这项工作是由美国国立卫生研究院资助DA15169和DA035224到下摆

所有的动物处理和组织收获是按照美国马萨诸塞大学医学院实验动物管理使用委员会大学（IACUC）的指引进行，继批准的协议＃A1506（Melikian，PI）。 所需的解决方案 人工脑脊液（学联） -使每日新鲜 125 mM氯化钠，2.5 mM的氯化钾，1.2毫米的NaH 2 PO 4，1.2毫摩尔MgCl 2，2.4 mM的氯化钙 ，26毫米的NaHCO 3，和11毫摩尔葡萄糖 注:准备学联为10倍原液，不含碳酸氢钠和葡萄糖。每天做从10倍股票1X工作溶液，用新鲜的碳酸氢钠和葡萄糖补充。 蔗糖补充学联（SACSF） -使每日新鲜 250毫米SUC升，2.5毫米氯化钾，1.2毫米的NaH 2 PO 4，1.2毫摩尔MgCl 2，2.4 mM的氯化钙 ，26毫米的NaHCO 3，和11毫摩尔葡萄糖 注:准备SACSF为10倍的贮备液，不含碳酸氢钠和葡萄糖。每天做从10倍股票1X工作溶液，用新鲜的碳酸氢钠和葡萄糖补充。 磺基-N-羟基琥珀酰基-SS-生物素 （磺基-NHS-SS-生物素，Pierce化学公司） 储备溶液应为200 mg / ml的DMSO中，并能抵抗多种冷冻/解冻循环。等分试样贮存于-20℃。琥珀酰酯水解迅速在水溶液中，使工作液应立即应用到片前做好准备。 片淬火解决方案 ACSF补充有100mM甘氨酸 RIPA裂解BuffeŘ 的10mM Tris，pH值7.4，150 mM氯化钠，1.0毫摩尔EDTA，1％的Triton-X-100，0.1％SDS，1％脱氧胆酸盐钠 RIPA与蛋白酶抑制剂（RIPA / PI） -使每日新鲜 RIPA补充了1μM的亮肽素，1μM的胃蛋白酶抑制剂，1μM的抑肽酶和1mM苯甲基磺酰氟。 1。准备脑片<ol><li>让新鲜的1X SACSF和1x学联</li><li>在一个小烧杯寒意SACSF冰上。这将被用于保持新鲜收获小鼠脑。 </li><li>通过用95％/ 5％的O 2 / CO 2，20分钟，在冰上鼓泡饱和的ACSF和SACSF与氧气。 </li><li> P30-38的小鼠应使用最佳的组织的生存能力。牺牲动物通过颈椎脱位和断头，迅速取出大脑进入预冷，含氧SACSF。 </li><li>用振动切片机，使感兴趣区域300μm的脑切片。 </li><li>如果需要的话，可以将它们进一步回收前解剖以富集特定脑区域或单独的左右半球分别作为对照组和实验的切片使用。 </li><li>用火抛光的巴斯德吸液管，转移切片网眼底套入24孔板腔。 </li><li>允许切片恢复为40分钟，31℃，含氧学联，具有持续，温和起泡。 </li></ol> 2。药物治疗（如适用）和生物素片<ol><li>以下恢复，洗净切片3倍于预热（37℃），含氧学联冒泡不断与95％/ 5％O 2 / CO 2。 </li><li>添加测试化合物，并培育具有连续充氧。 <ol><li>为方便起见，加10倍浓缩的药物一个1/10量，并通过轻轻颠倒混匀。 </li><li>摇板轻轻地在水浴中在所需的温度。 </li></ol></li><li>继药物治疗，迅速寒意片在冰冷学联洗3次。 </li></ol> 3。表面生物素化的蛋白质<ol><li>准备1.0毫克/毫升的前夕标签硫代NHS-SS-生物素在冰冷的学联。 </li><li>添加0.75毫升硫代NHS-SS-生物素片和切片孵育在冰上，45分钟。 </li><li>用冰冷学联快速洗片三次，然后孵育在冰冰冷学联10分钟。 </li><li>用冰冷的切片淬灭缓冲液冲洗切片3次，并培育与0.75毫升片骤冷缓冲液2次，25分钟，在冰上骤冷自由磺基-NHS-SS-生物素。 </li></ol> 4。准备组织裂解液<ol><li>在冰冷学联洗净切片三次，用火抛光的巴斯德吸管每片转移到离心管中。 </li><li>轻轻离心200×g离心1分钟，小心吸出剩余学联沉淀切片。 </li><li>加入400μl冰冷的RIPA / PI和移液器向上打破了组织，下一次通过P200的点子ETTE。 </li><li>转移解离片/ RIPA到一个新的管和孵育30分钟，4℃，旋转，完成溶解。 </li><li>通过离心沉淀细胞碎片，18,000×g离心，15分钟，4°C。 </li><li>确定裂解物中的蛋白浓度使用BCA蛋白测定法，以牛血清白蛋白（BSA）作为标准。 </li></ol> 5。