我们感谢詹姆斯·翡翠的图示。这项工作得到了补助金从精神健康国家学院YZ支持

审批需要从家里机构开展的手术和影像学前须获得。在这个手稿中描述的实验是按照从加州大学，圣克鲁斯机构动物护理和使用委员会的准则和法规的规定执行。 1，手术<ol><li>高压灭菌所有手术器械和手术前彻底用70％的酒精消毒的工作区。 </li><li>通过腹膜内（IP）注射的KX麻醉剂溶液（200毫克/千克氯胺酮和20毫克/公斤赛拉嗪）麻醉小鼠根据鼠标的体重注:KX剂量可以根据应变，年龄调整后，与健康状态小鼠。兽医咨询，以确定最佳剂量还建议。 </li><li>定期通过按鼠标的脚趾和检查自反响应监测麻醉状态进行脚趾捏测试。确保鼠标在开始手术前完全麻醉注意:在长时间的成像会议，定期检查鼠标的麻醉状态，如有必要，管理附加KX。 </li><li>放置在加热垫的鼠标来维持手术期间体温。 </li><li>使用刀片，露出头皮剃鼠标的头部。 </li><li>擦拭用酒精交替垫和优碘皮肤消毒剃区域。除去残留的头发剪下。 </li><li>轻轻涂抹眼膏，以润滑双眼，从而防止在实验过程中因缺水造成永久性损坏。 </li><li>使沿头皮正中直切口，然后将皮肤横向对颅骨的边缘。 </li><li>清除附着在头骨的结缔组织。 </li></ol> 2，稀疏的头颅准备<ol><li>确定基于球面度的摄像区域。eotaxic坐标注:尽量避免大血管，从而阻挡光线穿透力和模糊的成像结构。血管是最好的观察时头骨是用湿润的无菌生理盐水。 </li><li>使用高速微钻颅骨（0.5-1.0毫米直径）的薄的圆形区域。平行移动的钻颅骨表面，而不是抱着它反对头骨和下压。钻，直到两个外密质骨层和中间松质骨层被删除。 注意:为避免因过热损坏，避免钻头和颅骨之间的长时间接触。 </li><li>继续在钻变薄区域中心的显微叶片变薄，内密质骨层。握住显微叶片以约45°角刮颅骨没有按下反对头骨，直到均匀变薄小区域（200-300微米直径），厚度为近似获得LY 20-30微米。由于自发荧光的头骨，厚度可通过扫描下双光子显微镜注颅骨上，下表面之间的距离来衡量:虽然小于20μm的颅骨厚度提供了良好的成像质量，所以不推荐，因为它使在随后的重新成像会议细化处理困难。避免过度变薄，需要在手术过程中定期检查（见讨论 ）的头骨的厚度。 </li></ol> 3，固定化<ol><li>小心地取出骨头碎片。 </li><li>将一小滴的氰基丙烯酸酯胶在无菌头板，已灭菌的中心开口的每个边沿（ 见图1）。紧紧握住头板对与位于中心开幕注减薄区域头骨:如果胶水污染变薄ŘEGION意外，随着显微外科刀片小心地将其删除。 </li><li>轻轻地从头骨的两侧拉扯皮肤的头部板的中央开口的边缘。 </li><li>等待大约10分钟，直到头部板以及附着在头骨。 </li><li>将头板的支承板的两个侧块，然后拧紧螺丝的头板的边缘以固定鼠标上的支撑板（ 见图1）注:请确保之间不存在皮肤或胡须头板和块拧紧螺丝。一个好的固定化制剂应显示颅骨没有明显的运动解剖显微镜下当动物的背部轻轻地拍了拍。 </li><li>冲洗暴露的头骨用生理盐水除去未聚合胶注:删除任何剩余的胶水非常重要，因为未聚合胶模糊的图像和损害显微镜物镜&lt;。/ LI&gt; </ol> 4。成像<ol><li>就拿暴露颅骨变薄地区地图1，这是用来重新定位成像区域在随后的成像会议（参见图2）的脉管系统的照片。 </li><li>将鼠标成像显微镜下。找到减薄下使用萤光一个10X空中目标，并移动最薄的区域到视图的中心。 </li><li>添加盐水对颅骨顶部下降并切换到60X物镜，它已经用无菌水漂洗。选择其中个别树突棘沿树突清晰可见的区域。确定并经啶视图和脉管照片之间比较血管标注相应区域的地图1。 </li><li>根据荧光团调谐双光子激光波长。例如，920纳米的YFP; 890 nm的绿色荧光蛋白; 1,000 nm处的红色荧光蛋白和tdTomato 24。 </li><li>获取的图像与栈2沿使用60X物镜z轴微米的步骤。此图像堆栈包括一个约200微米×200微米的面积（512×512像素），并用作图2为在随后的成像会话重定位（参见图2）。 </li><li>收购中地图2 9图像堆栈使用3X数码变焦。每个图像堆栈占地70微米×70微米（512×512像素）的近似区域，具有0.7微米的步骤沿z轴注意:当在样品上测量，以减少光毒性的激光的强度应低于40毫瓦。 </li></ol> 5。恢复<ol><li>以下成像，轻轻地从头骨取下头板。 </li><li>彻底清洁头骨和皮肤去除所有剩余的胶水注意:在皮肤上的任何残胶会造成刺激，减缓皮肤的愈合，而在头骨的任何剩余胶水将导致颅骨侵蚀和ngiogenesis在新骨生长层，使得后续的搬迁和重新成像困难。 </li><li>冲洗颅骨并用盐水多次皮肤。 </li><li>缝合头皮，用无菌手术缝合。 </li><li>保持动物在加热垫在单独的笼子。管理丁丙诺啡镇痛药（0.1毫克/千克），皮下注射，以减轻手术后疼痛。返回动物的笼子完全恢复后。密切监测动物（至少检查一次，每日），直到切口愈合，拆线。如果需要管理的丁丙诺啡镇痛的后续注射。 </li></ol> 6，重新制作映像<ol><li>重复步骤1.1-1.8。 </li><li>重复步骤3.1-3.6 </li><li>通过比较血管图案来映射1和树突分支模式地图2找到先前成像区域。 </li><li>如果1周内进行重新映像，请重复步骤2.3以去除薄层新骨生长对再细化顶部祗园采用显微外科刀片。如果在1周以上进行重新映像，重复步骤既2.2和2.3 注:新骨生长层由少简明结构相比原来的密质骨层，从而降低了图像质量。因此，为了获得相同的质量，有必要薄颅骨比以前的成像会议稍多。 </li><li>调整位置和方向，以获取匹配以前拍摄的图像栈的双光子显微镜下的图像堆栈。 </li><li>获取图像，如步骤4.6。 </li><li>成像后，重复步骤5.1-5.5。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

