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.

Environmental Manipulations as an Effective Alternative Treatment to Reduce Endometriosis Progression.

Treatments for endometriosis include pharmacological or surgical procedures that produce significant side effects. We aimed to determine how environmental enrichment (EE) could impact the progression of endometriosis using the autotransplantation rat model. Female rats were exposed to EE (endo-EE: toys and nesting materials, 4 rats per cage, larger area enclosure) or no enrichment (endo-NE: 2 rats per cage) starting on postnatal day 21. After 8 weeks, sham surgery or surgical endometriosis was induced by suturing uterine horn tissue next to the intestinal mesentery, then allowed to progress for 60 days during which EE or NE continued. At the time of killing, we measured anxiety behaviors, collected endometriotic vesicles and uterus, and processed for quantitative real-time polymerase chain reaction for corticotropin-releasing hormone (CRH), urocortin-1, CRH receptors type 1 and type 2, and glucocorticoid receptor (GR). Endometriosis did not affect anxiety-like behaviors, yet rats in enriched conditions showed lower basal anxiety behaviors than the nonenriched group. Importantly, the endo-EE group showed a 28% reduction in the number of endometriosis vesicles and the vesicles were significantly smaller compared to the endo-NE group. Endometriosis increased CRH and GR only in the vesicles of endo-NE, and this increase was dampened in the endo-EE. However, urocortin 1 was increased in the vesicles of the endo-EE group, suggesting different pathways of activation of CRH receptors in this group. Our results suggest that the use of multimodal complementary therapies that reduce stress in endometriosis could be an effective and safe treatment alternative, with minimal side effects.

Plasma urocortin-1 as a preoperative marker of endometriosis in symptomatic women.

Accurate noninvasive diagnostic tests for endometriosis are still missing. This study evaluated the predictive value of the neuropeptide urocortin 1 (Ucn1) to detect pelvic endometriosis in symptomatic women. We enrolled prospectively 97 consecutive women submitted to gynecologic laparoscopy for chronic or acute pelvic pain, infertility or adnexal mass. Preoperative blood samples were assayed for Ucn1 using enzyme immunoassay. Patients with endometriosis had higher plasma Ucn1 levels compared to patients with no lesions (median 59 vs. 34 pg/ml, p < .01, Dunn's test). Elevated plasma Ucn1 levels were found among all endometriosis phenotypes (superficial peritoneal lesions, ovarian endometrioma, and deep infiltrating endometriosis, p < .05 vs. no lesions). Receiver operating characteristics curve analysis identified plasma Ucn1 > 46 pg/mL as the best cutoff point to detect endometriosis vs. no lesions, with 76% sensitivity and 88% specificity (area under the curve [AUC] 0.827, 95% confidence interval [CI] 0.695 - 0.959), but no cutoff could accurately distinguish endometriosis from other pathological conditions (AUC 0.593 [95% CI 0.474 - 0.711]). In women with chronic pelvic pain, infertility, or both symptoms, the probability of endometriosis (positive predictive value) increased consistently with the increase of plasma Ucn1 levels. The present findings suggest that high plasma Ucn1 levels increase the likelihood of endometriosis in symptomatic women.

The blockage of ventromedial hypothalamus CRF type 2 receptors impairs escape responses in the elevated T-maze.

In a previous study, the administration of corticotrophin-releasing factor (CRF) into the dorsomedial hypothalamus (DMH), a region that modulates defensive reactions, was shown to facilitate elevated T-maze (ETM) avoidance responses, an anxiogenic-like effect. Intra-DMH administration of the CRF type 1 receptor (CRFR1) antagonist antalarmin induced anxiolytic-like effects and counteracted the anxiogenic effects of CRF. The present study further investigates the role played by CRF receptors of the medial hypothalamus in anxiety. For that, male wistar rats were treated with CRFR1 and CRFR2-modulating drugs in the DMH or VMH, another hypothalamic nucleus implicated with defensive and emotional behavior, and tested in the ETM for inhibitory avoidance and escape measurements. In clinical terms, these responses have been respectively related to generalized anxiety and panic disorder. All animals were tested in an open field, immediately after the ETM, for locomotor activity assessment. The results showed that intra-VMH CRF or antalarmin did not alter ETM avoidance or escape performance. Intra-VMH injection of the CRFR2 preferential antagonist antisauvagine-30 or of the selective CRFR2 antagonist astressin 2-B inhibited escape performance, a panicolytic-like effect, without altering avoidance reactions. The CRFR2 agonist urocortin-2 intra-VMH was by itself without effect but blocked the effects of astressin 2-B. None of the drugs administered into the DMH altered ETM measurements. Additionally, none of the compounds altered locomotor activity measurements. These results suggest that VMH CRFR2 modulate a defensive response associated with panic disorder and are of relevance to the better understanding of the neural mechanisms underlying this pathological condition.

