Anatomical and functional implications of corticotrophin-releasing hormone neurones in a septal nucleus of the avian brain: an emphasis on glial-neuronal interaction via V1a receptors in vitro.

Previously, we showed that corticotrophin-releasing hormone immunoreactive (CRH-IR) neurones in a septal structure are associated with stress and the hypothalamic-pituitary-adrenal axis in birds. In the present study, we focused upon CRH-IR neurones located within the septal structure called the nucleus of the hippocampal commissure (NHpC). Immunocytochemical and gene expression analyses were used to identify the anatomical and functional characteristics of cells within the NHpC. A comparative morphometry analysis showed that CRH-IR neurones in the NHpC were significantly larger than CRH-IR parvocellular neurones in the paraventricular nucleus of the hypothalamus (PVN) and lateral bed nucleus of the stria terminalis. Furthermore, these large neurones in the NHpC usually have more than two processes, showing characteristics of multipolar neurones. Utilisation of an organotypic slice culture method enabled testing of how CRH-IR neurones could be regulated within the NHpC. Similar to the PVN, CRH mRNA levels in the NHpC were increased following forskolin treatment. However, dexamethasone decreased forskolin-induced CRH gene expression only in the PVN and not in the NHpC, indicating differential inhibitory mechanisms in the PVN and the NHpC of the avian brain. Moreover, immunocytochemical evidence also showed that CRH-IR neurones reside in the NHpC along with the vasotocinergic system, comprising arginine vasotocin (AVT) nerve terminals and immunoreactive vasotocin V1a receptors (V1aR) in glia. Hence, we hypothesised that AVT acts as a neuromodulator within the NHpC to modulate activity of CRH neurones via glial V1aR. Gene expression analysis of cultured slices revealed that AVT treatment increased CRH mRNA levels, whereas a combination of AVT and a V1aR antagonist treatment decreased CRH mRNA expression. Furthermore, an attempt to identify an intercellular mechanism in glial-neuronal communication in the NHpC revealed that brain-derived neurotrophic factor (BDNF) and its receptor (TrkB) could be involved in the signalling mechanism. Immunocytochemical results further showed that both BDNF and TrkB receptors were found in glia of the NHpC. Interestingly, in cultured brain slices containing the NHpC, the use of a selective TrkB antagonist decreased the AVT-induced increase in CRH gene expression levels. The results from the present study collectively suggest that CRH neuronal activity is modulated by AVT via V1aR involving BDNF and TrkB glia in the NHpC.

Atypical role of sprouty in colorectal cancer: sprouty repression inhibits epithelial-mesenchymal transition.

This corrects the article DOI: 10.1038/onc.2015.365.

Atypical role of sprouty in colorectal cancer: sprouty repression inhibits epithelial-mesenchymal transition.

Sprouty (SPRY) appears to act as a tumor suppressor in cancer, whereas we demonstrated that SPRY2 functions as a putative oncogene in colorectal cancer (CRC) (Oncogene, 2010, 29: 5241-5253). We investigated the mechanisms by which SPRY regulates epithelial-mesenchymal transition (EMT) in CRC. SPRY1 and SPRY2 mRNA transcripts were significantly upregulated in human CRC. Suppression of SPRY2 repressed AKT2 and EMT-inducing transcription factors and significantly increased E-cadherin expression. Concurrent downregulation of SPRY1 and SPRY2 also increased E-cadherin and suppressed mesenchymal markers in colon cancer cells. An inverse expression pattern between AKT2 and E-cadherin was established in a human CRC tissue microarray. SPRY2 negatively regulated miR-194-5p that interacts with AKT2 3' untranslated region. Mir-194 mimics increased E-cadherin expression and suppressed cancer cell migration and invasion. By confocal microscopy, we demonstrated redistribution of E-cadherin to plasma membrane in colon cancer cells transfected with miR-194. Spry1(-/-) and Spry2(-/-) double mutant mouse embryonic fibroblasts exhibited decreased cell migration while acquiring several epithelial markers. In CRC, SPRY drive EMT and may serve as a biomarker of poor prognosis.

Distribution of the Vasotocin Subtype Four Receptor (VT4R) in the Anterior Pituitary Gland of the Chicken, Gallus gallus, and its Possible Role in the Avian Stress Response.

