Growth differentiation factor-15 stimulates the synthesis of corticotropin-releasing factor in hypothalamic 4B cells.

Growth differentiation factor-15 (GDF15) is a stress-activated cytokine that regulates cell growth and inflammatory and stress responses. We previously reported the role and regulation of GDF15 in pituitary corticotrophs. Dexamethasone increases Gdf15 gene expression levels and production. GDF15 suppresses adrenocorticotropic hormone synthesis in pituitary corticotrophs and subsequently mediates the negative feedback effect of glucocorticoids. Here, we analyzed corticotropin-releasing factor (Crf) promoter activity in hypothalamic 4B cells transfected with promoter-driven luciferase reporter constructs. The effects of time and GDF15 concentration on Crf mRNA levels were analyzed using quantitative real-time polymerase chain reaction. Glial cell-derived neurotrophic factor family receptor α-like (GFRAL) protein is expressed in 4B cells. GDF15 increased Crf promoter activity and Crf mRNA levels in 4B cells. The protein kinase A and C pathways also contributed to the GDF15-induced increase in Crf gene expression. GDF15 stimulates GFRAL, subsequently increasing the phosphorylation of AKT, an extracellular signal-related kinase, and the cAMP response element-binding protein. Therefore, GDF15-dependent pathways may be involved in regulating Crf expression under stressful conditions in hypothalamic cells.

CRH-R2 signalling modulates feeding and circadian gene expression in hypothalamic mHypoA-2/30 neurons.

The hypothalamic type 2 corticotropin releasing hormone receptor (CRH-R2) plays critical roles in homeostatic regulation, particularly in fine tuning stress recovery. During acute stress, the CRH-R2 ligands CRH and urocortins promote adaptive responses and feeding inhibition. However, in rodent models of chronic stress, over-exposure of hypothalamic CRH-R2 to its cognate agonists is associated with urocortin 2 (Ucn2) resistance; attenuated cAMP-response element binding protein (CREB) phosphorylation and increased food intake. The molecular mechanisms involved in these altered CRH-R2 signalling responses are not well described. In the present study, we used the adult mouse hypothalamus-derived cell line mHypoA-2/30 to investigate CRH-R2 signalling characteristics focusing on gene expression of molecules involved in feeding and circadian regulation given the role of clock genes in metabolic control. We identified functional CRH-R2 receptors expressed in mHypoA-2/30 cells that differentially regulate CREB and AMP-activated protein kinase (AMPK) phosphorylation and downstream expression of the appetite-regulatory genes proopiomelanocortin (Pomc) and neuropeptide Y (Npy) in accordance with an anorexigenic effect. We studied for the first time the effects of Ucn2 on clock genes in native and in a circadian bioluminescence reporter expressing mHypoA-2/30 cells, detecting enhancing effects of Ucn2 on mRNA levels and rhythm amplitude of the circadian regulator Aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), which could facilitate anorexic responses in the activity circadian phase. These data uncover novel aspects of CRH-R2 hypothalamic signalling that might be important in regulation of circadian feeding during stress responses.

Nesfatin-1 stimulates the hypothalamus-pituitary-interrenal axis hormones in goldfish.

Stress in vertebrates is mediated by the hypothalamus-pituitary-adrenal (in mammals)/interrenal (in fish) (HPA/I) axis, which produces the corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), and corticosteroids, respectively. Nesfatin-1, a novel anorexigenic peptide encoded in the precursor nucleobindin-2 (NUCB2), is increasingly acknowledged as a peptide that influences the stress axis in mammals. The primary aim of this study was to characterize the putative effects of nesfatin-1 on the fish HPI axis, using goldfish (Carassius auratus) as an animal model. Our results demonstrated that nucb2/nesfatin-1 transcript abundance was detected in the HPI tissues of goldfish, with most abundant expression in the pituitary. NUCB2/nesfatin-1-like immunoreactivity was found in the goldfish hypothalamus, pituitary, and interrenal cells of the head kidney. GPCR12, a putative receptor for nesfatin-1, was also detected in the pituitary and interrenal cells. NUCB2/nesfatin-1-like immunoreactivity was observed in ACTH-expressing pituitary corticotrophs. Acute netting and restraint stress upregulated nucb2/nesfatin-1 mRNA levels in the forebrain, hypothalamus, and pituitary, as well as crf and crf-r1 expression in the forebrain and hypothalamus. Intraperitoneal and intracerebroventricular administration of nesfatin-1 increased cortisol release and hypothalamic crf mRNA levels, respectively. Finally, we found that nesfatin-1 significantly stimulated ACTH secretion from dispersed pituitary cells in vitro. Collectively, our data provide the first evidence showing that nesfatin-1 is a stress responsive peptide, which modulates the stress axis hormones in fish.

