这项工作是慷慨神经疾病和中风（NIH / NINDS）资助1R01NS086981和康拉德希尔顿N.基金会健康/国立全国学院的支持。我们感谢洛朗邦托利拉博士和马修SCHIBLER博士及其与激光共聚焦显微镜宝贵的援助。我们感谢那些给CLARITY论坛（http://forum.claritytechniques.org）作出了贡献。我们特别感谢卡尔戴瑟罗特博士打开了他的实验室，教这个迷人的技术。笔者是从脑成像医学研究组织，脑成像支持基金会皮尔森 - 洛夫莱斯基金会，基金会阿曼森，资本集团慈善基金会，威廉·M和琳达R. Dietel慈善基金，以及基金北极星的慷慨支持表示感谢。研究本出版物中报也部分由国家研究资源中心和民族的主任办公室的支持在奖项数量C06RR012169，C06RR015431和S10OD011939卫生研究院人。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

所有实验均按照机构动物护理和使用委员会（IACUC）的指导方针进行。 THY1-YFP，PLP-EGFP和PV-TdTomato小鼠用于这些实验，但表达的荧光蛋白的任何小鼠可以成功地清除并成像。 1.组织准备注意:多聚甲醛（PFA）和丙烯酰胺是有毒的和刺激物。执行利用这些试剂在通风橱，并配备适当的个人防护装备（实验室外套，手套和护目镜）的实验。 <ol><li>牺牲安乐死腔动物提供〜1毫升异氟烷，直到呼吸停止（〜2 - 3分钟）。 </li><li>使用蠕动泵设置为〜5毫升/分钟，以心内，在4℃下灌注灌注溶液（ 表1）的鼠标，直到血液从组织中清除（5 - 10分钟）。 </li><li>改变灌注到固定液（ 见表1）在4℃，直至颈部和尾部已经显著硬化（5 - 10分钟）。 </li><li>结束灌注和杀头的动物。通过首先除去围绕颅骨结缔组织，然后从颅骨的腹侧表面去除骨和提起脑出（ 图1a），如在20中所述，小心解剖出从头骨大脑。 注:优抚周围小脑flocculonodular叶和嗅球切除骨。 <ol><li>轻轻插入脊髓和脊椎之间的精，尖剪刀尖和切割个别脊柱段走，如在描述21从解剖脊柱脊髓。 </li></ol></li><li>通过将它放置在一个塑料小鼠大脑基质并沿与基体的刀片的中线切割Hemisect大脑。 </li><li>将每个半球和脊髓分离的50ml离心桶与ES〜40毫升的水凝胶溶液（ 表1）。从这点出发盖用铝箔管，以在所有后续步骤保护荧光。 </li><li>轻微振荡（〜30转）7天孵育含有在水凝胶中的样品的管中，在4℃。 </li></ol> 2.聚合<ol><li>丢弃约25mL水凝胶，保存样品和约25mL的水凝胶中的离心管中。 </li><li>通过去除帽和放置离心管在干燥器罐和罐连接到真空脱氧样品。施加真空，至少10分钟，以允许溶解的气体出来溶液和形式气泡。轻轻摇动赶走气泡。 </li><li> 3分钟 - 与100％的氮气罐干燥器超过2更换真空。一旦压力均衡（一旦干燥器罐的顶部可以打开），迅速帽管，以防止氧气的放归。 </li><li>聚合物通过将管与样品中的37℃水浴中3小时，直至聚合反应完成IZE水凝胶。