BACKGROUND

Posttraumatic stress disorder affects 20%-30% of those exposed. Clinical studies employ stringent inclusion-exclusion criteria, yet animal studies include the entire exposed population as the study population. We examined the effect of grouping prestressed rats according to magnitude of response on the statistical analysis of results.

METHODS

Response magnitude to predator exposure was assessed and used to group the animals into "diagnostic" groups. Two extremes were studied (clearly "maladapted" and clearly "well adapted" rats) using arbitrarily selected cutoff behavioral criteria (CBC). The data for the middle group were discarded for reasons of clarity. Hypothalamic-pituitary-adrenal axis and heart-rate variability were analyzed for the entire exposed population and then according to the CBC.

RESULTS

A single 10-min exposure to a predator caused fear-related behaviors in only 25.3% of exposed rats. Compared with control subjects and well-adapted exposed rats, maladapted rats exhibited significantly higher plasma corticosterone and corticotropin concentrations, increased sympathetic activity, diminished vagal tone, and increased sympathovagal balance. These differences surfaced only when data were analyzed according to CBC. Animals respond to stress heterogeneously, resembling humans. Overlooking this heterogeneity may obscure the results of data analysis.

CONCLUSIONS

Animals can be divided into distinct groups according to magnitude of response and be studied accordingly.

A 37-kDa corticotropin releasing factor (CRF) binding protein (CRF-BP) was purified from human plasma by repeated affinity purification and subsequently sequenced and cloned. The human and rat CRF-BP cDNAs encode proteins of 322 amino acids with one putative signal sequence, one N-glycosylation site, and 10 conserved cysteines. Human CRF-BP binds human CRF with high affinity but has low affinity for the ovine peptide. In contrast, sheep CRF-BP binds human and ovine CRF with high affinity. The CRF-BP gene consists of seven exons and six introns and is located on chromosome 13 and loci 5q of the mouse and human genomes, respectively. CRF-BP inhibits the adrenocorticotrophic hormone (ACTH) releasing properties of CRF in vitro. CRF-BP dimerizes after binding CRF and clears the peptide from blood. This clearance mechanism protects the maternal pituitary gland from elevated plasma CRF levels found during the third trimester of human pregnancy. CRF-BP is expressed in the brains of all species so far tested but is uniquely expressed in human liver and placenta. In brain, CRF-BP is membrane associated and is predominantly expressed in the cerebral cortex and subcortical limbic structures. In some brain areas CRF-BP colocalizes with CRF and CRF receptors. The protein is also present in pituitary corticotropes, where it is under positive glucocorticoid control, and is likely to locally modulate CRF-induced ACTH secretion. The ligand requirements of the CRF receptor and the CRF-BP can be distinguished in that central human CRF fragments, such as CRF (6-33) and CRF (9-33), have high affinity for CRF-BP but low affinity for the CRF receptor. The binding protein's ability to inhibit CRF-induced ACTH secretion can be reversed by CRF (6-33) and CRF (9-33), suggesting that ligand inhibitors may have utility in elevating free CRF levels in disease states associated with decreased CRF. Thus, by controlling the amount of free CRF which activates CRF receptors, it is likely that the CRF-BP is an important modulator of CRF both in the CNS and in the periphery.

A number of clinical investigations and postmortem brain studies have provided evidence that excessive corticotropin-releasing hormone (CRH) secretion and neurotransmission is involved in the pathophysiology of depressive illness, and several studies have suggested that the hyperactivity in CRH neurotransmission extends beyond the hypothalamus involving several extra-hypothalamic brain regions. The present study was designed to test the hypothesis that CRH levels are increased in specific brainstem regions of suicide victims with a diagnosis of major depression. Frozen tissue sections of the pons containing the locus coeruleus and caudal raphe nuclei from 11 matched pairs of depressed suicide and control male subjects were processed for radioimmunocytochemistry using a primary antiserum to CRH and a ([125])I-IgG secondary antibody. The optical density corresponding to the level of CRH-immunoreactivity (IR) was quantified in specific pontine regions from the film autoradiographic images. The level of CRH-IR was increased by 30% in the locus coeruleus, 39% in the median raphe and 45% in the caudal dorsal raphe in the depressed suicide subjects compared to controls. No difference in CRH-IR was found in the dorsal tegmentum or medial parabrachial nucleus between the subject groups. These findings reveal that CRH-IR levels are specifically increased in norepinephrine- and serotonin-containing pontine nuclei of depressed suicide men, and thus they are consistent with the hypothesis that CRH neurotransmission is elevated in extra-hypothalamic brain regions of depressed subjects.

