高分辨率定量免疫膜受体在视网膜带状突触分析
Summary
The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.
Abstract
Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.
Introduction
谷氨酸是在视网膜1中的主要兴奋性神经递质。视网膜神经节细胞（RGC）中，从双极电池 2接收的谷氨酸突触输入，是视网膜的输出神经元发送视觉信息到大脑。生理学研究表明，视网膜神经节细胞突触激励是通过NMDA受体（NMDA受体）和AMPA受体（的AMPARs）3,4,5突触后介导的。虽然在视网膜神经节细胞兴奋性突触后电流（EPSCS）通过的AMPARs NMDA受体和介导视网膜神经节细胞-1,3,5,6,7,8-，自发微型EPSCS（mEPSCs）只呈现一个的AMPARs介导的分量4,5,9。然而，减少摄取谷氨酸自发EPSCS 5透露出一股NMDA受体成分，这表明在研资局树突NMDA受体可能位于兴奋性突触之外。膜相关鸟苷酸激酶（MAGUKs）如PSD-95该集群神经递质受体，包括谷氨酸盐受体和离子通道■在突触部位，也表现出明显的subsynaptic表达模式10,11,12,13,14。
近几十年来，共焦免疫组织化学和预嵌入型电子显微镜（EM）的免疫组化已被用于研究膜受体的表达。虽然共焦免疫染色揭示受体表达的宽图案，其较低的分辨率使得不可能使用来区分的亚细胞位置。在哺乳动物视网膜预嵌入-EM研究表明NMDA受体亚基存在于视锥双极细胞带状突触15,16,17突触后的元件。这是明显的对比，以生理学证据。然而，反应产物的扩散是在预嵌入免疫过氧化物酶方法的公知的构件。因此，这种方法通常不会给统计上可靠的数据和可以排除国产化的区分突触膜与膜突触外18,19,20,21。上另一方面，生理和解剖数据与的AMPARs对视网膜神经节细胞3,5,7,9,22突触定位相一致。因此，谷氨酸受体和MAGUKs在视网膜色带突触本地化不仅对突触后也给perisynaptic或突触外膜隔室。然而，仍然需要在视网膜色带突触这些膜蛋白的高分辨率定量分析。
在这里，我们开发了一种postembedding EM免疫技术检测NMDA受体亚基，AMPAR亚基和PSD-95接着在突触到大鼠视网膜神经节细胞估算这些蛋白的数量，密度和可变性的subsynaptic定位用霍乱毒素B亚基标记的（CTB）逆行追踪方法。
Protocol
Representative Results
Discussion
我们已经描述了四种技术对于成功的定量后包埋免疫EM：1）短和弱固定，2）冷冻取代，3）后包埋免疫染色，以及4）定量。
EM免疫允许在超薄组织切片的特定蛋白质的检测。标记有金颗粒的抗体可以用EM直接可视化。而在检测细胞膜受体的subsynaptic本地化功能强大，EM免疫可以技术上具有挑战性，并要求组织固定和加工方法严谨的优化。
罢工膜的完整性和…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是由神经疾病与中风国家研究所（NINDS）和国立耳聋与其他交流障碍（NIDCD），美国国立卫生研究院（NIH）的校内方案的支持。我们感谢NINDS EM设施和援助NIDCD先进的成像核心（代号＃ZIC DC 000081-03）。
Materials
| Paraformaldehyde | EMS | 15710 | |
| Glutarldehyde | EMS | 16019 | |
| NaH2PO4 | Sigma | S9638 | |
| Na2HPO4 | Sigma | 7782-85-6 | |
| CaCl2 | Sigma | C-8106 | |
| BSA | Sigma | A-7030 | |
| Triton X-100 | Sigma | T-8787 | |
| NaOH | Sigma | 221465 | |
| NaN3 | JT Baker | V015-05 | |
| Glycerol | Gibco BRL | 15514-011 | |
| Lowicryl HM 20 | Polysciences | 15924-1 | |
| Tris-Base | Fisher | BP151-500 | |
| Tris | Fisher | 04997-100 | |
| Anti-GluN2A | Millipore | AB1555P | Dilution 1/50 |
| Anti-GluN2B | Millipore | AB1557P | Dilution 1/30 |
| Anti-GluA2/3 | Millipore | AB1506 | Dilution 1/30 |
| Anti-PSD-95 | Millipore | MA1–046 | Dilution 1/100 |
| Donkey anti-rabbit IgG-10 nm gold particles | EMS | 25704 | Dilution 1/20 |
| Donkey anti-mouse IgG-10 nm gold particles | EMS | 25814 | Dilution 1/20 |
| Donkey anti-mouse IgG-5 nm gold particles | EMS | 25812 | Dilution 1/20 |
| Donkey anti-goat IgG-18 nm gold particles | Jackson ImmunoResearch | 705-215-147 | Dilution 1/20 |
| Formvar-Carbon coated nickel-slot grids. | EMS | FCF2010-Ni | |
| Uranyl acetate | EMS | 22400-1 | |
| Methanol | EMS | 67-56-1 | |
| Lead citrate | Leica | ||
| Leica EM AFS | Leica | ||
| Leica EM CPC | Leica | ||
| Ultromicrotome | Leica | ||
| JEOL 1200 EM | JEOL | ||
| liquid nitrogen | Roberts Oxygen | ||
| Propane | Roberts Oxygen | ||
| CTB | List Biological Laboratories | 104 | 1-1.2% |
| Anti-CTB | List Biological Laboratories | 703 | Dilution 1/4000 |
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