威康信托基金和财政支持的ERC，我们非常感谢。 IMGG是欧洲分子生物学研究员。 KLH的是BBSRC的资助博士生。我们感谢菲利普·鲁宾和帕特里克蒂德博尔的技术和细胞培养的支持和安德鲁·多尔蒂博士与显微镜的维护和协助。

1。细胞培养，病毒转导，蛋白表达<ol><li>文化高密度聚-L-赖氨酸涂层玻片为14-25天（DIV）， 在体外胚胎18天（E18）幼鼠海马神经元。 </li><li> 6 - 24小时前的现场实验，减毒辛德毕斯病毒的转导细胞中含有利益的膜蛋白，与黄道超pHluorin（SEP）的标签。 </li><li>直接添加pseudovirion含中等盖玻片含1毫升的空调媒体和文化的孵化器。滴度和病毒转导蛋白表达的时间将取决于病毒批次而有所不同，并应为每个活细胞实验开始前，一批确定。 </li></ol> 2。 FRAP还原-FLIP的活细胞成像<ol><li> 设备设置 <ol><li>蔡司Axiovert LSM的510的META共聚焦显微镜成像室的盖玻片转移。成像，以尽量减少在电源波动，确保显微镜已接通，至少有20分钟与100％的激光输出，前成像。 </li><li>立即更换培养基预温（37°）外的录音解决方案，其中包含140 mM氯化钠，氯化钾5毫米，1.5毫米氯化镁 ， 氯化钙 1.5毫米，15毫米葡萄糖，20-25毫米肝素钠（调pH至7.4用NaOH）和蔡司Axiovert预热阶段（37℃）放置室。 确保外的录音解决方案渗透压调整10培养基mOsM内到。提供无显着性的蒸发在实验timecourse发生，这种合作的2个独立的解决方案是适合短期实验（&lt;10小时）。 1-2毫米碳酸氢钠补充建议。 </li></ol></li><li> 定义成像捕获参数 <ol><li>首先，确定一个神经细胞表达的重组亲蛋白的利益，并使其成为焦点。 </li><li>随着63X石油反对，获得整个细胞，使用488nm激光激发激光功率低的形象。为了尽量减少漂白，使用快速的额定转速（7-9）和低像素的分辨率（512-512），保持总的扫描速度&lt;1秒。 </li><li>选择部分图像和缩放的枝蔓捕获一帧包含的投资回报率（〜1.5-2.5倍的光学变焦）。在可能的情况下，确保视野包含多个进程，这样可以得到参考树突测量，以确定如果非特定的漂白，由于收购发生。 </li><li>调整过滤器，针孔，扫描速度和检测器从最小的激光激发，但有限的饱和增益使最大荧光。一个大型的针孔直径的建议，以最大限度地提高光子收集（2μm的是适合刺和三元树突）。探测器的增益应该强大到足以检测小的荧光增量，例如，第一图像之前，漂白，不克服饱和像素的10％。 </li><li>保存前/后漂白剂和复苏阶段的实验中使用的配置。 </li><li>接下来定义的光漂白地区;选择一个初始光漂白和随后的重复漂白阶段的侧翼地区的投资回报率。确保足够宽，以防止扩散扫描（一般为5微米，见图2）之间恢复侧翼地区。 </li><li>调整漂白光漂白的投资回报为参数。初始光漂白应该迅速（0.1-0.5秒）需要1-5迭代之间，视光学变焦和投资回报率的体积。激光激发的侧翼地区，应进行调整，以确保侧翼地区连续漂白，但没有光毒性损害。 </li><li>作为一个准则，我们使用100％的初始光漂白激光激发，10％的重复漂白pH值ASE。 </li></ol></li><li> 图像采集 <ol><li>一旦所有参数已经确定，执行作为一个变量的时间推移图像序列FRAP还原翻转实验，在下面4块概述： 1座3-10预漂白基线图像，在激光功率低，没有时间延迟 2座：光漂白完整的激光功率的中央投资回报率在1-5迭代 3座3-10漂白后恢复映像 4座：激光功率中等的地区，典型的时间间隔为1侧翼图像采集重复的光漂白 - 5秒取决于正在调查中的蛋白质的回收率。 </li><li>最后，更换与PH6含有140 mM氯化钠，氯化钾5毫米，15mm的葡萄糖， 氯化钙 1.5毫米，1.5毫米氯化镁 ，MES（解渴荧光）20-25毫米或50毫米补充缓冲的录音解决方案外的录音解决方案氯化铵含有90 mM氯化钠，氯化钾5毫米，15毫米葡萄糖， 氯化钙 1.8毫米，0.8毫米MgCl 2的 20-25毫米肝素钠，PH7.4（揭示在低pH值的细胞内储存的蛋白质），（见图2）。 </li><li>至少有10至20个数据集，收集各重组蛋白，使统计分析。为了避免偏见的结果，确保成像条件保持一致跨越复制。丢弃任何数据集，其中不完全漂白，显着焦平面漂移或光毒性细胞损伤观察。 </li></ol></li></ol> 3。数据分析<ol><li> ImageJ的软件打开图像。 </li><li>对齐栈：刚体在xy平面上的小波动可能发生的整个时间系列使用Stackreg插件（插件→stackreg→改造→ <ok> ）。 </ok></li><li>对于蔡司聚焦拍摄的图像，使用LSM的工具箱插件，报告为一个文本文件的时间值（插件→LSM的工具箱→显示LSM的工具箱→ <apply stamps=""> → <apply t-stamp=""> ）分析电子表格导入这些值。 </apply></apply></li><li>来衡量FRAP还原倒装实验过程中的荧光波动，划分成单个像素区域（20pixels）photobleached段，并在每个时间点（F）的衡量这些投资回报的平均荧光。快速获取这些值，选择多个投资回报使用ImageJ的“投资回报率管理的分析工具（分析→工具→投资回报率经理）和报告使用的阴谋Z轴配置文件”命令（每个像素的平均荧光图像→堆叠→图Z轴轮廓）。 </li><li>重复此步骤来衡量的背景下，非荧光区的荧光强度。 </li><li>在每个时间点的荧光强度，减去背景值，以消除实验噪音，所有的值除以平均预漂白基线措施正常化。 </li><li>测量相邻的非photobleached树突荧光的强度，以评估非特异性水平漂白过程中采集。非特异性漂白修正气馁FRAP还原-FLIP的数据的解释，并应尽可能避免。 </li><li>来计算，如果已明显复苏中的投资回报率的地方，从第一（ΔF）5-10措施的平均值减去平均序列的最后5-10措施，并确定统计意义。分类的恢复和非恢复的投资回报来评估胞吐模式（即胞吐热点或脊椎与轴恢复）。 </li></ol> 4。代表结果 FRAP还原典型的倒装实验的结果显示图。 2。在这里，一个神经元表达GluA2亚基的AMPA型谷氨酸受体与SEP标签，已选择性photobleached沿枝的地区。图2.A说明的的枝蔓地区已成像，并表示已漂白（白框区域）选择的投资回报。日é白色大盒装面积漂白一次，其次是重复的侧翼盒装双头蓝色箭头突出的地区，漂白。红色箭头表示测得的投资回报率，高倍率在较低的面板中显示。 2.B图，显示在实验过程中测量投资回报率的荧光强度。在这个例子中，一分钟恢复期内录得的初始光漂白后允许棉纱受体进入横向扩散的投资回报率。当photobleached侧翼地区，从这个高度能动的分数受体荧光信号被遮挡从中部地区，这些区域内的稀释。因此，可以在观察荧光（ΔF）增加了“翻转”序列插入的SEP GluA2的树突状轴。低pH清洗和pH值7.4 +氯化铵除了分别确认，测量的荧光从表面派生蛋白质和揭示内，幽静的蛋白质的比例，在衡量投资回报率。 在FRAP还原力的对比，这种方法隔离恢复由于胞吐，大大降低了在一个荧光恢复在photobleached地区的水平。迄今为止还没有任何可靠的数学模型已经开发，以适应和分析恢复跟踪记录使用这种选择性漂白技术。然而，这是可能的，以适应与一个单一指数复苏复苏跟踪： F（T）= S - 0 E（-T /τ） 其中F（t）是在时间t的荧光， 一个 S是稳态值，0是在时间0的偏移量，τ是时间常数。恢复性增长是固定的一个给定的汇率达到平衡，相应的稳定状态之间的插入和扩散。重要的是，这是一个时间常数提取nalysis并不反映的胞吐的时间常数，并且只能用于个别蛋白的比较治疗。 此外，荧光恢复的预期模式很可能将在子域观察沿photobleached地区。小内photobleached段的投资回报率分析可能是必要的揭示胞吐的“热点”和分析可比长度之间的枝蔓这些斑点的密度变异的地区，将影响计算速度，这是明智的。 图3显示了此协议的扩展，应用光漂白视野中的多个树突大断面的投资回报率，其次是选择性重复只有一个枝漂白。