Urocortin-1 promotes colorectal cancer cell migration and proliferation and inhibits apoptosis via inhibition of the p53 signaling pathway.

PURPOSE: To investigate the effect of urocortin-1 (UCN-1) on growth, migration, and apoptosis in colorectal cancer (CRC) in vivo and vitro and the mechanism by which UCN-1 modulates CRC cells in vitro. METHODS: The correlation between UCN-1 and CRC was evaluated using The Cancer Genome Atlas (TCGA) database and a tissue microarray. The expression of UCN-1 in CRC cells was assessed using quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting. In vitro, the influence of UCN-1 on the proliferation, apoptosis, and migration of HT-29, HCT-116, and RKO cells was explored using the celigo cell counting assay or cell counting kit-8 (CCK8), flow cytometry, and wound healing or Transwell assays, respectively. In vivo, the effect of UCN-1 on CRC growth and progression was evaluated in nude mice. The downstream pathway underlying UCN-1-mediated regulation of CRC was determined using the phospho-kinase profiler array in RKO cells. Lentiviruses were used to knockdown or upregulate UCN-1 expression in cells. RESULTS: Both the TCGA and tissue microarray results showed that UCN-1 was strongly expressed in the tissues of patients with CRC. Furthermore, the tissue microarray results showed that the expression of UCN-1 was higher in male than in female patients, and high expression of UCN-1 was associated with higher risk of lymphatic metastasis and later pathological stage. UCN-1 knockdown caused a reduction in CRC cell proliferation, migration, and colony formation, as well as an increase in apoptosis. In xenograft experiments, tumors generated from RKO cells with UCN-1 knockdown exhibited reduced volumes and weights. A reduction in the expression of Ki-67 in xenograft tumors indicated that UCN-1 knockdown curbed tumor growth. The human phospho-kinase array showed that the p53 signaling pathway participated in UCN-1-mediated CRC development. The suppression in migration and proliferation caused by UCN-1 knockdown was reversed by inhibitors of p53 signal pathway, while the increase in cell apoptosis was suppressed. On the other hand, overexpression of UCN-1 promoted proliferation and migration and inhibited apoptosis in CRC cells. Overexpression of p53 reversed the effect of UCN-1 overexpression on CRC development. CONCLUSION: UCN-1 promotes migration and proliferation and inhibits apoptosis via inhibition of the p53 signaling pathway.

The Lack of TRPA1 Ion Channel Does Not Affect the Chronic Stress-Induced Activation of the Locus Ceruleus.

We have previously proven the involvement of transient receptor potential ankyrin 1 (TRPA1) in stress adaptation. A lack of TRPA1 affects both urocortin 1 (member of the corticotropin-releasing hormone (CRH) family) content of the Edinger-Westphal nucleus. The noradrenergic locus ceruleus (LC) is also an important player in mood control. We aimed at investigating whether the TRPA1 is expressed in the LC, and to test if the response to chronic variable mild stress (CVMS) is affected by a lack of TRPA1. The TRPA1 expression was examined via RNAscope in situ hybridization. We investigated TRPA1 knockout and wildtype mice using the CVMS model of depression. Tyrosine hydroxylase (TH) and FOSB double immunofluorescence were used to test the functional neuromorphological changes in the LC. No TRPA1 expression was detected in the LC. The TH content was not affected by CVMS exposure. The CVMS-induced FOSB immunosignal did not co-localize with the TH neurons. TRPA1 is not expressed in the LC. A lack of functional TRPA1 receptor neither directly nor indirectly affects the TH content of LC neurons under CVMS.

The peripheral corticotropin releasing factor family's role in vasculitis.

Corticotropin releasing factor family peptides (CRF peptides) include 4 members, corticotropin releasing hormone (CRH), Urocortin (UCN1), UCN2 and UCN3. CRF peptides function via the two distinct receptors, CRF1 and CRF2. Among them, CRH/CRF1 has been recognized to influence immunity/inflammation peripherally. Both pro- and anti-inflammatory effects of CRH are reported. Likewise, UCNs, peripherally in cardiovascular system have been documented to have both potent protective and harmful effects, with UCN1 acting on both CRF1 & CRF2 and UCN2 & UCN3 on CRF2. We and others also observe protective and detrimental effects of CRF peptides/receptors on vasculature, with the latter of predominantly higher incidence, i.e., they play an important role in the development of vasculitis while in some cases they are found to counteract vascular inflammation. The pro-vasculitis effects of CRH & UCNs include increasing vascular endothelial permeability, interrupting endothelial adherens & tight junctions leading to hyperpermeability, stimulating immune/inflammatory cells to release inflammatory factors, and promoting angiogenesis by VEGF release while the anti-vasculitis effects may be just the opposite, depending on many factors such as different CRF receptor types, species and systemic conditions. Furthermore, CRF peptides' pro-vasculitis effects are found to be likely related to cPLA2 and S1P receptor signal pathway. This minireview will focus on summarizing the peripheral effects of CRF peptides on vasculature participating in the processes of vasculitis.

