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Prenatal stress (PS) is a major risk factor for the development of emotional disorders in adulthood that may be mediated by an altered hypothalamic-pituitary-adrenal axis response to stress. Although the early onset of stress-related disorders is recognized as a major public health problem, to date, there are relatively few studies that have examined the incidence of early-life stressors in younger individuals. In this study, we assessed PS impact on the stress-coping response of juvenile offspring in behavioral tests and in the induced molecular changes in the hippocampus. Furthermore, we assessed if pregnancy stress could be driving changes in patterns of maternal behavior during early lactation. We found that PS modified stress-coping abilities of both sex offspring. In the hippocampus, PS increased the expression of bdnf-IV and crfr1 and induced sex difference changes on glucocorticoids and BDNF mRNA receptor levels. PS changed the hippocampal epigenetic landscape mainly in male offspring. Stress during pregnancy enhanced pup-directed behavior of stressed dams. Our study indicates that exposure to PS, in addition to enhanced maternal behavior, induces dynamic neurobehavioral variations at juvenile ages of the offspring that should be considered adaptive or maladaptive, depending on the characteristics of the confronting environment. Our present results highlight the importance to further explore risk factors that appear early in life that will be important to allow timely prevention strategies to later vulnerability to stress-related disorders., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Corticotropin-releasing factor (CRF) orchestrates our body's response to stressful stimuli. Pain is often stressful and counterbalanced by activation of CRF receptors along the nociceptive pathway, although the involvement of the CRF receptor subtypes 1 and/or 2 (CRF-R1 and CRF-R2, respectively) in CRF-induced analgesia remains controversial. Thus, the aim of the present study was to examine CRF-R1 and CRF-R2 expression within the spinal cord of rats with Freund's complete adjuvant-induced unilateral inflammation of the hind paw using reverse transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis. Moreover, the antinociceptive effects of intrathecal (i.t.) CRF were measured by paw pressure algesiometer and their possible antagonism by selective antagonists for CRF-R1 and/or CRF-R2 as well as for opioid receptors. Our results demonstrated a preference for the expression of CRF-R2 over CRF-R1 mRNA, protein, binding sites and immunoreactivity in the dorsal horn of the rat spinal cord. Consistently, CRF as well as CRF-R2 agonists elicited potent dose-dependent antinociceptive effects which were antagonized by the i.t. CRF-R2 selective antagonist K41498, but not by the CRF-R1 selective antagonist NBI35965. In addition, i.t. applied opioid antagonist naloxone dose-dependently abolished the i.t. CRF- as well as CRF-R2 agonist-elicited inhibition of somatic pain. Importantly, double immunofluorescence confocal microscopy of the spinal dorsal horn showed CRF-R2 on enkephalin (ENK)-containing inhibitory interneurons in close opposition of incoming mu-opioid receptor-immunoreactive nociceptive neurons. CRF-R2 was, however, not seen on pre- or on postsynaptic sensory neurons of the spinal cord. Taken together, these findings suggest that i.t. CRF or CRF-R2 agonists inhibit somatic inflammatory pain predominantly through CRF-R2 receptors located on spinal enkephalinergic inhibitory interneurons which finally results in endogenous opioid-mediated pain inhibition., (© 2021. The Author(s).)

Signaling pathways mediated by corticotropin-releasing factor and its receptor 1 (CRF1) play a central role in stress responses. Dysfunction of the CRF system has been associated with neuropsychiatric disorders. However, dynamic changes in the CRF system during brain development and aging are not well investigated. In this study, we characterized CRF1, CRF, and corticotropin-releasing factor binding protein (CRFBP) expression in different brain regions in both male and female C57BL/6J mice from 1 to 18 months of age under basal conditions as well as after an acute 2-hr-restraint stress. We found that CRF and CRF1 levels tended to increase in the hippocampus and hypothalamus, and to decrease in the prefrontal cortex with aging, especially at 18 months of age, whereas CRFBP expression followed an opposite direction in these brain areas. We also observed area-specific sex differences in the expression of these three proteins. For example, CRF expression was lower in females than in males in all the brain regions examined except the prefrontal cortex. After acute stress, CRF and CRF1 were up-regulated at 1, 6, and 12 months of age, and down-regulated at 18 months of age. Females showed more robust changes compared to males of the same age. CRFBP expression either decreased or remained unchanged in most of the brain areas following acute stress. Our findings suggest that brain CRF1, CRF, and CRFBP expression changes dynamically across the lifespan and under stress condition in a sex- and regional-specific manner. Sex differences in the CRF system in response to stress may contribute to the etiology of stress-related neuropsychiatric disorders., (© 2020 International Society for Neurochemistry.)

Chronic stress is a crucial public issue that occurs when a person is repetitively stimulated by various stressors. Previous researches have reported that chronic stress induces spermatogenesis dysfunction in the reproductive system, but its molecular mechanisms remain unclear. The nectin protein family, including nectin-1 to nectin-4, is Ca(2+)-independent immunoglobulin-like cell adhesion molecules, that are widely expressed in the hippocampus, testicular tissue, epithelial cells and other sites. Nectin-3 contributes to the sperm development at the late stage, and the abnormal expression of nectin-3 impairs spermatogenesis. Some recent studies have demonstrated that stress induces a decrease in nectin-3 expression in the hippocampus via corticotropin-releasing hormone (CRH) to corticotropin-releasing hormone receptor 1 (CRHR1) pathway. Here, we tested whether chronic stress also caused a reduction in nectin-3 expression in the testis. We established a chronic social defeat stress paradigm, which provides naturalistic and complex chronic stress inmale C57BL/6 mice. After 25 days of chronic social defeat stress, the mice showed weight loss, thymic atrophy and some other typical symptoms of chronic stress (e.g.anxiety-like behavior and social avoidance behavior). We found gonad atrophy, testicular histological structure changes and semen quality reductions in the stressed mice. The stressed male mice significantly spent more time to impregnate the female mice than the control male mice. Moreover, nectin-3 protein levels in stressed mice were significantly decreased in the testes compared with those in control mice. In addition, we found that the CRHR1 expression level was increased in the testes of stressed mice. Therefore, we demonstrated a decreased level of nectin-3 expression and an increase in CRHR1 expression in the testis after exposure to chronic stress, which may provide a potential therapeutic target for the spermatogenesis dysfunction induced by chronic stress.

The present study aimed to investigate the long-term effects of exercise before pregnancy on changes induced by prenatal stress. Female and male Balb/c mice were divided into three groups: control (CON), prenatal restraint stress (PNS), and exercise before the gestational period plus PNS (EX + PNS). As adult, fear/anxiety behavior, corticosterone secretion, expression of hypothalamic-pituitary-adrenal (HPA)-related genes, as well as epigenetic modifications were evaluated. Exercise before gestation did not prevent the increased fear/anxiety behavior in PNS mice. A nearly significant (p = .06) basal corticosterone increase was observed in PNS males and the exercise before pregnancy reduced the stress-induced corticosterone increase in PNS females. In addition, an increase on prefrontal cortex (PFC) CRHR1 gene expression was observed in PNS females, which was attenuated by the exercise before gestation. We have also found a glucocorticoid receptor (GR) gene expression decrease in the prefrontal cortex in PNS males, as well as a histone H3 acetylation decrease (p = .06) close to the significance level. In conclusion, pregestational exercise may attenuate developmental changes induced by prenatal stress in a sex-dependent manner., (© 2020 ISDN. Published by John Wiley & Sons Ltd.)

PTSD is heterogeneous disorder that can be long lasting and often has delayed onset following exposure to a traumatic event. Therefore, it is important to take a staging approach to evaluate progression of biological mechanisms of the disease. Here, we begin to evaluate the temporal trajectory of changes following exposure to traumatic stressors in the SPS rat PTSD model. The percent of animals displaying severe anxiety on EPM increased from 17.5% at one week to 57.1% two weeks after SPS stressors, indicating delayed onset or progressive worsening of the symptoms. The LC displayed prolonged activation, and dysbalance of the CRH/NPY systems, with enhanced CRHR1 gene expression, coupled with reduced mRNAs for NPY and Y2R. In the mediobasal hypothalamus, increased CRH mRNA levels were sustained, but there was a flip in alterations of HPA regulatory molecules, GR and FKBP5 and Y5 receptor at two weeks compared to one week. Two weeks after SPS, intranasal NPY at 300 µg/rat, but not 150 µg which was effective after one week, reversed SPS triggered elevated anxiety. It also reversed SPS elicited depressive/despair symptoms and hyperarousal. Overall, the results reveal time-dependent progression in development of anxiety symptoms and molecular impairments in gene expression for CRH and NPY systems in LC and mediobasal hypothalamus by SPS. With longer time afterwards only a higher dose of NPY was effective in reversing behavioral impairments triggered by SPS, indicating that therapeutic approaches should be adjusted according to the degree of biological progression of the disorder., (Copyright © 2019. Published by Elsevier B.V.)

