Mechanisms of Vesicle Docking and Priming for Striatal Dopamine Release

神经系统中的信号传递依赖于化学信号从一个神经元到下一个神经元的转移。那里 这些信号主要有两类：快速神经递质和神经调节剂。快速神经递质是 从特定的释放部位释放，并直接与相对突触后膜上的受体结合 瞬间改变突触后细胞的膜电位。相比之下，神经调节剂 缓慢释放，并在细胞外空间扩散。它们同时与几个细胞上的受体结合，从而 在一群神经元中施加长期的变化。促性腺激素分泌途径的机制 神经调节剂还没有被很好地理解。许多实验室对FAST的发布进行了广泛的研究 神经递质发现，突触前膜上被称为活动区的蛋白质复合体， 从形态上对接并在功能上启动突触小泡，以实现快速和精确的释放。在这里我将重点放在 多巴胺的释放机制，这是一种对运动、奖励和情绪至关重要的神经调节剂。我们有 最近发现，出人意料的快速释放多巴胺需要一种活性区状的蛋白质复合体。 对释放部位的要求强烈表明，多巴胺囊泡的位置离它们的未来很近。 释放的位置，并呈现释放准备，使人想起对接和启动突触小泡。我 假设多巴胺神经元中专门的、活跃区样释放部位既停靠又 启动多巴胺囊泡，以在动作电位触发时允许快速胞吐。我会仔细分析这些 在功能和结构层面上的两个进程。在目标1中，我将描述启动蛋白的作用， Munc13在多巴胺释放中。我的初步数据表明，Munc13对多巴胺的释放是必不可少的。这就做 使用碳纤维安培法、超分辨率和共聚焦显微镜以及小鼠遗传学系统地 研究Munc13在多巴胺神经元中的定位和功能。在目标2中，我将描述水泡的特征 在多巴胺轴突和缺乏潜在对接蛋白的突变小鼠中对接。明确地识别 多巴胺终末，我将使用辣根过氧化物酶(HRP)对细胞类型的囊泡进行条件标记 在电子显微镜图像中进行鉴定。然后，我将使用序列EM对纹状体多巴胺轴突进行3D重建。 总而言之，这里提出的实验将有助于从新的机制上理解 多巴胺分泌途径。我们的发现是，多巴胺的释放迅速而准确地发出信号 开始了多巴胺传递的范式转变。拟议的工作扩展到解剖制造- 多巴胺快速排泄机制的建立和功能。对多巴胺分泌的精确理解 这也将为多巴胺信号如何在神经系统疾病中分解提供新的见解。

Signaling in the nervous system relies on the transfer of chemical signals from one neuron to the next. There are two main classes of these signals: fast neurotransmitters and neuromodulators. Fast neurotransmitters are released from specialized release sites and bind directly to receptors on the opposing post-synaptic membrane to exert an instant change in the membrane potential of the post-synaptic cell. In contrast, neuromodulators are released slowly and diffuse through the extracellular space. They bind to receptors on several cells at once to exert long-lasting changes in a population of neurons. The mechanisms in the secretory pathway for neuromodulators are not well understood. Extensive research from many laboratories on the release of fast neurotransmitters has found that a complex of proteins at the presynaptic membrane, known as the active zone, morphologically docks and functionally primes synaptic vesicles for rapid and precise release. Here I focus on release mechanisms of dopamine, a neuromodulator critical for movement, reward, and emotion. We have recently found that an active zone-like complex of proteins is required for unexpectedly rapid dopamine release. The requirement of a release site strongly suggests that dopamine vesicles are positioned close to their future sites of release and are rendered release ready, reminiscent of the docking and priming of synaptic vesicles. I hypothesize that the specialized, active zone-like release site in dopamine neurons both docks and primes dopamine vesicles to allow for fast exocytosis upon action potential triggering. I will dissect these two processes on a functional and structural level. In aim 1, I will characterize the role of the priming protein, Munc13 in dopamine release. My preliminary data suggests that Munc13 is essential for dopamine release. I will use carbon fiber amperometry, super resolution and confocal microscopy, and mouse genetics to systematically characterize the localization and function of Munc13 in dopamine neurons. In aim 2, I will characterize vesicle docking in dopamine axons and in mutant mice that lack potential docking proteins. To unambiguously identify dopamine terminals, I will employ conditional tagging of vesicles with horseradish peroxidase (HRP) for cell-type identification in electron microscopic images. I will then use serial EM to 3D-reconstruct striatal dopamine axons. In summary, the experiments proposed here will contribute to a novel mechanistic understanding of the dopamine secretory pathway. Our finding that dopamine release occurs rapidly and precisely signals the beginning of a paradigm shift for dopamine transmission. The proposed work expands on dissecting the make- up and function of the rapid exocytotic machinery for dopamine. Precise understanding of dopamine secretion will also provide new insights into how dopamine signaling may break down in neurological disease.