要获得成功变薄，颅准备，在这个协议的几个步骤是至关重​​要的。 1）颅骨的厚度。颅骨具有夹层结构，具有高密度的致密骨2层和低密度海绵骨的中间层。而高速微型钻孔机是适用于除去密质骨和松质骨的外层，显微叶片是理想的减薄致密骨的内层。由于在开发过程中的头骨的厚度增加和刚度，成年小鼠成像需要多个骨，以便获得质量良好的图像被删除。变薄的区域呈现出透明固体的外观，并提供良好的成像质量，当头骨的厚度约为20-30微米。颅骨厚度应定期在细化过程中进行检查的过度变薄使得在随后的重新映像会话细化处理困难。 2）减薄区域的大小。它建立了稳定的薄化颅骨配置，其中一圆锥状空腔的实现是重要的。减薄区的具有适当大小的体系结构可以防止造成损害的皮质，并有助于细化在随后的再成像会话。它建议，使减薄区域的底部区域（约直径200-300微米）比顶部开口​​（约0.5-1.0毫米的直径）小。 3）图像的稳定性。良好的固定准备有助于提高通过降低呼吸引起的运动伪影的图像质量。重要的是要保持干燥颅骨和缺乏结缔组织和骨骼碎片的头板连接到前囟是很重要的。额外的胶也可以应用来填充头板和颅骨之间留下的空隙。此外，针对一个地区远离大血管也最大限度地减少所造成的血泵的运动。 的变化胸罩调查在活的动物使用双光子显微镜的高时空分辨率的要求产生的光学窗口。除了 ​​薄化颅骨制备，荧光标记的结构，也可以通过除去头骨和一个盖玻璃（一般为3-5毫米的直径），它提供一个清晰的成像窗10，13替换它可视化。虽然两者变薄，颅骨和开颅骨成像协议被广泛应用在活体成像研究中，每种方法都有自己的优点，适用于不同的实验设计。例如，开放式的头骨是准备为调查比较大的脑区（ 如直径5 mm），并且需要许多成像会话短的时间间隔（ 即天）的研究很有用。一旦颅窗口被成功植入，不需要额外的手术是必需的。然而，手术后的时间（通常约1个月）的第一成像之前，需要会议以减轻因手术​​造成潜在的炎症反应。重新成像变得不可能时颅窗口被阻止的骨和/或脑膜增厚的再生。与此相反，动物用稀疏颅骨制剂可立即成像的初始手术后，使之更适合于年轻的动物（ 例如 ，2个周龄）的慢性成像。它适合于调查与长成像时间间隔（ 即几个月到几年），作为骨再生和硬脑膜增厚是没有问题的。然而，通常需要重新变薄颅骨随后再成像会议（甚至有2天的时间间隔），由于骨再生。此外，新生长的骨层由相对于原来的密质骨较少缩合结构的，大大减少了稀疏头骨区域的透明度。因此，要获得的图像相同的质量，有必要完全除去新生长的骨层。而这样的ttempts通常导致颅骨在随后的成像比以前的制备更薄的最终厚度。由于颅骨厚度已经准备20-30微米的初始成像会议，只剩下有限的空间再减薄，减薄头骨制剂的总成像时间一般不超过5次。近日，一个名为抛光和增强新的实验方法变薄，颅（端口）已经发展到同时结合了减薄和开颅骨制剂11，26，27。 到目前为止，大多数在体内成像的皮层中一直使用下神经元特异性启动子THY1表达EGFP或YFP的转基因小鼠系进行。这些转基因小鼠表达荧光蛋白在人烟稀少的一个子集，但皮层神经元25的混合种群。由于证据多行显示，经验依赖性结构可塑性发生在塞莱明确定义的电路，莫如类型的细胞，在细胞类型特异性的方式标记和图像是重要的。迄今为止，许多方法已被应用到实现这一目标。例如，在不同的皮质层锥体神经元可以通过在定义的胚胎阶段28，29执行在子宫内电有针对性。同样，注射改造以表达荧光蛋白的病毒载体也可以用于在不同脑区30标记的细胞。另外，已经产生了转基因小鼠的多线驱动下的细胞类型特异性启动子31,32 Cre重组酶的表达。注射病毒载体，如腺伴随病毒携带荧光报告基因下列一个两侧装接loxP的终止密码子插入这些小鼠提供了可能的目标指定的类神经元在限制区33。通过结合这些新的标签与我们的减薄-S接近库尔制备，改变皮层神经元的突触连接，可通过双光子的体内成像在一个细胞类型和/或电路的特定方式的影响。 以转基因动物的增加可用性与荧光标记的细胞群体和此处描述的体内标记技术，类似程序的快速发展，也可以应用于调查其他类型的细胞（神经胶质细胞）和脉管系统中的活体脑。结合行为的操作和疾病模型，双光子体内成像将极大地拓展我们的相关脑功能的分子，细胞和电路机制的理解。

