这项工作是支持的国家眼科研究所R01EY017925和R21EY020985的资金从达纳白厅基础PK我们也感谢马修·彼得雷拉外科手术的帮助，格雷斯·迪翁示于图5A追踪树突补助; PRATIK Chhatbar为的手稿上的意见。

1。手术准备<ol><li>诱导麻醉，并持续监测心跳率，结束潮汐CO 2，脑电图，及温度。南卡罗来纳大学医疗机构动物管理和使用委员会批准了所有的程序，是基于我们以前出版9,15。 </li><li>公开的背用手术刀切割皮肤表面的头骨。解剖的结缔组织，覆盖骨使用一个Brudon的刮匙。用棉签涂抹和棉花纱布清洁骨。应用骨蜡（如需要）的头骨，偶尔通过小使者静脉的出血停止。 </li><li>钛或不锈钢头板连接到所述颅骨（超过其中将执行开颅景点的区域）通过用黑色涂料（见材料 ）混合使用牙科用粘固剂。头板的中心的矩形开口中提供访问开颅和光学成像。将一个金属腔的头板。将作为该室的流体容器与水浸泡物镜成像。 </li><li>固定头板使用金属柱的 xy阶段。 </li><li>骨的大面积的薄的磁头板的矩形开口内，通过使用牙钻。被摊薄的区域必须远远超出计划开颅手术的边界。必须减薄高分辨率的图像时，取得的新皮层的，因为在开颅手术，骨的厚度将决定之间的盖玻片厚度的琼脂糖和新皮层表面的非啮齿类哺乳动物的厚头骨（见下文） 。 </li><li>钻一个2×2平方毫米的轮廓骨开颅手术。定期清洗室，用新鲜的人工脑脊液（ACSF）去除骨碎片，减少热能散发到底层的皮质。涂上骨蜡，偶尔出血停止。 </li><li> LIFT＃3钳骨瓣。 </li><li>删除所有底层的硬脑膜，用细镊子（＃5CO）和Vannas春风似剪刀。硬膜在非啮齿类哺乳动物是厚的和不透明的，但可以在不同的层中除去。出血是罕见的，但可以停止任何血液残余物，应以明胶海绵和轻轻地从其余层的硬脑膜钳和冲洗学联的。最后一层是透明的硬膜，并通过软脑膜血管清晰可见。离开这最后一层硬脑膜完整的内在信号成像过程中，会提高成功率，为以后的双光子成像阶段的​​实验荧光钙指标的批量加载（见讨论）。 </li><li>滴一滴温暖的2％琼脂糖（溶解在ACSF），并立即将一个玻璃罩了开颅手术。检查开颅确保呼吸系统和心血管脉动是最小的通过使用外科解剖显微镜或两个目镜光子显微镜明视场模式。 </li></ol> 2。内源信号的光学成像<ol><li>添加凝胶泡沫浸泡学联的盖片玻璃的外侧周围，以防止琼脂糖从干燥期间内在信号成像。 </li><li>将成像CCD相机的特性信号的标准C型安装的双光子显微镜（ 图1）的顶部的端口。将照明用光源附近的 xy阶段（ 图1A）上的气动工作台。定位和固定的光导过开颅手术和磁头板/室（ 图1B）。缩回主分色和反射镜下面的C型安装的端口，使得反射的红色光所需要的固有的信号将通过直接从开颅的CCD摄像机（ 图1C-D）。使用4倍的空中目标（0.13 NA，17毫米WD），2×2毫米视野是内在的信号成像。 </li><li>将热过滤器在L的飞行路径，以防止从加热皮质。使用一个过滤器，通过绿光（546纳米中心λ）和记录数字的参考图像的皮质表面血管。 </li><li>将在 z焦点移动到500微米以下的皮质表面。使用红色过滤器（630纳米中心λ）来照亮皮层内在的信号测量。位置使得皮质的表面被均匀地照射的导光体。屏蔽开颅从所产生的视觉刺激的显示的光。目前的视觉刺激和记录反射率数据的图像序列。在10分钟之内要产生一个定向地图，收集对应于4个重复序列8刺激（4方向和每个方向的两个方向）的数据。 </li><li>分析的特性的信号数据，以产生的假彩色定向地图。选择潜在目标区域的神经元的双光子的标签， 例如，方向风车奇异性-在地图上点所有的择优取向收敛（ 图2A）。叠加定向地图和皮质表面血管的图像（ 图2B-C）。使用的布局，功能结构域和大型水面舰艇的位置，选择一个目标位置，避免与大血管和动脉15。 </li></ol> 3。批量装载荧光钙指标<ol><li>准备20％的Pluronic / DMSO的溶液通过添加1 g的Pluronic于5μl的DMSO。加热该混合物，在60℃下10分钟，以完全溶解的Pluronic。允许该混合物冷却至室温。 4微升普朗尼克/ DMSO混合添加的荧光钙指示剂俄勒冈绿488 BAPTA-1 AM（OGB-AM）到50微克小瓶。然后加入1微升的红色染料（的Alexa Fluor 594，2mM的原液）和35微升溶液移液管（见材料 ）的最终浓度为1 mM的OGB-1 AM。