Previous studies have shown reduced hypothalamo-pituitary-adrenal responses to both acute and chronic restraint stressors in rats allowed to ingest highly palatable foods (32% sucrose +/- lard) prior to restraint. In this study we tested the effects of prior access (7 d) to chow-only, sucrose/chow, lard/chow, or sucrose/lard/chow diets on central corticotropin-releasing factor (CRF) expression in rats studied in two experiments, 15 and 240 min after onset of restraint. Fat depot, particularly intraabdominal fat, weights were increased by prior access to palatable food, and circulating leptin concentrations were elevated in all groups. Metabolite concentrations were appropriate for values obtained after stressors. For unknown reasons, the 15-min experiment did not replicate previous results. In the 240-min experiment, ACTH and corticosterone responses were inhibited, as previously, and CRF mRNA in the hypothalamus and oval nucleus of the bed nuclei of the stria terminalis were reduced by palatable foods, suggesting strongly that both neuroendocrine and autonomic outflows are decreased by increased caloric deposition and palatable food. In the central nucleus of the amygdala, CRF was increased in the sucrose-drinking group and decreased in the sucrose/lard group, suggesting that the consequence of ingestion of sucrose uses different neural networks from the ingestion of lard. The results suggest strongly that ingestion of highly palatable foods reduces activity in the central stress response network, perhaps reducing the feeling of stressors.

It has been claimed that factors favoring the development or maintenance of animal or human obesity may include increases in glucocorticoid production or hyperresponsiveness of the hypothalamic-pituitary-adrenal axis. In normal rats, glucocorticoids have been shown to be necessary for chronic intracerebroventricular infusion of neuropeptide Y to produce obesity and related abnormalities. Conversely, glucocorticoids inhibited the body weight-lowering effect of leptin. Such dual action of glucocorticoids may occur within the central nervous system, since both neuropeptide Y and leptin act within the hypothalamus. The aim of this study was to determine the effects of glucocorticoids (dexamethasone) given intracerebroventricularly to normal rats on body weight homeostasis and hypothalamic levels of neuropeptide Y and corticotropin-releasing hormone. Continuous central glucocorticoid infusion for 3 days resulted in marked sustained increases in food intake and body weight relative to saline-infused controls. The infusion abolished endogenous corticosterone output and produced hyperinsulinemia, hypertriglyceridemia, and hyperleptinemia, three salient abnormalities of obesity syndromes. Central glucocorticoid infusion also produced a marked decrease in the expression of uncoupling protein (UCP)-1 and UCP-3 in brown adipose tissue and UCP-3 in muscle. Finally, chronic central glucocorticoid administration increased the hypothalamic levels of neuropeptide Y and decreased those of corticotropin-releasing hormone. When the same dose of glucocorticoids was administered peripherally, it resulted in decreases in food intake and body weight, in keeping with the decrease in hypothalamic neuropeptide Y levels. These results suggest that glucocorticoids induce an obesity syndrome in rodents by acting centrally and not peripherally.

