Interaction between the hypothalamo-pituitary-adrenal and thyroid axes during immobilization stress.

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.

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.

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.

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.

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.