Possible roles of brain derived neurotrophic factor and corticotropin releasing hormone neurons in the nucleus of hippocampal commissure functioning within the avian neuroendocrine regulation of stress.

Corticotropin releasing hormone (CRH) neurons located in the nucleus of hippocampal commissure (NHpC) have been proposed to be involved in the avian neuroendocrine regulation of stress and appeared to respond prior to CRH neurons in the hypothalamic paraventricular nucleus (PVN) when food deprivation stress was applied. Since the response of the NHpC was rapid and short-lived, was it regulated differentially from CRH neurons in the PVN? We, therefore, applied immobilization stress to test whether the NHpC response was stressor specific. Gene expression of CRH and stress-related genes in the NHpC, PVN, anterior pituitary (APit) as well as plasma corticosterone (CORT) were determined. Furthermore, brain derived neurotrophic factor (BDNF) and glucocorticoid receptor (GR) were examined regarding their possible roles in the regulation of CRH neurons. Data showed that rapid activation of CRH mRNA in the NHpC occurred and preceded a slower gene activation in the PVN, upregulation of proopiomelanocortin (POMC) transcripts in the APit and significant increases of CORT concentrations. Results suggested BDNF's role in negative feedback between CRH and CRHR1 in the NHpC and positive feedback between CRH and CRHR1 in the PVN. In the APit, V1bR activation appeared responsible for sustaining CORT release when stress persisted. Overall, data suggest that the NHpC functions as part of the HPA axis of birds and perhaps a comparable extra-hypothalamic structure occurs in other vertebrates.Lay SummaryThe nucleus of the hippocampal commissure, a structure outside of the hypothalamus, shows rapidly increased neural gene expression that appears to contribute to the early activation of the traditional hypothalamic-pituitary-adrenal (HPA) axis responsible for the production of stress hormones.

Effects of light intensity and dual light intensity choice on plasma corticosterone, central serotonergic and dopaminergic activities in birds, Gallus gallus.

Light intensity plays an important role in the regulation of growth, behavior, reproduction, and welfare of avian species. Light intensity preference behavior has been suggested to be involved in welfare of birds. This study aims to investigate the effects of different light intensity and dual light intensity choice (DLIC) lighting program on plasma corticosterone (CORT), and tryptophan hydroxylase 2 (TPH2, the rate-limiting enzyme of serotonin biosynthesis) and tyrosine hydroxylase (TH, the rate-limiting enzyme of dopamine biosynthesis) gene expression in the brainstem of male chickens. Day old broilers were housed in two commercial houses, and placed in 24 pens. All the treatment groups were provided with 23 h light (L) /1 h dark (D) and 30 lx (lx) light intensity during the first week and then 18L:6D (10 lx) from day 7 to 14. Blood and brain were sampled at 14 days of age (10 lx) before the onset of light treatments. On day 15, four treatments (2, 10, 20, and 100 lx), and DLIC treatment (2/20 lx) were initiated. Samples were collected on days 15, 16, 17, 30 and 41. TPH2 expression in the dorsal raphe nucleus (DRN) and caudal raphe nucleus (CRN) of brainstem, and TPH2 and TH expression in ventral tegmental areas (VTN) of the midbrain were determined by qPCR. Results showed that bright light and DLIC lighting program temporarily attenuated plasma CORT, suggesting the short-term stress attenuating effect of bright light and DLIC lighting program. Differential TPH2 expression in the DRN and CRN observed in the DLIC birds indicate a significant effect of DLIC lighting program on the serotonergic activity in the avian brainstem. At the 41 days of age, the significant downregulation of TPH2 and TH expression occurred in the VTA of DLIC treated birds compared to the other group of birds. Taken together, temporal and spatial regulation of TPH2 and TH expression by DLIC lighting program indicate that compensatory regulation of serotonergic and dopaminergic activities might be involved in the light intensity preference behavior of birds, suggesting a possible beneficial effect of the DLIC lighting program on broiler welfare.

Differential delayed responses of arginine vasotocin and its receptors in septo-hypothalamic brain structures and anterior pituitary that sustain hypothalamic-pituitary-adrenal (HPA) axis functions during acute stress.

