The main objectives of this study are to identify the hormones, peptides and neurotransmitters that regulate biosynthesis of TRH in the paraventricular nucleus (PVN) of the hypothalamus, the principal source of neurons that regulate anterior pituitary TSH secretion, and to elucidate their mechanisms of action. The factors responsible for the cell-specific feedback effects of thyroid hormone (TH) on proTRH mRNA in the PVN will be investigated by nuclear run-off assays to determine whether TH regulates the rate of transcription and/or stability of proTRH mRNA; quantitating the amount of (125I)T3 produced from (125I)T4 in the PVN to determine whether TH effects are dependent upon local 5'- monodeiodination of T4 or exerted directly by circulating levels of T3; and in situ hybridization and immunocytochemistry using c- erbA probes as markers of the TH receptor to determine if TH has direct or indirect effects on TRH-synthesizing neurons. The origin and identification of peptides and neurotransmitter substances which make synaptic contacts on TH-responsive PVN neurons will be determined by double immunolabeling techniques including anterograde tracing with PHA-L and immunocytochemistry at light and ultrastructural levels. The role of each of these factors (agonists and/or antagonists) in regulating TRH gene expression will be demonstrated in vivo after stereotaxic implantation unilaterally into the hypothalamus using semiquantitative in situ hybridization, immunocytochemistry and computer image analysis. Similar techniques will be used to determine whether the nyctohemeral variation of TSH secretion and changes in TH levels associated with glucocorticoids, cold exposure and caloric deprivation are mediated through effects on proTRH mRNA and whether TH establishes the permanent set point for TRH secretion at a critical time during development. The significance and functional regulation of another group of TRH-synthesizing. TH-unresponsive PVN neurons will also be determined by retrograde and anterograde tracing studies and immunocytochemistry to demonstrate the topography of innervation of the median eminence, its afferent input and neurotransmitter/neuropeptide mediators.
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