TRH REGULATION/BIOSYNTHESIS AND PARAVENTRICULAR NUCLEUS

这些研究的主要目的是阐明 控制促甲状腺激素释放激素（TRH）的生物合成， 下丘脑室旁核的离散神经元群 (PVN)对调节垂体前叶促甲状腺激素至关重要 并确定这种调节系统是如何被扰乱的， 以循环甲状腺功能低下为特征的疾病状态和病症 激素和不适当低TSH（病态甲状腺功能正常综合征）。是 建议通过结合分子生物学方法来实现这些目标。 与经典神经解剖技术杂交， 免疫细胞化学光镜和超微结构观察。甲状腺 促垂体TRH神经元中的激素受体（TR）亚型以及 在大脑其他区域的神经元中， 使用针对TR α 1的特异性抗血清进行免疫细胞化学标记， TR β 1、TR β 2和TR变体TR α 2，以及抗血清 TRH前体。每个受体在介导反馈效应中的作用 甲状腺激素将进一步评估半定量原位 杂交组织化学和计算机图像分析 用特异性反义硫代磷酸酯抑制TR表达 寡脱氧核苷酸（S-oligos）。S-oligos的功效将是 通过体外生物测定系统（包括体外翻译）验证 和瞬时转染测定，然后注射到第三个细胞中。 心室或邻近PVN。一种材料， TRbeta2样免疫反应性代表一种新的甲状腺激素受体 在促垂体TRH神经元中的表达将通过表达克隆来解决， 鉴定其全长cDNA。TRs的亚细胞定位及其在细胞内的分布 将通过电子显微镜鉴定与T3的结合， 放射自显影术，目的是为 TRs在神经元过程中作为顺行转运蛋白的作用。 血脑屏障与血-CSF屏障在脑梗死中的作用 有助于反馈调节TRH生物合成的PVN将是 半定量原位杂交组织化学和图像分析 抑制全身施用的T4的运动后的分析 通过饱和甲状腺素运载蛋白结合进入甲状腺功能减退动物的CSF CSF中含有类黄酮EMD 21388的位点。的机制 甲状腺激素对室旁核TRH神经元的正常反馈作用 被导致病态甲状腺功能正常综合征的疾病所取代， 将在禁食动物中进行研究，并在中心给药后 炎性细胞因子（IL-1 β、IL-6、TNF α）。的作用 糖皮质激素，神经肽Y，生长抑素，促肾上腺皮质激素释放 激素和TR浓度变化介导这些反应 将使用类似的神经解剖学方法来确定。

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.