Victor Viau

The posterior bed nuclei of the stria terminalis (BST) are important neural substrate for relaying limbic influences to the paraventricular nucleus (PVN) of the hypothalamus to inhibit hypothalamic-pituitary-adrenal (HPA) axis responses to emotional stress. Androgen receptor-expressing cells within the posterior BST have been identified as projecting to the PVN region. To test a role for androgen receptors in the posterior BST to inhibit PVN motor neurons, we compared the effects of the non-aromatizable androgen dihydrotestosterone (DHT), the androgen receptor antagonist hydroxyflutamide (HF), or a combination of both drugs implanted unilaterally within the posterior BST. Rats bearing unilateral implants were analyzed for PVN Fos induction in response to acute-restraint stress and relative levels of corticotrophin-releasing hormone and arginine vasopressin (AVP) mRNA. Glutamic acid decarboxylase (GAD) 65 and GAD 67 mRNA were analyzed in the posterior BST to test a local involvement of GABA. There were no changes in GAD expression to support a GABA-related mechanism in the BST. For PVN neuropeptide expression and Fos responses, basic effects were lateralized to the sides of the PVN ipsilateral to the implants. However, opposite to our expectations of an inhibitory influence of androgen receptors in the posterior BST, PVN AVP mRNA and stress-induced Fos were augmented in response to DHT and attenuated in response to HF. These results suggest that a subset of androgen receptor-expressing cells within the posterior BST region may be responsible for increasing the biosynthetic capacity and stress-induced drive of PVN motor neurons.

Testosterone contributes to sex differences in hypothalamic–pituitary–adrenal (HPA) function in humans and rodents, but the central organization of this regulation remains unclear. The medial preoptic nucleus (MPN) stands out as an important candidate in this regard because it contains androgen receptors and projects to forebrain nuclei integrating cognitive–affective information and regulating HPA responses to homeostatic threat. These include the HPA effector neurons of the paraventricular nucleus (PVN) of the hypothalamus, medial amygdala, and lateral septum. To test the extent to which androgen receptors in the MPN engage these cell groups, we compared in adult male rats the effects of unilateral microimplants of testosterone and the androgen receptor antagonist hydroxyflutamide into the MPN on acute restraint induced activation and/or neuropeptide expression levels. The basic effects of these implants were lateralized to the sides of the nuclei ipsilateral to the implants. Testosterone, but not hydroxyflutamide implants, decreased stress-induced Fos and arginine vasopressin (AVP) heteronuclear RNA expression in the PVN, as well as Fos expression in the lateral septum. In unstressed animals, AVP mRNA expression in the PVN decreased and increased in response to testosterone and hydroxflutamide MPN implants, respectively. The differential influences of these implants on AVP mRNA expression were opposite in the medial amygdala. These results confirm a role for androgen receptors in the MPN to concurrently modulate neuropeptide expression and activational responses in the PVN and its extended circuitries. This suggests that the MPN is capable of bridging converging limbic influences to the HPA axis with changes in gonadal status.

Secretion of glucocorticoid hormones during stress produces an array of physiological changes that are adaptive and beneficial in the short term. In the face of repeated stress exposure, however, habituation of the glucocorticoid response is essential as prolonged glucocorticoid secretion can produce deleterious effects on metabolic, immune, cardiovascular, and neurobiological function. Endocannabinoid signaling responds to and regulates the activity of the hypothalamic–pituitary–adrenal (HPA) axis that governs the secretion of glucocorticoids; however, the role this system plays in adaptation of the neuroendocrine response to repeated stress is not well characterized. Herein, we demonstrate a divergent regulation of the two endocannabinoid ligands, N -arachidonylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG), following repeated stress such that AEA content is persistently decreased throughout the corticolimbic stress circuit, whereas 2-AG is exclusively elevated within the amygdala in a stress-dependent manner. Pharmacological studies demonstrate that this divergent regulation of AEA and 2-AG contribute to distinct forms of HPA axis habituation. Inhibition of AEA hydrolysis prevented the development of basal hypersecretion of corticosterone following repeated stress. In contrast, systemic or intra-amygdalar administration of a CB ₁ receptor antagonist before the final stress exposure prevented the repeated stress-induced decline in corticosterone responses. The present findings demonstrate an important role for endocannabinoid signaling in the process of stress HPA habituation, and suggest that AEA and 2-AG modulate different components of the adrenocortical response to repeated stressor exposure.