分离蛋白质生物素<ol><li>优化珠/总蛋白的比例。 注:个别蛋白表达水平可以有很大的不同在不同的脑区。除非所有的组织裂解液的一定量的生物素化的蛋白质的被捕获，这是不可能精确地检测在表面上表达的任何潜在的变化。因此，就必须根据经验确定特定蛋白质/大脑区域产生一个新的切片生物素化研究着手之前最佳珠/总蛋白比率。 <ol><li>孵育25μL链亲和素琼脂糖珠的增加量组织裂解液（大致范围25-200微克），然后第继续进行如下所述的结合，洗涤和洗脱步骤。 </li><li>量化所产生的免疫反应带，并选择绑定，这将允许增加或减少蛋白质表面表达准确定量的线性范围珠/裂解液的比例。 </li></ol></li><li>准备链霉亲和琼脂糖珠<ol><li>确定所需的所有样品总珠体积。准备了充足的珠子体积的金额加上一个额外的（ 即 4个样品在25μL/样本= 100μL珠+一个额外= 125μL珠。 </li><li>旋涡链霉亲珠股票和吸管出所需的音量。如果使用的是P200的移液器，切断尖底，以防止堵塞或损坏珠。 </li><li>在0.5-1.0毫升RIPA / PI珠洗3次离心18,000×g离心1分钟以去除防腐剂，每RIPA加入后涡旋和清洗间收集珠子，在室温脾气ATURE。 </li><li>吸过使用连接到一个保温瓶玻璃巴斯德吸管清洗之间的缓冲区。吸气时，附着在玻璃巴斯德吸管的末端的塑料P200尖提供更精细的控制。它没有必要完全除去所有的缓冲器的第2次洗涤，如它的风险珠吸进移液管。最后一次洗涤后，去除尽可能多的多余的缓冲尽量不吸珠。 </li><li>加入RIPA / PI带来珠回到他们原来的音量，上下吹打重悬。避免涡流在这一点上，作为珠会坚持到管壁。 </li><li>分装成小珠用P200与尖端的离心管切断。吸管向上和向下采样之间数次，以确保珠保持均匀悬浮分散管中。 </li></ol></li><li>结合生物素蛋白链亲和素磁珠<ol><li>分发细胞裂解物，以含有的琼脂糖珠的管中。对于实验磨片<html>再多个样品进行比较，确保使用的蛋白质相同数量的每个样品进行精确的比较。 </li><li>添加额外的RIPA / PI带给样品200μl的最小体积。这保证了样品将在孵化过程中充分混合，也统一了整个样品的蛋白浓度。 </li><li>放置在管的管肩和搅拌过夜，在4℃下</li><li>在单独的管中，分配用于每个样品的总裂解物体积的等效。另外，如果样品体积都高，总裂解物体积的一小部分可以被保留，而不是，以适应在SDS-PAGE凝胶上的最大负载量。这些将被用于归一化的表面表达的总蛋白量。 </li><li>加2倍的任一个等于或体积的1/5体积的6倍SDS-PAGE样品缓冲液并任孵育在4℃下，在平行珠的样品，或储存在-20℃直至样品进行了分析。 </li></ol></li></ol>ove_title"&gt; 6。洗脱和样品分析<ol><li>通过离心18,000×g离心2分钟，在室温下沉淀的珠子。 </li><li>吸上清，洗珠三次0.75毫升RIPA。为了尽量减少损失珠，留下缓冲的一个小头体积上面珠洗之间。最后一次洗涤后，除去尽可能多的RIPA尽可能不破坏珠粒料。 </li><li>通过减少二硫键洗脱生物素蛋白链菌素的磁珠。加25μl的2×SDS-PAGE还原样品缓冲液，涡旋以及与旋转样品30分钟，室温。 注意:许多膜蛋白具有较高的聚集倾向时，在SDS-PAGE样品缓冲液煮沸，严重影响其电泳迁移率。如果这是正在研究的蛋白质的情况下，避免加热样品，相反，洗脱在室温下缓慢。如果煮沸是绝对必要的（ 例如 ，如果在平行评估的另一种蛋白，需要煮沸），SAMPLë缓冲器可以用2M尿素（终浓度）进行补充，以减少聚集。而有效地减少聚集在某些情况下，尿素往往损害带的外观。 </li><li>通过免疫印迹分析样品。 </li><li>解冻总裂解物样品，并旋转的同时，胎圈的样品，30分钟，室温。 </li><li>分离的蛋白质在SDS-PAGE凝胶。 </li><li>通过免疫印迹鉴定目的蛋白（S）。 <ol><li>可以肯定的是，乐队在检测的正确量化的线性范围检测。 </li><li>以确保该生物素化试剂并没有通过受损/受损细胞得以进入细胞内的蛋白质。这最好是通过免疫印迹法在平行于特定于被研究的细胞类型的细胞内蛋白质来实现的。 </li><li>量化带的密度，并计算相对蛋白质表面密度为总蛋白的表达水平的百分比。 </li></ol></li></ol>

