conversely CRH to circulating glucocorticoids, whether differences in endocrine profiles might explain these neuropeptide differences remains to be seen. Nonetheless, the present study provides several new entry points for testing gonadal influences on stress-specific HPA habituation.

Effects of prenatal ethanol exposure on regulation of basal hypothalamic-pituitary-adrenal activity and hippocampal 5-HT1A receptor mRNA levels in female rats across the estrous cycle.

Prenatal ethanol exposure, like other early adverse experiences, is known to alter hypothalamic-pituitary-adrenal (HPA) activity in adulthood. The present study examined the modulatory effects of the gonadal hormones on basal HPA regulation and serotonin Type 1A receptor (5-HT(1A)) mRNA levels in adult female rats prenatally exposed to ethanol (E) compared to that in females from pair-fed (PF) and ad libitum-fed control (C) conditions. We demonstrate, for the first time, long-lasting consequences of prenatal ethanol exposure for basal corticosterone (CORT) regulation and basal levels of hippocampal mineralocorticoid (MR), glucocorticoid (GR) and serotonin Type 1A (5-HT(1A)) receptor mRNA, as a function of estrous cycle stage: (1) basal CORT levels were higher in E compared to C females in proestrus but lower in E and PF compared to C females in estrus; (2) there were no differences among groups in basal levels of adrenocorticotropin (ACTH), estradiol or progesterone; (3) hippocampal MR mRNA levels were decreased in E compared to PF and C females across the estrus cycle, with the greatest effects in proestrus, whereas E (but not PF or C) females had higher hippocampal GR mRNA levels in proestrus than in estrous and diestrus; (4) 5-HT(1A) mRNA levels were increased in E compared to PF and C females in diestrus. That alterations were revealed as a function of estrous cycle stage suggests a role for the ovarian steroids in mediating the adverse effects of ethanol. Furthermore, it appears that ethanol-induced nutritional effects may play a role in mediating at least some of the effects observed. The resetting of HPA activity by early environmental events could be one mechanism linking early life experiences with long-term health consequences. Thus, changes in basal CORT levels, a shift in the MR/GR balance and alterations in 5-HT(1A) receptor mRNA could have important clinical implications for understanding the secondary disabilities, such as an increased incidence of depression, in children with FASD.

Prenatal ethanol exposure alters the effects of gonadectomy on hypothalamic-pituitary-adrenal activity in male rats.

Prenatal ethanol exposure has marked effects on development of the hypothalamic-pituitary-adrenal (HPA) and -gonadal (HPG) axes. In adulthood, ethanol-treated rats show altered gonadal hormone responses and reproductive function, and increased HPA responsiveness to stressors. Importantly, prenatal ethanol differentially alters stress responsiveness in adult males and females, raising the possibility that the gonadal hormones play a role in mediating prenatal ethanol effects on HPA function. To examine a possible testicular influence on HPA activity in males, we compared the effects of gonadectomy on HPA stress responses of adult male offspring from ethanol, pair-fed (PF) and ad libitum-fed control dams. Intact ethanol-treated rats showed increased adrenocorticotrophic hormone (ACTH) but blunted testosterone and luteinising hormone (LH) responses to restraint stress, and no stress-induced elevation in arginine vasopressin (AVP) mRNA levels compared to those observed in PF and/or control rats. Gonadectomy: (i) significantly increased ACTH responses to stress in control but not ethanol-treated and PF males; (ii) eliminated differences among groups in plasma ACTH and AVP mRNA levels; and (iii) altered LH and gonadotrophin-releasing hormone responses in ethanol-treated males. Taken together, these findings suggest that central regulation of both the HPA and HPG axes are altered by prenatal ethanol exposure, with normal testicular influences on HPA function markedly reduced in ethanol-treated animals. A decreased sensitivity to inhibitory effects of androgens could contribute to the HPA hyperresponsiveness typically observed in ethanol-treated males.

Both estrogen receptor alpha and estrogen receptor beta agonists enhance cell proliferation in the dentate gyrus of adult female rats.

