Kuenzel Lab

Department of Poultry Science

Examples of Immunohistochemistry in chick brains and anterior pituitaries

In vertebrates, two types of neurons regulate the neuroendocrine stress pathway. In birds the two neuronal types are 1) corticotropin releasing hormone (CRH) and 2) arginine vasotocin (AVT) neurons. The primary site of the neurons regulating stress is the hypothalamus, specifically the paraventricular nucleus (PVN; Fig. 1). CRH neurons are in red and AVT neurons are green and both types are found in the PVN. Each neuron type has been shown to have two receptors involved in stress. The peptide CRH released by CRH neurons binds to CRH receptor 1 and 2 (CRHR1 and CRHR2), while AVT binds to vasotocin receptors. In birds four distinct vasotocin receptors have been cloned and sequenced, however, the specific functions of the original 4 vasotocin receptors have not been elucidated. Mammalian studies demonstrated that the two main vasopressin receptors involved in stress were the vasopressin 1b (V1b) and vasopressin 1a (V1a) receptors. We therefore focused on finding the possible role of the vasotocin II (VT2) and IV (VT4) receptors due to sequence similarity between the mammalian V1b and V1a receptors, respectively. Our data showed that both the avian VT2 (Fig. 2B) and VT4 (Fig. 2C, 2D) receptors were located on corticotropes in the anterior pituitary (Fig. 2). Corticotropes are specialized cell types that produce the necessary pituitary hormone adrenocorticotropic hormone (ACTH) from its precursor pro-opiomelanocortin (POMC) that is transported from the anterior pituitary to the adrenal gland where the ultimate stress hormone, corticosterone (CORT) is produced and released into the vasculature. We reported that different stressors imposed upon birds resulted in significant changes in gene expression of AVT mRNA in the PVN, the VT2 and VT4 receptors in the anterior pituitary and elevated plasma CORT levels. Our neuroendocrine anatomical and physiological data support the original genomic sequence data that the avian VT2 and VT4 receptors are homologous to the mammalian V1b and V1a receptors, respectively. We therefore recommended that the old nomenclature be dropped and avian V1b and V1a replace their previous names in order to align better the avian names with mammalian terminology.

Current Nucleus Being Investigated is the Nucleus of the Hippocampal Commissure (NHpC)
The NHpC May Be Part of the Traditional Hypothalamo-Pituitary-Adrenal (HPA) Axis

Recently we found a nucleus, the nucleus of the hippocampal commissure (NHpC), located in the septum contains a large group of CRH neurons (Fig. 1). Note that the septum is a brain region separate from the hypothalamus. Currently we are examining the NHpC to determine whether or not the CRH neurons within that structure function along with the CRH and AVT neurons in the PVN to regulate
the stress pathway in birds. Our data show that septal CRH gene expression is activated early and displays peak activation prior to CRH peak expression in the PVN. We are currently examining the neural and cellular contents of the NHpC (Fig. 3) and its functional role with the avian hypothalamo-pituitary-adrenal (HPA) axis.

Fig. 1. The paraventricular nucleus (PVN) in the hypothalamus is the major nucleus regulating stress. It contains corticotropin releasing hormone (CRH, red) and arginine vasotocin (AVT, green) neurons (Nagarajan et al., 2017a). The nucleus of the hippocampal commissure (NHpC), located in the septal brain region, has a significant population the CRH neurons (red).

Fig. 2. A. Side view of the avian anterior pituitary gland showing the cephalic (Cp) and caudal (Cd) lobes. The Cp lobe contains corticotropes (Fig. A,C). The avian V1b receptor (Red) is found in the cell membrane of corticotropes (green) (Fig 2B). The avian V1a receptor (Red) is found both in the cytoplasm and cell membrane of corticotropes (red, Fig. D & E) (Kuenzel et al., 2013).
Fig. 3. Higher magnification of the NHpC nucleus in poultry. CRH neurons found in the dorsal region (Fig. A). The V1a receptor is located in glia (Fig B, green); AVT terminals are likewise identified (Fig. B, magenta) (Nagarajan et al., 2017b).