Funding was provided by the Austrian Science Fund FWF grant No. P-22969-B11 to F. Ferraguti, and by the Charitable Hertie Foundation and the Werner Reichardt Centre for Integrative Neuroscience and by the DFG (CIN-Exc. 307) to I. Ehrlich.

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The authors declare that they have no competing financial interests.

冷冻断裂电子显微镜已在超微结构研究的重大技术超过40年。然而，由于缺乏有效的手段来研究膜的分子组成产生了它的使用显著下跌。近来，出现了在冷冻断裂电子显微术的一个主要复兴由于通过免疫标记1,2，即FRIL技术以显示完整的膜蛋白的有效方法的发展。 该FRIL技术比其他免疫超微结构方法具有几个优点。首先，蛋白质是抗体增加的敏感度容易接近。第二，细胞膜特，如突触后膜，副本的二维表面上的大的部分的曝光允许感兴趣的分子的空间分布和物理邻接的检查而不斯里的费力和耗时的重建人超薄切片。第三，质膜的两半的可用性增加了可被标记为每个单独的结构蛋白的数量，提供合适的抗体是可用的。在压裂，分裂膜的疏水面涂覆有碳 - 铂该entrenches残留在断裂表面上的蛋白质结构域。这可以防止在这些结构域抗原的抗体的访问。例如在一个复制品的P面仅面向原浆空间表位可以通过抗体而在E-面仅表位朝向可被抗体结合的exoplasmic空间进行检测，（ 见图2）。 在另一方面，FRIL技术也从一定的局限性2受损。由于骨折随机发生的，它可能是很难瞄准特定的细胞或结构。这也可能导致一个抽样偏差， 例如，突触集合中，考虑到不同的概率离的沿结构具有不同的曲率（ 例如，刺对轴）的膜cturing。此外，膜蛋白的两个面中的一个的分配是不可预测的。因此，蛋白质为P-面或E-面的分布，特别是用于定量研究，应认真使用抗体反应的细胞内和细胞外结构域检查。最后，在某些结构，例如突触前轴突终端的复制品的识别，仅基于形态特征时是很困难的。然而，对于标志蛋白的特异性抗体的使用或标记整合膜蛋白或使用病毒载体提供额外的工具通道的转导，以便于裂隙膜的鉴定。例如，本研究了的ChR2-YFP的丘脑神经元的转导的优点，以确定他们的轴突传出神经在杏仁核或标记为μ阿片受体揭示突触后箱ITC神经元mbranes。 为了成功地进行FRIL技术，应特别注意关于组织固定服用。强组织固定（&gt; 2％多聚甲醛）可导致交叉骨折率很高，并在贴标签的灵敏度的降低。另一方面，弱注视使组织处理和加工（ 例如，切割部分）的困难。同样重要的是，以控制修剪块的厚度的双面胶带的厚度相匹配。如果检体的厚度比带的下部，所述组织的表面可能不附着到两个金属载体的表面上，从而将冷冻试样不破裂。