The ability of stress to trigger cocaine seeking in humans and rodents is variable and is determined by the amount and pattern of prior drug use. This study examined the role of a corticotropin releasing factor (CRF)-regulated dopaminergic projection from the ventral tegmental area (VTA) to the prelimbic cortex in shock-induced cocaine seeking and its recruitment under self-administration conditions that establish relapse vulnerability. Male rats with a history of daily long-access (LgA; 14 × 6 h/d) but not short-access (ShA; 14 × 2 h/d) self-administration showed robust shock-induced cocaine seeking. This was associated with a heightened shock-induced prelimbic cortex Fos response and activation of cholera toxin b retro-labeled VTA neurons that project to the prelimbic cortex. Chemogenetic inhibition of this pathway using a dual virus intersectional hM4Di DREADD (designer receptor exclusively activated by designer drug) based approach prevented shock-induced cocaine seeking. Both shock-induced reinstatement and the prelimbic cortex Fos response were prevented by bilateral intra-VTA injections of the CRF receptor 1 (CRFR1) antagonist, antalarmin. Moreover, pharmacological disconnection of the CRF-regulated dopaminergic projection to the prelimbic cortex by injection of antalarmin into the VTA in one hemisphere and the D1 receptor antagonist, SCH23390, into the prelimbic cortex of the contralateral hemisphere prevented shock-induced cocaine seeking. Finally, LgA, but not ShA, cocaine self-administration resulted in increased VTA CRFR1 mRNA levels as measured using in situ hybridization. Altogether, these findings suggest that excessive cocaine use may establish susceptibility to stress-induced relapse by recruiting CRF regulation of a stressor-responsive mesocortical dopaminergic pathway. SIGNIFICANCE STATEMENT Understanding the neural pathways and mechanisms through which stress triggers relapse to cocaine use is critical for the development of more effective treatment approaches. Prior work has demonstrated a critical role for the neuropeptide corticotropin releasing factor (CRF) in stress-induced cocaine seeking. Here we provide evidence that stress-induced reinstatement in a rat model of relapse is mediated by a CRF-regulated dopaminergic projection from the ventral tegmental area (VTA) that activates dopamine D1 receptors in the prelimbic cortex. Moreover, we report that this pathway may be recruited as a result of daily cocaine self-administration under conditions of extended drug access/heightened drug intake, likely as a result of increased CRFR1 expression in the VTA, thereby promoting susceptibility to stress-induced cocaine seeking., (Copyright © 2018 the authors 0270-6474/18/3810658-15$15.00/0.)

Background: Nicotine addiction supports tobacco smoking, a main preventable cause of disease and death in Western countries. It develops through long-term neuroadaptations in the brain reward circuit by modulating intracellular pathways and regulating gene expression. This study assesses the regional expression of the transcripts of the CRF transmission in a nicotine sensitization model, since it is hypothesised that the molecular neuroadaptations that mediate the development of sensitization contribute to the development of addiction., Methods: Rats received intraperitoneal nicotine administrations (0.4 mg/kg) once daily for either 1 day or over 5 days. Locomotor activity was assessed to evaluate the development of sensitization. The mRNA expression of CRF and CRF1 and CRF2 receptors was measured by qPCR in the ventral mesencephalon, ventral striatum, dorsal striatum (DS), prefrontal cortex (PFCx), and hippocampus (Hip)., Results: Acute nicotine administration increased locomotor activity in rats. In the sub-chronic group, locomotor activity progressively increased and reached a clear sensitization. Significant effects of sensitization on CRF mRNA levels were detected in the DS (increasing effect). Significantly higher CRF1 and CRF2 receptor levels after sensitization were detected in the Hip. Additionally, CRF2 receptor levels were augmented by sensitization in the PFCx, and treatment and time-induced increases were detected in the DS. Nicotine treatment effects were observed on CRF1R levels in the DS., Conclusions: This study suggests that the CRF transmission, in addition to its role in increasing withdrawal-related anxiety, may be involved in the development of nicotine-habituated behaviours through reduced control of impulses and the aberrant memory plasticity characterising addiction., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The LIM domain only protein LMO3 is a transcriptional regulator that has been shown to regulate several behavioral responses to alcohol. Specifically, Lmo3 null (Lmo3 Z ) mice consume more ethanol in a binge-drinking test and show enhanced ethanol-induced sedation. Due to the high comorbidity of alcohol use and anxiety, we investigated anxiety-like behavior in Lmo3 Z mice. Lmo3 Z mice spent more time in the open arms of the elevated plus maze compared with their wild-type littermates, but the effect was confounded by reduced locomotor activity. To verify the anxiety phenotype in the Lmo3 Z mice, we tested them for novelty-induced hypophagia and found that they also showed reduced anxiety in this test. We next explored the mechanism by which LMO3 might regulate anxiety by measuring mRNA and protein levels of corticotropin releasing factor (encoded by the Crh gene) and its receptor type 1 (Crhr1) in Lmo3 Z mice. Reduced Crhr1 mRNA and protein was evident in the basolateral amygdala (BLA) of Lmo3 Z mice. To examine whether Lmo3 in the amygdala is important for anxiety-like behavior, we locally reduced Lmo3 expression in the BLA of wild type mice using a lentiviral vector expressing a short hairpin RNA targeting the Lmo3 transcript. Mice with Lmo3 knockdown in the BLA exhibited decreased anxiety-like behavior relative to control mice. These results suggest that Lmo3 promotes anxiety-like behavior specifically in the BLA, possibly by altering Crhr1 expression. This study is the first to support a role for Lmo3 in anxiety-like behavior., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Rationale: The corticotropin-releasing hormone (CRH) system is a key mediator of stress-induced responses in alcohol-seeking behavior. Recent research has identified the central nucleus of the amygdala (CeA), a brain region involved in the regulation of fear and stress-induced responses that is especially rich in CRH-positive neurons, as a key player in mediating excessive alcohol seeking. However, detailed characterization of the specific influences that local neuronal populations exert in mediating alcohol responses is hampered by current limitations in pharmacological and immunohistochemical tools for targeting CRH receptor subtype 1 (CRHR1)., Objective: In this study, we investigated the effect of cell- and region-specific overexpression of CRHR1 in the CeA using a novel transgenic tool., Methods: Co-expression of CRHR1 in calcium-calmodulin-dependent kinase II (αCaMKII) neurons of the amygdala was demonstrated by double immunohistochemistry using a Crhr1-GFP reporter mouse line. A Cre-inducible Crhr1-expressing adeno-associated virus (AAV) was site-specifically injected into the CeA of αCaMKII-Cre ERT2 transgenic rats to analyze the role of CRHR1 in αCaMKII neurons on alcohol self-administration and reinstatement behavior., Results: Forty-eight percent of CRHR1-containing cells showed co-expression of αCaMKII in the CeA. AAV-mediated gene transfer in αCaMKII neurons induced a 24-fold increase of Crhr1 mRNA in the CeA which had no effect on locomotor activity, alcohol self-administration, or cue-induced reinstatement. However, rats overexpressing Crhr1 in the CeA increased responding in the stress-induced reinstatement task with yohimbine serving as a pharmacological stressor., Conclusion: We demonstrate that CRHR1 overexpression in CeA-αCaMKII neurons is sufficient to mediate increased vulnerability to stress-triggered relapse into alcohol seeking.

The activation of the HPA axis is the endocrine measure of stress responsiveness that is initiated by corticotropin-releasing hormone (CRH). CRH exerts its effects via CRHR1 and CRH-R2 receptors coupled to the cAMP signaling system and this process involves transcription factor cAMP-responsive element-binding protein (CREB).This study investigated the role of CRH and the possible involvement of CREB in gene regulation of CRH receptor, under basal conditions and after stress application in the pituitary. We used wild type (wt +/+) controls and CRH knock-out (CRH-KO -/-) male mice. Using CRH-deficient mice, we were able to investigate the consequences of the lack of the CRH on the expression of CRH receptors and transcriptional regulation mediated by CREB. We estimated the effect of acute (IMO 1×) and repeated (IMO 7×) restraint stressors lasting 30 and 120 min on the expression of mRNA CREB, CRH-R1, and CRH-R2 by qPCR. We found very significant difference in the expression of these peptides under the effect of single and repeated stress in control and CRH-KO mice. Our results indicate that both CRH receptors and CREB might be involved in the regulation of stress response in the pituitary of mice. We propose that regulation of the stress response may be better understood if more were known about the mechanisms of CRH receptor signal transduction and involvement of CREB system.

