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

 描述（由申请人提供）：学习依赖于突触可塑性的完整性，并且假设随着年龄和阿尔茨海默病（AD）的认知功能下降是由于突触可塑性受损。用于研究海马突触可塑性的分子机制的最广泛使用的模型之一是NMDA受体依赖性长时程增强（LTP），其是学习和记忆的细胞类似物。我们建议，分离的突触体，它包括一个突触前终端连接到突触后单位，可以提供一个强大的系统，在生化水平上的突触可塑性的研究。我们的方法包括从海马分离突触体，并化学诱导长时程增强（cLTP），以驱动突触的活动依赖性变化。我们通过免疫染色确定可塑性的关键标志物，如谷氨酸受体亚基GluR 1，然后通过荧光细胞术（流式细胞术）选择具有阳性GluR 1表面染色的突触体，我们将这种方法称为“化学刺激长期增强的荧光评估”（FACS-LTP）。在这项提案中，我们的目标是建立在我们的初始数据基础上，FACS-LTP可以应用于突触体，以研究正常动物中的活性依赖性生化修饰，以及这些修饰在衰老和阿尔茨海默病（AD）中的变化。在本提案中，我们的目标是：1）评估突触体的cLTP处理是否涉及与在具有电生理学诱导的切片和培养物中发生的类似的生化途径，2）评估突触体-FACS-LTP方法是否检测到突触可塑性随年龄、AD和病理学存在而下降（特别是关注IL-1β驱动的炎症），并且可用于药物筛选以鉴定促进或损害突触可塑性的试剂。总的来说，我们的模型系统，它使用突触体结合LTP研究可塑性，介绍了一种新的功能测定的行为研究，机制研究，和筛选药物。

 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.