Super-Resolution Microscopy of Neuronal Synapses with Advanced Imaging Tools

意义：在纳米尺度上测量神经元通讯的分子机制的能力 空间尺度将对基础生物科学产生巨大影响，并可能对未来的临床神经科学产生影响。在……里面 特别是，AMPA和NMDA型谷氨酸受体(称为iGluR)动态参与神经元- 通过薄(≈30 nm)突触的神经元间通讯；当神经退行性疾病调节失调时 结果，如阿尔茨海默氏症和帕金森氏症。我们和其他人已经追踪到了这些事件 使用超分辨率荧光显微镜(SRFM)的纳米分辨率。使用小探测器-量子 Dot(≈12 nm直径)和其他光稳定荧光团，是我们实验室在上一次资助中开发的 想出了一些惊喜。我们发现，很大一部分AMPAR驻留在突触中，它们的 移动性受到限制；在长时程增强(LTP，记忆形成的分子基础)期间，我们已经 量化了它们的数量，并发现在它们的维护阶段，它们的横向扩散很少；NMDAR 有突触外的纳米结构域，这可能会阻止它们的数量在LTP期间上升。但这些是不是 其他结果，对吗？为了验证这些初步结果，我们将测量 IGluR，主要是AMPAR，使用三种不同的SRFM技术，每种技术都有自己的优势和 劣势。我们还将确定突触的3D方向，探针大小和类型的影响， LTP激活的细节，并定量确定每个突触上的iGluR数量。结果是 这三种技术之间将进行比较。 创新：每种SRFM技术都有新的方面，特别是在神经科学方面。首先，我们将 改进Palm/Storm技术(SRFM的一种)以测试iGluRs的分布和动态 准确地说。我们将使用新的探针-纳米抗体和单链抗体-来对抗突触后蛋白和iGluR，以及 针对iGluRs测试新的sQD和新的交联剂。我们还将确定方向和位置 通过标记神经连接素和各种突触前蛋白，如Bason和RIM1/2，首先 在基础条件下，再用化学LTP(CLTP)。第二，我们将使用和开发油漆，另一个 SRFM的形式，最近被证明具有100倍的速度提升和出色的空间 分辨率-0.2秒内≈5纳米。我们将展示定量绘制可以应用于固定的神经元 并可用于测量单个突触上的cLTP。我们将第一次将油漆用于谋生 神经元在生理条件下测量AMPAR动力学。有了油漆，我们将能够测试 每个突触都有许多iGluR，无论它们是突触还是突触外的，以及iGluR的数量如何 使用cLTP进行更改。第三，我们将利用一种带有iGluRs的荧光激活蛋白(FAP)，并证明 可以在没有背景的情况下，以纳米分辨率测量活的神经元中的受体数量，并且 对cLTP的潜在快速响应。因此，这种方法将为iGluR结构和动力学提供另一种测试。

Significance: The ability to measure the molecular mechanisms of neuronal communication at the nanometer spatial scale will have enormous impact on basic bioscience and likely to future clinical neuroscience. In particular, AMPA- and NMDA-type glutamate receptors (known as iGluRs) are dynamically involved in neuron- to-neuron communication across the thin (≈30 nm) synapse; when dysregulated, neurodegenerative diseases result, such as Alzheimer’s and Parkinson’s diseases. We—and others—have tracked these events with nanometric resolution using super-resolution fluorescence microscopy (SRFM). Using small probes—quantum dots (≈12 nm diameter) and other photostable fluorophores, developed in our lab in the preceding grant—we came up with some surprises. We find that a large fraction of the AMPARs reside in the synapse where their mobility is restricted; during long-term-potentiation (LTP, a molecular underpinning of memory formation), we’ve quantified their numbers and find during their maintenance phase that their lateral diffusion is rare; NMDARs have extra-synaptic nanodomains which may keep their numbers from rising during LTP. But are these, and other results, correct? To validate these preliminary results, we will measure the placement and diffusion of the iGluRs, primarily AMPARs, using three different SRFM techniques, each one having its own advantages and disadvantages. We will also determine the 3D-orientation of the synapse, the effect of probe size and type, the details of LTP activation, and quantitatively determine the number of iGluRs at each synapse. The results between the three techniques will be compared. Innovation: Each SRFM technique has new aspects, particularly with respect to neuroscience. First, we will improve the PALM/STORM technique (one type of SRFM) to test the distribution and dynamics of iGluRs more accurately. We will use new probes—nanobodies and scFv’s—against post-synaptic proteins and iGluRs, and test new sQDs and new cross-linking reagents against iGluRs. We will also determine the orientation and position of the synaptic zone by labeling neuroligin and various presynaptic proteins, such as Bassoon and RIM1/2, first under basal conditions and then with chemical LTP (cLTP). Second, we will use and develop PAINT, another form of SRFM, which has recently been shown to have a 100× increase in speed with excellent spatial resolution—≈5 nanometers in 0.2 sec. We will show that quantitative-PAINT can be applied to fixed neurons and can be used to measure cLTP on an individual synapse. And for the first time, we will apply PAINT to a living neuron under physiological conditions to measure AMPAR dynamics. With PAINT, we will be able to test how many iGluRs there are per synapse, whether they are synaptic or extra-synaptic, and how the number of iGluRs change with cLTP. Third, we will utilize a fluorogenic activating protein (FAP) with iGluRs and show that the number of receptors can be measured in living neurons with nanometric resolution, no background, and potentially fast response to cLTP. This method will therefore provide another test of iGluR structure & dynamics.