这项工作是由德国联邦教育与研究部（BMBF），（对系统生物学亥姆霍兹联盟）信令在巨蟹星座系统生物学和德国癌症研究中心（DKFZ）的支持。

所有动物实验均按照DKFZ的机构准则进行。 注意：为防止样品的RNA酶的污染，采取基本的预防措施，以避免RNA酶的污染，并准备所有的缓冲区用DEPC处理过的水。 蔗糖梯度的1。准备<ol><li>准备17.5，25.6，33.8，41.9和50％蔗糖溶液（ 见表1）。开始慢慢加入2ml的50％蔗糖溶液，以一个异质同超速离心管的底部制备梯度。通过将管20分钟，在-80°C冻结层</li><li>添加后续层在减少蔗糖的百分比与冷冻之间的步骤，结束了17.5％的蔗糖。保持此外蔗糖溶液的过程中，为了防止下层的层的融化冰梯度。 </li><li>准备蔗糖梯度刚使用前一天，并保持在4°C。 AlternativelY，实验前准备梯度提前，储存在-80°C和解冻，4℃过夜。 </li></ol> 2，组织准备（脊髓和/或脑） <ol><li>麻醉小鼠通过赛拉嗪和氯胺酮在NaCl的过量和用20毫升的汉克的含有200微克/毫升放线菌酮的固定不变的相关联的转录的核糖体的平衡盐溶液（HBSS）transcardially灌注。非反应到脚趾捏确认正确的麻醉。 </li><li>快速提取组织。打开颅骨背侧和提取大脑。执行整个胸腰椎脊柱椎板和提取脊髓。使用全脑（〜400毫克）或4厘米的脊髓（〜90毫克），分别。 </li><li>组织转移到冰冷的匀浆缓冲液A（ 见表1）（脊髓：1毫升;脑：4毫升）和骰子切成小块用干净的手术刀，使放线菌酮的摄取增加。 PERFORM冰的所有进一步的步骤。 </li><li>孵育15分钟，并机械地用Dounce匀浆器匀化组织。仔细控制均质化是必要的，以确保核保持完整。对待所有样品完全相同的方式，以保证可比性。注：对于脑组织：5招用紧紧贴合杵扰乱组织。对于脊髓组织：5招用松配合杵，其次是5再招一个紧密配合的杵破坏。 </li><li>采取等分试样（400微升用于脑和200μl的脊髓），闪存冷冻并储存在-80℃下进行总RNA提取。 </li><li>通过离心分离，在500×g离心在4℃下除去细胞核和不间断的细胞和组织碎片10分钟低速离心防止核糖体的损失。 </li><li>在细胞核上清液30分钟，裂解膜上的片段，加入NP-40和脱氧胆酸钠洗涤剂（各以1％的终浓度）。 </li></ol><ol><li>这个步骤从其中将另有掩蔽的信号中的多核糖更新的样本去除脂肪酸组分。首先，预冷超速离心桶和转子在4℃，并准备2 M，1.1 M和0.9M的蔗糖溶液（ 见表1）。 </li><li>混合溶胞产物用1.22体积的2M蔗糖溶液，并转入聚乙烯超速离心管。填满所有试管于10毫升的1.1M的蔗糖溶液等体积，然后小心地用0.9M的蔗糖溶液覆盖它。 </li><li>将超速离心管放入预先冷却的超速离心机桶。离心3小时以100,000×g离心在4℃下在此过程中成为核糖​​体沉积在沉淀而脂肪酸成分浮起，并保持在上清液中。 </li></ol> 4，蔗糖梯度分级<ol><li>除去上清液，沉淀物溶解在匀浆缓冲液B（ 见表1 </st荣&gt;）。测量260nm处的吸光度为使用NanoDrop或等效装置，每个样本和正常化量根据吸光度值来加载。 </li><li>放置蔗糖梯度（其制备的前一节中所述）插入到预先冷却的超速离心机桶。仔细层样品在梯度上。添加一个空的蔗糖梯度技术控制。 </li><li>调整每个桶匀浆缓冲液的重量。在4℃下离心样品1.5小时，于285,000×g离心</li><li>启动离心结束前准备伊斯科分馏塔30分钟。如果使用分馏塔的另一种模式，遵循制造商的协议。 <ol><li>设置适当的灵敏度（0.2 AUFS脑或0.05 AUFS脊髓）的紫外线灯和打开它的热身。装配在管穿孔器，通过经由管穿孔轧制泵至60％蔗糖溶液连接起来。 </li><li>抽蔗糖（流量大鼠测试设置E：1毫升/分钟），特别是要确保有一个在其中将引入气泡成梯度的油管没有泄漏。 </li><li>空白的背景吸收通过手动泵梯度缓冲到紫外检测和纠正的基准。 </li></ol></li><li>仔细沉积样品从转子取出含有蔗糖梯度超速离心桶，将它们放在冰上。避免梯度的任何碰撞，防止丧失决议。 </li><li>运行梯度上的分馏塔。首先，评估背景吸收，并确保适当的技术安装运行空梯度。 <ol><li>附加梯度的UV检测器和刺穿管的底部与管子穿孔。从60％的蔗糖的泵送，这将向上通过UV检测器和进滴分配器置换梯度与沉积样品。 </li><li>在260nm处的吸光度是记录以产生吸收曲线的样品，峰值指示沉积作用tion与核糖体亚基，monosomes，随后越来越多核糖体相关的mRNA。 </li></ol></li><li>通过下拉饮水机（每600微升，在2毫升试管）收集样本为20个部分。 </li></ol> 5，从个人分数RNA提取<ol><li>加10％的SDS至1％，以单个组分的终浓度，并以展开蛋白质和游离核糖体拌匀。在这点上的样品可以在-80℃下冷冻取决于要解决的问题，组分可以根据更新的吸光度进行汇集。 </li><li>分离RNA用酸性苯酚/氯仿提取，这也消除DNA的污染痕迹。 <ol><li>添加酸性苯酚/氯仿1卷（预热至RT）到每个样品中，加热，在65℃10分钟，并在通风橱下之后释放压力。 </li><li>离心样品20分钟，在17000×g离心于RT。水相小心转移到一个新的1.5 ml管。注意在致密蔗糖级分，其中所述水相可能在底部离心后倒相的。 </li><li>以沉淀RNA的添加1倍体积的异丙醇，1/9体积的醋酸钠（pH5.2）和1μl的GlycoBlue到各样品中。采样保持至少1小时，在-80°C </li><li>离心30分钟，在17000×g离心和4℃下GlycoBlue提供粒料的可视化。除去上清液，用冰冷的80％乙醇和空气干燥的颗粒洗一次沉淀。在无RNA酶的水溶解沉淀。 </li></ol></li><li>量化的RNA量的NanoDrop和由生物分析仪芯片分析RNA的完整性。这是由所提出的协议获得的RNA适用于艺术的高通量检测，包括基因芯片和深度测序的所有状态。 </li></ol>