Several recent studies on corticotropin-releasing factor (CRF) have suggested that this neuropeptide may play a role in depression. Consequently, CRF receptor antagonists have been proposed as potential new agents for the treatment of this condition. This study investigated the effects of a 4-week treatment with the well-known CRF(1) receptor antagonist, antalarmin, and the prototypical selective serotonin reuptake inhibitor (SSRI), fluoxetine, in the chronic mild stress (CMS) model in BALB/c mice. Animals were exposed to 9 weeks of CMS which rapidly (within 2 weeks) produced decrease of physical state (PS), body weight gain and blunted emotional response in the light/dark test. Chronic treatment with antalarmin (10 mg/kg ip) and fluoxetine (10 mg/kg ip) led to an improvement of CMS-induced modifications. These results suggest that CRF(1) receptor antagonists may represent potential antidepressants.

Chronic stress is widely regarded as a key risk factor for a variety of diseases. A large number of paradigms have been used to induce chronic stress in rodents. However, many of these paradigms do not consider the etiology of human stress-associated disorders, where the stressors involved are mostly of social nature and the effects of the stress exposure persist even if the stressor is discontinued. In addition, many chronic stress paradigms are problematic with regard to stress adaptation, continuity, duration and applicability. Here we describe and validate a novel chronic social stress paradigm in male mice during adolescence. We demonstrate persistent effects of chronic social stress after 1 week of rest, including altered adrenal sensitivity, decreased expression of corticosteroid receptors in the hippocampus and increased anxiety. In addition, pharmacological treatments with the antidepressant paroxetine (SSRI) or with the corticotropin-releasing hormone receptor 1 antagonist DMP696 were able to prevent aversive long-term consequences of chronic social stress. In conclusion, this novel chronic stress paradigm results in persistent alterations of hypothalamus-pituitary-adrenal axis function and behavior, which are reversible by pharmacological treatment. Moreover, this paradigm allows to investigate the interaction of genetic susceptibility and environmental risk factors.

The authors measured CSF concentrations of corticotropin-releasing hormone (CRH) and arginine vasopressin in nine depressed patients before and after fluoxetine treatment. They found significant decreases in CSF CRH, CSF arginine vasopressin, and Hamilton depression ratings. Thus, the therapeutic effect of this serotonin-uptake inhibitor may be related to diminution of these arousal-promoting neuropeptides.

Stress-like symptoms are an integral part of acute and protracted drug withdrawal, and several lines of evidence have shown that dysregulation of brain stress systems, including the extrahypothalamic corticotropin-releasing factor (CRF) system, following long-term drug use is of major importance in maintaining drug and alcohol addiction. Recently, two other neuropeptide systems have attracted interest, the nociceptin/orphanin FQ (N/OFQ) and orexin/hypocretin (Orx/Hcrt) systems. N/OFQ participates in a wide range of physiological responses, and the hypothalamic Orx/Hcrt system helps regulate several physiological processes, including feeding, energy metabolism, and arousal. Moreover, these two systems have been suggested to participate in psychiatric disorders, including anxiety and drug addiction. Dysregulation of these systems by chronic drug exposure has been hypothesized to play a role in the maintenance of addiction and dependence. Recent evidence demonstrated that interactions between CRF-N/OFQ and CRF-Orx/Hcrt systems may be functionally relevant for the control of stress-related addictive behavior. The present review discusses recent findings that support the hypotheses of the participation and dysregulation of these systems in drug addiction and evaluates the current understanding of interactions among these stress-regulatory peptides.

OBJECTIVE

We suggested fibromyalgia (FM) is a disorder associated with an altered functioning of the stress-response system. This was concluded from hyperreactive pituitary adrenocorticotropic hormone (ACTH) release in response to corticotropin-releasing hormone (CRH) and to insulin induced hypoglycemia in patients with FM. In this study, we tested the validity and specificity of this observation compared to another painful condition, low back pain.