The expression of corticotropin-releasing hormone family peptides in premalignant and malignant vulvar lesions.

OBJECTIVES: To examine the relation of corticotropin-releasing hormone (CRH) family peptides with inflammatory processes and oncogenesis, emphasizing in vulvar inflammatory, premalignant and malignant lesions, as well as to investigate the possibility of lesion cells immunoescaping, utilizing FAS/FAS-L complex. METHODS: Immunohistochemical expression of CRH, urocortin (UCN), FasL and their receptors CRHR1, CRHR2 and Fas was studied in vulvar tissue sections obtained from patients with histologically confirmed diagnosis of lichen, vulvar intraepithelial neoplasia (VIN) and vulvar squamous cell carcinoma (VSCC). The patient cohort was selected from a tertiary teaching Hospital in Greece, between 2005 and 2015. For each of the disease categories, immunohistochemical staining was evaluated and the results were statistically compared. RESULTS: A progressive increase of the cytoplasmic immunohistochemical expression of CRH and UCN, from precancerous lesions to VSCC was observed. A similar increase was detected for Fas and FasL expression. Nuclear localization of UCN was demonstrated in both premalignant and VSCC lesions, with staining being significantly intensified in carcinomas, particularly in the less differentiated tumor areas or in the areas at invasive tumor front. CONCLUSIONS: Stress response system and CRH family peptides seem to have a role in inflammation maintenance and progression of vulvar premalignant lesions to malignancy. It seems that stress peptides may locally modulate the stroma through Fas/FasL upregulation, possibly contributing to vulvar cancer development.

Urocortin2 attenuates diabetic coronary microvascular dysfunction by regulating macrophage extracellular vesicles.

Diabetic patients develop coronary microvascular dysfunction (CMD) and exhibit high mortality of coronary artery disease. Methylglyoxal (MGO) largely accumulates in the circulation due to diabetes. We addressed whether macrophages exposed to MGO exhibited damaging effect on the coronary artery and whether urocortin2 (UCN2) serve as protecting factors against such diabetes-associated complication. Type 2 diabetes was induced by high-fat diet and a single low-dose streptozotocin in mice. Small extracellular vesicles (sEV) derived from MGO-treated macrophages (MGO-sEV) were used to produce diabetes-like CMD. UCN2 was examined for a protective role against CMD. The involvement of arginase1 and IL-33 was tested by pharmacological inhibitor and IL-33-/- mice. MGO-sEV was capable of causing coronary artery endothelial dysfunction similar to that by diabetes. Immunocytochemistry studies of diabetic coronary arteries supported the transfer of arginase1 from macrophages to endothelial cells. Mechanism studies revealed arginase1 contributed to the impaired endothelium-dependent relaxation of coronary arteries in diabetic and MGO-sEV-treated mice. UCN2 significantly improved coronary artery endothelial function, and prevented MGO elevation in diabetic mice or enrichment of arginase1 in MGO-sEV. Diabetes caused a reduction of IL-33, which was also reversed by UCN2. IL-33-/- mice showed impaired endothelium-dependent relaxation of coronary arteries, which can be mitigated by arginase1 inhibition but can't be improved by UCN2 anymore, indicating the importance of restoring IL-33 for the protection against diabetic CMD by UCN2. Our data suggest that MGO-sEV induces CMD via shuttling arginase1 to coronary arteries. UCN2 is able to protect against diabetic CMD via modulating MGO-altered macrophage sEV cargoes.

Musclin prevents depression-like behavior in male mice by activating urocortin 2 signaling in the hypothalamus.