如果组织是厚，它会与不可避免结构扭曲压缩两个铜载波的三明治制成时。在该试样断裂（在此协议-115℃，）中的温度也起着重要的作用上的副本的结构。较高的温度可能会产生伪像的一个更高的速率，如在组织的表面上的水蒸汽的冷凝之前或蒸发期间。较低的温度（&lt;-125℃）压裂过程中可能会增加分裂的危险掉的材料。这种材料可落入到样品的表面上或保持连接到它。材料的这些片也铜版纸和对比产生图像上的黑斑。在较低温度下压裂也可以影响交骨折的频率特别是对于小的精细结构如树突棘。在副本的制备进一步关键步骤是该洗涤剂消化。如果消化不完全，未消化的组织出现在副本的暗斑，在TEM混杂结构的分析。此外，未消化的组织可以非特异性陷阱或绑定的抗体，增加了背景标记。另一方面，使用洗涤剂的S表示组织消化可变性关联到副本改变其二级和三级结构的分子。因此，对于某些抗原可能需要逐渐稀释SDS浓度额外洗涤步骤。 对于免疫标记，不同大小的缀合到二级抗体的金颗粒的可用性允许在同一时间进行检测，但只有定性，多种蛋白质，甚至在质膜的特定微区，如突触后专业化。然而，由于空间位阻，定量研究一般仅限于在检测只有一个分子。前述金颗粒的尺寸也可影响标签功效。 在FRIL标记的解释，应该记住的是，在免疫粒子可位于任何地方与从该抗原20-25纳米的半径的半球内由于柔性复杂的形式由初级和二级抗体26编有关理论和FRIL及其相关技术的实践提供进一步的信息，我们将为读者也给其他方法的第27条，28。 的FRIL技术最近已用于在不同的突触种群29,30。此外，AMPA-Rs中的FRIL技术的检测灵敏度估计高达每一个功能的AMPA 1免疫粒子谷氨酸受体定 ​​位的高分辨率定量分析-R通道29。因此，这种方法是整体非常有用的量化和分析AMPA-R和NMDA-R的在中央突触突触后表达的图案。在这里，我们展示了其在PIN / MGN-ITC突触适用性，一个网站最有可能的，生怕调节过程中传达美国的信息很重要。使用针对AMPA受体亚基谷氨酸的高度保守的细胞外的氨基酸残基产生的抗体A1-GluA4，我们发现对应于突触后膜专业进出口集群内连金颗粒的分布。相比于通过PIN / MGN丘脑传入针对性轴突触AMPA-R的在ITC棘密度为显著高。在两个脊柱和轴突触，检测标记为AMPA-R和突触后区之间的正相关性，常见于其他谷氨酸突触25的功能。在PIN / MGN-ITC突触AMPA-R的密度低方差指示类似于丘脑传出神经形成7突触等，但不同于皮层突触25的均匀分布。相反，NMDA-R的密度为更多变和脊椎和轴突触暗示比AMPA-R的不同规定之间没有差异。在未来，FRIL技术的高再现性将不仅允许评估中央突触的基础分子组成，但可有利于的c检测hanges在离子型谷氨酸受体的数量和分布subsynaptic恐惧学习后，补充这些投入前和突触后性能的离体记录。 总之，这种方法可以由其他研究者用来洞察特定输入兴奋性突触的结构与功能的关系中的许多其他的神经回路，其中解开输入的原点和突触后元件的性质和组合物是至关重要的，但问题。