Altered functionality of the corticotrophin-releasing hormone receptor-2 in the hypothalamic paraventricular nucleus of hyperphagic maternally separated rats.

Early-life stress induces endocrine and metabolic alterations that increase food intake and overweight in adulthood. The stress response activates the corticotropin-releasing hormone (CRH) and urocortins' (Ucns) system in the hypothalamic paraventricular nucleus (PVN). These peptides induce anorexic effects through CRH-R2 receptor activation; however, chronic stressed animals develop hyperphagia despite of high PVN CRH expression. We analyzed this paradoxical behavior in adult rats subjected to maternal separation (MS) for 180min/daily during post-natal days 2-14, evaluating their body weight gain, food intake, serum corticosterone and vasopressin concentrations, PVN mRNA expression of CRH-R1, CRH-R2, CRH, Ucn2, Ucn3, vasopressin and CRH-R2 protein levels. MS adults increased their feeding, weight gain as well as circulating corticosterone and vasopressin levels, evincing chronic hyperactivity of the stress system. MS induced higher PVN CRH, Ucn2 and CRH-R2 mRNA expression and protein levels of CRH-R2 showed a tendency to decrease in the cellular membrane fraction. An intra-PVN injection of the CRH-R2 antagonist antisauvagine-30 in control adults increased receptor's mRNA expression, mimicking the observed PVN receptor's up-regulation of early-life MS adults. An injection of Ucn-2 directly into the PVN reduced food intake and increased PVN pCREB/CREB ratio in control animals; in contrast, Ucn-2 was unable to reduce food intake and enhance phosphorylated-CREB levels in PVN of MS rats. In conclusion, the chronic hyperactivity of the stress axis and PVN CRH-R2 resistance to Ucn2 effects, supported impaired receptor functionality in MS animals, probably due to its chronic stimulation by CRH or Ucn2, induced by early-life stress.

The activation and blockage of CRF type 2 receptors of the medial amygdala alter elevated T-maze inhibitory avoidance, an anxiety-related response.

Previous results show that the activation of CRF type 1 (CRFR1) receptors of the medial amygdala (MeA) induces anxiogenic-like effects. The present study investigates the role played by medial amygdala CRF type 2 receptors (CRFR2) in the modulation of anxiety and panic-related responses. Male Wistar rats were administered into the MeA with the CRFR2 agonist urocortin 2 (0.5 e 1.0µg/0.2µl, experiment 1) or with the CRFR2 antagonist astressin 2-B (60ng/0.2µl, experiment 2) and 10min later tested in the elevated T-maze (ETM) for inhibitory avoidance and escape measurements. In clinical terms, these responses have been respectively related to generalized anxiety and panic disorder. In a third experiment, the effects of the combined treatment with urocortin 2 (1.0µg/0.2µl) and a sub-effective dose of astressin 2-B (30ng/0.2µl) were also investigated. All animals were tested in an open field, immediately after the ETM, for locomotor activity assessment. Results showed that urocortin 2, in the highest dose administered (1.0µg/0.2µl), facilitated ETM avoidance, an anxiogenic-like effect. Astressin 2-B, also in the highest dose (60ng/0.2µl), significantly decreased avoidance latencies, an anxiolytic-like effect. The lower dose of astressin 2-B (30ng/0.2µl) did not induce anxiolytic-like effects but was able to counteract the anxiogenic-like effects of urocortin 2. None of the compounds administered altered escape responses or locomotor activity measurements. These results suggest that CRFR2 in the medial amygdala, as CRFR1, selectively modulate an anxiety-related response.

