Corticotropin-releasing hormone (CRH)-binding protein: reduction in the adrenocorticotropin-releasing activity of placental but not hypothalamic CRH

This study is the first to use CRH-binding protein (CRH-BP) purified from human plasma to investigate how the CRH-BP affects the physiological activity of CRH. After incubation at 37 C for 15 min, purified CRH-BP reduced the ACTH-releasing activity of synthetic human (h) CRH in a dose-dependent fashion; using hCRH and CRH-BP at concentrations commonly found in late gestational maternal plasma, (1.5 and 100 ng/mL, respectively) an average 76% reduction in ACTH release was obtained. No effect was observed under the same conditions on ACTH release induced by ovine (o) CRH, which does not bind to CRH-BP. Kinetic estimations are presented to show that the extent of binding of CRH to its BP (and, hence, the reduction in its bioactivity) depends on the time available for binding and the concentration of reactants. Equilibrium between CRH and its BP at the concentrations used in the bioactivity studies take place within 400 s. Since long term secretion of placental CRH into the peripheral circulation occurs during the third trimester of pregnancy, we suggest that the presence of circulating CRH BP may partly explain how markedly elevated plasma levels of CRH coexist with normal ACTH levels at this time. We also propose that stress-induced CRH release will not be similarly quenched by the CRH-BP in the hypothalamic portal system, as the concentration of CRH released will be high, and the exposure time before reaching pituitary corticotropes will be low. Using pituitary cells constantly bathed in BP premixed with hCRH or BP alone (to mimic the situations in pregnancy and nonpregnancy, respectively), we show that short concentrated pulses of synthetic CRH (10 ng in 5 s) or rat stalk median eminence extract (one tenth stalk median eminence in 5 s) retain their ability to induce ACTH secretion despite the presence of CRH-BP. It is, thus, possible that CRH can still exert its role as a stress hormone in late gestation.     

Agouti-related protein containing nerve terminals innervate thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus.

Gene expression for agouti-related protein (AGRP), an endogenous antagonist of melanocortin receptors, has been localized to the hypothalamic arcuate nucleus, where it colocalizes with neuropeptide Y (NPY). Having reported that the NPY innervation of hypophysiotropic TRH neurons in the hypothalamic paraventricular nucleus (PVN) originates primarily from NPY-producing neurons in the arcuate nucleus, here we examined the possibility that TRH neurons in the PVN are similarly innervated by AGRP nerve terminals. Using immunohistochemistry, AGRP-containing cell bodies were found almost exclusively in the arcuate nucleus, but their projections were distributed widely in the hypothalamus, most conspicuously in the paraventricular (PVN), arcuate and dorsomedial nuclei, and the posterior hypothalamic area. Ablation of the arcuate nucleus by the neonatal administration of monosodium glutamate obliterated nearly all AGRP-immunoreactivity in the hypothalamus. In the PVN, double-labeling light and electron microscopic immunohistochemistry revealed that TRH neurons receive dense innervation by AGRP nerve terminals, with the frequent occurrence of axosomatic and axodendritic synapses (mainly of the symmetrical type). These findings provide morphological basis to hypothesize a role for AGRP in the arcuato-paraventricular pathway, in the down-regulation of the hypothalamic-pituitary-thyroid axis, which occurs as an adaptive response to starvation.     

Corticotropin-releasing hormone (CRH) requirement in Clostridium difficile toxin A-mediated intestinal inflammation

Clostridium difficile, the causative agent of antibiotic-associated colitis, mediates inflammatory diarrhea by releasing toxin A, a potent 308-kDa enterotoxin. Toxin A-induced inflammatory diarrhea involves many steps, including mucosal release of substance P (SP) corticotropin-releasing hormone (CRH) and neutrophil transmigration. Here we demonstrate that, compared with wild type, mice genetically deficient in CRH (Crh(-/-)) have dramatically reduced ileal fluid secretion, epithelial cell damage, and neutrophil transmigration 4 h after intraluminal toxin A administration. This response is associated with diminished mucosal activity of the neutrophil enzyme myeloperoxidase compared with that of wildtype mice. In wild-type mice, toxin A stimulates an increase in intestinal SP content compared with buffer administration. In contrast, toxin A administration in Crh(-/-) mice fails to result in an increased SP content. Moreover, immunohistochemical experiments showed that CRH and SP are colocalized in some enteric nerves of wild-type mice, and this colocalization is more evident after toxin A administration. These results provide direct evidence for a major proinflammatory role for CRH in the pathophysiology of enterotoxin-mediated inflammatory diarrhea and indicate a SP-linked pathway.     