这种方法使传统的“FRAP还原”和“FRAP还原翻转”并行采集的数据。海马神经元在这个例子中，已感染的红藻氨酸类的谷氨酸受体亚基独立日标签GluK2。 PRIOr来成像，这些神经元的治疗cylcohexamide（在200μg/ml2小时），阻止蛋白质的合成。正因为如此，在各自的衡量投资回报（图3.B）的荧光恢复显示恢复的比例，由于横向扩散和FRAP还原枝的标准与回收FRAP还原，翻转“协议的枝蔓单独回收。 FRAP还原与FRAP还原-FLIP的“恢复曲线，回收的相对贡献（ΔFREC = 11.05％）与横向扩散（ΔF 差异 = 9.35％）比较ΔF值可以推断。与图2不同的是，经济复苏不保持一个稳定的状态，而是由于抑制蛋白质的合成，显示了一个短暂的增加和随后的信号，相应的落客耗尽可用的受体池（约20％的跌幅观察在录音期间）。 <img alt="图1" src="/files/ftp_upload/3747/3747fig1.jpg" /></P&gt; 图1。FRAP还原与FRAP还原-FLIP的协议，这个原理说明了一个与“FRAP还原翻转”协议定期FRAP还原力的结果，使用sep标签受体校长的示意图。荧光恢复传统FRAP还原显示在左侧。在中央投资回报率的测量荧光恢复归因于横向扩散F非photobleached SEP-标签通过回收和/或从头到树突状轴的胞吐以外的photobleached的ROI和插入受体的受体结合。相比之下，在右侧所示的侧翼的ROI重复photobleached，显示FRAP还原翻转“协议修改后的”沉默的复苏，由于横向扩散。因此，任何测得的荧光恢复，可以归因于在投资回报率的直接插入。 <img alt="图2" src="/files/ftp_upload/3747/3747fig2.jpg" /> 图2。插入到细胞膜上的树突状轴SEP-GluA2 A）海马神经元表达SEP-GluA2，选择性地photobleached沿着枝蔓地区。原理说明枝地区已成像，而左上角的手面板突出已选定为漂白的投资回报。白色大盒装面积漂白一次，其次是重复的侧翼盒装双头蓝色箭头突出的地区，漂白。此面板显示的枝蔓前漂白。红色箭头表示测得的投资回报率，高倍率在较低的面板中显示。 B）显示的时间在实验过程中测得的投资回报率在荧光强度，绘制灰度等级在投资回报率（不归）。黑色箭头突出的时间点的初始光漂白和绿色箭头表示开始重复漂白。 ΔF表示日ê增加在恢复期内观察荧光，由于的SEP GluA2的树突状轴插入。随后的低pH值和pH值7.4 +，NH 4氯洗确认，恢复荧光涉及到表面表达的受体。 <img alt="图3" src="/files/ftp_upload/3747/3747fig3.jpg" /> 图3对树突状轴cyclohexamide治疗后的回收和横向扩散的SEP-GluK2 VS回收。 A）海马神经元表达SEP-GluK2，选择性photobleached平行FRAP还原力和FRAP还原翻转“恢复执行协议沿着单独的树突状的ROI。这个神经元是受预处理，与cyclohexamide，阻止蛋白质的合成，在200微克/毫升（2小时）。原理说明的的枝蔓地区已成像，而左手面板突出的漂白已选定的ROI。日é白色大盒装面积漂白一次，重复的侧翼盒装地区的低枝双头蓝色箭头强调，漂白。此面板显示前漂白树突。红色箭头突出高倍率在 B所示的投资回报）说明FRAP还原传统的荧光恢复与“FRAP还原翻转”协议。在复苏后期（第三小组）的荧光强度水平的比较结果显示，独自与回收的横向扩散和回收的贡献。）显示荧光强度，在实验过程中测量的ROI在作为归强度绘制值。黑色箭头突出的时间点的初始光漂白和绿色箭头表示开始重复漂白。 ΔF 建议表示FRAP还原/翻盖枝确定由于受体再造，瞬态恢复，而ΔF差异措施纳入树突轴“FRAP还原唯一的投资回报率横向扩散的SEP GluK2的贡献。瞬时增加，其次是逐步下降的信号，相应的可用受体运行cyclohexamide治疗。随后的低pH值和pH值7.4 +，NH 4氯洗确认，恢复荧光涉及到表面表达的受体。

