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

描述（由申请人提供）：突触是学习和记忆形成过程中修饰的基本细胞单位。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、突触前活动区、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。虽然这些结构中的一些是使用电子显微镜（EM）可分辨的，但是EM不能在活组织或真实的时间中测定结构，并且具有识别特定分子和样品制备伪影的显著问题。这些限制阻止观察突触在作为记忆形成基础的可塑性事件期间如何变化，例如长时程增强（LTP）和抑郁（LTD），以及在神经退行性疾病期间发生的突触变化，例如阿尔茨海默病或在神经发育疾病期间发生的突触变化，例如自闭症谱系障碍。在这个建议中，不同的超分辨率荧光显微镜技术被应用到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 nm），其允许进入20-30 nm宽的突触裂缝。相比之下，在该领域广泛使用的更大的商业量子点（直径约21 nm）被发现无法进入突触裂缝。使用超分辨率技术，将测试的假设，即两个突触后突触神经递质受体，AMPA型和NMDA型谷氨酸受体（AMPAR和NMDAR），具有独特的分布和流动性内PSD，因为它们与支架蛋白，PSD-95和SAP 97在PSD的不同的相互作用。附着在AMPAR和NMDAR上的小量子点将允许跟踪突触内外神经递质受体的运动。为了验证这一假设，提出了三个具体的目标;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（使用常规染料进行控制）。第一个目的是测量PSD-95和SAP 97在PSD中的分布及其随LTP和LTD的变化。第二个目的是测量PSD中AMPAR和NMDAR的分布和动力学及其随LTP和LTD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。

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.