Adolescence is a time period in development when the brain undergoes substantial remodeling in response to the environment. To determine whether a stressful experience during adolescence affects adult behavior, we exposed adolescent male and female C57BL/6J mice to chronic unpredictable stress (CUS) for 12 days starting at postnatal day 28 (PND28). We also exposed adult male and female mice to CUS for 12 days beginning at PND70 to determine whether adolescence is a sensitive time period when stress can have long-lasting effects on behavior. Regardless of when mice were exposed to stress, they were all tested exactly 30 days later in the marble burying task, elevated zero maze, acoustic startle response, and forced swim test. Adolescent stress exposure increased anxiety-like behaviors in adult male and female mice and decreased acoustic startle response in a sex-dependent manner. However, adult stress exposure did not change anxiety or response to an acoustic tone in adult male or female mice as compared with nonstressed animals. Of interest, increased depression-like behavior in the forced swim test was observed in all mice, regardless of when the stress occurred. Gene expression analysis showed significant upregulation of corticotropin releasing factor receptor 2 (CrfR2) in the amygdala of males subjected to CUS during adolescence, but not in males that experienced CUS during adulthood. In contrast, females, regardless of when they were exposed to CUS, were not affected. These data support clinical evidence suggesting that early-life stress may predispose individuals to increased anxiety and depression later in life.

Exposure to trauma is a potential contributor to anxiety; however, the molecular mechanisms responsible for trauma-induced anxiety require further clarification. In this study, in an aim to explore these mechanisms, we observed the changes in the hypothalamic pituitary adrenal (HPA) axis using a radioimmunoassay and the changes in anxiety-like behavior using the open field test and elevated plus maze test in a rat model following intervention with NBI‑27914, a specific corticotropin‑releasing hormone receptor 1 (CRHR1) antagonist. CRHR1 was found to be involved in trauma‑induced anxiety. We then applied bioinformatic analysis to screen microRNAs (miRNAs or miRs) that target CRHR1, and miR‑34b was determined to negatively regulate CRHR1 mRNA in primary hypothalamic neurons. The overexpression of miR‑34b in the paraventricular nucleus (PVN) by a miRNA agomir using a drug delivery system decreased the hyperactivity of the HPA axis and anxiety‑like behavior. Overall, the involvement of the HPA axis in trauma‑induced anxiety was demonstrated, and trauma-induced anxiety was attenuated by decreasing the hyperactivity of the HPA axis via miR‑34b by targeting CRHR1.

Maternal separation (MS) in neonates can lead to intestinal injury. MS in neonatal mice disrupts mucosal morphology, induces colonic inflammation and increases trans-cellular permeability. Several studies indicate that intestinal epithelial stem cells are capable of initiating gut repair in a variety of injury models but have not been reported in MS. The pathophysiology of MS-induced gut injury and subsequent repair remains unclear, but communication between the brain and gut contribute to MS-induced colonic injury. Corticotropin-releasing hormone (CRH) is one of the mediators involved in the brain-gut axis response to MS-induced damage. We investigated the roles of the CRH receptors, CRHR1 and CRHR2, in MS-induced intestinal injury and subsequent repair. To distinguish their specific roles in mucosal injury, we selectively blocked CRHR1 and CRHR2 with pharmacological antagonists. Our results show that in response to MS, CRHR1 mediates gut injury by promoting intestinal inflammation, increasing gut permeability, altering intestinal morphology, and modulating the intestinal microbiota. In contrast, CRHR2 activates intestinal stem cells and is important for gut repair. Thus, selectively blocking CRHR1 and promoting CRHR2 activity could prevent the development of intestinal injuries and enhance repair in the neonatal period when there is increased risk of intestinal injury such as necrotizing enterocolitis.

Stress is a trigger factor for seizure initiation which activates hypothalamic pituitary adrenal (HPA) axis as well other brain areas. In this respect, corticotropin releasing hormone (CRH) and lateral hypothalamus (LH) orexinergic system are involved in seizure occurrence. In this study, we investigated the role of LH area and orexin expression in (mediation of) stress effect on pentylenetetrazol (PTZ) -induced seizures with hippocampal involvement. Two mild foot shock stresses were applied to intact and adrenalectomized animals; with or without CRHr1 blocking (NBI 27914) in the LH area. Then, changes in orexin production were evaluated by RT-PCR. Intravenous PTZ infusion (25 mg/ml) -induced convulsions were scored upon modified Racine scale. Finally, hippocampal glutamate and GABA were evaluated to study excitability changes. We demonstrated that the duration and severity of convulsions in stress-induced as well as adrenalectomized group were increased. Plasma corticosterone (CRT) level and orexin mRNA expression were built up in the stress and/or seizure groups. Furthermore, glutamate and GABA content was increased and decreased respectively due to stress and seizures. In contrast, rats receiving CRHr1 inhibitor showed reduced severity and duration of seizures, increased GABA, decreased glutamate and corticosterone and also orexin mRNA compared to the inhibitor free rats. Stress and adrenalectomy induced augmenting effect on seizure severity and duration and the subsequent reduction due to CRHr1 blocking with parallel orexin mRNA changes, indicated the likely involvement of CRH1r induced orexin expression of the LH in gating stress effect on convulsions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Dysregulation of the corticotropin-releasing factor (CRF) system has been observed in rodent models of binge drinking, with a large focus on CRF receptor 1 (CRF-R1). The role of CRF-binding protein (CRF-BP), a key regulator of CRF activity, in binge drinking is less well understood. In humans, single-nucleotide polymorphisms in CRHBP are associated with alcohol use disorder and stress-induced alcohol craving, suggesting a role for CRF-BP in vulnerability to alcohol addiction., Methods: The role and regulation of CRF-BP in binge drinking were examined in mice exposed to the drinking in the dark (DID) paradigm. Using in situ hybridization, the regulation of CRF-BP, CRF-R1, and CRF mRNA expression was determined in the stress and reward systems of C57BL/6J mice after repeated cycles of DID. To determine the functional role of CRF-BP in binge drinking, CRF-BP knockout (CRF-BP KO) mice were exposed to 6 cycles of DID, during which alcohol consumption was measured and compared to wild-type mice., Results: CRF-BP mRNA expression was significantly decreased in the prelimbic (PL) and infralimbic medial prefrontal cortex (mPFC) of C57BL/6J mice after 3 cycles and in the PL mPFC after 6 cycles of DID. No significant changes in CRF or CRF-R1 mRNA levels were observed in mPFC, ventral tegmental area, bed nucleus of the stria terminalis, or amygdala after 3 cycles of DID. CRF-BP KO mice do not show significant alterations in drinking compared to wild-type mice across 6 cycles of DID., Conclusions: These results reveal that repeated cycles of binge drinking alter CRF-BP mRNA expression in the mPFC, a region responsible for executive function and regulation of emotion and behavior, including responses to stress. We observed a persistent decrease in CRF-BP mRNA expression in the mPFC after 3 and 6 DID cycles, which may allow for increased CRF signaling at CRF-R1 and contribute to excessive binge-like ethanol consumption., (Copyright © 2016 by the Research Society on Alcoholism.)

Urocortins (Ucns) 1, 2, and 3 and corticotropin-releasing factor receptor 2 (CRF2) mRNA are prominently expressed in various layers of the upper gut. We tested whether Ucns and CRF2 variants are also expressed in the different layers of the rat colon, regulated by LPS (100 μg/kg ip) and play a modulatory role in the colonic immune response to LPS. Transcripts of Ucns and CRF2b, the most common isoform in the periphery, were detected in all laser microdissected layers, including myenteric neurons. LPS increased the mRNA level of Ucn 1, Ucn 2, and Ucn 3 and decreased that of CRF2b in both the colonic mucosa and submucosa + muscle (S+M) layers at 2, 6, and 9 h after injection with a return to basal at 24 h. In addition, CRF2a, another variant more prominent in the brain, and a novel truncated splice variant CRF2a-3 mRNA were detected in all segments of the large intestine. LPS reciprocally regulated the colonic expression of these CRF2 variants by decreasing both CRF2a and CRF2b, while increasing CRF2a-3 in the mucosa and S+M. The CRF2 antagonist astressin2-B further enhanced LPS-induced increase of mRNA level of interleukin (IL)-1β, TNF-α, and inducible nitric oxide synthase in S+M layers and IL-1β in the mucosa and evoked TNF-α expression in the mucosa. These data indicate that Ucns/CRF2 variants are widely expressed in all colonic layers and reciprocally regulated by LPS. CRF2 signaling dampens the CD14/TLR4-mediated acute inflammatory response to Gram-negative bacteria in the colon.

