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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，作为国家卫生研究院的延伸授予#RO1 DK-37021，以确定可卡因和可卡因之间的解剖关系安非他明调节转录本(CART)和促垂性TRH神经元下丘脑室旁核。现建议与家长资助，这些研究将阐明CART在促垂体促肾上腺皮质激素释放激素的调节以及CART如何整合到作为瘦素作用的中介的中枢控制系统下丘脑-垂体-甲状腺轴。CART合成神经元的起源该投射到室旁核的TRH神经元将被识别通过一个两步的程序。首先，大脑中包含 CART合成神经元和投射到室旁核将是通过双标记免疫荧光技术鉴定逆行运输的标志物霍乱的立体定向注射毒素B亚单位(CTB)，分为室旁核的亚群。其次，通过共聚焦显微镜，将确定是否生产CART CART-IR神经元聚集CTB的每个区域的神经元，项目特别是对室旁核中的TRH神经元。这将是通过三重标记荧光技术实现，在该技术中顺次运输的标记物质、PHAL和CART将通过接触proTRH mRNA神经元的轴突终末的免疫荧光在室旁核中，并且proTRH mRNA将通过荧光、非同位素原位杂交组织化学。因为购物车是在脑室旁的大多数产生TRH的神经元中细胞核，一种双重标记的免疫荧光技术也将用于探讨CART对脑内TRH神经元的神经支配作用室旁核可能起源于超短反馈环促垂体神经元本身。CART和TRH的亚细胞组织将通过超微结构免疫细胞化学对这些神经元进行检查确定这两种物质是否包装在相同的囊泡中，以及他们的包装是否同样受到禁食的影响。最后，效果禁食和瘦素对禁食动物CART基因表达的影响在垂体视TRH神经元和CART产生神经元中，室旁核内含有TRH的神经元，将用原位杂交的同位素和非同位素联合技术组织化学和计算机图像分析。