This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (23700412, 25122707 and 26670090 to S.Y.).

涉及动物主题程序已在滨松医科大学获批准的机构动物护理和使用委员会。 1.矩阵的制备<ol><li>煮沸4-8硅氧烷基质在微波或热板上5分钟，使它们能够完全干燥下层流（条纹面朝上）1小时。 注意:下面的程序应在层流下进行。 </li><li>吹压缩空气或用透明胶带从矩阵的条纹侧上的灰尘。保持条纹侧干净，因此可以牢固地附着在板面。 </li><li>小心用手指（;条纹面朝下每皿矩阵）按压放置矩阵到6厘米的塑料盘中。避免让基质与盘之间的空气气泡。标记上的培养皿的底侧的条纹的位置。 </li><li>如果矩阵未能附加，从步骤1.2重复。 </li></ol>2.条纹的产生和层粘连蛋白涂层的神经文化<ol><li>制备荧光标记的重组蛋白质（25微升每注射[步骤2.2]或每对的放置方法[步骤2.2.1] 100微升）通过混合与Fc标签的蛋白（10-50微克/毫升）和荧光染料 - 缀合的抗人Fc抗体（1至3的比例:30-150微克/毫升）在磷酸盐缓冲盐水（PBS）。然后，孵育该混合物在RT下30分钟。 </li><li>使用22-G的注射器，通过对基体的侧面的小孔注入25μl的在上一步（2.1）中制备的重组蛋白（箭头图1A，1C和1E）。 <ol><li>可替代地，将100微升的重组蛋白的插入矩阵的顶部狭缝和抽吸大约一半从小孔的溶液（在图1A，1C和1E的箭头），因此，重组蛋白质保留在条纹区域。 </li></ol></li><li>孵育30英里的菜在细胞培养培养箱Ñ在37℃。 </li><li>放置300微升的PBS顶入狭缝（ 图1B）和抽吸从位于矩阵侧的小孔（ 图1A，C和E的箭头），以除去未连接的重组蛋白。不要完全吸出PBS，因为蛋白质会干。重复此步骤两次。 （3次总） </li><li>小心地取出矩阵，立即将约100微升控制蛋白质（10-50微克/毫升; Fc）的覆盖整个区域的条纹，创建备用涂层。然后，孵育盘30分钟在37℃下在细胞培养孵化器。 </li><li>用PBS三次，大衣呢〜洗餐具表面用100μl20μg/ ml的层粘连蛋白在PBS中后。 注:应层粘连蛋白在冰上解冻，以防止凝固。 </li><li>在细胞培养培养箱孵育1小时培养皿在37℃。 </li><li>用PBS洗涤餐具表面三次加文化中介嗯。放置在37℃，直到用在5％CO 2培养箱培养介质涂覆的培养皿中。 </li></ol> 3.培养海马神经元从E15.5小鼠胚胎<ol><li>通过颈椎脱位安乐死的母亲和隔离3 E15.5胚胎。 </li><li>切开皮肤及颅骨矢状在头用剪刀中线，并用小汤匙取出大脑。小心转移大脑装有3毫升Hank氏平衡盐溶液（HBSS）中的60毫米培养皿中。 </li><li>使用手术刀，切出的半球包括皮质，海马和纹状体（ 图2A）。然后，通过仔细地除去脑膜解剖出来的海马区，并解剖海马组织转移到含有2毫升的HBSS溶液15毫升离心管中，在冰上（ 图2B-C）。从3个胚胎中的管，其足以在8道菜中培养的神经元收集6海马。 </li><li>替换轰BSS溶液用胰蛋白酶/ EDTA溶液（2毫升）中，并在37℃反应管在水浴中15分钟。 注:吸HBSS不离心，不通过倾斜管倒出HBSS解决方案。 </li><li>通过加入500微升的胎牛血清（FBS）的中和胰蛋白酶活性。 </li><li>离心在100 xg离心混合物5分钟。除去上清液并用2ml培养基洗涤沉淀。重复此步骤一次。 </li><li>吸管使用1000微升尖端具有大孔的海马上下10次培养基（切尖）以解离的组织。 </li><li>更改为普通（未切割）1000微升尖端和吸管游离组织上下获得单细胞悬液。 </li><li>通过将其通过80-100微米网状细胞过滤分离出单个细胞。 </li><li>离心在150 xg离心过滤的单细胞悬浮液5分钟。 </li><li>通过抽吸去除上清，并悬浮佩尔等（神经元）在2ml培养基中。 </li><li>算使用血球台盼蓝染色来确定密度和生存力的细胞。然后，板在150微升培养基10000神经元为每一组stripes.The悬浮液应小心放置，以覆盖整个条纹区域。 </li></ol> 4.可视化使用抗TAU1抗体细胞体和免疫荧光染色轴突<ol><li>培养24小时后，吸出介质，而不会干扰贴壁细胞，并用温热的预4％低聚甲醛（PFA）/ 4％蔗糖/ PBS中在RT下15分钟固定的神经元。 </li><li>用PBS洗涤该板表面的3倍。如果需要，可以使用防水用笔画出周围的条纹区域了一圈，以减少所需的抗体量。 </li><li>透用0.3％的Triton X-100 / PBS中的神经元5分钟。 </li><li>孵育用含3％驴血清和0.1％的Triton X-100用于阻断溶液中的神经元30分钟，随后温育与抗TAU1抗体（1:200），在1％的驴血清/ 0.1％的Triton X-100 / PBS中O / N在4℃。 </li><li>用PBS洗涤该板表面的3倍。孵化用荧光团缀合的抗小鼠IgG抗体的神经细胞在1％牛血清白蛋白（BSA）/0.1%的Triton X-100 / PBS中进行30分钟。 </li><li>用PBS洗涤该板表面的3倍。使用50微升抗褪色溶液中18毫米×18毫米遮玻璃盖的条纹区域。避免气泡，这与成像造成干扰。 </li><li>获得使用合适的系统的荧光显微镜， 例如 ，×10的物镜，过滤套的GFP和RFP，和1000毫秒的曝光时间（ 图3）的荧光图像。 </li></ol>