生物膜在纳米尺度的功能结构的定义已经由许多适于透射电镜免疫标记技术的发展，近年来挑战。然而，这些技术， 例如，前和后包埋免疫，有一些重要的限制，其中包括检测差抗原和/或膜结合蛋白的有限定量的评估。这些限制成为在神经系统中，其特点是具有高度细胞多样性和突触异质性的精细结构的调查尤为关键。这种异质性的结果从由前和突触后的元素和由差异表达，富集，或信号蛋白，如受体，转运，和效应分子的相互作用所决定的结构和功能的多样性。 直接immunolabe的新方法在洗涤剂溶解的冷冻断裂副本（FRIL）积分或交联的膜蛋白的灵最初是由藤本介绍二十年前1。这种原始的方法有，然而，一些限制， 即，复制品，这阻碍了在复杂的组织分别映射细胞如脑标记分子的有意义的相关性的严重碎裂。大约10年前，茂 ​​源和深泽逐步提高技术2。这是通过从另一组在科罗拉多州立大学的博尔德实验室，谁也显著提高的技术中，特别是用于间隙连接3的研究科学家努力平行。 在冷冻压裂协议和机器的改进，以及引入快速冷冻的（在高压下），现在允许调查者产生的相对大的尺寸和标本不间断复制品喜大多数细胞成分的GH品质的图像，而不限制和强大的化学生产的注视文物。 的FRIL技术提供一个高度定量的很大的优点，在映射组织学一种或多种蛋白质（同时）和细胞学鉴定细胞复合组织内，如大脑的原位鉴别，与前和突触后的平面视图的额外的优点在一个单一的复制品的元件。因此，FRIL技术，尽管它的许多技术难题，适用于一些非常显著科学突破的承诺，尤其是对个别突触的结构和功能特性的相关性。在过去的几十年里，已获得的大量信息的结构，分子组成，与突触的生理功能;但突触在形态和分子高度多样化取决于前和突触后PA租金神经元4。仅适用于突触类型的少数人结构与功能的研究，到目前为止5-7完成。这主要是由于该防止前和突触后元件的性质的精确识别技术约束。 超微结构分析提供了关键见解跨越不同突触联系两个突触大小和内容方面突触后膜特化在神经递质受体6的可变性，这对突触传递的强度和可塑性很大的影响。此外，大量的研究表明，在不同类型的突触表达离子型谷氨酸受体的数量在afferent-和目标依赖的方式7-10调节。 这里，一个方法概述允许突触后膜特化的结构和受体组成的分析与限定ð突触前元件和功能。这种方法需要最近开发的光敏感藻蛋白，如channelrhodopsin2（的ChR2）的突触前表达的优点，并且FRIL技术来分析的α氨基-3-羟基-5-甲基-4-异唑突触后表达的图案酸（AMPA-RS）和N-甲基-D-天冬氨酸（NMDA-RS）谷氨酸受体。这表现在从后丘脑，内侧膝状核（PIN / MGN）始发到杏仁核（ITC）的插层细胞群众的神经元轴突形成突触。 ITC神经元是在围绕基底外侧杏仁络合物（BLA）11,12簇组织小刺的GABA能细胞。ITC神经元已知从BLA主要神经元接收兴奋性输入和定位的中央核（CEA），从而作为抑制栅极运作对信息的BLA和CEA 12-15之间流动。 最近，我们证明了IT位于BLA和CEA之间的内-背集群用C神经元也收到来自感官丘脑及颞皮层区域，这是在巴甫洛夫的听觉恐惧条件16期间学习的恐惧直接修改和收敛的兴奋性投入。恐惧空调是联想学习的大脑机制方面的最好理解的形式之一。在恐惧条件，最初的中性条件刺激（CS， 例如，一个音）是搭配厌恶非条件刺激（US， 例如，一个温和的脚震动）造成了CS-美国协会和空调的恐惧反应17，18。兴奋来自丘脑和新皮质区，其中携带表示CS和美国信息输入，分别被称为会聚到杏仁核（LA）的外侧核的锥体神经元和经受塑性19。我们以前的工作表明，感觉输入的信息也传达给ITC的神经元并行<s达&gt; 16。 作为对个体感觉输入的一种机械的分子分析的第一步突触到ITC神经元，我们使用腺伴随病毒（AAV）来表达的ChR2标记与黄色荧光蛋白（YFP）。所述AAV注入PIN / MGN和轴突终端被他们的ChR2-YFP的表达鉴定。我们所使用的FRIL技术生成两个面，以评估在与ITC神经元的PIN / MGN轴突终末形成突触的突触后AMPA-R和NMDA-Rs的密度。

