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

使用流式细胞术研究分离突触体突触可塑性的新方法

• 批准号: 8891691
• 负责人: Carl Wayne Cotman
• 金额: $ 23.18万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8891691
• 关键词: AMPA Receptors; Age; Aging; Alzheimer' s Disease; Animals; Antibodies; Behavior Therapy; Behavioral; Biochemical; Biochemical Pathway; Biological Assay; Biological Models; Brain; Ca(2+)-Calmodulin Dependent Protein Kinase; Calcium; Complement; Cytometry; Data; Dendritic Spines; Dependence; Drug Evaluation; Environment; Exposure to; F-Actin; Flow Cytometry; Fluorescence; Functional disorder; Glutamate Receptor; Glycine; Goals; Head; Hippocampus (Brain); Hypersensitivity; Impaired cognition; Impairment; In Vitro; Inflammation; Intervention; LIMK1 gene; Learning; Long-Term Potentiation; MAPK14 gene; Mediating; Membrane; Memory; Memory Loss; Methods; Modeling; Modification; Molecular; Monitor; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; N-terminal; Neurons; Pathology; Pharmacologic Substance; Preclinical Drug Evaluation; Presynaptic Terminals; Process; Receptor Activation; Signal Transduction; Slice; Staining method; Stains; Study models; Surface; Synapses; Synaptic plasticity; Synaptosomes; System; Techniques; Tg2576; Time; Vertebral column; age related; analog; behavioral study; cognitive function; drug use screening; improved; in vivo; indexing; inhibitor/antagonist; mimetics; mouse model; normal aging; novel; novel strategies; postsynaptic; postsynaptic neurons; public health relevance; response; screening; small molecule; trafficking

 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.

 描述（由适用提供）：学习取决于合成可塑性的完整性，并假设认知功能随着年龄的增长和阿尔茨海默氏病（AD）的下降是由于合成可塑性受损而导致的。用于研究海马合成可塑性分子机制的最广泛使用的模型之一是NMDA受体依赖性长期增强（LTP），这是一种学习和记忆的细胞类似物。我们提出，孤立的突触小体由附着在突触后单元的突触前末端组成，可以为在生化水平上研究突触可塑性提供强大的系统。我们的方法涉及从海马隔离突触体和化学诱导的长期增强（CLTP）以驱动突触时活动依赖性变化。我们通过对可塑性的关键标记进行免疫染色（例如谷氨酸接收器亚基GLUR1）来鉴定活动诱导的变化，然后进行荧光细胞仪（流式细胞仪），以选择具有阳性GLUR1表面染色的突触体，这是一种方法，我们称为“我们称为“化学刺激的长期potentiation faces potentiation”（Faces-ltppppppp）的方法。在此提案中，我们的目标是基于我们的初始数据，即FACS-LTP可以应用于突触体，以调查正常动物的活动依赖性生化修饰，以及它们在衰老和阿尔茨海默氏病（AD）中的变化。在该提案中，我们的目的是：1）评估CLTP是否治疗突触体活动是否与在具有电生理诱导的切片和培养物中发生的类似的生化途径相似药物筛查以识别促进或损害合成可塑性的药物。总体而言，我们的模型系统将使用突触体与LTP结合使用来研究可塑性，并引入了一种用于行为研究，机理研究和筛查药物的新功能测定法。