Organization and Dynamics of PSD-bound Glutamate Receptors at Super-resolution

超分辨率下 PSD 结合谷氨酸受体的组织和动态

• 批准号: 8929506
• 负责人: WILLIAM GREEN
• 金额: $ 66.03万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2017-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8929506
• 关键词: Affect; Alzheimer' s Disease; Binding; Biological Assay; Blinking; Caliber; Cleaved cell; Color; Complex; DLG1 gene; DLG4 gene; Data; Diffuse; Disease; Dyes; Electron Microscopy; Event; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Fluorescent Probes; Glutamate Receptor; Health; Image; Immunoglobulin Fragments; Kinetics; Label; Learning; Life; Light; Location; Long-Term Potentiation; Measurement; Measures; Memory; Mental Depression; Microscopy; Molecular Conformation; Morphologic artifacts; Motion; Movement; N-Methylaspartate; Neurodegenerative Disorders; Neurons; Neurotransmitter Receptor; Peripheral; Preparation; Process; Protein Binding; Proteins; Quantum Dots; Relative (related person); Resolution; Role; Sampling; Scaffolding Protein; Structure; Synapses; Synaptic Cleft; Synaptic plasticity; Techniques; Testing; Time; Tissues; Vesicle; autism spectrum disorder; density; fluorophore; forgetting; improved; interest; nano; palmitoylation; postsynaptic; postsynaptic density protein; presynaptic; presynaptic density protein 95; prevent; receptor; research study; sensor; synaptic function; tomography; trafficking

DESCRIPTION (provided by applicant): Synapses are the basic cellular units modified during learning and memory formation. A major barrier to characterizing synaptic function and plasticity is that the synaptic structures of most interest-presynaptic vesicles, presynaptic active zones, the synaptic cleft, postsynaptic densities (PSDs) and postsynaptic receptors-are mostly below the diffraction-limit of light. While some of these structures are resolvable using electron microscopy (EM), EM cannot assay structures in live tissue or real time, and has significant problems identifying specific molecules and with sample preparation artifacts. These limitations prevent observing how synapses change during plasticity events that underlie memory formation, such as long-term potentiation (LTP) and depression (LTD), as well as synaptic changes occurring during neurodegenerative diseases, such as Alzheimer's Disease or during neurodevelopmental diseases, such as Autism Spectrum Disorders. In this proposal different super-resolution fluorescence microscopy techniques are applied to live excitatory CNS synapses. This approach will improve imaging accuracy and resolution to near that of EM. To apply super-resolution microscopy techniques to synapses, small quantum dots (~7 nm diameter) have been developed that allow access to synaptic clefts, 20-30 nm wide. In contrast, the bigger, commercial quantum dots (~21 nm diameter), which are widely used in the field, are found to be unable to enter synaptic clefts. Using super-resolution techniques, the hypothesis will be tested that the two postsynaptic synaptic neurotransmitter receptors, AMPA-type and NMDA-type glutamate receptors (AMPARs and NMDARs), have unique distributions and mobility within PSDs because of their different interactions with the scaffold proteins, PSD-95 and SAP97 in PSDs. The small quantum dots attached to AMPARs and NMDARs will allow tracking of the movements of the neurotransmitter receptors outside and within synapses. To test the hypothesis, three specific aims are proposed; all of them use super-resolution microscopy and generally rely on our unique small quantum dots (with controls using regular dyes). The first Aim is to measure PSD-95 and SAP97 distributions within PSDs and their changes with LTP and LTD. The second Aim is to measure AMPAR and NMDAR distribution and kinetics within PSDs and the changes that occur with LTP and LTD. The third Aim is to examine the role of PSD-95 palmitoylation cycle in AMPAR and NMDAR dynamics and the effects of LTP and LTD.

描述（申请人提供）：突触是在学习和记忆形成过程中发生变化的基本细胞单位。表征突触功能和可塑性的一个主要障碍是，大多数感兴趣的突触结构——突触前囊泡、突触前活跃区、突触间隙、突触后密度（psd）和突触后受体——大多低于光的衍射极限。虽然其中一些结构可以用电子显微镜（EM）分辨，但EM不能在活组织或实时中分析结构，并且在识别特定分子和样品制备伪影方面存在重大问题。这些限制阻碍了观察突触如何在记忆形成的可塑性事件中发生变化，如长期增强（LTP）和抑郁（LTD），以及在神经退行性疾病（如阿尔茨海默病）或神经发育性疾病（如自闭症谱系障碍）期间发生突触变化。在这个建议中，不同的超分辨率荧光显微镜技术应用于活的兴奋性中枢神经系统突触。这种方法将提高成像精度和分辨率，接近于EM。为了将超分辨率显微镜技术应用于突触，已经开发了小量子点（直径约7纳米），可以访问20-30纳米宽的突触间隙。相比之下，在该领域广泛使用的更大的商业量子点（直径~21 nm）被发现无法进入突触间隙。利用超分辨率技术，我们将验证两种突触后神经递质受体ampa型和nmda型谷氨酸受体（AMPARs和NMDARs）在psd中具有独特的分布和流动性，因为它们与psd中的支架蛋白PSD-95和SAP97有不同的相互作用。附着在ampar和NMDARs上的小量子点可以跟踪突触内外神经递质受体的运动。为了验证这一假设，提出了三个具体目标；它们都使用超分辨率显微镜，通常依赖于我们独特的小量子点（控制使用常规染料）。第一个目标是测量psd内的PSD-95和SAP97分布及其随LTP和LTD的变化。第二个目标是测量psd内AMPAR和NMDAR的分布和动力学以及LTP和LTD发生的变化。第三个目的是研究PSD-95棕榈酰化循环在AMPAR和NMDAR动力学中的作用以及LTP和LTD的影响。