Adequate maternal behavior in rats requires minimal corticotropin-releasing factor receptor (CRF-R) activation in the medial-posterior bed nucleus of the stria terminalis (mpBNST). Based on the architectural heterogeneity of the BNST and its distinct inter-neural connectivity, we tested whether CRF-R manipulation in another functional part, the anterior-dorsal BNST (adBNST), differentially modulates maternal behavior. We demonstrate that in the adBNST, activation of CRF-R1 reduced arched back nursing (ABN) and nursing, whereas activation of CRF-R2 resulted in an initial reduction in nursing but significantly increased the incidence of ABN 5h after the treatment. Following stressor exposure, which is detrimental to maternal care, ABN tended to be protected by CRF-R1 blockade. Maternal motivation, maternal aggression, and anxiety were unaffected by any manipulation. Furthermore, under basal and stress conditions, activation of adBNST CRF-R1 increased plasma ACTH and corticosterone concentrations, whereas stimulation of adBNST CRF-R2 increased basal plasma ACTH and corticosterone concentrations, but blocked the stress-induced increase in plasma corticosterone secretion. Moreover, both the CRF-R1 and -R2 antagonists prevented the stress-induced increase in plasma corticosterone secretion. Importantly, elevated levels of circulating corticosterone induced by intra-adBNST administration of CRF-R1 or -R2 agonist did not impact maternal care. Finally, Crf mRNA expression in the adBNST was increased during lactation; however, Crfr1 mRNA expression was similar between lactating and virgin rats. In conclusion, maternal care is impaired by adBNST CRF-R1 activation, and this appears to be the result of a central action, rather than an effect of elevated circulating levels of CORT. These data provide new insights into potential causes of disturbed maternal behavior postpartum., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Systemic inflammation induces neuroimmune activation, ultimately leading to sickness (e.g., fever, anorexia, motor impairments, exploratory deficits, and social withdrawal). In this study, we evaluated the role of protein kinase R (PKR), a serine-threonine kinase that can control systemic inflammation, on neuroimmune responses and sickness., Methods: Wild-type (WT) PKR+/+ mice and PKR-/- mice were subcutaneously injected with live Escherichia coli (E. coli) or vehicle. Food consumption, rotarod test performance, burrowing, open field activity, object investigation, and social interaction were monitored. Plasma TNF-α and corticosterone were measured by ELISA. The percentage of neutrophils in blood was deduced from blood smears. Inflammatory gene expression (IL-1β, TNF-α, IL-6, cyclooxygenase (COX)-2, iNOS) in the liver and the brain (hypothalamus and hippocampus) were quantified by real-time PCR. Blood and lavage fluid (injection site) were collected for microbiological plate count and for real-time PCR of bacterial 16S ribosomal DNA (rDNA). Corticotrophin-releasing hormone (CRH) expression in the hypothalamus was also determined by real-time PCR., Results: Deficiency of PKR diminished peripheral inflammatory responses following E. coli challenge. However, while the core components of sickness (anorexia and motor impairments) were similar between both strains of mice, the behavioral components of sickness (reduced burrowing, exploratory activity deficits, and social withdrawal) were only observable in PKR-/- mice but not in WT mice. Such alteration of behavioral components was unlikely to be caused by exaggerated neuroimmune activation, by an impaired host defense to the infection, or due to a dysregulated corticosterone response, because both strains of mice displayed similar neuroimmune responses, bacterial titers, and plasma corticosterone profiles throughout the course of infection. Nevertheless, the induction of hypothalamic corticotrophin-releasing hormone (CRH) by E. coli was delayed in PKR-/- mice relative to WT mice, suggesting that PKR deficiency may postpone the CRH response during systemic inflammation., Conclusions: Taken together, our findings show that (1) loss of PKR could alter E. coli-induced sickness behaviors and (2) this was unlikely to be due to exacerbated neuroimmune activation, (3) elevated bacterial load, or (4) dysregulation in the corticosterone response. Further studies can address the role of PKR in the CRH response together with its consequence on sickness.

Noise is a widespread stress resource that may lead to detrimental effects on the health. However, the molecular basis of the stress response caused by noise remains elusive. We have studied the effects of acute and chronic noise stress on stress-related molecules in the hypothalamus and hippocampus and also corticosterone responses. Sprague Dawley rats were randomized into control, acute and chronic noise stress groups. While the chronic noise stress group animals were exposed to 100 dB white noise for 4 h/a day during 30 days, the acute noise stress group of animals was exposed to the same level of stress once for 4 h. The expression profiles of corticotropin releasing hormone (CRH), CRH1, CRH2 receptors and glucocorticoid receptor (GR) mRNAs were analysed by RT-PCR. Chronic noise stress upregulated CRH mRNA levels in the hypothalamus. Both acute and chronic noise increased CRH-R1 mRNA in the hypothalamus but decreased it in the hippocampus. GR mRNA levels were decreased by chronic noise stress in the hippocampus. The present results suggest that while corticosterone responses have habituated to continuous noise stress, the involvement of CRH family molecules and glucocorticoid receptors in the noise stress responses are different and structure specific.

We previously found that daidzein decreased food intake in female rats. The present study aimed to elucidate the relationship between dynamics of appetite-mediated neuropeptides and the anorectic effect of daidzein. We examined appetite-mediated gene expression in the hypothalamus and small intestine during the 3 meals per day feeding method. Daidzein had an anorectic effect specifically at the second feeding. Neuropeptide-Y (NPY) and galanin mRNA levels in the hypothalamus were significantly higher after feeding in the control but not in the daidzein group, suggesting that daidzein attenuated the postprandial increase in NPY and galanin expression. The daidzein group had higher corticotrophin-releasing hormone (CRH) mRNA levels in the hypothalamus after feeding, and increased cholelcystokinin (CCK) mRNA levels in the small intestine, suggesting that CCK is involved in the hypothalamic regulation of this　anorectic effect. Therefore, daidzein may induce anorexia by suppressing expression of NPY and galanin and increasing expression of CRH in the hypothalamus.

Background: Alcohol exposure has adverse effects on stress physiology and behavioral reactivity. This is suggested to be due, in part, to the effect of alcohol on β-endorphin (β-EP)-producing neurons in the hypothalamus. In response to stress, β-EP normally provides negative feedback to the hypothalamic-pituitary-adrenal axis and interacts with other neurotransmitter systems in the amygdala to regulate behavior. We examined whether β-EP neuronal function in the hypothalamus reduces the corticosterone response to acute stress, attenuates anxiety-like behaviors, and modulates alcohol drinking in rats., Methods: To determine whether β-EP neuronal transplants modulate the stress response, anxiety behavior, and alcohol drinking, we implanted differentiated β-EP neurons into the paraventricular nucleus (PVN) of the hypothalamus of normal, prenatal alcohol-exposed, and alcohol-preferring (P) and alcohol-non-preferring (NP) rats. We then assessed corticosterone levels in response to acute restraint stress and other markers of stress response in the brain and anxiety-like behaviors in the elevated plus maze and open-field assays., Results: We showed that β-EP neuronal transplants into the PVN reduced the peripheral corticosterone response to acute stress and attenuated anxiety-like behaviors. Similar transplants completely reduced the hypercorticosterone response and elevated anxiety behaviors in prenatal alcohol-exposed adult rats. Moreover, we showed that β-EP reduced anxiety behavior in P rats with minimal effects on alcohol drinking during and following restraint stress., Conclusions: These data further establish a role of β-EP neurons in the hypothalamus for regulating physiological stress response and anxiety behavior and resemble a potential novel therapy for treating stress-related psychiatric disorders in prenatal alcohol-exposed children and those genetically predisposed to increased alcohol consumption., (Copyright © 2015 by the Research Society on Alcoholism.)

Corticotropin-releasing hormone (CRH) and CRH receptors (CRH-Rs) are expressed in the skin; CRH-R1 is the predominant receptor. Whether the CRH/CRH-R1 system plays a role in psoriasis has not yet been assessed. Immunohistochemistry, real-time RT-PCR, ELISA assay, and Western blot analysis were used to investigate the expression of CRH/CRH-R1 in patients with chronic plaque psoriasis and that of IL-18 in CRH-treated HaCaT cells. CRH and CRH-R1 were downregulated in patients with chronic plaque psoriasis. In vitro, CRH attenuated the expression of IL-18 by a mitogen-activated protein kinase signaling pathway through CRH-R1 in HaCaT cells. Thus, an aberrant cutaneous CRH/CRH-R1 system exists in lesions from chronic plaque psoriasis which might play a role in psoriasis and offers further evidence for the study of CRH in the skin.