Novel serotonin-boosting effect of incense smoke from Kynam agarwood in mice: The involvement of multiple neuroactive pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Stress is a state of feeling that inhibits one from responding properly in the face of a threat. Agarwood smoke has been used in traditional medicine as a sedative anti-anxious, and anti-restless therapy. Its scent emitted from heat induces people to enter a stable state; however, the underlying molecular effect is still unclear. AIM OF THE STUDY: This study analyzed novel biological events and gene expression signatures induced by agarwood incense smoke in mice. MATERIALS AND METHODS: Incense smoke was produced by heating at 150 °C for 30 min in a headspace autosampler oven. We treated mice with exposure to incense smoke from Kynam agarwood for 45 min/day for 7 consecutive days. After a 7-day inhalation period, the potent agarwood smoke affected-indicators in serum were measured, and the RNA profiles of the mouse brains were analyzed by microarray to elucidate the biological events induced by agarwood incense smoke. RESULTS: Chemical profile analysis showed that the major component in the incense smoke of Kynam was 2-(2-phenylethyl) chromone (26.82%). Incense smoke from Kynam induced mice to enter a stable state and increased the levels of serotonin in sera. The emotion-related pathways, including dopaminergic synapse, serotonergic synapse, GABAergic synapse, long-term depression and neuroactive ligand-receptor interaction, were significantly affected by incense smoke. Moreover, the expression of Crhr2 and Chrnd genes, involved with neuroactive ligand-receptor interaction pathway, was upregulated by incense smoke. CONCLUSIONS: By a newly-established incense smoke exposure system, we first identified that anti-anxious and anti-depressant effects of agarwood incense smoke were likely associated with the increase of serotonin levels and multiple neuroactive pathways in mice.

Prenatal infection with Mycoplasma pulmonis in rats exaggerates the angiotensin II pressor response in adult offspring.

Exposure to different stressors in utero is linked to adult diseases such as obesity and hypertension. In this study, the impact of prenatal infection (PNI) on adult body weight and cardiovascular function was evaluated using a naturally occurring rodent pathogen, Mycoplasma pulmonis (MP). Pregnant Sprague-Dawley rats were infected with MP on gestationalday 14 and gave birth naturally. Adult PNI offspring weighed more than controls, but resting mean arterial pressure (MAP) was unchanged. Subcutaneous injection of angiotensin II (10 µg/kg) elicited a rise in MAP that was greater in both male and female PNI offspring compared with controls (P < 0.03). The accompanying reflex bradycardia was similar to the controls, suggesting that PNI induced baroreflex dysfunction. Subcutaneous nicotine administration, a potent cardiorespiratory stimulus, also elicited a transient rise in MAP that was generally greater in the PNI group, but the change in MAP from baseline was only significant in the PNI females compared with controls (P < 0.03). Elevated body weight and cardiovascular reactivity in the PNI offspring was associated with an increase in the ratio of hypothalamic corticotrophin-releasing hormone receptors type 1 to type 2 gene expression in both sexes compared with controls. These findings support previous studies demonstrating that PNI induces alterations in cardiovascular function and body weight. Yet, unlike previous studies utilizing other models of PNI (e.g., endotoxin), MP PNI did not induce resting hypertension. Thus, our study provides a foundation for future studies evaluating the cardiovascular risks of offspring exposed to microbial challenges in utero.

Methanol extract of semen Ziziphi Spinosae attenuates ethanol withdrawal anxiety by improving neuropeptide signaling in the central amygdala.

