Early-life stress (ES) leads to cognitive dysfunction in female adolescents, but the underlying neural mechanisms remain elusive. Recent evidence suggests that the cell adhesion molecules NECTIN1 and NECTIN3 play a role in cognition and ES-related cognitive deficits in male rodents. In this study, we aimed to investigate whether and how nectins contribute to ES-induced cognitive dysfunction in female adolescents. Applying the well-established limited bedding and nesting material paradigm, we found that ES impairs recognition memory, suppresses prefrontal NECTIN1 and hippocampal NECTIN3 expression, and upregulates corticotropin-releasing hormone (Crh) and its receptor 1 (Crhr1) mRNA levels in the hippocampus of adolescent female mice. Genetic experiments revealed that the reduction of dorsal CA1 (dCA1) NECTIN3 mediates ES-induced object recognition memory deficits, as knocking down dCA1 NECTIN3 impaired animals' performance in the novel object recognition task, while overexpression of dCA1 NECTIN3 successfully reversed the ES-induced deficits. Notably, prefrontal NECTIN1 knockdown did not result in significant cognitive impairments. Furthermore, acute systemic administration of antalarmin, a CRHR1 antagonist, upregulated hippocampal NECTIN3 levels and rescued object and spatial memory deficits in stressed mice. Our findings underscore the critical role of dCA1 NECTIN3 in mediating ES-induced object recognition memory deficits in adolescent female mice, highlighting it as a potential therapeutic target for stress-related psychiatric disorders in women.
Animals ; Female ; Mice ; CA1 Region, Hippocampal/metabolism* ; Cell Adhesion Molecules/metabolism* ; CRF Receptor, Type 1/metabolism* ; Memory Disorders/etiology* ; Mice, Inbred C57BL ; Nectins/genetics* ; Receptors, Corticotropin-Releasing Hormone/antagonists & inhibitors* ; Recognition, Psychology/physiology* ; Stress, Psychological/complications*

Adverse experiences in early life have long-lasting negative impacts on behavior and the brain in adulthood, one of which is sleep disturbance. As the corticotropin-releasing hormone (CRH)-corticotropin-releasing hormone receptor 1 (CRHR1) system and nucleus accumbens (NAc) play important roles in both stress responses and sleep-wake regulation, in this study we investigated whether the NAc CRH-CRHR1 system mediates early-life stress-induced abnormalities in sleep-wake behavior in adult mice. Using the limited nesting and bedding material paradigm from postnatal days 2 to 9, we found that early-life stress disrupted sleep-wake behaviors during adulthood, including increased wakefulness and decreased non-rapid eye movement (NREM) sleep time during the dark period and increased rapid eye movement (REM) sleep time during the light period. The stress-induced sleep disturbances were accompanied by dendritic atrophy in the NAc and both were largely reversed by daily systemic administration of the CRHR1 antagonist antalarmin during stress exposure. Importantly, Crh overexpression in the NAc reproduced the effects of early-life stress on sleep-wake behavior and NAc morphology, whereas NAc Crhr1 knockdown reversed these effects (including increased wakefulness and reduced NREM sleep in the dark period and NAc dendritic atrophy). Together, our findings demonstrate the negative influence of early-life stress on sleep architecture and the structural plasticity of the NAc, and highlight the critical role of the NAc CRH-CRHR1 system in modulating these negative outcomes evoked by early-life stress.
Animals ; Mice ; Corticotropin-Releasing Hormone/metabolism* ; Nucleus Accumbens/metabolism* ; Receptors, Corticotropin-Releasing Hormone/metabolism* ; Sleep ; Sleep Wake Disorders ; Stress, Psychological/complications*