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没有利益冲突声明。

虽然多核糖体分馏是不是一种新技术，它仍然是一个特别具有挑战性的。根据所输入的材料，主要优化可以是必要的。这是特别适用于体内中枢神经系统的样品，其中材料的量通常是一个限制和脂肪组织成分阻碍翻译的mRNA的分离的情况。大多数已发表 ​​的分馏协议处理酵母或哺乳动物细胞系，并有既定协议的大脑12,13,14。与此相反，也有几乎没有任何描述脊髓的分馏出版物，和以前的协议需要脊髓从大量动物被汇集15。由于这些原因，一些必要的改动做了适应分馏协议中枢神经系统组织，包括单小鼠脊髓。动物灌注期间进行核糖体固定用放线菌酮，以避免核糖体的解离超级碗组织提取克漫长的过程。随后，多核糖体RNA的提取以特定方式最大化多核糖产率。首先，通过受控douncing组织的均质化，保持了核完好并防止DNA污染。细胞核，然后可靠地通过离心分离除去。洗涤剂NP-40和脱氧胆酸钠的组合确保了内质网和与其相关联的膜核糖体释放的裂解。此外，富含脂质的髓鞘内UV-吸收组分混淆多核糖访问。因此髓鞘浮选是必要的，其中致密的化合物如多聚核糖体被沉淀和更轻的化合物如髓鞘浮子和被除去16。多聚核糖体，然后沉淀在17.5％至50％的蔗糖梯度。而不是手动分层和冷冻每个蔗糖层，渐变，也可以使用梯度混合器制备。使用酸性酚/氯仿萃取组分的RNA允许dNA以最小的损耗的RNA被除去。然而，可能发生在密集的蔗糖分（以上分数16）相位反转。 该协议提供了优势，解决翻译水平等常规方法。例如，磷酸化S6（著名翻译标记）的两个测量以及新生肽链与放射性同位素标记给全球翻译水平的信息，但很少透露有关具体是什么正在翻译。多核糖体分离，另一方面，不仅允许评估全局翻译的也是，翻译的mRNA和相关的调节蛋白的识别。实时定量PCR技术可以对离体的RNA进行一个快速的读出选定的mRNA和微阵列和下一代RNA测序可以用于全基因组研究的进行。这里提出的优化允许与中枢神经系统组织中的最小量的使用的技术，因此相应地减少ING所需动物的数量，并通过减少组织处理时间提高整体实验的质量。另一方面，这种技术不能从翻译那些区分失速核糖体。成绩单携带停滞的核糖体将沉淀在重分数，这应该解释数据时，必须牢记。 有最近报道的技术，即RiboTaq和TRAP，其中核糖体中的细胞类型特异性的方式分别标有表位标签或记者和相关的mRNA分离，通过免疫沉淀17,18。这种技术可以被耦合到多核糖体分馏提供读出特定的细胞群体高的分辨率。核糖体尺印刷是另一个新技术是由核糖体保护的mRNA，涉及核酸酶消化，产生小碎片，或“脚印”。然后，从这些脚印生成和测序文库。这种方法省IDES上翻译的密码子特异性的全基因组分析和能够识别停滞核糖体，非AUG起始密码子和小上游开放阅读框架，它可以不通过多核糖分馏19来实现。然而，多核糖分馏可耦合到核糖体的剖面为包含单个核糖体消化的片段的分离，从而富集所需的库制剂，其中高纯度的样品，通常需要的分子种类。两者合计，多核糖分馏是一种灵活的方法，以持久的意义，并且可以耦合到各种下游应用，包括基因组范围的分析像新一代测序。

