Name: brain membrane fractionation: an ex vivo approach to assess subsynaptic protein localization
Uploaded: 2017-05-12T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization at JoVE.com

这项工作得到了经济部长萨洛德·卡洛斯三世（SAF2014-55700-P，PCIN-2013-019-C03-03和PIE14 / 00034）的支持，加拿大皇家科学院院士（ICREA Academia-2010） ）和代理商Innovatie门Wetenschap en Technologie（SBO-140028）到FC另外，X. M，VF-D。和FC属于"神经药理学和疼痛"认可研究组（Generalitat de Catalunya，2014 SGR 1251） 。这项工作也得到了CAPES（巴西）对FC的"Programa Pesquisador Visitante Especial-Ciênciasem Fronteiras"的支持

所有动物实验程序均由巴塞罗那动物使用和护理委员会（CEEA）批准，符合"实验动物护理和使用指南" 11和遵循欧洲共同体第86/609 / CCE，FELASA和ARRIVE指南。因此，将小鼠容纳在标准的笼子中， 随意获得食物和水，并保持在受控的标准条件下（从7:30 AM，22℃温度和66％湿度开始12小时黑暗/轻循环）。 1.使用不连续的蔗糖梯度获取小鼠脑突触体注意:此方法以前报告10 。 <ol><li>准备新鲜的分离缓冲液（IB），pH 7.4; 2M蔗糖; 1M蔗糖/ 0.1mM CaCl 2 ;和0.1mM CaCl 2 （ 参见表1 ）。在冰上冷却解决方案。 </li><li>萨通过颈椎脱位引起五只小鼠。斩首并迅速清除每只动物的大脑。解剖感兴趣的脑区域（即，海马，0.25-0.35g新鲜组织），并将其置于5mL Potter-Elvehjem玻璃管中，冰上加入1mL IB。 </li><li>使用均质搅拌器（10冲程，以700-900转/分钟）均质化组织，优选在冰浴中排除样品加温。 </li><li>将匀浆的组织（1mL）放入含有6mL 2M蔗糖和2.5mL 0.1mM CaCl 2的 15mL管中，4℃。通过翻转管慢慢混合。 注意:钙的添加对于维持细胞器之间的粘合剂相互作用是至关重要的，因此保持不同"密度"的结构。 </li><li>将溶液放入12 mL离心管中，缓慢加入2.5 mL 1 M蔗糖/ 0.1 mM CaCl 2置于各管顶部，形成蔗糖梯度。 注意:标记位置of使用永久标记在离心管上的梯度界面。 </li><li>称量和平衡离心管与IB溶液在他们相应的钢支架和他们的闭合盖。 </li><li>使用摆动式转子离心机将管子在100,000xg和4℃下离心3小时。用所有的钢支架完全充满转子（如果需要，甚至是空的）。 </li><li>丢弃含有髓磷脂的顶层。使用巴斯德吸管，收集对应于突触体部分的1.25-M和1-M蔗糖间期的白色环。 </li><li>使用IB在离心管中稀释9倍体积的突触体。 </li><li>称量和平衡离心管与IB溶液在他们相应的钢支架和他们的闭合盖。 </li><li>使用摆动转子离心机将管子以15,000xg和4℃离心30分钟。 </li><li>丢弃上清液并重悬用1.1mL IB溶液沉淀。收集100μL的这种突触体溶液，并在11,000 x g和4ºC下离心5分钟。 </li><li>将突触体沉淀重悬于5％十二烷基硫酸钠（SDS）中，并将该样品储存在-20℃。 注意:该样品将对应于进一步的Western印迹分析的总突触体分数。剩余的突触体部分（〜1 mL）可以在-20°C下冷冻直到使用或进行后续的亚突触分选。突触体或纯化的突触占总海马体积的1％和2％。 </li></ol>前，后和外突的隔离<ol><li>准备2x新鲜增溶缓冲液，pH 6.0; 1x溶解缓冲液，pH 6.0;溶解缓冲液，pH 8.0;和0.1mM CaCl 2 （ 参见表1 ）。在冰上冷却解决方案。 注意:这些溶液的pH应该是准确的y调整以实现良好的亚突触分选。 </li><li>用5 mL 0.1 mM CaCl 2缓慢稀释1 mL重悬注射突变体部分（见步骤1.12）。 </li><li>加入相同体积（5 mL）冰冷的2x溶解缓冲液（pH 6.0），并在冰上在高搅拌下，在冰上的烧杯中孵育50分钟。 注意:虽然1％的Triton X-100在pH6.0溶解了所有的质膜蛋白质，它保留了突触接触点或结点内的蛋白质（ 即，突触前和突触后结构）。 </li><li>将溶液置于离心管中。称重并平衡管中的等量体积的0.1mM CaCl 2和2x溶解缓冲溶液，pH 6.0，在其相应的钢支架内及其闭合盖。 </li><li>使用摆动式转子离心机将管子以40,000xg和4℃离心30分钟。所得上清液代表突触后部分，并且颗粒对应于突触结（ 即，突触前和突触后部分）。 </li><li>将含有超弹性组分的上清液浓缩至最终200μL体积，使用在4,000xg和4℃离心的15mL 10K过滤管。 </li><li>将所得的浓缩超弹性组分（200μL）在-20℃下沉淀5次（1mL）预冷（-20℃）丙酮过夜。 </li><li>第二天，以18,000 x g和-15°C离心超刺激级分30分钟。离心后，弃去丙酮上清液，干燥颗粒以除去任何痕量的丙酮。 </li><li>最后，用200μL的5％SDS重新悬浮含有突触前蛋白质的沉淀。超声处理颗粒，如果需要。 </li><li>在不破坏它的情况下，用2 mL 1x溶解缓冲液（pH 6.0）仔细清洗含有突触前和突触后部分的沉淀。丢弃缓冲区。 </li><li>重悬使用玻璃巴斯德吸管，用10mL的1x溶解缓冲液（pH8.0）沉淀。 注意:尽管1％Triton X-100 pH 8.0可溶解突触前专业化，但仍保留不溶性突触后密度4 。 </li><li>在高搅拌下，在冰上的烧杯中，将悬浮液在冰上孵育50分钟。 </li><li>将溶液置于离心管中。称重并平衡管子与1x溶解缓冲溶液，pH 8.0，在它们相应的钢支架内和与他们的封闭盖子。 </li><li>使用摆动转子离心机将管子以40,000xg和4℃离心30分钟;所得到的上清液对应于突触前部分和沉淀到突触后部分。 </li><li>处理含有突触前部分的上清液，如步骤2.6-2.9所述。 </li><li>用200μL的5％SDS重悬含有突触后部分的沉淀。超声处理颗粒，我f需要。 </li></ol> 3.通过免疫印迹分析样品以验证膜分级<ol><li>使用二金鸡宁酸蛋白测定法确定每个级分中的蛋白质的量（ 即，总，外，前和突触后部分）。 </li><li>从SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）样品缓冲液中取每个级分的20μg蛋白质并稀释至最终体积为50μL。在100ºC煮沸5分钟。 </li><li>通过SDS-PAGE电泳分离蛋白质10％，在还原条件下用4％浓缩凝胶分离。在恒定电压80 V下电泳，直到染料进入下凝胶，然后将电压提高到120 V. </li><li>将蛋白质转移到PVDF膜上，并在室温下连续摇动，用IB封闭溶液封闭45分钟。 </li><li>在4℃下用指定的一抗（ 即抗SNAP-25，抗PSD-95，抗突触泡蛋白或抗GPR37）在IB封闭溶液中连续振荡稀释。 </li><li>用IB洗涤溶液洗涤膜三次（每次10分钟），以消除未结合的一抗。 </li><li>在暗条件下，在室温下连续振荡，与指定的二抗孵育90分钟。 </li><li>用IB洗涤溶液洗涤膜三次（每次10分钟）以消除未结合的二抗。 </li><li>用化学发光底物孵育膜（在黑暗条件下制备混合物，并按制造商提供的溶液A和B的1:1比例）。 </li><li>分析化学发光检测装置中的膜。 </li></ol>

