Neurotransmitter receptor/redistribution/synaptogenesis

描述(由申请人提供)： 这项关于NIMH探索/发展资助(R21)的修订研究方案将探索分子和细胞生物学方法与电生理和光学成像技术在旨在了解突触功效变化的细胞机制的研究中的结合使用。我们建议使用新的分子工具来研究谷氨酸和GABA能受体在发育和可塑性过程中对突触的动态活性依赖的重新分布和插入。编码离子型谷氨酸受体亚基GluR1或GABAAAlpha1受体亚基的DNA已与增强型绿色荧光蛋白的pH敏感突变体(‘PHluorins’)融合，并在细胞系和原代培养的大鼠海马神经元中表达。与受体的胞外NH2末端区域融合的‘PHluorins’将被视为突触后囊泡融合介导的受体向突触传递的敏感和特异的指标。这些实验旨在光学监测活神经元细胞表面受体随时间的分布，对于评估突触后离子受体重新分布和/或插入到激活的突触是突触效能变化的主要机制这一假说至关重要。建议的研究不仅限于谷氨酸能突触，我们还对受体重新分配的平行机制是否在GABA能突触的可塑性中发挥作用感兴趣。这项工作为研究培养的海马神经元活微电路中突触形成和突触可塑性的动力学提供了一种新颖和创新的方法。对突触可塑性的基本机制的理解不仅将为学习和记忆所涉及的生理过程提供重要的洞察力，而且将为病理生理学疾病的病因提供重要的洞察力，这些疾病包括伴随着精神疾病、衰老和药物成瘾的认知功能恶化。

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.