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，?...

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1...

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 ...

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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.