We would like to acknowledge the technical assistance of Kristina Jurcic in the University of Western Ontario MALDI MS facility, as well as the National Sciences and Engineering Council (NSERC) for funding this work. The authors would also like to acknowledge the Caprioli group (Vanderbilt University, TN) and Chaurand group (Universit&#233; de Montreal, QC) for their advice in optimizing the sublimation technique presented in this manuscript.

坚持以下描述西方安大略省的动物护理委员会（2016-014）的大学所有的动物处理程序。 1.组织准备和剖分<ol><li>通过所谓的"新鲜冷冻萃取"（FFE）的过程中提取从大鼠脑组织。 <ol><li>安乐死大鼠的戊巴比妥钠的过量和监测四肢反射。一旦所有的反射都停止了，断绝使用断头台鼠的头上。 </li><li>仔细的肌肉和其他组织使用手术刀颅骨分离。使用骨切割钳打破颅骨以暴露脑，从头骨（枕骨大孔）的底部开始到前部。 </li><li>一旦大脑暴露出来，小心地舀出来用手术锅铲，立即放在闪存冷冻碎干冰。 注:大脑会很可塑性。因此，放置在大脑时要格外小心干冰。如果大脑被压碎或针对一个表面按压时，它会改变其形状和在该位置结冰。 </li></ol></li><li>取下新鲜冷冻组织（未用甲醛灌注或嵌入在OCT） - 80℃的冰箱中放干冰。 </li><li>放置几滴水上低温恒温器保持器和地点上或干冰或低温恒温器冻结栏。按组织上轻轻低温恒温器支架，因为它开始冻结并按住，直到组织的基地周围水结冰和主持人到位。添加额外的水，如果必要，以进一步固定在支架上的组织。 注:水是用来代替OCT介质中，以避免与这会影响所希望的信号的检测化合物的组织的污染安装组织。 </li><li>位置组织低温恒温器。部组织到所需的解剖位置。确保切割厚度为8之间 - 12微米。 注:当切片在公共组织的低温恒温器设备，当务之急是独立的材料，如刀片，刷子，防倾杆，并持有人按顺序使用，以避免与嵌入化合物污染。低温恒温器的内也应在使用前与乙醇彻底清洁。 </li><li>使用低温恒温器防倾杆，慢慢的部分扁平的组织切片。如果倾杆的位置不正确，或刀片变钝可以发生在组织孔或标记。移动至组织，使用干净的漆刷切片平台的中心。 </li><li>安装<ol><li>通过将它们放置在低温恒温器，而切片冷冻导电幻灯片或金属板。当滑动被完全冻结，小心移动切片组织上使用清洁漆刷滑动的导电表面。一旦所有的组织切片的幻灯片正确定位，请将手指滑动下，组织对面，按直到部分融化。 注:章节可以折叠或在解冻过程中，当卷曲使用这种安装方法。这可以通过解冻组织时保持滑动在低温恒温器被减少。如果这些问题成为问题，或者，预热安装方法如下。 </li><li>可替代地，采取室温铟锡氧化物（ITO）滑动（或金属板），并轻轻地向下压上的切片平台表面冷冻组织部分，导电性的一面朝下。这将导致该组织部分解冻均匀到具有小卷曲或组织的折叠滑动的表面上。 注意:使用这种方法可能导致某些蛋白质和脂质的损失时安装冷凝可能会出现部分的下方。 </li></ol></li><li>放置与组织切片中进行5干燥器 - 10分钟。 </li></ol> 2.升华器设备建立注:在通风橱中执行这些步骤。 <ol><li>放置在铝容器中的砂浴上热板，以在金属剪式升降热板适当的表面积（ 即比热板大）。开启热板，并设置温度至140℃。 注:熔点DAN矩阵是187之间 - Visual C 190°。不要超过在烤盘这个温度。 <ol><li>如果加热板配有一个温度反馈探针，用它来监视砂温度在整个实验，确保温度一致。此功能可以帮助实验在不同的升华实验重复性。 注:砂浴应铝容器如玻璃容器内被包含在高温下可能碎裂。 </li></ol></li><li>放置300毫克DAN基质上升华装置的底表面上。放置基质在该装置的中心，并在近似宽度和滑动的长度的偶数层摊开被升华。 注意:DAN矩阵是有毒的。因此，重要的是戴手套，口罩，和安全在任何时候都护目镜处理粉状基质时。 DAN应贮存在黑暗中，因为它是光敏感。 </li><li>带的金属板到具有制版与冷凝器的底部直接接触，以确保均匀的温度的升华期间横跨滑板的整个表面的冷却分布装置的内表面上。可替代地，砂冷凝器的底部，以确保一个平的表面。 <ol><li>放置胶带沿板的外边缘和附着到内玻璃器皿的侧面。