CART AND THE HYPOTHALAMIC-PITUITARY-THYROID AXIS

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。

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.