Corticosteroid-Binding Globulin (SERPINA6) Consolidates Sexual Dimorphism of Adult Rat Liver.

Produced by the liver, corticosteroid-binding globulin (CBG) regulates the plasma distribution and actions of glucocorticoids. A sex difference in pituitary growth hormone secretion patterns established during puberty in rats results in increased hepatic CBG production and 2-fold higher plasma corticosterone levels in females. Glucocorticoids control hepatic development and metabolic activities, and we have therefore examined how disrupting the SerpinA6 gene encoding CBG influences plasma corticosterone dynamics, as well as liver gene expression in male and female rats before and after puberty. Comparisons of corticosterone plasma clearance and hepatic uptake in adult rats, with or without CBG, indicated that CBG limits corticosterone clearance by reducing its hepatic uptake. Hepatic transcriptomic profiling revealed minor sex differences (207 differentially expressed genes) and minimal effect of CBG deficiency in 30-day-old rats before puberty. While liver transcriptomes in 60-day-old males lacking CBG remained essentially unchanged, 2710 genes were differentially expressed in wild-type female vs male livers at this age. Importantly, ∼10% of these genes lost their sexually dimorphic expression in adult females lacking CBG, including those related to cholesterol biosynthesis, inflammation, and lipid and amino acid catabolism. Another 203 genes were altered by the loss of CBG specifically in adult females, including those related to xenobiotic metabolism, circadian rhythm, and gluconeogenesis. Our findings reveal that CBG consolidates the sexual dimorphism of the rat liver initiated by sex differences in growth hormone secretion patterns and provide insight into how CBG deficiencies are linked to glucocorticoid-dependent diseases.

Analysis of a genetic region affecting mouse body weight.

Genetic factors affect an individual's risk of developing obesity, but in most cases each genetic variant has a small effect. Discovery of genes that regulate obesity may provide clues about its underlying biological processes and point to new ways the disease can be treated. Preclinical animal models facilitate genetic discovery in obesity because environmental factors can be better controlled compared with the human population. We studied inbred mouse strains to identify novel genes affecting obesity and glucose metabolism. BTBR T+ Itpr3tf/J (BTBR) mice are fatter and more glucose intolerant than C57BL/6J (B6) mice. Prior genetic studies of these strains identified an obesity locus on chromosome 2. Using congenic mice, we found that obesity was affected by a ∼316 kb region, with only two known genes, pyruvate dehydrogenase kinase 1 (Pdk1) and integrin α 6 (Itga6). Both genes had mutations affecting their amino acid sequence and reducing mRNA levels. Both genes have known functions that could modulate obesity, lipid metabolism, insulin secretion, and/or glucose homeostasis. We hypothesized that genetic variation in or near Pdk1 or Itga6 causing reduced Pdk1 and Itga6 expression would promote obesity and impaired glucose tolerance. We used knockout mice lacking Pdk1 or Itga6 fed an obesigenic diet to test this hypothesis. Under the conditions we studied, we were unable to detect an individual contribution of either Pdk1 or Itga6 to body weight. During our studies, with conditions outside our control, we were unable to reproduce some of our previous body weight data. However, we identified a previously unknown role for Pdk1 in cardiac cholesterol metabolism providing the basis for future investigations. The studies described in this paper highlight the importance and the challenge using physiological outcomes to study obesity genes in mice.

Activity in a prefrontal-periaqueductal gray circuit overcomes behavioral and endocrine features of the passive coping stress response.

