ABSTRACT
Amniotes feature two principal visual processing systems: the tectofugal and thalamofugal pathways. In most mammals, the thalamofugal pathway predominates, routing retinal afferents through the dorsolateral geniculate complex to the visual cortex. In most birds, the thalamofugal pathway often plays the lesser role with retinal afferents projecting to the principal optic thalami, a complex of several nuclei that resides in the dorsal thalamus. This thalamic complex sends projections to a forebrain structure called the Wulst, the terminus of the thalamofugal visual system. The thalamofugal pathway in birds serves many functions such as pattern discrimination, spatial memory, and navigation/migration. A comprehensive analysis of avian species has unveiled diverse subdivisions within the thalamic and forebrain structures, contingent on species, age, and techniques utilized. In this study, we documented the thalamofugal system in three dimensions by integrating histological and contrast-enhanced computed tomography imaging of the avian brain. Sections of two-week-old chick brains were cut in either coronal, sagittal, or horizontal planes and stained with Nissl and either Gallyas silver or Luxol Fast Blue. The thalamic principal optic complex and pallial Wulst were subdivided on the basis of cell and fiber density. Additionally, we utilized the technique of diffusible iodine-based contrast-enhanced computed tomography (diceCT) on a 5-week-old chick brain, and right eyeball. By merging diceCT data, stained histological sections, and information from the existing literature, a comprehensive three-dimensional model of the avian thalamofugal pathway was constructed. The use of a 3D model provides a clearer understanding of the structural and spatial organization of the thalamofugal system. The ability to integrate histochemical sections with diceCT 3D modeling is critical to better understanding the anatomical and physiologic organization of complex pathways such as the thalamofugal visual system.
Subject(s)
Imaging, Three-Dimensional , Visual Pathways , Animals , Visual Pathways/physiology , Thalamus/physiology , Prosencephalon/physiology , Chickens/physiology , MammalsABSTRACT
Image processing in amniotes is usually accomplished by the thalamofugal and/or tectofugal visual systems. In laterally eyed birds, the tectofugal system dominates with functions such as color and motion processing, spatial orientation, stimulus identification, and localization. This makes it a critical system for complex avian behavior. Here, the brains of chicks, Gallus gallus, were used to produce serial brain sections in either coronal, sagittal, or horizontal planes and stained with either Nissl and Gallyas silver myelin or Luxol fast blue stain and cresyl echt violet (CEV). The emerging techniques of diffusible iodine-based contrast-enhanced computed tomography (diceCT) coupled with serial histochemistry in three planes were used to generate a comprehensive three-dimensional (3D) model of the avian tectofugal visual system. This enabled the 3D reconstruction of tectofugal circuits, including the three primary neuronal projections. Specifically, major components of the system included four regions of the retina, layers of the optic tectum, subdivisions of the nucleus rotundus in the thalamus, the entopallium in the forebrain, and supplementary components connecting into or out of this major avian visual sensory system. The resulting 3D model enabled a better understanding of the structural components and connectivity of this complex system by providing a complete spatial organization that occupied several distinct brain regions. We demonstrate how pairing diceCT with traditional histochemistry is an effective means to improve the understanding of, and thereby should generate insights into, anatomical and functional properties of complicated neural pathways, and we recommend this approach to clarify enigmatic properties of these pathways.
