Mapping dopamine neuron cotransmission by proximity detection

 描述(申请人提供)：许多脑神经元释放一种混合的递质，但根据递质状态识别它们的突触一直是一项挑战。邻近连接分析(PLA)技术在光遗传小鼠中的应用提供了一种全面解决这些问题的方法，使得能够可视化已识别的神经元群体的所有突触及其递质状态。聚乳酸是一种免疫化学和原位杂交的混合方法，在这种方法中，两个相距约20 nm的选定表位产生离散的荧光信号。活动区的突触小泡是突触的一个独特的功能元件，当准备释放时，它们位于质膜20 nm以内。最近，我们用聚乳酸观察了纹状体神经元中多巴胺受体的寡聚化，并研究了多巴胺受体共定位的发展轨迹。在这个项目中，我们将使用聚乳酸来可视化多巴胺神经元中突触标志物的接近程度，以确定衡量多巴胺神经元突触功能的关键指标。具体目的是：显示多巴胺神经元突触释放的特异性部位，然后根据释放的递质显示多巴胺神经元的释放部位。这将在光遗传小鼠中完成，通过检测质膜中ChR2-EYFP与关键突触小泡蛋白的接近程度，在多巴胺神经元中有条件地表达外源性膜、轴突靶向蛋白ChR2-EYFP。&lt；2&gt；将解放军对靶区多巴胺神经元连接性的测量与功能连接性相关联，以确定解放军测量的功能读数。&lt；3&gt；展示了聚乳酸能够利用天然质膜和囊泡膜蛋白识别野生型小鼠组织中的突触释放位置。体视学将用于系统的图像采集和测绘。用解放军获得的定量信息将使人们能够问，突触连通性是如何变化的，具有区域特异性，超过发育，以及在疾病模型中？解放军将进一步解决诸如药物在大脑的哪个部位影响最大、影响时间和持续时间等问题？一旦在多巴胺神经元中建立了原型，本项目中开发的解放军方法可以扩展到其他主要的调制神经元组。这将为临床材料的尸检研究奠定基础，并使提问成为可能，例如，在疾病状态下，哪些突触连接受到的影响最大，治疗是否会影响连接性，治疗是否会导致代偿性变化或逆转病理变化？

 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?