A novel genetic and electrophysiological approach to study hippocampal neurons

研究海马神经元的新遗传和电生理学方法

• 批准号: 7586580
• 负责人: Tarik F Haydar
• 金额: $ 22.5万
• 依托单位: CHILDREN'S RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7586580
• 关键词: Alzheimer' s Disease; Amnesia; Brain; Brain-Derived Neurotrophic Factor; Cell Differentiation process; Cell physiology; Cells; Communication; Complex; Data; Development; Disease; Dominant-Negative Mutation; Down-Regulation; Electrophysiology (science); Electroporation; Embryonic Development; Engineering; Epilepsy; Functional disorder; Funding; Funding Mechanisms; Future; Gene Expression; Gene Proteins; Gene Transfer; Genes; Genetic; Genetic Recombination; Goals; Hand; Hippocampus (Brain); Interneurons; Learning; Length; Ligands; Limbic System; Measurement; Mediating; Memory; Mental Retardation; Methodology; Methods; Molecular; Mutate; Nerve Growth Factor Receptors; Neurons; Neurotrophic Tyrosine Kinase Receptor Type 2; Pathway interactions; Pattern; Phase; Physiology; Plasmid Cloning Vector; Plasmids; Proteins; Public Health; Pyramidal Cells; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Role; Signal Transduction; Site; Specificity; Staining method; Stains; Stem cells; Synapses; Synaptic Transmission; Synaptic plasticity; System; Tamoxifen; Techniques; Technology; Test Result; Testing; Time; Transgenic Organisms; Viral; Work; base; cell type; cost; cost effective; design; expression vector; hippocampal pyramidal neuron; hippocampal subregions; in utero; in vivo; inhibitory neuron; interdisciplinary approach; method development; migration; nervous system disorder; novel; patch clamp; postnatal; postsynaptic; prenatal; presynaptic; prevent; promoter; protein expression; rapid technique; relating to nervous system; research study; tool; transgene expression; vector

DESCRIPTION (provided by applicant): The hippocampus, part of the limbic system, mediates learning and memory and is implicated in epilepsy. We are developing a multidisciplinary approach to study development and function of the excitatory and inhibitory neurons in the hippocampus. Using electroporation to transfect the hippocampal stem cells in utero, we will introduce fluorescent protein markers as well as mutated versions of the TrkB tyrosine kinase receptor into specific subregions of the hippocampal circuitry. The expression of these exogenous genes will be controlled temporally by placing them under the control of inducible gene promoters so that their expression coincides with particular phases of neuronal development. The functional effects of these molecular changes will be assessed by characterizing cell differentiation using immunochemical staining and by direct electrophysiogical measurements using patch clamp recording. These experiments will define a novel and rapid method for assessing the development, allocation and synaptic physiology of specific subpopulations of hippocampal neurons by introducing molecular changes in their germinal cells. This method will result in a technical advance for the study of hippocampal function and will be useful for understanding the underlying causes of abnormal synaptic transmission in diseases including epilepsy and mental retardation. PUBLIC HEALTH RELEVANCE:In this project we seek to develop methods for tracking the development and changing the gene expression of neurons in the hippocampus to enable rapid and cost-effective functional tests. In particular we will be assessing the role of the TrkB neurotrophin receptor in hippocampal neuronal migration and synaptic plasticity. To study both prenatal and postnatal effects of TrkB perturbation, we have engineered inducible plasmid constructs with which we can initiate expression of the transgene upon administration of tamoxifen. This work will enhance our ability to elucidate normal developmental mechanisms and allow rapid testing on causes of epilepsy and mental retardation.

描述（由申请人提供）：海马体是边缘系统的一部分，调节学习和记忆，与癫痫有关。 我们正在开发一种多学科方法来研究海马兴奋性和抑制性神经元的发育和功能。 使用电穿孔转染子宫内的海马干细胞，我们将荧光蛋白标记物以及 TrkB 酪氨酸激酶受体的突变版本引入海马回路的特定亚区域。 通过将这些外源基因置于诱导型基因启动子的控制下，可以暂时控制这些外源基因的表达，从而使它们的表达与神经元发育的特定阶段相一致。 这些分子变化的功能影响将通过使用免疫化学染色表征细胞分化以及使用膜片钳记录进行直接电生理测量来评估。 这些实验将定义一种新颖且快速的方法，通过在生发细胞中引入分子变化来评估海马神经元特定亚群的发育、分配和突触生理学。 该方法将推动海马功能研究的技术进步，并将有助于了解癫痫和智力低下等疾病中突触传递异常的根本原因。 公共健康相关性：在这个项目中，我们寻求开发跟踪海马神经元发育和改变基因表达的方法，以实现快速且具有成本效益的功能测试。 我们将特别评估 TrkB 神经营养素受体在海马神经元迁移和突触可塑性中的作用。 为了研究 TrkB 扰动的产前和产后影响，我们设计了诱导型质粒构建体，用它我们可以在给予他莫昔芬后启动转基因的表达。 这项工作将增强我们阐明正常发育机制的能力，并允许快速检测癫痫和精神发育迟滞的原因。