Urocortin 1 expression and secretion by human umbilical vein endothelial cells: In vitro effects of interleukin 8, interferon γ, lipopolysaccharide, endothelin 1, prostaglandin F-2α, estradiol, progesterone and dexamethasone.

Urocortin 1 (Ucn1) is a 40-amino-acid peptide that has vasodilatory activity and displays immunomodulatory and antioxidant properties. Maternal and cord plasma Ucn1 levels are increased in preeclampsia and preterm labor, but the mechanisms of such increase are poorly known. Thus, we investigated Ucn1 localization in human umbilical cord and assessed some potential stimuli to Ucn1 release by human umbilical vein endothelial cells (HUVEC). Human umbilical cords were obtained at uncomplicated term pregnancy (n=11). Ucn1 localization was assessed by immunohistochemistry and quantified. HUVEC were grown in vitro to confluence, then incubated with serial concentrations of interleukin (IL)-8, interferon (INF)-γ, lipopolysaccharide (LPS), endothelin (ET)-1, prostaglandin (PG)F-2α, estradiol, progesterone and dexamethasone and Ucn1 concentrations were measured in the supernatants. Ucn1 was immunolocalized with similar intensity in umbilical cord arteries, vein and Wharton's jelly. Ucn1 mRNA was detected in all HUVEC cultures and Ucn1 peptide was detectable in culture medium from untreated cells at different time points. Incubation with IFN-γ increased Ucn1 secretion in a dose-dependent manner. Treatments with IL-8, LPS, ET-1 and dexamethasone were able to increase three to fourfold Ucn1 release from cultured endothelial cells. In conclusion, umbilical vessels express Ucn1 and may be a contributive source of Ucn1 release into fetal-placental circulation. IL-8, IFN-γ, LPS, ET-1 and dexamethasone promote Ucn1 secretion from cultured HUVEC.

Expression, localization and control of activin A release from human umbilical vein endothelial cells.

Activin-A is a member of the TGFß superfamily found in maternal and umbilical cord blood throughout gestation. We investigated whether human umbilical vein endothelial cells (HUVEC) express activin-A in vivo and tested the effects of vasoactive (endothelin-1), pro-inflammatory (interferon-γ, interleukin-8) and anti-inflammatory (dexamethasone, urocortin) factors on activin-A release by isolated HUVEC in vitro. Activin ßA subunit protein and mRNA were strongly localized in the endothelial cells of umbilical veins and were also detectable in scattered cells of the cord connective tissue. Dimeric activin-A was detected in the HUVEC culture medium at picomolar concentrations. Activin-A release by HUVEC decreased after cell incubation with urocortin (p < 0.01), whereas no effect was observed with interleukin-8, interferon-γ, endothelin-1 or dexamethasone. In summary, activin-A is present in the human umbilical vein endothelium in vivo and is produced and released by isolated HUVEC. Activin-A secretion is inhibited in vitro by urocortin, a neuropeptide with predominantly anti-inflammatory action.

CRF1 and CRF2 receptors in the bed nucleus of the stria terminalis modulate the cardiovascular responses to acute restraint stress in rats.

