O Brother, Where Art Thou - Interrogating Glutamate Co-transmission in Dopaminergic Neurons at Single-vesicle Level using Cell Reprograming and CRISPR

项目摘要 多巴胺（DA）神经元在各种脑功能中起着至关重要的作用，包括运动控制、奖励、 情绪和记忆，因此与许多神经系统疾病有关，如成瘾， 帕金森症精神分裂症。越来越多的证据表明，DA神经元也共同- 除了DA外，还释放其他神经递质，这种共传递具有行为学意义。 结果，使相关神经网络中DA能传递的观点复杂化。比如说， 最近的研究发现，中脑DA能神经元具有快速和强烈的抑制作用， 纹状体投射神经元（SPNs）。DA神经元的GABA能传递行为非常相似 与GABA能神经元的比例。有趣的是，GABA的释放依赖于囊泡单胺 VMAT转运体而不是囊泡GABA转运体，表明DA神经元释放DA 和GABA通过同一个突触囊泡池。然而，谷氨酸（Glu）的共同释放 DA神经元的情况要复杂得多。首先，Glu是一种快节奏的生电神经递质， DA是一种慢作用神经调质。第二，Glu通过转运进入DA终末的囊泡中， 囊泡谷氨酸转运蛋白2（VGluT 2），依赖于膜电位梯度（VEGF 2） 而不是VMAT所用的pH梯度（pH梯度）。第三，并非所有中脑DA的轴突终末 神经元释放谷氨酸。我们的初步研究表明，VMAT阳性和VGluT 2阳性的囊泡 即使在同一DA能轴突终末，也可能属于不同的群体， 群体行为不同。因此，我们假设DA和Glu被包装并释放， 来自DA能突触中不同的突触囊泡，具有不同的动力学，并受 不同的机制。为了验证这一点，我们将利用细胞重编程技术的最新进展 和体细胞基因修饰，以构建亚细胞和分子检测的体外平台 DA神经元中多种神经递质共传递。我们将重点关注（1） 表征DA神经元的单个突触处的Glu释放，和（2）阐明组织， 负责DA和/或Glu释放的突触囊泡的运输和释放动力学。的 这个项目的结果将导致深入了解突触共传递，一个共同的 大脑中众多类型的神经元之间的特性。更重要的是， 在这个项目中收集的知识将为解码纳米级的复杂性铺平道路。 微观突触内的神经传递。

PROJECT SUMMARY Dopamine (DA) neurons play a vital role in various brain functions including motor control, reward, emotion and memory, and thus are implicated in a number of neurological disorders, like addiction, Parkinson’s disease, schizophrenia. Accumulating evidence suggests that DA neurons also co- release other neurotransmitters in addition to DA and that such co-transmission bears behavioral outcomes, complicating the view of DAergic transmission in associated neural network. For example, recent studies have found that midbrain DA neurons have a rapid and strong inhibitory action on striatal projection neurons (SPNs). This GABAergic transmission of DA neurons behaves very similar to that of GABAergic neurons. Intriguingly, GABA release is dependent on vesicular monoamine transporter (VMAT) instead of vesicular GABA transporter, indicating that DA neurons release DA and GABA through the same pool of synaptic vesicles. However, the co-release of glutamate (Glu) from DA neurons is far more complex. First, Glu is a fast-pace electrogenic neurotransmitter whereas DA is a slow-action neuromodulator. Second, Glu is transported into vesicles in DA terminals via vesicular glutamate transporter 2 (VGluT2), which depends on a membrane potential gradient (∆ψ) instead of a pH gradient (∆pH) utilized by VMAT. Third, not all axonal terminals of midbrain DA neurons release Glu. Our pilot study has suggested that VMAT-positive and VGluT2-positve vesicles are likely belongs to different populations even in the same DAergic axonal terminals and those two groups behavior differently. Therefore, we hypothesize that DA and Glu are packed and released from different synaptic vesicles in DAergic synapses with distinct kinetics and regulated by different mechanisms. To test that, we will utilize recent advances in cell reprograming technology and somatic gene modification to build an in vitro platform for sub-cellular and molecular examination of multi-neurotransmitter co-transmission in DA neurons. In particular, we will focus on (1) characterize Glu release at single synapses of DA neurons, and (2) elucidate the organization, trafficking and release kinetics of synaptic vesicles responsible for DA and/or Glu release. The outcome of this project will lead to in-depth understanding of synaptic co-transmission, a common character among numerous types of neurons in the brain. More importantly, the new methodologies and knowledge gathered in this project will pave the way for decoding the nanoscopic complexity of neurotransmission within the microscopic synapse.