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：
6629368
负责人：
RONALD Michael LECHAN
金额：
$ 3.66万
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 2005-01-31
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的扩展授予＃RO1 DK-37021，以定义可卡因与可卡因之间的解剖学关系苯丙胺调节的转录本（CART）和下生理TRH神经元下丘脑室室核。有人提出与父母的授予，这些研究将阐明购物车在调节下生理TRH并确定推车如何整合到中央控制系统是瘦素在下丘脑 - 垂体 - 甲状腺轴。卡特合成神经元的起源将鉴定出对室室中TRH神经元的项目通过两个步骤。首先，包含大脑的区域卡车合成的神经元和向室室核的项目将是通过双重标记的免疫荧光技术鉴定立体定位注射逆转录的标记物质，霍乱毒素亚基B（CTB）进入阶室核的细分。其次，通过共聚焦显微镜，将确定是否生产购物车来自Cart-IR神经元积聚CTB的每个区域的神经元，项目专门针对椎间盘核中的TRH神经元。这将是通过三标签荧光技术完成直接运输的标记物质，phal和Cart将通过接触含有肌肉mRNA的神经元的轴突末端的免疫荧光在室室核中，将通过荧光，非同位素原位杂交组织化学。因为购物车是大多数产生TRH的神经元中包含核，双标记免疫荧光技术也将用于探索推车对TRH神经元的神经支配的可能性室室核可能是由来自下生理神经元本身。购物车和TRH的亚细胞组织在这些神经元中，将通过超微结构免疫细胞化学进行检查确定是否将两种物质都包装在相同的囊泡中，并且它们的包装是否受到禁食的影响。最后，效果在卡特基因表达上禁食和瘦素给予禁食动物的在降低主题为TRH神经元和产生推车的神经元中将使用二室核中含TRH的神经元进行研究原位杂交的同位素和非同位素技术组合组织化学和计算机图像分析。