Concurrent changes in photoperiod-induced seasonal phenotypes and hypothalamic CART peptide-containing systems in night-migratory redheaded buntings.

This study tested the hypothesis whether hypothalamic cocaine-and amphetamine-regulated transcript (CART)-containing systems were involved in photoperiod-induced responses associated with spring migration (hyperphagia and weight gain) and reproduction (gonadal maturation) in migratory songbirds. We specifically chose CART to examine neural mechanism(s) underlying photoperiod-induced responses, since it is a potent anorectic neuropeptide and involved in the regulation of changes in the body mass and reproduction in mammals. We first studied the distribution of CART-immunoreactivity in the hypothalamus of migratory redheaded buntings (Emberiza bruniceps). CART-immunoreactive neurons were found extensively distributed in the preoptic, lateral hypothalamic (LHN), anterior hypothalamic (AN), suprachiasmatic (SCN), paraventricular (PVN), dorsomedialis hypothalami (DMN), inferior hypothalamic (IH), and infundibular (IN) nuclei. Then, we correlated hypothalamic CART-immunoreactivity in buntings with photostimulated seasonal states, particularly winter non-migratory/non-breeding (NMB) state under short days, and spring premigratory/pre-breeding (PMB) and migratory/breeding (MB) states under long days. There were significantly increased CART-immunoreactive cells, and percent fluorescent area of CART-immunoreactivity was significantly increased in all mapped hypothalamic areas, except the SCN, PVN, AN, and DMN in photostimulated PMB and MB states, as compared to the non-stimulated NMB state. In particular, CART was richly expressed in the medial preoptic nucleus, LHN, IH and IN during MB state in which buntings showed reduced food intake and increased night-time activity. These results suggest that changes in the activity of the CART-containing system in different brain regions were associated with heightened energy needs of the photoperiod-induced seasonal responses during spring migration and reproduction in migratory songbirds.

Effects of star anise (Illicium verum Hook.f) oil on the nuclear factor E2-related factor 2 signaling pathway of chickens during subclinical Escherichia coli challenge.

We characterized the mechanism underlying star anise (Illicium verum Hook.f) oil (SAO)-mediated antioxidant status during subclinical Escherichia coli (E. coli) challenge. A total of 512 male birds (White Leghorn) at 30 wk of age with similar body weight (2.14 ± 0.02 kg) were randomly divided into 2 groups with 1 group being orally challenged with E. coli (every other day from day 15 to day 27) during the experiment. Each group of birds was then randomly allocated to dietary treatment of SAO supplementation at 0, 200, 400, or 600 mg/kg of basal diet (8 replicate cages during each treatment). The treatments were arranged a 4 × 2 factorial arrangement. The experiment comprised 1 wk of adaptation and 3 wks of data collection. There was no interaction (P > 0.05) between SAO supplementation and E. coli challenge for final body weight and average daily feed intake of birds. However, E. coli challenge resulted in a significant decrease (P < 0.001) in final body weight of birds as compared with unchallenged birds. There were interactions between SAO supplementation and E. coli challenge for the activity of glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) concentration in serum and for the activity of GSH-Px in the liver of birds. Supplementation of SAO enhanced the activities of antioxidant enzymes but decreased the MDA content in the serum and liver of birds, and it also enhanced the expression of genes including superoxide dismutase, catalase, and nuclear factor E2-related factor 2 (Nrf2) in the liver of the birds. Meanwhile, supplementation of SAO can also reduce E. coli challenge-induced oxidative stress in the serum and liver of birds, and the efficacy of SAO in birds during subclinical E. coli challenge is dose-dependent. In conclusion, the enhancement of antioxidant capacity by star anise or its effective compounds is through upregulation of Nrf2 signaling pathway. The optimum supplementation dose of SAO for protecting birds against E. coli challenge is 400 mg/kg.

Comparative proteomic analysis of hypothalamus tissue from Huoyan geese between pre-laying period and laying period using an iTRAQ-based approach.

The hypothalamus plays a central role in controlling poultry endocrine and reproductive activities. So far there is limited information focused on the proteome profiles of the hypothalamus from geese during different stages of the egg-laying cycle. In order to identify proteins regulating the egg-laying process of Huoyan geese, we investigated the proteome profiles of the hypothalamus from Huoyan geese during the laying period and pre-laying period by applying an isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic technology. A total number of 3,337 were identified and quantified, of which 18 were significantly up-regulated and 16 were significantly down-regulated. These differentially expressed proteins were subjected to bioinformatics analyses based on the Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway. Some of these were revealed to be involved in hormone and neurotransmitter secretion, exocytosis, calcium ion transport and synaptic transmission. Subsequently, excitatory amino acid transporter 2, complexin-1 and inositol 1,4,5-trisphosphate receptor, type 3 were confirmed at the messenger RNA level using quantitative real-time RT-PCR. Then, the abundance change of these proteins was verified further using Western blotting analysis. These data may aid in elucidating the molecular mechanism of higher laying performance in Huoyan geese.