BACKGROUND: Ethanol withdrawal (EtOHW) anxiety is a crucial risk factor for alcoholic relapse. The neuropeptide nociceptin/orphanin FQ (N/OFQ) acts upon its receptor (NOP) to antagonize corticotropin-releasing factor (CRF) and elicit anxiolytic actions. Semen Ziziphi Spinosae (SZS), a prototypical hypnotic-sedative herb in Oriental medicine, exhibits anxiolytic effects during nicotine withdrawal by improving amygdaloid CRF/CRF1 receptor (CRFR1) signaling. Therefore, we evaluated the effects of SZS on EtOHW anxiety and the involvement of amygdaloid CRF/CRFR1 and N/OFQ/NOP pathways. METHODS: Male Sprague Dawley rats received intraperitoneal injections of 2 g/kg EtOH (20% v/v) once daily for 28 d followed by a 3-d withdrawal. During EtOHW, the rats were given once-daily intragastric treatments of a methanol extract of SZS (MESZS, 60 or 180 mg/kg/d). Anxiety-like behaviors were measured with the open field (OF) and elevated plus maze (EPM) tests, and plasma corticosterone (CORT) levels were examined by an enzyme-linked immunosorbent assay. mRNA and protein expression levels of the neuropeptides and their receptors were determined by quantitative polymerase chain reaction and Western blot assays. RESULTS: MESZS increased the distance traveled in the center zone of the OF and dose-dependently elongated the duration of staying in the center zone in EtOHW rats. MESZS increased both the number of entries into and the time spent in the open arms of the EPM by EtOHW rats. And, MESZS inhibited the over secretion of plasma CORT during EtOHW. EtOHW enhanced CRF and CRFR1 gene and protein expression in the central nucleus of the amygdala (CeA), which were inhibited by 180 mg/kg/d MESZS. EtOHW increased amygdaloid NOP mRNA and protein expression but spared N/OFQ mRNA expression, and 180 mg/kg/d MESZS further promoted these increases. Additionally, a post-MESZS intra-CeA infusion of either CRF or the selective NOP antagonist UFP-101 abolished the expected anxiolytic effect of 180 mg/kg/d MESZS. CONCLUSIONS: These results suggest that MESZS ameliorates EtOHW anxiety by improving both CRF/CRFR1 and N/OFQ/NOP transmissions in the CeA.

Aggressive behavior and stress response after oxytocin administration in male Norway rats selected for different attitudes to humans.

Oxytocin (OXT) is known to influence on social behaviors, including intermale aggression and hypothalamic-pituitary-adrenal (HPA) axis activity. However, there are no data on the effects of oxytocin on intermale aggression and HPA axis activity in rats selected for elimination and enhancement of aggressiveness towards humans. The aim of this study is to elucidate the role of oxytocin in expression of aggressive behavior and stress response in Norway rats selected for elimination (tame) and enhancement (aggressive) of an aggressive-defensive reaction to humans. Oxytocin was administered to males via nasal applications once or for 5 days (daily). Resident-intruder test showed that in aggressive males, single oxytocin administration caused an increase in the latent period of aggressive interactions and a decrease in the percentage of direct aggression time (not including the time of lateral threat postures) as compared to the control aggressive rats administered with saline. After a 5-day oxytocin administration, aggressive animals demonstrated shorter time of aggressive interactions compared to the control rats. Resident-intruder test revealed no significant changes in behavior of tame rats after single oxytocin administration, while multiple administration caused an increase in aggressive behavior in tame rats. Oxytocin applications caused an elevation of corticosterone level after restriction in aggressive males, but did not affect expression of Crh, Crh1 and Crhr2 genes in hypothalamus in either tame or aggressive rats. The data obtained indicate significant role of oxytocinergic system in the behavior formed in the process of selection by reaction to humans.

Corticotropin-releasing factor receptor 2-deficiency eliminates social behaviour deficits and vulnerability induced by cocaine.