基因表达是通过转录，翻译和mRNA和蛋白的稳定的联合作用决定的，与翻译承载的最主要的作用1。它现在明显的是，每个步骤是高度管制。微核糖核酸，核糖核酸形成颗粒剂，替代和胞质多聚腺苷酸化是基因表达的2,3的转录后调控的一些例子。从翻译这些机制的解耦转录并影响蛋白质在感兴趣的生物系统。因此，毫不奇怪，单独的mRNA水平蛋白水平4的一个不完美的读数。定量蛋白质组学提供了基因表达的最直接的评价，但尽管最近的进展，但仍有灵敏度和蛋白质序列的分辨率相当的局限性。因此，解决translatome，翻译mRNA的剧目，提供学习的一个很好的妥协转录组和蛋白质组。它比在最终评估基因表达转录更准确，同时提供更高的覆盖率和序列分辨率比蛋白质组学。 在哺乳动物系统中，大多数翻译活动通过帽依赖性启动开始。一组真核起始因子与40S核糖体小亚基一起聚集在一个mRNA的5&#39;帽。复杂的然后扫描mRNA和在到达起始密码子八月，募集60S核糖体大亚基形成一个完整的80S核糖体。伸长阶段所得与核糖体沿着mRNA的移动，与延伸因子协助的氨基酸的掺入从加载的tRNA到新生肽链上。多核糖体可以沿着一个单一的mRNA同时进行和相关的核糖体的数目已证实与蛋白质合成5,6的速率。这使得核糖体装载一个可靠的指标TRANslation并允许积极地翻译基于沉降率的mRNA分离。除了翻译的mRNA的定量化，序列信息可以得到鉴定参与翻译调控基序。还核糖核酸结合蛋白和其他翻译因子可以从不同的级分进行分离，便于相关调控蛋白的研究和发现。 在神经系统，平移控制已链接到诸如mRNA的储存，运输和地方蛋白质的合成过程。生长锥已被证明怀有的mRNA从轴突7的其余部分不同的特定局部池。此外，轴突具备的能力，本地合成蛋白质8,9。因此，翻译的本地控制已成为研究的神经生物学一个重要课题。多核糖分馏的电位，以解决这已在几项研究中已经示出，其中，所述技术被用于探讨在脊髓发育轴突导向，并证明BDNF在脑中10,11的活性依赖性翻译。

