A Novel Approach to Study Synaptic Plasticity in Isolated Synaptosomes using Flow Cytometry

 描述(申请人提供)：学习依赖于突触可塑性的完整性，并假设认知功能随着年龄的增长而下降和阿尔茨海默病(AD)是由于突触可塑性受损。NMDA受体依赖的长时程增强(LTP)是研究海马区突触可塑性分子机制中应用最广泛的模型之一，是学习和记忆的细胞模拟。我们认为，分离的突触体由一个连接到突触后单位的突触前终末组成，可以为在生化水平上研究突触可塑性提供一个强大的系统。我们的方法包括从海马体中分离突触体，并通过化学诱导长时程增强(CLTP)来驱动突触的活性依赖变化。我们通过对可塑性的关键标志进行免疫染色，如谷氨酸受体亚单位GluR1，然后通过荧光细胞术(流式细胞术)选择GluR1表面染色阳性的突触体来识别活性诱导的变化，这种方法我们称为‘化学刺激长期放大的荧光评估’(FACS-LTP)。在这项提案中，我们的目标是基于我们的初步数据，即FACS-LTP可以应用于突触体，以研究正常动物中活性依赖的生化修饰，以及这些修饰在衰老和阿尔茨海默病(AD)中的变化。在这项建议中，我们的目标是：1)评估cLTP对突触小体的处理是否采用了与电生理诱导切片和培养中类似的生化途径；2)评估突触体-流式细胞仪-LTP方法是否可以检测到突触可塑性随着年龄、AD和病理的存在而下降(特别是关注IL-1β驱动的炎症)，并可用于药物筛选，以确定促进或损害突触可塑性的因素。总体而言，我们的模型系统使用突触体和LTP相结合来研究可塑性，为行为研究、机制研究和药物筛选引入了一种新的功能分析。

 DESCRIPTION (provided by applicant): Learning depends on the integrity of synaptic plasticity, and it is hypothesized that the decline in cognitive function with age and Alzheimer's disease (AD) is due to impaired synaptic plasticity. One of the most widely used models for studying molecular mechanisms of hippocampal synaptic plasticity is NMDA-receptor dependent long term potentiation (LTP), a cellular analogue of learning and memory. We propose that isolated synaptosomes, which consist of a presynaptic terminal attached to a postsynaptic unit, can provide a powerful system for the study of synaptic plasticity at a biochemical level. Our approach involves isolating synaptosomes from the hippocampus and chemically inducing long-term potentiation (cLTP) to drive activity-dependent changes at the synapse. We identify activity-induced changes by immunostaining for key markers of plasticity, such as the glutamate receptor subunit GluR1, followed by fluorescence cytometry (flow cytometry) to select synaptosomes with positive GluR1 surface staining, a method we refer to as `Fluorescent Assessment of Chemically-Stimulated Long-Term Potentiation' (FACS-LTP). In this proposal, our goal is to build on our initial data that FACS-LTP can be applied to synaptosomes to investigate activity-dependent biochemical modifications in normal animals, and how these change in aging and with Alzheimer's Disease (AD). In this proposal, we aim to: 1) evaluate if cLTP treatment of synaptosomes engages similar biochemical pathways as occurs in slices and cultures with electrophysiological induction, 2) evaluate if the synaptosome-FACS-LTP approach detects declines in synaptic plasticity with age, AD and in the presence of pathology (specifically focusing on IL-1β driven inflammation), and can be used for drug-screening to identify agents that facilitate or impair synaptic plasticity. Overall, our model system, which uses synaptosomes in combination with LTP to study plasticity, introduces a new functional assay for behavioral studies, mechanistic studies, and screening of pharmaceuticals.