哺乳动物的大脑皮层参与了许多脑功能，从感官知觉和运动控制，以抽象的信息处理和认知。各种皮质的功能建立在不同的神经回路，不同类型的神经元沟通和个别突触交换信息这是由。突触的结构和功能都一致地被修改以响应经验和病状。在成熟的大脑，突触可塑性需要双方力量的变化和突触的添加/删除的形式，发挥着重要作用的形成和维持功能的神经电路。树突棘是大多数哺乳动物大脑兴奋性突触的突触后成分。不断更替和棘形态变化被认为是作为在突触连接1-7修改一个很好的指标。 双光子激光扫描显微SCOPY透过厚厚的，不透明的准备和光毒性低，这使得它适合用在完整的大脑8实时成像提供深层渗透。与荧光标记的组合，双光子成像提供了一个强大的工具，不期而遇的活体脑并按照在具有高时空分辨率的单个突触的结构重组。各种方法已被用来制备老鼠实时成像9-13。在这里，我们描述了一个稀疏的头颅制剂在体内双光子成像研究在小鼠大脑皮层突触后树突棘的结构可塑性。使用这种方法，我们最近的研究已经描绘的树突棘变化动态画面响应于运动技能学习用的转基因动物与荧光标记的神经元亚群的体内标记技术的快速发展提高可用性，此处描述的类似的程序也可应用于到investiga德其他细胞类型和皮层区域，并结合其它操作，以及在疾病模型中使用16-23。