BACKGROUND AND PURPOSE: Poor social behaviour and vulnerability to stress are major clinical features of stimulant use disorders. The corticotropin-releasing factor (CRF) system mediates stress responses and might underlie substance use disorders; however, its involvement in social impairment induced by stimulant substances remains unknown. CRF signalling is mediated by two receptor types, CRF1 and CRF2 . In the present study we investigated the role of the CRF2 receptor in social behaviour deficits, vulnerability to stress and related brain alterations induced by cocaine administration and withdrawal. EXPERIMENTAL APPROACH: CRF2 receptor-deficient (CRF2 -/-) and littermate wild-type mice were repeatedly tested in the three-chamber task for sociability (i.e. preference for an unfamiliar conspecific vs. an object) and social novelty preference (SNP; i.e. preference for a novel vs. a familiar conspecific) before and after chronic cocaine administration. An in situ hybridization assay was used to assess gene expression of the stress-responsive arginine vasopressin (AVP) and oxytocin (OT) neuropeptides in the hypothalamus. KEY RESULTS: CRF2 receptor deficiency eliminated the sociability deficit induced by cocaine withdrawal. Moreover, CRF2 -/- mice did not show either the stress-induced sociability deficit or the increased AVP and OT expression associated with long-term cocaine withdrawal, indicating resilience to stress. Throughout, wild-type and CRF2 -/- mice displayed SNP, suggesting that cocaine withdrawal-induced sociability deficits were not due to impaired detection of social stimuli. CONCLUSIONS AND IMPLICATIONS: These findings demonstrate a central role for the CRF2 receptor in social behaviour deficits and biomarkers of vulnerability induced by cocaine withdrawal, suggesting new therapeutic strategies for stimulant use disorders.

Dietary macronutrient composition affects hypothalamic appetite regulation in chicks.

OBJECTIVE: The objective was to determine the effects of high-protein and high-fat diets, and fasting and refeeding, on appetite regulation in chicks. METHODS: Day of hatch chicks were fed one of four diets: basal, high protein (25% crude protein), and 15 and 30% high fat (15 and 30% metabolizable energy derived from soybean oil, respectively), and assigned to one of three treatments at 4 days: (1) access to feed, (2) 3 hours of fasting, or (3) fasting followed by 1 hour of refeeding. The hypothalamus was collected, total RNA isolated, and mRNA abundance measured. RESULTS: Food intake was reduced in chicks fed the high-protein and high-fat diets. Agouti-related peptide, neuropeptide Y (NPY), NPY receptors 1, 2, and 5, melanocortin receptors 3 and 4 (MC3R and 4R, respectively), mesotocin, corticotropin-releasing factor (CRF), and CRF receptor sub-type 2 (CRFR2) mRNAs were greatest in chicks that consumed the basal diet. Refeeding was associated with increased MC3R mRNA in the high-protein diet group. CRFR2 mRNA was increased by fasting and refeeding in chicks that consumed the high-protein diet. DISCUSSION: Food intake and hypothalamic gene expression of some important appetite-associated factors were reduced in chicks fed the high-protein or high-fat diets. Fasting and refeeding accentuated several differences and results suggest that the CRF and melanocortin pathways are involved.

Tectal corticotropin-releasing factor (CRF) neurons respond to fasting and a reactive stressor in the African Clawed Frog, Xenopus laevis.

It is well established that hypothalamic neurons producing the peptide corticotropin-releasing factor (CRF) play a key role in stress adaptation, including reduction of food intake when a threat or stressor is present. We have previously reported on the presence of an intrinsic CRF signaling system within the optic tectum (OT), a brain area that plays a key role in visually guided prey capture/predator avoidance decisions. To better understand the potential role of tectal CRF neurons in regulating adaptive behavior and energy balance during stress we examined evidence for modulation of tectal CRF neuronal activity after stressor exposure and food deprivation in the African clawed frog Xenopus laevis. We tested two predictions, 1) that exposure to categorically distinct stressors (ether vapors and shaking) will reduce food intake and modulate the activity of tectal CRF cells, and 2) that food deprivation will modulate the activity of tectal CRF cells. Exposure to ether increased tectal content of CRF and CRF transcript, but lowed CRFR1 transcript abundance. Two weeks of food deprivation reduced total fat stores in frogs and decreased tectal content of CRF content while having no effect on CRF and CRFR1 transcript abundance. Our data are consistent with a role for tectal CRF neurons in modulating food intake in response to certain stressors.

CRHR2/Ucn2 signaling is a novel regulator of miR-7/YY1/Fas circuitry contributing to reversal of colorectal cancer cell resistance to Fas-mediated apoptosis.