The corticotropin-releasing factor (CRF) is involved in behavioral and physiological responses to emotional stress through its action in several limbic structures, including the bed nucleus of the stria terminalis (BNST). Nevertheless, the role of CRF1 and CRF2 receptors in the BNST in cardiovascular adjustments during aversive threat is unknown. Therefore, in the present study we investigated the involvement of CRF receptors within the BNST in cardiovascular responses evoked by acute restraint stress in rats. For this, we evaluated the effects of bilateral treatment of the BNST with selective agonists and antagonists of either CRF1 or CRF2 receptors in the arterial pressure and heart rate increase and the decrease in tail skin temperature induced by restraint stress. Microinjection of the selective CRF1 receptor antagonist CP376395 into the BNST reduced the pressor and tachycardiac responses caused by restraint. Conversely, BNST treatment with the selective CRF1 receptor agonist CRF increased restraint-evoked arterial pressure and HR responses and reduced the fall in tail skin temperature response. All effects of CRF were inhibited by local BNST pretreatment with CP376395. The selective CRF2 receptor antagonist antisalvagine-30 reduced the arterial pressure increase and the fall in tail skin temperature. The selective CRF2 receptor agonist urocortin-3 increased restraint-evoked pressor and tachycardiac responses and reduced the drop in cutaneous temperature. All effects of urocortin-3 were abolished by local BNST pretreatment with antisalvagine-30. These findings indicate an involvement of both CRF1 and CRF2 receptors in the BNST in cardiovascular adjustments during emotional stress.

Diagnostic capability of biological markers in assessment of obstructive sleep apnea: a systematic review and meta-analysis.

OBJECTIVE: The purpose of this systematic review is to evaluate the diagnostic value of biological markers (exhaled breath condensate, blood, salivary and urinary) in the diagnosis of OSA in comparison to the gold standard of nocturnal PSG. METHODS: Studies that differentiated OSA from controls based on PSG results, without age restriction, were eligible for inclusion. The sample of selected studies could include studies in obese patients and with known cardiac disease. A detailed individual search strategy for each of the following bibliographic databases was developed: Cochrane, EMBASE, MEDLINE, PubMed, and LILACS. The references cited in these articles were also crosschecked and a partial grey literature search was undertaken using Google Scholar. The methodology of selected studies was evaluated using the 14-item Quality Assessment Tool for Diagnostic Accuracy Studies. RESULTS: After a two-step selection process, nine articles were identified and subjected to qualitative and quantitative analyses. Among them, only one study conducted in children and one in adults found biomarkers that exhibit sufficiently satisfactory diagnostic accuracy that enables application as a diagnostic method for OSA. CONCLUSION: Kallikrein-1, uromodulin, urocotin-3, and orosomucoid-1 when combined have enough accuracy to be an OSA diagnostic test in children. IL-6 and IL-10 plasma levels have potential to be good biomarkers in identifying or excluding the presence of OSA in adults.

Evaluation of the effect of natural peptide 'Urocortin' on corticotrophin releasing factor (CRF) receptor expression in ND7/23 cells

CRF receptors are involved in the stress management of the cells and are believed to have a cytoprotective role in the body. CRF receptors have been reported to be potential drug targets for the treatment of neurodegenerative disorders. The cell line used in the study is ND7/23 (mouse neuroblastoma and rat dorsal root ganglion neuron hybridoma). The aim of the study was to confirm the expression of CRF receptors in ND7/23 cells and to determine if urocortin (Ucn) can enhance the expression of CRF receptors. ND7/23 cells were cultured in RPMI 1640 media and cells grown after the second passage were used for the experiments. RNA was extracted from the cells and amplified by RT-PCR to confirm the presence of CRF receptors. The cells were then subjected to oxidative stress by hydrogen peroxide (0.00375%) and divided into two groups i.e. control and Ucn (10-8 μM) treated. Later RNA was extracted from both group of cells and PCR was performed. Finally, densitometry analysis was conducted on the agarose gel to determine the quantity of PCR product formed. PCR experiment confirmed the expression of both CRF-R1 and CRF-R2 in the cell line, but CRF-R1 was found to be expressed more strongly. Densitometry analysis of the PCR product and calculation of the relative expression of CRF receptors indicated a higher level of expression of CRF receptors in samples treated with Ucn as compared to those that were kept untreated. The results indicate that Ucn may be useful for the management of neuro-degenerative disorders and further studies may be carried out to establish its use as a therapeutic agent.