Recently, we proposed that corticotropin releasing hormone (CRH) neurons in the nucleus of hippocampal commissure (NHpC), located in the septum, function as a part of the traditional hypothalamic-pituitary-adrenal (HPA) axis in avian species. CRH and its receptor, CRHR1, are regulated differently in the NHpC compared to the paraventricular nucleus (PVN) following feed deprivation (FD). Therefore, we followed up our work by examining arginine vasotocin (AVT), the other major ACTH secretagogue, and its receptors, V1aR and V1bR, gene expression during FD stress in the NHpC, PVN, and ventral mediobasal hypothalamus/median eminence (MBHv/ME). The objectives were to 1) identify AVT perikarya, fibers and its two major receptors, V1aR and V1bR, in the NHpC, PVN, and MBHv/ME using immunohistochemistry, 2) determine the effect of stress on AVT, V1aR and V1bR mRNA expression in the same three brain structures, NHpC, PVN, and MBHv/ME; and, 3) ascertain the expression pattern of V1aR and V1bR mRNA in the anterior pituitary and measure plasma stress hormone, corticosterone (CORT), concentration following FD stress. Male chicks (Cobb 500), 14 days of age, were divided into six groups (10 birds/treatment) and subjected to different times of FD stress: (Control, 1 h, 2 h, 3 h, 4 h, and 8 h). For each bird, blood, brain, and anterior pituitary were sampled and frozen immediately. The NHpC, PVN, and MBHv/ME were micro-dissected for RT-PCR. Data were analyzed using one-way ANOVA followed by Tukey Kramer HSD test using a significance level of p < 0.05. Perikarya of AVT neurons were identified in the PVN but not in the NHpC nor MBHv/ME, and only V1aR-immunoreactivity (ir) was observed in the three structures, however, gene expression data for AVT and its two receptors were obtained in all structures. Both AVT and V1aR mRNA are expressed and increased significantly in the PVN following FD stress (p < 0.01). For the first time, V1bR mRNA was documented in the avian brain and specifically shown upregulated in the NHpC and PVN (p < 0.01) following stress. Additionally, delayed significant gene expression of AVT and its receptors in the PVN showed a positive feedback relationship responsible for maintaining CORT release. In contrast, a significant downregulation of AVT mRNA and upregulation of V1aR mRNA occurred in the NHpC (p < 0.01) during FD showing a negative feedback relationship between AVT and its receptors, V1aR and V1bR. Within the MBHv/ME and anterior pituitary, a gradual increase of AVT mRNA in PVN as well as MBHv/ME was associated with significant upregulation of V1bR (p < 0. 01) and downregulation of V1aR (p < 0.01) in both MBHv/ME and anterior pituitary indicating AVT regulates its receptors differentially to sustain CORT release and control overstimulation of the anterior pituitary during a stress response.

Characterization of stress response involved in chicken myopathy.

Myopathies (Woody Breast (WB) and White Striping (WS)) of broiler chickens have been correlated with fast growth. Recent studies reported that localized hypoxia and metabolic impairment may involve in these myopathies of birds. In order to better understand the stress response mechanisms affecting myopathies of broilers, the aim of this study was to examine effects of WB and both WB/WS on stress hormone corticosterone (CORT) levels and expressional changes of stress response genes including glucocorticoid (GC) receptor (GR), 11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1), DNA methylation regulators (DNMTs), and arginine vasotocin receptor 1a and 1b (V1aR, V1bR). Results of radioimmunoassay showed that CORT levels of WB and WB/WS birds were significantly higher compared to Con (p < 0.05), however, the combination of WB/WS was not significantly higher than WB birds, implying that the effects of WB and WS on CORT are not synergistic. Hepatic GR expression of both WB and WB/WS birds were significantly higher compared to Con (p < 0.05). However, GR expression levels in breast muscle of both WB and WB/WS birds were decreased compared to Con (p < 0.05). Hepatic 11ß-HSD1 expression was increased only in WB/WS birds compared to Con birds with no significant difference between Con and WB birds. 11ß-HSD1 expression was decreased and increased in WB and WB/WS birds compared to Con, respectively, in breast muscle (p < 0.05). DNMT1 expression was significantly decreased in both muscle and liver of WB birds, and in muscle of WB/WS birds, but not in liver of WB/WS birds, indicating differential effects of WS on the epigenetical stress response of muscle and liver compared to WB. V1aR expression was significantly increased in muscle of WB birds, and in liver of WB/WS birds compared to Con birds (p < 0.05). V1bR was not changed in muscle and liver of WB birds compared to Con birds. Taken together, results suggest that GC-induced myopathies occur in fast-growing broiler chickens and circulating CORT level might be a significant biochemical marker of myopathies (WB and WS) of birds. In addition, chronic stress responses in breast muscle and tissue-specific epigenetic changes of stress response genes by DNMTs may play a critical role in the occurrence of myopathies.

Deep-brain photoreceptors (DBPs) involved in the photoperiodic gonadal response in an avian species, Gallus gallus.

