CART AND THE HYPOTHALAMIC-PITUITARY-THYROID AXIS

购物车和下丘脑-垂体-甲状腺轴

基本信息

批准号：
6499501
负责人：
RONALD Michael LECHAN
金额：
$ 3.65万
依托单位：
TUFTS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-03-07 至 2004-01-31
项目状态：
已结题

项目摘要

DESCRIPTION This collaborative study will be performed in Hungary as an extension of NIH grant #RO1 DK-37021, to define the anatomical relationships between cocaine and amphetamine-regulated transcript (CART) and hypophysiotropic TRH neurons in the hypothalamic paraventricular nucleus. It is proposed that in conjunction with the parent grant, these studies will elucidate the role of CART in the regulation of hypophysiotropic TRH and determine how CART is integrated into the central control system as a mediator for the action of leptin on the hypothalamic-pituitary-thyroid axis. The origin of CART-synthesizing neurons that project to TRH neurons in the paraventricular nucleus will be identified by a two-step procedure. First, regions of the brain that contain CART-synthesizing neurons and project to the paraventricular nucleus will be identified by a double-labeling immunofluorescent technique following the stereotaxic injection of the retrogradely transported marker substance, cholera toxin subunit B (CTB), into subdivisions of the paraventricular nucleus. Second, by confocal microscopy, it will be determined whether CART-producing neurons from each of the regions where CART-IR neurons accumulate CTB, project specifically to TRH neurons in the paraventricular nucleus. This will be accomplished by a triple-labeling fluorescent technique in which the anterogradely transported marker substance, PHAL and CART will be identified by immunofluorescence in axon terminals contacting proTRH mRNA-containing neurons in the paraventricular nucleus, and proTRH mRNA will be identified by fluorescent, non-isotopic in situ hybridization histochemistry. Since CART is contained in the majority of TRH-producing neurons in the paraventricular nucleus, a double-labeling immunofluorescent technique will also be used to explore the possibility that the CART innervation to TRH neurons in the paraventricular nucleus may arise from an ultrashort feedback loop from hypophysiotropic neurons, itself. The subcellular organization of CART and TRH in these neurons will be examined by ultrastructural immunocytochemistry to determine whether both substances are packaged in the same vesicles, and whether their packaging is similarly affected by fasting. Finally, the effect of fasting and leptin administration to fasting animals on CART gene expression in hypophysitoropic TRH neurons and in CART-producing neurons that project to TRH-containing neurons in the paraventricular nucleus, will be studied using combined isotopic and non-isotopic techniques of in situ hybridization histochemistry and computerized image analysis.

描述这项合作研究将在匈牙利进行，作为 NIH 的延伸授予#RO1 DK-37021，定义可卡因和安非他明调节转录本 (CART) 和促垂体 TRH 神经元下丘脑室旁核。建议结合在家长资助下，这些研究将阐明 CART 在调节促垂体 TRH 并确定 CART 如何整合到中央控制系统作为瘦素作用的中介下丘脑-垂体-甲状腺轴。 CART 合成神经元的起源投射到室旁核 TRH 神经元的神经元将被识别通过两步程序。首先，大脑中包含的区域 CART 合成神经元并投射到室旁核将通过双标记免疫荧光技术进行鉴定立体定向注射逆行运输的标记物质霍乱毒素亚基 B (CTB)，分为室旁核的细分部分。其次，通过共聚焦显微镜，将确定是否产生 CART 来自 CART-IR 神经元积累 CTB 的每个区域的神经元，投射特别针对室旁核中的 TRH 神经元。这将是通过三重标记荧光技术完成，其中顺行运输的标记物质，PHAL和CART将被识别接触含有 proTRH mRNA 的神经元的轴突末端的免疫荧光在室旁核中，proTRH mRNA 将被识别荧光、非同位素原位杂交组织化学。由于 CART 是包含在室旁大部分产生TRH的神经元中细胞核，双标记免疫荧光技术也将用于探讨 CART 神经支配 TRH 神经元的可能性室旁核可能由超短反馈回路产生促生理神经元本身。 CART 和 TRH 的亚细胞组织这些神经元将通过超微结构免疫细胞化学进行检查确定两种物质是否包装在同一个囊泡中，并且他们的包装是否同样受到禁食的影响。最后是效果禁食和瘦素给药对禁食动物对 CART 基因表达的影响在垂体功能低下的 TRH 神经元和产生 CART 的神经元中，这些神经元投射到室旁核中含有 TRH 的神经元将使用以下方法进行研究原位杂交同位素和非同位素组合技术组织化学和计算机图像分析。