It is well established that increased excitability of the presympathetic neurons in the hypothalamic paraventricular nucleus (PVN) during hypertension leads to heightened sympathetic outflow and hypertension. However, the mechanism underlying the overactivation of PVN presympathetic neurons remains unclear. This study aimed to investigate the role of endogenous corticotropin-releasing factor (CRF) on the excitability of presympathetic neurons in PVN using Western blot, arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA) recording, CRISPR/Cas9 technique and patch-clamp technique. The results showed that CRF protein expression in PVN was significantly upregulated in spontaneously hypertensive rats (SHRs) compared with normotensive Wistar-Kyoto (WKY) rats. Besides, PVN administration of exogenous CRF significantly increased RSNA, heart rate and ABP in WKY rats. In contrast, knockdown of upregulated CRF in PVN of SHRs inhibited CRF expression, led to membrane potential hyperpolarization, and decreased the frequency of current-evoked firings of PVN presympathetic neurons, which were reversed by incubation of exogenous CRF. Perfusion of rat brain slices with artificial cerebrospinal fluid containing CRF receptor 1 (CRFR1) blocker, NBI-35965, or CRF receptor 2 (CRFR2) blocker, Antisauvagine-30, showed that blocking CRFR1, but not CRFR2, hyperpolarized the membrane potential and inhibited the current-evoked firing of PVN presympathetic neurons in SHRs. However, blocking CRFR1 or CRFR2 did not affect the membrane potential and current-evoked firing of presympathetic neurons in WKY rats. Overall, these findings indicate that increased endogenous CRF release from PVN CRF neurons enhances the excitability of presympathetic neurons via activation of CRFR1 in SHRs.
Rats ; Animals ; Rats, Inbred SHR ; Paraventricular Hypothalamic Nucleus/physiology* ; Receptors, Corticotropin-Releasing Hormone/metabolism* ; Rats, Inbred WKY ; Corticotropin-Releasing Hormone/metabolism* ; Neurons/physiology* ; Hypertension ; Sympathetic Nervous System

OBJECTIVE: Corticotropin-releasing hormone (CRH) is a crucial regulator of human pregnancy and parturition. Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels are important for regulating myometrial quiescence during pregnancy. We investigated regulatory effects of different concentrations of CRH on KATP channel expression in human myometrial smooth muscle cells (HSMCs) in in vitro conditions. METHODS: After treating HSMCs with different concentrations of CRH (1, 10, 102, 103, 104 pmol/L), mRNA and protein expression of KATP channel subunits (Kir6.1 and SUR2B) was analyzed by reverse transcription-polymerase chain reaction and western blot. We investigated which CRH receptor was involved in the reaction and measured the effects of CRH on intracellular Ca2+ concentration when oxytocin was administered in HSMCs using Fluo-8 AM ester. RESULTS: When HSMCs were treated with low (1 pmol/L) and high (103, 104 pmol/L) CRH concentrations, KATP channel expression significantly increased and decreased, respectively. SUR2B mRNA expression at low and high CRH concentrations was significantly antagonized by antalarmin (CRH receptor-1 antagonist) and astressin 2b (CRH receptor-2 antagonist), respectively; however, Kir6.1 mRNA expression was not affected. After oxytocin treatment, the intracellular Ca2+ concentration in CRH-treated HSMCs was significantly lowered in low concentration of CRH (1 pmol/L), but not in high concentration of CRH (103 pmol/L), compared to control. CONCLUSION: Our data demonstrated the regulatory effect was different when HSMCs were treated with low (early pregnancy-like) and high (labor-like) CRH concentrations and the KATP channel expression showed significant increase and decrease. This could cause inhibition and activation, respectively, of uterine muscle contraction, demonstrating opposite dual actions of CRH.
Adenosine Triphosphate ; Adenosine ; Animals ; Blotting, Western ; Corticotropin-Releasing Hormone ; Female ; Humans ; In Vitro Techniques ; KATP Channels ; Mice ; Myocytes, Smooth Muscle ; Myometrium ; Oxytocin ; Parturition ; Potassium Channels ; Potassium ; Pregnancy ; Receptors, Corticotropin-Releasing Hormone ; RNA, Messenger

Functional dyspepsia (FD) and irritable bowel syndrome (IBS) are common gastrointestinal (GI) diseases; however, there is frequent overlap between FD and IBS patients. Emerging evidence links the activation of corticotropin releasing factor (CRF) receptors with stress-related alterations of gastric and colonic motor function. Therefore, we investigated the effect of peripheral CRF peptide and water avoidance stress (WAS) on upper and lower GI transit in guinea pigs. Dosages 1, 3, and 10 µg/kg of CRF were injected intraperitoneally (IP) in fasted guinea pigs 30 minutes prior to the intragastric administration of charcoal mix to measure upper GI transit. Colonic transits in non-fasted guinea pigs were assessed by fecal pellet output assay after above IP CRF doses. Blockade of CRF receptors by Astressin, and its effect on GI transit was also analyzed. Guinea pigs were subjected to WAS to measure gastrocolonic transit in different sets of experiments. Dose 10 µg/kg of CRF significantly inhibited upper GI transit. In contrast, there was dose dependent acceleration of the colonic transit. Remarkably, pretreatment of astressin significantly reverses the effect of CRF peptide on GI transit. WAS significantly increase colonic transit, but failed to accelerate upper GI transit. Peripheral CRF peptide significantly suppressed upper GI transit and accelerated colon transit, while central CRF involved WAS stimulated only colonic transit. Therefore, peripheral CRF could be utilized to establish the animal model of overlap syndrome.
Acceleration ; Animals ; Charcoal ; Colon* ; Corticotropin-Releasing Hormone ; Dyspepsia ; Guinea Pigs* ; Guinea* ; Humans ; Irritable Bowel Syndrome ; Models, Animal ; Receptors, Corticotropin-Releasing Hormone ; Water*