Endometriosis is an inflammatory disease of women of reproductive age featured by the presence of ectopic endometrium and is strongly related to infertility. Galectins, carbonhydrate-binding proteins, have been found to have pro- or anti-inflammatory roles in the reproductive tract and in pathological conditions concerning infertility. Galectin-1, which is expressed at endometrium and decidua, plays a major role in implantation and trophoblast invasion. Also, the neuropeptides, corticotropin releasing hormone (CRH) and urocortin (UCN) and their receptors are expressed in eutopic and ectopic endometrium showing a differential expression pattern in endometriotic women compared to healthy ones. The aim of this study was to examine the galectin-1 expression in endometriotic lesions and compare its expression in eutopic endometrium of endometriotic and healthy women. Furthermore, we examined the effect of CRH and UCN in galectin-1 expression in Ishikawa cell line and macrophages and investigated the implication of CRHR1 in these responses. Eutopic and ectopic endometrium specimens, Ishikawa cell line and mice macrophages were used. Immunohistochemistry and western blot analysis were performed in order to identify galectin-1 expression in ectopic and eutopic endometrium of women with and without endometriosis and the regulatory effect of CRH and UCN on galectin-1 expression. This study presents for the first time that galectin-1 is overexpressed in endometriotic lesions compared to eutopic endometrium of endometriotic women and is more abundantly expressed in eutopic endometrium of disease women compared to healthy ones. Furthermore, it is shown that CRH and UCN upregulate galectin-1 expression in Ishikawa cell line and macrophages and this effect is mediated through CRHR1. These results suggest that galectin-1 might play an important role in endometriosis pathology and infertility profile of women suffering from endometriosis by being at the same time regulated by CRH and UCN interfering in the immune disequilibrium which characterizes this pathological condition.

We have recently developed a soluble mimic of the corticotropin-releasing factor receptor type 1 (CRF1), a membrane-spanning G protein-coupled receptor, which allowed investigations on receptor-ligand interactions. The CRF1 mimic consists of the receptor N-terminus and three synthetic extracellular loops (ECL1-3), which constitute the extracellular receptor domains (ECDs) of CRF1, coupled to a linear peptide template. Here, we report the synthesis of a modified CRF1 mimic, which is more similar to the native receptor possessing a cyclic template that displays the ECDs in a more physiological conformation compared with the initial linear design. In order to facilitate detailed biophysical investigations on CRF1 mimics, we have further established a cost-efficient access to the CRF1 mimic, which is suitable for isotopic labeling for NMR spectroscopy. To this end, the loop-mimicking cyclic peptide of the ECL2 of CRF1 was produced recombinantly and cyclized by expressed protein ligation. Cyclic ECL2 was obtained in milligram scale, and CRF1 mimics synthesized from this material displayed the same binding properties as synthetic CRF1 constructs., (Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.)

Aims: To investigate the regulating effects of catalpol on the hypothalamic-pituitary- adrenocortical-axis (HPA) in an Alzheimer's disease (AD) rat model., Methods: Healthy male Wistar Rats were selected. The AD model was generated by orthotopic injection of beta-amyloid 25-35 (Abeta25-35) into the right lateral ventricle. The animals were divided into five study groups: Catalpol at low dose (5 mg/kg), Catalpol at high dose (10 mg/kg), model control group and sham surgery control group, n = 9 respectively. The serum concentration of hydrocortisone (HYD), adrenocorticotropin (ACTH) and corticotropin releasing hormone (CRH) determined by Enzyme-Linked Immunosorbent Assay (ELISA). Structural alterations of the hypothalamus were examined by H&E stain and electron microscope. The CRH receptor 1 (CRHR1) positive neurons were detected with immunohistochemistry., Results: Serum HYD level was significantly increased (p < 0.01), and both ACTH and CRH were dramatically decreased (p < 0.01) in the AD model group rats compared with normal control rats at day 7. Catalpol treatment was able to improve the hormone secretion disorder in AD model group rats compared with the model group (p < 0.01 or p < 0.05) in particular at 21 days. Structure damage of hypothalamus in the AD rat as evidenced less CRHR1 positive neurons, rough endoplasmic reticulum dilation and degranulation, and mitochondrial swelling under electron microscope. Catalpol treatment at both high and low doses was able to alleviate the structure damage of the hypothalamus in the AD rats., Conclusions: Catalpol could improve the endocrine function of the HPA and alleviate the structural damage of hypothalamus in AD rats.

Peripheral corticotropin-releasing hormone receptors (CRHRs) are G protein-coupled receptors that play different roles depending on tissue types. Previously, we discovered the mechanism of CRHR-mediated apoptosis of mouse prostate cancer cell line (RM-1) to be a change of Bcl-2:Bax ratio, and CRH was found to inhibit transforming growth factor β migration of breast cancer cells via CRHRs. In the present study, we investigated cytosolic calcium-dependent phospholipase A2 (cPLA2) bridging CRHR activations and Bcl-2:Bax ratio and the effect of CRHR activation on cell migration. Silencing of cPLA2 attenuated a CRHR1 agonist, CRH-induced apoptosis, and the decrease of the Bcl-2:Bax ratio, whereas silencing of cPLA2 aggravated CRHR2 agonist, Urocortin 2 (Ucn2)-inhibited apoptosis, and the increase of the Bcl-2:Bax ratio. CRH in a time- and concentration-dependent manner increased cPLA2 expression mainly through interleukin 1β (IL1β) upregulation. Ucn2 decreased cPLA2 expression through neither tumor necrosis factor α nor IL1β. CRH-suppressed decay of cPLA2 mRNA and Ucn2 merely suppressed its production. Overexpression of CRHR1 or CRHR2 in HEK293 cells correspondingly upregulated or downregulated cPLA2 expression after CRH or Ucn2 stimulation respectively. In addition, both CRH and Ucn2 induced migration of RM-1 cells. Our observation not only established a relationship between CRHRs and cell migration but also for the first time, to our knowledge, demonstrated that cPLA2 participates in CRHR1-induced apoptosis and CRHR2-inhibited apoptosis.

Smoking cessation leads to a dysphoric state and this increases the risk for relapse. Animal studies indicate that the dysphoric state associated with nicotine withdrawal is at least partly mediated by an increase in corticotropin-releasing factor (CRF) release in the central nucleus of the amygdala (CeA). In the present study, we investigated whether a sustained overexpression of CRF in the CeA affects the dysphoric-like state associated with nicotine withdrawal. To study brain reward function, rats were prepared with intracranial self-stimulation (ICSS) electrodes in the medial forebrain bundle. An adeno-associated virus (AAV, pseudotype 2/5) was used to overexpress CRF or green fluorescent protein (GFP, control) in the CeA and minipumps were used to induce nicotine dependence. The AAV2/5-CRF vector induced a 40% increase in CRF protein and mRNA levels in the CeA. Administration of the nicotinic receptor antagonist mecamylamine (precipitated withdrawal) or nicotine pump removal (spontaneous withdrawal) led to elevations in ICSS thresholds. Elevations in ICSS thresholds are indicative of a dysphoric-like state. The overexpression of CRF did not affect baseline ICSS thresholds but diminished the elevations in ICSS thresholds associated with precipitated and spontaneous nicotine withdrawal. The real-time reverse transcriptase (RT)-PCR analysis showed that the overexpression of CRF led to a decrease in CRF1 mRNA levels and an increase in CRF2 mRNA levels in the CeA. In conclusion, the overexpression of CRF in the CeA diminishes the dysphoric-like state associated with nicotine withdrawal and this might be driven by neuroadaptive changes in CRF1 and CRF2 receptor gene expression.

