Name: improving the application of high molecular weight biotinylated dextran amine for thalamocortical projection tracing in the rat
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本研究由中国国家自然科学基金 (项目编号: 81373557; 81403327) 资助。

这项研究是由中国医学科学院伦理委员会 (参考编号 20160014) 批准的。所有程序都是按照国家卫生研究院关于实验室动物的护理和使用指南进行的 (国家科学院出版社, 华盛顿特区, 1996)。本研究采用四只成年雄性大鼠 (重量250-280 克)。所有动物都被安置在一个12小时的光/暗循环中, 控制着温度和湿度, 并允许免费获得食物和水。本研究中使用的仪器和材料显示在图 1中。 手术前, 所有仪器, 如立体定向框架和玻璃吸管, 都用70% 乙醇清洗。
1. 手术程序
<ol><li>使用立体定向地图集9 (图 2A) 确定 VPM 中感兴趣的坐标区域。</li>
	<li>准备一1µL 微型注射器, 配备玻璃微 (尖直径约10-20 µm) (图 1D), 并用液体石蜡进行测试。</li>
	<li>用腹腔注射麻醉7% 水合氯醛 (0.7 mL/100 g) 的大鼠。</li>
	<li>一旦深部麻醉证实与尾部捏和踏板撤退反射, 剃光头的动物的头部与电动剃刀和擦洗手术现场3倍, 10% 聚维酮碘其次70% 乙醇分别。</li>
	<li>把钝耳棒放到耳朵里, 把老鼠放到立体定向装置中, 把大鼠的上门牙放进嘴里 (图 1E), 然后在眼睛上涂抹眼膏。</li>
	<li>用70% 乙醇再次清洁手术部位的头部皮肤。使用无菌的外科手套和毛巾在不育条件下维持手术。</li>
	<li>用手术刀沿着矢状缝合 (图 1F) 在皮肤上作矢状切口。</li>
	<li>在整个手术过程中, 用无菌的棉尖喷头将肌肉和骨膜从头骨上刮掉, 以控制出血 (图 1F)。</li>
	<li>使用地图集的预定义坐标 (图 2A), 确定开颅手术 (图 1G) 的位置 (-3.3 毫米 Bregma 点, 2.6 毫米右至中线)。</li>
	<li>使用带有圆形尖端钻头 (#106) (图 1H) 的毛刺钻头进行开颅手术, 并在几分钟内继续钻至大约1毫米深度, 直至到达脑膜 (图 1I)。</li>
	<li>使用 microforceps 将硬脑膜置于注射部位, 使大脑皮层暴露 (图 1I)。</li>
	<li>用 10% BDA (1万分子量, 蒸馏水) 溶液 (图 1J) 改变微注射器中的液体石蜡。</li>
	<li>将注射器装入微注射装置并与微型泵连接 (图 1K)。 注: 注入量取决于微泵的速度。这里它被调整了到 30 nL/分钟 (图 1L)。</li>
	<li>在显微镜下, 将玻璃微与微注射装置手工插入 VPM, 通过大脑的皮质表面, 深度为5.8 毫米 (图 1M)。</li>
	<li>压力-注入 100 nL 10% BDA 进入 VPM 在3分钟 (35 nL/分钟) 与微型泵 (图 1L, M)。</li>
	<li>注射后, 把吸管放在原地, 再加5分钟, 然后慢慢地取出。</li>
	<li>用无菌螺纹缝合伤口 (图 1N)。遵循当地的动物护理委员会的指导方针, 为手术前和术后镇痛。</li>
	<li>把老鼠放在温暖的恢复区, 直到它苏醒并完全恢复。</li>
	<li>把恢复的老鼠送回笼子里去。</li>
</ol>2. 显影和章节
<ol><li>经过典型的10天的生存时间, 注射的动物与过量的10% 氨基甲酸乙酯 (2 mL/100 g) 的腹腔注射, 以诱导安乐死。</li>
	<li>灌注实验鼠的引擎盖 (图 1O)。<ol>
<li>一旦呼吸停止, 使用剪刀和镊子, 打开大鼠胸腔进入心脏。将静脉导管插入左心室向主动脉, 然后打开右耳廓。</li>
