CART AND THE HYPOTHALAMIC-PITUITARY-THYROID AXIS

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，以定义可卡因和 苯丙胺调节转录本（CART）和垂体促TRH神经元在 下丘脑室旁核建议结合 父母补助金，这些研究将阐明CART在 调节促垂体TRH，并确定CART如何整合到 中枢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 将确定投射到室旁核TRH神经元的 通过两步程序。首先，大脑中包含 CART合成神经元并投射到室旁核将被激活。 通过双标记免疫荧光技术鉴定， 立体定位注射逆行转运的标记物霍乱 毒素亚单位B（CT B），进入室旁核的亚单位。 第二，通过共聚焦显微镜，将确定是否CART产生 来自CART-IR神经元积累CTB的每个区域的神经元投射 尤其是室旁核的TRH神经元。这将是 通过三重标记荧光技术完成，其中 顺行转运的标记物、PHAL和CART将通过以下方法进行鉴别： 轴突终末接触含proTRH mRNA神经元的免疫荧光 在室旁核，和proTRH mRNA将被确定， 荧光、非同位素原位杂交组织化学。由于CART是 在室旁核的大多数TRH产生神经元中含有 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。 室旁核可能起源于一个超短反馈回路， 垂体神经元本身。CART和TRH的亚细胞结构 将通过超微结构免疫细胞化学检查这些神经元， 确定两种物质是否包装在相同的囊泡中，以及 它们的包装是否同样受到禁食的影响。最后，效果 禁食和瘦素给药对禁食动物CART基因表达的影响 在垂体后叶TRH神经元和CART产生神经元中， 室旁核中含TRH的神经元，将使用 同位素和非同位素原位杂交技术 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.