The neurohormone arginine vasotocin (AVT) in non mammalian vertebrates is homologous to arginine vasopressin (AVP) in mammals. Its actions are mediated via G protein-coupled receptors that belong to the vasotocin/mesotocin family. Because of the known regulatory effects of nonapeptide hormones on anterior pituitary functions, receptor subtypes in that family have been proposed to be located in anterior pituitary cells. Recently, an avian vasotocin receptor subtype designated VT4R has been cloned, which shares 69% sequence homology with a human vasopressin receptor, the V1aR. In the present study, a polyclonal antibody to the VT4R was developed and validated to confirm its specificity to the VT4R. The antibody was used to test the hypothesis that the VT4R is present in the avian anterior pituitary and is specifically associated with certain cell types, where its expression is modulated by acute stress. Western blotting of membrane protein extracts from pituitary tissue, the use of HeLa cells transfected with the VT4R and peptide competition assays all confirmed the specificity of the antibody to the VT4R. Dual-labelling immunofluorescence microscopy was utilised to identify pituitary cell types that contained immunoreactive VT4R. The receptor was found to be widely distributed throughout the cephalic lobe but not in the caudal lobe of the anterior pituitary. Immunoreactive VT4R was associated with corticotrophs. Approximately 89% of immunolabelled corticotrophs were shown to contain the VT4R. The immunoreactive VT4R was not found in gonadotrophs, somatotrophs or lactotrophs. To determine a possible functional role of the VT4R and previously characterised VT2R, gene expression levels in the anterior pituitary were determined after acute immobilisation stress by quantitative reverse transcriptase-polymerase chain reaction. The results showed a significant increase in plasma corticosterone levels (three- to four-fold), a significant reduction of VT4R mRNA and an increase of VT2R mRNA (P < 0.05) in acutely immobilised chicks compared to controls. The data suggest a role of the VT4R in the avian stress response.

Appetitive and consummatory sexual and agonistic behaviour elicits FOS expression in aromatase and vasotocin neurones within the preoptic area and bed nucleus of the stria terminalis of male domestic chickens.

Some components of male sexual and agonistic behaviours are considered to be regulated by the same neurocircuitry in the medial preoptic nucleus (POM) and the medial portion of bed nucleus of the stria terminalis (BSTM). To better understand this neurocircuitry, numbers of aromatase- (ARO) or arginine vasotocin- (AVT) immunoreactive (ir) neurones expressing immediate early gene protein FOS were compared in the POM and BSTM of male chickens following sexual or agonistic behaviours. Observations were made on males showing: (i) appetitive (courtship) and consummatory (copulation) sexual behaviours; (ii) only appetitive sexual behaviour, or (iii) displaying agonistic behaviour toward other males. Control males were placed on their own in the observation pen, or only handled. In the POM, appetitive sexual behaviour increased ARO+FOS colocalisation, whereas agonistic behaviour decreased the number of visible ARO-ir cells. In the dorsolateral subdivision of BSTM (BSTM1), appetitive sexual behaviour also increased ARO+FOS colocalisation, although the numbers of visible ARO-ir and AVT-ir cells were not altered by sexual or agonistic behaviours. In the ventromedial BSTM (BSTM2), appetitive sexual behaviour increased ARO+FOS and AVT+FOS colocalisation, and all behaviours decreased the number of visible ARO-ir cells, particularly in males expressing consummatory sexual behaviour. Positive correlations were found between numbers of cells with ARO+FOS and AVT+FOS colocalisation in both subdivisions of the BSTM. Waltzing frequency was positively correlated with ARO+FOS colocalisation in the lateral POM, and in both subdivisions of the BSTM in males expressing sexual behaviour. Waltzing frequency in males expressing agonistic behaviour was negatively correlated with the total number of visible ARO-ir cells in the lateral POM and BSTM2. These observations suggest a key role for ARO and AVT neurones in BSTM2 in the expression of appetitive sexual behaviour, and differential roles for ARO cells in the POM and BSTM in the regulation of components of sexual and agonistic behaviours.

Molecular neuroendocrine events during stress in poultry.

Magnocellular neurons in the hypothalamic paraventricular nucleus containing arginine vasotocin (AVT) project to the posterior pituitary and release the peptide peripherally to target tissues. Parvocellular neurons contain either corticotropin-releasing hormone (CRH), AVT, or both neuropeptides and project to the median eminence. Corticotropin-releasing hormone and AVT are then transported to the anterior pituitary where they bind to CRH1 or vasotocin VT2 receptors, many found co-localized on the same pituitary cell type, the corticotrope. Central administration of CRH compared with AVT is more effective in releasing the stress hormone corticosterone, whereas peripheral administration of AVT is more efficacious. Simultaneous, peripheral administration of CRH and AVT also resulted in a synergistic release of corticosterone. Cell culture studies demonstrated a synergistic release of the second messenger, cyclic adenosine monophosphate, when both CRH and AVT were added to cells transfected with CRH and VT2 receptors, providing a possible explanation for the enhanced release of corticosterone following combined peripheral administration of the 2 peptides. A social stress model, mature male-male interaction, demonstrated activation of neurons in the paraventricular nucleus and suggested that the posterior pituitary as well as the anterior pituitary are involved in a social stress response.

