A Molecular Dynamics Approach to GnRH Pulsatility

描述（由申请方提供）：下丘脑脉冲性释放促性腺激素释放激素（GnRH）是正常生殖功能所必需的。尽管它的重要性，非常少的信息是在细胞的基础上，这种现象，因为在研究中存在于下丘脑组织中的少数和分散的GnRH神经元的困难。最近开发的GT 1克隆神经元培养物，释放GnRH在脉动的方式提供了一个很好的模型系统来研究这一过程。我们在上一个资助期的努力是针对开发一系列的工具，可以与这些细胞一起使用，开始阐明有助于脉冲式GnRH释放的细胞成分。在这方面，我们开发的工具，使用启动子驱动的荧光素酶报告基因表达的测量，fura-2的细胞内钙变化的鉴定，和FM 1 -43的胞吐功能的评估。这些工具的独特之处在于（无论是单独应用还是按顺序应用），每个工具都被设计为仅测量单个细胞的活性并实时测量。使用这种方法，我们发现GT 1 -7细胞中的偶发性活动不仅限于分泌，而且钙变化和基因表达也发生在脉冲中。但更重要的是，我们的研究结果表明，GnRH的偶发性活动是由两个细胞过程控制的;单个神经元的内在脉动功能和多个神经元的协调，以实现不同的集体释放脉冲。这些发现提供了第一个实验证据，证明下丘脑神经元可能在间歇性GnRH释放中发挥关键作用。我们认为，研究这一作用的最有效手段是分别侧重于其中每一个组成部分。因此，我们的具体目标是：1）确定单个GnRH神经元间歇性活动的细胞和分子机制; 2）阐明GnRH神经元细胞同步化的过程。旨在实现这些目标的实验应提供重要的洞察力的原则，管理GnRH脉动的细胞基础。这些原则可能是非常宝贵的，在设计未来的治疗策略，可用于纠正生殖功能障碍所造成的异常GnRH释放。

DESCRIPTION (provided by applicant): Pulsatile release of gonadotropin-releasing hormone (GnRH) from the hypothalamus is necessary for proper reproductive function. Despite its importance, very little information is available on the cellular basis for this phenomenon because of the difficulty in studying the few and scattered GnRH neurons present in hypothalamic tissue. Recent development of GT1 clonal neuronal cultures that release GnRH in a pulsatile manner provided an excellent model system to study this process. Our efforts during the last grant period were directed toward developing a series of tools that could be used in conjunction with these cells to begin to elucidate the cellular components that contributed to pulsatile GnRH release. In this regard, we developed tools using a promoter-driven luciferase reporter for measurement of gene expression, fura-2 for identification of intracellular calcium changes, and FM1-43 for assessment of exocytotic function. These tools were unique in that (whether applied singly or in sequence) each was designed to measure activity only individual cells and in real-time. Using this approach, we found that episodic activity in GT1-7 cells was not just restricted to secretion, but that calcium changes and gene expression also occurred in pulses. More importantly though, our findings indicated that GnRH episodic activity was governed by two cell processes; an intrinsic pulsatile function of individual neurons and a coordination of multiple neurons to achieve distinct collective pulses of release. These findings provided the first experimental evidence of a process by which hypothalamic neurons may achieve the critical role of episodic GnRH release. We believe that the most effective means to study this role is by focusing individually on each of these components. Thus, our specific aims in this proposal are 1) to determine what cellular and molecular mechanisms underlie intermittent activity in individual GnRH neurons and 2) to elucidate the processes underlying GnRH neuronal cell synchronization. Experiments directed toward achieving these aims should provide important insight in to the principles governing the cellular basis for GnRH pulsatility. These principles may be invaluable in devising future therapeutic strategies that can be used to correct reproductive disfunction resulting from abnormal GnRH release.