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</ol>

The authors have nothing to disclose.

这个协议描述了使用重组蛋白质和分离的神经元从E15.5小鼠海马的条纹检测。该测定允许神经元的排斥力，吸引力，或中性反应于感兴趣的重组蛋白放置在条纹图案的有效观察。该协议的主要优点是用于产生条纹，其中蛋白质被直接印刷到塑料盘的表面上的简单的方法，相对于传统的方法，该方法需要特殊的基质，真空系统，和一个核孔膜20 21。为了使蛋白条带，标记的重组蛋白可通过一?...

轴突导向是由新形成的神经元的神经系统1,2开发过程中发出轴突到其目标的过程。显影轴突在其前端称为生长锥携带一个高度能动结构。生长锥检测外线索导航轴突的路径。导向分子，如狭缝，脑信号蛋白，肝配蛋白和，可以吸引或取决于它们与轴突1,3,4合适受体和共同受体相互作用击退轴突。该激活受体信号传送到影响其轴突和生长锥的运动细胞骨架组织生长锥。 各种方法已被开发来评估诱和驱避剂分子的作用。化疗诱和驱虫剂可施用与梯度浓度的生长/培养基（ 例如 ，唐恩的腔室或μ幻灯片）由微-对5,6，在一个高度集中点ipette（ 例如 ，车削试验）7，或通过浴应用程序（ 例如 ，生长锥塌陷测定法）8,9均匀浓度。 其它方法包括条纹测定或微接触印刷（μCP），其中一个化学引诱或斥涂覆的板的表面上作为底物10-12。 Thestripe法最初是由朋霍费尔和他的同事开发了在1987年以分析小鸡眼膜-顶盖系统13地形测绘。原始方法需要真空系统的外壳蛋白上使用的条纹和啮合矩阵聚碳酸酯核孔膜。在以后的版本中，重组蛋白用窄缝硅矩阵14,15直接印刷的培养板的表面上在一个条纹图案。近日，各研究小组已成功地应用这个条纹检测到轴突导向分子的活动16-21分析。 <p claSS ="jove_content"&gt;在这里，我们提出详细的协议为措施分离的海马神经元轴突导向分子的吸引或排斥的条纹检测。值得注意的是，这种方法可以在最低限度地配备实验室设置被应用。对于该测定，是在使用硅基质的塑料培养皿具有90微米缝隙产生并涂覆有层粘连蛋白的荧光标记的底物和对照蛋白的交替条纹。在我们的示范，解离从E15.5小鼠海马神经元分别在交替纤连蛋白和跨膜蛋白2（FLRT2）富含亮氨酸和控制Fc蛋白21的重组胞外结构域的条纹进行培养。培养24小时后，无论是轴突和神经元的细胞体强烈从FLRT2条纹排斥。用抗TAU1抗体染色揭示〜神经元的90％被分布在与Fc包被的区域，相比于上FLRT2-Fc的〜10％，这表明FLRT2具有很强的推斥力功能海马神经元21。

<table><tbody><tr><td>15 ml centrifuge tube</td><td>Violamo&amp;nbsp;</td><td>1-3500-01</td><td></td></tr><tr><td>4% Paraformaldehyde (PFA)</td><td>Nacalai</td><td>01954-85</td><td></td></tr><tr><td>Alexa Fluor 488 Goat anti-human IgG antibody</td><td>Thermo Scientific</td><td>A11013</td><td></td></tr><tr><td>Alexa Fluor 594 Donkey anti-mouse IgG antibody</td><td>Thermo Scientific</td><td>A-21203</td><td>Dilution 1/500</td></tr><tr><td>Anti-Tau1 antibody</td><td>Chemicon</td><td>MAB3420</td><td>Dilution 1/200</td></tr><tr><td>Antifade</td><td>Thermo Scientific</td><td>P7481</td><td>Alternative mounting media may be used</td></tr><tr><td>B27 supplement</td><td>Thermo Scientific</td><td>17504-044</td><td>Dilution 1/50</td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>01-2030-2</td><td></td></tr><tr><td>Cell strainer 100 um</td><td>BD Falcon</td><td>352360</td><td></td></tr><tr><td>Centrifugation machine</td><td>Kubota</td><td>2410</td><td></td></tr><tr><td>Cover