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没有利益冲突的声明。

我们描述了一个创新的战略，可视化组件的质膜蛋白质贩运。选择性漂白技术与SEP-标记蛋白相结合的方法，使细胞膜插入事件进行评估。通过不断漂白侧翼地区在恢复过程中的膜蛋白，FRAP还原，倒装的方法评估荧光恢复水泡贩运的贡献。这种新方法可直接录制蛋白膜插入，使副舱室的数量都在恢复观察，在稳定状态恢复的幅度（ΔF）待定。此外，FRAP还原力比较与不倒装允许恢复的比例应占计算的横向扩散。 此外，在相同的实验，相邻侧翼photobleached地区的的非photobleached枝的地区可定性评估在恢复过程中，在这些地区观察到的荧光损失是由于横向扩散photobleached受体对树突这些非photobleached的分部。 这种选择性漂白协议可以用来探讨各种excocytosis在质膜中定义的子区域特征（例如，刺或树突）或评估的横向扩散与插入进行并行FRAP还原的贡献和FRAP还原如蜂窝贩运过程沿相邻的树突-FLIP的“协议（图3）。 显然，已提出具体的指导方针，同时，每个实验室将需要根据特定的标本和设备的成像参数优化。重要的是，在所有的投资回报率表面受体需要photobleached，Z轴焦平面独立，但没有显着的光毒性损伤或非特异性漂白。我们ERS时，应谨慎试图在胞体这个协议，因为高比例的内舱相对较低的pH值通常是在这些地区的高背景荧光的结果。此外，虽然我们已经证明如何这项技术可以应用在不同的方式选择性漂白实验开始前，一个新的SEP-标签的构造，我们建议首先进行标签受体的初步鉴定，Ashby等2。 总的来说，这个方法是一个标准的FRAP还原协议的强大和灵活的适应，能够插入细胞膜近实时评估的事件。

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt;试剂名称</strong</td&gt;<td<strong&gt;公司</strong</td&gt;<td<strong&gt;产品目录号</strong</td&gt;<td<strong&gt;评论（可选）</strong</td&gt;</tr&gt;<tr&gt;<td&gt;24毫米盖玻片</td&gt;<td&gt; VWR国际</td&gt;<td&gt; 631-0161</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt;聚-L-赖氨酸</td&gt;<td&gt;西格玛</td&gt;<td&gt; P2636</td&gt;<td&gt; 1mg/ml的大衣盖玻片在硼酸盐缓冲</td&gt;</tr&gt;<tr&gt;<td&gt;的Neurobasal中等</td&gt;<td&gt; Invitrogen公司</td&gt;<td&gt; 21103</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; B27的</td&gt;<td&gt; Invitrogen公司</td&gt;<td&gt; 17504-044</td&gt;<td&gt; 2％的神经元的电镀和喂养中等</td&gt;</tr&gt;<tr&gt;<td&gt;青霉素链霉素</td&gt;<td&gt;西格玛</td&gt;<td&gt; P0781</td&gt;<td&gt; 1％，在神经电镀和饲养的媒介</td&gt;</tr&gt;<tr&gt;<td&gt; L-谷氨酰胺</td&gt;<td&gt; Invitrogen公司</td&gt;<td&gt; 25030</td&gt;<td&gt; 2毫米/ 0.8毫米，在电镀/喂养中等</td&gt;</tr&gt;<tr&gt;<td&gt;马血清</td&gt;<td&gt; Biosera</td&gt;<td&gt; DH-291H</td&gt;<td仅在神经电镀媒体&gt; 10％</td&gt;</tr&gt;<tr&gt;<td&gt; PSIN于白色念珠菌克隆载体</td&gt;<td&gt; Invitrogen公司</td&gt;<td&gt; K75001</td&gt;<td&gt;辛德毕斯病毒的生产</td&gt;</tr&gt;<tr&gt;<td&gt; LSM510 META共聚焦系统</td&gt;<td&gt;蔡司</td&gt;<td&gt;</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Image J软件</td&gt;<td&gt;国立卫生研究院</td&gt;<td&gt;</td&gt;<td&gt;开放式访问软件。所述的所有插件可在<a href="http://rsbweb.nih.gov/ij/plugins/"&gt; http://rsbweb.nih.gov/ij/plugins/</a</td&gt;</tr&gt;</tbody&gt;</table