The hypothalamic photoreceptors regulating seasonal reproduction in birds: a prime role for VA opsin.

Extraretinal photoreceptors located within the medio-basal hypothalamus regulate the photoperiodic control of seasonal reproduction in birds. An action spectrum for this response describes an opsin photopigment with a λmax of ∼ 492 nm. Beyond this however, the specific identity of the photopigment remains unresolved. Several candidates have emerged including rod-opsin; melanopsin (OPN4); neuropsin (OPN5); and vertebrate ancient (VA) opsin. These contenders are evaluated against key criteria used routinely in photobiology to link orphan photopigments to specific biological responses. To date, only VA opsin can easily satisfy all criteria and we propose that this photopigment represents the prime candidate for encoding daylength and driving seasonal breeding in birds. We also show that VA opsin is co-expressed with both gonadotropin-releasing hormone (GnRH) and arginine-vasotocin (AVT) neurons. These new data suggest that GnRH and AVT neurosecretory pathways are endogenously photosensitive and that our current understanding of how these systems are regulated will require substantial revision.

Gonadotropin-inhibitory hormone-stimulation of food intake is mediated by hypothalamic effects in chicks.

Gonadotropin-inhibitory hormone (GnIH), a 12 amino acid peptide, is expressed in the avian brain and inhibits luteinizing hormone secretion. Additionally, exogenous injection of GnIH causes increased food intake of chicks although the central mechanism mediating this response is poorly understood. Hence, the purpose of our study was to elucidate the central mechanism of the GnIH orexigenic response using 12 day post hatch layer-type chicks as models. Firstly, via mass spectrometry we deduced the chicken GnIH amino acid sequence: SIRPSAYLPLRFamide. Following this we used chicken GnIH to demonstrate that intracerebroventricular (ICV) injection of 2.6 and 7.8 nmol causes increased food intake up to 150 min following injection with no effect on water intake. The number of c-Fos immunoreactive cells was quantified in appetite-associated hypothalamic nuclei following ICV GnIH and only the lateral hypothalamic area (LHA) had an increase of c-Fos positive neurons. From whole hypothalamus samples following ICV GnIH injection abundance of several appetite-associated mRNA was quantified which demonstrated that mRNA for neuropeptide Y (NPY) was increased while mRNA for proopiomelanocortin (POMC) was decreased. This was not the case for mRNA abundance in isolated LHA where NPY and POMC were not affected but melanin-concentrating hormone (MCH) mRNA was increased. A comprehensive behavior analysis was conducted after ICV GnIH injection which demonstrated a variety of behaviors unrelated to appetite were affected. In sum, these results implicate activation of the LHA in the GnIH orexigenic response and NPY, POMC and MCH are likely also involved.

Identification of a cDNA encoding a novel small secretory protein, neurosecretory protein GL, in the chicken hypothalamic infundibulum.

To find novel neuropeptide and/or peptide hormone precursors in the avian brain, we performed a cDNA subtractive screen of the chicken hypothalamic infundibulum, which contains one of the feeding and neuroendocrine centers. After sequencing 596 clones, we identified a novel cDNA encoding a previously unknown protein. The deduced precursor protein consisted of 182 amino acid residues, including one putative small secretory protein of 80 amino acid residues. This small protein was flanked at the N-terminus by a signal peptide and at the C-terminus by a glycine amidation signal and a dibasic amino acid cleavage site. Because the predicted C-terminal amino acids of the small protein were Gly-Leu-NH2, the small protein was named neurosecretory protein GL (NPGL). Quantitative RT-PCR analysis demonstrated specific expression of the NPGL precursor mRNA in the hypothalamic infundibulum. Furthermore, the mRNA levels in the hypothalamic infundibulum increased during post-hatching development. In situ hybridization analysis showed that the cells containing the NPGL precursor mRNA were localized in the medial mammillary nucleus and infundibular nucleus within the hypothalamic infundibulum of 8- and 15-day-old chicks. Subcutaneous infusion of NPGL in chicks increased body weight gain without affecting food intake. To our knowledge, this is the first report to describe the identification and localization of the NPGL precursor mRNA and the function of its translated product in animals. Our findings indicate that NPGL may participate in the growth process in chicks.

Differential roles of hypothalamic serotonin receptor subtypes in the regulation of prolactin secretion in the turkey hen.

