Optical study of secretion in mammalian nerve terminals

描述(申请人提供)：分泌物是最普遍的细胞过程之一。调控胞吐作用是信息处理、生殖、运动、体温调节、代谢、免疫反应和信号转导等多种生物学功能的核心，但其机制的阐明仍然是细胞生理学家面临的一个挑战(S)。神经垂体是一种经典的体外制剂，仅与研究多肽激素的诱发释放有关。来自下丘脑视上核和室旁核的大细胞神经元通过正中隆起和漏斗柄以纤维束的形式投射轴突，广泛分布并终止于神经垂体，神经垂体是神经末梢的准纯集合，在那里神经肽催产素和加压素被释放到血液循环中。尽管在识别其释放所必需的大分子机械组件方面取得了显著进展，但直接实时观察电刺激之后但在膜融合之前的终端内事件被证明是极其困难的。为了识别这些步骤，我们开发了生物物理技术武器库，具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)，研究哺乳动物神经末梢中伴随神经肽分泌的快速但非常小(~1.0？)的机械事件(“尖峰”和“凹陷”)。2)实时检测整个终末和近膜区域的钙瞬变，以识别钙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。这些技术在我们实验室已经处于不同的开发阶段，将在这项提议中结合起来，在一个单独的实验中捕获连续的、尽管部分重叠的事件，这些事件是兴奋-分泌耦合的基本组成部分。 与公共健康相关：神经垂体分泌的激素，催产素和加压素，涉及多种功能，如调节血压、水分平衡、排乳、社会认知、性行为和情绪控制。因此，例如，更深入地了解精氨酸加压素胞吐功能，可能会增强我们治疗高血压的能力。此外，由于许多神经疾病涉及神经递质释放的障碍，提高我们对其突触前成分的理解是开发新方法治疗突触传递障碍的关键，这些疾病涉及破坏性较大的疾病，如Lambert-Eaton综合征、精神分裂症、阿尔茨海默病和抑郁症。

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. PUBLIC HEALTH RELEVANCE: The hormones secreted by the neurohypophysis, oxytocin and vasopressin, are implicated in functions as diverse as blood pressure regulation, water balance, milk ejection, social cognition, sexual behavior, and mood control. Therefore, greater insight into arginine vasopressin exocytosis, for example, may enhance our ability to treat hypertension~ furthermore, since many neurological ailments involve an impairment of neurotransmitter release, advancing our understanding of its presynaptic component is key to the development of new approaches to the treatment of disorders of synaptic transmission implicated in ailments as devastating as, to name a few, Lambert-Eaton Syndrome, schizophrenia, Alzheimer's disease and depression.