超声波清洗半小时的解决方案冷藏水浴用0.45μm的过滤器，以除去杂质，然后离心。 </li><li>拉出长锥形吸移管具有外尖端直径为2.0-2.5微米。将5μl的染料混合物进入移液管。 </li><li>卸下的最后一层硬膜，以便吸移管插入和获得最高质量的双光子图像。 </li><li>确定为了达到其目标的皮质层的2/3，移液管必须放置在皮层表面上的确切位置。我们预定的皮质的目标是一个风车取向图（ 图2）中的奇异性。吸移管必须被驱动到皮质，在一个30°的角度，腔室的壁和目标之间传递。因此，标记的目标区域200微米以下的皮质表面，皮质表面上的吸液管进入位置将是约350微米的目标位置（ 图3中A，B）的侧方。 </li><li>移动的 xy阶段（该动物和pipette显微操纵器被放置）的中心的目标（ 图3C）的皮质表面下进入位置。使用20倍或40倍物镜的工作距离（WD）的至少3毫米（见材料 ），以确保吸液管将很容易地输入不接触的目标或骨沿着壁的开颅皮层。一旦条目的位置为中心的目标下，提高目标的Z位置由2毫米（ 图3D）。随着绿色外延荧光灯打开，以可视化的红色那样Alexa染料中的移液管，使用的显微操作的对角线轴，使移液管，直到它的端部的中心（和集中）根据以上皮质表面条目位置目标直接（ 图3E）。 </li><li>降低吸移管的吸移管的垂直方向（ 沿 z轴），直到到达皮质表面（ 图3F），而连续地观看通过移液管尖的OCU拉斯显微镜。用明视场照明，皮质表面血管和剩余脑膜膜（软脑膜和蛛网膜）是很容易看到的吸管接近皮质表面。如果吸移管未达到所需条目的位置，提高移液管并重新校准定位基于皮层的表面上的血管上的界标。 </li><li>删除的目的，灯芯过多的脑脊液从开颅手术，并使用无绒的吸收矛干燥相邻的骨。滴一滴温暖的3％琼脂糖（溶解在学联）以稳定的皮质通过开颅（ 图3G）是可见的。在非啮齿类动物中，3％琼脂糖以抑制脉动的神经元的荧光染料成功的摄取是必要的。对于内源信号光学成像是必要的，因为只有2％的琼脂糖结合的2％琼脂糖和盖玻片提供必要的稳定性。在这里，没有盖玻片用于染料加载步骤3％琼脂糖必要的。琼脂糖凝固后，将40倍的目标（0.8 NA，3.3毫米WD），并重新填写室学联。 </li><li>从明场切换双光子观赏和定位移液器吸头。使用显微操纵的对角线轴，使移液管，直到其前端到达皮层（ 图3H）中的所需深度。偶尔压力喷射喷的的染料混合物（几磅，每个脉冲0.1秒）将有助于防止吸头堵塞。由于OGB-1进入细胞后变得可见，红色Alexa的染料吸头内皮层的可视化是至关重要的。 </li><li>尝试加载皮层神经元的三分之二与全剂量的染料，重新聚焦的目标上的皮质表面确认的移液管尖端部的深度，并确保的前端是在目标位置的基础上的布局之前表面的血管。如果吸移管是在层2/3的染料混合物，小喷应该照亮细胞外空间，并揭示黑暗的阴影圆形细胞体密集的组装。 </li><li>加载神经元胞体皮质300-600微米直径的球体，染料混合物注入到细胞外空间使用30-90的脉冲，每个脉冲1.0秒，2-10磅。不应该是如此强烈，组织移动的脉冲压力。注射完成后，等待几分钟，然后取出的吸管和目标。等待1小时，以允许OGB-1 AM是充分考虑了由神经元。 </li><li>取出琼脂糖用于染料荷载和冲洗的记录室。将一小滴2-3％琼脂糖表面上，并迅速在开颅手术中放置一个玻璃罩。由于荧光染料的光敏感，避免过度曝光的皮质亮场或落射荧光光。 使用 xy阶段的目标到目标支架和中心标记的皮质下区域，将高NA（1.0）20倍的目标。屏蔽开颅手术前traneous光源， 如视觉刺激显示，使用多图层的遮光材料。 </li></ol> 4。单细胞电穿孔的荧光染料<ol><li>为了研究树突状形态，准备的100μM的Alexa Fluor 594（1:20稀释2毫米的Alexa Fluor 594股票学联）的解决方案。功能成像的树突OGB盐可用于16,17。 </li><li>拉出长锥形移液管的尖端尺寸为1μm，并加载到移液管的5微升染料。为目标的功能域的所有其他步骤是相同的批量加载方法（ 图1-3）。相邻的神经元细胞体膜的吸头定位，监控电极阻抗的增加和施加压力，喷染，照亮细胞外空间，并了解细胞体的阴影在双光子成像。 </li><li>当吸头相邻的神经元细胞体膜是，使用Axoporator的800A应用1-3脉冲（-8到-10 V，10毫秒的脉冲）。的神经元与染料的直接填充与双光子显微镜容易地可视化。取出吸管，并收集在体内的树突和轴突的高清晰度图像。 </li></ol>

