Mechanisms for somatodendritic dopamine release in the midbrain

摘要 许多中枢神经元释放神经调节递质，例如单胺、神经肽、 以及神经营养因子，来自它们的体细胞和树突。这些递质的释放之后是G-蛋白 偶联受体激活，而这些神经调节通路对大脑发育和 功能。腹侧中脑中的多巴胺信号是这种传递方式的一个突出例子，它是 对于应对药物滥用尤为重要。 对躯体树突状细胞释放和G蛋白偶联受体介导的信息传递的认识还很滞后 它落后于突触信号，但通常被认为是缓慢和不精确的。这样做的长期目标是 该项目是为了确定介导躯体树突状细胞多巴胺传递的分子机制。 我们假设躯体树突状细胞的多巴胺分泌是由机械专门化的机械来调节的。 用于快速和同步释放。我们建议将这台机器组装成发布热点，以产生 针对靶细胞上G蛋白偶联受体结构域的快速动力学信号，以及 结构非常适合于强健的受体激活。我们最近的工作在这方面取得了初步进展 进球。我们发现，RIM是一种对轴突递质释放的时空精度很重要的蛋白质， 是诱发的躯体树突状多巴胺释放所必需的。此外，我们还发现，突触素-1，一种快速的 CA2感受器，介导钙离子触发这种形式的释放。 我们在此建议确定躯体树突状细胞多巴胺释放机制的组织和功能 采用条件性小鼠基因敲除、电生理学、成像和超分辨显微镜。在目标1中， 我们建议解剖中脑多巴胺神经元的躯体树突状释放部位结构。我们会 系统检测控制速度和速度的五个关键活性区带蛋白家族的必要性和局限性 经典突触的胞吐作用位置。在目标2中，我们建议确定钙来源和传感器 躯体树突状细胞释放多巴胺。我们将评估缺乏钙通道或钙离子的条件性小鼠突变 分泌缺陷的传感器蛋白质，并将评估钙触发所需蛋白质的定位 在躯体树突间。这些目标将定义钙离子的触发机制，并可能 确定类似活动区域的释放热点。我们的工作将进一步揭示共享和独特的释放机制 在多巴胺神经元的躯体树突和轴突内。 这个多PI项目将促进对中脑中躯体树突状细胞多巴胺信号的理解 具体地说，以及G蛋白偶联受体介导的一般传递。它结合了 约翰·威廉姆斯和帕斯卡·凯瑟尔的实验室。对这些机制的深入研究将使我们能够 得出这些神经元分泌途径的原理和规格，并最终可能有助于 治疗多巴胺功能紊乱的疾病，例如吸毒成瘾。

Summary Many central neurons release neuromodulatory transmitters, for example monoamines, neuropeptides, and neurotrophins, from their somata and dendrites. Release of these transmitters is followed by G-protein coupled receptor activation, and these neuromodulator pathways are essential for brain development and function. Dopamine signaling in the ventral midbrain is a prominent example of this transmission mode, and it is particularly important for the response to drugs of abuse. The knowledge of somatodendritic release and G-protein coupled receptor-mediated transmission lags far behind that of synaptic signaling, but it is often considered slow and imprecise. The long-term goal of this project is to determine the molecular mechanisms that mediate somatodendritic dopamine transmission. We hypothesize that somatodendritic dopamine secretion is mediated by mechanistically specialized machinery for fast and synchronous release. We propose that this machinery is assembled into release hotspots to generate a signal with rapid kinetics that is directed towards G-protein coupled receptor domains on target cells, an architecture ideally suited for robust receptor activation. Our recent work has made first progress towards this goal. We have found that RIM, a protein important for the spatiotemporal precision of axonal transmitter release, is essential for evoked somatodendritic dopamine release. Furthermore, we found that synaptotagmin-1, a fast Ca2+ sensor, mediates Ca2+-triggering of this form of release. We here propose to determine the organization and function of somatodendritic dopamine release machinery using conditional mouse gene knockout, electrophysiology, imaging and superresolution microscopy. In aim 1, we propose to dissect somatodendritic release site architecture in midbrain dopamine neurons. We will systematically test the necessity and localization of five key active zone protein families that control speed and location of exocytosis at classical synapses. In aim 2, we propose to identify Ca2+ sources and sensors for somatodendritic dopamine release. We will assess conditional mouse mutants that lack Ca2+ channel or Ca2+ sensor proteins for secretory deficits and will assess the localization of the proteins needed for Ca2+-triggering in the somatodendritic compartments. These aims will define mechanisms of Ca2+-triggering and are likely to identify active zone-like release hotspots. Our work will further reveal shared and distinct release mechanisms in somatodendritic and axonal compartments of dopamine neurons. This multi-PI project will advance the understanding of somatodendritic dopamine signaling in the midbrain specifically, and of G-protein coupled receptor-mediated transmission in general. It combines the expertise of the laboratories of John Williams and Pascal Kaeser. In-depth studies of these mechanisms will allow us to derive principles and specifications of these neuronal secretory pathways and may ultimately help advancing treatments for diseases with disrupted dopamine function, for example drug addiction.