Mycotoxins are chemicals produced by fungus and many of them are toxic to humans. Zeranol is a mycotoxin used to promote growth in cattle in North America; yet such a practice draws concern about the residual compound in meat in European countries. In the present study, the toxicity of zeranol was tested in a mouse model for reproduction. Pregnant ICR mice were given p.o. daily doses of zeranol at 0, 1, 10, 100mg/kg for 4 days (from E13.5 to E16.5). Increased rates of fetal resorption at late gestation (E17.5) and preterm birth (

Objective: Expression of corticotropin-releasing factor type 1 receptor (CRFR1) has been shown on pancreatic β cells, and its activation potentiates glucose-stimulated insulin secretion (GSIS). However, the roles of CRFR1 in energy metabolism beyond insulin release remain elusive., Materials/methods: We characterized the metabolic phenotypes of mice lacking CRFR1 (CRFR1KO mice) under conditions of energy excess., Results: When fed a normal diet, the glucose profile of CRFR1KO mice in response to a glucose tolerance test was similar to that of wild-type (WT) mice, while serum insulin levels were significantly lower in CRFR1KO mice, reflecting high insulin sensitivity in part due to very low glucocorticoid levels. Histology of the pancreas revealed islet hypoplasia in CRFR1KO mice, suggesting a role of CRFR1 in maintaining the β cell mass in a manner similar to incretins. In response to a high-fat diet, CRFR1KO mice showed insulin resistance, but serum insulin levels during glucose challenge remained at a low level, indicating defective GSIS. In addition, CRFR1KO mice showed resistance to diet-induced obesity and hepatic steatosis. Although total food intake was not different between CRFR1KO and WT mice, oxygen consumption was significantly increased in CRFR1KO mice. The increased energy expenditure may explain the lean phenotype of CRFR1KO mice under conditions of energy excess., Conclusions: Our results suggest that CRFR1 plays important roles in whole body energy homeostasis, providing compelling evidence of the close relationship between energy homeostasis and the function of the hypothalamic-pituitary-adrenal axis., (© 2013 Elsevier Inc. All rights reserved.)

Urocortin 2 (Ucn2) is a member of the corticotropin-releasing factor peptide family and is expressed by various tissues, including reproductive tissues such as the uterus, ovary, and placenta. However, the regulatory mechanisms of Ucn2 expression and the physiological significance of Ucn2 in these tissues remain unclear. We previously showed that passive immunization of immature female rats by i.p. injection of anti-Ucn2 IgG induces earlier onset of puberty. Therefore, this study was designed to clarify the site and regulatory mechanisms of Ucn2 expression in the uterus. Expression levels of Ucn2 mRNA in the uterus were higher in immature (2- and 4-week-old) and aged (17-month-old) rats than in mature (9-week-old) rats in the proestrus phase. In 9-week-old rats, mRNA expression levels and contents in the uterus were lower in the proestrus phase than in the diestrus phase, while plasma Ucn2 concentrations did not differ between the two phases. Ucn2-like immunoreactivitiy was detected in the endometrial gland epithelial cells of the uterus. S.c. injection of estradiol benzoate or an estrogen receptor α (ERα) agonist significantly reduced mRNA expression levels and contents of Ucn2 in the uterus when compared with vehicle-injected ovariectomized rats. By contrast, estradiol benzoate increased Ucn2 mRNA expression levels in the lung. Thus, estrogens downregulate Ucn2 expression in the uterus in a tissue-specific manner, and Ucn2 may play a role in the regulatory mechanisms of maturation of the uterus through ERα and estrous cycle.

Background: The mechanism underlying estrogen cardioprotection remains largely unknown. Urocortin (UCN), a member of corticotropin-releasing hormone (CRH) family, is one of endogenous cardioprotective factors. The goal of present study is to investigate whether estrogens regulate UCN and its receptor CRH receptor type 2 (CRHR2) in female rat heart., Methods: 17β-estradiol (E2) was subcutaneously administrated to ovariectomized (OVX) rats for eight weeks. UCN was administrated before simulated myocardial ischemia/reperfusion (I/R). Cell damage was assessed by measurement of infarct size, activity of serum creatine kinase (CK) and lactate dehydrogenase (LDH) and percentage of TUNEL staining in myocardium. The mRNA and protein levels of UCN and CRHR2 were determined in sham operated and OVX rats with or without E2 replacement. DNA methylation frequency of CRHR2 gene promoter was determined by bisulfite-sequencing., Results: UCN administration reduced infarct size, LDH and CK level and percentage of TUNEL staining upon I/R injury. The cardioprotective effects of UCN were abrogated in OVX rats and E2 replacement restored UCN-induced cardioprotection.CRHR2 mRNA and protein expression were down-regulated more than 40% in OVX rats, both of which were restored by E2 replacement. UCN mRNA and protein levels were not affected by ovariectomy and E2 replacement. Hypermethylation in CRHR2 promoter was found in OVX rats, and two of the methylated CpG sites were seated at cis-acting elements. Hypermethylation induced by OVX could also be ameliorated by E2 replacement., Conclusion: Estrogens maintain CRHR2 expression in myocardium, which may through an epigenetic mechanism, and enhance UCN-induced cardioprotective effects against I/R injury., (© 2013.)

The corticotropin-releasing hormone (CRH) family regulates the endocrine stress response. Here, we examined the effect of immobilization stress (IMO) on gene expression of adrenomedullary CRH family members. Urocortin 2 (Ucn2) has the highest basal gene expression and is increased by > 30-fold in response to single IMO and about 10-fold after six daily repeated IMO. IMO also induced a smaller rise in CRH (six-fold) and CRH receptor type 1 (CRHR1; two-fold with single IMO). The influence of glucocorticoids was examined. Dexamethasone (DEX) or corticosterone greatly increased Ucn2 mRNA levels in PC12 cells in a dose-dependent manner. The DEX elicited rise in Ucn2 was abolished by actinomycin D pre-treatment, indicating a transcriptionally mediated response. DEX also triggered a rise in CRHR1 and lowered CRH mRNA levels. In CRH-knockout mice, where the IMO-induced rise in corticosterone was attenuated, the response of IMO on Ucn2, as well as CRHR2 mRNAs was absent. Overall, the results suggest that the stress-triggered rise in glucocorticoids is involved in the large induction of Ucn2 mRNA levels by IMO, which may allow Ucn2 to act in an autocrine/paracrine fashion to modulate adrenomedullary function, or act as an endocrine hormone., (© 2013 International Society for Neurochemistry.)

Increasing estradiol concentrations during the late follicular phase stimulate sexual behavior and the GnRH/LH surge, and it is known that kisspeptin signaling is essential for the latter. Administration of LPS can block these events, but the mechanism involved is unclear. We examined brain tissue from intact ewes to determine: i) which regions are activated with respect to sexual behavior, the LH surge and LPS administration, ii) the location and activation pattern of kisspeptin cells in control and LPS treated animals, and iii) whether CRFR type 2 is involved in such disruptive mechanisms. Follicular phases were synchronized with progesterone vaginal pessaries and control animals were killed at 0 h, 16 h, 31 h or 40 h (n=4-6/group) after progesterone withdrawal (time zero). At 28 h, other animals received endotoxin (LPS; 100 ng/kg) and were subsequently killed at 31 h or 40 h (n=5/group). LH surges only occurred in control ewes, during which there was a marked increase in c-Fos expression within the ventromedial nucleus (VMN), arcuate nucleus (ARC), and medial preoptic area (mPOA), as well as an increase in the percentage of kisspeptin cells co-expressing c-Fos in the ARC and mPOA compared to animals sacrificed at all other times. Expression of c-Fos also increased in the bed nucleus of the stria terminalis (BNST) in animals just before the expected onset of sexual behavior. However, LPS treatment increased c-Fos expression within the VMN, ARC, mPOA and diagonal band of broca (dBb), along with CRFR type 2 immunoreactivity in the lower part of the ARC and median eminence (ME), compared to controls. Furthermore, the percentage of kisspeptin cells co-expressing c-Fos was lower in the ARC and mPOA. Thus, we hypothesize that in intact ewes, the BNST is involved in the initiation of sexual behavior while the VMN, ARC, and mPOA as well as kisspeptin cells located in the latter two areas are involved in estradiol positive feedback only during the LH surge. By contrast, disruption of sexual behavior and the LH surge after LPS involves cells located in the VMN, ARC, mPOA and dBb, as well as cells containing CRFR type 2 in the lower part of the ARC and ME, and is accompanied by inhibition of kisspeptin cell activation in both the ARC and mPOA., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Rationale: Stress is a common antecedent reported by people suffering major depression. In these patients, extrahypothalamic brain areas, like the hippocampus and basolateral amygdala (BLA), have been found to be affected. The BLA synthesizes CRF, a mediator of the stress response, and projects to hippocampus. The main hippocampal target for this peptide is the CRF subtype 1 receptor (CRF1). Evidence points to a relationship between dysregulation of CRF/CRF1 extrahypothalamic signaling and depression., Objective: Because selective serotonin reuptake inhibitors (SSRIs) are the first-line pharmacological treatment for depression, we investigated the effect of chronic treatment with the SSRI fluoxetine on long-term changes in CRF/CRF1 signaling in animals showing a depressive-like behavior., Methods: Male Wistar rats were exposed to the learned helplessness paradigm (LH). After evaluation of behavioral impairment, the animals were treated with fluoxetine (10 mg/kg i.p.) or saline for 21 days. We measured BLA CRF expression with RT-PCR and CRF1 expression in CA3 and the dentate gyrus of the hippocampus with in situ hybridization. We also studied the activation of one of CRF1's major intracellular signaling targets, the extracellular signal-related kinases 1 and 2 (ERK1/2) in CA3., Results: In saline-treated LH animals, CRF expression in the BLA increased, while hippocampal CRF1 expression and ERK1/2 activation decreased. Treatment with fluoxetine reversed the changes in CRF and CRF1 expressions, but not in ERK1/2 activation., Conclusion: In animals exposed to the learned helplessness paradigm, there are long-term changes in CRF and CRF1 expression that are restored with a behaviorally effective antidepressant treatment.