Three primitive photoreceptors [melanopsin (Opn4), neuropsin/opsin5 (Opn5) and vertebrate ancient opsin (VAOpn)] were reported as possible avian deep-brain photoreceptors (DBPs) involved in the perception of photoperiodic information affecting the onset and development of reproduction. The objective of this study was to determine the effect of long-day photostimulation and/or sulfamethazine treatment (SMZ, a compound known to advance light-induced testes development) on gene expression of DBPs and key hypothalamic and pituitary genes involved in avian reproductive function. Two-week old chicks were randomly selected into four experimental groups: short-day control (SC, LD8:16), short-day+SMZ (SS, LD8:16, 0.2% diet SMZ), long-day control (LC, LD16:8), and long-day+SMZ (LS, LD16:8, 0.2% diet SMZ). Birds were sampled on days 3, 7, and 28 after initiation of a long-day photoperiod and/or SMZ dietary treatments. Three brain regions [septal-preoptic, anterior hypothalamic (SepPre/Ant-Hypo) region, mid-hypothalamic (Mid-Hypo) region, posterior-hypothalamic (Post-Hypo) region], and anterior pituitary gland were dissected. Using quantitative real-time RT-PCR, we determined changes of expression levels of genes in distinct brain regions; Opn4 and Opn5 in SepPre/Ant-Hypo and Post-Hypo regions and, VAOpn in the Mid-Hypo region. Long-day treatment resulted in a significantly elevated testes weight on days 7 and 28 compared to controls, and SMZ augmented testes weight in both short- and long-day treatment after day 7 (P<0.05). Long-day photoperiodic treatment on the third day unexpectedly induced a large 8.4-fold increase of VAOpn expression in the Mid-Hypo region, a 15.4-fold increase of Opn4 and a 97.8-fold increase of Opn5 gene expression in the Post-Hypo region compared to SC birds (P<0.01). In contrast, on days 7 and 28, gene expression of the three DBPs was barely detectable. LC group showed a significant increase in GnRH-1 and TRH mRNA in the Mid-Hypo compared to SC on day 3. Pituitary LHß and FSHß mRNA were significantly elevated in LC and LS groups compared to SC on days 3 and 7 (P<0.05). On days 3 and 7, TSHß mRNA level was significantly elevated by long-day treatment compared to the SC groups (P<0.05). Results suggest that long-day photoperiodic activation of DBPs is robust, transient, and temporally related with neuroendocrine genes involved in reproductive function. Additionally, results indicate that two subsets of GnRH-1 neurons exist based upon significantly different gene expression from long-day photostimulation and long-day plus SMZ administration. Taken together, the data indicate that within 3 days of a long-day photoperiod, an eminent activation of all three types of DBPs might be involved in priming the neuroendocrine system to activate reproductive function in birds.

Exploring avian deep-brain photoreceptors and their role in activating the neuroendocrine regulation of gonadal development.

In the eyes of mammals, specialized photoreceptors called intrinsically photosensitive retinal ganglion cells (ipRGC) have been identified that sense photoperiodic or daylight exposure, providing them over time with seasonal information. Detectors of photoperiods are critical in vertebrates, particularly for timing the onset of reproduction each year. In birds, the eyes do not appear to monitor photoperiodic information; rather, neurons within at least 4 different brain structures have been proposed to function in this capacity. Specialized neurons, called deep brain photoreceptors (DBP), have been found in the septum and 3 hypothalamic areas. Within each of the 4 brain loci, one or more of 3 unique photopigments, including melanopsin, neuropsin, and vertebrate ancient opsin, have been identified. An experiment was designed to characterize electrophysiological responses of neurons proposed to be avian DBP following light stimulation. A second study used immature chicks raised under short-day photoperiods and transferred to long day lengths. Gene expression of photopigments was then determined in 3 septal-hypothalamic regions. Preliminary electrophysiological data obtained from patch-clamping neurons in brain slices have shown that bipolar neurons in the lateral septal organ responded to photostimulation comparable with mammalian ipRGC, particularly by showing depolarization and a delayed, slow response to directed light stimulation. Utilizing real-time reverse-transcription PCR, it was found that all 3 photopigments showed significantly increased gene expression in the septal-hypothalamic regions in chicks on the third day after being transferred to long-day photoperiods. Each dissected region contained structures previously proposed to have DBP. The highly significant increased gene expression for all 3 photopigments on the third, long-day photoperiod in brain regions proposed to contain 4 structures with DBP suggests that all 3 types of DBP (melanopsin, neuropsin, and vertebrate ancient opsin) in more than one neural site in the septal-hypothalamic area are involved in reproductive function. The neural response to light of at least 2 of the proposed DBP in the septal/hypothalamic region resembles the primitive, functional, sensory ipRGC well characterized in mammals.

Regulation of gene expression of vasotocin and corticotropin-releasing hormone receptors in the avian anterior pituitary by corticosterone.