Receptores de CRF estão envolvidos na gestão do estresse das células e são acreditados para ter um papel de cito-proteção no organismo. Os receptores do CRF têm sido relatados como alvos potenciais de fármacos para o tratamento de doenças neurodegenerativas. A linhagem celular utilizada no estudo é ND7/23 (neuroblastoma de camundongo e hibridoma de raíz dorsal do neurônio ganglionar de rato). O objetivo do estudo foi confirmar o que a expressão de receptores de CRF em células ND7/23 determinar se urocortina (Ucn) pode aumentar a expressão de receptores de CRF. Cultivaram-se células ND7/23 em meio RPMI 1640 e as células que cresceram após a segunda passagem foram usadas para os experimentos. O RNA foi extraído células e amplificado por RT-PCR para confirmar a presença de receptores de CRF. As células foram, então, submetidas a estresse oxidativo por peróxido de hidrogênio (0.00375 %) e divididas em dois grupos, ou seja, controle e tratadas com UCN (10-8 µM). Em seguida, o RNA foi extraído de ambos os grupo de células e realizou-se o PCR. Finalmente, realizou-se análise densitométrica em gel de agarose para determinar a quantidade de produto formado por PCR. O PCR confirmou a expressão de CRF-R1 e CRF-R2 na linhagem celular, mas o CRF-R1 expresso mais fortemente. A análise densitométrica do produto de PCR e o cálculo da expressão relativa de receptores de CRF indicaram um nível mais elevado de expressão de receptores de CRF em amostras tratadas com Ucn, em comparação com aqueles sem tratamento. Os resultados indicam que a Ucn pode ser útil no tratamento de doenças neurodegenerativas e mais estudos podem ser realizados para estabelecer seu uso como agente terapêutico.

Effects of dorsal periaqueductal gray CRF1- and CRF2-receptor stimulation in animal models of panic.

An increasing amount of evidence suggests that dysregulation of corticotrophin-releasing factor (CRF) signaling may contribute to the etiology of anxiety disorders such as post-traumatic stress disorder and panic. The dorsal periaqueductal gray matter (dPAG) in the midbrain has been considered a key region involved in the physiopathology of anxiety and panic. Administration of CRF in this structure enhances the expression of anxiety-related defensive behaviors in different animal models. Controversial results have been obtained regarding the involvement of CRF1 and CRF2 receptors in the regulation of panic-related responses. We report here that CRF (0.0625-1 µg) in the dPAG facilitates escape expression in two animal models that associate this behavior with panic, the elevated T-maze and the electrical stimulation of the dPAG. This effect, equally observed after CRF injection in the dorsomedial and dorsolateral columns of the PAG, is due to the activation of CRF1 receptors as revealed by its blockade by the CRF1 receptor antagonist antalarmin. In the elevated T-maze, CRF also facilitates inhibitory avoidance acquisition, suggesting an anxiogenic effect. Local administration of urocortin 2 (0.01-0.1 µg), a preferential CRF2 receptor agonist, failed to change escape expression, but impaired avoidance learning, indicating an anxiolytic effect. The results indicate that CRF1 receptors in the dPAG play a pervasive role in the regulation of defensive responses associated with both generalized anxiety and panic. Recruitment of CRF2 receptors only impacts upon the former type of behaviors, leading to an effect opposed to that caused by CRF1 receptor activation.

CRF family peptides are differently altered by acute restraint stress and chronic unpredictable stress.

Corticotropin-releasing factor (CRF) acts to promote stress-like physiological and behavioral responses and is mainly expressed in the paraventricular hypothalamic nucleus (PVN). Urocortin 1 (Ucn1) is also a ligand to CRF type 1 and 2 receptors that has been associated with the stress response. Ucn1 neurons are primarily found in the Edinger-Westphal (EW) nucleus. It has been previously proposed that CRF and Ucn1 differently modulate stress responses to distinct types of stressors. The present study used male Wistar rats to compare the effects of acute restraint stress and unpredictable chronic stress (UCS) through Fos-immunoreactivity (Fos-ir) on CRF-containing neurons of PVN and Ucn1-containing EW centrally projecting neurons. Results showed that PVN neurons responded to both acute restraint and UCS. Also for the PVN, unspecific variables, dependent on the time animals remained in the laboratory, do not seem to alter Fos-ir, since no significant differences between acute and chronic control groups were found. On the other hand, EW neurons were only activated in response to acute restraint stress. Also, for this nucleus a significant difference was found between acute and chronic control groups, suggesting that unspecific variables, dependent on the time animals remain in the laboratory, interfere with the nucleus activation. These results suggest that CRF/Ucn1 neuronal circuits encompass two interconnected systems, which are coordinated to respond to acute stressors, but are differentially activated during chronic unpredictable stress.

mRNA expression of corticotropin-releasing factor and urocortin 1 after restraint and foot shock together with alprazolam administration.