<ol>
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没有利益冲突的声明。

我们提出一个方法来定位标签在预先确定的功能性微域的新皮层的神经元细胞体（或树突和轴突）。合并内在信号光学成像与双光子显微镜提供的可能性确定哪些突触和细胞有助于微域的任何功能的地图，是否选择性神经元的神经元中的功能图的位置，以及神经元电路相关组件这种变化的视觉体验7或应用临床治疗的药物18。 据报道，在视觉皮层的成年野生型?...

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td></td><td></td><td></td><td> 1。生命支持/实验准备 </td></tr><tr><td>异氟醚</td><td>韦伯斯特兽医</td><td> NDC 57319-474-05 </td><td></td></tr><tr><td>异氟醚蒸发器</td><td> MIDMARK </td><td> VIP 3000 </td><td></td></tr><tr><td>反馈调节加热毯</td><td>哈佛设备</td><td> 50-7079F </td><td></td></tr><tr><td>心电监护仪</td><td> Digicare生物医学</td><td> LifeWindow精简版</td><td></td></tr><tr><td>脑电放大器</td><td> AM系统</td><td> 1800 </td><td></td></tr><tr><td>脑显示器</TD&gt; <td>惠普</td><td> 78304A </td><td></td></tr><tr><td>呼气末CO 2显示器</td><td>伟</td><td> Novametrix呼气Capnoguard 1265 </td><td>优化通风</td></tr><tr><td>硬质合金钻头钻骨毛刺</td><td>亨利施恩</td><td>罚款（0.5毫米头），粗（1.25毫米头） </td><td></td></tr><tr><td>水泥磁头板/室</td><td>登士柏</td><td> 675571，675572 </td><td></td></tr><tr><td>黑火药滕佩拉漆</td><td>萨金特艺术公司</td><td> 22-7185 </td><td>巩固提高遮光，减少反射</td></tr><tr><td>琼脂糖 - Ⅲ型-A </td><td>西格玛</td><td> A9793 </td><td>用于最小化内在信号和双光子成像过程中的脉动</td></tr><tr><td>盖玻片:5或8毫米，直径0.17毫米的厚度</td><td>世界精密剂量率仪</td><td> 502040，502041 </td><td>为了最大限度地减少脉动在成像过程中，根据需要可能会被切除玻璃罩</td></tr><tr><td> Brudon刮匙</td><td>乔治·蒂曼</td><td> 105-715-0，105-715-3 </td><td>清洁颅骨表面</td></tr><tr><td>骨蜡</td><td>爱惜</td><td> W31G </td><td>快速止血</td></tr><tr><td>棉签</td><td>电子显微镜科学</td><td> 72308-05 </td><td>清洁，干燥，骨面</td></tr><tr><td>杜蒙5CO钳</td><td>精细科学工具</td><td> 11295-20 </td><td>抓住各个层硬脑膜或软</td></tr><tr><td> Vannas春风似剪刀</td><td>精细科学工具</td><td> 15000-03 </td><td>切硬膜</td></tr><tr><td>明胶海绵</td><td>辉瑞公司</td><td> 09-0396-05 </td><td>要停止硬脑膜上出血</td></tr><tr><td>吸收矛</td><td>精细科学工具</td><td> 18105-01 </td><td>超快速和无绒排汗CSF </td></tr><tr><td>停电材料</td><td> Thorlabs公司</td><td> BK5 </td><td>盾开颅手术</td></tr><tr><td></td><td></td><td></td><td> 2。