The main objectives of these studies are to elucidate the mechanisms of control of the biosynthesis of thyrotropin-releasing hormone (TRH) in a discrete population of neurons in the hypothalamic paraventricular nucleus (PVN) that is critical for the regulation of anterior pituitary TSH secretion and to determine how this regulatory system is perturbed in disease states and disorders characterized by lows circulating thyroid hormone and inappropriately low TSH (sick euthyroid syndrome). It is proposed to accomplish these goals by combining methods of molecular hybridization with classical neuroanatomical techniques and immunocytochemistry at the light and ultrastructral levels. The thyroid hormone receptor (TR)subtypes in hypophysiotropic TRH neurons as well as in neurons in other regions of the brains will be identified by double- labeling immunocytochemistry using specific antiseria to TRalpha1, TRbeta1, TRbeta2, and the TR variant, TRalpha2, respectively and antiserum to the TRH precursor. The role each receptor in mediating feedback effects of thyroid hormone will be further assessed by semiquanitative in situ hybridization histochemistry and computerized image analysis after inhibiting TR expression with specific, antisense, phosphorothioate oligodeoxynucleotides (S-oligos). The efficacy of S-oligos will be validated by in in vitro bioassay systems including in vitro translation and transient transfection assays before injection into the third ventricle or adjacent to the PVN. The possibility that a material with TRbeta2-like immunoreactivity represents a novel thyroid hormone receptor in hypophysiotropic TRH neurons will be addressed by expression cloning to identify its full length cDNA. The subcellular location of TRs and their association with T3 will be identified by electron microscopy and autoradiography with the intent of providing anatomical evidence for a role of TRs as anterogradely transported proteins in neuronal processes. The role of the blood-brain barrier versus the blood-CSF barrier in contributing to feedback regulation of TRH biosynthesis in the PVN will be assessed by semiquanitative in situ hybridization histochemistry and image analysis after inhibiting the movement of systemically administered T4 into the CSF of hypothyroid animals by saturating transthyretin binding sites in the CSF with the flavonoid, EMD 21388. The mechanisms by which the normal feedback effects of thyroid hormone on TRH neurons in the PVN is superseded by conditions that result in the sick euthyroid syndrome, will be studied in fasted animals and following central administration of inflammatory cytokines (Il-1beta, IL-6, TNFalpha). The role of glucocorticoids, neuropeptide-Y, somatostatin, corticotropin-releasing hormone and alternations in TR concentration in mediating these responses will be determined using similar neuroanatomic methods.
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