The question of how the brain links behavioral and biological features of defensive responses has remained elusive. The importance of this problem is underscored by the observation that behavioral passivity in stress coping is associated with elevations in glucocorticoid hormones, and each may carry risks for susceptibility to a host of stress-related diseases. Past work implicates the medial prefrontal cortex (mPFC) in the top-down regulation of stress-related behaviors; however, it is unknown whether such changes have the capacity to buffer against the longer-lasting biological consequences associated with aversive experiences. Using the shock probe defensive burying test in rats to naturalistically measure behavioral and endocrine features of coping, we observed that the active behavioral component of stress coping is associated with increases in activity along a circuit involving the caudal mPFC and midbrain dorsolateral periaqueductal gray (PAG). Optogenetic manipulations of the caudal mPFC-to-dorsolateral PAG pathway bidirectionally modulated active (escape and defensive burying) behaviors, distinct from a rostral mPFC-ventrolateral PAG circuit that instead limited passive (immobility) behavior. Strikingly, under conditions that biased rats toward a passive coping response set, including exaggerated stress hormonal output and increased immobility, excitation of the caudal mPFC-dorsolateral PAG projection significantly attenuated each of these features. These results lend insight into how the brain coordinates response features to overcome passive coping and may be of importance for understanding how activated neural systems promote stress resilience.

Corticosteroid-binding Globulin (SERPINA6) Establishes Postpubertal Sex Differences in Rat Adrenal Development.

Encoded by SerpinA6, plasma corticosteroid-binding globulin (CBG) transports glucocorticoids and regulates their access to cells. We determined how CBG influences plasma corticosterone and adrenal development in rats during the pubertal to adult transition using CRISPR/cas9 to disrupt SerpinA6 gene expression. In the absence of CBG, total plasma corticosterone levels were ∼80% lower in adult rats of both sexes, with a greater absolute reduction in females than in males. Notably, free corticosterone and adrenocorticotropic hormone were comparable between all groups. Between 30 and 90 days of age, wild-type female rats showed increases in adrenal weight and the size of the corticosterone-producing region, the zona fasciculata (zf), in tandem with increases in plasma CBG and corticosterone concentrations, whereas no such changes were observed in males. This sex difference was lost in rats without CBG, such that adrenal growth and zf expansion were similar between sexes. The sex-specific effects of CBG on adrenal morphology were accompanied by remarkable changes in gene expression: ∼40% of the adrenal transcriptome was altered in females lacking CBG, whereas almost no effect was seen in males. Over half of the adrenal genes that normally exhibit sexually dimorphic expression after puberty were similarly expressed in males and females without CBG, including those responsible for cholesterol biosynthesis and mobilization, steroidogenesis, and growth. Rat adrenal SerpinA6 transcript levels were very low or undetectable. Thus, sex differences in adrenal growth, morphology and gene expression profiles that emerge during puberty in rats are dependent on concomitant increases in plasma CBG produced by the liver.

Sex Differences in Serotonin 5-HT 1A Receptor Responses to Repeated Restraint Stress in Adult Male and Female Rats.

BACKGROUND: Male and female rats were exposed to repeated restraint to determine how changes in serotonin (5-hydroxytryptamine; 5-HT) 1A receptors associate with stress hypothalamic-pituitary-adrenal (HPA) axis habituation. METHODS: In response to 2-hour episodes of restraint, repeated daily for 5 consecutive days, males and females displayed reliable declines in HPA output, indicated by diminished adrenocorticotropin and corticosterone secretory responses. Using the 5-HT 1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) as a pharmacological challenge for inducing hypothermia and elevations in plasma corticosterone, males displayed sensitized hypothermal responses after repeated restraint, whereas corticosterone responses to 8-OH-DPAT were enhanced in both sexes following single or repeated exposure. RESULTS: Only males showed elevations in 5-HT 1A receptor G-protein coupling responses in the dorsal raphe after repeated restraint, whereas only females showed an increase in 5-HT 1A receptor responses in the hippocampus following single or repeated exposure. G-protein coupling responses within both regions correlated positively with 5-HT 1A receptor binding capacity. Thus, despite expressing similar capacities for stress HPA axis habituation, males and females emerged from repeated restraint to show region-specific changes in 5-HT 1A receptor function that may be explained, at least in part, by changes in receptor availability. CONCLUSIONS: Based on the hypothermal and corticosteroid responses to 8-OH-DPAT, the present data suggest that stress habituation is met by an increase in the sensitivity of presynaptic 5-HT 1A receptors in males and by an increase in the sensitivity of a population of postsynaptic receptors in both sexes.