如果磁带被放置在板下，并附着在内层玻璃表面的底部，温度分布可能不连，并可能导致在横跨滑板的表面凹凸不平矩阵分布。 </li><li>带的空白（测试）跨越与再次放置在滑动件的外边缘带的金属板的表面上对角滑动。 </li></ol></li><li>连接装置的顶部和底部，W第i个中间橡胶O型圈，以确保完全密封。 </li><li>放置一个金属U型关节周围的装置的中心并拧紧虎钳直到该装置的顶部和底部一半被密封紧密在一起。在金属O型圈的沙浴以上的地方。 </li><li>以碎冰一把，并将其放置在冷凝器中。填写冷凝器¼到½充分用冷水创建冰凌。冰雪泥会冷却在装置的内部，并且随后将滑动附着于它的金属板。等待至少5分钟，使温度达到稳定状态。 </li><li>倒300毫升乙醇中的冷阱容器和玻璃器皿放置到容器中。降2 - 3小块干冰入乙醇中的冷阱容器的底部。冷阱输入具有运行在气缸底部的玻璃管，而不会输出。 </li><li>连接真空泵使用橡胶管冷阱输出。用另一块橡胶油管冷阱输入连接到升华设备。确保油管（如果可用）使用金属夹子关紧。 </li><li>使用真空泵以提供30的真空 - 为50mT。允许泵为至少5分钟，使压力平衡运行。升华装置的U形环上的虎钳可能必须被重新拧紧，一旦真空泵被接通，因为在该装置中的压力降低上。 注:强烈建议真空计被连接到泵以监测在实验过程中的真空的压力和以达到实验之间的最高可能的再现性来测试泄漏的压力。然而，大多数的泵被设计成维持恒定的压力，因此该计可能不是经验丰富的用户是必不可少的。此外，真空密封润滑脂可用于帮助维持真空压力。 </li></ol> 3.升华<ol><li>确保砂浴温度具有STABIL源化在140℃，并且该装置被固定在该金属O型环的砂浴具有连接所有管路的上方和真空接通。 </li><li>设置计时器7分钟，但不启动定时器。慢慢地将剪叉式升降机和沙浴直至升华设备，直至升华的万向节是很好的金属O型圈以上。这额外的空间允许在砂设备的调整。 <ol><li>迅速轻轻按下升华的砂表面上的，以确保该装置是坐在均匀在沙滩上，然后立即启动定时器。 </li></ol></li><li>当计时器的声音，关闭真空泵，并小心地将剪叉式升降机，直到升华不再接触沙浴，并在金属O型圈安全坐着。 <ol><li>缓慢地松开微排气阀来释放在装置的压力。松开周围的升华设备的橡皮管金属夹，慢慢地开始放松管。一旦RUBBER管已略有松动，弯曲管一侧，使残余压力逃脱。 </li><li>当环境压力的回报，小心地取出从升华设备的橡胶管。 </li></ol></li><li>松开装置的万向节的恶习，并删除。仔细分离升华装置的两个半部，并从内玻璃器皿的顶部拉断的幻灯片。检查幻灯片确认连矩阵分布。 注意:如果矩阵是不均匀的，在升华的底部粉状基质可以重新定位或升华器设备所用的砂为下一幻灯片重新定位。升华工序可以重复使用相同的矩阵数次，但是，基质最终将成为反复热暴露和粉末的量更暗将减小，使得升华时间将必须稍微调整以补偿。出于这个原因，重要的是监测矩阵贝的量是非常重要的纳克实验后升华，以确保幻灯片之间的基质沉积的一致性</li><li>如果基质的升华的分布和数量是足够的，用胶带与组织新的幻灯片到升华器，并重复第3节的内侧。 注:对于质量控制（QC）的目的，升华基质的量可以在每个实验后通过称量滑动之前和升华后除以与滑动2的表面积升华基质的重量计算，其应注意的是由于缺乏超过4小数点和鳞之间的变异最秤的精度，这些测量应仅用于最佳基质沉积的引导，而不是质量控制的一个完全量化手段，除非使用高精密天平（ 见图3）。 </li></ol> 4.组织存储/补液<ol><li>升华后，储存滑动在密封容器或小卡斯ETTE在密封的塑料袋中。 </li><li>孵育在-20℃冷冻载玻片2小时或过夜。 注:冷冻过程的目标是充当组织材料的解吸到矩阵的复水的步骤。冷冻过程也被证明当储存在-80℃下12以防止脂质的信号的劣化长达1周。出于这个原因，时间的样值存储在冰箱的量可以改变，以一定的程度，以满足实验者的需要而不显著改变的信号检测（如在我们的实验室观察到的）。但是，我们已经注意到矩阵的一些变色当样品在-20℃冷冻的时间超过24小时。因此，我们建议成像升华组织该时间之前或在-80℃储存组织更长的潜伏期。 </li><li> 10分钟 - 在5干燥器卸下从冰箱（集装箱），地点幻灯片。 </li></ol> 5.成像和Analysis <ol><li>从干燥器中取出的幻灯片和应用仪器标准，以确保在成像MALDI仪器的质量精确度。的类型的标准将取决于仪器而有所不同。标准一般横跨整个幻灯片施加均匀，周围组织将被成像（ 图3D）。 </li><li>将滑入MALDI仪器，并按照制造商的成像实验说明（仪器方法应针对1000进行优化 - 2000质量范围）。代表性的结果（ 图4）在反射器负模式获得具有70微米的光栅和20张/谱（采集时间〜2小时）。 </li></ol>