聚合水凝胶将采取的凝胶状稠度（ 图1b）。注意:在顶部未聚合水凝胶的少量是可接受的。 </li><li>用刮刀以除去由离心管聚合样品，然后切去从样品中多余的水凝胶。 </li><li>通过将样品在软绒布擦拭，并轻轻揉并在其上滚动的组织，留下的样品聚合水凝胶的只有薄薄的一层去除剩余的水凝胶。 </li></ol> 3.脂质去除注:脂质清除的组织是脆弱的，小心处理。引流管时，使用细胞过滤器，以避免丢失样本。此外，该样品将膨胀整个结算处理，但是，肿胀可以在折射率匹配过程颠倒。 注意:SODI嗯十二烷基硫酸钠（SDS）和硼酸是刺激物。执行利用它们在通风橱，并配备适当的个人防护装备（实验室外套，手套和护目镜）的实验。 <ol><li>转移聚合样品到一个干净的50ml离心管中，加入清除缓冲液（ 表1）45毫升孵育在45℃下轻轻摇动（〜30转）。通过浇灭了用于清除缓存为化学废物和添加〜45毫升新鲜缓冲结算日更改缓冲区最初7天。保持含在铝箔覆盖，以保护荧光团的样品的离心管中。 </li><li>最初7天后，在40℃和交换结算缓冲器孵育样品每2 - 3天。继续结算，直到所有的脂类是溶解和组织达到透明度（ 图1c）。这将是4 - 6周的小鼠大脑半球2 - 4周为一个脊髓。 </li><li>一旦组织被清除，转移样品到一个新的50ml离心管中并在40℃轻微振荡（〜30转）在PBST中（ 表1）清洗4次超过24小时，以除去残余的SDS。 </li></ol> 4.染色分子注意:4&#39;，6-二脒基-2-苯二盐酸盐（DAPI）是一种刺激性和利用它应该在通风橱，并配备适当的个人防护装备（实验室外套，手套和护目镜）进行任何实验。 <ol><li>样品转移到一个干净的15ml离心管，用1微克/毫升的DAPI在1X PBST，pH值7.5填补。在室温下孵育（RT）下24小时。保持含在铝箔覆盖，以保护荧光团的样品的离心管中。 </li><li>在室温洗PBST 3次，每次至少6小时/洗。 </li></ol> 5.折射率匹配注意:2,2&#39;-硫代二乙醇（TDE）是一种刺激和利用它的任何实验SH乌尔德在通风橱，并配备适当的个人防护装备（实验室外套，手套和护目镜）来完成。 <ol><li>将样品转移到一个干净的15ml离心管中，用30％在1×PBS（体积/体积）TDE，pH值7.5填。孵育24小时40℃轻轻摇动（〜30转）。 </li><li>交换30％TDE用63％（V / V）的1×PBS中，pH值7.5 TDE。在40℃孵育轻微振荡24小时。 注意:示例将退缩到约原来的大小。 </li><li>在平坦，清洁的表面滚动的一块可重复使用的粘合剂的与均匀的直径只比所述样品的厚度稍大的圆筒。 </li><li>莱可重复使用的胶粘剂缸开放式圆（〜25内径在顶部一个小口）上有40毫米的玻璃底菜。如果修正需要进行，从玻璃移除筒和用刀片切割。 </li><li>使用P1000枪头通过创建可重复使用的粘合剂和玻璃之间的密封滚动它周围的筒的外缘。 </li><li>地方〜100微升63％TDE到玻璃菜，传遍覆盖可重复使用的胶粘剂的圆内的玻璃表面。 </li><li>使用刮刀，小心地在圆圈的中间样品，注意不要形成泡沫。 </li><li>移液器另外100微升63％TDE直接到样品，然后立即放置40mm的圆形玻璃盖在可重复使用的粘合剂。 </li><li>使用索引，中间和两手的无名指，小心地按左右的圆的周长，直到盖玻璃已经取得与样品接触。 </li><li>缓慢将玻璃底培养皿到直立位置靠在一个表面上，然后添加63％TDE用吸管直到溶液到达在上面的小开口。使用无尘擦拭擦干枪头使得溶液不会对玻璃滴落。以避免在该室的气泡，从未空吸管一路。 </li><li>至小开，加硅弹性体包围的圈子，并创建一个封闭室。等待5分钟，它的成像之前干燥。 </li></ol> 6.显微镜注意:在激光共聚焦，单一平面照明和光片显微镜使用的激光器是非常强大的，可以导致失明。任何利用激光器实验后应广泛的培训和十分谨慎和适当的保护眼睛进行。 <ol><li>放置成像室里面上的激光扫描共聚焦显微镜的阶段样品。 </li><li>打开成像软件（见材料表）。要打开氩激发激光，单击该程序，然后激光图标的上方配置选项卡。勾选旁边氩供电激光移动滑块规模的30％。观察指针慢慢温热至所选择的位置。 <ol><li>返回到获取标签的顶部。在大盒子"光束路径设置＆＃34;进入加载/保存设置框，然后选择下拉菜单中选择一个合适的波长信道图像YFP（527纳米）。 </li></ol></li><li>下的通道设置框:通过定位标记为"当前对象客观"红色超链接选择适当的成像目标。点击选项卡上打开所有可用的目标和选择会自动旋转炮塔。 <ol><li>使用的目标（ 例如 ，10X）具有大的工作距离（大于组织的厚度大要成像， 例如，第11毫米）和大数值孔径（ 例如 ，0.3），以最大限度地提高面内的图像分辨率和最小化光学切片厚度。 </li></ol></li><li>在下拉菜单，打开"XY:解析"的左栏窗口设置采集矩阵，所谓的"格式"，1024×1024或适当的目标的横向分辨率极限值。设置缩放因子尽可能低（ 例如 ，10.7）。 </li><li>在同一窗口，通过相应地在下降了单独标记菜单中设置8行平均1帧的平均参数。使用较大的值要高信噪比和小的值的快速采集。 注:8号线均线和1架平均将获得小鼠大脑半球的高品质的图像约4小时。 </li><li>找到使用阶段控制样本。一旦有代表性的领域是可见的，设置增益和偏移。 注意:增益和偏移基于所述组织类型，荧光团将基本上不同，和/或正被成像的解剖特征。对YFP，从760至780为增益和从-1.0到-1.5的偏移设定值。 </li><li>选择"瓷砖扫描"，并选择感兴趣的区域的边界。要获取设置的Z堆叠的上限和下限。设定基于所述物镜的光学部分分辨率极限的光学部分的厚度。 注:客观的光学部分分辨率极限主要由其数值孔径限定。所使用的大多数显微镜控制软件会自动计算出适当的值的目标。要10X物镜具有0.3的数值孔径，这将是大约11微米。 </li><li>开始采集。 注意:这可能需要多个小时。 </li><li>使用高倍目标从感兴趣的特定区域收集更高分辨率的图像。 </li></ol>