Cellular and serotonergic correlates of habituated neuroendocrine responses in male and female rats.

Male and females appear equally capable of showing habituated hypothalamic-pituitary-adrenal (HPA) axis output responses to repeated exposures of the same challenge. Whether this reflects, within males and females, common mechanisms of decreased neuronal activity within stress responding, afferents to the paraventricular hypothalamic nucleus (PVH), the final common pathway to the HPA axis, has not been examined. Here we compared in adult male and female rats the extent to which declines in HPA axis responses to repeated restraint are met by habituated cellular (Fos) responses, in addition to changes in serotonin (5-hydroxytryptamine; 5-HT) expression and signaling, which normally stimulates the HPA axis. Thus, alterations in this component of HPA axis drive could provide an underlying basis for sex differences in adaptive responses. Males and females showed reliable declines in ACTH and corticosterone responses after 10 daily episodes of repeated restraint, recapitulated, in largest part, by similar regional patterns of Fos habituation, including within the PVH, several stress sensitive cell groups of the limbic forebrain, as well as within the raphe nucleus. Serotonin staining in the dorsal raphe and terminal profiles in the forebrain continued to reflect a higher pre-synaptic capacity for the 5-HT system in females. The sexual dimorphism encountered within the lateral septum and medial preoptic area of control animals was less distinguished in the repeat condition, however, whereas 5-HT varicosities in the PVH increased after repeated restraint only in females. Relative to their singly restrained counterparts, males displayed an increase in 5-HT 1 A receptor expression in the raphe nucleus after repeated restraint, whereas females showed a decrease in 5-HT 1 A mRNA levels in the hippocampus and in the zona incerta, representing the most proximal of cell groups expressing the 5-HT 1 A receptor in the vicinity of the PVH. In conclusion, similar regional profiles of cellular habituation in males and females suggest common CNS substrates of neuroendocrine adaptation. However, this process may be met by underlying sex differences in serotonergic control, given the respective roles for pre- and postsynaptic 5-HT 1 A receptors in mediating serotonin availability and signal transfer.

Liver at the nexus of rat postnatal HPA axis maturation and sexual dimorphism.

Normal function of the hypothalamic-pituitary-adrenal (HPA) axis is critical for survival, and its development is choreographed for age-, sex- and context-specific actions. The liver influences HPA ontogeny, integrating diverse endocrine signals that inhibit or activate its development. This review examines how developmental changes in the expression of genes in the liver coordinate postnatal changes in multiple endocrine systems that facilitate the maturation and sexual dimorphism of the rat HPA axis. Specifically, it examines how the ontogeny of testicular androgen production, somatostatin-growth hormone activities, and hypothalamic-pituitary-thyroid axis activity intersect to influence the hepatic gene expression of insulin-like growth factor 1, corticosteroid-binding globulin, thyroxine-binding globulin, 11ß-hydroxysteroid dehydrogenase type 1 and 5α-reductase type 1. The timing of such molecular changes vary between mammalian species, but they are evolutionarily conserved and are poised to control homeostasis broadly, especially during adversity. Importantly, with the liver as their nexus, these diverse endocrine systems establish the fundamental organization of the HPA axis throughout postnatal development, and thereby ultimately determine the actions of glucocorticoids during adulthood.

Prefrontal-Bed Nucleus Circuit Modulation of a Passive Coping Response Set.

