Regulation of NMDA receptor activation by postsynaptic nanostructure

突触后纳米结构对 NMDA 受体激活的调节

• 批准号: 8784845
• 负责人: Sarah Wein Ransom Metzbower
• 金额: $ 3.73万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8784845
• 关键词: AMPA Receptors; Area; Binding; Biological Assay; Brain; Cells; Characteristics; Cognition Disorders; Computer Simulation; DLG4 gene; Data; Development; Disease; Event; Functional Imaging; Glutamates; Hippocampus (Brain); Image; Imaging Techniques; Individual; Lead; Learning Disorders; Left; Life; Light; Link; Maps; Measurement; Measures; Mediating; Memory Disorders; Mental disorders; Methods; Monitor; N-Methyl-D-Aspartate Receptors; Nanostructures; Neurologic Dysfunctions; Neurons; Peptides; Physiology; Play; Positioning Attribute; Probability; Proteins; Publishing; Receptor Activation; Regulation; Relative (related person); Resolution; Role; Scaffolding Protein; Signal Pathway; Simulate; Site; Source; Structure; Synapses; Synaptic Transmission; Synaptic plasticity; Techniques; Testing; Variant; Vertebral column; computer studies; density; fluorophore; ifenprodil; insight; interest; mutant; nanoscale; nervous system disorder; neurotransmitter release; novel; novel strategies; patch clamp; postsynaptic; presynaptic; presynaptic density protein 95; public health relevance; receptor; receptor function; research study; response; role model; single molecule; small hairpin RNA; spatiotemporal; synaptic function; theories; transmission process

DESCRIPTION (provided by applicant): Understanding what regulates variations in synaptic strength is of great interest, since changes in synaptic function are a hallmark of many psychiatric and neurological diseases. At glutamatergic synapses, AMPA and NMDA receptors (NMDARs) are positioned across from presynaptic active zones by retention within the postsynaptic density (PSD). It is well established that altering the number of postsynaptic receptors is a key mechanism of synaptic plasticity. However, theory and computational modeling suggest that aside from the importance of receptor number, the distribution of receptors within the PSD may have a dramatic impact on synapse physiology, by governing the likelihood that released neurotransmitter will activate the receptors. In my lab, single-molecule mapping of the PSD (using an imaging technique called PALM) has led to the identification of nanodomains within the PSD that contain a relatively high density of the scaffold protein PSD95 and the NMDAR subunit GluN2B. These data suggest that the nanoscale organization of the synapse could play a critical role in synaptic function. Thus, I hypothesize that nanodomains of PSD-95 control receptor activation through subsynaptic localization of GluN2B. Previously, it has not been feasible to test this idea in live cells due to the lack of imaging resolution. However, by combining our recently published assays with a new approach I have developed, I propose to test this how this novel feature influences synaptic transmission. To determine the relationship between PSD nanoscale organization and NMDAR activation, I co-transfected cultured hippocampal neurons with a photoactivatable version of PSD-95 and the genetically encoded Ca2+ indicator GCaMP6f. This permitted me to combine PALM imaging of synapse nanostructure with measurement of miniature spontaneous Ca2+ transients in the same individual spines. This approach revealed a significant correlation between NMDAR- mediated Ca2+ transients at single spines and the fractional area of the PSD that was within nanodomains. There was no relationship between the Ca2+ transient and PSD area, consistent with previous observations that synaptic NMDAR number is only weakly correlated with PSD size. To better understand the mechanism responsible for this relationship, I will first use a combination of imaging and electrophysiological measures of NMDAR function at synapses whose nanostructure is monitored via PALM. Second, GluN2A and GluN2B receptor subtypes are predicted to vary in the spatiotemporal aspects of their activation, and GluN2B is preferentially enriched in PSD-95 high-density nanodomains. Therefore, I will probe the relative contribution of each subunit to the relationship between receptor activation and PSD nanostructure. Finally, I will assess the role of NMDAR interaction with PSD-95 using peptides or PSD-95 mutants that cannot bind NMDARs, each of which I expect to abolish or weaken the relationship between nanostructure and NMDAR activation. Fine tuning of NMDAR activation through nanoscale manipulations of receptor distribution could have a profound impact on synaptic physiology and the induction of synaptic plasticity.

描述（由申请人提供）：了解什么调节突触强度的变化是非常有趣的，因为突触功能的变化是许多精神和神经疾病的标志。在突触能突触处，AMPA和NMDA受体（NMDAR）通过保留在突触后密度（PSD）内而定位在突触前活动区的对面。众所周知，改变突触后受体的数量是突触可塑性的关键机制。然而，理论和计算模型表明，除了受体数量的重要性之外，PSD内受体的分布可能对突触生理学产生巨大影响，通过控制释放的神经递质激活受体的可能性。在我的实验室中，PSD的单分子映射（使用称为PALM的成像技术）已经导致PSD内含有相对高密度的支架蛋白PSD 95和NMDAR亚基GluN 2B的纳米结构域的鉴定。这些数据表明，突触的纳米级组织可能在突触功能中发挥关键作用。因此，我假设PSD-95的纳米结构域通过GluN 2B的突触下定位来控制受体激活。以前，由于缺乏成像分辨率，在活细胞中测试这一想法是不可行的。然而，通过将我们最近发表的试验与我开发的新方法相结合，我建议测试这种新功能如何影响突触传递。为了确定PSD纳米级组织和NMDAR激活之间的关系，我用PSD-95的光活化版本和遗传编码的Ca 2+指示剂GCaMP 6 f共转染培养的海马神经元。这使我能够将突触纳米结构的联合收割机成像与同一个体脊柱中微型自发Ca 2+瞬变的测量结合起来。该方法揭示了单个棘处NMDAR介导的Ca 2+瞬变与纳米域内PSD的分数面积之间的显著相关性。钙瞬变和PSD面积之间没有关系，这与以前的观察结果一致，即突触NMDAR数量与PSD大小仅弱相关。为了更好地理解这种关系的机制，我将首先使用NMDAR功能的成像和电生理学测量的组合在突触的纳米结构是通过PALM监测。其次，预测GluN 2A和GluN 2B受体亚型在其激活的时空方面变化，并且GluN 2B优先富集在PSD-95高密度纳米结构域中。因此，我将探测每个亚基对受体活化和PSD纳米结构之间关系的相对贡献。最后，我将使用不能结合NMDAR的肽或PSD-95突变体来评估NMDAR与PSD-95相互作用的作用，我希望每种肽或PSD-95突变体都能消除或削弱纳米结构与NMDAR激活之间的关系。通过对受体分布的纳米级操纵来微调NMDAR激活可能对突触生理学和突触可塑性的诱导产生深远的影响。