Corticotropin-releasing hormone (CRH) depresses n-methyl-D-aspartate receptor-mediated current in cultured rat hippocampal neurons via CRH receptor type 1

CRH, the primary regulator of the neuroendocrine responses to stress, has been shown to modulate synaptic efficacy and the process of learning and memory in hippocampus. However, effects of CRH on N-methyl-d-aspartate (NMDA) receptor, the key receptor for synaptic plasticity, remain unclear. In primary cultured hippocampal neurons, using the technique of whole-cell patch-clamp recordings, we found that CRH (1 pmol/liter to 10 nmol/liter) inhibited NMDA-induced currents in a dose-dependent manner. This effect was reversed by the CRH receptor type 1 (CRHR1) antagonist antalarmin but not by the CRHR2 antagonist astressin-2B, suggesting that CRHR1 mediated the inhibitory effect of CRH. Investigations on the signaling pathways of CRH showed that CRH dose-dependently induced phosphorylated phospholipase C (PLC)-beta3 expression and increased intracellular cAMP content in these cells. Blocking PLC activity with U73122 prevented CRH-induced depression of NMDA current, whereas blocking protein kinase A (H89) and adenylate cyclase (SQ22536) failed to affect the CRH-induced depression of NMDA current. Application of inositol-1,4,5-triphosphate receptor (IP(3)R) antagonist, Ca(2+) chelators or protein kinase C (PKC) inhibitors also mainly blocked CRH-induced depression of NMDA currents, suggesting involvement of PLC/IP(3)R/Ca(2+)and PLC/PKC signaling pathways in CRH down-regulation of NMDA receptors. Our results suggest that CRH may exert neuromodulatory actions on hippocampus through regulating NMDA receptor function.     

Corticotropin-releasing hormone (CRH)-induced thyrotropin release is directly mediated through CRH receptor type 2 on thyrotropes

CRH is known as the main stimulator of ACTH release. In representatives of all nonmammalian vertebrates, CRH has also been shown to induce TSH secretion, acting directly at the level of the pituitary. We have investigated which cell types and receptors are involved in CRH-induced TSH release in the chicken (Gallus gallus). Because a lack of CRH type 1 receptors (CRH-R1) on the chicken thyrotropes has been previously reported, two hypotheses were tested using in situ hybridization and perifusion studies: 1) TSH secretion might be induced in a paracrine way involving melanocortins from the corticotropes; and 2) thyrotropes might express another type of CRH-R. For the latter, we have cloned a partial cDNA encoding the chicken CRH-R2. Neither alpha-melanotropin (alpha-MSH) nor its powerful analog Nle4,d-Phe7-MSH could mimic the in vitro TSH-releasing effect of ovine CRH. The nonselective melanocortin receptor blocker SHU91199 did not influence CRH- or TRH-induced TSH secretion. On the other hand, we have found that thyrotropes express CRH-R2 mRNA. The involvement of this CRH receptor in the response of thyrotropes to CRH was further confirmed by the fact that TSH release was stimulated by human urocortin III, a CRH-R2-specific agonist, whereas the TSH response to CRH was completely blocked by the CRH-R blocker astressin and the CRH-R2-specific antagonist antisauvagine-30. We conclude that CRH-induced TSH secretion is mediated by CRH-R2 expressed on thyrotropes.     

Corticotropin-releasing hormone (CRH) directly stimulates corticosterone secretion by the rat adrenal gland.

Corticotropin-releasing hormone (CRH) acute ip administration (10 micrograms) significantly increased the blood concentration of corticosterone (B) in hypophysectomized rats, without inducing any rise in the level of circulating ACTH. CRH (10(-6) M) did not affect B production by isolated rat adrenocortical cells, but notably enhanced that by adrenal slices including both cortex and medulla. This last effect of CRH was blocked by corticotropin inhibiting peptide (CIP), at a concentration (10(-6) M) which was found to completely annul B response of adrenal slices to ACTH (10(-8) M). In light of many findings indicating that adrenal medulla contains and releases CRH and numerous POMC-derived peptides, the hypothesis is advanced that an intra-adrenal CRH/ACTH mechanism may be operative in the control of adrenocortical steroid-hormone secretion.     

