SECRETION FROM INDIVIDUAL VESICLES

描述：(改编自申请人的摘要)研究的目标 本申请中提出的计划是研究摄取和释放 单个细胞水平的神经递质和相关物质。这个 实验方法将是双重的：现有的神经化学工具将是 用来进一步描述和理解这两个基本过程， 并将开发新的工具，允许在单个 细胞水平。这个实验室开发了第一种检测胞吐的方法。 单个小泡的分泌物。我们还开发了第一项技术来 观察从大脑向神经元摄取多巴胺的非常快的时间过程 细胞外液。因此，我们有相当多的专业知识来量化和 评估释放和摄取。技术与机械相结合 这里提出的方法应该会导致对神经细胞手段的更全面的看法 通过多种物质调节神经传递。具体项目 将接受调查的有： 表征单胺的储存和释放之间的相互作用 含有囊泡的。这将通过检查时间进程来完成 胞吐后的个别分泌事件。 确定囊泡单胺转运体(VMAT)在储存中的作用 然后释放。这些实验将在具有遗传基因的小鼠身上进行。 修改后，它们缺少VMAT2。 探讨细胞内钙离子与释放的关系。这些 实验将在单细胞水平上用安培法和 用荧光染料监测钙离子内流。 以表征急性分离的多巴胺能神经元的释放。从… 这样的原代培养，对影响胞吐的因素有清晰的认识 将获得释放。 在单细胞水平上表征多巴胺的摄取。微量采样 技术将适用于这一应用。

DESCRIPTION: (Adapted from the Applicant's Abstract) The goal of the research plan set forth in this application is to investigate uptake and release of neurotransmitters and related substances at the level of individual cells. The experimental approach will be two-fold: existing neurochemical tools will be used to further characterize and understand these two fundamental processes, and new tools will be developed that allow such characterization at the single cell level. This laboratory developed the first method to examine exocytotic secretion from single vesicles. We also developed the first technique to observe the very rapid time course of dopamine uptake into neurons from brain extracellular fluid. Thus, we have considerable expertise in quantifying and evaluating both release and uptake. The combined technological and mechanistic approach proposed here should lead to a more complete view of neuronal means of regulating neurotransmission by a variety of substances. The specific projects that will be investigated are: To characterize the interplay between storage and release in monoamine containing vesicles. This will be done by examining the time course of individual secretory events following exocytosis. To determine the role of the vesicular monoamine transporter (VMAT) on storage and release. These experiments will be done in mice that have been genetically altered so that they lack VMAT2. To characterize the relationship between intracellular Ca2+ and release. These experiments will be done at the single cell level with amperometry and fluorescent dyes to monitor Ca2+ entry. To characterize release from an acutely dissociated dopaminergic neuron. From such primary cultures, a clear view on the factors that affect exocytotic release will be obtained. To characterize dopamine uptake at the single cell level. Microsampling techniques will be adapted for this application.