Mapping dopamine neuron cotransmission by proximity detection

 描述（由申请人提供）：许多脑神经元释放混合的递质，但根据递质状态识别它们的突触一直是一个挑战。在光遗传学小鼠中应用邻近连接测定（PLA）技术提供了一种全面解决这些问题的方法，使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，其中彼此在约20 nm内的两个选择的表位产生离散的荧光信号。活动区的突触囊泡是突触的独特功能元件，当其准备释放时，其在质膜的20 nm内。与解放军，我们最近可视化多巴胺受体寡聚化纹状体神经元，并解决了多巴胺受体共定位的发展轨迹。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物的接近程度，以确定多巴胺神经元突触功能的关键措施。具体目标是：<1>具体地可视化多巴胺神经元突触释放位点，然后基于释放的递质可视化多巴胺神经元释放位点。这将在多巴胺神经元中条件性表达外源性膜、轴突靶向蛋白ChR 2-EYFP的光遗传学小鼠中通过检测质膜中ChR 2-EYFP与关键突触囊泡蛋白的接近度来完成。<2>将目标区域中多巴胺神经元连接的PLA测量与功能连接相关联，以确定PLA测量的功能读数。<3>利用天然质膜和囊泡膜蛋白的接近性，证明PLA在野生型小鼠组织中识别突触释放位点的能力。体视学将用于系统图像采集和标测。用PLA获得的定量信息将使我们能够询问突触连接如何改变，区域特异性，过度发育和疾病模型？PLA将进一步帮助解决诸如药物对大脑中哪个部位的影响最大、影响时间和持续时间等问题。一旦在多巴胺神经元中原型化，本项目中开发的PLA方法可以扩展到其他主要的调节神经元组。这将为临床材料的尸检研究奠定基础，并能够提出诸如疾病状态下哪些突触连接受影响最大，治疗是否影响连接，治疗是否诱导代偿性变化或逆转病理变化等问题。

 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?