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.

Androgen receptors are distributed throughout the central nervous system and are contained by a variety of nuclei that are known to project to or regulate the paraventricular nucleus (PVN) of the hypothalamus, the final common pathway by which the brain regulates the hypothalamic–pituitary–adrenal (HPA) response to homeostatic threat. Here we characterized androgen receptor staining within cells identified as projecting to the PVN in male rats bearing iontophoretic or crystalline injections of the retrograde tracer FluoroGold aimed at the caudal two‐thirds of the nucleus, where corticotropin‐releasing hormone‐expressing neurons are amassed. Androgen receptor (AR) and FluoroGold (FG) double labeling was revealed throughout the limbic forebrain, including scattered numbers of cells within the anterior and posterior subdivisions of the bed nuclei of the stria terminalis; the medial zone of the hypothalamus, including large numbers of AR‐FG‐positive cells within the anteroventral periventricular and medial preoptic cell groups. Strong and consistent colabeling was also revealed throughout the hindbrain, predominantly within the periaqueductal gray and the lateral parabrachial nucleus, and within various medullary cell groups identified as catecholaminergic, predominantly C1 and A1 neurons of the ventral medulla. These connectional data predict that androgens can act on a large assortment of multimodal inputs to the PVN, including those involved with the processing of various types of sensory and limbic information, and provide an anatomical framework for understanding how gonadal status could contribute to individual differences in HPA function. J. Comp. Neurol. 503:717–740, 2007.

Activation of the hypothalamic–pituitary–adrenal (HPA) axis is initiated by neurosecretory neurons residing within the medial parvicellular part of the hypothalamic paraventricular nucleus (PVN). Despite the potency by which sex steroids operate on HPA and medial parvocellular responses to stress, previous topographic and phenotypic studies suggest that gonadal steroid hormone receptors are scarcely, if at all, expressed by PVN neurons controlling anterior pituitary corticotropes. Guided by the pattern of retrograde accumulation of fluorogold, we used a direct connectional approach to define the distribution of androgen receptors (AR) and estrogen‐beta receptors (ER‐β) within populations of neurosecretory vs. nonneurosecretory neurons in the PVN. Juxtaposition of AR‐immunoreactivity (ir) and ER‐β mRNA to the pattern of intravenous fluorogold labeling showed these steroid hormone receptors to be concentrated within portions of the PVN devoid of neurosecretory neurons. Superimposing receptor profiles onto the pattern of spinal retrograde labeling confirmed a selective distribution of AR‐ir within autonomic‐related cells of the medial parvocellular division, including its dorsal, lateral, and ventral medial components. ER‐β mRNA expression was likewise concentrated within regions accumulating spinal tracer, highest within the ventral aspect of the PVN. These results indicate a direct influence of gonadal hormones on preautonomic effector neurons and remain in keeping with an indirect influence of androgens on adrenocorticotropin‐regulating neurons in the PVN. J. Comp. Neurol. 499:911–923, 2006.