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The authors have nothing to disclose.

水凝胶包埋组织（无源净度）的被动结算是用于清除大块组织的一个简单和廉价的方法。这种方法不要求专用设备，并且可以很容易地在一个温度控制的振荡器进行。在几个星期的跨度，即使是大的，高度髓组织，例如整个脑或脊髓，将成为透明，适用于显微镜。尽管这份报告集中在CNS组织中的清除，被动净度可应用于任何组织。 的光透明的技术的相对优点和弱点已在文献13,17,22先前所论述，但最清楚地23布置。其中，样品的大量被同时清除和可重复性是很重要的被动净度是非常适合的实验。使用水凝胶，以提供额外的支持，组织有利于米ultiple探测组织。在这份报告中，分子量小的染料，DAPI，被证明，但是多轮抗体探测的也是可能的，这种方法。 上述方法从我们先前公布的协议15日在一对夫妇的显著偏离的方式。首先，在当前的方法中，标本不心内与水凝胶灌注。这是由于这样的事实，该水凝胶可过早聚合在管道和灌注装置的针，导致不完全的灌注和不一致的结算。在水凝胶温育的多聚甲醛固定的组织7天是与原始净度协议14相一致，虽然1天温育用于1 -在无源净度技术（PACT）17 3毫米厚的组织切片。其次，这种方法在以前报道15-17温度稍高执行脂质去除。高temperaturÈ加速脂质去除，但必须针对组织​​完整性（熔化）的灾难性损失的可能性进行平衡。通过8℃下温育温度的升高（至45℃）下放置7天，然后通过3℃（〜40℃）的脂质去除处理的其余部分，组织清除加快，但没有并发风险组织完整性的损失。这种温度上升是在原来的净度协议电泳组织结算系统（ETC）（50℃）14中报道的温度是一致的。此外，在转基因表达的荧光团也不在DAPI染色降低的强度没有降低已经观察到。 脱氧是在创造水凝胶的一个重要步骤。所述水凝胶可以聚合在氧溶解在水凝胶溶液中的存在，但在我们手中的速率和聚合的完整性是更变量无脱氧。如果Hydrogel不聚合，最常见的原因是氧的在水凝胶的存在。 光学结算过程是非常容易实现的，但是，随后的显微镜可以相当复杂。至关重要的是用于图像的组织的目标。客观的工作距离限定的深度，它可以像转变成组织样品。为2毫米的工作距离的物镜只能图像2.毫米深处的样品，因此一个客观的工作距离必须按顺序比样品的厚度大到图像它完全。同样，物镜的数值孔径一个限制的特征的尺寸，它可以解决两个平面内，并且它可以有意义地获得光学切片的厚度。选择性平面照明显微镜（SPIM）和轻表显微镜（LSM）可以通过照明的性质克服光学部厚度的限制，但这些万分之一COPES并不普及。高数值孔径，大的工作距离的目标是罕见的，昂贵的，但所必需的高品质成像的光学大清除部分。 一旦图像已被获得的，它们往往是非常大的，从几百MB用于低分辨率（都在平面和z方向）的图像，以用于收集在一个LSM高分辨率图像数结核病。用于图像分析的计算机需要不仅存储了大量的，但大量的用于图像处理的随机存取存储器（RAM）的。适合大光清图像卷的分析软件包包括阿米拉，了Imaris和ImageJ的14-16。 之间的这种方法的局限性，被动净度需要更多的时间来清除组织比其他光透明的方法。减少的组织的体积，以通过修整它或切片它成小块可以ACCEL被清除中心提供全方位脂去除。此外，在水凝胶减少多聚甲醛的浓度将依次减少交联和提高水凝胶的孔隙大小，导致更快速的交换17，尽管这可能导致更大的组织扩张，从个人实验者可以发现，这是值得的结算的减少时间。此外，用TDE作为清除剂相结合，整体组织扩张的量可能是有限的。 虽然这份报告描述了小分子量染料，抗体探测是一个很自然的下一个步骤。水凝胶的存在不仅提供物理支撑，以被清除的组织中，但也作为抗体的穿透深入到组织的障碍。降低的丙烯酰胺浓度或低聚甲醛浓度将增加孔径在水凝胶，便利（和从而加速）抗体渗透17，但这对做卷烟产生有害影响UE扩张。然而，抗体探测的组织块可以在几天和全脑的物质在一使用在原来的报告14中描述的丙烯酰胺和低聚甲醛浓度周来完成。 光透明技术允许一个深入观察到大脑，促进其结构和功能的理解。这些新工具将使研究人员能够可视化在细胞和分子的决议大脑，提高我们的器官的这个最复杂的理解。