染料制备/注射 </td></tr><tr><td>二甲基亚砜（DMSO）的</td><td>西格玛</td><td> D2650 </td><td></td></tr><tr><td>普朗尼克</td><td>西格玛</td><td> P2443 </td><td></td></tr><tr><td>俄勒冈绿488 BAPTA-AM </td><td> Invitrogen公司</td><td> O6807 </td><td>钙指示剂</td></tr><tr><td>的Alexa Fluor 594 </td><td> Invitrogen公司</td><td> A10438 </td><td></td></tr><tr><td>离心式过滤器（0.45μm孔径） </td><td>密理博</td><td> UFC30HV00 </td><td>注射前要除去杂质</td></tr><tr><td>玻璃吸管拉马</td><td>萨特仪器</td><td> P97 </td><td></td></tr><tr><td>高硼硅玻璃filamented毛细管（1.5毫米外径） </td><td>世界精密仪器</td><td> 1B150F-4 </td><td>染料弹射移液管</td></tr><tr><td> Microloader </td><td> Eppendorf公司</td><td> 5242 956 003 </td><td>加载染料吸管</td></tr><tr><td>微操作机器人</td><td>萨特仪器</td><td> MP-285 </td><td>要定位移液管</td></tr><tr><td>压力脉冲控制器</td><td>派克汉尼汾</td><td> PicoSpritzer III </td><td>对于染料的压力注入</td></tr><tr><td>单细胞电穿孔</td><td> Molecular Devices公司</td><td> Axoporator 800A </td><td>有关染料的电穿孔</td></tr><tr><td></td><td></td><td></td><td> 3。本质IMA晋</td></tr><tr><td> 4倍的目标（0.13 NA，17毫米WD） </td><td>奥林巴斯</td><td> UPLFLN4X </td><td></td></tr><tr><td>内在的硬件/软件</td><td>光学成像公司</td><td>成像仪3001 / VDAQ </td><td>情节成像软件是用于VDAQ </td></tr><tr><td> CCD相机</td><td> Adimec公司</td><td> Adimec公司-1000 </td><td></td></tr><tr><td>光源电源</td><td> KEPCO </td><td>吃了15-15M </td><td></td></tr><tr><td>光源</td><td>光学成像公司</td><td> HAL 100 </td><td>在皮层表面的光照强度为3-5毫瓦</td></tr><tr><td>绿色过滤器（用于血管图像） </td><td>光学成像公司</td><td> λ= 546 nm处（带通30纳米） </td><td>对于参考图像的表面脉管</td></tr><tr><td>红色过滤器（内在信号） </td><td>光学意马纳克公司</td><td> λ= 630 nm处（带通30纳米） </td><td> ，要收集内在的信号</td></tr><tr><td>热过滤器</td><td>光学成像公司</td><td> KG-1 </td><td></td></tr><tr><td></td><td></td><td></td><td> 4。双光子台/影像 </td></tr><tr><td>双光子显微镜和软件</td><td>草原技术</td><td></td><td> Shen 等人 2012年的光路，过滤器和激光功率</td></tr><tr><td>掺钛蓝宝石激光器</td><td>光谱物理公司</td><td>麦态XF </td><td></td></tr><tr><td> 20倍（0.5 NA，3.5毫米WD） </td><td>奥林巴斯</td><td> UMPLFLN20X </td><td>仅用于对准移液器超过开颅0.5 NA物镜（未用于两个光子成像） </td></tr><tr><td> 20X（1.0 NA，2.0毫米WD） </td><td>奥林巴斯</td><td> XLUMPLFLN20X </td><td></td></tr><tr><td> 40X（0.8 NA; WD3.3毫米） </td><td>奥林巴斯</td><td> LUMPLFLN40X/IR </td><td></td></tr><tr><td>航空表</td><td>新港</td><td> ST-200 </td><td>从外部振动隔离准备</td></tr><tr><td> XY阶段</td><td>迈克机械有限公司（Attleboro，麻萨诸塞州） </td><td></td><td>放置在 xy阶段的实验对象和Sutter微操作机器人</td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td colspan="2"> 食谱 </td></tr><tr><td>人工脑脊髓液</td><td>氯化钠（135毫摩尔），氯化钾（5.4毫摩尔），的MgCl 2（1.0毫摩尔），的CaCl 2（1.8毫摩尔），HEPES（5毫摩尔），pH为7.4 </td></tr><tr><td>液14 </td><td>氯化钠（150毫摩尔），氯化钾（2.5毫摩尔），HEPES（10mM的），pH值7.4 </td></tr></tbody>
			</table>
			</td>
		</tr>
	</tbody>
</table>