METHODS

We recruited 40 patients with primary FM (F:M 36:4), 28 patients (25:3) with chronic noninflammatory low back pain (LBP), and 14 (12:2) healthy, sedentary controls. A standard 100 microg CRH challenge test was performed with measurement of ACTH and cortisol levels at 9 time points. They were also subjected to an overnight dexamethasone suppression test, followed by injection of synthetic ACTH1-24. At 9 AM, the patients divided in 2 groups, received either 0.025 or 0.100 microg ACTH/kg body weight to test for adrenocortical sensitivity. Basal adrenocortical function was assessed mainly by measurement of 24 h urinary excretion of free cortisol.

RESULTS

Compared to the controls, the patients with FM displayed a hyperreactive ACTH release in response to CRH challenge (ANOVA interaction effect p = 0.001). The mean ACTH response of the patients with low back pain appeared enhanced also, but to a significantly lesser extent (p = 0.02 at maximum level) than observed in the patients with FM. The cortisol response was the same in the 3 groups. Following dexamethasone intake there were 2 and 4 nonsuppressors in the FM and LBP groups, respectively. The very low and low dose of exogenous ACTH1-24 evoked a dose and time dependent cortisol response, which, however, was not significantly different between the 3 groups. The 24 h urinary free cortisol levels were significantly lower (p = 0.02) than controls in both patient groups; patients with FM also displayed significantly lower (p < 0.05) basal total plasma cortisol than controls.

CONCLUSIONS

The present data validate and substantiate our preliminary evidence for a dysregulation of the HPA axis in patients with FM, marked by mild hypocortisolemia, hyperreactivity of pituitary ACTH release to CRH, and glucocorticoid feedback resistance. Patients with LBP also display hypocortisolemia, but only a tendency toward the disrupted HPA features observed in the patients with FM. We propose that a reduced containment of the stress-response system by corticosteroid hormones is associated with the symptoms of FM.

An increase in corticotropin-releasing factor (CRF) is a putative factor in the pathophysiology of stress-related disorders. As CRF expression in the central nucleus of the amygdala (CeA) is important in adaptation to chronic stress, we hypothesized that unrestrained synthesis of CRF in CeA would mimic the consequences of chronic stress exposure and cause dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, increase emotionality and disrupt reproduction. To test this hypothesis, we used a lentiviral vector to increase CRF-expression site specifically in CeA of female rats. Increased synthesis of CRF in CeA amplified CRF and arginine vasopressin peptide concentration in the paraventricular nucleus of the hypothalamus, and decreased glucocorticoid negative feedback, both markers associated with the pathophysiology of depression. In addition, continuous expression of CRF in CeA also increased the acoustic startle response and depressive-like behavior in the forced swim test. Protein levels of gonadotropin-releasing hormone in the medial preoptic area were significantly reduced by continuous expression of CRF in CeA and this was associated with a lengthening of estrous cycles. Finally, sexual motivation but not sexual receptivity was significantly attenuated by continuous CRF synthesis in ovariectomized estradiol-progesterone-primed females. These data indicate that unrestrained CRF synthesis in CeA produces a dysregulation of the HPA axis, as well as many of the behavioral, physiological and reproductive consequences associated with stress-related disorders.Molecular Psychiatry (2009) 14, 37-50; doi:10.1038/mp.2008.91; published online 12 August 2008.

OBJECTIVE

Endogenous Cushing syndrome in children is a rare disorder that is most frequently caused by pituitary or adrenocortical tumors. Diagnostic criteria have generally been derived from studies of adult patients despite significant differences in both the physiology of the hypothalamic-pituitary-adrenal axis and the epidemiology of Cushing syndrome in childhood. The purpose of this study was to identify the tests that most reliably and efficiently diagnose pituitary or adrenal tumors in a large cohort of pediatric patients with Cushing syndrome.

METHODS

A retrospective review of clinical data of children who were referred to a tertiary care center for evaluation for Cushing syndrome during the years 1997 to 2005 was conducted. A total of 125 consecutive children were studied retrospectively; 105 were found to have Cushing syndrome, which was confirmed histologically; and 20 children who did not have Cushing syndrome or any other endocrinopathy served as the control group. The following tests were performed in all children: midnight and morning cortisol, corticotropin hormone, urinary free cortisol and 17-hydroxycorticosteroid levels, ovine corticotropin-releasing hormone stimulation test, and overnight high-dosage dexamethasone suppression test. Imaging of the pituitary and adrenal glands was also obtained. The main outcome measure was the sensitivity of these parameters for the diagnosis and differential diagnosis of Cushing syndrome at 100% specificity.