Introduction: Physical activity is recommended as an alternative treatment for depression. Myokines, which are secreted from skeletal muscles during physical activity, play an important role in the skeletal muscle-brain axis. Musclin, a newly discovered myokine, exerts physical endurance, however, the effects of musclin on emotional behaviors, such as depression, have not been evaluated. This study aimed to access the anti-depressive effect of musclin and clarify the connection between depression-like behavior and hypothalamic neuropeptides in mice. Methods: We measured the immobility time in the forced swim (FS) test, the time spent in open arm in the elevated-plus maze (EPM) test, the mRNA levels of hypothalamic neuropeptides, and enumerated the c-Fos-positive cells in the paraventricular nucleus (PVN), arcuate nucleus (ARC), and nucleus tractus solitarii (NTS) in mice with the intraperitoneal (i.p.) administration of musclin. Next, we evaluated the effects of a selective corticotropin-releasing factor (CRF) type 1 receptor antagonist, selective CRF type 2 receptor antagonist, melanocortin receptor (MCR) agonist, and selective melanocortin 4 receptor (MC4R) agonist on changes in behaviors induced by musclin. Finally we evaluated the antidepressant effect of musclin using mice exposed to repeated water immersion (WI) stress. Results: We found that the i.p. and i.c.v. administration of musclin decreased the immobility time and relative time in the open arms (open %) in mice and increased urocortin 2 (Ucn 2) levels but decreased proopiomelanocortin levels in the hypothalamus. The numbers of c-Fos-positive cells were increased in the PVN and NTS but decreased in the ARC of mice with i.p. administration of musclin. The c-Fos-positive cells in the PVN were also found to be Ucn 2-positive. The antidepressant and anxiogenic effects of musclin were blocked by central administration of a CRF type 2 receptor antagonist and a melanocortin 4 receptor agonist, respectively. Peripheral administration of musclin also prevented depression-like behavior and the decrease in levels of hypothalamic Ucn 2 induced by repeated WI stress. Discussion: These data identify the antidepressant effects of musclin through the activation of central Ucn 2 signaling and suggest that musclin and Ucn 2 can be new therapeutic targets and endogenous peptides mediating the muscle-brain axis.

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.

Effects of Urocortin 2 Gene Transfer on Glucose Disposal in Insulin-Resistant db/db Mice on Metformin.

The study was designed to determine whether urocortin 2 (Ucn2) gene transfer is as safe and effective as metformin in insulin-resistant mice. Four groups of insulin-resistant db/db mice and a nondiabetic group were studied: (1) metformin; (2) Ucn2 gene transfer; (3) metformin + Ucn2 gene transfer; (4) saline; and (5) nondiabetic mice. After completion of the 15-week protocol, glucose disposal was quantified, safety assessed, and gene expression documented. Ucn2 gene transfer was superior to metformin, providing reductions in fasting glucose and glycated hemoglobin and enhanced glucose tolerance. The combination of metformin + Ucn2 gene transfer provided no better glucose control than Ucn2 gene transfer alone and was not associated with hypoglycemia. Metformin alone, Ucn2 gene transfer alone, and metformin + Ucn2 gene transfer together reduced fatty infiltration of the liver. Serum alanine transaminase concentration was elevated in all db/db groups (vs. nondiabetic controls), but the metformin + Ucn2 gene transfer combined group had the lowest alanine transaminase levels. No group differences in fibrosis were detected. In a hepatoma cell line, activation of AMP kinase showed a rank order of combined metformin + Ucn2 peptide > Ucn2 peptide > metformin. We conclude (1) The combination of metformin + Ucn2 gene transfer does not result in hypoglycemia. (2) Ucn2 gene transfer alone provides superior glucose disposal versus metformin alone. (3) The combination of Ucn2 gene transfer and metformin is safe and has additive effects in reducing serum alanine transaminase concentration, activating AMP kinase activity, and increasing Ucn2 expression, but is no more efficacious than Ucn2 gene transfer alone in reducing hyperglycemia. These data indicate that Ucn2 gene transfer is more effective than metformin in the db/db model of insulin resistance and combined treatment with metformin + Ucn2 gene transfer appears to have favorable effects on liver function and Ucn2 expression.

Chronic UCN2 treatment desensitizes CRHR2 and improves insulin sensitivity.