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作者没有什么可以披露的。

这里提出的方案构成了一个强大的生物化学工具，用于研究任何脑区域内特异性蛋白质亚突触分布。然而，在技术上固有的一些缺点在此值得强调。例如，主要的限制之一是净化合理量的蛋白质所需的相对较大量的组织以便对所有亚突触部分进行免疫印迹分析。这个问题可能与突触（ 即突触体）仅占总海马体积的1-2％的事实有关。实际上，需要1到1.5g的新鲜组织（ 即海马体）来进行...

突触传播取决于突触的身体完整性，早在1897年由福斯特和谢林顿1设想的概念。因此，了解关键的神经传递组分（ 例如离子通道，受体等 ）的分布对于在正常和病理状况下阐明突触功能至关重要。电子显微镜（EM）对原型中枢神经系统（CNS）突触的当前超微结构概念作出了巨大贡献。以这种方式，EM已经精细地建立了突触前和突触后密度之间的差异，这些差异被相当均匀的距离（〜25nm） 2的裂缝隔开。有趣的是，突触后装置在其质膜下方表现出相对连续的电子致密增厚，即所谓的突触后密度或PSD2。相反，在突触前的装置，一个警告e不连续的细胞质网络布置在质膜正下方，这对于突触囊泡与质膜活性区3的对准和对接是必不可少的。因此，EM构成调查结构保守的CNS突触中蛋白质分布的黄金实验方法。然而，由电子显微照片提供的信息是静态的。实际上，积累的证据表明， 体内突触是非常动态的，因此在持续突触传播时经历剧烈的结构变化。此外，突触的形态和组成可以在不同的CNS区域以及发育，成熟，老化和神经病理学病症的发展中发生变化。总的来说，一项专注于分离属于不同突触室的蛋白质的生物条件的方案代表了一种更全面的突触功能研究的宝贵工具。 等人 （2001） 4 ，基于pH偏移，其削弱在突触前和突触后装置中发生的粘合相互作用。首先，通过在pH6.0下使用温和的洗涤剂，可以辨别保持突触前和突触后装置的粘附连接，并且可以从突触外膜结构域维持，其可以被溶解并因此可以从突触接触中提取。随后，在温和的洗涤剂存在下，将pH从6.0升高至8.0减弱了将突触前活性区域紧密结合到突触后密度的粘附连接点的强度。因此，突触前隔室是如此活化并且可以与突触后密度分离，这主要是由于所用洗涤剂的浓度不能促进其溶解而保留4 。有趣的是，最终高于90％的分离效率可以通过不同的亚突触标记来证实: i ）突触前活动区的突触体相关蛋白25（SNAP-25）; ii ）来自突触后部分的突触素（ 即活性区外，包括微粒体）;和iii ）突触后密度蛋白95（PSD-95）。值得注意的是，这种脑膜分离方法已被成功应用。因此，可以精确地确定不同受体的亚突触定位，例如α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）受体5 ，腺苷A 1受体（A 1 R） 6 ，腺苷A 2A受体（A 2A R） 7 ，三磷酸腺苷（ATP）P2受体8 ，烟碱乙酰胆碱受体亚基9和帕金森病相关受体GPR37 10 。然而，许多限制可能阻碍对特定神经元蛋白的突触分布的适当评估。因此，在这个过程中，我们不仅完整地描述了整个方案，而且还强调了要考虑的一些关键点，例如需要相当大量的组织，低蛋白质产量以及验证效率的强制性要求在进行确定的实验之前进行每次分离。