This study investigated the involvement of estrogen receptors alpha and beta in estradiol-induced enhancement of hippocampal neurogenesis in the adult female rat. Subtype selective estrogen receptor agonists, propyl-pyrazole triol (estrogen receptor alpha agonist) and diarylpropionitrile (estrogen receptor beta agonist) were examined for each receptor's contribution, individual and cooperative, for estradiol-enhanced hippocampal cell proliferation. Estradiol increases hippocampal cell proliferation within 4 h [Ormerod BK, Lee TT, Galea LA (2003) Estradiol initially enhances but subsequently suppresses (via adrenal steroids) granule cell proliferation in the dentate gyrus of adult female rats. J Neurobiol 55:247-260]. Therefore, animals received s.c. injections of estradiol (10 microg), propyl-pyrazole triol and diarylpropionitrile alone (1.25, 2.5, 5.0 mg/0.1 ml dimethylsulfoxide) or in combination (2.5 mg propyl-pyrazole triol+2.5 mg diarylpropionitrile/0.1 ml dimethylsulfoxide) and 4 h later received an i.p. injection of the cell synthesis marker, bromodeoxyuridine (200 mg/kg). Diarylpropionitrile enhanced cell proliferation at all three administered doses (1.25 mg, P<0.008; 2.5 mg, P<0.003; 5 mg, P<0.005), whereas propyl-pyrazole triol significantly increased cell proliferation (P<0.0002) only at the dose of 2.5 mg. Our results demonstrate both estrogen receptor alpha and estrogen receptor beta are individually involved in estradiol-enhanced cell proliferation. Furthermore both estrogen receptor alpha and estrogen receptor beta mRNA was found co-localized with Ki-67 expression in the hippocampus albeit at low levels, indicating a potential direct influence of each receptor subtype on progenitor cells and their progeny. Dual receptor activation resulted in reduced levels of cell proliferation, supporting previous studies suggesting that estrogen receptor alpha and estrogen receptor beta may modulate each other's activity. Our results also suggest that a component of estrogen receptor-regulated cell proliferation may take place through alternative ligand and/or cell-signaling mechanisms.

Distinct temporal activity patterns in the rat M1 and red nucleus during skilled versus unskilled limb movement.

The mammalian motor system contains multiple interconnected supraspinal networks, but little is known about their relative roles in producing different movements and behaviors, particularly given their apparently fused activity in primates. We tested whether the task context, as well as using a phylogenetically older mammal, rats, could distinguish the separate contributions of these networks. We obtained simultaneous multi-single neuron recordings from the forelimb motor cortex and magnocellular red nucleus as rats performed two contextually different, but kinematically similar, forelimb reach-like tasks: highly learned, skilled reaching for food through a narrow slot, a task requiring extensive training, versus the swing phases of treadmill locomotion. In both the M1 and the mRN, large subpopulations of neurons peaked in their spike firing rates near the onset and the end of the swing phase during treadmill locomotion. In contrast, neural subgroups in the two areas displayed different temporal sequences of activity during the skilled reaching task. In the mRN, the majority of task-modulated neurons peaked in their firing rate in the middle of the reach when the rat was preparing to project the arm through the slot, whereas large subgroups of M1 neurons displayed elevated firing rates during the initial and terminal phases of the reach. These results suggest that motor-behavioral context can alter the degree of overlapping activity in different supraspinal sensorimotor networks. Moreover, results for the skilled reaching task in rats may have highlighted a distinct processing role of the rubral complex: adapting natural muscle synergies across joints and limbs to novel task demands, in concert with cortically based learning.

Alpha1 adrenoreceptors mediate the stimulatory effects of oestrogen on stress-related hypothalamic-pituitary-adrenal activity in the female rat.