RESULTS

A midnight cortisol value of>> or = 4.4 microg/dL confirmed the diagnosis of Cushing syndrome in almost all children, with a sensitivity of 99% and a specificity of 100%. Suppression of morning cortisol levels>> 20% in response to an overnight, high-dosage dexamethasone test excluded all patients with adrenal tumors and identified almost all patients with pituitary tumors (sensitivity: 97.5%; specificity: 100%).

CONCLUSIONS

Our study suggests that among children who were referred for the evaluation of possible Cushing syndrome, a single cortisol value at midnight followed by overnight high-dosage dexamethasone test led to rapid and accurate confirmation and diagnostic differentiation, respectively, of hypercortisolemia caused by pituitary and adrenal tumors.

The stress response in a healthy organism is generally viewed as a warning and thus a protective reaction to a threat. However, the response may be deleterious if it is linked to an inflammatory stimulus or if it proceeds an inflammatory event. Prior stress enhances the response to an inflammatory stimulus by a mechanism that is independent of the release of hypothalamic corticotropin-releasing factor (CRF) or arginine vasopressin. Putative mechanisms include an increase in intestinal permeability as well as the release of the proinflammatory neuropeptide substance P. Stress may also reactivate previous inflammation when applied in conjunction with a small luminal stimulus. This reactivation involves increased permeability and requires the presence of T lymphocytes. Inflammatory mediators activate hypothalamic pathways, and a negative feedback loop, mediated by CRF release, has been proposed because animals with impaired hypothalamic CRF responses are more susceptible to inflammatory stimuli. Together, these experimental observations provide insights into the expression of inflammatory disorders in humans, including inflammatory bowel disease and postinfective irritable bowel syndrome.

The functional role of central catecholamines in regulation of ACTH secretion remains controversial. In the present report, the nature of catecholaminergic influences was directly assessed by measurement of hypophysial-portal plasma immunoreactive CRF (irCRF) levels after activation of endogenous aminergic pathways by electrical stimulation or administration of norepinephrine (NE). Electrical stimulation of the ventral noradrenergic ascending bundle, a fiber system primarily carrying catecholaminergic fibers arising from brainstem regions, resulted in a 2.9-fold elevation of portal irCRF levels. Pretreatment with the alpha 1-adrenergic receptor antagonist coryanthine, but not the beta-adrenergic antagonist propranolol, blocked the facilitatory effect of electrical stimulation and reduced prestimulation irCRF levels by 34.7 +/- 4.2% (P less than 0.05). Intracerebroventricular administration of 0.1-5.0 nmol NE resulted in a dose-dependent facilitation of portal plasma irCRF levels which could be blocked by pretreatment with coryanthine. Alternatively, administration of greater than or equal to 5 nmol NE caused a dose-dependent inhibition of irCRF release which could be prevented by pretreatment with propranolol. Finally, irCRF secretion evoked by nitroprusside-induced hypotension was also blocked by pretreatment with coryanthine, but not propranolol. These observations provide strong evidence in favor of a predominantly stimulatory action of NE (and possibly epinephrine) at the hypothalamic level to evoke secretion of CRF and thus to activate the pituitary-adrenal axis.