Urocortin 2 (UCN2) acts as a ligand for the G protein-coupled receptor corticotropin-releasing hormone receptor 2 (CRHR2). UCN2 has been reported to improve or worsen insulin sensitivity and glucose tolerance in vivo. Here we show that acute dosing of UCN2 induces systemic insulin resistance in male mice and skeletal muscle. Inversely, chronic elevation of UCN2 by injection with adenovirus encoding UCN2 resolves metabolic complications, improving glucose tolerance. CRHR2 recruits Gs in response to low concentrations of UCN2, as well as Gi and ß-Arrestin at high concentrations of UCN2. Pre-treating cells and skeletal muscle ex vivo with UCN2 leads to internalization of CRHR2, dampened ligand-dependent increases in cAMP, and blunted reductions in insulin signaling. These results provide mechanistic insights into how UCN2 regulates insulin sensitivity and glucose metabolism in skeletal muscle and in vivo. Importantly, a working model was derived from these results that unifies the contradictory metabolic effects of UCN2.

Possible Correlation between Urocortin 1 (Ucn1) and Immune Parameters in Patients with Endometriosis.

The etiology of endometriosis (EMS) has not been clearly elucidated yet, and that is probably the reason why its diagnostic process is frequently long-lasting and inefficient. Nowadays, the non-invasive diagnostic methods of EMS are still being sought. Our study aimed to assess the serum and peritoneal fluid levels of urocortin 1 (Ucn1) in patients with EMS and healthy women. Moreover, considering the immune background of the disease, the association between Ucn1 and several immune parameters was studied in both groups. We found that the serum Ucn1 level was significantly upregulated in women with EMS compared to healthy patients. Moreover, higher serum Ucn1 levels tended to correspond with more advanced stages of the disease (p = 0.031). Receiver operating characteristic (ROC) analysis revealed that based on serum Ucn1 levels, it is possible to distinguish deep infiltrating endometriosis (DIE) from among other EMS types. Together, these results indicate Ucn1 as a possible promising biomarker of EMS: however, not in isolation, but rather to enhance the effectiveness of other diagnostic methods.

Aging Changes the Efficacy of Central Urocortin 2 to Induce Weight Loss in Rats.

Middle-aged obesity and aging cachexia present healthcare challenges. Central responsiveness to body-weight-reducing mediators, e.g., to leptin, changes during aging in a way, which may promote middle-aged obesity and aging cachexia. Leptin is connected to urocortin 2 (Ucn2), an anorexigenic and hypermetabolic member of the corticotropin family. We aimed to study the role of Ucn2 in middle-aged obesity and aging cachexia. The food intake, body weight and hypermetabolic responses (oxygen consumption, core temperature) of male Wistar rats (3, 6, 12 and 18 months) were tested following intracerebroventricular injections of Ucn2. Following one central injection, Ucn2-induced anorexia lasted for 9 days in the 3-month, 14 days in the 6-month and 2 days in the 18-month group. Middle-aged 12-month rats failed to show anorexia or weight loss. Weight loss was transient (4 days) in the 3-month, 14 days in the 6-month and slight but long-lasting in the 18-month rats. Ucn2-induced hypermetabolism and hyperthermia increased with aging. The age-dependent changes in the mRNA expression of Ucn2 detected by RNAscope in the paraventricular nucleus correlated with the anorexigenic responsiveness. Our results show that age-dependent changes in Ucn2 may contribute to middle-aged obesity and aging cachexia. Ucn2 shows potential in the prevention of middle-aged obesity.

Urocortin 2 - a protective effect in hypertension?

Urocortin 2, an endogenous selective ligand for the corticotropin-releasing hormone receptor type 2, has been suggested to exert cardioprotective effects. We analyzed the possible relationship between the level of Ucn2 and specific indicators of cardiovascular risk factors in patients with untreated hypertension and in healthy subjects. Sixty seven subjects were recruited: 38 with newly diagnosed treatment-naive hypertension (with no pharmacological treatment - HT group) and 29 healthy subjects without hypertension (nHT group). We evaluated ambulatory blood pressure monitoring, Ucn2 levels and metabolic indices. Multivariable regression analyses were performed to assess the effects of gender, age, and Ucn2 levels on metabolic indices or blood pressure (BP) level. Log of Ucn2 levels were higher in healthy subjects than in hypertensive patients (2.44±0.7 versus 2.09±0.66, p<.05) and correlated inversely with 24-hour diastolic blood pressure, and both night-time systolic and diastolic blood pressure regardless of age and gender (R2=0.06; R2=0.06; R2=0.052; respectively). Furthermore, Ucn2 levels inversely correlated with cholesterol and low-density cholesterol (LDL) concentrations in healthy subjects only. Ucn2 was independently related to total cholesterol (but not to LDL) regardless of age, gender and the presence of hypertension (R2=0.18). However, we did not find any relationship between urocortin 2, body mass index or waist-hip ratio as well as parameters of glucose metabolism. Our data indicates that higher levels of urocortin 2 are related to more favorable lipid profiles and lower blood pressure.