BACKGROUND/AIMS: Dendritic cells (DCs) are a significant contributor to the pathology of numerous chronic inflammatory autoimmune disorders; however, the effects of Corticotropin-releasing factor (CRF) on intestinal DCs are poorly understood. In this study, we investigated the role of CRF in alterations of intestinal dendritic cell phenotype and function. METHODS: Mouse mesenteric lymph node dendritic cells (MLNDCs) were obtained using magnetic bead sorting. Surface expression of CRF receptor type 1 (CRF-R1) and CRF-R2 was determined by double-labeling immunofluorescence and quantitative polymerase chain reaction (qPCR) and MLNDCs were subsequently exposed to CRF in the presence or absence of CRF-R1 and CRF-R2 antagonists. Expression of surface molecules (MHC-I and MHC-II) and co-stimulatory molecules (CD80 and CD86) was determined by flow cytometric and western blot analyses, and the T cell stimulatory capacity of MLNDCs was evaluated by mixed lymphocyte reaction. RESULTS: Immunofluorescent staining and quatitative polymerase chain reaction indicated that both the CRF receptors (CRF-R1 and CRF-2) are expressed on the surface of MLNDCs. Exposure to CRF increased the expression of MHC-II on MLNDCs as well as their capacity to stimulate T cell proliferation. MLNDCs treated with CRF-R1 antagonist exhibited a phenotype characterized by a less activated state and reduced surface expression of MHC-II, and consequently showed reduced capacity to stimulate T cells. In contrast, treatment of MLNDCs with CRF-R2 antagonist yielded an opposite result. CONCLUSIONS: CRF can alter the phenotype and function of intestinal DCs through direct action on CRF-R1 and CRF-R2, and activation of the CRF-R1 and CRF-R2 pathways yields opposing outcomes.
Animals ; Blotting, Western ; Cell Proliferation ; Corticotropin-Releasing Hormone* ; Dendritic Cells* ; Fluorescent Antibody Technique ; Immunity, Cellular ; Lymph Nodes* ; Lymphocyte Culture Test, Mixed ; Mice* ; Pathology ; Phenotype* ; Polymerase Chain Reaction ; Receptors, Corticotropin-Releasing Hormone ; T-Lymphocytes

The family of corticotropin-releasing factor (CRF) composed of 4 ligands including CRF, urocortin (Ucn) 1, Ucn2, and Ucn3 is expressed both in the central nervous system and the periphery including the gastrointestinal tract. Two different forms of G protein coupled receptors, CRF1 and CRF2, differentially recognize CRF family members, mediating various biological functions. A large body of evidence suggests that the CRF family plays an important role in regulating inflammation and angiogenesis. Of particular interest is a contrasting role of the CRF family during inflammatory processes. The CRF family can exert both pro- and anti-inflammatory functions depending on the type of receptors, the tissues, and the disease phases. In addition, there has been a growing interest in a possible role of the CRF family in angiogenesis. Regulation of angiogenesis by the CRF family has been shown to modulate endogenous blood vessel formation, inflammatory neovascularization and cardiovascular function. This review outlines the effect of the CRF family and its receptors on 2 major biological events: inflammation and angiogenesis, and provides a possibility of their application for the treatment of inflammatory vascular diseases.
Angiogenesis Inducing Agents ; Blood Vessels ; Central Nervous System ; Corticotropin-Releasing Hormone* ; Gastrointestinal Tract ; Humans ; Inflammation* ; Ligands ; Negotiating ; Receptors, G-Protein-Coupled ; Urocortins ; Vascular Diseases

