Architecture and function of striatal dopamine release machinery

摘要 多巴胺是一种重要的神经调节剂，而多巴胺信号转导的病理过程是大脑的标志 神经变性、药物滥用和精神分裂症等疾病。尽管这些角色对 值得注意的是，人们对多巴胺释放的分子机制知之甚少。因为多巴胺起作用 作为一种体积递质，目前尚不清楚多巴胺的释放是否涉及到保证 释放的空间和时间精度。另一种选择是，多巴胺的释放可以散布在 轴突，与体积传递相一致。经典发射器的释放依赖于一个活动的 区域，一种高度组织化的蛋白质结构，包含支架蛋白，如RIM和ELKS和 决定了突触囊泡胞吐的精确定位、速度和准确性。活动区还 提供在可塑性过程中调节释放的机制。我们的初步实验表明， 突触前支架蛋白RIM是小鼠纹状体释放多巴胺所必需的，但 ELKs对于多巴胺的释放是必不可少的。这与经典的快速突触不同，在经典的快速突触中 任何一种蛋白质家族都会导致50%-80%的释放减少。因此我们假设多巴胺的释放 这就需要专门的机械释放场所。这一假说得到了超分辨率的支持 纹状体脑片的显微镜观察显示，几个释放部位的支架蛋白聚集在一起 在多巴胺轴突内。我们采取双管齐下的方法来解决这一核心假设。在目标一号中，我们 在小鼠急性纹状体脑片中使用严格的条件小鼠遗传学和电生理学来 系统地阐述了支架蛋白、启动蛋白和钙离子通道纽带的必要性 多巴胺的释放以及多巴胺神经元中GABA和谷氨酸的共同释放。这是第一次关于 分子支架对多巴胺分泌的要求，并将导致全面评估 多巴胺释放机制。在第二个目标中，我们评估支架蛋白是否介导多巴胺。 分泌物作为可溶性释放因子，或者它们是否聚集在聚集的释放部位来靶向 多巴胺释放到特定的膜区。后一种可能性得到了我们初步的支持 数据。我们将结合超分辨显微镜、亚细胞分离、电子显微镜和小鼠 遗传学研究小鼠纹状体中多巴胺释放部位的存在和组成。我们会 评估多巴胺释放部位如何与囊泡团、多巴胺受体和 共同的递质GABA和谷氨酸，与胆碱能神经支配，有力地触发多巴胺 放手。这些实验将确定多巴胺释放的存在、外观和组成 纹状体突触微环路中的位置及其结构排列。我们的方法是第一个 剖析多巴胺分泌途径的综合方法。我们希望能找到新的 支持多巴胺释放的机制，并揭示神经调节的一般原理。

Summary Dopamine is an important neuromodulator and pathologies in dopamine signaling are a hallmark of brain diseases such as neurodegeneration, substance abuse, and schizophrenia. Despite these important roles for dopamine, remarkably little is known about the molecular mechanisms of its release. Because dopamine acts as a volume transmitter, it is not clear whether dopamine release involves molecular machinery that warrants spatial and temporal precision for release. Alternatively, dopamine release could be spread over the surface of an axon, which is consistent with volume transmission. The release of classical transmitters relies on an active zone, a highly organized protein structure that contains scaffolding proteins such as RIM and ELKS and determines the precise localization, speed and accuracy of synaptic vesicle exocytosis. The active zone also provides mechanisms for regulation of release during plasticity. Our preliminary experiments reveal that the presynaptic scaffolding protein RIM is absolutely required for dopamine release in the mouse striatum, but that ELKS is dispensable for dopamine release. This is different from classical fast synapses, where knockout of either protein family leads to a reduction of 50-80% of release. We thus hypothesize that dopamine release necessitates mechanistically specialized release sites. This hypothesis is bolstered by superresolution microscopy in striatal brain slices, which shows that several release site scaffolding proteins are clustered inside dopamine axons. We pursue a two-pronged approach to address this central hypothesis. In aim one, we use rigorous conditional mouse genetics and electrophysiology in acute brain slices of the mouse striatum to systematically address the necessity of scaffolding proteins, priming proteins and Ca2+ channel tethers in dopamine release and in co-release of GABA and glutamate from dopamine neurons. This is the first study on the requirements of molecular scaffolds for dopamine secretion and it will lead to a comprehensive assessment of the dopamine release machinery. In aim two, we assess whether scaffolding proteins mediate dopamine secretion as soluble release factors, or whether they are assembled in clustered release sites to target dopamine release to specific membrane domains. The latter possibility is strongly supported by our preliminary data. We will combine superresolution microscopy, subcellular fractionation, electron microscopy and mouse genetics to study the existence and composition of dopamine release sites in the mouse striatum. We will assess how dopamine release sites are associated with vesicle clusters, with receptors for dopamine and for the co-transmitters GABA and glutamate, and with cholinergic innervation, which powerfully triggers dopamine release. These experiments will establish the existence, appearance and composition of dopamine release sites and their structural arrangement into striatal synaptic microcircuits. Our approach is the first comprehensive approach to dissect the secretory pathway for dopamine. We expect to identify new mechanisms that support dopamine release and to uncover general principles for neuromodulation.