Neurotransmitter receptor/redistribution/synaptogenesis

描述（由申请人提供）： 这项修订后的 NIMH 探索/发展资助 (R21) 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联合使用，旨在了解突触功效变化背后的细胞机制。我们建议应用新的分子工具来检查发育和可塑性过程中谷氨酸和 GABA 受体在突触中的动态活性依赖性重新分布和插入。编码离子型谷氨酸受体亚基 GluR1 或 GABAAα 1 受体亚基与增强型绿色荧光蛋白（“pHluorins”）的 pH 敏感突变体融合的 DNA 构建体已经生成，并在大鼠海马神经元的细胞系和原代培养物中表达。与受体胞外 NH2 末端区域融合的“pHluorins”将被检查为突触后囊泡融合介导的受体向突触递送的敏感且特异的指标。这些实验旨在随着时间的推移以光学方式监测活神经元细胞表面上的受体分布，对于评估突触后离子型受体重新分布和/或插入激活的突触是突触功效变化的主要机制这一假设至关重要。提出的研究不仅限于谷氨酸能突触，因为我们还对受体重新分布的平行机制是否在 GABA 能突触的可塑性中发挥作用感兴趣。所提出的工作是一种新颖且创新的方法，用于研究培养物中海马神经元活体微电路中突触形成和突触可塑性的动力学。了解突触可塑性的基本机制不仅将为学习和记忆所涉及的生理过程提供重要的见解，而且还将为病理生理障碍的病因学提供重要的见解，这些障碍涉及伴随精神疾病、衰老和药物成瘾而导致的认知功能恶化。

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.