Subject(s)
Imaging, Three-Dimensional , Visual Pathways , Animals , Visual Pathways/diagnostic imaging , Visual Pathways/physiology , Chickens/metabolism , Prosencephalon , Sense OrgansABSTRACT
The location of corticotropin-releasing hormone receptor 2 (CRH-R2) on thyrotropes within the avian anterior pituitary (APit) and its activation by different stressors indicate a possible communication between hypothalamo-pituitary-adrenal (HPA) and thyroid (HPT) axes. Therefore, an experiment was designed to 1) compare the timing of major components of the HPT axis to those of the HPA axis; 2) address whether stressors activating the HPA axis may simultaneously upregulate components of the HPT axis. Blood, brain, and APit were sampled from chicks prior to stress (control) and 15, 30, 60, 90, and 120 min following immobilization (IM) stress. The nucleus of the hippocampal commissure (NHpC) and paraventricular nucleus (PVN) were cryo-dissected from brains for RT-qPCR. Gene expression of thyrotropin-releasing hormone (TRH) and its receptors (TRH-R1 and TRH-R3), urocortin3 (UCN3), deiodinase 2 (D2), and the second type of corticotropin-releasing hormone (CRH2) within the NHpC and PVN was measured. Additionally, gene expression of TRH receptors, thyroid stimulating hormone subunit beta (TSHß), and D2 was determined in the APit and corticosterone assayed in blood. In brains, a significant upregulation in examined genes occurred at different times of IM. Specifically, UCN3 and CRH2 which have a high affinity to CRH-R2 showed a rapid increase in their mRNA levels that were accompanied by an early upregulation of TRHR1 in the NHpC. In the APit, a significant increase in gene expression of TSHß and TRH receptors was observed. Therefore, results supported concurrent activation of major brain and APit genes associated with the HPA and HPT axes following IM. The initial neural gene expression originating within the NHpC resulted in the increase of TSHß mRNA in the APit. Specifically, the rapid upregulation of UCN3 in the NHpC appeared responsible for the early activation of TSHß in the APit. While sustaining TSHß activation appeared to be due to both CRH2 and TRH. Therefore, data indicate that CRH-producing neurons and corticotropes as well as CRH- and TRH-producing neurons and thyrotropes are activated to produce the necessary energy required to maintain homeostasis in birds undergoing stress. Overall, data support the inclusion of the NHpC in the classical avian HPA axis and for the first time show the concurrent activation of the HPA axis and components of the HPT axis following a psychogenic stressor.
ABSTRACT
Woody breast (WB) myopathy results in poor muscle quality. The increasing incidence of WB over the last several years indicates a need for improved prediction or early diagnosis. We hypothesized that the use of body fluids, including blood, may be more suitable than breast muscle tissue in developing a minimally invasive diagnostic tool for WB detection. To identify potential early-age-biomarkers that may represent the potential onset of WB, blood samples were collected from 100, 4 wks old commercial male broilers. At 8 wks of age, WB conditions were scored by manual palpation. A total of 32 blood plasma samples (eight for each group of WB and non-WB control birds at two time points, 4 wks and 8 wks) were subjected to shotgun proteomics and untargeted metabolomics to identify differentially abundant plasma proteins and metabolites in WB broilers compared to non-WB control (Con) broilers. From the proteomics assay, 25 and 16 plasma proteins were differentially abundant (p < 0.05) in the 4 and 8 wks old samples, respectively, in WB compared with Con broilers. Of those, FRA10A associated CGG repeat 1 (FRAG10AC1) showed >2-fold higher abundance in WB compared with controls. In the 8 wks old broilers, 4 and 12 plasma proteins displayed higher and lower abundances, respectively, in WB compared with controls. Myosin heavy chain 9 (MYH9) and lipopolysaccharide binding protein (LBP) showed more than 2-fold higher abundances in WB compared with controls, while transferrin (TF) and complement C1s (C1S) showed more than 2-fold lower abundances compared with controls. From the untargeted metabolomics assay, 33 and 19 plasma metabolites were differentially abundant in birds at 4 and 8 wks of age, respectively, in WB compared with controls. In 4 wks old broilers, plasma 3-hydroxybutyric acid (3-HB) and raffinose concentrations showed the highest and lowest fold changes, respectively, in WB compared with controls. The blood plasma 3-HB and raffinose concentrations were confirmed with targeted biochemical assays. Blood biomarkers, such as 3-HB and raffinose, may be suitable candidate targets in the prediction of WB onset at early ages.
ABSTRACT
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.
