Date of Graduation

5-2012

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Biological Sciences

Advisor

Wayne J. Kuenzel

Committee Member

Suresh K. Thallapuranam

Second Committee Member

Narayan C. Rath

Third Committee Member

Alexander Jurkevic

Keywords

Biological sciences; Chickens; Circumventricular organs; Glial cells; Pituitary; Stress; Vasotocin receptors

Abstract

The present study investigated the distribution of the vasotocin subtype four receptor (VT4R) in brain and pituitary gland of the chicken, Gallus gallus. The anterior pituitary cell types associated with the VT4R were also determined. Two polyclonal antibodies were raised in rabbit against a cocktail of peptides, 15 amino acids from the amino terminal region and 17 amino acids from the carboxy terminal region of VT4R receptor. The antibody was validated utilizing the Western blot and immunocytochemistry. A single band at 47KDa utilizing membrane protein extracts of chicken brain and pituitary tissues was shown for the VT4R antibody. A peptide preadsorption or blocking experiment was performed to confirm the specificity of the antibody to the VT4R. Second, the VT4R distribution in brain and pituitary were studied using immunocytochemistry. The VT4R antibody immunostained both neuronal and non-neuronal elements present in the brain. The VT4R was widely distributed throughout the brain. Intense cell body staining was observed in the following neural structures: supraoptic nucleus, paraventricular nucleus, periventricular hypothalamic nucleus, Edinger Westphal and other oculomotor nuclei, nucleus tractus solitarius, hypoglossal nucleus, trigeminal nucleus, sensory nucleus of the trigeminal nerve, nucleus of the abducens nerve, nucleus supraspinalis and magnocellular cochlear nucleus. Intense immunoreactivity of the VT4R in glial cells was evident in circumventricular organs. Radial glial cells in hippocampus, astrocytes in optic tectum and tanycytes in median eminence were prominently immunostained with the VT4R. In addition, the regional distribution of the VT4R was determined in the pituitary gland of the chicken. The VT4R was located in the cephalic lobe of the anterior pituitary. The phenotype of pituitary cells was revealed using dual labelled immunofluorescence microscopy. The VT4R immunoreactive cells were located in both corticotropes and gonadotropes. About 89% and 12% of corticotropes and luteinizing hormone (LH) containing gonadotropes, respectively, displayed the VT4R. The VT4R was not detected in lactotropes nor somatotropes. Based on its neuroanatomical distribution in brain and colocalization studies of the VT4R in anterior pituitary, the proposed functions of the VT4R was suggested to be associated with stress responses, maintenance of homeostasis and lutenizing hormone release from a subgroup of pituitary gonadotropes. The receptor could also have a plausible role in auditory function and motor regulation of the eye based upon intense immunoreactivity of neurons associated with the fifth and third cranial nerves, respectively.

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