<table border="0" cellpadding="0" cellspacing="0" width="1253">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Ketamine</td>
			<td style="width:181px;">Bioniche Pharma</td>
			<td style="width:108px;">67457-034-10</td>
			<td style="width:775px;">Mixed with xylazine for anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Xylazine</td>
			<td style="width:181px;">Lloyd laboratories</td>
			<td style="width:108px;">139-236</td>
			<td>Mixed with ketamine for anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Saline</td>
			<td style="width:181px;">Hospira</td>
			<td style="width:108px;">0409-7983-09</td>
			<td>0.9% NaCl for injection and imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">Razor blades</td>
			<td style="width:181px;">Electron microscopy sciences</td>
			<td style="width:108px;">72000</td>
			<td>Double-edge stainless steel razor blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Alcohol pads</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:108px;">06-669-62</td>
			<td>Sterile alcohol prep pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Eye ointment</td>
			<td style="width:181px;">Henry Schein</td>
			<td style="width:108px;">102-9470</td>
			<td>Petrolatum ophthalmic ointment sterile ocular lubricant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">High-speed micro drill</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:108px;">18000-17</td>
			<td>The high-speed micro drill is suitable for thinning the outer layer of compact bone and targeting a small area</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Micro drill steel burrs</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:108px;">19007-14</td>
			<td>1.4 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Microsurgical blade</td>
			<td style="width:181px;">Surgistar</td>
			<td style="width:108px;">6961</td>
			<td>The microsurgical blade is suitable for thinning the inner layer of compact bone and middler layer of spongy bone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Cyanoacrylate glue</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:108px;">NC9062131</td>
			<td>Fix the head plate onto the skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Suture</td>
			<td style="width:181px;">Havard Apparatus</td>
			<td style="width:108px;">510461</td>
			<td>Non-absorbale, sterile silk suture, 6-0 monofilament</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Dissecting microscope</td>
			<td>Olympus</td>
			<td style="width:108px;"></td>
			<td>SZ61</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">CCD camera</td>
			<td>Infinity</td>
			<td style="width:108px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Two-photon microscope</td>
			<td style="width:181px;">Prairie Technologies</td>
			<td style="width:108px;"></td>
			<td style="width:775px;">Ultima IV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">10X objective</td>
			<td style="width:181px;">Olympus</td>
			<td style="width:108px;"></td>
			<td>NA 0.30, air</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">60X objective</td>
			<td style="width:181px;">Olympus</td>
			<td style="width:108px;"></td>
			<td>NA 1.1, IR permeable, water immersion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Ti-sapphire laser</td>
			<td style="width:181px;">Spectra-Physics</td>
			<td style="width:108px;"></td>
			<td>Mai Tai HP</td>
		</tr>
	</tbody>
</table>

two-photon in vivo imaging of dendritic spines in the mouse cortex using a thinned-skull preparation

在哺乳动物大脑皮层，神经元形态极为复杂的网络，并在突触交换信息。改变突触强度，以及突触的添加/删除，发生在经验依赖性方式，提供了神经元可塑性的结构基础。如在皮层中最兴奋性突触的突触后的部件，树突棘被认为是突触的一个良好指标。小鼠遗传学和荧光标记技术，单个神经元和突触的结构，以优势可以在标签完整的大脑。在这里，我们使用双光子激光扫描显微术跟随荧光标记的突触后树突棘随着时间在体内引入颅成像协议。这个协议利用一个稀疏颅骨制备，这使颅骨完好，并避免所引起的脑膜的曝光和皮质抗炎作用。因此，图像可立即苏收购后rgery被执行。的实验步骤可重复进行以上不同的时间间隔范围从小时到数年。此制剂中的应用也可以被扩展以调查不同的皮层区域，层，以及其它类型的细胞，生理和病理条件下。

在YFP-H线的小鼠25，黄色荧光蛋白表达在V层锥体神经元，其突出的顶端树突在皮质的表面层的一个子集。透过稀疏的头颅准备，荧光标记树突段可重复双光子显微镜下成像在不同成像时间间隔，从小时到几个月。这里，我们表明在1个月大的小鼠，其中个别棘以及丝状伪足可以沿着枝晶可清晰显示运动皮层8天以上相同的树突四时间摄像的一个例子。通常，图像堆栈的深度是从软脑膜表面约100-200微米。各种分析可以基于这些图像来进行。例如，该书脊形成，消除和周转可以通过从不同的会话比较图像进行量化。棘密度可以通过将刺的数目由dendrit的长度来计算集成电路分部。也可以分析脊柱活力和形态的变化。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51520/51520fig1highres.jpg" src="/files/ftp_upload/51520/51520fig1.jpg" /> 图1稀疏的头颅准备双光子体内成像的定做固定板（A）头板，它是由粘在一起，所涵盖的带尖角两个或三个刀片的照片。 （二）固定板，它由1不锈钢板，2个不锈钢块，2个螺丝和2垫片的照片。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51520/51520fig2highres.jpg" width="500" /> 图2通过一个稀疏颅骨制剂在小鼠运动皮层的经颅双光子成像 <sTRONG&gt;显示超过八天的树突棘的动态。（A）与颅骨变薄的区域（图1）的血管图案的CCD图像。黑色方块表示在那里被收购双光子的体内图像的区域。 （二）树突分支的一个1个月大的鼠标（图2）运动皮质的低倍率最大投影。同样的树突段（C）重复图像显示新形成的棘（箭头），消除棘（箭头），并在第0天，2，4，8和丝状伪足（星级）。左侧面板中的更高倍率的看法在（B）中的盒装区域显示的树突段。标尺:500微米（一），20微米（B）和2微米（C）。

Watch this Scientific Journal Video about Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation at JoVE.com

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

two-photon-vivo-imaging-dendritic-spines-mouse-cortex-using-thinned

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

17.8K 次观看.  University of California, Santa Cruz. Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

视频: Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

科研

神经科学

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

抛光和钢筋，颅骨变薄窗口，为长期的小鼠脑成像

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

稀疏的头骨皮层的窗口技术<em在体内</em光学相干断层成像

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.