Variation in challenge-induced adrenocorticotropin hormone (ACTH) release over the oestrous cycle occurs in response to fluctuations in circulating concentrations of oestrogen and progesterone. However, how these ovarian steroids interact to regulate the principal ACTH cosecretagogues, corticotropin-releasing hormone (CRH) and arginine vasopressin is not understood. Here, we measured median eminence CRH and vasopressin content in intact cycling female rats, and in ovariectomized (OVX) females steroid-replaced in a manner that approximates the relative release patterns of oestrogen and progesterone seen over the oestrous cycle. Intact cycling females showed significantly higher median eminence CRH and vasopressin concentrations during proestrous and oestrous compared to the diestrous phase. In OVX rats, a single 10 micro g injection of oestrogen failed to mimic this increase in median eminence CRH and vasopressin. However, this dose significantly elevated CRH and vasopressin content in OVX rats previously exposed to diestrous concentrations of oestrogen and progesterone. Moreover, oestrogen priming enhanced restraint-induced depletion of CRH and vasopressin from the median eminence, but only against a background of low oestrogen and progesterone replacement. Oestrogen-induced elevations in median eminence vasopressin (but not CRH) content were reduced by peripheral administration of the alpha1 adrenoreceptor antagonist prazosin. Finally, plasma ACTH concentrations following central injection of the alpha1 receptor agonist, phenylephrine, were significantly higher in rats during proestrous compared to diestrous. These results indicate that the stimulatory effect of oestrogen on both the expression and stress-induced release of ACTH cosecretagogues is exerted only against a background of low oestrogen and progesterone levels, and is mediated, in part, via the alpha1 adrenoreceptor.

Functional cross-talk between the hypothalamic-pituitary-gonadal and -adrenal axes.

Under normal conditions, the adrenal glucocorticoids, the endproduct of the hypothalamic-pituitary-adrenal (HPA) axis, provide a frontline of defence against threats to homeostasis (i.e. stress). On the other hand, chronic HPA drive and glucocorticoid hypersecretion have been implicated in the pathogenesis of several forms of systemic, neurodegenerative and affective disorders. The HPA axis is subject to gonadal influence, indicated by sex differences in basal and stress HPA function and neuropathologies associated with HPA dysfunction. Functional cross-talk between the gonadal and adrenal axes is due in large part to the interactive effects of sex steroids and glucocorticoids, explaining perhaps why several disease states linked to stress are sex-dependent. Realizing the interactive nature by which the hypothalamic-pituitary-gonadal and HPA systems operate, however, has made it difficult to model how these hormones act in the brain. Manipulation of one endocrine system is not without effects on the other. Simultaneous manipulation and assessment of both endocrine systems can overcome this problem. This dual approach in the male rat reveals that testosterone can act and interact on different aspects of basal and stress HPA function. Basal adrenocorticotropic hormone (ACTH) release is regulated by testosterone-dependent effects on arginine vasopressin synthesis, and corticosterone-dependent effects on corticotropin-releasing hormone (CRH) synthesis in the paraventricular nucleus (PVN) of the hypothalamus. In contrast, testosterone and corticosterone interact on stress-induced ACTH release and drive to the PVN motor neurones. Candidate structures mediating this interaction include several testosterone-sensitive afferents to the HPA axis, including the medial preoptic area, central and medial amygdala and bed nuclei of the stria terminalis. All of these relay homeostatic information and integrate reproductive and social behaviour. Because these modalities are affected by stress in humans, a dual systems approach holds great promise in establishing further links between the neuroendocrinology of stress and the central bases of sex-dependent disorders, including psychiatric, cardiovascular and metabolic disease.

Androgens alter corticotropin releasing hormone and arginine vasopressin mRNA within forebrain sites known to regulate activity in the hypothalamic-pituitary-adrenal axis.