Background: Urocortin and corticotropin-releasing factors (CRFs) and their receptors are expressed in many organs, including the central nervous system. In this study, the expression of mRNAs of urocortin 1, 2, 3, and CRF and CRF receptors 1 and 2 in malignant glioma, was examined., Materials and Methods: The RNAs of human and rat glioma cell lines were isolated. Transcripts in these cells were analyzed using cDNA. In addition, the effects of proliferative and cytotoxic stimulation by serum supplementation, ionizing radiation, and the antineoplastic agent temozolomide were investigated., Results: Human and rat cells transcribed urocortin. CRF receptors were detected in human glioma cells. When human KNS42 cells were exposed to stimulation, transcription was altered according to the specific condition., Conclusion: Expression of mRNAs of urocortin and CRF receptors was confirmed in human glioma cell lines. Although the quantities of transcripts varied with the proliferative and cytotoxic stimulation, the overall transcription pattern was not influenced by these stimuli.

During cortical development, cell proliferation and cell cycle exit are carefully regulated to ensure that the appropriate numbers of cells are produced. Urocortin (UCN) is a member of the corticotrophin releasing hormone (CRH) family of neuropeptides that regulates stress responses. UCN is widely distributed in adult rat brain. However, the expression and function of UCN in embryonic brain is, as yet, unclear. Here, we show that UCN is endogenously expressed in proliferative zones of the developing cerebral cortex and its receptors are exhibited in neural stem cells (NSCs), thus implicating the neuropeptide in cell cycle regulation. Treatment of cultured NSCs or organotypic slice cultures with UCN markedly reduced cell proliferation. Furthermore, blocking of endogenous UCN/CRHRs system either by treatment with CRHRs antagonists or by neutralization of secreted UCN with anti-UCN antibody increased NSCs proliferation. Cell cycle kinetics analysis demonstrated that UCN lengthened the total cell cycle duration via increasing the G1 phase and accelerated cell cycle exit. UCN directly inhibited the histone deacetylase (HDAC) activity and induced a robust increase in histone H3 acetylation levels. Using pharmacological and RNA interference approaches, we further demonstrated that antiproliferative action of UCN appeared to be mediated through a HDAC inhibition-induced p21 upregulation. Moreover, UCN treatment in vitro and in vivo led to an increase in neuronal differentiation of NSCs. These findings suggest that UCN might contribute to regulate NSCs proliferation and differentiation during cortical neurogenesis., (Copyright © 2012 AlphaMed Press.)

Peripheral activation of corticotropin-releasing factor receptor type 2 (CRF(2)) by urocortin 1, 2, or 3 (Ucns) exerts powerful effects on gastric function; however, little is known about their expression and regulation in the stomach. We investigated the expression of Ucns and CRF(2) isoforms by RT-PCR in the gastric corpus (GC) mucosa and submucosa plus muscle (S+M) or laser captured layers in naive rats, their regulations by lipopolysaccharide (LPS, 100 μg/kg ip) over 24 h, and the effect of the CRF(2) antagonist astresssin(2)-B (100 μg/kg sc) on LPS-induced delayed gastric emptying (GE) 2-h postinjection. Transcripts of Ucns and CRF(2b,) the most common wild-type CRF(2) isoform in the periphery, were expressed in all layers, including myenteric neurons. LPS increased Ucn mRNA levels significantly in both mucosa and S+M, reaching a maximal response at 6 h postinjection and returning to basal levels at 24 h except for Ucn 1 in S+M. By contrast, CRF(2b) mRNA level was significantly decreased in the mucosa and M+S with a nadir at 6 h. In addition, CRF(2a), reportedly only found in the brain, and the novel splice variant CRF(2a-3) were also detected in the GC, antrum, and pylorus. LPS reciprocally regulated these variants with a decrease of CRF(2a) and an increase of CRF(2a-3) in the GC 6 h postinjection. Astressin(2)-B exacerbated LPS-delayed GE (42-73%, P < 0.001). These data indicate that Ucn and CRF(2) isoforms are widely distributed throughout the rat stomach and inversely regulated by immune stress. The CRF(2) signaling system may act to counteract the early gastric motor alterations to endotoxemia.

Posttraumatic stress disorder (PTSD) is a debilitating disease, which affects 8-10% of the population exposed to traumatic events. The factors that make certain individuals susceptible to PTSD and others resilient are currently unknown. Corticotropin-releasing factor receptor type 2 (CRFR2) has been implicated in mediating stress coping mechanisms. Here, we use a physiological PTSD-like animal model and an in-depth battery of tests that reflect the symptomology of PTSD to separate mice into subpopulations of "PTSD-like" and "Resilient" phenotypes. PTSD-like mice are hypervigilant, hyperalert, insomniac, have impaired attention and risk assessment, as well as accompanying attenuated corticosterone levels. Intriguingly, PTSD-like mice show long-term robust upregulation of BNST-CRFR2 mRNA levels, and BNST-CRFR2-specific lentiviral knockdown reduces susceptibility to PTSD-like behavior. Additionally, using a BNST mRNA expression array, PTSD-like mice exhibit a general transcriptional attenuation profile, which was associated with upregulation of the BNST-deacetylation enzyme, HDAC5. We suggest PTSD to be a disease of maladaptive coping.

Glucose transport across the placenta is mediated by glucose transporters (GLUT), which is critical for normal development and survival of the fetus. Regulatory mechanisms of GLUT in placenta have not been elucidated. Placental CRH has been implicated to play a key role in the control of fetal growth and development. We hypothesized that CRH, produced locally in placenta, could act to modulate GLUT in placenta. To investigate this, we obtained human placentas from uncomplicated term pregnancies and isolated and cultured trophoblast cells. GLUT1 and GLUT3 expressions in placenta were determined, and effects of CRH on GLUT1 and GLUT3 were examined. GLUT1 and GLUT3 were identified in placental villous syncytiotrophoblasts and the endothelium of vessels. Treatment of cultured placental trophoblasts with CRH resulted in an increase in GLUT1 expression while a decrease in GLUT3 expression in a dose-dependent manner. Cells treated with either CRH antibody or nonselective CRH receptor (CRH-R) antagonist astressin showed a decrease in GLUT1 and an increase in GLUT3 expression. CRH-R1 antagonist antalarmin decreased GLUT1 expression while increased GLUT3 expression. CRH-R2 antagonist astressin2b increased the expression of both GLUT1 and GLUT3. Knockdown of CRH-R1 decreased GLUT1 expression while increased GLUT3 expression. CRH-R2 knockdown caused an increase in both GLUT1 and GLUT3 expression. Our data suggest that, in placenta, CRH produced locally regulates GLUT1 and GLUT3 expression, CRHR1 and CRHR2-mediated differential regulation of GLUT1 and GLUT3 expression. Placental CRH may regulate the growth of fetus and placenta by modulating the expression of GLUT in placenta during pregnancy.