The effect of chronic stress (CS) on gene expression of the chicken arginine vasotocin (AVT) and corticotropin-releasing hormone (CRH) receptors [VT2R, VT4R, CRH-R1, and CRH-R2] was examined by measuring receptor mRNA levels in the anterior pituitary gland of the chicken after chronic immobilization stress compared to acute stress (AS). Radioimmunoassay results showed that blood circulating corticosterone (CORT) levels in the CS group were significantly decreased compared to that of birds in the AS group (P<0.05). The VT2R and CRH-R2 mRNA in CS birds were significantly decreased to that of controls. The VT4R mRNA was significantly decreased compared to controls in AC birds and was further decreased in the CS group compared to controls (P<0.05). The CRH-R1 mRNA was significantly decreased in the AS birds compared to controls. However, there was no significant difference of CRH-R1 mRNA between acute stress and chronic stress birds. Using primary anterior pituitary cell cultures, the effect of exogenous CORT on VT/CRH receptor gene expression was examined. Receptor mRNA levels were measured after treatment of CORT followed by AVT/CRH administration. The CORT pretreatment resulted in a dose-dependent decrease of proopiomelanocortin heteronuclear RNA, a molecular marker of a stress-induced anterior pituitary. Without CORT pretreatment of anterior pituitary cell cultures, the VT2R, VT4R and CRH-R1mRNA levels were significantly increased within 15 min and then decreased at 1 h and 6 h by AVT/CRH administration (P<0.05). Pretreatment of CORT in anterior pituitary cells induced a dose-dependent increase of VT2R, VT4R and CRH-R2 mRNA levels, and a significant decrease of CRH-R1 mRNA levels at only the high dose (10 ng/ml) of CORT (P<0.05).Taken together, results suggest a modulatory role of CORT on the regulation of VT/CRH receptor gene expression in the avian anterior pituitary gland dependent upon CORT levels.

Identification of arginine vasotocin (AVT) neurons activated by acute and chronic restraint stress in the avian septum and anterior diencephalon.

Effects of acute and chronic psychological stress in the brain of domestic avian species have not been extensively studied. Experiments were performed using restraint stress to determine groups of neurons activated in the septum and diencephalon of chickens. Using FOS immunoreactivity six brain structures were shown activated by acute stress including: the lateral hypothalamic area (LHy), ventrolateral thalamic nucleus (VLT), lateral septum (LS), lateral bed nucleus of the stria terminalis (BSTL), nucleus of the hippocampal commissure (NHpC) and the core region of the paraventricular nucleus (PVNc). Additionally, the LHy and PVNc showed increased FOS immunoreactive (-ir) cells in the birds chronically stressed when compared to controls. In contrast, the NHpC showed decreased FOS-ir cells following the 10day chronic stress imposed. Thereafter, restraint stress experiments were performed to identify activated arginine vasotocin (AVT) neurons (parvocellular or magnocellular) using immunocytochemistry. Of the six FOS activated structures, the PVN was known to contain distinct size groups of AVT-ir neurons, parvocellular (small), medium sized and magnocellular (large). Using dual immunostaining (AVT/FOS), AVT-ir parvocellular neurons in the PVNc were found activated in both acute and chronic stress. To determine whether these AVT-ir parvocellular neurons are co-localized with corticotropin releasing hormone (CRH), an attempt was made to visualize CRH-ir neurons using colchicine. Although AVT-ir and CRH-ir parvocellular neurons occur in the PVNc, only a few neurons were shown co-localized with AVT and CRH after acute restraint stress. Results of this study suggest that the NHpC, LS, VLT, BSTL, LHy and AVT-ir parvocellular neurons in the PVNc are associated with psychological stress in birds.

Effects of Environmental Enrichments on Welfare and Hepatic Metabolic Regulation of Broiler Chickens.

The aims of this study were to find suitable environmental enrichment (EE) and evaluate the combined effect of two EEs, variable light intensity (VL) lighting program and EH, on mental health and hepatic metabolic regulation in commercial broilers. To find the advantageous EEs for broilers, three different EEs (board, hut, and ramp) were tested in trial 1. EEs were placed and the engagement of birds to EEs, dustbathing behavior, and daily physical activity were observed. Birds treated with huts showed higher engagement than the board- or ramp-treated birds (p < 0.05). The results of dustbathing behavior and daily physical activity indicated that the environmental hut (EH) is the most favorable enrichment for broilers. In the second trial, to test the effect of EHs on mental health and hepatic metabolic conditions, the brain and liver were sampled from the four treatment birds (20 lx_Con, 20 lx_Hut, VL_Con and VL_Hut) on day 42. The lower expression of TPH2 (tryptophan hydroxylase 2) of VL_Hut birds than those of VL_Con and 20 lx_Hut treated birds suggests the combining effect of EHs with the VL lighting program on the central serotonergic homeostasis of broilers. Reduced expressions of TH (tyrosine hydroxylase), GR (glucocorticoid receptor), BDNF (brain-derived neurotrophic factor) of VL_Hut treated birds compared to those of VL_Con and 20 lx_Hut birds suggest lower stress, stress susceptibility, and chronic social stress in VL_Hut treated birds. The expression of CPT1A (carnitine palmitoyl transferase 1) increased over three-fold in the liver of VL_Con birds compared to 20 lx_Con birds (p < 0.05). EHs treatment in VL birds (VL_Hut) significantly decreased CPT1A but not in 20 lx birds (20 lx_Hut). The expression of ACCα (acetyl-CoA carboxylase alpha) was significantly decreased in VL_Con birds compared to 20 lx_Con birds. There was no significant difference in the hepatic FBPase (fructose-1,6-bisphosphatase), GR, and 11ß-HSD1 (11 ß-hydroxysteroid dehydrogenease-1) expression between 20 lx_Con and VL_Con birds, but EHs significantly stimulated GR in 20 lx_Hut birds, and stimulated FBPase and 11ß-HSD1 expression in the VL_Hut birds compared to 20 lx_Con birds, suggesting that the VL lighting program reduced fatty acid synthesis and increased fatty acid ß-oxidation in the broilers' liver and VL_Hut improved the hepatic de novo glucose production. Taken together, the results suggest that the stimulated voluntary activity by EHs in the light-enriched broiler house improved mental health and hepatic metabolic function of broilers and may indicate that the improved hepatic metabolic function contributes to efficient nutritional support for broilers.