The ability to image complete neuroanatomical structures is immensely valuable for understanding the brain in health and disease. Traditionally, 3D imaging has required tissue sectioning to provide the axial resolution and to visualize deep anatomical structures. This approach can produce high-resolution data sets, but requires sophisticated image reconstruction techniques and is very labor intensive. As a result, it has been limited to the imaging of small volumes of tissue1-3. Optical sectioning, on the other hand, is well suited for the creation of high-resolution 3D images of fluorescently labeled tissues. Since optical sectioning is inherently three dimensional, it does not require extensive computation to produce a 3D image volume. However, light scattering and tissue opacity limit the depth at which tissues can be optically sectioned. The depth of imaging is limited to about 150 &#181;m in laser scanning confocal microscopy and to less than 800 &#181;m using two-photon excitation microscopy4-8.
In order to overcome these limitations, several optical clearing techniques have been recently developed and then further refined to permit the deep microscopic imaging of intact tissues. The use of benzyl alcohol and benzyl benzoate (BABB) to render fixed tissues transparent was among one of the earliest approaches9. However, this approach was limited by the quenching of fluorescence in the samples and incomplete clearing of highly myelinated structures10. Refinements of this technique, such as 3D Imaging of Solvent Cleared Organs (3DISCO), have led to very rapid and complete tissue clearing, but still suffer from rapid loss of fluorescent signals, especially yellow fluorescent protein (YFP)10. Water-based clearing solutions, such as Sca/e11 and SeeDB12, preserve fluorescent signals, but do not completely clear highly myelinated tissues. Clear, Unobstructed Brain Imaging Cocktails and Computational analysis (CUBIC) is a promising new optical clearing technology that appears to overcome the limitations of previously developed water-based clearing solutions13. In contrast with other optical clearing techniques, Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY)14 embeds the brain in a porous matrix that provides structural integrity to proteins, nucleic acids and small molecules, while leaving lipids unbound. The lipids are then removed electrophoretically, culminating in an optically cleared brain that can be easily visualized and equally easily probed using commonly available techniques.
Electrophoretic tissue clearing (ETC) is used to remove lipids from hydrogel embedded tissues in CLARITY, however, ETC can be difficult to implement consistently and tissues subject to electrophoresis can exhibit tissue distortion, browning, loss of fluorescence and antibody reactivity. Passive lipid clearing avoids these limitations, but requires increased clearing times15-17. Passive clearing also permits the clearing of large numbers of samples in parallel, since it is not limited by the number of ETC apparatuses available. Decreasing the concentration of paraformaldehyde and excluding bis-acrylamide from the hydrogel have led to great increases in the speed of clearing at the expense of greater tissue expansion16. Less expensive alternatives to the refractive index matching solution used in the original CLARITY technique have been developed17-19. 2,2'-thiodiethanol (TDE) is an inexpensive and rapid brain clearing agent that reverses some of the tissue expansion that occurs during lipid removal18. This report incorporates passive clearing of hydrogel embedded tissue and the use of TDE as a refractive index matching solution to the original CLARITY technique to produce a highly reproducible and inexpensive protocol for the optical clearing of the mouse central nervous system.