The association between psychological and environmental stress with functional gastrointestinal disorders, especially irritable bowel syndrome (IBS), is well established. However, the underlying pathogenic mechanisms remain unknown. We aimed to probe chronic psychosocial stress as a primary inducer of intestinal dysfunction and investigate corticotropin-releasing factor (CRF) signaling and mitochondrial damage as key contributors to the stress-mediated effects. Wistar-Kyoto rats were submitted to crowding stress (CS; 8 rats/cage) or sham-crowding stress (SC; 2 rats/cage) for up to 15 consecutive days. Hypothalamic-pituitary-adrenal (HPA) axis activity was evaluated. Intestinal tissues were obtained 1h, 1, 7, or 30 days after stress exposure, to assess neutrophil infiltration, epithelial ion transport, mitochondrial function, and CRF receptors expression. Colonic response to CRF (10 μg/kg i.p.) and hyperalgesia were evaluated after ending stress exposure. Chronic psychosocial stress activated HPA axis and induced reversible intestinal mucosal inflammation. Epithelial permeability and conductance were increased in CS rats, effect that lasted for up to 7 days after stress cessation. Visceral hypersensitivity persisted for up to 30 days post stress. Abnormal colonic response to exogenous CRF lasted for up to 7 days after stress. Mitochondrial activity was disturbed throughout the intestine, although mitochondrial response to CRF was preserved. Colonic expression of CRF receptor type-1 was increased in CS rats, and negatively correlated with body weight gain. In conclusion, chronic psychosocial stress triggers reversible inflammation, persistent epithelial dysfunction, and colonic hyperalgesia. These findings support crowding stress as a suitable animal model to unravel the complex pathophysiology underlying to common human intestinal stress-related disorders, such as IBS., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In several species, individuals from the same population behave differently from each other. A functional link between variation in personality traits and the stress response has been suggested by studies in artificial selection lines in fish, birds and mammals. The aim of this study was to test whether the expression of genes involved in the stress response co-varies with personality traits in a natural population. Four personality traits, excreted cortisol level and brain expression of six candidate genes (CRF, CRF-R2, POMC1, GR1, GR2, MR) were measured in non-stressed wild-caught threespine sticklebacks (Gasterosteus aculeatus). We found correlations between variation in personality traits and variation in the expression of genes involved in the stress response. Aggressiveness was negatively correlated with cortisol levels. Boldness and aggressiveness formed a behavioural syndrome and were both positively correlated with brain expression of glucocorticoid receptors (GR1 and GR2). Boldness and exploration were positively correlated with expression of POMC1 but not with each other. Our results are compatible with a model that suggests that the aggressiveness-boldness behavioural syndrome could be the consequence of a physiological pleiotropic effect of glucocorticoid receptors, which are involved in the stress response and behaviour variation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Because overactivation of the hypothalamic-pituitary-adrenal (HPA) axis occurs in Alzheimer's disease (AD), dysregulation of stress neuromediators may play a mechanistic role in the pathophysiology of AD. However, the effects of stress on tau phosphorylation are poorly understood, and the relationship between corticosterone and corticotropin-releasing factor (CRF) on both β-amyloid (Aβ) and tau pathology remain unclear. Therefore, we first established a model of chronic stress, which exacerbates Aβ accumulation in Tg2576 mice and then extended this stress paradigm to a tau transgenic mouse model with the P301S mutation (PS19) that displays tau hyperphosphorylation, insoluble tau inclusions and neurodegeneration. We show for the first time that both Tg2576 and PS19 mice demonstrate a heightened HPA stress profile in the unstressed state. In Tg2576 mice, 1 month of restraint/isolation (RI) stress increased Aβ levels, suppressed microglial activation, and worsened spatial and fear memory compared with nonstressed mice. In PS19 mice, RI stress promoted tau hyperphosphorylation, insoluble tau aggregation, neurodegeneration, and fear-memory impairments. These effects were not mimicked by chronic corticosterone administration but were prevented by pre-stress administration of a CRF receptor type 1 (CRF(1)) antagonist. The role for a CRF(1)-dependent mechanism was further supported by the finding that mice overexpressing CRF had increased hyperphosphorylated tau compared with wild-type littermates. Together, these results implicate HPA dysregulation in AD neuropathogenesis and suggest that prolonged stress may increase Aβ and tau hyperphosphorylation. These studies also implicate CRF in AD pathophysiology and suggest that pharmacological manipulation of this neuropeptide may be a potential therapeutic strategy for AD.

Previous studies have demonstrated that various type of stressors modulate messenger ribonucleic acid (mRNA) for type 1 corticotropin-releasing hormone (CRH) receptor (CRH-R1 mRNA) and type 2 CRH receptor (CRH-R2 mRNA). The purpose of this study was to explore the effect of social isolation stress of varying durations on the CRH, CRH-R1 and CRH-R2 mRNAs expression in the hypothalamus, hippocampus and pituitary of socially monogamous female and male prairie voles (Microtus ochrogaster). Isolation for 1h (single isolation) or 1h of isolation every day for 4 weeks (repeated isolation) was followed by a significant increase in plasma corticosterone levels. Single or repeated isolation increased hypothalamic CRH mRNA expression, but no changes in CRH-R1 mRNA in the hypothalamus were observed. Continuous isolation for 4 weeks (chronic isolation) showed no effect on hypothalamic CRH or CRH-R1 mRNAs in female or male animals. However, hypothalamic CRH-R2 mRNA was significantly reduced in voles exposed to chronic isolation. Single or repeated isolation, but not chronic isolation, significantly increased CRH-R1 mRNA and decreased CRH-R2 mRNA in the pituitary. Despite elevated CRH mRNA expression, CRH-R1 and CRH-R2 mRNAs were not modulated in the hippocampus following single or repeated isolation. Although, chronic isolation did not affect hippocampal CRH or CRH-R1 mRNAs, it did increase CRH-R2 mRNA expression in females and males. The results of the present study in prairie voles suggest that social isolation has receptor subtype and species-specific consequences for the modulation of gene expression for CRH and its receptors in brain and pituitary. Previous studies have revealed a female-biased increase in oxytocin in response to chronic isolation; however, we did not find a sex difference in CRH or its receptors following single, repeated or chronic social isolation, suggesting that sexually dimorphic processes beyond the CRH system, possibly involving vasopressin, might explain this difference., (Published by Elsevier Ltd.)

The expression of corticotropin-releasing hormone (CRH) receptor 1 messenger RNA in stages of follicle growth was examined by reverse transcriptase-polymerase chain reaction in long-term cultures of early preantral mouse follicles with and without CRH addition. Corticotropin-releasing hormone receptor 1 is present in stages of mouse follicle growth, whereas 10(-9), 10(-7), and 10(-6) mol/L CRH inhibits oocyte maturation in vitro, an effect reversed by antalarmin addition., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Fallopian tube (FT) and endometrial urocortin 1 (Ucn1) and corticotropin-releasing hormone (CRH)-receptor (CRH-R1/CRH-R2) expression were examined using quantitative real-time polymerase chain reaction (RT-PCR) and immunohistochemistry in nonpregnant and pregnant women (intrauterine, IUP; ectopic pregnancy, EP). Tubal Ucn1 messenger RNA (mRNA) expression was higher in luteal compared to follicular phase (P < .01) and equivalent to follicular phase in FT from EP. Tubal CRH-R1/CRH-R2 mRNA was lower in luteal phase (P < .05) and in FT from EP compared to follicular phase (P < .01). Ucn1 mRNA was lower in endometrium from EP compared to IUP (P < .05). Corticotropin-releasing hormone-R1 mRNA was higher in endometrium from EP compared to viable IUP (P < .05). No differences were observed in CRH-R2 expression. Corticotropin-releasing hormone-R1 protein was primarily localized to epithelium of FT and endometrium. Quantitative analysis of tubal CRH-R1 protein expression reflected that seen at the mRNA level but endometrial expression was equivocal. The demonstration of attenuated tubal/endometrial Ucn1/CRH-R expression in EP further supports a role of the CRH-family in embryo implantation.

Context: The pathogenesis of preterm delivery (PTD) is not clear, although inflammation/infection play a major role. Corticotropin releasing-hormone (CRH) and Urocortins (Ucns) are involved in the pathophysiology of PTD., Objective: This study evaluates trophoblast mRNA expression of CRH, Ucn, Ucn2, Ucn3, and their receptors [CRH-type 1 receptor (CRH-R1), CRH-R2] in infective conditions. To determine whether infection or glucocorticoids contribute to change their placental mRNA expression, the effects of lipopolysaccharide or dexamethasone was evaluated., Design: Placentas were obtained from spontaneous PTD; premature rupture of membranes (pPROM) and pPROM with chorioamnionitis., Setting: Placental specimens were collected from women receiving perinatal care at our Division of Obstetrics and Gynecology., Patients or Other Participants: Pregnant women delivered preterm were enrolled., Interventions: mRNA expression was evaluated by RT-PCR., Main Outcome Measure: Because CRH and Ucns are involved in immunological functions we evaluated their involvement in PTD with or without infection., Results: CRH, Ucn2, and CRH-R1 mRNA expression were higher, while Ucn and CRHR-2 were lower in pPROM with chorioamnionitis than in PTD and pPROM. Ucn3 mRNA expression was lower in pPROM with and without chorioamnionitis than in PTD. The addition of lipopolysaccharide in trophoblast explants decreased Ucn, Ucn3, and CRH-R2 and increased CRH, Ucn2, and CRH-R1 mRNA expression in a dose-dependent manner. Dexamethasone increased CRH and decreased Ucn2 mRNA expression in a dose dependent manner., Conclusions: Our findings showed a significant impact of pPROM with chorioamnionitis on placental CRH peptides and receptors, suggesting that placental expression of stress-related pathways is activated in infective process.