To reveal direct effects of androgens, independent of glucocorticoids, we studied the effects of gonadectomy (GDX) in adrenalectomized (ADX) rats with or without androgen replacement on corticotropin releasing hormone (CRH) and arginine vasopressin (AVP) mRNA expression within various forebrain sites known to regulate the hypothalamic-pituitary-adrenal axis. These included the medial parvocellular portion of the paraventricular nucleus of the hypothalamus (mp PVN), the central and medial nuclei of the amygdala and bed nuclei of the stria terminalis (BNST). In the mp PVN, ADX stimulated both CRH and AVP mRNA expression. Combined ADX + GDX inhibited only AVP, and testosterone and dihydrotestosterone (DHT) restored AVP mRNA. In the central nucleus of the amygdala, ADX decreased CRH mRNA expression, and this response was unaffected by GDX +/- testosterone or DHT replacement. In the medial amygdala, AVP mRNA expression was decreased by ADX, abolished by ADX + GDX, and restored by androgen replacement. ADX had no effect on CRH and AVP mRNA expression in the BNST. GDX + ADX, however, reduced CRH mRNA expression only within the fusiform nuclei of the BNST and reduced the number of AVP-expressing neurones in the posterior BNST. Androgen replacement reversed both responses. In summary, in ADX rats, AVP, but not CRH mRNA expression in the amygdala and mp PVN, is sensitive to GDX +/- androgen replacement. Both CRH- and AVP-expressing neurones in the BNST respond to GDX and androgen replacement, but not to ADX alone. Because androgen receptors are not expressed by hypophysiotropic PVN neurones, we conclude that glucocorticoid-independent, androgenic influences on medial parvocellular AVP mRNA expression are mediated upstream from the PVN, and may involve AVP-related pathways in the medial amygdala, relayed to and through CRH- and AVP-expressing neurones of the BNST.

Distribution of mRNAs encoding CRF receptors in brain and pituitary of rat and mouse.

Two G protein-coupled receptors have been identified that bind corticotropin-releasing factor (CRF) and urocortin (UCN) with high affinity. Hybridization histochemical methods were used to shed light on controversies concerning their localization in rat brain, and to provide normative distributional data in mouse, the standard model for genetic manipulation in mammals. The distribution of CRF-R1 mRNA in mouse was found to be fundamentally similar to that in rat, with expression predominating in the cerebral cortex, sensory relay nuclei, and in the cerebellum and its major afferents. Pronounced species differences in distribution were few, although more subtle variations in the relative strength of R1 expression were seen in several forebrain regions. CRF-R2 mRNA displayed comparable expression in rat and mouse brain, distinct from, and more restricted than that of CRF-R1. Major neuronal sites of CRF-R2 expression included aspects of the olfactory bulb, lateral septal nucleus, bed nucleus of the stria terminalis, ventromedial hypothalamic nucleus, medial and posterior cortical nuclei of the amygdala, ventral hippocampus, mesencephalic raphe nuclei, and novel localizations in the nucleus of the solitary tract and area postrema. Several sites of expression in the limbic forebrain were found to overlap partially with ones of androgen receptor expression. In pituitary, rat and mouse displayed CRF-R1 mRNA signal continuously over the intermediate lobe and over a subset of cells in the anterior lobe, whereas CRF-R2 transcripts were expressed mainly in the posterior lobe. The distinctive expression pattern of CRF-R2 mRNA identifies additional putative central sites of action for CRF and/or UCN. Constitutive expression of CRF-R2 mRNA in the nucleus of the solitary tract, and stress-inducible expression of CRF-R1 transcripts in the paraventricular nucleus may provide a basis for understanding documented effects of CRF-related peptides at a loci shown previously to lack a capacity for CRF-R expression or CRF binding. Other such "mismatches" remain to be reconciled.

A cholecystokinin-mediated pathway to the paraventricular thalamus is recruited in chronically stressed rats and regulates hypothalamic-pituitary-adrenal function.

Chronic stress alters hypothalamic-pituitary-adrenal (HPA) responses to acute, novel stress. After acute restraint, the posterior division of the paraventricular thalamic nucleus (pPVTh) exhibits increased numbers of Fos-expressing neurons in chronically cold-stressed rats compared with stress-naive controls. Furthermore, lesions of the PVTh augment HPA activity in response to novel restraint only in previously stressed rats, suggesting that the PVTh is inhibitory to HPA activity but that inhibition occurs only in chronically stressed rats. In this study, we further examined pPVTh functions in chronically stressed rats. We identified afferent projections to the pPVTh using injection of the retrograde tracer fluorogold. Of the sites containing fluorogold-labeled cells, neurons in the lateral parabrachial, periaqueductal gray, and dorsal raphe containing fluorogold also expressed cholecystokinin (CCK) mRNA. We then examined whether these CCKergic inputs to the pPVTh were involved in HPA responses to acute, novel restraint after chronic stress. We injected the CCK-B receptor antagonist PD 135,158 into the PVTh before restraint in control and chronically cold-stressed rats. ACTH responses to restraint stress were augmented by PD 135,158 only in chronically stressed rats but not in controls. In addition, CCK-B receptor mRNA expression in the pPVTh was not altered by chronic cold stress. We conclude that previous chronic stress specifically facilitates the release of CCK into the pPVTh in response to acute, novel stress. The CCK is probably secreted from neurons in the lateral parabrachial, the periaqueductal gray, and/or the dorsal raphe nuclei. Acting via CCK-B receptors in pPVTh, CCK then constrains facilitated ACTH responses to novel stress in chronically stressed but not naive rats. These results demonstrate clearly that chronic stress recruits a new set of pathways that modulate HPA responsiveness to a novel stress.