Subject(s)
Corticotropin-Releasing Hormone , Hypothalamo-Hypophyseal System , Animals , Birds/metabolism , Brain-Derived Neurotrophic Factor/genetics , Brain-Derived Neurotrophic Factor/metabolism , Corticotropin-Releasing Hormone/genetics , Corticotropin-Releasing Hormone/metabolism , Fornix, Brain/metabolism , Hypothalamo-Hypophyseal System/metabolism , Neurons/metabolism , Paraventricular Hypothalamic Nucleus/metabolism , Pituitary-Adrenal System/metabolism , Stress, Physiological , Stress, PsychologicalABSTRACT
Mitigation of stress is of great importance in poultry production, as chronic stress can affect the efficiency of production traits. Selective breeding with a focus on stress responses can be used to combat the effects of stress. To better understand the genetic mechanisms driving differences in stress responses of a selectively bred population of Japanese quail, we performed genomic resequencing on 24 birds from High Stress (HS) and Low Stress (LS) lines of Japanese quail using Illumina HiSeq 2 × 150 bp paired end read technology in order to analyze Single Nucleotide Polymorphisms (SNPs) within the genome of each line. SNPs are common mutations that can lead to genotypic and phenotypic variations in animals. Following alignment of the sequencing data to the quail genome, 6,364,907 SNPs were found across both lines of quail. 10,364 of these SNPs occurred in coding regions, from which 2886 unique, non-synonymous SNPs with a SNP% ≥ 0.90 and a read depth ≥ 10 were identified. Using Ingenuity Pathway Analysis, we identified genes affected by SNPs in pathways tied to immune responses, DNA repair, and neurological signaling. Our findings support the idea that the SNPs found within HS and LS lines of quail could direct the observed changes in phenotype.
Subject(s)
Coturnix/genetics , Polymorphism, Single Nucleotide/genetics , Stress, Physiological/genetics , Animals , DNA Repair/genetics , Genome/genetics , Genomics/methods , Genotype , PhenotypeABSTRACT
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.
Subject(s)
Chickens/physiology , Muscular Diseases/physiopathology , Muscular Diseases/veterinary , Stress, Physiological , 11-beta-Hydroxysteroid Dehydrogenase Type 1/genetics , 11-beta-Hydroxysteroid Dehydrogenase Type 1/metabolism , Animals , Body Weight , Chickens/blood , Chickens/genetics , Corticosterone/blood , DNA Methylation/genetics , Female , Gene Expression Regulation, Developmental , Liver/metabolism , Mammary Glands, Animal/metabolism , Muscles/metabolism , Muscular Diseases/blood , Muscular Diseases/genetics , Organ Specificity , Receptors, Glucocorticoid/metabolism , Receptors, Vasopressin/genetics , Receptors, Vasopressin/metabolismABSTRACT
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.
Subject(s)
Hypothalamo-Hypophyseal System/metabolism , Pituitary-Adrenal System/metabolism , Receptors, Vasopressin/metabolism , Stress, Physiological/physiology , Vasotocin/metabolism , Animals , Birds , ChickensABSTRACT
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), 14â¯days of age, were divided into six groups (10 birds/treatment) and subjected to different times of FD stress: (Control, 1â¯h, 2â¯h, 3â¯h, 4â¯h, and 8â¯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 pâ¯<â¯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 (pâ¯<â¯0.01). For the first time, V1bR mRNA was documented in the avian brain and specifically shown upregulated in the NHpC and PVN (pâ¯<â¯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 (pâ¯<â¯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 (pâ¯<â¯0. 01) and downregulation of V1aR (pâ¯<â¯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.
Subject(s)
Hypothalamo-Hypophyseal System/metabolism , Pituitary Gland, Anterior/metabolism , Pituitary-Adrenal System/metabolism , Stress, Physiological/physiology , Vasotocin/metabolism , Acute Disease , Animals , Chickens , MaleABSTRACT
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 23â¯h light (L) /1â¯h dark (D) and 30â¯lx (lx) light intensity during the first week and then 18L:6D (10â¯lx) from day 7 to 14. Blood and brain were sampled at 14â¯days of age (10â¯lx) before the onset of light treatments. On day 15, four treatments (2, 10, 20, and 100â¯lx), and DLIC treatment (2/20â¯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 41â¯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.
Subject(s)
Chickens/blood , Chickens/metabolism , Corticosterone/blood , Dopamine/metabolism , Light , Serotonin/metabolism , Animals , Brain Stem/metabolism , Brain Stem/radiation effects , Chickens/growth & development , Gene Expression Regulation, Enzymologic/radiation effects , Male , Raphe Nuclei/metabolism , Raphe Nuclei/radiation effects , Tryptophan Hydroxylase/genetics , Tryptophan Hydroxylase/metabolism , Tyrosine 3-Monooxygenase/genetics , Tyrosine 3-Monooxygenase/metabolism , Ventral Tegmental Area/metabolismABSTRACT
Copy number variation (CNV) is a major driving factor for genetic variation and phenotypic diversity in animals. To detect CNVs and understand genetic components underlying stress related traits, we performed whole genome re-sequencing of pooled DNA samples of 20 birds each from High Stress (HS) and Low Stress (LS) Japanese quail lines using Illumina HiSeq 2×150 bp paired end method. Sequencing data were aligned to the quail genome and CNVnator was used to detect CNVs in the aligned data sets. The depth of coverage for the data reached to 41.4x and 42.6x for HS and LS birds, respectively. We identified 262 and 168 CNV regions affecting 1.6 and 1.9% of the reference genome that completely overlapped 454 and 493 unique genes in HS and LS birds, respectively. Ingenuity pathway analysis showed that the CNV genes were significantly enriched to phospholipase C signaling, neuregulin signaling, reelin signaling in neurons, endocrine and nervous development, humoral immune response, and carbohydrate and amino acid metabolisms in HS birds, whereas CNV genes in LS birds were enriched in cell-mediated immune response, and protein and lipid metabolisms. These findings suggest CNV genes identified in HS and LS birds could be candidate markers responsible for stress responses in birds.