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作者没有财务利益，这项工作冲突。

尽管长期以来知识内吞贩卖大脑危重影响突触信号，它已被证明具有挑战性的定量测量在成年神经元中的变化的蛋白质的表面表达。在这项工作中，我们提出一个可靠的方法来治疗急性脑片表面标记蛋白体外 。脑片制剂有实用的电生理记录的悠久历史，因为他们保持突触连接和细胞存活率高达小时的准备后。另外，切片的策略可以进行优化，以保持各种感兴趣的大脑区域之间的特定突触连...

内吞贩运是微调的各种整合膜蛋白的细胞膜呈现一个无处不在的细胞机制。内吞作用提供重要的营养物质向细胞内环境1和麻痹受体信号响应受体激活2。内吞再循环回至质膜可以通过增加蛋白质的表达水平在细胞表面3还增强细胞信号传导。此外，膜运输扰动有牵连的许多疾病和病理条件下4,5，强调需要调查支配蛋白质内吞贩运分子机制。而许多蛋白质​​利用经典的网格蛋白依赖的内化机制，越来越多的证据，在过去数年证明了多个网格蛋白独立的内吞机制支配的增加数组的内吞潜在蛋白质6,7。因此，有必要探讨促进生理系统有关拐卖的内吞机制已有很大的增长。 在大脑中，受体，离子通道和神经递质转运蛋白的内吞贩运在建立突触可塑性8-11和应对滥用药物12-15，最终影响神经细胞的兴奋性和突触反应的基础性作用。两者都不迄今为止，大多数神经元拐卖的研究都依赖于异源表达系统或原代培养的神经元，可以可靠地反映在作怪成年神经元的机制。在这里，我们报告说，使用表面生物素定量测量表面蛋白水平在成年啮齿类动物源性急性脑切片的方法。使用这种方法，我们提出了证明小鼠纹状体多巴胺转运蛋白迅速内化在数据重响应对佛波酯介导的蛋白激酶C（PKC）的活化。

<table><tbody><tr><td>sulfo NHS-SS-biotin</td><td>Pierce</td><td>21331</td><td></td></tr><tr><td>Streptavidin agarose</td><td>Pierce</td><td>20347</td><td></td></tr><tr><td>IgG-free, Protease-free bovine serum albumin</td><td>Sigma</td><td>A3059</td><td></td></tr><tr><td>Vibrating microtome sectioner</td><td>various</td><td></td><td></td></tr><tr><td>Shaking water bath</td><td>various</td><td></td><td></td></tr><tr><td>Millicell mesh-bottomed inserts (8 &amp;micro;m pore size)</td><td>Millipore</td><td>PI8P 012 50</td><td>These can be washed by hand and reused</td></tr></tbody></table>

brain slice biotinylation: an ex vivo approach to measure region-specific plasma membrane protein trafficking in adult neurons