		<li>首先灌注与0.9% 磷酸盐缓冲盐水 (PBS) 在生理温度 (37 °c) 大约1-2 分钟直到血液从心脏撤出是清楚的, 然后继续与250-300 毫升4% 多聚甲醛在 0.1 M 磷酸盐缓冲 (PB, pH 7.4)。</li>
	</ol>
</li>
	<li>灌注后, 切开头部皮肤, 打开头骨, 然后解剖大鼠大脑。后修复解剖的大脑在4% 多聚甲醛2小时室温 (26 °c), 然后 cryoprotect 30% 蔗糖在0.1 米 PBS (pH 7.4) 3 天4摄氏度, 直到大脑沉浸在解决方案(图 1P)。</li>
	<li>一旦大脑沉浸在溶液中, 将大脑分成三个块, 在大脑矩阵的日冕方向上 (图 1Q)。中心块包含 VPM 和 S1。</li>
	<li>在40µm 上, 在一个冰冻阶段的切片系统中, 将大脑的中心块切开到日冕方向。用0.1 米 PBS (pH 7.4) (图 1RS) 在6井盘中有序地收集这些部分。</li>
</ol>3. 标准 ABC 染色
注意: 从大脑的每第三个日冕部分自由浮动部分用于可视化 BDA 标签与标准 ABC 过程10。
<ol><li>将0.1 米 PBS 中的剖面冲洗约1分钟。</li>
	<li>将 1% ABC 溶液中的剖面孵育为0.1 米 PB (pH 7.4), 在室温下含有0.3% 海卫 X-100 1 小时。</li>
	<li>在50毫米的三个缓冲 (pH 7.4) 洗涤部分三次。</li>
	<li>将包含 0.02% 33 '-diaminobenzidine tetrahydrochloride (民建) 和 0.01% H2O2的解决方案中的各部分着色为50毫米, 在室温下约为2-5 分钟。</li>
	<li>在50毫米的三个缓冲 (pH 7.4) 洗涤部分三次。</li>
	<li>使用标准的组织化学技术 (图 1T) 将切片安装在显微镜幻灯片上; 请参阅补充视频文件 III、灌注和切片)。</li>
	<li>在室温下, 将空气中的部分干燥一夜。</li>
	<li>在一系列酒精 (50%、70%、95%、100%) 溶液中简要地脱水切片。将每个解决方案中的幻灯片淹没大约十五年代。不要让幻灯片在每个步骤之间晾干。</li>
	<li>将二甲苯中的剖面清除三次, 约20分钟。</li>
	<li>将2或3滴苦瓜放在切片上, 然后将盖玻片放在各部分。</li>
</ol>4. 双荧光染色对 BDA 和大脑皮层的基本神经元
注: 对比中, 采用双荧光染色法观察了 streptavidin-AF594 和复染与荧光尼斯尔染色 AF500/525 在邻段中的 BDA 标记和基本神经元的相关性。
<ol><li>将0.1 米 PBS 中的剖面冲洗约1分钟。</li>
	<li>streptavidin-AF594 (1:500) 和 AF500/525 绿色荧光尼斯尔染色 (1:1, 000) 在0.1 米 PBS (pH 7.4) 的混合溶液中孵化剖面, 在室温下含有0.3% 的海卫 X-100 2 小时。</li>
	<li>用0.1 米 PB (pH 7.4) 冲洗三次。</li>
	<li>使用标准的组织化学技术将切片安装在显微镜幻灯片上。将空气中的部分干燥约1小时。</li>
	<li>观察前将盖玻片应用于蒸馏水中50% 甘油的荧光切片。</li>
</ol>5. 双荧光染色对中间神经元和脑皮质的促进
注: 采用双荧光染色法观察中间神经元与 streptavidin-AF594、光伏免疫化学在皮质靶区代表性切片上的相关性。
<ol><li>冲洗0.1 米 PBS (pH 7.4) 的代表性部分约1分钟。</li>
	<li>在包含3% 正常山羊血清和0.3% 海卫 X-100 0.1 米 PBS 的阻塞溶液中孵化部分30分钟。</li>
	<li>将这些部分转移到一个小鼠单克隆抗 PV IgG (1:1, 000) 的解决方案, 0.1 米 PBS (pH 7.4) 包含1% 正常山羊血清和0.3% 海卫 X-100 为隔夜在4°c。</li>
	<li>第二天, 在0.1 米 PBS (pH 7.4) 上冲洗三次。</li>
