Architecture and function of striatal dopamine release machinery

总结 多巴胺是一种重要的神经调质，多巴胺信号传导中的病理是脑损伤的标志。 神经变性、药物滥用和精神分裂症等疾病。尽管这些重要的角色， 对于多巴胺，人们对其释放的分子机制知之甚少。因为多巴胺 作为一种音量传递器，尚不清楚多巴胺的释放是否涉及保证 释放的空间和时间精度。或者，多巴胺的释放可以扩散到大脑皮层的表面， 一个轴突，这与体积传输相一致经典发射器的释放依赖于一种活性的 区，一种高度组织化的蛋白质结构，含有支架蛋白，如RIM和ELKS， 决定了突触囊泡胞吐作用的精确定位、速度和准确性。活动区还 提供了可塑性过程中释放的调节机制。我们的初步实验表明， 突触前支架蛋白RIM是小鼠纹状体中多巴胺释放所必需的，但是， ELKS是多巴胺释放的标志。这与经典的快速突触不同，其中敲除 任一蛋白质家族导致释放减少50-80%。因此我们假设多巴胺的释放 需要机械上专门的释放场所。这一假设得到了超分辨率的支持 在纹状体脑切片的显微镜下，这表明几个释放位点的支架蛋白聚集在一起， 在多巴胺轴突中我们采取双管齐下的方法来解决这个中心假设。目标一，我们 在小鼠纹状体的急性脑切片中使用严格的条件小鼠遗传学和电生理学， 系统地阐述了支架蛋白、引发蛋白和Ca 2+通道栓系在 多巴胺释放以及GABA和谷氨酸从多巴胺神经元的共释放。这是第一个研究 多巴胺分泌的分子支架的要求，它将导致一个全面的评估 释放多巴胺的机制在目标二中，我们评估支架蛋白是否介导多巴胺 分泌作为可溶性释放因子，或者它们是否组装在簇状释放位点以靶向 多巴胺释放到特定的膜区域。后一种可能性得到了我们初步的有力支持。 数据我们将联合收割机、超分辨显微镜、亚细胞分离、电子显微镜和小鼠 遗传学研究的存在和组成的多巴胺释放位点在小鼠纹状体。我们将 评估多巴胺释放位点如何与囊泡簇、多巴胺受体和 共同递质GABA和谷氨酸，并与胆碱能神经支配，这有力地触发多巴胺 release.这些实验将建立多巴胺释放的存在、外观和组成 位点和它们的结构排列成纹状体突触微电路。我们的方法是第一个 全面的方法来剖析多巴胺的分泌途径。我们希望能发现新的 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.