<table><tbody><tr><td>&lt;strong&gt;Surgery&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Stereotactic frame</td><td>Stoelting, USA</td><td>51670</td><td>can be replaced by other stereotactic frame for mice</td></tr><tr><td>Stereotactic frame mouse adaptor</td><td>Stoelting, USA</td><td>51625</td><td></td></tr><tr><td>Gas anesthesia mask for mice</td><td>Stoelting, USA</td><td>50264</td><td>no longer available, replaced by item no. 51609M</td></tr><tr><td>Pressure injection device, Toohey Spritzer</td><td>Toohey Company, USA</td><td>T25-2-900</td><td>other pressure injection devices (e.g. Picospritzer) can be used</td></tr><tr><td>Kwik Fill glass capillaries</td><td>World Precision Instruments, Germany</td><td>1B150F-4</td><td></td></tr><tr><td>Anesthesia machine, IsoFlo</td><td>Eickemeyer, Germany</td><td>213261</td><td></td></tr><tr><td>DC Temperature Controler and heating pad</td><td>FHC, USA</td><td>40-90-8D</td><td></td></tr><tr><td>Horizontal Micropipette Puller Model P-1000</td><td>Sutter Instruments, USA</td><td>P-1000</td><td></td></tr><tr><td>Surgical tool sterilizer, Sterilizator 75</td><td>Melag, Germany</td><td>08754200</td><td></td></tr><tr><td>rAAV-hSyn-ChR2(H134R)-eYFP (serotype 2/9)</td><td>Penn Vector Core, USA</td><td>AV-9-26973P</td><td></td></tr><tr><td>fast green</td><td>Roth, Germany</td><td>0301.1</td><td></td></tr><tr><td>Isoflurane Anesthetic, Isofuran CP (1 ml/ml)</td><td>CP Pharma, Germany</td><td></td><td></td></tr><tr><td>Antiseptic, Betadine (providone-iodine)</td><td>Purdure Products, USA</td><td>BSOL32</td><td>can be replaced by other disinfectants</td></tr><tr><td>Analgesic, Metacam Solution (5 mg/ml meloxicam)</td><td>Boehringer Ingelheim, Germany</td><td></td><td>can be replaced by other analgesics</td></tr><tr><td>Bepanthen eye ointment</td><td>Bayer, Germany</td><td>0191</td><td>can be replaced by other eye ointments</td></tr><tr><td>Drill NM3000 (SNKG1341 and SNIH1681)</td><td>Nouvag, Switzerland</td><td></td><td></td></tr><tr><td>Sutranox Suture Needle</td><td>Fine Science Tools, Germany</td><td>12050-01</td><td></td></tr><tr><td>Braided Silk Suture</td><td>Fine Science Tools, Germany</td><td>18020-60</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Tissue preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde EM grade</td><td>Agar Scientific Ltd., United Kingdom</td><td>AGR1018</td><td></td></tr><tr><td>Saturated picric acid solution</td><td>Sigma-Aldrich, USA</td><td>P6744-1GA</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;-2H&lt;sub&gt;2&lt;/sub&gt;0</td><td>Merck Millipore, Germany</td><td>1065860500</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;-2H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Merck Millipore, Germany</td><td>1063451000</td><td></td></tr><tr><td>NaCl</td><td>Merck Millipore, Germany</td><td>1064041000</td><td></td></tr><tr><td>4N NaOH</td><td>Carl Roth, Germany</td><td>T198.1</td><td></td></tr><tr><td>Thiopental</td><td>Sandoz, Austria</td><td>5,133</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich, USA</td><td>G5516-500ML</td><td></td></tr><tr><td>GenPure ultrapure water system</td><td>Thermo Fisher Scientific, USA</td><td>50131235</td><td></td></tr><tr><td>Peristaltic pump</td><td>ISMATEC, Germany</td><td>ISM 930C</td><td></td></tr><tr><td>Filter Paper</td><td>MACHEREY-NAGEL, Germany</td><td>MN 615 1/4</td><td></td></tr><tr><td>Vibroslicer, VT1000S</td><td>Leica Microsystems, Austria</td><td></td><td></td></tr><tr><td>Ophthalmic scalpel</td><td>Alcon Laboratories, USA</td><td></td><td>can be replaced by other ophthalmic scalpels</td></tr><tr><td>Perfusion cannula</td><td>Vieweg, Germany</td><td>F560088-1</td><td>can be replaced by similar items from other companies</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;High-pressure&amp;nbsp; Freezing&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Copper carriers</td><td>Engineering Office M. Wohlwend, CH</td><td>528</td><td></td></tr><tr><td>Sidol Polish</td><td>Henkel, Germany</td><td></td><td>can be replaced by same item from other companies</td></tr><tr><td>Chamois skin</td><td>Household supply store</td><td></td><td></td></tr><tr><td>Hole punch, 1,5mm</td><td>Stubai, Austria</td><td></td><td>can be replaced by same item from other companies</td></tr><tr><td>Denatured ethanol</td><td>Donauchem, Austria</td><td></td><td>can be replaced by same item from other companies</td></tr><tr><td>Acetone</td><td>Roth, Germany</td><td>9372.5</td><td>CAUTION!