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		<col />
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		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Hank&#8217;s balanced salts solution</td>
			<td style="width:147px;">Gibco&#160;</td>
			<td style="width:119px;">14170-138</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube, Thinwall, Polyallomer, 14 mL, 14 x 95mm</td>
			<td>Beckman Coulter</td>
			<td>331374</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diethylpyrocarbonate&#160;</td>
			<td>Sigma</td>
			<td>D5758</td>
			<td>caution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cycloheximide</td>
			<td>Sigma</td>
			<td>C7698</td>
			<td>danger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol</td>
			<td>Sigma</td>
			<td>43815</td>
			<td>warning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cOmplete Protease Inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>11697498001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenylmethanesulfonyl fluoride solution</td>
			<td>Sigma</td>
			<td>93482</td>
			<td>danger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNasin Plus RNase Inhibitor</td>
			<td>Promega</td>
			<td>N2611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nonidet P-40</td>
			<td>Roche</td>
			<td>11332473001</td>
			<td>danger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium deoxycholate</td>
			<td>Sigma</td>
			<td>D6750</td>
			<td>warning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)</td>
			<td>Ambion</td>
			<td>AM9722</td>
			<td>danger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlycoBlue</td>
			<td>Invitrogen</td>
			<td>AM9516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Equipment</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dounce Tissue Grinder, 1mL/7ml</td>
			<td>Wheaton</td>
			<td>357538/357542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanodrop 2000</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW 40 Ti Rotor, Swinging Bucket, Titanium, 6 x 14 mL, 40,000 rpm, 285,000 x&#160;g</td>
			<td>Beckman Coulter</td>
			<td>331302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Density Gradient Fractionator</td>
			<td>Teledyne Isco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">2100 Bioanalyzer</td>
			<td style="width:147px;">Agilent Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo interrogation of central nervous system translatome by polyribosome fractionation

多个进程中涉及的基因表达，包括基因和蛋白质的转录，翻译和稳定性。其中的每个步骤都受到严格调控，影响蛋白质丰度的最终动力。各种监管机制的转换步骤存在，使mRNA水平单独基因表达的一种不可靠的指标。此外，mRNA翻译的地方性法规已特别牵连的神经元功能，移动性&#39;translatomics&#39;来关注在神经生物学的焦点。该方法可用于桥梁转录组学和蛋白质组学。 在这里，我们描述了必要的修改，以多核糖体分离的技术的基础上，积极的mRNA翻译到多个核糖体的沉降差在蔗糖梯度协会并询问该translatome。传统上，正在与体内样品特别的CENTRA，升中枢神经系统（CNS），已被证明是由于材料的限制量和脂肪组织成分的存在挑战性。为了解决这个问题，所描述的协议是专为与中枢神经系统的材料最小量的使用进行优化，就证明了使用单个小鼠脊髓和大脑。简要地，中枢神经系统组织中提取和翻译的核糖体上固定的mRNA与放线菌酮。然后进行浮选髓鞘去除脂质丰富的组件。分馏是，蔗糖梯度，其中的mRNA是根据其核糖体装载分开进行。孤立的分数是适用于多种下游检测，包括新的全基因组分析技术。