One of the challenges facing neuroscience entails localization of circuits and mechanisms accounting for how multiple features of stress responses are organized to promote survival during adverse experiences. The rodent medial prefrontal cortex (mPFC) is generally regarded as a key site for cognitive and affective information processing, and the anteroventral bed nuclei of the stria terminalis (avBST) integrates homeostatic information from a variety of sources, including the mPFC. Thus, we proposed that the mPFC is capable of generating multiple features (endocrine, behavioral) of adaptive responses via its influence over the avBST. To address this possibility, we first optogenetically inhibited input to avBST from the rostral prelimbic cortical region of mPFC and observed concurrent increases in immobility and hypothalamo-pituitary-adrenal (HPA) output in male rats during tail suspension, whereas photostimulation of this pathway decreased immobility during the same challenge. Anatomical tracing experiments confirmed projections from the rostral prelimbic subfield to separate populations of avBST neurons, and from these to HPA effector neurons in the paraventricular hypothalamic nucleus, and to aspects of the midbrain periaqueductal gray that coordinate passive defensive behaviors. Finally, stimulation and inhibition of the prelimbic-avBST pathway, respectively, decreased and increased passive coping in the shock-probe defensive burying test, without having any direct effect on active coping (burying) behavior. These results define a new neural substrate in the coordination of a response set that involves the gating of passive, rather than active, coping behaviors while restraining neuroendocrine activation to optimize adaptation during threat exposure.SIGNIFICANCE STATEMENT The circuits and mechanisms accounting for how multiple features of responses are organized to promote adaptation have yet to be elucidated. Our report identifies a prefrontal-bed nucleus pathway that organizes a response set capable of gating passive coping behaviors while concurrently restraining neuroendocrine activation during exposure to inescapable stressors. These data provide insight into the central organization of how multiple features of responses are integrated to promote adaptation during adverse experiences, and how disruption in one neural pathway may underlie a broad array of maladaptive responses in stress-related psychiatric disorders.

Pannexin1 knockout and blockade reduces ischemic stroke injury in female, but not in male mice.

The membrane channel Pannexin 1 (Panx1) mediates apoptotic and inflammatory signaling cascades in injured neurons, responses previously shown to be sexually dimorphic under ischemic conditions. We tested the hypothesis that Panx1 plays an underlying role in mediating sex differences in stroke outcome responses. Middle-aged, 8-9 month old male and female wild type and Panx1 KO mice were subjected to permanent middle cerebral artery (MCA) occlusion, and infarct size and astrocyte and microglia activation were assessed 4 days later. The sexually dimorphic nature of Panx1 deletion was also explored by testing the effect of probenecid a known Panx1 blocker to alter stroke volume. Panx1 KO females displayed significantly smaller infarct volumes (~ 50 % reduction) compared to their wild-type counterparts, whereas no such KO effect occurred in males. This sex-specific effect of Panx1 KO was recapitulated by significant reductions in peri-infarct inflammation and astrocyte reactivity, as well as smaller infarct volumes in probenecid treated females, but not males. Finally, females showed overall, higher Panx1 protein levels than males under ischemic conditions. These findings unmask a deleterious role for Panx1 in response to permanent MCA occlusion, that is unique to females, and provide several new frameworks for understanding sex differences in stroke outcome.

Chronic stress and brain plasticity: Mechanisms underlying adaptive and maladaptive changes and implications for stress-related CNS disorders.

Stress responses entail neuroendocrine, autonomic, and behavioral changes to promote effective coping with real or perceived threats to one's safety. While these responses are critical for the survival of the individual, adverse effects of repeated exposure to stress are widely known to have deleterious effects on health. Thus, a considerable effort in the search for treatments to stress-related CNS disorders necessitates unraveling the brain mechanisms responsible for adaptation under acute conditions and their perturbations following chronic stress exposure. This paper is based upon a symposium from the 2014 International Behavioral Neuroscience Meeting, summarizing some recent advances in understanding the effects of stress on adaptive and maladaptive responses subserved by limbic forebrain networks. An important theme highlighted in this review is that the same networks mediating neuroendocrine, autonomic, and behavioral processes during adaptive coping also comprise targets of the effects of repeated stress exposure in the development of maladaptive states. Where possible, reference is made to the similarity of neurobiological substrates and effects observed following repeated exposure to stress in laboratory animals and the clinical features of stress-related disorders in humans.

Central vasopressin V1A receptor blockade alters patterns of cellular activation and prevents glucocorticoid habituation to repeated restraint stress exposure.