</td></tr><tr><td>High Pressure Freezing Machine HPM 010</td><td>BalTec, CH; now Leica Microsystems</td><td>HPM010</td><td>not produced any more, substituted by LeicaEM HPM100</td></tr><tr><td>Stereo-microscope</td><td>Olympus, Japan</td><td>SZX10</td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td>CAUTION!</td></tr><tr><td>Cryo-vials</td><td>Roth, Germany</td><td>E309.1</td><td>can be replaced by same item from other companies</td></tr><tr><td>CryoCane</td><td>Nalge Nunc International,USA</td><td>5015-0001</td><td>can be replaced by same item from other companies</td></tr><tr><td>CryoSleeve</td><td>Nalge Nunc International,USA</td><td>5016-0001</td><td>can be replaced by same item from other companies</td></tr><tr><td>Liquid nitrogen storage vessel</td><td>Cryopal, France</td><td>GT38</td><td>can be replaced by same item from other companies</td></tr><tr><td>Non-ionic detergent (Lavocid)</td><td>Werner &amp;amp; Mertz Professional, Germany</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Freeze-fracture and Replication&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sandblaster, Mikromat 200-1</td><td>JOKE Joisten &amp;amp; Kettenbaum, Germany</td><td>SANDURET 2-K</td><td>can be replaced by same item from other companies</td></tr><tr><td>Siliciumcarbid SIC 360, grain size 25 - 21&amp;micro;</td><td>JOKE Joisten &amp;amp; Kettenbaum, Germany</td><td>955932</td><td></td></tr><tr><td>Freeze Fracture System BAF 060</td><td>BalTec, CH; now Leica Microsystems</td><td>BAF060</td><td></td></tr><tr><td>Ceramic 12 well plate</td><td>Gr&amp;ouml;pel, Austria</td><td>14511</td><td>can be replaced by same item from other companies</td></tr><tr><td>Trizma base</td><td>SIGMA, USA</td><td>T1503</td><td>can be replaced by same item from other companies</td></tr><tr><td>Trizma hydrochloride</td><td>SIGMA, USA</td><td>T3253</td><td>can be replaced by same item from other companies</td></tr><tr><td>Sodium chloride</td><td>Merck, Germany</td><td>1,064,041,000</td><td>can be replaced by same item from other companies</td></tr><tr><td>SDS, Sodium lauryl sulfate</td><td>Roth, Germany</td><td>5136.1</td><td>CAUTION! ;&amp;nbsp; can be replaced by same item from other companies</td></tr><tr><td>Sucrose</td><td>Merck, Germany</td><td>1,076,871,000</td><td>can be replaced by same item from other companies</td></tr><tr><td>TRIS</td><td>Roth, Germany</td><td>5429.3</td><td>can be replaced by same item from other companies</td></tr><tr><td>Universal Hybridization Oven</td><td>Binder, Germany</td><td>7001-0050</td><td>can be replaced by same item from other companies</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Immunolabelling&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BSA</td><td>SIGMA, USA</td><td>A9647</td><td>can be replaced by same item from other companies</td></tr><tr><td>Anti-GFP Antibody</td><td>Molecular Probes, USA</td><td>A11122</td><td></td></tr><tr><td>Anti-pan-AMPAR Antibody</td><td>Frontier Institute, Japan</td><td>pan AMPAR-GP-Af580-1</td><td></td></tr><tr><td>Anti-NMDAR1 Antibody, clone 54.1</td><td>Merck Millipore, Germany</td><td>MAB363</td><td></td></tr><tr><td>Opioid Receptor-Mu (MOR) Antibody</td><td>ImmunoStar, USA</td><td>24216</td><td></td></tr><tr><td>EM goat anti-guinea pig, 5 nm; secondary antibody</td><td>BBInternational, UK</td><td>EM.GAG5</td><td></td></tr><tr><td>EM goat anti-rabbit, 15 nm; secondary antibody</td><td>BBInternational, UK</td><td>EM.GAR15</td><td></td></tr><tr><td>Donkey anti-rabbit, 10nm, secondary antibody</td><td>AURION, Netherlands</td><td>DAR 10nm</td><td></td></tr><tr><td>Copper grids, 100 Parallel Bar</td><td>&amp;nbsp;Agar scientific, UK</td><td>G2012C</td><td></td></tr><tr><td>Incubator</td><td>Major Science, USA</td><td>MO-RC</td><td>can be replaced by same item from other companies</td></tr><tr><td>Pioloform Powder</td><td>&amp;nbsp;Agar scientific, UK</td><td>R1275</td><td></td></tr><tr><td>Chloroform</td><td>Roth, Germany</td><td>3313.1</td><td>CAUTION! ;&amp;nbsp; can be replaced by same item from other companies</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;EM analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Philips CM120 TEM</td><td>Philips/FEI</td><td></td><td></td></tr><tr><td>Morada CCD camera</td><td>Soft Imaging Systems, Germany</td><td></td><td></td></tr><tr><td>iTEM Ver. 5.2, imaging software</td><td>Soft Imaging Systems, Germany</td><td></td><td></td></tr></tbody></table>