Individual variations in hypothalamic-pituitary-adrenal (HPA) function are most evident at or beyond the time of puberty, when marked changes in sex steroid release occur. To explore the nature by which gender differences in HPA function emerge we examined in prepubertal (∼30-d-old) and postpubertal (∼60-d-old) male and female rats HPA activity under basal conditions and in response to 30 min of restraint. Within the ACTH-regulating, medial parvocellular portion of the paraventricular nucleus, restraint-induced Fos protein and arginine vasopressin heteronuclear RNA were lower in 60- than in 30-d-old males. No such age-related shift in the response of these synaptic and transcriptional markers of cellular activation occurred in female rats. Basal CRH mRNA expression levels in the paraventricular nucleus increased with age in female but not male rats. Conversely, only male rats showed an age-related increase in basal CRH mRNA in the central amygdala, suggesting that neuronal and neurosecretory CRH-expressing cell types are subject to different pubertal and gender influences. We conclude that gonadal regulation of the HPA axis develops via distinct mechanisms in males and females. Puberty-related shifts in parvocellular neurosecretory function in males are emphasized by stress-induced shifts in neuronal activation, whereas biosynthetic alterations dominate in female rats.

Hypothalamic-pituitary-adrenal (HPA) activity is governed by glucocorticoid negative feedback and the magnitude of this signal is determined, in part, by variations in plasma corticosteroid-binding globulin (CBG) capacity. Here, in gonadectomized male rats we examine the extent to which different testosterone replacement levels impact on CBG and HPA function. Compared with gonadectomized rats with low testosterone replacement ( approximately 2 ng/ml), plasma adrenocorticotropin and beta-endorphin/beta-lipotropin responses to restraint stress were reduced in gonadectomized rats with high testosterone replacement ( approximately 5 ng/ml). Plasma CBG levels also varied negatively as a function of testosterone concentration. Moreover, glucocorticoid receptor binding in the liver was elevated by higher testosterone replacement, suggesting that testosterone acts to enhance glucocorticoid suppression of CBG synthesis. Since pituitary intracellular CBG (or transcortin) is derived from plasma, this prompted us to examine whether transcortin binding was similarly responsive to different testosterone replacement levels. Transcortin binding was lower in gonadectomized rats with high plasma testosterone replacement ( approximately 7 ng/ml) than in gonadectomized rats with low testosterone replacement ( approximately 2 ng/ml). This testosterone-dependent decrease in pituitary transcortin was associated, in vitro, with an enhanced nuclear uptake of corticosterone. These findings indicate that the inhibitory effects of testosterone on corticotrope responses to stress may be linked to decrements in plasma and intrapituitary CBG. This could permit greater access of corticosterone to its receptors and enhance glucocorticoid feedback regulation of ACTH release and/or proopiomelanocortin processing.

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 µg 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 α₁ adrenoreceptor antagonist prazosin. Finally, plasma ACTH concentrations following central injection of the α₁ 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 α₁ adrenoreceptor.

To gauge the strength by which the testes influence stress-induced activation of neurosecretory neurons in the paraventricular nucleus, we studied within medial parvocellular neurons the effects of gonadectomy on restraint-induced Fos-immunoreactivity and on CRH and arginine vasopressin (AVP) heteronuclear (hn) RNA expression levels. Relative to intact male rats (sham-gonadectomized), gonadectomized rats showed a significantly greater number of medial parvocellular neurons recruited to express Fos protein evident at 0.5 h and from 1-4 h after the onset of 30-min restraint exposure. Restraint provoked a transient increase in hnCRH levels that was maximal at the end of restraint and this was significant only in gonadectomized rats. Both intact and gonadectomized rats displayed an increase in AVP hnRNA expression levels in response to restraint exposure; however, it was significantly greater in gonadectomized rats. All of these responses were accompanied by a higher corticosterone response in gonadectomized compared with intact rats and negatively correlated with plasma testosterone concentrations, with the exception of stress-induced CRH transcription. These findings indicate an inhibitory role for testosterone on stress-induced indexes of synaptic (Fos) and transcriptional (AVP hnRNA) activation among hypophysiotropic paraventricular neurons and provide meaningful end points with which to pursue how and where androgens operate on stress-related input to the paraventricular nucleus motor neurons.