Corticotropin-releasing factor (CRF) is expressed in the paraventricular nucleus of the hypothalamus (PVN), and act centrally to provoke stress-like autonomic and behavioral responses. Urocortins 1-3 are additional ligands to the CRF receptors 1 and 2. Ucn 1 neurons are primarily concentrated in the Edinger-Westphal (EW) nucleus and also have been associated with stress responses. It is also known that UCN 1 respond in different ways depending on the stressor presented. Benzodiazepines can act via the CRF peptidergic system and chronic administration of alprazolam does not interfere with CRF mRNA expression in the PVN, but significantly increase Ucn 1 mRNA expression in the EW. The aim of our study was to investigate the relationship between different stressor stimuli, foot shock (FS) and restraint (R), and the mRNA expression of CRF and Ucn 1 in the PVN and EW using alprazolam (A). We employed fos activation and in situ hybridization. Restraint group presented increased fos-ir and CRF mRNA expression in the PVN compared to FS group. The stress responses of R group were prevented by A. In the EW, fos-ir was higher in the FS group than in the R group, whereas Ucn 1 mRNA expression was higher in the R group than in the FS group. Alprazolam significantly increased fos-ir and Ucn 1 mRNA expression in both groups. Our results show that PVN and EW respond in different ways to the same stressors. Furthermore, EW of stressed animals replies in a complementary way comparing to PVN with the use of Alprazolam.

Increase of core temperature induced by corticotropin-releasing factor and urocortin: a comparative study.

This study compared the ability of CRF and UCN1 to induce a thermoregulatory response when centrally injected into rats. The effects of antipyretic drugs and CRF receptor antagonists (CRF1 and CRF2) on the temperature (T) changes induced by these peptides were also investigated. Rectal (rT) and tail skin (T(sk)) temperatures were measured with a thermistor probe while body (bT) temperature was measured with a battery-operated biotelemetry transmitter in male Wistar rats (200 g) every 30 min over a period of 6h, after intracerebroventricular (i.c.v.) injection of 1 nmol of either CRF or UCN1. Rats were pre-treated with indomethacin (2 mg kg⁻¹, i.p.) or celecoxib (5 mg kg⁻¹, p.o.), dexamethasone (0.5 mg kg⁻¹, s.c.), astressin (a CRF1/CRF2 antagonist, 7 nmol, i.c.v.) or antalarmin (a CRF1 antagonist, 20 mg kg⁻¹, i.p.). The increase in body temperature induced by CRF was accompanied by a reduction in T(sk) while the response induced by UCN1 was accompanied by an elevation in T(sk). Indomethacin or celecoxib did not change the increases in rT caused by either CRF or UCN1. Although dexamethasone attenuated the increase in rectal temperature in response to CRF, dexamethasone did not modify the response induced by UCN1. Astressin blocked the UCN1-induced hyperthermia and reduced CRF-induced fever. Antalarmin did not modify the hyperthermia in response to UCN1, but reduced the fever evoked by CRF. This study demonstrated that CRF by acting on the CRF1 receptor induces a prostaglandin-independent fever which seems to depend, at least in part, on the synthesis of other mediators while UCN1 acts on the CRF2 receptor, promoting a hyperthermic response which seems to be independent on synthesis/release of any mediator.

Male and female odors induce Fos expression in chemically defined neuronal population.