Colorectal cancer (CRC) responds poorly to immuno-mediated cytotoxicity. Underexpression of corticotropin-releasing-hormone-receptor-2 (CRHR2) in CRC, promotes tumor survival, growth and Epithelial to Mesenchymal Transition (EMT), in vitro and in vivo. We explored the role of CRHR2 downregulation in CRC cell resistance to Fas/FasL-mediated apoptosis and the underlying molecular mechanism. CRC cell sensitivity to CH11-induced apoptosis was compared between Urocortin-2 (Ucn2)-stimulated parental and CRHR2-overexpressing CRC cell lines and targets of CRHR2/Ucn2 signaling were identified through in vitro and ex vivo analyses. Induced CRHR2/Ucn2 signaling in SW620 and DLD1 cells increased specifically their sensitivity to CH11-mediated apoptosis, via Fas mRNA and protein upregulation. CRC compared to control tissues had reduced Fas expression that was associated with lost CRHR2 mRNA, poor tumor differentiation and high risk for distant metastasis. YY1 silencing increased Fas promoter activity in SW620 and re-sensitized them to CH11-apoptosis, thus suggesting YY1 as a putative transcriptional repressor of Fas in CRC. An inverse correlation between Fas and YY1 expression was confirmed in CRC tissue arrays, while elevated YY1 mRNA was clinically relevant with advanced CRC grade and higher risk for distant metastasis. CRHR2/Ucn2 signaling downregulated specifically YY1 expression through miR-7 elevation, while miR-7 modulation in miR-7high SW620-CRHR2+ and miR-7low HCT116 cells, had opposite effects on YY1 and Fas expressions and cell sensitivity to CH11-killing. CRHR2/Ucn2 signaling is a negative regulator of CRC cell resistance to Fas/FasL-apoptosis via targeting the miR-7/YY1/Fas circuitry. CRHR2 restoration might prove effective in managing CRC response to immune-mediated apoptotic stimuli.

The behavioral and molecular evaluation of effects of social instability stress as a model of stress-related disorders in adult female rats.

The study aimed to test the hypotheses that chronic social instability stress (CSIS) alters behavioral and physiological parameters and expression of selected genes important for stress response and social behaviors. Adult female Sprague-Dawley rats were subjected to the 4-week CSIS procedure, which involves unpredictable rotation between phases of isolation and overcrowding. Behavioral analyses (Experiment 1) were performed on the same rats before and after CSIS (n = 16) and physiological and biochemical measurements (Experiment 2) were made on further control (CON; n = 7) and stressed groups (CSIS; n = 8). Behaviors in the open field test (locomotor and exploratory activities) and elevated-plus maze (anxiety-related behaviors) indicated anxiety after CSIS. CSIS did not alter the physiological parameters measured, i.e. body weight gain, regularity of estrous cycles, and circulating concentrations of stress hormones and sex steroids. QRT-PCR analysis of mRNA expression levels was performed on amygdala, hippocampus, prefrontal cortex (PFC), and hypothalamus. The main finding is that CSIS alters the mRNA levels for the studied genes in a region-specific manner. Hence, expression of POMC (pro-opiomelanocortin), AVPR1a (arginine vasopressin receptor), and OXTR (oxytocin receptor) significantly increased in the amygdala following CSIS, while in PFC and/or hypothalamus, POMC, AVPR1a, AVPR1b, OXTR, and ERß (estrogen receptor beta) expression decreased. CSIS significantly reduced expression of CRH-R1 (corticotropin-releasing hormone receptor type 1) in the hippocampus. The directions of change in gene expression and the genes and regions affected indicate a molecular basis for the behavior changes. In conclusion, CSIS may be valuable for further analyzing the neurobiology of stress-related disorders in females.

Hypothalamo­hypophysial system in rats with autotransplantation of the adrenal cortex.