Sex differences in plasma corticosterone release in undisturbed chickens (Gallus gallus) in response to arginine vasotocin and corticotropin releasing hormone.

In birds, two neuropeptides, corticotropin releasing hormone (CRH) and arginine vasotocin (AVT), are major regulators of the hypothalamo-pituitary-adrenal axis (HPA) during the stress response. In birds, however, the relative efficacy of CRH and AVT to stimulate the HPA axis in males and females remains unknown. The purpose of this study was to determine the time course of CORT release following central CRH and AVT administration to male and female chickens. Chickens were fitted with a stainless steel cannula surgically implanted in the lateral ventricle and a catheter chronically inserted in the jugular vein. Birds were housed individually in cages behind a one-way glass partition and unnecessary noise was avoided during the sampling period. Each bird received a single 5.0microtracerebroventricular (ICV) injection of either saline (SAL), AVT (10 and 100pmol), or CRH (10 and 100pmol). Blood was sampled remotely every 15min for 2h and plasma CORT was determined by radioimmunoassay. There was a significant increase in plasma CORT concentration in males injected with 100pmol AVT beginning at 15min post-injection through 2h compared with SAL injected birds. In males, injection of 100pmol CRH was significantly more effective in releasing CORT compared to an equal molar concentration of AVT or SAL. In females, ICV injection of 100pmol AVT induced moderate increase in CORT levels. In contrast, 100pmol CRH significantly increased plasma CORT compared to SAL injected controls but the CORT response was nearly 50% less than that obtained in males.

Vasotocin and reproductive functions of the domestic chicken.

The neurohypophyseal hormone arginine vasotocin (AVT) combines both antidiuretic and reproductive activities. In the domestic chicken AVT produces assimetric effects on the reproductive functions of males and females. AVT synthesized in magnocellular diencephalic neurons is released into circulation in a highly coordinated manner contributing to the peripheral control of oviposition in hens. Conversely, parvocellular AVT cells located in the limbic system (bed nucleus of stria terminalis (BST)) are quite different in their properties and, possible, functions. In domestic chickens these cells express AVT in a sexually dimorphic manner and are found solely in males. This sexually dimorphic part of the AVT system is sensitive to gonadal steroids. Experimental data demonstrated that AVT modulates different aspects of reproductive behavior including courtship vocalization and copulation. Sexual differentiation of these limbic vasotocinergic cells show striking correlation with sexual differentiation of masculine behavior. Evidences coming from physiological, anatomical and ethological studies suggest strong implication of the vasotocinergic system in the control of reproductive functions.

Gender-related changes in the avian vasotocin system during ontogeny.

The arginine vasotocin (AVT) system of the avian brain includes a sexually dimorphic part that extends from the caudal part of preoptic region through the medial part of the bed nucleus of stria terminalis (BSTm) to the lateral septum. It is composed of the parvocellular neurons located in the BSTm and the dense innervation of the medial preoptic region and lateral septum. In this part of the brain, AVT expression is stronger in males than in females in a few bird species investigated to date. This review focuses on the ontogeny of sexual differences in the vasotocinergic system of two gallinaceous species, domestic chicken and Japanese quail, and on the role of gonadal hormones in organizing during development and maintaining in adulthood these differences. Parvocellular AVT neurons become discernible in the BSTm of males and females during the second half of embryonic development. These cells undergo a profound and irreversible sexual differentiation during ontogenetic development. Recent findings demonstrate a dual role of estrogens in the organization and activation of sex differences in the AVT system. During the embryonic period of ontogeny, estrogens differentiate the AVT system in a sexually dimorphic manner in parallel with the differentiation of sexual behavior, while in adulthood estrogens, locally produced from testosterone in the male brain, activate AVT synthesis in the BSTm. The sexually dimorphic part of the AVT system is sensitive to a number of abiotic factors such as light, temperature, and water availability. It is suggested that sex dimorphic vasotocinergic systems could be implicated in processes of social recognition in various behavioral contexts.

Development of sexually dimorphic vasotocinergic system in the bed nucleus of stria terminalis in chickens.