The human placenta expresses the genes for proopiomelanocortin and the major stress hormone, corticotropin-releasing hormone (CRH), profoundly altering the "fight or flight" stress system in mother and fetus. As pregnancy progresses, the levels of these stress hormones, including maternal cortisol, increase dramatically. These endocrine changes are important for fetal maturation, but if the levels are altered (e.g., in response to stress), they influence (program) the fetal nervous system with long-term consequences. The evidence indicates that fetal exposure to elevated levels of stress hormones (i) delays fetal nervous system maturation, (ii) restricts the neuromuscular development and alters the stress response of the neonate, (iii) impairs mental development and increases fearful behavior in the infant, and (iv) may result in diminished gray matter volume in children. The studies reviewed indicate that fetal exposure to stress peptides and hormones exerts profound programming influences on the nervous system and may increase the risk for emotional and cognitive impairment.

Interactions between central corticotropin-releasing factor (CRF) and serotonergic systems are believed to be important for mediating fear and anxiety behaviors. Recently we demonstrated that infusions of CRF into the rat dorsal raphe nucleus result in a delayed increase in serotonin release within the medial prefrontal cortex that coincided with a reduction in fear behavior. The current studies were designed to study the CRF receptor mechanisms and pathways involved in this serotonergic response. Infusions of CRF (0.5 microg/0.5 microL) were made into the dorsal raphe nucleus of urethane-anesthetized rats following either inactivation of the median raphe nucleus by muscimol (25 ng/0.25 microL) or antagonism of CRF receptor type 1 or CRF receptor type 2 in the dorsal raphe nucleus with antalarmin (25-50 ng/0.5 microL) or antisauvagine-30 (2 microg/0.5 microL), respectively. Medial prefrontal cortex serotonin levels were measured using in-vivo microdialysis and high-performance liquid chromatography with electrochemical detection. Increased medial prefrontal cortex serotonin release elicited by CRF infusion into the dorsal raphe nucleus was abolished by inactivation of the median raphe nucleus. Furthermore, antagonism of CRF receptor type 2 but not CRF receptor type 1 in the dorsal raphe nucleus abolished CRF-induced increases in medial prefrontal cortex serotonin. Follow-up studies involved electrical stimulation of the central nucleus of the amygdala, a source of CRF afferents to the dorsal raphe nucleus. Activation of the central nucleus increased medial prefrontal cortex serotonin release. This response was blocked by CRF receptor type 2 antagonism in the dorsal raphe. Overall, these results highlight complex CRF modulation of medial prefrontal cortex serotonergic activity at the level of the raphe nuclei.

The corticotropin releasing factor (CRF) type 1alpha receptor, a member of the G protein-coupled receptor (GPCR) subfamily B, is involved in the aetiology of anxiety and depressive disorders. In the present study, we examined the internalization and trafficking of the CRF1alpha receptor in both human embryonic kidney (HEK)293 cells and primary cortical neurons. We found that CRF1alpha receptor activation leads to the selective recruitment of beta-arrestin2 in both HEK293 cells and neurons. We observed distinct distribution patterns of CRF1alpha receptor and beta-arrestin2 in HEK293 cells and cortical neurons. In HEK293 cells, beta-arrestin2-green fluorescent protein (GFP) co-localized with CRF1alpha receptor in vesicles at the plasma membrane but was dissociated from the receptor in endosomes. In contrast, in primary cortical neurons, beta-arrestin2 and CRF1alpha receptor were internalized in distinct endocytic vesicles. By bioluminescence resonance energy transfer, we demonstrated that beta-arrestin2 association with CRF1alpha receptor was increased in cells transfected with G protein-coupled receptor kinase (GRK)3 and GRK6 and decreased in cells transfected with GRK2 and GRK5. In both HEK293 cells and cortical neurons, internalized CRF1alpha receptor transited from Rab5-positive early endosomes to Rab4-positive recycling endosomes and was not targeted to lysosomes. However, CRF1alpha receptor resensitization was blocked by the overexpression of wild-type, but not dominant-negative, Rab5 and Rab4 GTPases. Taken together, our results suggest that beta-arrestin trafficking differs between HEK293 cells and neurons, and that CRF1alpha receptor resensitization is regulated in an atypical manner by Rab GTPases.