图2显示了分馏后代表多核糖体轮廓。脑和脊髓的剖面显示特征吸收曲线，如前所述为细胞系。的mRNA结合到核糖体小亚基（40S）的沉积物在较轻馏分和第一个出现在分布的峰值，随后是核糖体大亚基（60S）和monosome（80S）绑定的mRNA。的mRNA绑定到多个核糖体沉积物重质馏分，与后来的峰表明越来越多核糖体结合的。全球翻译可以从配置文件中进行评估。作为一个例子，脑组织中具有较高的多核糖到monosome比率比脊髓组织，这表明更活跃的翻译。 从单个组分提取的RNA被生物分析仪评估，显示出18S和28S rRNA的分布。 18S rRNA基因较早出现在配置文件中，按照核糖体小亚基沉降较轻SUCR<html> OSE分数。总RNA的典型产量为10-20微克的大脑和2-4微克脊髓。从单个组分的RNA产率高达4微克和0.8微克对大脑和脊髓分别取决于分数。空白蔗糖梯度已经显示了一些背景吸收在260 nm处，由于DTT的存在。这样的背景下可以在数据分析，以标准化样本值中减去。 EDTA处理倒塌的多核糖体峰，表明沉积分布是由于翻译。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51255/51255fig1highres.jpg" width="500" /> 图1的工作流程和体内多核糖分馏的潜在应用。&gt;请点击这里查看该图的放大版本。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51255/51255fig2highres.jpg" src="/files/ftp_upload/51255/51255fig2.jpg" /> 空梯度和大脑和无图2（A）脑和脊髓，与生物分析仪提取的RNA进行评估的多核糖体的典型剖面（B）GAPDH mRNA的分布馏分定量RT-PCR（C）多核糖体型材EDTA处理。 <a href="https://www.jove.com/files/ftp_upload/51255/51255fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td> 1.1 </td><td> 2个梯度缓冲液</td><td> 30毫米的Tris-HCl pH7.4的，30毫米MgCl 2，600 mM氯化钠，200微克/毫升放线菌酮（CHX），2mM的二硫苏糖醇（DTT）的</td><td></td></tr><tr><td> 1.1 </td><td>蔗糖溶液17.5％ </td><td> 8.67克蔗糖，将25ml 2倍梯度缓冲液，最多至50ml DEPC H 2 O中</td><td></td></tr><tr><td> 1.1 </td><td>蔗糖溶液25.6％ </td><td> 12.8克蔗糖，将25ml 2倍梯度缓冲液，最多至50ml DEPC H 2 O中</td><td></td></tr><tr><td> 1.1 </td><td>蔗糖溶液33.8％ </td><td> 16.9克蔗糖，将25ml 2倍梯度缓冲液，最多至50ml DEPC H 2 O中</td><td></td></tr><tr><td> 1.1 </td><td>蔗糖溶液41.9％ </td><td> 20.95克蔗糖，将25ml 2倍梯度缓冲液，最多至50ml DEPC H 2 O中</td><td></td></tr><tr><td> 1.1 </td><td>蔗糖溶液50％ </td><td> 25克蔗糖，将25ml 2倍梯度缓冲液，最多至50ml DEPC H 2 O中</td><td></td></tr><tr><td> 2.3 </td><td>同质化缓冲液A </td><td> 0.25M蔗糖，50毫米的Tris /盐酸，pH7.4）中，5毫米氯化镁2，25毫米氯化钾</td><td> 200μg/ ml的CHX，1个罗氏完全蛋白酶抑制剂，1 mM的DTT，1mM的phenylmethanesulfonylfluoride（PMSF），100 U / ml的核糖核酸酶</td></tr><tr><td> 3.1 </td><td> 2M蔗糖溶液</td><td> 68.4％蔗糖，50mM的Tris-HCl，pH7.4的，5毫米MgCl 2，25毫米氯化钾</td><td> 100μg/ ml的CHX，1个罗氏完全蛋白酶抑制剂，1 mM的DTT，1 mM的PMSF </td></tr><tr><td> 3.1 </td><td> 1.1M蔗糖溶液</td><td> 38.5％蔗糖，50mM的Tris-HCl，pH7.4的，5毫米MgCl 2，25毫米氯化钾</td><td> 100μg/ ml的CHX，1个罗氏完全蛋白酶抑制剂，1 mM的DTT，1 mM的PMSF </td></tr><tr><td> 3.1 </td><td> 0.9M蔗糖溶液</td><td> 30.8％蔗糖，50mM的Tris-HCl，pH7.4的，5毫米MgCl 2，25毫米氯化钾</td><td> 100μg/ ml的CHX，1个罗氏完全蛋白酶抑制剂，1 mM的DTT，1 mM的PMSF </td></tr><tr><td> 4.1 </td><td> ħomogenization缓冲液B </td><td>的0.25M蔗糖，50mM的Tris / HCl，pH7.4的，5毫摩尔MgCl 2，25mM的氯化钾，1％NP-40，1％脱氧胆酸钠</td><td> 200μg/ ml的CHX，1个完整的罗氏公司的蛋白酶抑制剂，1mM的DTT，1 mM的PMSF，100 U / ml的核糖核酸酶</td></tr></tbody></table>表1 列出缓冲器和解决方案。

Watch this Scientific Journal Video about In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation at JoVE.com

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

这个协议说明为了研究体内中枢神经系统的样本translatome多核糖分馏的必要修改。它允许翻译和转录调控的全球评估通过隔离和核糖体结合的RNA成分的总RNA的比较。

在中枢神经系统Translatome由多核糖体分馏体内拷问

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

in-vivo-interrogation-central-nervous-system-translatome-polyribosome

Research

JoVE Journal

Neuroscience

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

12.2K 次观看.  German Cancer Research Center (DKFZ). 这个协议说明为了研究体内中枢神经系统的样本translatome多核糖分馏的必要修改。它允许翻译和转录调控的全球评估通过隔离和核糖体结合的RNA成分的总RNA的比较。 

视频: In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

视神经横断：一个成人在中枢神经系统的神经元凋亡模型

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

科研

神经科学

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

调查中枢神经系统修复中受伤范式<em&gt;果蝇</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

生物神经毒素在茎的网络文化功能评估细胞衍生的中枢神经系统神经元

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

鼠标中枢神经系统的光透明使用被动CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

第三星果蝇中枢神经系统活动的电生理记录

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...