combined optogenetic and freeze-fracture replica immunolabeling to examine input-specific arrangement of glutamate receptors in the mouse amygdala

冷冻断裂电子显微镜已在超微结构研究的重大技术超过40年。然而，由于缺乏有效的手段来研究膜的分子组成产生了它的使用显著下跌。近来，出现了在冷冻断裂电子显微镜得益于通过免疫标记以显示完整的膜蛋白的有效方法的发展的一个主要的复兴。这些方法之一被称为洗涤剂溶解的冷冻断裂副本免疫标记（FRIL）。 与光遗传学的FRIL技术的组合允许中央突触的结构和功能特性的一个相关的分析。使用这种方法，可以鉴定和由它们各自的一个标记channelrhodopsin的表达和特定的分子标志物表征前和突触后神经元。 glutamat的突触后膜专业化的独特的外观GABA能突触进一步允许，当离子型谷氨酸受体的标记，来量化和分析这些受体的intrasynaptic分布。在这里，我们给的准备配对副本和如何immunolabel它们所需的程序一步一步的说明。我们也将讨论FRIL技术的警告和限制，特别是与潜在的采样偏见有关的。的FRIL技术的高再现性和通用性，当与光遗传学相结合，提供了用于在大脑识别的神经元微电路突触传递的不同方面表征一个非常强大的方法。 在这里，我们提供了一个例子这种方法是如何用在鼠标杏仁核的细胞插群众的神经元洞察兴奋性突触的结构与功能的关系。特别是，我们已经调查离子型谷氨酸受体的表达在确定的输入或从丘脑后部板内核和内侧膝​​状核iginated。这些突触被证明传递感觉信息有关的恐惧学习和对恐惧条件经受塑性变化。