<table border="0" cellpadding="0" cellspacing="0" width="750">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10X phosphate buffered saline (PBS)</td>
			<td style="width:212px;">Fisher</td>
			<td style="width:119px;">BP399-1</td>
			<td style="width:203px;">buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">32% paraformaldehyde (PFA)</td>
			<td style="width:212px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15714-5</td>
			<td>perfusion and hydrogel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2,2&#39;-thiodiethanol (TDE)</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:119px;">166782-500G</td>
			<td>refractive index matching solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40% acrylamide</td>
			<td style="width:212px;">Bio-Rad</td>
			<td style="width:119px;">161-0140</td>
			<td>hydrogel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2% bis-acrylamide</td>
			<td style="width:212px;">Bio-Rad</td>
			<td style="width:119px;">161-0142</td>
			<td>hydrogel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VA-044 initiator</td>
			<td>Wako Pure Chemical Industries, Ltd.</td>
			<td>VA044</td>
			<td>hydrogel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">boric acid</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:119px;">B7901</td>
			<td>clearing buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium dodecyl sulfate (SDS)</td>
			<td style="width:212px;">Fisher</td>
			<td style="width:119px;">BP166-5</td>
			<td>clearing buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium hydroxide</td>
			<td style="width:212px;">Fisher</td>
			<td style="width:119px;">55255-1</td>
			<td>buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium azide</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:119px;">52002-100G</td>
			<td>preservative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">triton x-100</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:119px;">X100-500G</td>
			<td>buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">heparin</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3393-50KU</td>
			<td>perfusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium nitrate</td>
			<td style="width:212px;">Fisher</td>
			<td style="width:119px;">BP360-500</td>
			<td>perfusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DAPI</td>
			<td style="width:212px;">Molecular Probes</td>
			<td style="width:119px;">D1306</td>
			<td>nuclear stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">99.9% isoflurane</td>
			<td style="width:212px;">Phoenix</td>
			<td style="width:119px;">57319-559-06</td>
			<td>anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">hydrocholoric acid</td>
			<td style="width:212px;">Fisher</td>
			<td style="width:119px;">A1445-500</td>
			<td>buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">glass bottom dish</td>
			<td style="width:212px;">Willco</td>
			<td style="width:119px;">HBSB-5040</td>
			<td>Willco dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">reusable adhesive</td>
			<td style="width:212px;">Bostick</td>
			<td style="width:119px;">371351</td>
			<td>Blu-Tack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">silicon elastomer</td>
			<td style="width:212px;">World Precision Instruments</td>
			<td style="width:119px;">KWIK-CAST</td>
			<td>Kwik-Cast</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">lint-free wipe</td>
			<td style="width:212px;">Kimberly-Clark</td>
			<td style="width:119px;">34120</td>
			<td>KimWipe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mouse brain matrix</td>
			<td style="width:212px;">Roboz</td>
			<td style="width:119px;">SA-2175</td>
			<td>sectioning tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">matrix blades</td>
			<td style="width:212px;">Roboz</td>
			<td style="width:119px;">RS-9887</td>
			<td>sectioning tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">peristaltic pump</td>
			<td style="width:212px;">Cole-Parmer</td>
			<td style="width:119px;">77122-24</td>
			<td>pefusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">laser scanning confocal microscope</td>
			<td>Leica</td>
			<td>SP5</td>
			<td>microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">imaging software</td>
			<td style="width:212px;">Leica</td>
			<td style="width:119px;">LAS AF</td>
			<td>microscopy</td>
		</tr>
	</tbody>
</table>

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.