Starvation: early signals, sensors, and sequelae.

To identify the sequences of changes in putative signals, reception of these and responses to starvation, we sampled fed and starved rats at 2- to 6-h intervals after removal of food 2 h before dark. Metabolites, hormones, hypothalamic neuropeptide expression, fat depots, and leptin expression were measured. At 2 h, insulin decreased, and FFA and corticosterone (B) increased; by 4 h, leptin and glucose levels decreased. Neuropeptide Y messenger RNA (mRNA) increased 6 h after food removal and thereafter. Adrenal and plasma B did not follow ACTH and were elevated throughout, with a nadir at the dark-light transition. Leptin correlated inversely with adrenal B. Fat stores decreased during the last 12 h. Leptin mRNA in perirenal and sc fat peaked during the dark period, resembling plasma leptin in fed rats. We conclude that 1) within the first 4 h, hormonal and metabolic signals relay starvation-induced information to the hypothalamus; 2) hypothalamic neuropeptide synthesis responds rapidly to the altered metabolic signals; 3) catabolic activity quickly predominates, reinforced by elevated B, not driven by ACTH, but possibly to a minor extent by leptin, and more by adrenal neural activity; and 4) leptin secretion decreases before leptin mRNA or fat depot weight, showing synthesis-independent regulation.

Independent and overlapping effects of corticosterone and testosterone on corticotropin-releasing hormone and arginine vasopressin mRNA expression in the paraventricular nucleus of the hypothalamus and stress-induced adrenocorticotropic hormone release.

Adrenocorticotropin (ACTH) release is regulated by both glucocorticoids and androgens; however, the precise interactions are unclear. We have controlled circulating corticosterone (B) and testosterone (T) by adrenalectomy (ADX) +/- B replacement and gonadectomy (GDX) +/- T replacement, comparing these to sham-operated groups. We hoped to reveal how and where these neuroendocrine systems interact to affect resting and stress-induced ACTH secretion. ADX responses. In gonadal-intact rats, ADX increased corticotropin-releasing factor (CRH) and vasopressin (AVP) mRNA in hypothalamic parvocellular paraventricular nuclei (PVN) and ACTH in pituitary and plasma. B restored these toward normal. GDX blocked the increase in AVP but not CRH mRNA and reduced plasma, but not pituitary ACTH in ADX rats. GDX+T restored increased AVP mRNA in ADX rats, although plasma ACTH remained decreased. Stress responses. Restraint-induced ACTH responses were elevated in ADX gonadally intact rats, and B reduced these toward normal. GDX in adrenal-intact and ADX+B rats increased ACTH responses. Without B, T did not affect ACTH; together with B, T restored ACTH responses to normal. The magnitude of ACTH responses to stress was paralleled by similar effects on the number of c-fos staining neurons in the hypophysiotropic PVN. We conclude that gonadal regulation of ACTH responses to ADX is determined by T dependent effects on AVP biosynthesis, whereas CRH biosynthesis is B-dependent. Stress-induced ACTH release is not explained by B and T interactions at the PVN, but is determined by B- and T-dependent changes in drive to PVN motorneurons.

Warning! Nearby construction can profoundly affect your experiments.