Olfactory information modulates innate and social behaviors in rodents and other species. Studies have shown that the medial nucleus of the amygdala (MEA) and the ventral premammillary nucleus (PMV) are recruited by conspecific odor stimulation. However, the chemical identity of these neurons is not determined. We exposed sexually inexperienced male rats to female or male odors and assessed Fos immunoreactivity (Fos-ir) in neurons expressing NADPH diaphorase activity (NADPHd, a nitric oxide synthase), neuropeptide urocortin 3, or glutamic acid decarboxylase mRNA (GAD-67, a GABA-synthesizing enzyme) in the MEA and PMV. Male and female odors elicited Fos-ir in the MEA and PMV neurons, but the number of Fos-immunoreactive neurons was higher following female odor exposure, in both nuclei. We found no difference in odor induced Fos-ir in the MEA and PMV comparing fed and fasted animals. In the MEA, NADPHd neurons colocalized Fos-ir only in response to female odors. In addition, urocortin 3 neurons comprise a distinct population and they do not express Fos-ir after conspecific odor stimulation. We found that 80% of neurons activated by male odors coexpressed GAD-67 mRNA. Following female odor, 50% of Fos neurons coexpressed GAD-67 mRNA. The PMV expresses very little GAD-67, and virtually no colocalization with Fos was observed. We found intense NADPHd activity in PMV neurons, some of which coexpressed Fos-ir after exposure to both odors. The majority of the PMV neurons expressing NADPHd colocalized cocaine- and amphetamine-regulated transcript (CART). Our findings suggest that female and male odors engage distinct neuronal populations in the MEA, thereby inducing contextualized behavioral responses according to olfactory cues. In the PMV, NADPHd/CART neurons respond to male and female odors, suggesting a role in neuroendocrine regulation in response to olfactory cues.

Involvement of urocortinergic neurons below the midbrain central gray in the physiological response to restraint stress in pigeons.

The present study was carried out to identify the diencephalic and midbrain neurons in pigeons that respond to stress (using restraint as the stressor) and determine if the urocortinergic neurons (expressing urocortin 1, Ucn1) below the midbrain central gray are among those activated. Immunolabeling for the immediate early gene Egr-1 was used to identity stress-responsive neurons, following 1-3 h of restraint. A large increase in nuclear Egr-1 immunolabeling was observed in several dorsomedial thalamic nuclei, and in a stream of neurons extending from below the mesencephalic central gray (overlapping the nucleus of Darkschewitsch at these levels) to just anterior to the nucleus of Edinger-Westphal. A more modest increase in neuronal nuclear Egr-1 was observed in the medial posterior hypothalamic area, the mesencephalic periventricular area, the ventral tegmental area, the inferior colliculus, the nucleus paramedianus of the midbrain, and the intercollicular nucleus. The distribution and abundance of urocortin-immunolabeled neurons coincided with that of the stress-responsive neurons below the mesencephalic periaqueductal gray, and about 50% of these urocortin neurons were activated by stress. These results suggest that, as in some mammals, the urocortinergic neurons of the paramedian subgriseal mesencephalon respond to stress. In those mammals, in which the boundaries of the nucleus of Edinger-Westphal are indistinct, the caudal part of the homologous field of urocortinergic neurons has been referred to as the nucleus of Edinger-Westphal. In pigeons, in which the nucleus of Edinger-Westphal is cytoarchitectonically well-defined, the caudal part of this urocortinergic field clearly does not include the nucleus of Edinger-Westphal.

Intron retention as an alternative splice variant of the rat urocortin 1 gene.

