Optical study of secretion in mammalian nerve terminals

描述（由申请人提供）：分泌是最普遍的细胞过程之一。受调节的胞吐作用在信息处理、生殖、运动、温度调节、代谢、免疫应答和信号转导等多种生物学功能中起着重要作用，但其机制的阐明仍然是细胞生理学家面临的一个挑战。神经垂体是一个经典的体外制备，独特的相关研究肽类激素的诱发释放。来自下丘脑视上核和室旁核的大细胞神经元将其轴突投射为纤维束，穿过正中隆起和漏斗柄，以广泛地分支并终止于神经垂体，神经垂体是神经末梢的准纯集合，其中神经肽催产素和加压素被释放到循环中。尽管在识别大分子机械的释放所必需的组分方面取得了显着进展，但在真实的时间内直接观察电激发之后但在膜融合之前的终末内事件已被证明是极其困难的。为了确定这些步骤，我们开发了一个生物物理技术的武器库，拥有从微秒到分钟的时间尺度，重点放在前者。其中三个尤其构成了我们每个特定目标的核心：1）高带宽动态原子力显微镜（HBDAFM），用于研究快速但非常小（~1.0 º）的机械事件（“尖峰”和“下降”）哺乳动物神经末梢中伴随神经肽分泌的。2)实时检测整个末端以及近膜区域的Ca 2+瞬变，以识别Ca 2+进入和胞吐之间的中间步骤。3)分泌物本身的毫秒时间分辨光学检测。这些技术，已经在不同的发展阶段，在我们的实验室，将结合在这个建议，在一个单一的实验中，捕捉顺序，虽然部分重叠的事件，这是兴奋分泌耦合的重要组成部分。

DESCRIPTION (provided by applicant): Secretion is one of the most ubiquitous of cellular processes. Regulated exocytosis is central to such diverse biological functions as information processing, reproduction, motility, temperature regulation, metabolism, the immune response, and signal transduction, but the elucidation of its mechanism(s) remains a challenge to cell physiologists. The neurohypophysis is a classic in vitro preparation, uniquely relevant for studying evoked release of peptide hormones. Magnocellular neurons from the supraoptic and paraventricular nuclei in the hypothalamus project their axons as bundles of fibers through the median eminence and infundibular stalk to arborize extensively and terminate in the neurohypophysis, a quasi-pure collection of nerve terminals, where the neuropeptides oxytocin and vasopressin are released into the circulation. Despite remarkable progress in identifying components of the macromolecular machinery essential for their release, direct observation in real time of the intraterminal events that follow electrical excitation, but precede membrane fusion, has proven extremely difficult. To identify these steps we have developed an arsenal of biophysical techniques possessing time scales ranging from microseconds to minutes, with emphasis on the former. Three of them, in particular, constitute the core of each of our specific aims: 1) High Bandwidth Dynamic Atomic Force Microscopy (HBDAFM), to study the rapid, but very small (~1.0 ¿), mechanical events ("spike" and "dip") which accompany neuropeptide secretion in mammalian nerve terminals. 2) Real-time detection of Ca2+-transients, from whole terminals as well as from near- membrane regions, to identify intermediate steps between Ca2+-entry and exocytosis. 3) Millisecond time resolved optical detection of secretion per se. These technologies, already at different stages of development in our laboratory, will be combined in this proposal to capture, in a single experiment, sequential, albeit partially overlapping events, which are essential components of excitation-secretion coupling.