A confocal microscopic study of gonadotropin-releasing hormone (GnRH) neuron inputs to dopaminergic neurons containing estrogen receptor alpha in the arcuate nucleus of GnRH-green fluorescent protein transgenic mice

The purpose of the present study was to determine whether the septo-preoptico-tuberoinfundibular gonadotropin-releasing hormone (GnRH) pathway comes in close juxtaposition with tyrosine hydroxylase immunoreactive (TH-IR) neurons in the arcuate nucleus of female mice. Immunohistochemical staining with a TH monoclonal antibody coupled with confocal microscopy was employed on vibratome-cut brain sections of female GnRH-green fluorescent protein (GFP) transgenic mice to evaluate possible appositions between GnRH and tuberoinfundibular dopaminergic (TIDA) neurons. TH-IR neurons of the arcuate nucleus received GnRH neuronal appositions in adult female mice at proestrus and estrus stages. In contrast, no GnRH appositions were observed in adult females at diestrus. Subsequently, double immunohistochemical staining for TH and estrogen receptor-alpha (ERalpha) was performed to examine the role of estradiol on this relationship. We found that most TH-IR neurons contacted by GnRH fibers were immunoreactive for ERalpha. Our observations suggest that GnRH neurons communicate directly with TIDA neurons in the adult female. Furthermore, ERalpha activation in TIDA neurons may be involved in the formation of connections between GnRH neurons and TIDA neurons.     

Growth hormone-releasing hormone (GHRH) neurons in GHRH-enhanced green fluorescent protein transgenic mice: a ventral hypothalamic network

The hypothalamic GHRH neurons secrete pulses of GHRH to generate episodic GH secretion, but little is known about the mechanisms involved. We have made transgenic mice expressing enhanced green fluorescent protein (eGFP) specifically targeted to the secretory vesicles in GHRH neurons. GHRH cells transported eGFP from cell bodies in the arcuate nucleus to extensively arborized varicose fiber terminals in the median eminence. Patch clamp recordings from visually identified GHRH cells in mature animals showed spontaneous action potentials, often firing in short bursts up to 10 Hz. GHRH neurons received frequent synaptic inputs, as demonstrated by the recording of abundant inward postsynaptic currents, but spikes were followed by large after-hyperpolarizations, which limited their firing rate. Because many GHRH neurons lie close to the ventral hypothalamic surface, this was examined by wide-field binocular epifluorescence stereomicroscopy. This approach revealed an extensive horizontal network of GHRH cells at low power and individual fiber projections at higher power in the intact brain. It also showed the dense terminal projections of the GHRH cell population in the intact median eminence. This model will enable us to characterize the properties of individual GHRH neurons and their structural and functional connections with other neurons and to study directly the role of the GHRH neuronal network in generating episodic secretion of GH.     

Corticotropin-releasing factor-overexpressing mice exhibit reduced neuronal activation in the arcuate nucleus and food intake in response to fasting

Corticotropin-releasing factor (CRF) overexpressing (OE) mice are a genetic model that exhibits features of chronic stress. We investigated whether the adaptive feeding response to a hypocaloric challenge induced by food deprivation is impaired under conditions of chronic CRF overproduction. Food intake response to a 16-h overnight fast and ip injection of gut hormones regulating food intake were compared in CRF-OE and wild type (WT) littermate mice along with brain Fos expression, circulating ghrelin levels, and gastric emptying of a nonnutrient meal. CRF-OE mice injected ip with saline showed a 47 and 44% reduction of 30-min and 4-h cumulative food intake response to an overnight fast, respectively, compared with WT. However, the 30-min food intake decrease induced by ip cholecystokinin (3 microg/kg) and increase by ghrelin (300 microg/kg) were similar in CRF-OE and WT mice. Overnight fasting increased the plasma total ghrelin to similar levels in CRF-OE and WT mice, although CRF-OE mice had a 2-fold reduction of nonfasting ghrelin levels. The number of Fos-immunoreactive cells induced by fasting in the arcuate nucleus was reduced by 5.9-fold in CRF-OE compared with WT mice whereas no significant changes were observed in other hypothalamic nuclei. In contrast, fasted CRF-OE mice displayed a 5.6-fold increase in Fos-immunoreactive cell number in the dorsal motor nucleus of the vagus nerve and a 34% increase in 20-min gastric emptying. These findings indicate that sustained overproduction of hypothalamic CRF in mice interferes with fasting-induced activation of arcuate nucleus neurons and the related hyperphagic response.     

