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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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.