Angiogenesis is the formation of new blood vessels from existing ones and an underlying cause of numerous human diseases, including cancer and inflammation. A large body of evidence indicates that angiogenic inhibitors have therapeutic potential in the treatment of vascular diseases. However, detrimental side effects and low efficacy hinder their use in clinical practice. Members of the corticotropin-releasing hormone (CRH) family, which comprises CRH, urocortin I-III, and CRH receptors (CRHR) 1 and 2, are broadly expressed in the brain and peripheral tissues, including the intestine and cardiovascular system. The CRH family regulates stress-related responses through the hypothalamic-pituitary-adrenal axis. Therapeutic agents that target CRH family members offer a new approach to the treatment of various gastrointestinal disorders, including irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and colorectal cancer. Since the discovery that CRHR 2 has anti-angiogenic activity during postnatal development in mice, studies have focused on the role of the CRH system in the modulation of blood vessel formation and cardiovascular function. This review will outline the basic biological functions of the CRH family members and the implications for the development of novel anti-angiogenic therapies.
Angiogenesis Inhibitors ; Animals ; Axis, Cervical Vertebra ; Biodiversity* ; Blood Vessels ; Brain ; Cardiovascular System ; Colorectal Neoplasms ; Corticotropin-Releasing Hormone* ; Humans ; Inflammation ; Inflammatory Bowel Diseases ; Intestines ; Irritable Bowel Syndrome ; Mice ; Receptors, Corticotropin-Releasing Hormone ; Urocortins ; Vascular Diseases

OBJECTIVE: To observe the expression of corticotropin-releasing factor-1 receptor in hippocampus of rats model of salicylate induced tinnitus. METHOD: Twenty-four rats were randomly divided into three groups, eight for each group. For Group A and Group B, 10% salicylic sodium solution was intraperitoneal injected each day at the dose of 350 mg/kg for 21 days in Group A and 14 days in Group B. Group C received intraperitoneal injection with the same amount of saline solution each day for 14 days. ABR were tested 2 days before, and 2 hours after the first administration and after the last injection. Immunohistochemical test and Western Blot were utilized to detect the expression of CRF1R in hippocampus for each group. RESULT: ABR thresholds tested 2 days before the first administration of the 3 groups showed no statistically significant difference (P > 0.05). At the time point of 2 hours after the first injection, the ABR thresholds of Group A and Group B rose by 25.90 dB SPL and 25.03 dB SPL compared with that before the administration, respectively (P < 0. 01). After the last administration, the ABR thresholds of Group A and Group B rose 34.91 dB SPL and 32.62 dB SPI. compared with that before the administration, respectively (P < 0.01). The ABR thresholds of Group C showed no significant statistical difference at all the tested time points (P > 0.05). Immunohistochemical test and Western Blot revealed that the expression level of CRF1R in the hippocampus was A > B > C (P < 0.05). CONCLUSION The expression of CRF1R in the hippocampus of salicylate induced tinnitus rat increased with the injection time, illustrating that CRF1R may participate in the mechanism of tinnitus involving the limbic system.
Animals ; Auditory Threshold ; Disease Models, Animal ; Hippocampus ; metabolism ; Injections, Intraperitoneal ; Rats ; Receptors, Corticotropin-Releasing Hormone ; metabolism ; Salicylates ; adverse effects ; Tinnitus ; chemically induced ; metabolism

OBJECTIVETo explore the expression level of corticotropin-releasing hormone receptor type-1 (CRHR-1) in intrahepatic cholestatic placental (ICP) tissue.

METHODSHuman placental samples were collected from 10 ICP patients and 10 healthy controls after parturition at 37-40 weeks of gestation. CRHR-1 protein and mRNA expression was assessed by western blotting and nested-real-time fluorescence quantitative PCR, respectively. Normally distributed data were summarized as mean +/- standard deviation, and intergroup comparisons were made by two-tailed Student's t-test. Non-normally distributed data were presented as median with interquartile range, and intergroup comparisons were made by Wilcoxon test. For all statistical analyses, a two-tailed P-value of less than 0.05 was considered statistically significant.

RESULTSThe CRHR-1 fluorescence intensity was lower in ICP tissues (1.55 +/- 0.28) than in placental tissues from healthy controls (1.60 +/- 0.37), but the difference did not reach statistical significance (t = 0.349, P = 0.732). The CRHR-1 mRNA content was slightly higher in the ICP tissues [0.139(0.268)] than in the placental tissues from healthy controls [0.031(0.245)], but the difference did not reach statistical significance (t = 1.504, P = 0.136).

CONCLUSIONCRHR-1 expression is decreased in ICP tissues, which may lead to a smaller volume of placental lobular villi vessels and restrict the CRH positive feedback loop, ultimately promoting acute hypoxic stress and possible harm to the fetus.

Adult ; Case-Control Studies ; Cholestasis, Intrahepatic ; metabolism ; Female ; Humans ; Liver Cirrhosis, Biliary ; metabolism ; Placenta ; metabolism ; Pregnancy ; Pregnancy Complications ; metabolism ; Receptors, Corticotropin-Releasing Hormone ; metabolism