A possible mechanism contributing to the synergistic action of vasotocin (VT) and corticotropin-releasing hormone (CRH) receptors on corticosterone release in birds.

Arginine vasotocin (AVT) and corticotropin-releasing hormone (CRH) are two neuronal regulators in the hypothalamic-pituitary-adrenal (HPA) axis that modulate biological responses to stress in avian species. When AVT and CRH are administered together in vitro or in vivo, levels of adrenocorticotropic hormone (ACTH) or plasma corticosterone (CORT) are released, respectively, in a synergistic manner. The underlying mechanism of this greater than additive stress response was investigated by expressing the vasotocin receptor type 2 (VT2R) and CRH receptor type 1 (CRH-R1), both G-protein coupled receptors, in HeLa cells. Fluorescence resonance energy transfer (FRET) analysis provided the evidence for heterodimerization of the VT2R/CRH-R1 in the presence of their respective ligands, AVT and CRH. The VT2R and CRH-R1 were tagged at the C-terminal ends with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), and a VT2R chimera was constructed by replacing the fourth transmembrane region (TM4) of the VT2R with TM-IV of the ß2-adrenergic receptor (ß2AR). When VT2R/ß2AR chimera and CRH-R1 were expressed in HeLa cells, heterodimerization was partly disrupted. Taken together, these data indicate that TM-IV of the VT2R may provide an important interface for effective receptor dimerization, suggesting that direct molecular interaction between VT2R and CRH-R1 receptors plays a role in mediating an enhanced interaction between these two receptors. Their interaction at the anterior pituitary level may potentiate the endocrine output of the avian HPA system.

Neuroendocrine regulation of stress in birds with an emphasis on vasotocin receptors (VTRs).

The neuroendocrine stress response of vertebrates, particularly mammals, comprises at least two types of neuropeptide containing neurons, corticotropin-releasing hormone (CRH) and vasopressin (VP) neurons, and four receptors [CRH receptor one (CRH-R1) and two (CRH-R2) and VP receptor 1a (V1aR) and 1b (V1bR)]. The avian neuropeptide CRH, a 41-amino acid peptide, has been shown to have the same amino acid sequence as humans while nonapeptide neurohormone arginine-vasotocin (AVT) is regarded as highly conserved having a single amino acid substitution compared to mammalian arginine vasopressin. Similar to mammals, birds have two receptor subtypes (CRH-R1 and CRH-R2) for CRH, however, four vasotocin receptors have been identified. Less is known about the functions of the four avian vasotocin receptors compared to homologous ones found in mammals and other vertebrate classes. Recently, chicken vasotocin receptor two (VT2R) and four (VT4R) have been characterized utilizing immunocytochemistry and an imposed stress test. The purpose of this review is to present evidence that the VT2R and VT4R are involved in the avian stress response and that the cephalic lobe of the anterior pituitary appears specialized for this function as it contains the major population of corticotropes and necessary neuroendocrine receptors to respond to stressors impacting avian species.

Effects of a variable light intensity lighting program on the welfare and performance of commercial broiler chickens.

Our previous variable-light intensity lighting program studies indicate the light intensity preference behavior of broilers for their daily activity including eating and resting. To evaluate the effects of variable-light intensity lighting program on performance and welfare of broilers, four commercial trials were conducted for looking at behaviors, mortality, leg-health, performance, and brain welfare indicator genes including tryptophan hydroxylase 2 and tyrosine hydroxylase (TH), glucocorticoid receptor (GR), brain-derived neurotropic factor (BDNF), and melanopsin (Opn4) gene expression. One-day-old broilers were housed in four commercial broiler houses. Each quadrant (section) of the house was placed with 4,800 chicks. A total of four lighting programs began on day 7 with 5 lux (lx), 20 lx, natural light (NL, 480 lx), and variable light (2-5/40 lx) using LED lights on a 16L:8D photoperiod. In the variable-light house, the number of dustbathing holes was significantly higher than that in natural-light houses and 5-lx and 20-lx houses. Daily physical activities, footpad condition, fear response to novel objects, body weight, feed conversion ratio, and the number of leg-problem induced culled birds were affected by the variable-light intensity lighting program. Expression of tryptophan hydroxylase 2 in the DRN and VTA of variable-light treated birds was lower than that of 5-lx- and 20-lx-treated birds on day 42 (p < 0.05). Higher expression of VTA-TH in 5-lx-treated birds than that in 20-lx-, NL-, and variable-light-treated birds suggests the high stress-susceptibility of 5-lx treated birds. Lower VTA-GR expression in 20-lx- and variable-light-treated birds indicates lower stress than that in NL- and 5-lx-treated birds (p < 0.05). The VTA-BDNF expression of NL-treated birds was 2.5 fold higher than that of 5-lx-, 20-lx-, and variable-light-treated birds (p < 0.05), and variable-light-treated birds showed the lowest level of BDNF expression (p < 0.05), suggesting the chronic social defeat stress in NL-treated birds. The result of VTA-Opn4 expression on day 42 suggests the possible role of VTA-Opn4 in broiler welfare through central light perception. Taken together, the variable-light intensity lighting program increased volunteer natural behaviors and physical activity, which may improve footpad condition and leg health of birds, consequently. Performance data including the increased daily weight gain and the lowered feed conversion ratio and results of brain welfare indicator gene expression showed the beneficial effect of the variable-light intensity lighting program on the performance and welfare of commercial broilers.