Subject(s)
Coturnix/genetics , Coturnix/physiology , DNA Copy Number Variations , Stress, Physiological/genetics , Whole Genome Sequencing , Animals , PhenotypeABSTRACT
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 4â¯h of FD compared to controls. A significant increase in plasma CORT levels occurred at 2â¯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.
Subject(s)
Corticotropin-Releasing Hormone/metabolism , Receptors, Corticotropin-Releasing Hormone/metabolism , Stress, Physiological/physiology , Adrenocorticotropic Hormone/blood , Animals , Brain-Derived Neurotrophic Factor/metabolism , Cell Nucleus/metabolism , Chickens/metabolism , Corticosterone/blood , Corticotropin-Releasing Hormone/genetics , Food Deprivation/physiology , Fornix, Brain/metabolism , Hypothalamo-Hypophyseal System/metabolism , Male , Neurons/metabolism , Paraventricular Hypothalamic Nucleus/metabolism , Pituitary-Adrenal System/metabolism , Pro-Opiomelanocortin/analysis , RNA, Messenger/metabolism , Receptors, Corticotropin-Releasing Hormone/genetics , Receptors, Glucocorticoid/metabolismABSTRACT
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.
Subject(s)
Antidiuretic Hormone Receptor Antagonists/chemistry , Models, Molecular , Molecular Conformation , Receptors, Vasopressin/chemistry , Amino Acid Sequence , Animals , Antidiuretic Hormone Receptor Antagonists/pharmacology , Cells, Cultured , Chickens , Gene Expression , Ligands , Male , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Structure , Protein Binding , Receptors, Vasopressin/genetics , Receptors, Vasopressin/metabolism , Stress, PhysiologicalABSTRACT
Autophagy, a highly conserved intracellular self-digestion process, plays an integral role in maintaining cellular homeostasis. Although emerging evidence indicate that the endocrine system regulates autophagy in mammals, there is still a scarcity of information on autophagy in avian (non-mammalian) species. Here, we show that intracerebroventricular administration of leptin reduces feed intake, modulates the expression of feeding-related hypothalamic neuropeptides, activates leptin receptor and signal transducer and activator of transcription (Ob-Rb/STAT) pathway, and significantly increases the expression of autophagy-related proteins (Atg3, Atg5, Atg7, beclin1, and LC3B) in chicken hypothalamus, liver, and muscle. Similarly, leptin treatment activates Ob-Rb/STAT pathway and increased the expression of autophagy-related markers in chicken hypothalamic organotypic cultures, muscle (QM7) and hepatocyte (Sim-CEL) cell cultures as well as in Chinese Hamster Ovary (CHO-K1) cells-overexpressing chicken Ob-Rb and STAT3. To define the downstream mediator(s) of leptin's effects on autophagy, we determined the role of the master energy sensor AMP-activated protein kinase (AMPK). Leptin treatment significantly increased the phosphorylated levels of AMPKα1/2 at Thr172 site in chicken hypothalamus and liver, but not in muscle. Likewise, AMPKα1/2 was activated by leptin in chicken hypothalamic organotypic culture and Sim-CEL, but not in QM7 cells. Blocking AMPK activity by compound C reverses the autophagy-inducing effect of leptin. Together, these findings indicate that AMPK mediates the effect of leptin on chicken autophagy in a tissue-specific manner.