glass 18mmx18mm</td><td>Matsunami</td><td>18x18 mm No. 1</td><td></td></tr><tr><td>DAKO pen</td><td>DAKO</td><td>S2002</td><td>Alternative water-repellent pen may be used</td></tr><tr><td>Disposable scalpel</td><td>Feather</td><td>2975#11</td><td></td></tr><tr><td>FBS</td><td>Thermo Scientific</td><td>10437-028</td><td></td></tr><tr><td>Fluorecent microscope</td><td>Nikon</td><td>E600</td><td></td></tr><tr><td>Forceps No. 5</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>GlutaMAX</td><td>Thermo Scientific</td><td>35050-061</td><td>Dilution 1/100</td></tr><tr><td>Hamilton Syringe</td><td>Hamilton</td><td>805N</td><td>22 gauge, 50 uL</td></tr><tr><td>HBSS</td><td>Thermo Scientific</td><td>14170-112</td><td></td></tr><tr><td>Human IgG, Fc Fragment</td><td>Jackson</td><td>009-000-008</td><td></td></tr><tr><td>Laminin</td><td>Thermo Scientific</td><td>23017-015</td><td></td></tr><tr><td>Neurobasal</td><td>Thermo Scientific</td><td>21103-049</td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Jackson</td><td>017-000-121</td><td></td></tr><tr><td>PBS</td><td>Nacalai</td><td>14249-24</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Scientific</td><td>15070-063</td><td>Dilution 1/100</td></tr><tr><td>Plastic culture dish, 60 mm</td><td>Thermo Scientific</td><td>150288</td><td></td></tr><tr><td>Silicone Matrices</td><td></td><td></td><td>Available and purchasable from Prof. Martin Bastmeyer (bastmeyer@kit.edu)</td></tr><tr><td>Stereo Microscope</td><td>Olympus</td><td>SZ61</td><td></td></tr><tr><td>Tip, 1000 uL</td><td>Watson</td><td>125-1000S</td><td></td></tr><tr><td>Transparent sticky tape</td><td>Tesa</td><td>57315</td><td>Alternative sticky tape may be used</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Trypan blue, 0.4%</td><td>Bio-Rad</td><td>145-0013</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Thermo Scientific</td><td>25300-054</td><td></td></tr><tr><td>Culture medium</td><td></td><td></td><td>Neurobasal supplemented with B27, GlutaMAX and Penicillin-Streptomycin.</td></tr></tbody></table>

