THE PRESYNAPTIC VESICLE CYCLE FOR SLOW ACTING NEUROTRANSMITTER SECRETION

大脑皮层和基底节的多巴胺(DA)释放与CMS功能的调节密切相关 和行为，并被认为是通过各种潜在的分泌部位发生的。其中包括轴突 小而透明的突触小泡呈突触前曲张状聚集的投射 典型的快速神经递质分泌终端。鉴于DA在更长的时间尺度上起作用 除了快速作用的神经递质，参与控制突触前机制的机制可能 这与那些更典型的“快速”突触有所不同。在这里，我们建议研究 这些多巴胺能释放部位的突触前囊泡周期。这项建议的长期目标是 描述小透明多巴胺能突触前囊泡周期的控制机制 水车。我们将利用实验室以前开发的技术来检查以下多个方面 皮层和海马区培养的突触前囊泡周期的分子和生物物理性质。 这些方法严重依赖使用外源有机探针的光学技术，如FM染料家族 成员以及突触前蛋白的基因编码标签，允许动态和定量 关于囊泡周期的信息将被获得。这些细胞将适应原代分离的细胞培养。 腹侧被盖区(VTA)的中脑神经元。我们提出了实现这一目标的三个具体目标 多巴胺能细胞生物学、生理学和生物物理方面的初步表征 囊泡循环。这些包括以耗尽率的顺序表征囊泡池的特性， 补给率，以及池周转对不同钙浓度刺激的敏感性 作为内吞作用的动力学。最后，我们将利用检测多巴胺成分的能力 直接使用碳纤维安培法检查囊泡周期的细节如何影响神经递质 放手。

Dopamine (DA) release in the cortex and basal ganglia is strongly implicated in modulation of CMS function and behavior and is thought to occur through a variety of potential secretory sites. Among these are axonal projections where small clear synaptic vesicles appear clustered in varicosities that resemble presynaptic terminals for typical fast-acting neurotransmitter secretion. Given that DA acts on much longer time scales than fast-acting neurotransmitters, the mechanism involved in controlling the presynaptic machinery may well be different than for those more typical "fast" synapses. Here we propose to examine details of the presynaptic vesicle cycle for these dopaminergic release sites. The long term objective of this proposal is to characterize the mechanism that control the presynaptic vesicle cycle for small clear dopaminergic veshicles. We will make use of technologies previously developed in the lab to examine many aspects of the molecular and biophysical nature of the presynaptic vesicle cycle in cortical and hippocampal cultures. These approaches rely heavily on optical techniques using exogenous organic probes such FM dye family members as well as genetically-encoded tags of presynaptic proteins that allow dynamic and quantitative information about the vesicle cycle to be obtained. These will be adapted to primary dissociated cell cultures of mid-brain neurons from the ventral tegmental area (VTA). We propose 3 specific aims to accomplish this initial characterization of the cell biological, physiological and biophysical aspects of the dopaminergic vesicle cycle. These include characterizing the properties of the vesicle pool in turns of depletion rates, replenishment rates, the sensitivity of pool turnover to stimulation at varied calcium concentrations, as well as the kinetics of endocytosis. Finally we will take advantage of the ability to detect dopamine sectretion directly using carbon-fiber amperometry to examine how details of the vesicle cycle impact neurotransmitter release.