Urocortin stimulates the ERK1/2 signaling pathway and the proliferation of HeLa cells via CRF receptor 1.

Corticotropin-releasing factor (CRF) stimulates adrenocorticotropic hormone (ACTH) secretion from the pituitary gland and is an essential regulator of the hypothalamic-pituitary-adrenocortical axis. Isoforms of CRF receptor are known to mediate the effects of urocortin stress ligands on the regulation of stress responses, anxiety, and feeding behavior; however, urocortin stress ligands also influence cell proliferation. In view of the tumor-promoting capacity of prolonged stress, here we investigated (a) the effect of urocortin on cell proliferative signaling via extracellular signal-regulated kinase 1/2, (b) the expression and cellular distribution of the specific CRF receptor isoforms, and (c) the intracellular localization of phosphorylated ERK1/2 in HeLa cells. Stimulation of cell proliferation was observed in the presence of 10 nm urocortin. Our data also suggest that MAP kinase MEK, the transcription factors E2F-1 and p53, and PKB/Akt are involved in this process. These findings may have therapeutic relevance for the targeted treatment of various malignancies.

A Sleep-Specific Midbrain Target for Sevoflurane Anesthesia.

Sevoflurane has been the most widely used inhaled anesthetics with a favorable recovery profile; however, the precise mechanisms underlying its anesthetic action are still not completely understood. Here the authors show that sevoflurane activates a cluster of urocortin 1 (UCN1+ )/cocaine- and amphetamine-regulated transcript (CART+ ) neurons in the midbrain involved in its anesthesia. Furthermore, growth hormone secretagogue receptor (GHSR) is highly enriched in sevoflurane-activated UCN1+ /CART+ cells and is necessary for sleep induction. Blockade of GHSR abolishes the excitatory effect of sevoflurane on UCN1+ /CART+ neurons and attenuates its anesthetic effect. Collectively, their data suggest that anesthetic action of sevoflurane necessitates the GHSR activation in midbrain UCN1+ /CART+ neurons, which provides a novel target including the nucleus and receptor in the field of anesthesia.

Urocortin-positive nerve fibres and cells are present in the human choroid.

BACKGROUND: Choroidal vascular regulation is mediated by the autonomic nervous system in order to gain proper blood flow control. While the mechanisms behind this control are unknown, neuroregulatory peptides are involved in this process. To better understand choroidal function, we investigate the presence of urocortin-1 (UCN), a neuroregulatory peptide with vascular effects, in the human choroid and its possible intrinsic and extrinsic origin. METHODS: Human choroid and eye-related cranial ganglia (superior cervical ganglion- SCG, ciliary ganglion-CIL, pterygopalatine ganglion-PPG, trigeminal ganglion-TRI) were prepared for immunohistochemistry against UCN, protein-gene product 9.5 (PGP9.5), substance P (SP), tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (VAChT). For documentation, confocal laser scanning microscopy was used. RESULTS: In choroidal stroma, UCN-immunoreactivity was present in nerve fibres, small cells and intrinsic choroidal neurons (ICN). Some UCN+ nerve fibres colocalised for VAChT, while others were VAChT. A similar situation was found with SP: some UCN+ nerve fibres showed colocalisation for SP, while others lacked SP. Colocalisation for UCN and TH was not observed. In eye-related cranial ganglia, only few cells in the SCG, PPG and TRI were UCN+, while many cells of the CIL displayed weak UCN immunoreactivity. CONCLUSION: UCN is part of the choroidal innervation. UCN+/VAChT+ fibres could derive from the few cells of the PPG or cells of the CIL, if these indeed supply the choroid. UCN+/SP+ fibres might originate from ICN, or the few UCN+ cells detected in the TRI. Further studies are necessary to establish UCN function in the choroid and its implication for choroidal autonomic control.

Urocortin-3 neurons in the perifornical area are critical mediators of chronic stress on female infant-directed behavior.