的FRIL技术，当与微生物来源21， 即信道集成在质膜和有效地沿轴突顺行输送的光遗传学致动器的表达相结合，允许在限定的亚组来定量检验AMPA-R和NMDA-Rs的突触后表达突触。这显示在这里从不同丘脑核（如 PIN / MGN）始发到杏仁核神经元ITC轴突。这种方法使个体感觉输入突触到ITC神经元，一组细胞的已耐火仔细的解剖和分子表征到目前为止的分子分析。 立体定向注射的rAAV-的ChR2-YFP的入PIN / MGN 4周后，的ChR2-YFP阳性轴突密集支配洛杉矶，所述amygdalostriatal过渡区域（Astria）和内侧paracapsular我<html>在杏仁核的TC簇，与以前的跟踪完全一致的图案研究16,22。我们还检测到强烈的金上从腺相关病毒的ChR2-YFP-在冷冻断裂复制品轴突和终端的P-面免疫标记为的ChR2-YFP注射的小鼠（ 图5A），但不是从非注射的小鼠的副本。在一个复制品谷氨酸突触的突触后膜专业化可以观察到作为质膜2，23的E-面膜内颗粒（IMP）中的集群，并且常伴有其突触前质膜7的P面（ 图5B-C）。允许识别由PIN / MGN轴突终末形成谷氨酸突触的突触后专业化这些功能（ 图5和6）。我们标记AMPA-R的与识别所有四个亚基（GluA1-4），而使用抗必要NR1亚基的抗体检测NMDA-R的抗体。 ntent"FO:保together.within页="1"&gt;由于缺乏工具来检测相同的副本是否这些突触用树突棘或ITC神经元的轴做，我们标注相应的副本脸μ阿片受体，作为ITC的神经元表达突触后高水平这些受体24，这使用了采用标策略所需的相同的突触后轮廓的识别的两个副本（ 图5C-F和图6A-D）（ 图4E） 。 <img alt="图5" src="/files/ftp_upload/53853/53853fig5.jpg" /> 图5的ChR2-YFP和离子型谷氨酸受体的检测通过免疫颗粒副本。（A）横断裂轴突终端（浅蓝色）和标记有15nm的金颗粒检测的ChR2-YFP其P-面的一小部分。在终端内，膜Ô˚F众多突触小泡可以观察（SV）。注意免疫标记的限制的质膜很大一部分的特异性。标记为的ChR2识别终端作为从PIN / MGN始发。该终端形成具有脊的不对称突触。突触后膜专业化（PSD）在E-面显示膜内粒子的特性簇，并标记为5nm的金颗粒揭示AMPA-卢比。 （B）一种的ChR2表达轴突（标有15纳米的金颗粒）的P面所示接壤2树突的它们具有标记为5nm的金颗粒露出的NMDA-R的2的PSD之一。 （CD）一个PIN / MGN-ITC突触前和突触后膜的相对面。 （C）的端子的P面表达的ChR2（标有15纳米的金粒子）并延伸超过两树突轴，包含标记为AMPA-卢比（5纳米的金颗粒）二的PSD其中之一的E面。 （ <strong&gt;的D）的两个树突的相应的P-面标记为μ阿片受体（10纳米的金粒子）。 （EF）由虚线所概述的区域的放大图。比例尺:500纳米<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53853/53853fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 为在PIN / MGN-ITC突触AMPA-卢比Immunoparticles发现所有的IMP簇以上，这表明突触后专业化（ 图5E）中的均匀分布。在PIN / MGN观察到显著较高（非配对t检验P &lt;0.018）AMPA-R标记密度突触到ITC棘（715±38金颗粒/微米2，N = 32）相比，突触到ITC树突（590 ±44金颗粒/微米2，N = 32）。总体而言，在PIN / MGN-ITC突触AMPA-R的密度呈相对较低方差（方差的系数，CV = 0.37）与均匀分布是一致的。 为在PIN / MGN-ITC突触的NMDA-R的Immunoparticles常常突触后的IMP簇（ 图5B）内观察到的分布不均。 NMDA-R标记的密度相似（非配对t检验p = 0.39）之间的PIN / MGN突触到ITC棘（1070±153金颗粒/微米2，N = 8）和ITC树突（812±183金粒子/微米2，N = 9）。不像所观察为AMPA-卢比，在PIN / MGN-ITC突触的NMDA-Rs的密度是高度变量（CV = 0.54）。 <img alt="图6" src="/files/ftp_upload/53853/53853fig6.jpg" /> 图6. AMPA-R和NMDA-R的免疫标记在鉴定PIN / MGN-ITC突触。 （AB）一个PIN / MGN-ITC的前和突触后膜的相对面作出到树突棘其中终端的P面表达的ChR2突触（标有15纳米的金颗粒）和在PSD到树突棘标记为AMPA-卢比（5纳米金粒子）。 （CD）由虚线所概述的区域的放大图。这些区域已经被旋转约45°逆时针允许的PSD更好的视野。 （E） - AMPA-R颗粒对在ITC的脊骨和树突突触区的数目的散点图。在这两种结构中，一个正相关已经观察到。 （F）对在ITC刺和树突突触区的NMDA-R的粒子数的散点图。检测只在树突棘一个显著的正相关关系。比例尺:500纳米<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53853/53853fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 因为部分常常覆盖在突触后的IMP簇突触前质膜的P面，我们可以估算的只有30％的突触的突触区（棘:平均面积0.032微米2，范围:0.007〜0.063微米2中，n = 8;树突:0.047 平方微米，范围:0.024至0.166微米2，N = 11）。这是在一个类似的范围内前面所分析的端脑谷氨酸突触25。 在这两种刺和树突，金immunoparticles在个别突触AMPA-Rs的数目呈正突触区域相关（斯皮尔曼，棘:R = 0.88，树突:R = 0.60，P &lt;0.0001）（ 图6E）。相反，发现金immunoparticles为NMDA-Rs的数目在棘（斯皮尔曼，棘:R = 0.90，P &lt;0.002）与突触区域关联，但不是在树突性（r = 0.21，P = 0.29）（ 图6F ）。