Our previous experiments implicated a role for the arginine vasopressin (AVP) V1A receptor subtype in mediating the normal decline (habituation) of hypothalamic-pituitary-adrenal (HPA) axis responses to repeated restraint exposure. To explore pathways mediating the endogenous effects of central AVP on stress HPA axis habituation, here we compared cellular (Fos) and hormone responses in male rats receiving chronic icv infusion of vehicle or a V1A receptor antagonist that began 7 d before stress testing, continued through the duration of acute and repeat restraint exposure. As a group, rats with V1A antagonism displayed a modest reduction in ACTH habituation, whereas the decline in corticosterone was completely prevented. V1A antagonized rats also showed reduced evidence of habituated Fos responses in the paraventricular nucleus of the hypothalamus, medial amygdala, and within the anterior division of the bed nucleus of the stria terminalis. Based on these cellular and neuroendocrine responses, we then examined whether repeated restraint is reflected by changes in V1A receptor binding. Relative to stress naïve animals, repeatedly exposed rats showed lower levels of V1A binding in the dentate gyrus of the hippocampus, thalamus and central amygdala, but higher levels in the septum and anterior BST. Taken together, these findings suggest that AVP may act within multiple targets to regulate the normal decline in stress-induced drive to the HPA axis, and that this may involve the net of V1A receptor stimulatory and inhibitory influences on neuroendocrine habituation.

Sex differences in the HPA axis.

The hypothalamic-pituitary-adrenal (HPA) axis is a major component of the systems that respond to stress, by coordinating the neuroendocrine and autonomic responses. Tightly controlled regulation of HPA responses is critical for maintaining mental and physical health, as hyper- and hypo-activity have been linked to disease states. A long history of research has revealed sex differences in numerous components of the HPA stress system and its responses, which may partially form the basis for sex disparities in disease development. Despite this, many studies use male subjects exclusively, while fewer reports involve females or provide direct sex comparisons. The purpose of this article is to present sex comparisons in the functional and molecular aspects of the HPA axis, through various phases of activity, including basal, acute stress, and chronic stress conditions. The HPA axis in females initiates more rapidly and produces a greater output of stress hormones. This review focuses on the interactions between the gonadal hormone system and the HPA axis as the key mediators of these sex differences, whereby androgens increase and estrogens decrease HPA activity in adulthood. In addition to the effects of gonadal hormones on the adult response, morphological impacts of hormone exposure during development are also involved in mediating sex differences. Additional systems impinging on the HPA axis that contribute to sex differences include the monoamine neurotransmitters norepinephrine and serotonin. Diverse signals originating from the brain and periphery are integrated to determine the level of HPA axis activity, and these signals are, in many cases, sex-specific.

Sex differences in serotonin (5-HT) 1A receptor regulation of HPA axis and dorsal raphe responses to acute restraint.

The serotonin (5-HT) 1A receptor subtype has been implicated as an important mediator for the stimulatory influence of serotonin on stress hypothalamic-pituitary-adrenal (HPA) activity, at least in males. Females show greater HPA axis responses to stress compared to males. To determine the nature by which the 5-HT 1A receptor contributes to the sex difference in stress, we examined neuroendocrine and cellular (Fos) responses in male and female rats receiving systemic injections of the 5-HT 1A receptor antagonist, WAY 100635, prior to acute restraint exposure. WAY decreased the corticosterone response in males, but not in females. In the paraventricular nucleus of the hypothalamus (PVH), WAY produced similar decrements in the restraint-induced activation (Fos) of neuroendocrine neurons in males and females. In contrast to the PVH, WAY administration increased total Fos activation in the dorsal raphe nucleus, but only in males. WAY also provoked higher Fos responses within subsets of dorsal raphe cells identified as serotonergic (tryptophan hydroxylase-, TPH-ir) in both males and females. These data provide evidence to suggest a differential influence of presynaptic 5-HT 1A receptors to regulate the stress-induced recruitment of non-serotonergic dorsal raphe neurons in males and females. At present, we cannot rule out a possible role for estrogen in females to alter 5-HT outflow to the HPA axis. There was a negative correlation between estrogen and Fos responses within TPH-positive cells in the dorsal raphe of WAY-administered females, whereas a positive correlation was found between estrogen and 5-HT 1A mRNA expression localized to the region of the zona incerta in close proximity to the PVH. As the raphe complex and 5-HT system impinge on several central autonomic, behavioral and neuroendocrine control systems, the current findings provide an important framework for future studies directed at sex differences in adaptive homeostatic responses.