Urocortin 1, highly conserved metazoan gene of the corticotropin-releasing hormone family, is a simple gene structured in two exons and the corresponding intron. The urocortin 1 prepropeptide is entirely coded in the second exon. Preliminary non-isotopic in situ hybridization experiments with an oligonucleotide complementary to an intron sequence of the urocortin 1 gene showed a significant cytoplasmic-like staining, suggesting the occurrence of an intron-retained urocortin 1 transcript. This observation prompted us to study whether the urocortin 1 gene presents alternative splicing by intron retention event. Confocal fluorescent in situ hybridization for urocortin 1 RNA and the use of the specific DNA dye TOPRO-3 allowed us to show significant expression of the intron-retained urocortin 1 transcript that did not colocalize with TOPRO-3 staining indicating a cytoplasmic localization for the intron-retained urocortin 1 transcript. The natural occurrence of a polyadenylated intron-retained urocortin 1 RNA was further documented by reverse transcriptase polymerase chain reaction (PCR), primed with oligo(dT), of total RNA extracted from three brain regions, a midbrain region containing the Edinger-Westphal nucleus, cerebellum and prefrontal cortex. In the three brain regions studied, it was possible to amplify both intron-less as well as intron-retained urocortin 1 transcripts. The use of PCR primers that simultaneously amplify both urocortin 1 transcripts allowed us to show that the expression of both urocortin 1 transcripts differs among the brain regions analyzed, suggesting a tissue specific regulation of this alternative splicing. In silico analysis of the five known mammalian urocortin 1 genomic sequences showed high conservation of the urocortin 1 intron sequence. Further studies should investigate the regulation of this intron retention event and its consequence for the functionality of the urocortin 1 gene.

Estrogen receptors alpha and beta differentially regulate the transcriptional activity of the Urocortin gene.

Urocortin (Ucn), a highly conserved metazoan gene, is related to stress and feeding, behaviors with significant gender differences. We investigated whether estrogens regulate the expression of the Ucn gene using transient transfection in PC12 cells with the human Ucn (hUcn) promoter coupled to luciferase and either alpha or beta estrogen receptors (ERalpha or ERbeta, respectively). The results demonstrate that estradiol (E2) increases the activity of the hUcn promoter via ERalpha, and decreases hUcn promoter activity through ERbeta. Deletions of the hUcn promoter show that the increase in promoter activity mediated by E2-ERalpha depends on a promoter region containing a half-estrogen response element and an Sp1 site, and the decrease mediated by E2-ERbeta depends on a proximal promoter region containing a cAMP response element. Ucn and ERs coexist in neurons of rat hypothalamic nuclei, giving anatomical support for a direct effect of estrogen receptors on the Ucn gene. By in situ hybridization, we observed that cycling female rats have a higher number of cells expressing Ucn mRNA than males in the paraventricular nucleus of the hypothalamus (PVN) and the septum. Both of these brain nuclei are related to stress behaviors and express moderate levels of Ucn. Furthermore, Ucn mRNA was significantly decreased in the PVN and increased in the septum 30 d after ovariectomy. Acute E2 administration to ovariectomized rats significantly increased Ucn mRNA expression in the PVN and septum. In conclusion, our in vitro and in vivo evidence suggests that estrogens exert a direct and differential transcriptional regulation of the Ucn gene.

Distribution of urocortin 3 neurons innervating the ventral premammillary nucleus in the rat brain.

Urocortin 3 (Ucn 3) is a recently described peptide of the corticotropin-releasing factor family. Neurons expressing Ucn 3 mRNA and peptide are distributed in specific brain areas, including the median preoptic nucleus, the perifornical area (PFx), and the medial nucleus of the amygdala (MEA). Fibers immunoreactive to Ucn 3 are confined to certain brain nuclei, being particularly dense in the ventral premammillary nucleus (PMV). In studies involving electrolytic lesions and analysis of Fos distribution according to behavioral paradigms, the PMV has been potentially implicated in conspecific aggression and sexual behavior. However, the role that Ucn 3 plays in this pathway has not been explored. Therefore, we investigated the origins of the urocortinergic innervation of the PMV of Wistar rat in an attempt to map the brain circuitry and identify likely related functions. We injected the retrograde tracer cholera toxin b subunit into the PMV and found that 88% of the Ucn 3-immunoreactive fibers in the PMV originate in the dorsal MEA, and that few originate in the PFx. As a control, we injected the anterograde tracer biotin dextran amine into both regions. We observed that the PMV is densely innervated by the MEA, and scarcely innervated by the PFx. The MEA is a secondary relay of the vomeronasal system and projects amply to hypothalamic nuclei related to hormonal and behavioral adjustments, including the PMV. Although physiological studies should also be performed, we hypothesize that Ucn 3 participates in such pathways, conveying sensory information to the PMV, which in turn modulates behavioral and neuroendocrine responses.