Neurotransmitter receptor/redistribution/synaptogenesis

描述（由申请人提供）： NIMH探索/发展补助金（R21）的修订研究提案将探索分子和细胞生物学方法与电生理学和光学成像技术的结合使用，旨在了解突触功效变化的细胞机制。我们建议应用新的分子工具，对检查动态活动依赖性的重新分配和插入的突触发育和可塑性的谷氨酸能和GABA能受体。已经产生了编码与增强型绿色荧光蛋白（“pHluorins”）的pH敏感突变体融合的离子型谷氨酸受体亚基GluR 1或GABAA α 1受体亚基的DNA构建体，并在大鼠海马神经元的细胞系和原代培养物中表达。融合到受体的细胞外NH 2-末端区域的“pHluorins”将作为突触后囊泡融合介导的受体向突触递送的敏感和特异性指标进行检查。这些实验旨在光学监测受体分布的细胞表面上的活神经元随着时间的推移是至关重要的假设，重新分配和/或插入突触后离子型受体激活的突触是一个主要的机制，突触效能的变化。提出的研究不仅限于GABA能突触，因为我们也感兴趣的是，受体再分布的平行机制是否在GABA能突触的可塑性中发挥作用。这项工作是一种新的和创新的方法来研究动态的突触形成和突触可塑性的海马神经元在文化的生活微电路。对突触可塑性的基本机制的理解不仅将为学习和记忆中涉及的生理过程提供重要的见解，而且还将为涉及伴随精神疾病、衰老和药物成瘾的认知功能恶化的病理生理障碍的病因学提供重要的见解。

DESCRIPTION (provided by applicant): This revised research proposal for an NIMH Exploratory/Developmental Grant (R21) will explore the combined use of molecular and cell biological methods with electrophysiological and optical imaging techniques in studies aimed at understanding the cellular mechanisms underlying changes in synaptic efficacy. We propose to apply the use of new molecular tool towards examining the dynamic activity dependent redistribution and insertion of glutamatergic and GABAergic receptors to synapses during development and plasticity. DNA constructs encoding the ionotropic glutamate receptor subunit GluR1 or GABAAalpha 1 receptor subunits fused with the pH-sensitive mutants of enhanced green fluorescent protein ('pHluorins') have been generated, and expressed in cell lines and primary cultures of rat hippocampal neurons. The 'pHluorins' fused to the extracellular NH2-terminal region of the receptors will be examined as sensitive and specific indicators of postsynaptic vesicle fusion-mediated delivery of receptors to the synapse. These experiments designed to optically monitor receptor distributions on the cell surface in living neurons over time are critical for evaluating the hypothesis that redistribution and/ or insertion of postsynaptic ionotropic receptors to activated synapses is a major mechanism underlying changes in synaptic efficacy. Studies proposed are not limited solely to glutamatergic synapses, as we are also interested in whether parallel mechanisms of receptor redistribution play a role in plasticity of GABAergic synapses. The proposed work is a novel and innovative approach to studying the dynamics of synapse formation and synaptic plasticity in living microcircuits of hippocampal neurons in culture. An understanding of fundamental mechanisms underlying synaptic plasticity will provide critical insight to not only physiological processes involved in learning and memory, but also to the etiology of pathophysiological disorders involving the deterioration of cognitive function accompanying mental illness, aging and drug addiction.