可视化脑的结构组织为理解形态学和连通如何影响在健康和疾病的脑功能的关键。光透明的技术使得有可能在三维图像的细胞群在完整组织，使我们能够研究形态学和连接内聚。 小鼠表达荧光蛋白通过特异于亚群的启动子驱动的细胞允许一个梳理开神经连接的大脑中的复杂的图案。 THY1-YFP的转基因小鼠进行了广泛的光透明文献，因为这些小鼠在驻留在大脑皮质和海马投射神经元，以及其他结构的大脑中的（ 图2a和b）一种子集表达YFP被使用。此外，YFP +皮质V层神经元的项目，通过在s皮质脊髓束皮纳尔线（ 图2c＆D），使得它们在评价脊髓轴突损伤和/或损伤对皮质神经元15的效果非常有用的。通过使用光清组织共聚焦显微镜和光学切片的人可以轻松地评估在大型解剖结构的神经元密度差异。 除了THY1-YFP的转基因小鼠，任何数量的转基因小鼠进行成像。 PLP-EGFP转基因小鼠表达整个大脑和脊髓（ 图3a）表达蛋白脂质蛋白（PLP）成熟的少突胶质细胞增强绿色荧光蛋白（EGFP）。光伏TdTomato转基因小鼠表达TdTomato在小清蛋白（PV）的一个子集表示在小脑，纹状体和海马（ 图3b）的interneurons。甚至在小鼠中不表达荧光转基因，小分子量的荧光染料，如D的API，可以容易地扩散到水凝胶包埋组织，并允许细胞核的评价在大脑中（ 图4）。 <img alt="图1" src="/files/ftp_upload/54025/54025fig1.jpg" /> 图 1:已经从头骨取出THY1-YFP +小鼠大脑脑结算照片的不同阶段 （一）。水凝胶嵌入式THY1-YFP +小鼠大脑半球（B）的照片。脂质去除（c）在THY1-YFP +小鼠大脑半球的照片。比例尺为5毫米，每块面板。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54025/54025fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54025/54025fig2.jpg" /> 图2: 在大脑中THY1-YFP表达在大脑皮层次脊髓。THY1-YFP +神经元和海马在放大5倍成像和三维重建（一）。大脑皮层展示皮质V层锥体神经元（B）的树突分支的10X图像堆栈的最大强度投影。在一个完整的颈，胸段脊髓THY1-YFP表达成像在放大5倍，并在3D（三）重建。脊髓展示个人轴突（D）的10X图像堆栈的最大强度投影。比例尺为1毫米（A），100微米（B），2毫米（C），和100微米（D）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54025/54025fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54025/54025fig3.jpg" /> 图3:PLP-EGFP 和PV-TdTomato大脑中表达。 </stron在放大5倍成像在2毫米厚的冠状脑切片g&gt;的PLP-EGFP表达（A）。这个图像是从组织部分的表面约1毫米。在10X放大率（二）成像的完整大脑半球的小脑光伏TdTomato表达。此图片是从矢状切面大约为0.5毫米。比例尺为1毫米（a）和250微米（b）所示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54025/54025fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54025/54025fig4.jpg" /> 图 4: 在在放大5倍下拍摄的完整大脑半球的小脑的脑 DAPI表达DAPI染色 。该图像是从正中矢状平面约1毫米。插图显示细节在该成像深度可见的在10倍放大倍率的水平。比例尺一个重250微米的主面板和50微米的插图。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54025/54025fig4large.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> 缓冲 </td><td> 化学 </td><td> 最终浓度 </td></tr><tr><td> 灌注液 </td><td></td><td></td></tr><tr><td></td><td> 10倍磷酸盐缓冲盐水</td><td> 1X </td></tr><tr><td></td><td>硝酸钠</td><td> 0.5％w / v的</td></tr><tr><td></td><td>肝素</td><td> 10单位/毫升</td></tr><tr><td> 固定液 </td><td></td><td></td></tr><tr><td></td><td> 10倍磷酸盐缓冲盐水</td><td> 1X </td></tr><tr><td></td><td> Paraformaldehy德</td><td> 4％体积/体积</td></tr><tr><td> 水凝胶的解决方案 </td><td></td><td></td></tr><tr><td></td><td> 10倍磷酸盐缓冲盐水</td><td> 1X </td></tr><tr><td></td><td>多聚甲醛</td><td> 4％体积/体积</td></tr><tr><td></td><td>丙烯酰胺</td><td> 4％体积/体积</td></tr><tr><td></td><td>双丙烯酰胺</td><td> 0.05％体积/体积</td></tr><tr><td></td><td> VA-044引发</td><td> 0.25％w / v的</td></tr><tr><td> 清除缓冲区 </td><td></td><td></td></tr><tr><td></td><td>硼酸</td><td> 200毫米</td></tr><tr><td></td><td>十二烷基硫酸钠</td><td> 4％w / v的</td></tr><tr><td> PBST </td><td></td><td></td></tr><tr><td></td><td> 10倍磷酸盐缓冲盐水</td><td> 1X </td></tr><tr><td></td><td>的Triton X-100 </td><td> 0.1％体积/体积</td></tr><tr><td></td><td>叠氮化钠</td><td> 0.1％w / v的</td></tr></tbody></table> 表1: 缓冲器和解决方案的列表。

Watch this Scientific Journal Video about Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY at JoVE.com

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

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 ...

optical-clearing-mouse-central-nervous-system-using-passive

Nanyang Technological University

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JoVE Journal

Neuroscience

13.4K 次观看.  University of California, Los Angeles. 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.

视频: Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

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 ...

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 ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

一种新的用于全中枢神经系统光透明快速生成的无源清除方法

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

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 ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

将化学遗传工程皮质内神经内生前生移植到早期产后小鼠大脑

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

大鼠中枢神经系统中反义寡核苷酸的细胞内传递

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...