Patients with bilateral pheochromocytoma often require an adrenalectomy. Autotransplantation of the adrenal cortex is an alternative therapy that could potentially be performed instead of receiving glucocorticoid replacement following adrenalectomy. Adrenal cortex autotransplantation aims to avoid the side effects of long­term steroid treatment and adrenal insufficiency. Although the function of the hypothalamo­hypophysial system is critical for patients who have undergone adrenal cortex autotransplantation, the details of that system, with the exception of adrenocorticotropic hormone in the subjects with adrenal autotransplantation, have been overlooked for a long time. To clarify the precise effect of adrenal autotransplantation on the pituitary gland and hypothalamus, the current study examined the gene expression of hormones produced from the hypothalamus and pituitary gland. Bilateral adrenalectomy and adrenal autotransplantation were performed in 8 to 9­week­old male rats. The hypothalamus and pituitary tissues were collected at 4 weeks after surgery. Transcriptional regulation of hypothalamic and pituitary hormones was subsequently examined by reverse transcription­quantitative polymerase chain reaction. Proopiomelanocortin, glycoprotein hormone α polypeptide, and thyroid stimulating hormone ß were significantly elevated in the pituitary gland of autotransplanted rats when compared with sham­operated rats. In addition, there were significant differences in the levels of corticotropin releasing hormone receptor 1 (Crhr1), Crhr2, nuclear receptor subfamily 3 group C member 1 and thyrotropin releasing hormone receptor between the sham­operated rats and autotransplanted rats in the pituitary gland. In the hypothalamus, corticotropin releasing hormone and urocortin 2 mRNA was significantly upregulated in autotransplanted rats compared with sham­operated rats. The authors identified significant alterations in the function of not only the hypothalamus­pituitary­adrenal axis, but also the adenohypophysis thyrotropes in autotransplanted rats. In the future, it will be important to examine other tissues affected by glucocorticoids following adrenal cortex autotransplantation.

Corticotropin-releasing factor overexpression in mice abrogates sex differences in body weight, visceral fat, and food intake response to a fast and alters levels of feeding regulatory hormones.

BACKGROUND: Corticotropin-releasing factor overexpressing (CRF-OE) male mice showed an inhibited feeding response to a fast, and lower plasma acyl ghrelin and Fos expression in the arcuate nucleus compared to wild-type (WT) mice. We investigated whether hormones and hypothalamic feeding signals are impaired in CRF-OE mice and the influence of sex. METHODS: Male and female CRF-OE mice and WT littermates (4-6 months old) fed ad libitum or overnight fasted were assessed for body, adrenal glands and perigonadal fat weights, food intake, plasma hormones, blood glucose, and mRNA hypothalamic signals. RESULTS: Under fed conditions, compared to WT, CRF-OE mice have increased adrenal glands and perigonadal fat weight, plasma corticosterone, leptin and insulin, and hypothalamic leptin receptor and decreased plasma acyl ghrelin. Compared to male, female WT mice have lower body and perigonadal fat and plasma leptin but higher adrenal glands weights. CRF-OE mice lost these sex differences except for the adrenals. Male CRF-OE and WT mice did not differ in hypothalamic expression of neuropeptide Y (NPY) and proopiomelanocortin (POMC), while female CRF-OE compared to female WT and male CRF-OE had higher NPY mRNA levels. After fasting, female WT mice lost more body weight and ate more food than male WT, while CRF-OE mice had reduced body weight loss and inhibited food intake without sex difference. In male WT mice, fasting reduced plasma insulin and leptin and increased acyl ghrelin and corticosterone while female WT showed only a rise in corticosterone. In CRF-OE mice, fasting reduced insulin while leptin, acyl ghrelin and corticosterone were unchanged with no sex difference. Fasting blood glucose was higher in CRF-OE with female > male. In WT mice, fasting increased hypothalamic NPY expression in both sexes and decreased POMC only in males, while in CRF-OE mice, NPY did not change, and POMC decreased in males and increased in females. CONCLUSIONS: These data indicate that CRF-OE mice have abnormal basal and fasting circulating hormones and hypothalamic feeding-related signals. CRF-OE also abolishes the sex difference in body weight, abdominal fat, and fasting-induced feeding and changes in plasma levels of leptin and acyl ghrelin.

Loss of hypothalamic corticotropin-releasing hormone markedly reduces anxiety behaviors in mice.