Corticotropin-releasing hormone (CRH) stimulation in Nelson's syndrome: response of adrenocorticotropin secretion to pulse injection and continuous infusion of CRH

Nelson's syndrome develops in 10-15% of patients with Cushing's disease who undergo bilateral adrenalectomy. Whether the pituitary tumors of Nelson's syndrome are autonomous or are regulated by hypothalamic signals or glucocorticoids is controversial. We, therefore, compared the plasma ACTH responses to synthetic ovine corticotropin-releasing hormone (CRH) in 11 patients with Nelson's syndrome, 1 patient with Cushing's disease who had had bilateral adrenalectomy and did not have Nelson's syndrome, 14 patients with Cushing's disease, and 27 normal subjects. The plasma ACTH response to CRH in 10 patients with Nelson's syndrome was markedly increased and prolonged compared to the responses of normal subjects or patients with Cushing's disease. In 4 patients with Nelson's syndrome, plasma ACTH and cortisol concentrations also were determined at frequent intervals for 10-24 h during continuous infusions of 0.15 M saline or CRH (1 microgram/kg X h). There was no desensitization of ACTH secretion during short term continuous infusion of CRH. Exogenous cortisol inhibited CRH-stimulated ACTH secretion. These findings suggest that the ACTH response to CRH of patients with ACTH-secreting tumors of Nelson's syndrome differs from the response of those who have the microadenomas of Cushing's disease in two ways: the magnitude is greater, and the response is prolonged. These differences can be explained by the greater size of the tumor and the reduced glucocorticoid feedback in adrenalectomized patients with Nelson's syndrome.     

Functional hypothalamic amenorrhea due to increased CRH tone in melanocortin receptor 2-deficient mice

Exposure to chronic stressors results in dysregulation of the hypothalamic-pituitary-adrenal axis and a disruption in reproduction. CRH, the principal regulator of the hypothalamic-pituitary-adrenal axis induces the secretion of ACTH from the pituitary, which stimulates adrenal steroidogenesis via the specific cell-surface melanocortin 2 receptor (MC2R). Previously, we demonstrated that MC2R(-/-) mice had undetectable levels of corticosterone despite high ACTH levels. Here, we evaluated the reproductive functions of female MC2R(-/-) mice and analyzed the mechanism of the disrupted cyclicity of these mice. The expression of CRH in the paraventricular nucleus was significantly increased in MC2R(-/-) mice under nonstressed conditions. Although MC2R(-/-) females were fertile, they showed a prolonged estrous cycle. After hormonal stimulation, MC2R(-/-) females produced nearly-normal numbers of eggs, but slightly less than MC2R(+/-) females, and showed near-normal ovarian histology. During diestrus, the number of GnRH-positive cells in the medial preoptic area was significantly reduced in MC2R(-/-) females. CRH type 1 receptor antagonist restored estrous cyclicity in MC2R(-/-) females. Kisspeptin-positive areas in the arcuate nucleus were comparable, whereas kisspeptin-positive areas in the anteroventral periventricular nucleus in MC2R(-/-) females were significantly reduced compared with MC2R(+/-) females, suggesting that arcuate nucleus kisspeptin is not involved, but anteroventral periventricular nucleus kisspeptin may be involved, in the maintenance of estrous cyclicity. Our findings show that high levels of hypothalamic CRH disturb estrous cyclicity in the female animals and that the MC2R(-/-) female is a unique animal model of functional hypothalamic amenorrhea.     