This is meant to alert people to potentially major effects of construction projects on research results. Because we study the effects of stress on regulation of ACTH and corticosterone secretion and of serotonin receptors and stress on energy balance, we serve as an early warning system when things go awry. Most of our experiments include taking daily, or twice daily, measurements of rat or mouse weights and food intake as well as stress hormone levels. We are highly sensitized to environmental disruption and we've shown previously the effects of construction on stress hormones (1). However, we did not anticipate the change and disruption in energy balance that may occur in response to environmental perturbation. We provide two examples of these, below.

The inhibitory effect of testosterone on hypothalamic-pituitary-adrenal responses to stress is mediated by the medial preoptic area.

In gonadectomized (GDX) animals replaced with subcutaneous steroid implants supplying physiological levels of testosterone (T; 1-10 ng/ml), the magnitude of adrenocorticotropic hormone (ACTH) and corticosterone (B) responses to restraint was negatively correlated with the level of T replacement, reflecting the inhibitory influence of T on hypothalamic-pituitary-adrenal (HPA) responses to stress. Although T had no effect on resting-state levels of corticotropin-releasing hormone (CRH) in the median eminence, arginine vasopressin (AVP) levels were significantly lower in GDX animals replaced with higher T levels, and the magnitude of the ACTH response to restraint was strongly correlated with median eminence levels of AVP. High physiological levels of T increased glucocorticoid receptor binding in the medial preoptic area (MPOA), with no effect on mineralocorticoid receptor binding or on glucocorticoid receptor binding in other regions. Crystalline T or B implants in the MPOA significantly reduced plasma ACTH and B responses to 10 min of restraint stress compared with cholesterol-implanted controls. Moreover, B or T MPOA implants also decreased resting-state levels of AVP but not CRH in the median eminence, and these effects were correlated with ACTH responses to restraint. Finally, lesioning the MPOA blocked the inhibitory effects of high peripheral T levels on ACTH and B responses to restraint. Thus, variations in the magnitude of HPA responses to stress among males are explained in part by individual differences in circulating T levels, and the MPOA is a critical site for this effect via the inhibition of hypophysial AVP.

Changes in plasma adrenocorticotropin, corticosterone, corticosteroid-binding globulin, and hippocampal glucocorticoid receptor occupancy/translocation in rat pups in response to stress.

Pituitary-adrenal responses to stress in the neonatal rat have been reported to be substantially reduced compared to older animals (i.e. a stress hyporesponsive period). This supposed period of endocrine quiescence is characterized by reduced stress-induced increases in both plasma ACTH and corticosterone. At the same time a number of authors have noted the decreased plasma corticosteroid-binding globulin (CBG) levels of the neonate, and there is evidence for an increased percentage of free corticosterone as well as age-related changes in the volume of distribution for corticosterone. These findings suggest that the reduced CBG levels might enhance the biological significance of existing glucocorticoid levels, beyond that assumed on the basis of plasma total corticosterone levels. We examined this question by estimating hippocampal glucocorticoid receptor occupancy and 'translocation' in Day 6, Day 15, and adult animals under basal and stressful conditions. The results showed that: 1) plasma ACTH levels were elevated in Day 6 animals in response to acute exposure to ether, maternal separation, and maternal separation + ether, however, ACTH responses were substantially lower than in Day 15 or adult animals; 2) Plasma total corticosterone levels followed a similar pattern; most noteworthy was the potent glucocorticoid response in Day 15 animals to the combination of maternal separation + ether; 3) Plasma CBG levels in Day 6 animals were extremely low (< 3% adult values); by Day 15 CBG levels were about 25% of adult levels. Interestingly, maternal separation was associated with a substantial decrease in plasma CBG levels; 4) Hippocampal glucocorticoid receptor occupancy/translocation was similar at all ages under both basal and stress conditions. The only notable exception occurred during maternal separation in Day 15 animals, where the percentage of hippocampal glucocorticoid receptor occupancy/translocation was higher than that observed at any time in either Day 6 or adult animals. This finding is likely related to the decrease in plasma CBG that occurs following separation of Day 15 pups from the dam. Thus, despite the higher corticosterone level in the adult, the increase in glucocorticoid receptor occupancy/translocation was generally comparable across all ages either under basal conditions, or following stress. These receptor data underscore the importance of developmental changes in plasma CBG levels.

Stimulation of CRH-mediated ACTH secretion by central administration of neurotensin: evidence for the participation of the paraventricular nucleus.