A long-standing paradigm posits that hypothalamic corticotropin-releasing hormone (CRH) regulates neuroendocrine functions such as adrenal glucocorticoid release, whereas extra-hypothalamic CRH has a key role in stressor-triggered behaviors. Here we report that hypothalamus-specific Crh knockout mice (Sim1CrhKO mice, created by crossing Crhflox with Sim1Cre mice) have absent Crh mRNA and peptide mainly in the paraventricular nucleus of the hypothalamus (PVH) but preserved Crh expression in other brain regions including amygdala and cerebral cortex. As expected, Sim1CrhKO mice exhibit adrenal atrophy as well as decreased basal, diurnal and stressor-stimulated plasma corticosterone secretion and basal plasma adrenocorticotropic hormone, but surprisingly, have a profound anxiolytic phenotype when evaluated using multiple stressors including open-field, elevated plus maze, holeboard, light-dark box and novel object recognition task. Restoring plasma corticosterone did not reverse the anxiolytic phenotype of Sim1CrhKO mice. Crh-Cre driver mice revealed that PVHCrh fibers project abundantly to cingulate cortex and the nucleus accumbens shell, and moderately to medial amygdala, locus coeruleus and solitary tract, consistent with the existence of PVHCrh-dependent behavioral pathways. Although previous, nonselective attenuation of CRH production or action, genetically in mice and pharmacologically in humans, respectively, has not produced the anticipated anxiolytic effects, our data show that targeted interference specifically with hypothalamic Crh expression results in anxiolysis. Our data identify neurons that express both Sim1 and Crh as a cellular entry point into the study of CRH-mediated, anxiety-like behaviors and their therapeutic attenuation.

Effects of Paternal Predation Risk and Rearing Environment on Maternal Investment and Development of Defensive Responses in the Offspring.

Detecting past experiences with predators of a potential mate informs a female about prevailing ecological threats, in addition to stress-induced phenotypes that may be disseminated to offspring. We examined whether prior exposure of a male rat to a predator (cat) odor influences the attraction of a female toward a male, subsequent mother-infant interactions and the development of defensive (emotional) responses in the offspring. Females displayed less interest in males that had experienced predator odor. Mothers that reared young in larger, seminaturalistic housing provided more licking and grooming and active arched back-nursing behavior toward their offspring compared with dams housed in standard housing, although some effects interacted with paternal experience. Paternal predation risk and maternal rearing environment revealed sex-dependent differences in offspring wean weight, juvenile social interactions, and anxiety-like behavior in adolescence. Additionally, paternal predator experience and maternal housing independently affected variations in crf gene promoter acetylation and crf gene expression in response to an acute stressor in offspring. Our results show for the first time in mammals that variation among males in their predator encounters may contribute to stable behavioral variation among females in preference for mates and maternal care, even when the females are not directly exposed to predator threat. Furthermore, when offspring were exposed to the same threat experienced by the father, hypothalamic crf gene regulation was influenced by paternal olfactory experience and early housing. These results, together with our previous findings, suggest that paternal stress exposure and maternal rearing conditions can influence maternal behavior and the development of defensive responses in offspring.

Fish oil enhances intestinal barrier function and inhibits corticotropin-releasing hormone/corticotropin-releasing hormone receptor 1 signalling pathway in weaned pigs after lipopolysaccharide challenge.

Stress induces injury in intestinal barrier function in piglets. Long-chain n-3 PUFA have been shown to exhibit potential immunomodulatory and barrier protective effects in animal models and clinical trials. In addition, corticotropin-releasing hormone (CRH)/CRH receptor (CRHR) signalling pathways play an important role in stress-induced alterations of intestinal barrier function. We hypothesised that fish oil could affect intestinal barrier function and CRH/CRHR signalling pathways. In total, thirty-two weaned pigs were allocated to one of four treatments. The experiment consisted of a 2×2 factorial design, and the main factors included immunological challenge (saline or lipopolysaccharide (LPS)) and diet (5 % maize oil or 5 % fish oil). On d 19 of the trial, piglets were treated with saline or LPS. At 4 h after injection, all pigs were killed, and the mesenteric lymph nodes (MLN), liver, spleen and intestinal samples were collected. Fish oil decreased bacterial translocation incidence and the number of translocated micro-organisms in the MLN. Fish oil increased intestinal claudin-1 protein relative concentration and villus height, as well as improved the intestinal morphology. In addition, fish oil supplementation increased intestinal intraepithelial lymphocyte number and prevented elevations in intestinal mast cell and neutrophil numbers induced by LPS challenge. Moreover, fish oil tended to decrease the mRNA expression of intestinal CRHR1, CRH and glucocorticoid receptors. These results suggest that fish oil supplementation improves intestinal barrier function and inhibits CRH/CRHR1 signalling pathway and mast cell tissue density.