Novel expression of neuropeptide Y (NPY) mRNA in hypothalamic regions during development: region-specific effects of maternal deprivation on NPY and Agouti-related protein mRNA

During development there is novel expression of NPY mRNA in the dorsomedial hypothalamic nucleus (DMH) and perifornical region (PFR), in addition to the arcuate nucleus (ARH). Furthermore, NPY mRNA levels peak in all regions on postnatal d 16 (P16) and decrease to adult levels by P30. The purpose of the present study was to determine whether NPY and agouti-related protein (AGRP) mRNA expression in the different hypothalamic regions on P11 and P16 are similarly affected by fasting. An examination of the full rostral to caudal extent of the hypothalamus revealed two additional regions displaying novel NPY mRNA expression, the parvocellular division of the paraventricular nucleus (PVH) and lateral hypothalamus (LH). Maternal deprivation for 36 h, used to bring about a fast, similarly increased (23-29%) NPY and AGRP mRNA expression in the ARH on P11 and P16. In contrast, NPY expression in the DMH and PFR were significantly decreased (19-30% and 48-53%, respectively), whereas NPY mRNA levels in the PVH and LH were not altered by this treatment. The increase in NPY and AGRP mRNA expression in the ARH in response to maternal deprivation suggests that these neuronal populations respond to signals of energy balance. In contrast, NPY expression in the DMH, PFR, PVH, and LH is differentially regulated by maternal deprivation or other factors associated with maternal separation.     

Involvement of the beta-endorphin neurons of the hypothalamic arcuate nucleus in ethanol-induced place preference conditioning in mice

Background: Increasing evidence indicates that mu‐ and delta‐opioid receptors are decisively involved in the retrieval of memories underlying conditioned effects of ethanol. The precise mechanism by which these receptors participate in such effects remains unclear. Given the important role of the proopiomelanocortin (POMc)‐derived opioid peptide beta‐endorphin, an endogenous mu‐ and delta‐opioid receptor agonist, in some of the behavioral effects of ethanol, we hypothesized that beta‐endorphin would also be involved in ethanol conditioning. Methods: In this study, we treated female Swiss mice with estradiol valerate (EV), which induces a neurotoxic lesion of the beta‐endorphin neurons of the hypothalamic arcuate nucleus (ArcN). These mice were compared to saline‐treated controls to investigate the role of beta‐endorphin in the acquisition, extinction, and reinstatement of ethanol (0 or 2 g/kg; intraperitoneally)‐induced conditioned place preference (CPP). Results: Immunohistochemical analyses confirmed a decreased number of POMc‐containing neurons of the ArcN with EV treatment. EV did not affect the acquisition or reinstatement of ethanol‐induced CPP, but facilitated its extinction. Behavioral sensitization to ethanol, seen during the conditioning days, was not present in EV‐treated animals. Conclusions: The present data suggest that ArcN beta‐endorphins are involved in the retrieval of conditioned memories of ethanol and are implicated in the processes that underlie extinction of ethanol‐cue associations. Results also reveal a dissociated neurobiology supporting behavioral sensitization to ethanol and its conditioning properties, as a beta‐endorphin deficit affected sensitization to ethanol, while leaving acquisition and reinstatement of ethanol‐induced CPP unaffected.     

Novel ontogenetic patterns of sexual differentiation in arcuate nucleus GHRH neurons revealed in GHRH-enhanced green fluorescent protein transgenic mice

GH secretion and growth rates are developmentally regulated and sexually dimorphic, but the neuroregulatory mechanisms between birth and puberty are unclear. Using the GHRH-enhanced green fluorescent protein (eGFP) transgenic mouse, in which eGFP provides a strong surrogate signal for identifying GHRH neurons, we showed that numbers in the male arcuate nucleus were double those seen in females at x postnatal day (P)1 and P10, during which time numbers increased 2- to 3-fold. Thereafter (P20, P30, P60, P365) there was a significant trend for numbers to decrease in males and increase in females, such that sex differences were, surprisingly, absent in young and late adulthood. Conversely, we identified the emergence of male-dominant sex differences in the number of processes extended per GHRH perikarya across puberty. Intriguingly, prepubertal gonadectomy (P28), unlike adult gonadectomy, caused a dramatic 40% loss of GHRH cells in both sexes in adulthood and a significant (30%) increase in processes emanating from cell bodies only in females. These findings establish a novel ontogenetic profile for GHRH neurons and suggest previously undiscovered roles for peripubertal gonadal factors in establishing population size in both sexes. They also provide the first demonstration of emergent sex-specific GHRH architecture, which may signal the onset of sex-dependent regulation of activity reported for adult GHRH-eGFP neurons, and its differential regulation by gonadal factors in males and females. This information adds to our knowledge of processes that underpin the emergence of sex-specific GH secretory dynamics and hence biological activity of this pleiotropic hormone.     