<table><tbody><tr><td>Sucrose</td><td>Pancreac Qu&amp;iacute;mica SL, Barcelona, Spain</td><td>1,316,211,211</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Pancreac Qu&amp;iacute;mica SL, Barcelona, Spain</td><td>2,112,211,210</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H2O</td><td>Pancreac Qu&amp;iacute;mica SL, Barcelona, Spain</td><td>1,313,961,210</td><td></td></tr><tr><td>Protease inhibitor cocktail Set III</td><td>Millipore, Darmstadt, Germany</td><td>535140</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma, St. Louis, MO, USA</td><td>T1503</td><td></td></tr><tr><td>Tris-HCl</td><td>Pancreac Qu&amp;iacute;mica SL, Barcelona, Spain</td><td>1,236,541,209</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma, St. Louis, MO, USA</td><td>X100</td><td></td></tr><tr><td>SDS</td><td>Sigma, St. Louis, MO, USA</td><td>L3771</td><td></td></tr><tr><td>Glycerol</td><td>Sigma, St. Louis, MO, USA</td><td>G5516</td><td></td></tr><tr><td>Bromophenol Blue</td><td>Pancreac Qu&amp;iacute;mica SL, Barcelona, Spain</td><td>1,311,651,604</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma, St. Louis, MO, USA</td><td>D0632</td><td></td></tr><tr><td>Tween 20</td><td>Sigma, St. Louis, MO, USA</td><td>P2287</td><td></td></tr><tr><td>Non fat dry milk</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Pancreac Qu&amp;iacute;mica SL, Barcelona, Spain</td><td>1,216,591,211</td><td></td></tr><tr><td>KCl</td><td>Pancreac Qu&amp;iacute;mica SL, Barcelona, Spain</td><td>1,314,941,210</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>4873</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Pancreac Qu&amp;iacute;mica SL, Barcelona, Spain</td><td>1,316,781,211</td><td></td></tr><tr><td>Basic 20 pH</td><td>Crison, Alella, Spain</td><td></td><td></td></tr><tr><td>Polytron VDI 12 Adaptable Homogenizer</td><td>VWR, Radnor, PA, USA.</td><td></td><td></td></tr><tr><td>Ultra-Clear Tubes (14x89mm)</td><td>Beckman Coulter, Hospitalet de Llobregat, Barcelona</td><td>344059</td><td>Tubes should be filled almost completely when used to prevent collapsing due to ultracentrifugation.</td></tr><tr><td>Amicon Ultra-15 Centrifugal filters Ultracel -10K</td><td>Merck Millipore, Darmstadt, Germany</td><td>UFC901008</td><td></td></tr><tr><td>Centrifuge 5430R</td><td>Eppendorf, Hamburgo, Germany</td><td></td><td></td></tr><tr><td>Optima L-90K Ultracentrifuge</td><td>Beckman Coulter, Hospitalet de Llobregat, Barcelona</td><td></td><td></td></tr><tr><td>Sonifier 250</td><td>Branson, Danbury, Connecticut</td><td></td><td></td></tr><tr><td>Amersham Imager 600</td><td>GE Healthcare Europe GmbH, Barcelona, Spain</td><td></td><td></td></tr><tr><td>Disposable Glass Pasteur Pippetes 230 mm</td><td>VWR, Radnor, PA, USA</td><td>612-1702</td><td></td></tr><tr><td>Compact Balance EK-610</td><td>A&amp;amp;D, Tokyo, Japan</td><td></td><td></td></tr><tr><td>Pierce&amp;trade; BCA Protein Assay Kit</td><td>Pierce Biotechnology, Rockford, IL, USA</td><td></td><td></td></tr><tr><td>SuperSignal west pico chemiluminescent substrate</td><td>Thermo Fisher Scientific Inc., Waltham, MA, USA</td><td></td><td></td></tr><tr><td>GR 200 Precision Balance</td><td>A&amp;amp;D, Tokyo, Japan</td><td></td><td></td></tr><tr><td>Anti-GPR37</td><td>Homemade antibody anti-GPR37 produced and validated in Francisco Ciruela Laboratory.</td><td></td><td>Primary antibodies used at a final concentration of 0.250 &amp;mu;g/mL</td></tr><tr><td>Anti-SNAP-25, anti-PSD-95, anti-synaptophysin</td><td>Abcam, Cambridge, United Kingdom</td><td></td><td>Primary antibodies diluted 1:10,000</td></tr><tr><td>HRP-conjugated goat anti-mouse IgG</td><td>Thermo Fisher Scientific, Waltham, MA, USA.</td><td></td><td>Secondary antibody diluted 1:10,000</td></tr><tr><td>HRP-conjugated goat anti-rabbit IgG</td><td>Thermo Fisher Scientific, Waltham, MA, USA.</td><td></td><td>Secondary antibody diluted 1:30,000</td></tr></tbody></table>