Central Nervous System Associated With Light Perception and Physiological Responses of Birds.

Environmental light that animal receives (i.e., photoperiod and light intensity) has recently been shown that it affects avian central nervous system for the physiological responses to the environment by up or downregulation of dopamine and serotonin activities, and this, in turn, affects the reproductive function and stress-related behavior of birds. In this study, the author speculated on the intriguing possibility that one of the proposed avian deep-brain photoreceptors (DBPs), i.e., melanopsin (Opn4), may play roles in the dual sensory-neurosecretory cells in the hypothalamus, midbrain, and brain stem for the behavior and physiological responses of birds by light. Specifically, the author has shown that the direct light perception of premammillary nucleus dopamine-melatonin (PMM DA-Mel) neurons is associated with the reproductive activation in birds. Although further research is required to establish the functional role of Opn4 in the ventral tegmental area (VTA), dorsal raphe nucleus, and caudal raphe nucleus in the light perception and physiological responses of birds, it is an exciting prospect because the previous results in birds support this hypothesis that Opn4 in the midbrain DA and serotonin neurons may play significant roles on the light-induced welfare of birds.

Serotonin receptor subtypes influence prolactin secretion in the turkey.

Serotonin (5-HT) stimulation of prolactin (PRL) secretion is mediated through the dopaminergic (DAergic) system, with 5-HT ligands having no direct effect on pituitary PRL release. Infusion of 5-HT into the third ventricle (ICV) or electrical stimulation (ES) of the medial preoptic area (POM) or the ventromedial nucleus (VMN) induces an increase in circulating PRL in the turkey. These increases in PRL do not occur when a selective antagonist blocks the D(1) dopamine (DA) receptors in the infundibular area (INF). In this study, the ICV infusion of (R)(-)-DOI hydrochloride (DOI), a selective 5-HT(2A) eceptor agonist, caused PRL to increase. Pretreatment with Ketanserin tartrate salt (KETAN), a selective 5-HT(2A) receptor antagonist, blocked DOI-induced PRL secretion, attesting to the specificity of the response. DOI-induced PRL secretion was prevented when the D(1) DA receptors in the INF were blocked by the D(1) DA receptor antagonist, R(+)-SCH-23390 hydrochloride microinjection, suggesting that the DAergic activation of the vasoactive intestinal peptide (VIP)/PRL system is mediated by a stimulatory 5-HT(2A) receptor subtype. The DOI-induced PRL increase did not occur when (+/-)-8-OH-DPAT (DPAT) was concurrently infused. DPAT is a 5-T(1A) receptor agonist which appears to mediate the inhibitory influence of 5-HT on PRL secretion. When DPAT was microinjected directly into the VMN, it blocked the PRL release affected by ES in the POM. These data suggested that when 5-HT(2A) receptors are activated, they influence the release of DA to the INF. When 5-HT(1A) receptors are stimulated, they somehow inhibit the PRL-releasing actions of 5-HT(2A) receptors. This inhibition could take place centrally, or it could occur postsynaptically at the pituitary level. It is known that D(2) DA receptors in the pituitary antagonize PRL-releasing effect of VIP. A release of DA to the pituitary, initiated by 5-HT(1A) receptors, could effectively inhibit PRL secretion.

The vasotocinergic system and its role in the regulation of stress in birds.

The regulation of stress in birds includes a complex interaction of neural systems affecting the hypothalamic-pituitary-adrenal (HPA) axis. In addition to the hypothalamic paraventricular nucleus, a structure called the nucleus of the hippocampal commissure likewise affects the output of pituitary stress hormones and appears to be unique to avian species. Within the anterior pituitary, the avian V1a and V1b receptors were found in corticotropes. Based on our studies with central administration of hormones in the chicken, corticotropic releasing hormone (CRH) is a more potent ACTH secretagogue than arginine vasotocin (AVT). In contrast, when applied peripherally, AVT is more efficacious. Co-administration of AVT and CRH peripherally, resulted in a synergistic stimulation of corticosterone release. Data suggest receptor oligomerization as one possible mechanism. In birds, vasotocin receptors associated with stress responses include the V1a and V1b receptors. Three-dimensional, homology-based structural models of the avian V1aR were built to test agonists and antagonists for each receptor that were screened by molecular docking to map their binding sites on each receptor. Additionally, binding affinity values for each available peptide antagonist to the V1aR and V1bR were determined. An anterior pituitary primary culture system was developed to determine how effective each antagonist blocked the function of each receptor in culture when stimulated by a combination of AVT/CRH administration. Use of an antagonist in subsequent in vivo studies identified the V1aR in regulating food intake in birds. The V1aR was likewise found in circumventricular organs of the brain, suggesting a possible function in stress.