Central vasopressin V1A receptor blockade impedes hypothalamic-pituitary-adrenal habituation to repeated restraint stress exposure in adult male rats.

Previous studies suggest that central arginine vasopressin (AVP) signaling can inhibit the hypothalamic-pituitary-adrenal (HPA) axis. To test a role for the AVP V1A receptor in stress HPA axis habituation, adult male rats were exposed to 5 consecutive days of 3 h restraint with or without continuous intracerebroventricular infusion of the V1A receptor antagonist d(CH2)5Tyr(Me)AVP (10 µg/day). Assessment of neuropeptide expression and HPA output under basal conditions revealed no effects of V1A receptor antagonism in stress naive animals. Between the first and last day of restraint exposure, controls showed marked declines in ACTH and corticosterone responses, and maintained plasma concentrations of testosterone. In contrast, V1A receptor antagonized animals displayed significantly smaller declines in ACTH and corticosterone responses, and a decrease in plasma testosterone. Despite their reduced expression of HPA axis habituation, antagonized animals continued to show stress-induced increases in AVP mRNA in the hypothalamic paraventricular nucleus and bed nucleus of the stria terminalis, and even higher levels of AVP expression in the medial amygdala relative to controls. The data leave open the nature and extent to which these and other AVP-containing pathways are recruited during repeated restraint, but nevertheless reveal a critical role for central V1A receptors in stress adaptation. As the effects of V1A receptor antagonism were restricted to the repeated restraint condition, we conclude that normal adaptation to stress involves a shift toward enhanced AVP utilization and/or V1A receptor signaling.

Acute stress induces selective alterations in cost/benefit decision-making.

Acute stress can exert beneficial or detrimental effects on different forms of cognition. In the present study, we assessed the effects of acute restraint stress on different forms of cost/benefit decision-making, and some of the hormonal and neurochemical mechanisms that may underlie these effects. Effort-based decision-making was assessed where rats chose between a low effort/reward (1 press=2 pellets) or high effort/reward option (4 pellets), with the effort requirement increasing over 4 blocks of trials (2, 5, 10, and 20 lever presses). Restraint stress for 1 h decreased preference for the more costly reward and induced longer choice latencies. Control experiments revealed that the effects on decision-making were not mediated by general reductions in motivation or preference for larger rewards. In contrast, acute stress did not affect delay-discounting, when rats chose between a small/immediate vs larger/delayed reward. The effects of stress on decision-making were not mimicked by treatment with physiological doses of corticosterone (1-3 mg/kg). Blockade of dopamine receptors with flupenthixol (0.25 mg/kg) before restraint did not attenuate stress-induced effects on effort-related choice, but abolished effects on choice latencies. These data suggest that acute stress interferes somewhat selectively with cost/benefit evaluations concerning effort costs. These effects do not appear to be mediated solely by enhanced glucocorticoid activity, whereas dopaminergic activation may contribute to increased deliberation times induced by stress. These findings may provide insight into impairments in decision-making and anergia associated with stress-related disorders, such as depression.

Postnatal aromatase blockade increases c-fos mRNA responses to acute restraint stress in adult male rats.