Estrogens exert important actions on fear and anxiety-like behavior both in humans and non-human animals. Currently, the mechanisms underlying estrogenic modulation of fear are not known. However, evidence suggests that estrogens may exert their influence on fear within the amygdala. The purpose of the present study was to examine effects of estrogen on fear conditioning. Specifically, the present study examined whether long-term estrogen treatment in ovariectomized female mice via Silastic capsule implantation would facilitate both contextual and cued fear conditioning. In a separate set of experiments, we then examined whether estrogen treatment in ovariectomized female mice would modulate corticotropin-releasing hormone (CRH) gene expression within the amygdala. Long-term estrogen treatment facilitated both contextual and cued fear. Ovariectomized mice treated with estrogen froze significantly more to a context as well as to a discrete auditory cue. In addition, estrogen treatment significantly increased CRH mRNA expression within the central nucleus of the amygdala as measured by in situ hybridization and quantitative PCR. These data raise the possibility that estrogens could influence fear responses in females through their actions in the amygdala.

The immunoperoxidase technique was used to study the effect of adrenalectomy on vasopressin (VP) immunoreactivity in the hypothalamic paraventricular nucleus of rat. In control animals, relatively few VP-immunostained parvocellular neurons were found in addition to a large population of magnocellular VP neurons. Seven to 14 days after bilateral adrenalectomy, VP immunostaining increased markedly in specific subdivisions of the paraventricular nucleus. In contrast to normal animals, VP immunoreactivity was localized in a large number of parvocellular neurons. Colchicine treatment, on the other hand, did not significantly increase the number of VP-immunostained parvocellular neurons found in control rats. These observations suggest that adrenalectomy increases the number of VP-positive neurons and appears to increase the intensity of VP immunoreactivity specifically in parvocellular neurons. VP parvocellular neurons are confined to those paraventricular nucleus subdivisions that are known to project to the external zone of the median eminence. Moreover, their distribution pattern is very similar, if not identical, to that of the corticotropin-releasing factor (CRF) immunoreactive cells. Parvocellular neurons on adjacent thin sections could be stained for both CRF and VP. Thus, adrenalectomy seems to increase VP staining in CRF immunoreactive parvocellular neurons, which innervate the external zone of the median eminence.

The neuropeptide corticotropin-releasing factor (CRF) is believed to play a role in a number of psychiatric conditions, including anxiety disorders and depression. In the present study, male Sprague Dawley rats were used to examine the behavioral effects of altering dopamine transmission on CRF-enhanced startle, a behavioral assay believed to reflect stress- or anxiety-like states. Systemic administration of the selective dopamine D1 receptor antagonist SCH 23390 [R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride] (0, 0.01, 0.05, 0.1, 0.5 mg/kg) dose dependently blocked the effect of CRF (1 microg, i.c.v.) on startle at doses that had no effect on baseline startle response. Immunohistochemical studies showed that most CRF-containing cells in the dorsolateral division of the bed nucleus of the stria terminalis (BSTld), part of the critical brain area mediating CRF-enhanced startle, are surrounded by a dense plexus of tyrosine hydroxylase (TH)-positive fibers. Intra-BSTld injections of the retrograde tracer Fluorogold (FG) into the TH field identified neurons in the major dopaminergic areas (A8-A10), but not the major noradrenergic areas [A5, A6 (locus ceruleus), A7], as a significant source of TH-positive innervation. The majority of FG-filled cells double-labeled for TH were found in the dorsocaudal A10 cell group (A10dc) located in the periaqueductal gray area. Together, these data suggest that neuronal regulation of the BSTld by specific dopaminergic pathways and receptors may be an important mechanism for controlling CRF-dependent moods and affective states. These data also suggest that compounds with D1 receptor antagonist properties might have anxiolytic-like effects that could be useful for treating conditions associated with hyperactive CRF systems.