Dopamine-melatonin neurons in the avian hypothalamus and their role as photoperiodic clocks.

A timing mechanism in the brain governs reproduction in seasonally breeding temperate zone birds by triggering gonad development in response to long days in the spring. The neural mechanism(s) responsible for the timing and induction of reproductive activity by this clock are unknown. Utilizing in situ hybridization, immunocytochemistry and reverse transcriptase-polymerase chain reaction techniques, a group of dopamine (DA) neurons in the premammillary nucleus (PMM) of the caudal turkey hypothalamus that synthesize and colocalize both DA and melatonin (MEL) were identified. In addition, these neurons are found to express clock genes and the circadian photoreceptor melanopsin. DA-MEL neurons reach threshold activation (c-fos expression) when a light pulse is given during the photosensitive phase. This is associated with increases in the number of gonadotropin releasing hormone-I (GnRH-I) neurones activated, as well as an up-regulation of GnRH-I mRNA expression. The expression of tyrosine hydroxylase (TH; the rate limiting enzyme in DA biosynthesis) and tryptophan hydroxylase 1, (TPH1; the first enzyme in MEL biosynthesis) and consequently DAergic-MELergic activities are associated with the daily light-dark cycle. TPH1 mRNA expression shows low levels during the light phase and high levels during the dark phase of the light/dark illumination cycle and is 180 degrees out of phase with the rhythm of TH mRNA expression. Hypothalamic DA-MEL neurons may constitute a critical cellular process involved in the generation and expression of seasonal reproductive rhythms and suggests a previously undescribed mechanism(s) by which light signals gain access to neural targets.

Differential and temporal expression of corticotropin releasing hormone and its receptors in the nucleus of the hippocampal commissure and paraventricular nucleus during the stress response in chickens (Gallus gallus).

Recently, in addition to the paraventricular nucleus (PVN), the nucleus of the hippocampal commissure (NHpC) have been proposed to regulate stress in birds due to the discovery of corticotropin releasing hormone (CRH) neurons in the NHpC. Expression of CRH, CRHR1, CRHR2 and glucocorticoid receptors (GRs) were determined within the NHpC compared to the PVN. Additionally, two levels of the hypothalamo-pituitary-adrenal (HPA) axis: 1) anterior pituitary and 2) adrenal gland were examined following food deprivation (FD) stress including proopiomelanocortin (POMC) expression and plasma corticosterone (CORT), respectively. CRH expression in the NHpC increased rapidly, however it quickly returned to control levels, showing a negative feedback with CRHR1. In contrast, CRH expression in the PVN and its receptor CRHR1, steadily increased throughout the sampling period showing a positive feedback with CRH. Of interest, brain-derived neurotrophic factor (BDNF) mRNA was significantly elevated in the PVN, while no significant change in BDNF mRNA was observed in the NHpC. The rapid increase in BDNF expression that matched the pattern shown by CRHR1 in the PVN may play a role in the positive feedback of CRH and its receptor. GRs were downregulated in both the NHpC and PVN throughout the study. POMC hnRNA and mRNA were significantly elevated from 1 to 4 h of FD compared to controls. A significant increase in plasma CORT levels occurred at 2 h and persisted to the end of the experiment, suggesting that CRH neurons in the NHpC initiated, while PVN CRH neurons sustained the early response of the HPA axis to stress.

Identification of avian vasotocin receptor subtype-specific antagonists involved in the stress response of the chicken, Gallus gallus.

Vasotocin 1a and 1b receptors (V1aR and V1bR) have been shown to play important roles in the neuroendocrine regulation of stress responses via the anterior pituitary (AP) of birds. To identify effective subtype-specific antagonists for the chicken V1aR (cV1aR) and cV1bR, potential antagonists to the mammalian V1R were screened against the cV1aR and cV1bR 3D structural models by molecular docking analysis with determination of binding pocket/amino acid residues involved in the interaction. The antagonistic effects of the selected ligands were examined by measuring pro-opiomelanocortin (POMC) heteronuclear RNA (hnPOMC) levels following the in vitro stress administration to primary chicken AP cells. Results of in silico analysis showed that the Manning compound and several other antagonists were bound to cV1bR with higher affinity than the natural agonist, arginine vasotocin (AVT). Similarities and differences in the antagonist-receptor binding interface with receptors were characterized for each ligand. Non-peptide mammalian V1bR antagonists, SSR-149415 and L-368899, were shown to be effective and had an additive effect in blocking POMC hnRNA expression in pituitary cell culture studies. SR-49059 antagonized the effect(s) of AVT/CRH on the downregulation of the cV1aR and the upregulation of the cCRH-R2 expression but not the cV1bR and cCRH-R1. The Manning compound antagonized the downregulation of cV1aR, cV1bR and cCRH-R1 and the upregulation of cCRH-R2 expression. The specificity of antagonists apparently resulted from unique differences in the interacting residues and their binding affinities. Collectively, these results provide valuable leads for future development of novel compounds capable of blocking or attenuating the AP stress response of avian species and perhaps other non-mammalian vertebrates as well.

