Mechanisms for somatodendritic dopamine release in the midbrain

总结 许多中枢神经元释放神经调节递质，例如单胺，神经肽， 和神经营养素。这些递质的释放之后是G蛋白 偶联受体激活，这些神经调节剂通路对大脑发育至关重要， 功能中脑腹侧的多巴胺信号是这种传输模式的一个突出例子， 对应对药物滥用问题尤为重要。 对体树突释放和G蛋白偶联受体介导的传递的认识远远滞后 在突触信号传导之后，但它通常被认为是缓慢和不精确的。长期目标是 项目是确定介导体树突多巴胺传递的分子机制。 我们假设体树突多巴胺的分泌是由机械性的专门机制介导的 用于快速同步释放。我们建议，这种机制组装成释放热点，以产生 具有快速动力学的信号，其指向靶细胞上的G蛋白偶联受体结构域， 结构非常适合于强大的受体激活。我们最近的工作在这方面取得了初步进展 目标.我们已经发现，RIM是一种对轴突递质释放的时空精确性很重要的蛋白质， 对诱发的体树突多巴胺释放至关重要。此外，我们发现，突触结合蛋白-1，一种快速的 Ca 2+传感器介导这种形式的释放的Ca 2+触发。 我们在这里提出，以确定组织和功能的体树突多巴胺释放机制， 使用条件小鼠基因敲除、电生理学、成像和超分辨率显微镜。在目标1中， 我们建议解剖中脑多巴胺神经元的体树突释放位点结构。我们将 系统地测试控制速度的五个关键活性区蛋白家族的必要性和定位， 胞吐作用在典型突触的位置。在目标2中，我们建议确定Ca 2+源和传感器， 体树突多巴胺释放。我们将评估缺乏Ca 2+通道或Ca 2+通道的条件性小鼠突变体。 分泌缺陷的传感器蛋白，并将评估Ca 2+触发所需蛋白的定位 在体树突区室中。这些目标将定义Ca 2+触发机制，并可能 识别类似活动区释放热点。我们的工作将进一步揭示共享和不同的释放机制 多巴胺神经元的体树突和轴突隔室。 这个多PI项目将推进对中脑体树突多巴胺信号传导的理解 具体地，和一般的G蛋白偶联受体介导的传递。它结合了 约翰威廉姆斯和帕斯卡尔凯瑟的实验室。深入研究这些机制将使我们能够 推导出这些神经元分泌途径的原理和规格，并可能最终有助于推进 治疗多巴胺功能受损的疾病，例如药物成瘾。

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.