调节内吞贩运是中央机制，促进多种神经调节活动，通过动态控制在分钟时间尺度受体，离子通道和转运细胞表面呈现。还有就是控制个别蛋白质的内吞机制拐卖了广泛的多样性。研究调查贩卖人口的分子基础主要依赖于表面生物素定量测量响应外源性刺激和基因操作改变膜蛋白的表面表达。然而，这种方法已被主要限于培养的细胞，这可能不是忠实地反映在游戏中成年神经元的生理学相关的机制。此外，培养细胞的方法可能低估贩运机制特定地区的差异。在这里，我们描述了一个扩展的细胞表面生物素的急性脑片制备的方法。我们表明，该方法提供了高精确度的方法来测量在膜表面蛋白水平的快速变化在成年神经元。这种方法可能在神经元内吞贩运领域广泛的实用性。

神经元的多巴胺转运蛋白被内响应于PKC活化在细胞系16-20。尽管许多报告表明在各种细胞系和表达系统的PKC诱导的DAT表面的损失，已经具有挑战性证实这一发现在培养的多巴胺能神经元21-23。我们用小鼠纹状体切片直接测试DAT是否内化响应于PKC活化在成年多巴胺能神经元。以下切片制备，切片沿着从相同平面上的中线和切片切成对半收治±1μM佛波醇肉豆蔻酸酯-13乙酸酯（PMA）处理30...

Watch this Scientific Journal Video about Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons at JoVE.com

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

神经元膜贩运动态控制细胞膜蛋白的可用性和显著影响神经传递。迄今为止，它已经挑战来衡量成人的神经元的神经元内吞贩运。在这里，我们描述了一个非常有效的，量化的方法来衡量在急性脑切片的表面蛋白表达体外快速变化。

大脑切片生物素化:一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

brain-slice-biotinylation-an-ex-vivo-approach-to-measure-region

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

12.9K 次观看.  University of Massachusetts Medical School. 神经元膜贩运动态控制细胞膜蛋白的可用性和显著影响神经传递。迄今为止，它已经挑战来衡量成人的神经元的神经元内吞贩运。在这里，我们描述了一个非常有效的，量化的方法来衡量在急性脑切片的表面蛋白表达体外快速变化。 

视频: Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

脑膜分离法<em&gt;体检</em评估亚突触蛋白定位的方法

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

科研

生物化学

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 ...

神经科学

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

生物学

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 ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD) and Huntington&#8217;s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

初级量子神经元轴突运输的实时成像点标记的BDNF

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

对于神经化学标记用biocytin填充和加工部分的免疫染色

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

使用生物素化测定原代星形细胞培养物中蛋白质的细胞表面表达和吞噬率

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A reproducible protocol was developed to directly&#160;mono-biotinylate BDNFAvi with the enzyme BirA in a tube. In this reaction, mono-biotinylated BDNFAvi retains its biological activity.
Neurotrophins are target-derived growth factors playing a role in neuronal development and maintenance. They require rapid transport mechanisms along the endocytic pathway to allow long-distance signaling between different neuronal compartments. The development of molecular tools to study the trafficking of neurotrophins has enabled the precise tracking of these proteins in the cell using in vivo recording. In this protocol, we developed an optimized and cost-effective procedure for the production of mono-biotinylated BDNF. A recombinant BDNF variant containing a biotinylable avi sequence (BDNFAvi) is produced in HEK293 cells in the microgram range and then purified in an easily scalable procedure using affinity chromatography. The purified BDNF can then be homogeneously mono-biotinylated by a direct in vitro reaction with the enzyme BirA in a tube. The biological activity of the mono-biotinylated BDNF (mbtBDNF) can be conjugated to streptavidin-conjugated to different fluorophores. BDNFAvi and mbtBDNF retain their biological activity demonstrated through the detection of downstream phosphorylated targets using western blot and activation of the transcription factor CREB, respectively. Using streptavidin-quantum dots, we were able to visualize mbtBDNF internalization concomitant with activation of CREB, which was detected with a phospho-CREB specific antibody. In addition, mbtBDNF conjugated to streptavidin-quantum dots was suitable for retrograde transport analysis in cortical neurons grown in microfluidic chambers. Thus, in tube produced mbtBDNF is a reliable tool to study physiological signaling endosome dynamics and trafficking in neurons.

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a cost-effective manner and is purified by affinity chromatography. BDNFavi is then directly mono-biotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF.

大脑切片生物素化:一个

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脑膜分离法