In the turkey, exogenous serotonin (5-hydroxytryptamine, 5-HT) increases prolactin (PRL) secretion by acting through the dopaminergic (DAergic) system. In the present study, infusion of the 5-HT(2C) receptor agonist, (R)(-)-DOI hydrochloride (DOI), into the third ventricle stimulates PRL secretion, whereas the 5-HT(1A) receptor agonist, (+/-)-8-OH-DPAT hydrobromide (DPAT), inhibits PRL secretion. Using the immediate-early gene, c-fos, as an indicator of neuronal activity, in situ hybridization histochemistry showed preferential c-fos co-localization within tyrosine hydroxylase immunoreactive neurons (the rate limiting enzyme in DA synthesis) in the areas of the nucleus preopticus medialis (POM) and the nucleus premammillaris (PMM), in response to DPAT and DOI, respectively. To clarify the involvement of 5-HT(1A) and 5-HT(2C) receptors in PRL regulation, their mRNA expression was determined on hypothalamic tissue sections from birds in different reproductive stages. A significant difference in 5-HT1A receptor was observed, with the POM of hypoprolactinemic short day and photorefractory birds showing the highest expression. 5-HT2C receptors mRNA did not change during the reproductive cycle. The data presented support the notion that DA neurons in the PMM and POM mediate the stimulatory and inhibitory effects of 5-HT, respectively, on PRL secretion and the 5-HTergic system can both stimulate and inhibit PRL secretion.

Insight on trace element detoxification in the Black-tailed Godwit (Limosa limosa) through genetic, enzymatic and metallothionein analyses.

Trace element concentrations (Ag, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, Zn) were investigated in the liver, kidneys, muscle and feathers of 31 black-tailed godwits (Limosa limosa) accidentally killed during catches by mist net in the Pertuis Charentais, Atlantic coast of France. Analyses of carbon and nitrogen stable isotope ratios were carried out in liver, muscle and feathers in order to elucidate dietary patterns and to determine whether differences in diet explained the variation in elemental uptake. This study also aimed to have a preliminary assessment of sub-lethal effects triggered by trace elements through the investigation of gene expressions by quantitative real-time PCR, antioxidant enzyme activities (catalase, superoxide dismutase, glutathione peroxidase), and metallothionein (MT) levels. The results showed that Cr and Ni concentrations in tissues of adults were lower than in juveniles in part because adults may have eliminated these trace elements through moulting. Except for Cd and Ni, trace element concentrations were negatively correlated to the body mass of godwits. Ag, As, Hg and Se concentrations were positively linked with the trophic position of birds. The diet could be considered as a fundamental route of exposure for these elements demonstrating therefore the qualitative linkage between dietary habits of godwits and their contaminant concentrations. Our results strongly suggest that even though trace element concentrations were mostly below toxicity threshold level, the elevated concentrations of As, Ag, Cd, Cu, Fe and Se may however trigger sub-lethal effects. Trace elements appear to enhance expression of genes involved in oxidative stress defence, which indicates the production of reactive oxygen species. Moreover, birds with the highest concentrations appeared to have an increased mitochondrial metabolism suggesting that the fight against trace element toxicity requires additional energetic needs notably to produce detoxification mechanisms such as metallothioneins.

A new key neurohormone controlling reproduction, gonadotropin-inhibitory hormone (GnIH): Biosynthesis, mode of action and functional significance.

Identification of novel neurohormones that play important roles in the regulation of pituitary function is essential for the progress of neurobiology. The decapeptide gonadotropin-releasing hormone (GnRH) is the primary factor responsible for the hypothalamic control of gonadotropin secretion. Gonadal sex steroids and inhibin inhibit gonadotropin secretion via feedback from the gonads, but a neuropeptide inhibitor of gonadotropin secretion was, until recently, unknown in vertebrates. In 2000, a novel hypothalamic dodecapeptide that inhibits gonadotropin release was identified in quail and termed gonadotropin-inhibitory hormone (GnIH). This was the first demonstration of a hypothalamic neuropeptide inhibiting gonadotropin release in any vertebrate. GnIH acts on the pituitary and GnRH neurons in the hypothalamus via a novel G protein-coupled receptor for GnIH to inhibit gonadal development and maintenance by decreasing gonadotropin release and synthesis. GnIH neurons express the melatonin receptor and melatonin stimulates the expression of GnIH. Because GnIH exists and functions in several avian species, GnIH is considered to be a new key neurohormone controlling avian reproduction. From a broader perspective, subsequently the presence of GnIH homologous peptides has been demonstrated in other vertebrates. Mammalian GnIH homologous peptides also act to inhibit reproduction by decreasing gonadotropin release in several mammalian species. Thus, the discovery of GnIH has opened the door to a new research field in reproductive neurobiology. This review summarizes the advances made in our understanding of the biosynthesis, mode of action and functional significance of GnIH, a newly discovered key neurohormone, and its homologous peptides.