SECRETION FROM INDIVIDUAL VESICLES

单个囊泡的分泌

• 批准号: 2881196
• 负责人: Robert Mark Wightman
• 金额: $ 25.26万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2881196
• 关键词: acidity /alkalinity; body fluid osmolarity; calcium flux; calcium indicator; calcium ion; dopamine; dopamine transporter; electrochemistry; exocytosis; fluorescent dye /probe; genetically modified animals; laboratory mouse; microelectrodes; neurotransmitter transport; synaptic vesicles; tissue /cell culture

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.

描述：（改编自申请人摘要）研究目标 在本申请中提出的计划是研究吸收和释放 神经递质和相关物质在单个细胞的水平。的 实验方法将是双重的：现有的神经化学工具将 用于进一步表征和理解这两个基本过程， 并将开发新的工具，使这种表征在单一的 细胞水平。该实验室开发了第一种检测胞吐的方法， 单个囊泡的分泌物。我们还开发了第一种技术， 观察多巴胺从大脑进入神经元的非常快的时间过程 细胞外液因此，我们在量化和 评估释放和吸收。技术和机械的结合 这里提出的方法应该导致一个更完整的观点神经元的手段， 通过多种物质调节神经传递。具体项目 将被调查的是： 表征单胺中储存和释放之间的相互作用 含有囊泡的。这将通过检查时间过程来完成， 胞吐作用后的单个分泌事件。 确定囊泡单胺转运蛋白（VMAT）在储存中的作用 然后释放这些实验将在遗传上 改变，使他们缺乏VMAT 2。 探讨细胞内Ca ~（2+）与释放的关系。这些 实验将在单电池水平上用电流分析法进行， 荧光染料来监测Ca 2+进入。 表征急性分离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一个清晰的看法。 将获得释放。 在单细胞水平表征多巴胺摄取。微量脉冲 技术将适用于这种应用。