lateral diffusion and exocytosis of membrane proteins in cultured neurons assessed using fluorescence recovery and fluorescence-loss photobleaching

<p class="jove_content"&gt;如受体和离子通道的膜蛋白进行高度极化和形态复杂的神经元，这是积极的贩运。这直接贩运提供，保持或删除突触蛋白具有根本的重要性。</p&gt;<p class="jove_content"&gt;黄道超pHluorin（SEP），是一种绿色荧光蛋白的pH敏感的衍生工具，已被广​​泛用于细胞膜蛋白的活细胞成像<sup&gt; 1-2</sup&gt;。在低pH值下降的SEP质子光子吸收和消除荧光。作为最内的贩卖事件发生在低pH值，九月荧光黯然失色，从9月标签蛋白的荧光信号的车厢主要是从质膜SEP是一个中性的pH值外环境。在高强度九月亮起时，每一个荧光染料一样，是不可逆转的光损伤（photobleached）<sup&gt; 3-5</sup&gt;。重要的是，因为低pH值淬火光子吸收，只有表面表达，而细胞内的SEP是由高强度照明的影响，SEP可以photobleached<sup&gt; 6-10</sup&gt;。</p&gt;<p class="jove_content"&gt; FRAP还原的SEP标签蛋白（荧光漂白后恢复）是为方便和强大的技术评估在细胞膜上的蛋白质动力学。当荧光标记的蛋白质，在一个地区的利益photobleached率（ROI）的发生是由于荧光恢复到漂白区域的运动本色SEP-tagged蛋白。这可以通过横向扩散和/从非photobleached受体胞吐提供或发生<em&gt;从头</em&gt;合成或再造（见图1）。静止和移动的蛋白质的一小部分，可以决定和扩散分数的流动性和动力学激动剂应用程序或如神经元激活NMDA受体或氯化钾申请的刺激，如基底和刺激的条件下可以讯问<sup&gt; 8,10</sup&gt;。</p&gt;

Watch this Scientific Journal Video about Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching at JoVE.co

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

这份报告介绍了利用活细胞成像和光漂白技术，以确定表面的表达，运输途径和贩运外生表示，pH敏感的绿色荧光蛋白标记的蛋白质在神经元细胞膜动力学。

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

lateral-diffusion-exocytosis-membrane-proteins-cultured-neurons

Research

JoVE Journal

Neuroscience

82.9K 次观看.  University of Bristol. 这份报告介绍了利用活细胞成像和光漂白技术，以确定表面的表达，运输途径和贩运外生表示，pH敏感的绿色荧光蛋白标记的蛋白质在神经元细胞膜动力学。

视频: Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

科研

神经科学

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

活大鼠皮层神经元线粒体膜电位和活性氧的测定

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

在培养的中枢神经系统的神经元树突棘形态分析

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

诱导培养的交感神经元的树突状增长

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

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 ...

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 ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

亚细胞钙荧光和生物发光成像<sup&gt; 2+</sup&gt;老年海马神经元

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...