Recent evidence suggests that the aromatization of testosterone to estrogen is important for the organizing effects of neonatal testosterone on neuroendocrine responses to acute challenges. However, the extent to which neonatal inhibition of aromatase alters the stress-induced activation of neural pathways has not been examined. Here we assessed central patterns of c-fos mRNA induced by 30 min of restraint in 65-d-old adult male rats that were implanted with sc capsules of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD), introduced within 12 h of birth and removed on d 21 of weaning. Neonatal ATD decreased the expression of arginine vasopressin within extrahypothalamic regions in adults, confirming reduced estrogen exposure during development. As adults, ATD-treated animals showed higher corticosterone responses at 30 min of restraint exposure compared with control animals as well as higher c-fos expression levels in the paraventricular nucleus of the hypothalamus. ATD treatment also increased stress-induced c-fos within several limbic regions of the forebrain, in addition to areas involved in somatosensory processing. Based on these results, we propose that the conversion of testosterone to estrogen during the neonatal period exerts marked, system-wide effects to organize adult neuroendocrine responses to homeostatic threat.

Recruitment of prefrontal cortical endocannabinoid signaling by glucocorticoids contributes to termination of the stress response.

The mechanisms subserving the ability of glucocorticoid signaling within the medial prefrontal cortex (mPFC) to terminate stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis are not well understood. We report that antagonism of the cannabinoid CB(1) receptor locally within the mPFC prolonged corticosterone secretion following cessation of stress in rats. Mice lacking the CB(1) receptor exhibited a similar prolonged response to stress. Exposure of rats to stress produced an elevation in the endocannabinoid 2-arachidonoylglycerol within the mPFC that was reversed by pretreatment with the glucocorticoid receptor antagonist RU-486 (20 mg/kg). Electron microscopic and electrophysiological data demonstrated the presence of CB(1) receptors in inhibitory-type terminals impinging upon principal neurons within layer V of the prelimbic region of the mPFC. Bath application of corticosterone (100 nm) to prefrontal cortical slices suppressed GABA release onto principal neurons in layer V of the prelimbic region, when examined 1 h later, which was prevented by application of a CB(1) receptor antagonist. Collectively, these data demonstrate that the ability of stress-induced glucocorticoid signaling within mPFC to terminate HPA axis activity is mediated by a local recruitment of endocannabinoid signaling. Endocannabinoid activation of CB(1) receptors decreases GABA release within the mPFC, likely increasing the outflow of the principal neurons of the prelimbic region to contribute to termination of the stress response. These data support a model in which endocannabinoid signaling links glucocorticoid receptor engagement to activation of corticolimbic relays that inhibit corticosterone secretion.

Androgen receptors in the posterior bed nucleus of the stria terminalis increase neuropeptide expression and the stress-induced activation of the paraventricular nucleus of the hypothalamus.

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.

Postnatal blockade of androgen receptors or aromatase impair the expression of stress hypothalamic-pituitary-adrenal axis habituation in adult male rats.

Sex steroid hormones during development permanently alter, or organize, the brain and behavior, while during adulthood they act to reversibly modulate, or activate, physiology and behavior. Testosterone exerts both organizational and activational effects on the magnitude of the hypothalamic-pituitary-adrenal (HPA) axis response to acute stress. What has never been approached is how testosterone can organize habituation of the HPA axis, in which stress induced elevations in ACTH and corticosterone release decline over repeated exposures to the same stimulus. In the current study we examined HPA responses to repeated psychogenic stress in 65-day-old, adult male rats that received subcutaneous capsules containing the antiandrogen flutamide or the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD), introduced within 12h of birth and removed on day 21 of weaning. An additional group of castrated, adult male rats were used to differentiate organizational from activational effects of testosterone. All treatment groups displayed smaller declines in ACTH in response to repeated restraint compared to control animals. Remarkably, the normal decline in corticosterone failed to occur in flutamide- and ATD-treated animals. By contrast, males that were castrated as adults showed a significant reduction in corticosterone after repeated stress. Taken together, these findings underscore an organizing influence of both androgen receptors and estrogen conversion on HPA habituation to repeated psychogenic stress, which appears to occur independent of the activational effects of testosterone.

The medial preoptic nucleus integrates the central influences of testosterone on the paraventricular nucleus of the hypothalamus and its extended circuitries.

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.