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The authors have nothing to disclose.

这项工作细节矩阵升华对组织的标准化协议，用于检测带负电荷的脂类，如神经节苷脂，在MALDI IMS实验。用于MALDI IMS的样品制备是高度可变的，并且必须被定制，以适应所关注分析物的独特性能。矩阵的上组织样品表面上的应用程序是在样品制备过程的与问候结果在IMS中的质量的一个关键方面。应特别注意选择矩阵时，确保该矩阵是与所关注的分析物相容并且是自由的频谱矩阵衍生工件作出。升?...

基质辅助激光解吸/电离（MALDI）成像质谱（IMS）正在成为一种高度寻求技术为脂质，肽和蛋白质穿过完好样品表面的空间分布的可视化之后。 MALDI IMS的以前被称为预纯化的分析物的分析技术，但在最近几年中，已经在因为具有高分辨率视觉/解剖参考点，而质谱的精确度结合的能力许多其他学科提请注意需要任何外部标记。作为利用这种技术的研究的科学池继续增长，所需要增加的标准化，易于后续的协议，以协助IMS实验开发和优化。神经节苷脂，一组膜脂质在中枢神经系统高度丰富的，是理想的MALDI IMS实验作为它们的位置，嵌入在膜中，使某些特定上课不可能使用常规的免疫标记以检测。此外，我们已经表明，使用MALDI IMS中，这些脂质，其充当细胞信号的调节剂，除其他事项外，有在被脑损伤3，4，5后改变了健康啮齿动物脑独特的解剖分布模式。神经节苷脂位于较高质量范围相比，大多数脂质种类，并因此最适合于MALDI成像平台。 <img alt="图1" src="/files/ftp_upload/55254/55254fig1.jpg" /> 图1:MALDI IMS实验的工作流程。使用升华在MALDI IMS实验的一般流程的图。组织在-80℃冷冻于低温恒温器被分段和10微米的部分是安装在导电的ITO载玻片解冻。然后将载玻片置于干燥器直到升华。 SLIDES插入升华装置和偶数层矩阵施加到组织样本表面。样品在-20℃冷冻然后置于干燥器中10分钟，冷冻过夜。一旦标准被应用，将样品插入到其中激光跨越引起解吸分子在基质中以电离所述组织指示MALDI仪器。离子行进向下根据它们的质量（时间飞行/ TOF）直到它们到达了检测器的飞行管和分离。上一个预定的质量 - 电荷（M / Z）范围内的离子丰度分析物的信息显示为两个分子图像和质谱。这个数据可以用来既可视化和量化的成像组织内感兴趣的分析物的离子丰度。 <a href="http://ecsource.jove.com/files/ftp_upload/55254/55254fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 样品制备用于MALDI IMS是高度可变的过程的每个步骤必须进行定制，以所关注的分析物。基于MALDI-实验限定特征是使用沉积到分析前的样品表面上的基质的涂层。除了吸收和在消融过程中从激光转印辐射能量的作用，该基质也用于各种分析物从样品中分离，从而有利于感兴趣6,7化合物的分析。基质样品表面的均匀的应用是在样品制备过程中最关键的步骤。不正确的基质沉积可导致大量异构基质晶体形态和文物的发展，低离子信号，重复性差7。 由于某些矩阵的亲和力来分离特定的分析物，被选择用于实验矩阵的类型可以显著改变的结果。用于蛋白质和肽的成像矩阵常常不同于用于成像脂质不同，此过程是通过需要额外的方法，如为了从组织成功地检测信号的洗涤和再水合步骤进一步复杂化。虽然脂质信号8的增强存在洗涤步骤，它们不是用于检测最脂质种类的一个先决条件。当选择的脂成像实验的矩阵，它考虑的兴趣，因为这脂质的极性将缩小合适矩阵的范围是重要的。例如，神经节苷脂含有唾液酸残基这给他们一个整体的负极性。有许多的矩阵，可以有效地解吸和电离来自组织神经节苷脂;然而，因素，如在频谱并在真空下基质的稳定性基质衍生的峰必须在考虑服用。 1,5-diaminonapthalene（DAN）矩阵是仪器的真空条件对于大多数成像应用下足够稳定，并表现出高度的脂质解吸灵敏度和可用于在正和负离子模式2脂质的分析。 DAN基质，相对于其他负脂质亲和基质如二羟基苯甲酸（DHB），9-氨基吖啶（9-AA）和5-氯-2-巯基苯并（CMBT）时，能够最有效地解吸从大鼠脑神经节苷脂组织在负离子模式（手稿准备中）。 选择基质沉积的适当的方法是同等重要选择矩阵本身。湿基质沉积方法，其中所述固体基质溶解在有机溶剂中，并通过气动沉积或自动喷雾器或检举，作为液体渗透的样品，以允许额外的用于蛋白质和肽的解吸特别有效化合物和共结晶与基质的ction。尽管这些技术也可用于脂质应用，分析物离域和不均匀的基质晶体地层是屡见不鲜由于脂质的在溶剂中的高丰度和溶解度，特别是在组织2,9。因为脂质易于从组织电离，干燥基质沉积技术，如升华，提供一种简单的，成本效益的替代喷雾器而绕过许多这些技术的缺点的。