Watch this Scientific Journal Video about Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala at JoVE.com

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

结合光遗传学和冷冻断裂免疫标记副本研究在老鼠杏仁核谷氨酸受体的特定投入安排

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

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11.0K 次观看.  Medical University of Innsbruck. This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

视频: Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

嵌入后突触蛋白的免疫标记海马切片培养

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

科研

神经科学

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

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.

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.

高分辨率定量免疫膜受体在视网膜带状突触分析

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

在脑片恐惧电路体外光遗传学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

抗体喂养方法研究分离原发性希马培养物中的谷氨酸受体贩运

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

正常小鼠和亨廷顿小鼠急性脑切片中谷氨酸释放和服用的单突触指标

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Immunolabeling of Neuronal Membrane Proteins in a Freeze-fractured Specimen of Mouse Brain Tissue

<a href='https://app-jove-com.bdigitaluss.remotexs.co/v/53853/combined-optogenetic-freeze-fracture-replica-immunolabeling-to'>Source: Sch&#246;nherr, S., et. al. Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala. J. Vis. Exp. (2016)</a>This video demonstrates the immunolabelling of freeze-fractured replicas of mouse brain tissue for electron microscopy. The fractured and coated replicas are digested to expose receptors. Primary and gold-tagged secondary antibodies are added to label specific neuronal membrane receptors. The replica is mounted on a grid and visualized under an electron microscope.

Source: Sch&#246;nherr, S., et. al. Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate ...

JoVE Encyclopedia of Experiments

Neuropathology

Epilepsy and Seizure Disorders

Measuring Protein Expression in the Rodent Brain Using Near-Infrared Fluorescence and High-Resolution Scanning 

Source:&#160;<a style='text-decoration:none;' href='https://app-jove-com.bdigitaluss.remotexs.co/v/59685/using-near-infrared-fluorescence-and-high-resolution-scanning-to-measure-protein-expression-in-the-rodent-brain'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Kimmelmann-Shultz, B.,&#160;et. al.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>&#160;<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>&#160;(2019)</a> &#160;This video demonstrates a near-infrared fluorescence and high-resolution scanning method for measuring protein expression in the rodent brain.

Source:&#160;Kimmelmann-Shultz, B.,&#160;et. al.&#160;Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the ...

Neuroimaging

Synaptic & Cellular Imaging

Visualization and Mapping of Neuronal Pathways in an Amygdala Brain Slice

Source: Bosch, D., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53628">Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices.</a> J. Vis. Exp. (2016).
This video demonstrates a procedure for analyzing synaptic connectivity between medial prefrontal cortex (mPFC) and amygdala neurons using acute amygdala brain slices from mice. The mPFC neurons in these mice express channelrhodopsin fused to a fluorescent protein. The channelrhodopsins facilitate ion influx and action potential generation in the mPFC neurons upon light activation. Consequently, mPFC neurons release neurotransmitters that bind to postsynaptic amygdala neurons, triggering postsynaptic currents that are recorded to visualize mPFC-amygdala connectivity.

杏仁核脑切片中神经元通路的可视化和映射

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Visualization and ...

Neurophysiology

Imaging and Optical Techniques

An In Vitro Technique to Study Glutamate Receptor Trafficking in Hippocampal Neurons

Source: Chiu, A. M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/59982">An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures.</a> J. Vis. Exp. (2019)
This video demonstrates an antibody-feeding approach to study the trafficking of glutamate receptors in primary hippocampal neuronal cultures. This approach allows the observation of receptor populations at both the plasma and internal membranes. Discrimination between these subsets is achieved by labeling the receptors before and after membrane permeabilization, using the same primary antibody but secondary antibodies conjugated to different fluorophores.

一种研究海马神经元中谷氨酸受体运输的体外技术

1. Preparation Before Labeling

	Preparation and maintenance of primary hippocampal cultures&#8203;
	
		Prepare primary hippocampal cultures at a density ...

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

Generation of Tilted Amygdala Slices from a Mouse Brain

Source: Bosch, D., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53628">Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices.</a> J. Vis. Exp. (2016)
The video demonstrates the generation of tilted amygdala slices from a mouse brain. The brain is positioned at an angle on the vibratome specimen stage to obtain slices containing the amygdala and axons from the medial prefrontal cortex that form synaptic connections with the amygdala. The brain slices are maintained at the physiological temperature to preserve the tissue for further experiments.

从小鼠大脑中生成倾斜的杏仁核切片

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Acute Slices

	...