Infant avoidance and aggression are promoted by activation of the Urocortin-3 expressing neurons of the perifornical area of hypothalamus (PeFAUcn3) in male and female mice. PeFAUcn3 neurons have been implicated in stress, and stress is known to reduce maternal behavior. We asked how chronic restraint stress (CRS) affects infant-directed behavior in virgin and lactating females and what role PeFAUcn3 neurons play in this process. Here we show that infant-directed behavior increases activity in the PeFAUcn3 neurons in virgin and lactating females. Chemogenetic inhibition of PeFAUcn3 neurons facilitates pup retrieval in virgin females. CRS reduces pup retrieval in virgin females and increases activity of PeFAUcn3 neurons, while CRS does not affect maternal behavior in lactating females. Inhibition of PeFAUcn3 neurons blocks stress-induced deficits in pup-directed behavior in virgin females. Together, these data illustrate the critical role for PeFAUcn3 neuronal activity in mediating the impact of chronic stress on female infant-directed behavior.

Stress-induced bladder hypersensitivity: Effect of corticotropin releasing factor receptors assessed by spinal neurophysiology and neurochemistry.

Corticotropin releasing factor (CRF) receptors have been implicated in stress-induced hyperalgesia. The present study examined the role of CRF receptors Type 1&2 (CRFR1, CRFR2) in stress-induced bladder hyperalgesia in female rats by quantifying changes in receptor and agonist content following chronic (CFS, 7 daily episodes), acute (AFS, single episode) and control (NFS, no episodes) footshock protocols. ELISAs demonstrated that CFS lead to an increase in spinal thoracolumbar and lumbosacral spinal cord CRFR2 content and a decrease in lumbosacral spinal cord CRFR1 content. Content of the endogenous CRFR2 agonist, urocortin 2, was also increased in lumbosacral spinal cord and bladder tissues of CFS-pretreated rats, but urocortin 3 was decreased. Correlative single unit studies of lumbosacral dorsal horn neurons excited by bladder distension, in anesthetized rats that had undergone CFS, AFS or NFS protocols, used a before-after methodology with administration of a CRFR1 antagonist (antalarmin, 24 µg), CRFR2 antagonist (aSVG30, 12 µg) or normal saline topically to the exposed spinal cord following primary characterization. aSVG30 produced a reduction of neuronal responses evoked by bladder distension in CFS-pretreated rats but no statistically significant effects of aSVG30, antalarmin or vehicle were noted in other groups tested with the exception that antalarmin had an inhibitory effect on spontaneous activity in NFS-pretreated rats. The present findings are consistent with previous experiments using reflex responses to bladder distension as endpoints and further support a role for CRFR2-related mechanisms in stress-induced bladder hypersensitivity. This suggests CRFR2 antagonists may have efficacy in the treatment of bladder pain.

Posterodorsal Medial Amygdala Urocortin-3, GABA, and Glutamate Mediate Suppression of LH Pulsatility in Female Mice.

The posterodorsal subnucleus of the medial amygdala (MePD) is an upstream modulator of the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-adrenal (HPA) axes. Inhibition of MePD urocortin-3 (Ucn3) neurons prevents psychological stress-induced suppression of luteinizing hormone (LH) pulsatility while blocking the stress-induced elevations in corticosterone (CORT) secretion in female mice. We explore the neurotransmission and neural circuitry suppressing the gonadotropin-releasing hormone (GnRH) pulse generator by MePD Ucn3 neurons and we further investigate whether MePD Ucn3 efferent projections to the hypothalamic paraventricular nucleus (PVN) control CORT secretion and LH pulsatility. Ucn3-cre-tdTomato female ovariectomized (OVX) mice were unilaterally injected with adeno-associated virus (AAV)-channelrhodopsin 2 (ChR2) and implanted with optofluid cannulae targeting the MePD. We optically activated Ucn3 neurons in the MePD with blue light at 10 Hz and monitored the effect on LH pulses. Next, we combined optogenetic stimulation of MePD Ucn3 neurons with pharmacological antagonism of GABAA or GABAB receptors with bicuculline or CGP-35348, respectively, as well as a combination of NMDA and AMPA receptor antagonists, AP5 and CNQX, respectively, and observed the effect on pulsatile LH secretion. A separate group of Ucn3-cre-tdTomato OVX mice with 17ß-estradiol replacement were unilaterally injected with AAV-ChR2 in the MePD and implanted with fiber-optic cannulae targeting the PVN. We optically stimulated the MePD Ucn3 efferent projections in the PVN with blue light at 20 Hz and monitored the effect on CORT secretion and LH pulses. We reveal for the first time that activation of Ucn3 neurons in the MePD inhibits GnRH pulse generator frequency via GABA and glutamate signaling within the MePD, while MePD Ucn3 projections to the PVN modulate the HPG and HPA axes.