brain membrane fractionation: an ex vivo approach to assess subsynaptic protein localization

评估突触蛋白的组成和功能是神经科学中的重要挑战。然而，评估突触内发生的神经传递是不容易的，因为它被动态蛋白质 - 蛋白质相互作用和磷酸化事件高度调控。因此，当使用任何方法研究突触传播时，主要目标是保留这些瞬时生理修饰。在这里，我们提出一个脑膜分离方案，代表一个强大的程序来分离属于不同突触室的蛋白质。换句话说，该方案描述了从突触前，突触后和突触后室进行蛋白质富集的生物化学方法。首先，突触体或突触末端通过不连续的蔗糖梯度从包含所有突触室的神经元获得。值得注意的是，这种初始突触膜制备的质量是critical。随后，通过在不同pH条件下使用温和的去污剂进行轻溶解来实现不同亚突触室的分离。这允许通过梯度和等离子体离心分离。最后，通过使用良好表征的突触蛋白标记（ 即， SNAP-25，PSD-95和突触素蛋白）的免疫印迹分析验证了不同亚突触间隔区（ 即，前，后和再突触后膜部分）的蛋白质富集，分别），从而能够直接评估任何特定神经元蛋白的突触分布。

所描述的方法主要用于神经元蛋白的亚突触分析，特别是用于突触受体5,6,7,8,9的分离和生物化学表征。有趣的是，这里显示的代表性结果显示了该实验程序对孤立G蛋白偶联受体的亚突触海马分布的分析的有用性，即帕金森病相关受体GPR37 10 。 GPR37最初是通过搜索内皮素和铃蟾肽受体13的同系物来鉴定的，尽管发现它不结合内皮素或相关?...

Watch this Scientific Journal Video about Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization at JoVE.com

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

在这里，我们提出一个脑膜分离方案，代表一个强大的程序来分离属于不同突触室的蛋白质。

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 ...

brain-membrane-fractionation-an-ex-vivo-approach-to-assess

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

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

10.3K 次观看.  Universitat de Barcelona, L'Hospitalet de Llobregat. 在这里，我们提出一个脑膜分离方案，代表一个强大的程序来分离属于不同突触室的蛋白质。 

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

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

准备使用一个不连续Percoll蔗糖密度梯度的从小鼠皮质Synaptoneurosomes

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

科研

神经科学

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

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

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

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

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 ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

突触质膜和突触后密度蛋白采用不连续蔗糖梯度准备

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

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 ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