Tissue-Specific Expression of DNA Methyltransferases Involved in Early-Life Nutritional Stress of Chicken, Gallus gallus.

DNA methylation was reported as a possible stress-adaptation mechanism involved in the transcriptional regulation of stress responsive genes. Limited data are available on effects of psychological stress and early-life nutritional stress on DNA methylation regulators [DNMTs: DNA (cytosine-5)-methyltransferase 1 (DNMT1), DNMT1 associated protein (DMAP1), DNMT 3 alpha (DNMT3A) and beta (DNMT3B)] in avian species. The objectives of this study were to: (1) investigate changes in expression of DNMT1, DMAP1, DNMT3A, and DNMT3B following acute (AS) or chronic immobilization stress (CS); (2) test immediate effect of early-life nutritional stress [food deprivation (FD) for 12 h (12hFD) or 36 h (36hFD) at the post-hatching period] on expression of DNA methylation regulators and glucocorticoid receptor (GR), and the long-term effect of early-life nutritional stress at 6 weeks of age. Expression of DNMTs and plasma corticosterone (CORT) concentration decreased by CS compared to AS (p < 0.05), indicating differential roles of DNA methylation regulators in the stress response. Plasma CORT at 12hFD and 36hFD birds increased compared to control birds (12hF and 36hF), but there were no significant differences in plasma CORT of 12hFD and 36hFD birds at 6 weeks of age compared to 6 week controls. DNMT1, DMAP1, and DNMT3B expression in the anterior pituitary increased by 12hFD, but decreased at 36hFD compared to their controls (P < 0.05). In liver, DNMT1, DNMT3A, and DNMT3B expression decreased by 12hFD, however, no significant changes occurred at 36hFD. Expression of DMAP1, DNMT3A, and DNMT3B in anterior pituitary and DMAP1 and DNMT3A expression in liver at 6 weeks of age were higher in 36hFD stressed birds compared to controls as well as 12hFD stressed birds. Hepatic GR expression decreased by 12hFD and increased by 36hFD (p < 0.05). Expression patterns of GR in the liver of FD stress-induced birds persisted until 6 weeks of age, suggesting the possible lifelong involvement of liver GR in early-life nutritional stress response of birds. Taken together, results suggest that DNA methylation regulator genes are tissue-specifically responsive to acute and chronic stress, and hepatic GR may play a critical role in regulating the early-life nutritional stress response of birds. In addition, the downregulation of DNMT1 and DMAP1 may be one of the adaptive mechanisms to chronic early-life nutritional stress via passive demethylation.

Functional evidence that the nucleus of the hippocampal commissure shows an earlier activation from a stressor than the paraventricular nucleus: Implication of an additional structural component of the avian hypothalamo-pituitary-adrenal axis.

Despite extensive data addressing the regulation of the hypothalamo-pituitary-adrenal (HPA) axis in vertebrates, the neuroendocrine regulation of stress in birds remains incomplete. The paraventricular nucleus (PVN) contains the key neuropeptides, corticotropin releasing hormone (CRH) and arginine vasotocin (AVT), containing neurons. However, another population of CRH neurons was recently identified in a septal nucleus called the nucleus of the hippocampal commissure (NHpC). Therefore, the current study investigated changes in gene expression of CRH and AVT in the PVN and CRH in the NHpC, as well as changes in plasma corticosterone concentrations following a stressor, food deprivation. In the NHpC, a rapid increase in CRH mRNA levels was observed as early as 2h, while relative CRH mRNA expression in the PVN increased thereafter from 4 to 12h of food deprivation. On the other hand, relative mRNA levels of AVT in the PVN were not observed until 8h and significantly increased at 12 and 24h following food deprivation. Furthermore, at the level of the anterior pituitary, relative expression of proopiomelanocortin transcripts followed gene expression patterns of CRH and AVT in the brain. In the absence of food, the pattern of plasma CORT showed an initial rise at 2h and a fourfold increase was measured at 4h that was sustained through 24h. Taken together, results from this study suggest that (1) CRH neurons in the NHpC appear to be the first responsive neurons to stress stimuli compared to those in the PVN, (2) CRH is predominantly functional in the early phase of stress while AVT is involved in the later phase of the stress period and (3) in birds, CRH neurons in the NHpC appear to be part of the classical HPA axis.