ABSTRACT
There has been remarkable progress in discoveries made in the avian brain, particularly over the past two decades. This review first highlights some of the discoveries made in the forebrain and credits the Avian Brain Nomenclature Forum, responsible for changing many of the terms found in the cerebrum and for stimulating collaborative research thereafter. The Forum facilitated communication among comparative neurobiologists by eliminating confusing and inaccurate names. The result over the past 15yearshas been a standardized use of avian forebrain terms. Nonetheless, additional changes are needed. The goal of the paper is to encourage a continuing effort to unify the nomenclature throughout the entire avian brain. To emphasize the need for consensus for a single name for each neural structure, I have selected specific structures in the septum and hypothalamus that our laboratory has been investigating, to demonstrate a lack of uniformity in names applied to conservative brain regions compared to the forebrain. The specific areas reviewed include the distributions of gonadotropin-releasing hormone neurons and their terminal fields in circumventricular organs, deep-brain photoreceptors, gonadotropin inhibitory neurons and a complex structure and function of the nucleus of the hippocampal commissure.
Subject(s)
Brain Mapping , Chickens/physiology , Hypothalamus/physiology , Septum of Brain/physiology , Animals , Gonadotropin-Releasing Hormone/metabolism , Hypothalamus/anatomy & histology , Neurons/metabolism , Septum of Brain/anatomy & histologyABSTRACT
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.
ABSTRACT
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.
Subject(s)
Corticotropin-Releasing Hormone/metabolism , Food Deprivation/physiology , Hypothalamo-Hypophyseal System/physiopathology , Paraventricular Hypothalamic Nucleus/physiopathology , Pituitary-Adrenal System/physiopathology , Septum of Brain/physiopathology , Stress, Physiological/physiology , Vasotocin/metabolism , Animals , Chickens , Corticosterone/blood , Corticotropin-Releasing Hormone/genetics , Gene Expression , Hypothalamo-Hypophyseal System/metabolism , Male , Neurons/metabolism , Paraventricular Hypothalamic Nucleus/metabolism , Pituitary Gland/metabolism , Pituitary Gland/physiopathology , Pituitary-Adrenal System/metabolism , Pro-Opiomelanocortin/genetics , Pro-Opiomelanocortin/metabolism , RNA, Messenger , Septum of Brain/metabolism , Time Factors , Vasotocin/geneticsABSTRACT
Past studies have shown that the avian vasotocin 1a receptor (V1aR) is involved in immobilization stress. It is not known whether the receptor functions in osmotic stress, and if sensory circumventricular organs may be involved. An experiment was designed with four treatment groups including a 1h immobilization acute stress (AS) group, an unstressed acute control (AC), a third given an intraperitoneal (ip) hypertonic saline injection (HS) and isotonic saline controls (IC) administered ip. One set of chick brains was perfused for immunohistochemistry while a second was sampled for quantitative RT-PCR. Plasma corticosterone (CORT) and arginine vasotocin (AVT) concentrations were significantly increased in the immobilized and hypertonic saline groups (p<0.01) compared to controls. Intense staining of the V1aR occurred throughout the organum vasculosum of the lamina terminalis (OVLT) and subseptal organ (SSO)/subfornical organ (SFO). The immunostaining allowed the boundaries of the two circumventricular organs (CVOs) to be described for the first time in avian species. Both treatment groups showed marked morphological changes in glia within the OVLT and SSO/SFO. The avian V1aR, angiotensin II type 1 receptor (AT1R), and transient receptor potential vanilloid receptor 1 (TRPV1) mRNA levels were increased in the SSO/SFO in hypertonic saline treated birds compared to isotonic controls. In contrast, the latter two genes (AT1R and TRPV1) were significantly decreased in the OVLT of birds subjected to hyperosmotic stress, while all three genes were significantly up-regulated after immobilization. Taken together, results show a possible differential function for the same receptors in two anatomically adjacent CVOs.