Noise Stress-Induced Changes in mRNA Levels of Corticotropin-Releasing Hormone Family Molecules and Glucocorticoid Receptors in the Rat Brain.

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.

Chronic all-trans retinoic acid administration induces CRF over-expression accompanied by AVP up-regulation and multiple CRF-controlling receptors disturbance in the hypothalamus of rats.

Clinical reports suggest a potential link between excess retinoids and development of depression. Corticotropin-releasing factor (CRF) produced in the hypothalamic paraventricular nucleus (PVN) is considered the central driver of the hypothalamic-pituitary-adrenal (HPA) axis and plays a key role in the pathogenesis of depression. Although we had shown that chronic all-trans retinoic acid (ATRA) administration induced hypothalamic CRF over-expression and hyperactivity of HPA axis in rats, further insight into how ATRA modulate CRF expression is lacking. The activity of CRF neurons is under close control of vasopressinergic system and three-paired receptors (corticosteroid receptors, sex hormone receptors and CRF receptors). Here we show that ATRA-induced CRF over-expression is accompanied by arginine-vasopressin (AVP) up-regulation and apparent gene expression disturbances of CRF-controlling receptors. ATRA was applied to rats by daily intraperitoneal injection for 6 weeks. Chronic ATRA treatment induced significantly increased expression of CRF and AVP in the PVN. Moreover, the transcript levels of CRF receptor 1 (CRFR1), estrogen receptor-ß (ERß) and mineralocorticoid receptor (MR), three genes involved in the activation of CRF neurons, were significantly increased in the hypothalamus, and the expression ratio of GRα/MR was markedly decreased. Correlation analysis indicated that the alteration of multiple CRF-controlling receptors is highly correlated with depression-related behaviors of rats in the forced swimming test. These findings support that in addition to the 'classic' retinoic acid receptor α-mediated transcriptional control of CRF expression, disruption in CRF-modulating systems constitutes a novel pathway that underlies ATRA-induced HPA axis hyperactivity in vivo.

Sympathetic activity induced by naloxone-precipitated morphine withdrawal is blocked in genetically engineered mice lacking functional CRF1 receptor.

There is large body evidence indicating that stress can lead to cardiovascular disease. However, the exact brain areas and the mechanisms involved remain to be revealed. Here, we performed a series of experiments to characterize the role of CRF1 receptor (CRF1R) in the stress response induced by naloxone-precipitated morphine withdrawal. The experiments were performed in the hypothalamic paraventricular nucleus (PVN) ventrolateral medulla (VLM), brain regions involved in the regulation of cardiovascular activity, and in the right ventricle by using genetically engineered mice lacking functional CRF1R levels (KO). Mice were treated with increasing doses of morphine and withdrawal was precipitated by naloxone administration. Noradrenaline (NA) turnover, c-Fos, expression, PKA and TH phosphorylated at serine 40, was evaluated by high-performance liquid chromatography (HPLC), immunohistochemistry and immunoblotting. Morphine withdrawal induced an enhancement of NA turnover in PVN in parallel with an increase in TH neurons expressing c-Fos in VLM in wild-type mice. In addition we have demonstrated an increase in NA turnover, TH phosphorylated at serine 40 and PKA levels in heart. The main finding of the present study was that NA turnover, TH positive neurons that express c-Fos, TH phosphorylated at serine 40 and PKA expression observed during morphine withdrawal were significantly inhibited in CRF1R KO mice. Our results demonstrate that CRF/CRF1R activation may contribute to the adaptive changes induced by naloxone-precipitated withdrawal in the heart and in the brain areas which modulate the cardiac sympathetic function and suggest that CRF/CRF1R pathways could be contributing to cardiovascular disease associated to opioid addiction.