targeted labeling of neurons in a specific functional micro-domain of the neocortex by combining intrinsic signal and two-photon imaging

在非啮齿类哺乳动物初级视觉皮层的神经元聚集根据自己的喜好刺激的功能，如方向1-4，5-7方向，眼优势8,9和双眼视差9。方位选择性是最被广泛研究的功能，并具有准周期的布局为择优取向的连续映射在整个初级视觉皮层10,11是本。整合的突触，手机和网络的贡献，在这些功能的地图，以的刺激选择性的反应，需要杂交成像技术，跨越亚微米到毫米的空间尺度。与传统的内源信号光学成像，整体布局功能地图的整个表面上的视觉皮层可确定12。 在体内双光子显微镜使用钙敏感染料的发展使一个确定SYNAPT到达13或记录个人的树突棘IC输入端的活动，同时从数以百计的单个神经元细胞体6,14。因此，结合特性的信号与亚微米的空间分辨率的双光子显微镜成像提供准确地确定树突状段和细胞有助于在新皮质的任何功能的地图的微域的可能性。在这里，我们展示了一个高收益的方法快速获得皮质的方向图，针对一个特定的微域在这个功能的地图，用荧光染料标记细胞在非啮齿类哺乳动物。用于双光子成像具有相同的显微镜中，我们首先生成一个定向地图使用固有的信号的光学成像。然后，我们如何定位的利益，用微装有染料为标签的微域人口的神经元细胞体或标签的单个神经元的树突棘和轴突中是可见的体内。我们比以前的方法的改进，促进神经元的结构与功能的关系与亚细胞分辨率的新皮层的功能体系结构的框架中的一个考试。

为了说明我们的染料标记方法的精度，我们针对最小的微域非啮齿类动物的新皮质中的任何已知的功能图。在初级视觉皮层的方向图是稀疏穿插在整个奇点。这些发生在所有优选方位点收敛等，在优选的方向，周围的区域"风车"（ 图2A-B）的奇异性的样子的假彩色地图。每一个风车开颅手术选择染料标记（绿色圆圈在图2C），一定要避免大血管和小动脉，特别是15。</sup...

Watch this Scientific Journal Video about Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging at JoVE.com

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

用荧光染料标记细胞在预定的新皮层的功能性微域的一种方法。首先，本征信号光学成像用来获得一个功能图。双光子显微镜被用于标签和图像的神经元内微域的地图。

有针对性的特定功能的微域的新皮质相结合的内源信号和双光子成像标记的神经元

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

targeted-labeling-neurons-specific-functional-micro-domain-neocortex

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

13.6K 次观看.  Medical University of South Carolina. 用荧光染料标记细胞在预定的新皮层的功能性微域的一种方法。首先，本征信号光学成像用来获得一个功能图。双光子显微镜被用于标签和图像的神经元内微域的地图。

视频: Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

在体内2光子层钙成像对小鼠的2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

科研

生物学

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

神经科学

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 ...

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal&#8217;s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.

This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.

警报，头抑制大鼠子皮层的大脑结构中Juxtacellular监测和本地化单神经元

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures ...

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

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

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 ...

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.

同时双光子<em&gt;在体内</em&gt;突触输入的影像和突触后目标中的鼠标Retrosplenial的Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

小鼠大脑皮层细胞组织的大尺度三维成像

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

有针对性的特定功能的微域的新皮质相结合的内源信号和双光子成像标记的神经元

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在体内2光子层钙成像对小鼠的2 / 3

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元

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

警报，头抑制大鼠子皮层的大脑结构中Juxtacellular监测和本地化单神经元

双光子

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脑切片神经元树突的双光子钙成像

小鼠大脑皮层细胞组织的大尺度三维成像

体内新生小鼠皮层神经元的双光子成像

在Vivo脑内立体注射中，用于小鼠脑切片中远距离输入的光遗传学刺激