升华的MALDI IMS实验的成功归功于功能，如微晶矩阵的形态这增加了矩阵分析物结合的表面面积，增加基质纯度和均匀的基质的沉积，从而增加可重复性比较潮湿的矩阵技术1，10。 升华invoLVES正下方产生的固体，粉状基质随后沉积在组织样品表面的气相过渡冷却的样品表面加热真空下粉状基质。期间升华，基质沉积可通过改变因素例如时间，温度和压力，以提供高度可重复的结果来控制。单个升华试验可以在任何地方需要5至20分钟取决于矩阵的选择的类型，处理前可重复使用数次。该装置可商购于自动喷雾器的价格的几分之一处购买，并且很容易被拆开进行清洁和维护。成本低，这种基质沉积技术相对简单使其非常适用于开始后，或MALDI IMS脂质成像应用拓展的研究人员。虽然详细说明了组织对IMS的升华协议的信息已报告11，少数标准化协议存在W¯¯HICH重点涉及进行升华实验在负离子模式成像高质量脂质，使得难以建立的技术中没有广泛的试验和错误的基本工作流程。下面是一个试验性协议旨在填补DAN矩阵的升华到大鼠脑切片用于高分辨率成像和神经节苷脂的检测该间隙。 <img alt="图2" src="/files/ftp_upload/55254/55254fig2.jpg" /> 图2:升华设备。照片（A）和升华装置的示意图（ 二 ）。真空泵由橡胶管连接到冷阱充满300毫升乙醇。冷阱，然后由橡胶管的升华装置相连。该装置是由两个独立的玻璃器皿的件所用的金属U形接头密封在一起。升华器的上半部分包含填充有冰凌冷凝器。该样品板贴到所述冷凝器的底部，所述密封的玻璃装置内。升华设备的下半部分包含DAN矩阵，摊开均匀面临的样板。期间升华，玻璃装置被放置在砂浴中加热至140℃用热板正下方。温度探头有助于维持整个升华试验通过沙浴温度的反馈相比为实验的预设温度稳定的温度。 <a href="http://ecsource.jove.com/files/ftp_upload/55254/55254fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sublimator</td>
			<td>Chemglass Life Sciences</td>
			<td>CG3038-01</td>
			<td></td>
		</tr>
		<tr>
			<td>1,5- Diaminonapthalene (DAN) matrix</td>
			<td>Sigma-Aldrich</td>
			<td>D21200&#160;</td>
			<td>100 G</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Thermo-Fisher Scientific</td>
			<td></td>
			<td>CryoStar NX50</td>
		</tr>
		<tr>
			<td>Hot plate with temperature feedback</td>
			<td>Thermo-Fisher Scientific</td>
			<td>HP88857290</td>
			<td>Isotemp ADVD 7x7 HP 100-120v</td>
		</tr>
		<tr>
			<td>Stainless Steel Jack</td>
			<td>Thermo-Fisher Scientific</td>
			<td>2216479</td>
			<td>10x10</td>
		</tr>
		<tr>
			<td>Cold Trap</td>
			<td></td>
			<td></td>
			<td>Custom built on site</td>
		</tr>
		<tr>
			<td>Vacuum Pump</td>
			<td>Franklin Electric</td>
			<td>1102180403</td>
			<td>Savant VP100 Two Stage</td>
		</tr>
		<tr>
			<td>Indium-tin-oxide (ITO) Slides</td>
			<td>Hudson Surface Technology</td>
			<td>PSI 1111000</td>
			<td>type II, 1.1mm/25 each</td>
		</tr>
		<tr>
			<td>MALDI TOF/TOF 5800 Instrument</td>
			<td>AB Sciex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>Sigma-Aldrich</td>
			<td>D2797</td>
			<td>tabletop desiccator</td>
		</tr>
	</tbody>
</table>