ABSTRACT
Recently, it was found that the avian central vasotocin receptor (V1aR) is associated with the regulation of food intake. To identify V1aR-containing brain structures regulating food intake, a selective V1aR antagonist SR-49059 that induced food intake was administrated intracerebroventricularly in male chickens followed by detection of brain structures using FOS immunoreactivity. Particularly, the hypothalamic core region of the paraventricular nucleus, lateral hypothalamic area, dorsomedial hypothalamic nucleus, a subnucleus of the central extended amygdalar complex [dorsolateral bed nucleus of the stria terminalis], medial septal nucleus and caudal brainstem [nucleus of the solitary tract] showed significantly increased FOS-ir cells. On the other hand, the supraoptic nucleus of the preoptic area and the nucleus of the hippocampal commissure of the septum showed suppressed FOS immunoreactivity in the V1aR antagonist treatment group. Further investigation revealed that neuronal activity of arginine vasotocin (AVT-ir) magnocellular neurons in the supraoptic nucleus, preoptic periventricular nucleus, paraventricular nucleus and ventral periventricular hypothalamic nucleus and most likely corticotropin releasing hormone (CRH-ir) neurons in the nucleus of the hippocampal commissure were reduced following the antagonist treatment. Dual immunofluorescence labeling results showed that perikarya of AVT-ir magnocellular neurons in the preoptic area and hypothalamus were colabeled with V1aR. Within the nucleus of the hippocampal commissure, CRH-ir neurons were shown in close contact with V1aR-ir glial cells. Results of the present study suggest that the V1aR plays a role in the regulation of food intake by modulating neurons that synthesize and release anorectic neuropeptides in the avian brain.
Subject(s)
Appetite Regulation/physiology , Avian Proteins/metabolism , Diencephalon/metabolism , Eating/physiology , Receptors, Vasopressin/metabolism , Septum of Brain/metabolism , Animals , Antidiuretic Hormone Receptor Antagonists/pharmacology , Appetite Regulation/drug effects , Appetitive Behavior/drug effects , Appetitive Behavior/physiology , Avian Proteins/antagonists & inhibitors , Central Nervous System Agents/administration & dosage , Chickens , Diencephalon/cytology , Diencephalon/drug effects , Eating/drug effects , Indoles/pharmacology , Male , Motor Activity/drug effects , Motor Activity/physiology , Neuroglia/cytology , Neuroglia/drug effects , Neuroglia/metabolism , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Neuropeptide Y/administration & dosage , Proto-Oncogene Proteins c-fos/metabolism , Pyrrolidines/pharmacology , Random Allocation , Septum of Brain/cytology , Septum of Brain/drug effectsABSTRACT
Previous studies identified SR-49059 as a most effective antagonist of the avian vasotocin 4 receptor (VT4R) compared to other candidate blockers including the Manning compound using in silico 3 dimensional (3D) modeling/docking analysis of the chicken VT4R and an in vitro anterior pituitary cell culture study. The present experiments were designed to validate whether SR-49059 and the Manning compound would likewise be effective in vivo in blocking the VT4R when applied intracerebroventricularly (ICV) to chicks. Two treatments were tested, a stressor (immobilization) and administration of neuropeptide Y (NPY), a potent orexigenic compound. In the first experiment, birds were given the Manning compound, SR-49059 or physiological saline ICV followed by immobilization stress. Blood samples were taken and corticosterone (CORT) was determined by radioimmunoassay. It was hypothesized that both antagonists would reduce the stress response. A second experiment examined the role of the VT4R in food intake regulation. The Manning compound, SR-49059 or physiological saline was administered prior to NPY and food intake was monitored for 1h. It was hypothesized that each of the two antagonists coupled with NPY would augment food intake above the intake resulting from saline plus NPY administration. Related to the second experiment was a third that examined the difference between the effect of central administration of NPY versus SR-49059 in releasing CORT. Results of the first study showed that the Manning compound or SR-49059 prior to stress decreased CORT levels compared to controls while the second experiment showed that SR-49059 or the Manning compound plus NPY, enhanced food intake above that of the experimental group given saline and NPY. The last study showed that NPY increased plasma CORT above birds given SR-49059 centrally or saline administered controls. Taken together, results suggest that the avian VT4R is involved in the central neuroendocrine stress response as well as functions in appetite regulation by mediating an anorexigenic effect similar to what has been reported in mammals for the V1aR. In conclusion, similar to the past in silico and in vitro tests, the current in vivo experiments showed SR-49059 to be a most efficacious avian vasotocin receptor antagonist. Therefore based upon results of functional tests utilizing a highly specific mammalian antagonist, SR-49059, to the mammalian V1aR that likewise was most effective in blocking the avian VT4R and past reported high sequence homology between the mammalian V1aR and the VT4R, it is recommended that the chicken VT4R be renamed the avian V1aR to facilitate better communication among scientists involved in comparative studies.