Central administration of neurotensin (NT) stimulates hypothalamic-pituitary-adrenal (HPA) activity in freely-moving rats. Increases in adrenocorticotropin hormone (ACTH) and corticosterone (B) were observed 15 min following central NT administration and remained elevated for up to 4 h. Of the two NT fragments tested, NT1-8 and NT8-13, only NT8-13 was found to significantly elevate ACTH and B levels. Moreover, NT8-13 activated the HPA axis with a temporal profile similar to NT1-13, suggesting an interaction with the pharmacologically and molecularly characterized NT receptor. Animals pre-treated intravenously with the corticotropin-releasing hormone (CRH) antagonist, alpha-helical CRH, showed attenuated plasma ACTH and B responses to central NT administration. This indicates that CRH receptor activation is necessary for the stimulatory effects of NT on HPA function. Bilateral lesions of the paraventricular nucleus (PVN) of the hypothalamus significantly reduced NT-induced stimulation of ACTH and B release suggesting that the PVN is essential for NT's stimulatory action. Median eminence content studies indicated that acute central NT administration stimulates CRH, but not arginine vassopressin (AVP), release in animals examined 60 min following NT injection. Taken together, these findings suggest that the stimulatory effects of NT on HPA activity occur via specific NT receptors and that one site of action of NT is likely at the level of the PVN where NT elicits the release of CRH.

The role of the medial prefrontal cortex (cingulate gyrus) in the regulation of hypothalamic-pituitary-adrenal responses to stress.

In the studies reported here we have examined the role of the medial prefrontal cortex (MpFC) in regulating hypothalamic-pituitary-adrenal (HPA) activity under basal and stressful conditions. In preliminary studies we characterized corticosteroid receptor binding in the rat MpFC. The results revealed high-affinity (Kd approximately 1 nM) binding with a moderate capacity (42.9 +/- 3 fmol/mg) for 3H-aldosterone (with a 50-fold excess of cold RU28362; mineralocorticoid receptor) and high-affinity (Kd approximately 0.5-1.0 nM) binding with higher capacity (183.2 +/- 22 fmol/mg) for 3H-RU 28362 (glucocorticoid receptor). Lesions of the MpFC (cingulate gyrus) significantly increased plasma levels of both adrenocorticotropin (ACTH) and corticosterone (CORT) in response to a 20 min restraint stress. The same lesions had no effect on hormone levels following a 2.5 min exposure to ether. Implants of crystalline CORT into the same region of the MpFC produced a significant decrease in plasma levels of both ACTH and CORT with restraint stress, but again, there was no effect with ether stress. Neither MpFC lesions nor CORT implants had any consistent effect on A.M. or P.M. levels of plasma ACTH or CORT. Manipulations of MpFC function were not associated with changes in the clearance rate for CORT or in corticosteroid receptor densities in the pituitary, hypothalamus, hippocampus, or amygdala. Taken together, these findings suggest that MpFC is a target site for the negative-feedback effects of glucocorticoids on stress-induced HPA activity, and that this effect is dependent upon the nature of the stress.

Molecular basis for the development of individual differences in the hypothalamic-pituitary-adrenal stress response.

1. Several years ago, investigators described the effects of infantile handling on the development of hypothalamic-pituitary-adrenal (HPA) responses to stress in the rat. Rat pups exposed to brief periods of innocuous handling early in life showed reduced HPA responses to a wide variety of stressors, and the effect persists throughout the life of the animal. These effects are robust and provide an excellent model for understanding how early environmental stimuli, which are external to the organism, alter neural differentiation and, thus, neuroendocrine responsivity to stress. 2. This paper reviews the endocrine mechanisms affected by early handling and our current understanding of the neural transduction of environmental events and their effects at the level of the target neurons (in the hippocampus and frontal cortex). 3. In brief, handling serves to increase glucocorticoid receptor gene transcription, increasing sensitivity to glucocorticoid negative feedback regulation and, thus, altering the activity within hypothalamic corticotropin-releasing factor/vasopressin neurons. Together these changes serve to determine neuroendocrine responsivity to stress.