Co-localization of growth hormone secretagogue receptor and NPY mRNA in the arcuate nucleus of the rat

Growth hormone secretagogues (GHS) are small, synthetic compounds which have the potential of releasing growth hormone (GH) from the pituitary. The mechanism of action of GHS has not been fully elucidated. A specific GHS receptor (GHS-R) is expressed in the pituitary gland and in several areas of the brain including the hypothalamus. We have characterized the GHS-R-mRNA-expressing neurons with respect to co-expression of selected neurotransmitters in the hypothalamus. This was done by dual chromogenic and autoradiographic in situ hybridization with riboprobes for GHS-R mRNA and neuropeptide Y (NPY), pro-opiomelanocortin (POMC), somatostatin (SRIH) or GH-releasing hormone (GHRH) mRNA. In the arcuate nucleus, GHS-R mRNA was expressed in 94 +/- 1% of the neurons expressing NPY, 8 +/- 2% of those expressing POMC and 30 +/- 6% expressing SRIH mRNA. 20-25% of the GHRH- mRNA-expressing neurons contained GHS-R mRNA, whereas the vast majority of the arcuate GHS-R-mRNA-containing cells did not contain GHRH mRNA. The finding of a significant co-expression of GHS-R and NPY mRNA in the arcuate nucleus is in accordance with the previous demonstration by Dickson et al. that c-Fos is induced in NPY neurons following GHS administration. These results indicate that GHS have other effects on neuroendocrine regulation than GH release via GHRH neurons. Stimulation of the arcuate NPY neurons via GHS-R may explain the increased appetite and the cortisol release seen after administration of some GHS compounds.     

Neuropeptide Y gene expression in the arcuate nucleus is increased during preovulatory luteinizing hormone surges.

Recent studies have suggested that neuropeptide Y (NPY) plays an important role in the induction of the preovulatory LH surge. The present study was performed in order to determine if a change in NPY gene expression within arcuate nucleus NPY neurons is associated with the generation of the preovulatory LH surge. In Exp 1, in situ hybridization was used to measure NPY messenger RNA (mRNA) levels in the arcuate nucleus of female rats at 0900 h and every 2 h from 1400-2200 h on the day of proestrus (PRO). Comparisons between groups showed a clear, stepwise increase in NPY gene expression throughout the day of PRO. At 1600 h, when LH values were significantly greater than 0900 h values, NPY mRNA labeling intensities in the arcuate nucleus were significantly greater than 0900 h levels (P < 0.01). By 1800 h, the time at which the LH surge peaked, NPY mRNA levels also peaked and were nearly 3-fold greater than levels observed at 0900 h (P < 0.01). NPY mRNA levels at 2000 h and 2200 h remained elevated above 0900 h levels (P < 0.01) but by 2000 h had decreased significantly from 1800 h levels (P < 0.05). In Exp 2, NPY mRNA levels were measured once again at 0900 h and 1800 h on PRO, and then at 0900 h and 1800 h on metestrus (MET), in order to determine if the change in gene expression seen in Exp 1 was unique to the day of PRO, or if it simply reflected a daily rhythm of gene expression in the nucleus. Analysis of mRNA levels showed no difference in NPY mRNA levels between 0900-1800 h on MET. Also, NPY mRNA levels at 0900 h and 1800 h on MET were significantly less than levels at 1800 h on PRO (P < 0.01). These results are consistent with the hypothesis that NPY neurons participate in the generation of LH surges through increased production of NPY and subsequent potentiation of the release and/or actions of LHRH.     

地方性甲状腺肿大鼠子代下丘脑弓状核的实验形态学研究

本文应用半薄切片,超薄切片对地甲肿大鼠5个月子代下丘脑弓状核进行了光镜和电镜观察。结果表明,实验组大鼠弓状核神经细胞中尼氏小体含量显著增加,核仁样小体大小不等,形态各异,不仅出现在细胞质中,在树突中也可见到。粗面内质网丰富,分泌颗粒增多,溶酶体体积增大,电子密度增强,以次级溶酶体居多。