Mapping dopamine neuron cotransmission by proximity detection

通过邻近检测绘制多巴胺神经元共传递

• 批准号: 8985749
• 负责人: STEPHEN RAYPORT
• 金额: $ 25.72万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8985749
• 关键词: Address; Affect; Area; Axon; Biological Assay; Brain; Cell membrane; Corpus striatum structure; Detection; Development; Disease; Disease model; Dopamine; Dopamine Receptor; Electron Microscopy; Elements; Epitopes; Fluorescence; Functional disorder; Genetic; Glutamates; Hybrids; Image; Imagery; In Situ Hybridization; Label; Ligation; Maps; Measurement; Measures; Membrane; Membrane Proteins; Mental disorders; Modification; Molecular; Mus; Neurons; Neurotransmitters; Parkinson Disease; Pharmaceutical Preparations; Phenotype; Population; Presynaptic Terminals; Proteins; Reading; Rhodopsin; Role; Schizophrenia; Signal Transduction; Site; Slice; Specificity; Synapses; Synaptic Vesicles; Technology; Time; Tissues; Transgenic Organisms; Vesicle; Wild Type Mouse; addiction; base; cholinergic neuron; clinical material; density; dopaminergic neuron; gamma-Aminobutyric Acid; neuropsychiatry; neurotransmitter release; noradrenergic; optogenetics; psychostimulant; public health relevance; synaptic function; tomography; vesicle-associated membrane protein; vesicular SNARE proteins; vesicular glutamate transporter 2; vesicular monoamine transporter 2

 DESCRIPTION (provided by applicant): Many brain neurons release a mix of transmitters, but it has been challenging to identify their synapses based on transmitter status. The application of proximity ligation assay (PLA) technology in optogenetic mice offers a way to address these issues comprehensively, enabling visualization of all synapses of an identified population of neurons and their transmitter status. PLA is a hybrid immunochemical and in situ hybridization approach in which two selected epitopes, which are within about 20 nm of each other, generate a discrete fluorescence signal. Synaptic vesicles at the active zone are a distinctive functional element of synapses, which are within 20 nm of the plasmalemma when poised for release. With PLA, we have recently visualized dopamine receptor oligomerization in striatal neurons and addressed the developmental trajectory of dopamine receptor colocalization. In this project, we will use PLA to visualize the proximity of synaptic markers in dopamine neurons to determine key measures of dopamine neuron synaptic function. The specific aims are to: <1> Visualize dopamine neuron synaptic release sites specifically, then visualize dopamine neuron release sites based on the transmitter released. This will be done in optogenetic mice conditionally expressing the exogenous membrane, axon-targeted protein ChR2-EYFP in dopamine neurons by detecting proximity of ChR2-EYFP in the plasmalemma to key synaptic vesicle proteins. <2> Correlate PLA measurements of dopamine neuron connectivity in target areas with functional connectivity to determine the functional readout of the PLA measurements. <3> Demonstrate the ability of PLA to identify synaptic release sites in tissue from wild type mice using proximity of native plasmalemma and vesicular membrane proteins. Stereology will be used for systematic image acquisition and mapping. The quantitative information obtained with PLA will enable asking how does synaptic connectivity change, with regional specificity, over development, and in disease models? PLA will further enable addressing questions such as, where in the brain do drugs impact most, over what time, and for how long? Once prototyped in the dopamine neurons, the PLA approaches developed in this project could be extended to the other major modulatory neuron groups. This will lay the groundwork for post-mortem studies in clinical material, and enable asking, questions such as, which synaptic connections are most affected in disease states, do treatments impact connectivity, are treatments inducing compensatory changes or reversing pathological changes?

 描述（由申请人提供）：许多大脑神经元释放多种递质，但根据递质状态识别其突触一直具有挑战性。邻近结扎分析（PLA）技术在光遗传学小鼠中的应用提供了一种全面解决这些问题的方法，使已识别的神经元群体的所有突触及其递质状态可视化。 PLA 是一种混合免疫化学和原位杂交方法，其中两个选定的表位（彼此相距约 20 nm）产生离散的荧光信号。活性区的突触小泡是突触的独特功能元件，当准备释放时，突触小泡位于质膜 20 nm 范围内。通过 PLA，我们最近观察到了纹状体神经元中的多巴胺受体寡聚化，并解决了多巴胺受体共定位的发育轨迹。在这个项目中，我们将使用 PLA 来可视化多巴胺神经元中突触标记的接近程度，以确定多巴胺神经元突触功能的关键指标。具体目标是： <1>特异性地可视化多巴胺神经元突触释放位点，然后根据释放的递质可视化多巴胺神经元释放位点。这将在光遗传学小鼠中进行，通过检测质膜中 ChR2-EYFP 与关键突触小泡蛋白的接近度，在多巴胺神经元中条件性表达外源膜、轴突靶向蛋白 ChR2-EYFP。 <2> 将目标区域中多巴胺神经元连接性的 PLA 测量值与功能连接性相关联，以确定 PLA 测量值的功能读数。 <3> 展示 PLA 利用天然质膜和囊泡膜蛋白的邻近性来识别野生型小鼠组织中突触释放位点的能力。体视学将用于系统图像采集和绘图。通过 PLA 获得的定量信息将有助于探究突触连接如何随着区域特异性、过度发育和疾病模型而变化？ PLA 将进一步解决诸如药物对大脑的哪个部位影响最大、作用时间和持续时间等问题。一旦在多巴胺神经元中形成原型，该项目中开发的 PLA 方法就可以扩展到其他主要调节神经元组。这将为临床材料的尸检研究奠定基础，并能够提出以下问题：哪些突触连接在疾病状态下受影响最大、治疗是否会影响连接、治疗是否会引起代偿性变化或逆转病理变化？