stripe assay to study the attractive or repulsive activity of a protein substrate using dissociated hippocampal neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

从E15.5小鼠分离的海马神经元接种并培养24 h上的条纹的荧光标记控制的Fc（ 图3A-C）或FLRT2-Fc的（ 图3D-F），与非标记的对照Fc的交替。在这两种情况下，神经元聚集和扩展它们的轴突束为。在控制的Fc / Fc的条纹，神经元均匀地分布在荧光标记和非标记的条纹，它们扩展它们的轴突在随机的方向（ 图3A-C）。与此相反，在FLRT-2FC / Fc的条?...

Watch this Scientific Journal Video about Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons at JoVE.com

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

条纹分析来研究蛋白质底物使用游离海马神经元的吸引力或令人反感的活动

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

stripe-assay-to-study-attractive-or-repulsive-activity-protein

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.6K 次观看.  Hamamatsu University School of Medicine. Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons. 

视频: Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

检测蛋白质棕榈酰化培养海马神经元通过免疫沉淀和酰基-生物素交易所（ABE）

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

科研

神经科学

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. Therefore, recent research invented different tools to target protein expression in mammalian cell lines or even animal models, including RNA and antibody interference. While the first strategy has gathered much attention during the past two decades, peptides &#160;mediating a translocation of antibody cargos across cellular membranes and into cells, obtained much less interest. In this publication, we provide a detailed protocol how to utilize a peptide carrier named Chariot in human embryonic kidney cells as well as in primary hippocampal neurons to perform antibody interference experiments and further illustrate the application of three-dimensional reconstructions in analyzing protein function. Our findings suggest that Chariot is, probably due to its nuclear localization signal, particularly well-suited to target proteins residing in the soma and the nucleus. Remarkably, when applying Chariot to primary hippocampal cultures, the reagent turned out to be surprisingly well accepted by dissociated neurons.

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

研究蛋白质功能和改变的蛋白的表达由源抗体干扰和三维重建中的作用

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions ...

生物工程

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

细胞表面和内蛋白质的现场培养神经细胞的抗体后喂养差异化标记

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

从海马CA1区片核富含分的分离，研究突触核信使蛋白质的活性依赖进口核

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

海马神经元中突触囊泡吞的测量

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

一种用于监测 ampa 受体贩运的高含量检测方法

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

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 ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

脑切片和原细胞培养中金字塔神经元的弹道标记

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

<meta charset="utf8"></head><body>通过活细胞成像可视化原代海马神经元中的蛋白质

Using Live&#45;Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-&#946;, &#945;-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions.
Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellular&#160;domain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport.
Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

<meta charset="utf8"></head><body>作者聚焦：了解病理蛋白对神经退行性疾病轴突转运的影响

Here, we demonstrate how to combine transfection of primary hippocampal rodent neurons with live-cell confocal imaging to analyze pathological protein-induced effects on axonal transport and identify mechanistic pathways mediating these effects.