sublimation of dan matrix for the detection and visualization of gangliosides in rat brain tissue for maldi imaging mass spectrometry

样品制备为最佳检测和基质辅助激光解吸/电离（MALDI）成像质谱（IMS）的实验分析物的可视键。确定适当的协议遵循整个试样制备过程中可能是困难的，因为每个步骤必须进行优化以符合与所关注分析物的独特的特点。这一过程不仅涉及发现能解吸和有效地电离感兴趣的分子相容的基质，而且选择合适的基质沉积技术。例如，一个湿基质沉积技术，这需要在溶剂中的基体溶解，优于大多数蛋白质和肽的解吸，而干燥基质沉积技术对于脂质的电离特别有效。升华已被报告为干燥基质沉积用于检测通过MALDI IMS在组织脂类的一种高效的方法，因为该homogenei相比于许多湿沉降方法1，2基质晶体沉积和最小的离域分析的TY。广泛地说，它包括将一个样品和粉状基质与按压冷的表面的采样的真空密封室中。然后该装置被降低到加热浴（砂或油），结果在粉状基质的升华到冷却组织样品表面。在这里，我们描述了使用1,5-二氨基萘（DAN）矩阵为使用MALDI IMS在大鼠脑神经节苷脂的检测和可视化升华协议。

经升华实验结束后，将玻璃仪器的两半在通风柜和滑动分离，贴在冷凝器，可以去除（ 图3A）。此时，滑动应检查在沙浴中的装置的不均匀矩阵分布和时间，温度，或位置可能必须调整为下一幻灯片。一个成功的DAN升华实验将导致甚至涂层沿着那里的组织的解剖特征可以清楚地显现在滑动和载玻片上基质的量的表面灰色/褐色矩阵的类似于量在组织上（ ?...

Watch this Scientific Journal Video about Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry at JoVE.com

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

DAN矩阵升华为神经节苷脂对大鼠脑组织的检测和可视化MALDI成像质谱

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

sublimation-dan-matrix-for-detection-visualization-gangliosides-rat

Research

JoVE Journal

Neuroscience

10.7K 次观看.  The University of Western Ontario. A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

视频: Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

科研

神经科学

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

特定脑区的兴奋毒性损伤的成年大鼠立体定向手术治疗

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

光纤植入慢性Optogenetic刺激脑组织

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which&#160;11C-AcAc and 18F-fluorodeoxyglucose (18F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of 11C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of 11C-AcAc and 18F-FDG brain uptake (cerebral metabolic rate; &#956;mol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer&#39;s and Parkinson&#39;s diseases.

Small-animal positron emission tomography enables the assessment of the brain&#39;s two main energy substrates:&#160;glucose and ketones. In the present method, 11C-acetoacetate and 18F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

非侵入性的脑实质，血管和代谢高频超声和光声大鼠深脑成像

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

矩阵辅助激光去离子成像质谱法显示人脑中的淀粉样β矿床

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.

Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding.

使用 PRIMO 微模式系统对蛋白质的光诱导分子吸附，研究细胞对细胞外基质蛋白的反应

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

使用天然多酚姜黄素对脑组织淀粉样斑块进行标记和成像

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...