The bed nucleus of stria terminalis (BnST) of the domestic fowl contains two groups of parvicellular vasotocinergic neurons that are sexually dimorphic. In adult cockerels, arginine vasotocin (AVT) synthesis is well expressed in the dorsolateral and ventromedial portions of the BnST, whereas in corresponding brain areas of hens, AVT synthesis is completely lacking. In the present study, in situ hybridization and immunocytochemical methods were used to compare the ontogeny of sexually dimorphic AVT gene expression in the BnST of male and female chickens from day 12 of embryonic development (E12) until the onset of sexual maturation. By E12, both parvicellular groups of AVT-immunoreactive (AVT-ir) perikarya in the developing BnST can be distinguished in some males, whereas in females their presence is questionable. A quantitative analysis, beginning at E14, showed that the parvicellular dorsolateral portion of the BnST of male embryos had more AVT perikarya compared with females. In contrast, no evident sex difference in distribution pattern and number of AVT mRNA containing neurons in this BnST portion was observable by in situ hybridization at E15. At E18, as well as on the first and second days posthatch (D1 and D2), no differences in the number of AVT synthesizing cells and intensity of immunoreactive staining in male versus female chickens were found. Between D2 and D7, the number of AVT-ir cells in the BnST declined rapidly in both sexes until it disappeared completely in females before D35. In males, another increase in sexually dimorphic AVT-ir cells and innervation of the lateral septum was associated with the onset of puberty and fully matched a pattern observed in adult fowls. These results demonstrate that the sexually dimorphic part of the AVT system undergoes sexual differentiation during early stages of ontogeny.

Sexual dimorphism of arg-vasotocin gene expressing neurons in the telencephalon and dorsal diencephalon of the domestic fowl. An immunocytochemical and in situ hybridization study.

A strong sex dimorphism in the distribution of immunoreactive arginine-vasotocin (AVT) and AVT mRNA was observed in telencephalic and dorsal diencephalic areas of the domestic fowl using immunocytochemistry and in situ hybridization. Two subgroups of immunoreactive parvocellular perikarya surrounded by dense plexus of immunoreactive fibres were found within the bed nucleus of the stria terminalis and the dorsal part of the diencephalic paraventricular region of males. No signs of immunoreactivity were observed within corresponding regions of the female brain. Instead, in females a few scattered weakly stained perikarya were observed rostrally to the level of the anterior commissure, juxtapositioned to the nucleus accumbens and the floor of the lateral ventricle. The distribution of AVT mRNA containing cell profiles fully confirmed the immunocytochemical findings. Osmotic stress induced by water deprivation for 48 h had no influence on the number of immunoreactive or AVT mRNA containing parvocellular cell bodies. However, it resulted in an increase of immunoreactive cell area in the bed nucleus of the stria terminalis and dorsal diencephalon of 5. 9 and 11.7%, respectively. We suggest that the sexually dimorphic vasotocinergic circuit may be involved in the co-ordination of behavioural and autonomic functions in response to environmental stress.

Intracerebral sex differences in the vasotocin system in birds: possible implication in behavioral and autonomic functions.

The brain vasotocinergic system demonstrates clear sexual dimorphism in birds investigated so far. This paper examines the evidence obtained in studies on gallinaceous (domestic fowl, Japanese quail) and passerine (canary, junco, zebra finch) birds. Vasotocin (VT)-immunoreactive parvocellular neurons are present in the nucleus of stria terminalis of males, but they are less abundant or absent in the corresponding structure of females. A similar difference has been observed in the dorsal paraventricular area of domestic fowl. Sex-related differences in VT-gene expression have been confirmed by in situ hybridization. Moreover, overall brain content of VT mRNA in cockerels is about twice that of hens, suggesting that VT synthesis may also be sexually dimorphic in other brain areas where morphological sex differences have not yet been revealed. The vasotocinergic system in birds is implicated in body fluid homeostasis, and during ontogeny it starts to respond to osmotic challenges in a sexually dimorphic way. Photoperiod, aging, or castration--all associated with changes in circulating testosterone levels--affect sexually dimorphic VT pathways and cell clusters. Sexually dimorphic vasotocinergic circuits are distributed in regions containing steroid-concentrating cells and are closely intermingled with aromatase-containing neurons that may mediate activational effects of gonadal steroids on this peptidergic system. However, it remains undetermined whether the observed neuroanatomical sex differences are related to sexually dimorphic autonomic and behavioral effects induced by VT. Most likely, VT in birds has a modulatory rather than a specific regulatory function in control of male sexual behavior and vocalization.