Stress may be a contributing factor in intestinal inflammatory disease; however, the underlying mechanisms have not been elucidated. We previously reported that acute stress altered jejunal epithelial physiology. In this study, we examined both physical and psychological stress-induced functional changes in colonic mucosa. Colonic mucosal tissue from rats subjected to either 2 hr of cold-restraint stress or 1 hr of water-avoidance stress demonstrated altered ionic transport as well as significantly elevated baseline conductance (ionic permeability) and flux of horseradish peroxidase (macromolecular permeability). Intraperitoneal pretreatment with the corticotropin-releasing hormone (CRH) antagonist, a helical CRH(9-41), inhibited the stress-induced abnormalities, while exogenous intraperitoneal administration of CRH, to control rats, mimicked the stress responses and in vitro CRH increased the macromolecular permeability. These results suggest that peripheral CRH mediates stress-induced colonic pathophysiology. We speculate that a stress-induced barrier defect may allow uptake of immunogenic substances into the colonic mucosa, initiating or exacerbating intestinal inflammation.

Stress affects the gastrointestinal tract as part of the visceral response. Various stressors induce similar profiles of gut motor function alterations, including inhibition of gastric emptying, stimulation of colonic propulsive motility, and hypersensitivity to colorectal distension. In recent years, substantial progress has been made in our understanding of the underlying mechanisms of stress's impact on gut function. Activation of corticotropin-releasing factor (CRF) signaling pathways mediates both the inhibition of upper gastrointestinal (GI) and the stimulation of lower GI motor function through interaction with different CRF receptor subtypes. Here, we review how various stressors affect the gut, with special emphasis on the central and peripheral CRF signaling systems.

Corticotropin-releasing hormone (CRH) and urocortins (Ucn) bind with various affinities to two G-protein-coupled receptors, CRHR1 and CRHR2, which are expressed in brain and in peripheral tissues, including immune cells. CRHR2-deficient mice display anxiety-like behavior, hypersensitivity to stress, altered feeding behavior and metabolism, and cardiovascular abnormalities. However, the phenotype of these mice in inflammatory responses has not been determined. In the present study we found that compared with wild-type CRHR2-null mice developed substantially reduced intestinal inflammation and had lower intestinal mRNA expression of the potent chemoattractants keratinocyte chemokine and monocyte chemoattractant protein 1 following intraluminal exposure to Clostridium difficile toxin A, a potent enterotoxin that mediates antibiotic-associated diarrhea and colitis in humans. This effect was recapitulated by administration of astressin 2B, a selective CRHR2 antagonist, before toxin A exposure. Moreover, Ab array analysis revealed reduced expression of several inflammatory chemokines, including keratinocyte chemokine and monocyte chemoattractant protein 1 in toxin A-exposed mice pretreated with astressin 2B. Real-time RT-PCR of wild-type mouse intestine showed that only UcnII, but not other Ucn, was significantly up-regulated by ileal administration of toxin A at 4 h compared with buffer exposure. We also found that human colonic epithelial HT-29 cells express CRHR2alpha mRNA, whereas expression of beta and gamma spliced variants was minimal. Moreover, treatment of HT-29 cells with UcnII, which binds exclusively to CRHR2, stimulated expression of IL-8 and monocyte chemoattractant protein 1. Taken together, these results provide direct evidence that CRHR2 mediates intestinal inflammatory responses via release of proinflammatory mediators at the colonocyte level.

The class B family of G-protein-coupled receptors (GPCRs) regulates essential physiological functions such as exocrine and endocrine secretions, feeding behaviour, metabolism, growth, and neuro- and immuno-modulations. These receptors are activated by endogenous peptide hormones including secretin, glucagon, vasoactive intestinal peptide, corticotropin-releasing factor and parathyroid hormone. We have identified a common structural motif that is encoded in all class B GPCR-ligand N-terminal sequences. We propose that this local structure, a helix N-capping motif, is formed upon receptor binding and constitutes a key element underlying class B GPCR activation. The folded backbone conformation imposed by the capping structure could serve as a template for a rational design of drugs targeting class B GPCRs in several diseases.