鼠脑区的高分辨率定量突触蛋白质双向听觉辨别学习后

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS induces generalized tonic-clonic seizures with low mortality and neuronal death and is a widely-used model to screen anti-epileptic drugs. Here, we describe an ECS induction method in which a brief 55-mA current is delivered for 0.5 s to male rats 200 - 250 g in weight via ear-clip electrodes. Such bilateral stimulation produced stage 4 - 5 clonic seizures that lasted about 10 s. After the cessation of acute or chronic ECS, most rats recovered to be behaviorally indistinguishable from sham &#34;no seizure&#34; rats. Because ECS globally elevates brain activity, it has also been used to examine activity-dependent alterations of synaptic proteins and their effects on synaptic strength using multiple methods. In particular, subcellular fractionation of the postsynaptic density (PSD) in combination with Western blotting allows for the quantitative determination of the abundance of synaptic proteins at this specialized synaptic structure. In contrast to a previous fractionation method that requires large amount of rodent brains, we describe here a small-scale fractionation method to isolate the PSD from the hippocampi of a single rat, without sucrose gradient centrifugation. Using this method, we show that the isolated PSD fraction contains postsynaptic membrane proteins, including PSD95, GluN2B, and GluA2. Presynaptic marker synaptophysin and soluble cytoplasmic protein &#945;-tubulin were excluded from the PSD fraction, demonstrating successful PSD isolation. Furthermore, chronic ECS decreased GluN2B expression in the PSD, indicating that our small-scale PSD fractionation method can be applied to detect the changes in hippocampal PSD proteins from a single rat after genetic, pharmacological, or mechanical treatments.

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy for severe depression. ECS globally stimulates activity in the hippocampus, leading to synaptogenesis and synaptic plasticity. Here, we describe methods for ECS induction in rats and for subcellular fractionation of their hippocampi to examine seizure-induced changes in synaptic proteins.

大鼠电癫痫发作与海马的分离检测突触后密度蛋白的变化

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

免疫荧光定量小鼠脑突触蛋白的异种分布

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

Microtransplantation of Synaptic Membranes to Reactivate Human Synaptic Receptors for Functional Studies

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal communication. Therefore, alterations in their abundance, function, and relationships with other synaptic elements have been observed as a major correlate of alterations in brain function and cognitive impairment in neurodegenerative diseases and mental disorders. Understanding how the function of excitatory and inhibitory synaptic receptors is altered by disease is of critical importance for the development of effective therapies. To gain disease-relevant information, it is important to record the electrical activity of neurotransmitter receptors that remain functional in the diseased human brain. So far this is the closest approach to assess pathological alterations in receptors' function. In this work, a methodology is presented to perform microtransplantation of synaptic membranes, which consists of reactivating synaptic membranes from snap frozen human brain tissue containing human receptors, by its injection and posterior fusion into the membrane of Xenopus laevis oocytes. The protocol also provides the methodological strategy to obtain consistent and reliable responses of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and &#947;-aminobutyric acid (GABA) receptors, as well as novel detailed methods that are used for normalization and rigorous data analysis.

The protocol demonstrates that by performing microtransplantation of synaptic membranes into Xenopus laevis oocytes, it is possible to record consistent and reliable responses of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and &#947;-aminobutyric acid receptors.

突触膜的微移植以重新激活人突触受体以进行功能研究

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal ...

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The characterization of synaptic sub-compartments by biochemical isolation is, therefore, a powerful method to elucidate the molecular bases of synaptic processes, both in health and disease. This protocol describes the isolation of synaptic terminals and synaptic sub-compartments from mouse brains by subcellular fractionation. First, sealed synaptic terminal structures, known as synaptosomes, are isolated following brain tissue homogenization. Synaptosomes are neuronal pre- and post-synaptic compartments with pinched-off and sealed membranes. These structures retain a metabolically active state and are valuable for studying synaptic structure and function. The synaptosomes are then subjected to hypotonic lysis and ultracentrifugation to obtain synaptic sub-compartments enriched for synaptic vesicles, synaptic cytosol, and synaptic plasma membrane. Fraction purity is confirmed by electron microscopy and biochemical enrichment analysis for proteins specific to sub-synaptic compartments. The presented method is a straightforward and valuable tool for studying the structural and functional characteristics of the synapse and the molecular etiology of various brain disorders.

This protocol presents a robust, detailed method to obtain highly pure synaptosomes, synaptic vesicles, and other synaptic fractions from the mouse brain. This method enables the evaluation of synaptic processes, including the biochemical analysis of protein localization and function with compartmental resolution.

亚细胞分离用于从小鼠脑中分离突触成分

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

外周相关蛋白血浆膜分型的测定

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

脑膜分离法

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