TRH REGULATION/BIOSYNTHESIS AND PARAVENTRICULAR NUCLEUS

这些研究的主要目的是阐明 促甲状腺激素释放激素(TRH)生物合成的调控 下丘脑室旁核内神经元的离散群 (PVN)对垂体前叶TSH的调节至关重要 并确定这种调节系统是如何被扰乱的 以低循环甲状腺为特征的疾病状态和疾病 激素和不适当的低TSH(病态正常甲状腺综合征)。它是 建议通过结合分子生物学的方法来实现这些目标 与经典神经解剖学技术和 光镜和超微结构水平的免疫细胞化学。甲状腺 促垂体激素释放激素神经元的激素受体亚型 在大脑其他区域的神经元中，将通过双倍- 用TRAlpha1特异性抗血清标记免疫细胞化学， 分别为TRbeta1、TRbeta2和TRR变异体TRalpha2和抗血清 与TRH前体有关。每种受体在调节反馈效应中的作用 甲状腺激素的含量将通过半定量原位进一步评估 手术后的杂交组织化学和计算机图像分析 用特异性、反义、硫代磷酸盐抑制TR的表达 寡核苷酸(S-寡聚核苷酸)。S的药效将是寡头 通过包括体外翻译在内的体外生物测定系统进行验证 并在注射到第三代细胞之前进行瞬时转染检测 脑室或与室旁核相邻。一种材料有可能 TRbeta2样免疫反应代表一种新的甲状腺激素受体 在促垂体素释放激素神经元中，将通过表达克隆 对其全长cDNA进行鉴定。TRs及其受体的亚细胞定位 与T3的关联将通过电子显微镜和 放射自显影，目的是提供解剖学证据 受体作为顺向转运蛋白在神经元突起中的作用。 血脑屏障与血脑脊液屏障在脑出血中的作用 对PVN中TRH生物合成的反馈调节的贡献将是 半定量原位杂交组织化学和图像分析 全身给药T4抑制运动后的分析 通过饱和转甲状腺素结合进入甲状腺功能低下动物的脑脊液 脑脊液中含有类黄酮的部位，EMD为21388。它们的作用机制 甲状腺激素对PVN内TRH神经元的正常反馈作用 被导致病态正常甲状腺综合征的疾病所取代， 将在禁食动物身上进行研究，并在中央给药后 炎性细胞因子(IL-1β、IL-6、TNFα)。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 调节这些反应的激素和激素受体浓度的变化 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.