	<li>在2米 PBS (pH 1:40,000) 中, anti-mouse-AF488 山羊的二次抗体 (1:500)、streptavidin-AF594 (1:500) 和 4 '、6-diamidino-0.1-苯基吲哚盐酸盐 (DAPI、7.4) 的混合溶液, 包括1% 条正常山羊血清和0.3% 海卫。X-100 1 小时。</li>
	<li>重复步骤4.3 到4.5。</li>
</ol>6. 观察
<ol><li>拍摄 VPM、丘脑-皮质轴突和 corticothalamic 神经元的图像。<ol>
<li>观察荧光样品与共焦成像系统装备目标透镜 (4x, na: 0.13; 10x, na: 0.40; 和 40x, na: 0.95)。使用激发和发射波长 405 (蓝色), 488 (绿色) 和 559 (红色) nm。 注: 在这里, 共焦针孔是152µm (4x, 10x) 和105µm (40x)。图像捕获的空间分辨率为 1024 x 1024 像素 (4x、10x) 和 640 x 640 像素 (40x)。</li>
		<li>在2µm 的连续帧中拍摄二十幅图像, 每节的厚度为40µm (Z 系列)。</li>
		<li>将图像与共焦图像处理软件系统集成到一个单一的聚焦图像中, 进行三维分析, 如下所示: 设置起始焦平面→设置端部焦平面→设置步长→选择深度图案→图像捕获→ Z 系列。</li>
	</ol>
</li>
	<li>用装有数码相机的光镜拍摄 brightfield 图像 (4x, na: 0.13; 10x, na: 0.40; 40x, na: 0.95 透镜)。使用500毫秒的曝光时间. 使用照片编辑软件调整图像的亮度和对比度, 并添加标签。</li>
</ol>

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作者没有什么可透露的。

选择合适的示踪剂是成功的神经追踪实验的关键步骤。在 bda 家族中, 建议通过顺神经通路优先输送高分子量的 bda (1万分子量), 与低分子量 bda (3000 分子量)2,3,11,12,13. 然而, 许多研究还表明, 高分子量的 BDA 也可能会与顺追踪1,2,3, 同时用于逆行路径跟踪。通过 VPM 和 S1 之间的相互神经通路, 我们提供了进一步的证据, 支持高分子量 BDA 适用于双向跟踪追踪4的观点。同样在这个领域, 高分子量的 BDA 也被用于视觉和听觉系统, 用于检查背侧膝状体与视觉皮层以及内侧膝状体和听觉皮层之间的相互连接。3,14。
在神经追踪实验中, 另一个重要的考虑因素是神经标记染色方法的选择。在以往的大多数研究中, 传统的 ABC 协议和荧光链亲和素染色法都已用于检测 BDA 标签, 并揭示了类似形态 模式1, 2, 3,4,5. 在此, 我们发现 streptavidin-AF594 不仅是 BDA 标签的正确选择, 更适合与其他神经标记, 如荧光尼斯尔和 PV 抗原相结合, 为观察 bda 的相关性提供了新的契机。标记和其他神经元素。虽然对 BDA 和其他神经标记的传统双染色方法也经常采用双色民建联程序14,15, 但不适合使用三维分析系统在共焦激光扫描显微镜。从技术的角度来看, 本研究为我国的 BDA 标签与其他标签的比较提供了有价值的参考。
神经道追踪技术最初用于揭示神经系统中注射部位与靶点之间的神经元连接;然而, 研究人员仍然在追求理想的神经标记结构, 正确的跟踪16,17。与其他示踪物, 如辣根过氧化物酶和霍乱毒素亚单位 B 相比, 高分子量的 BDA 产生了更高质量的神经标记来量化轴突和神经元树突的数量16,17.虽然在本研究中没有对 bda 标签进行定量分析, 但随着 bda 的神经标记质量的提高, 为进一步了解标记轴突和神经元的形态学特征提供了更多的机会。枝 晶。
类似于许多其他示踪剂的应用, 本试验过程中应考虑一系列重要问题, 包括: 用于注射的 BDA 的浓度和容积、正确的注射部位、玻璃吸管的尖端直径、手术过程, 最佳生存时间, 灌注, 切片, 观察下显微镜下。它直接关系到我们所期望的 BDA 标签的质量。除了上面提到的关键步骤之外, 还有一些技术上的限制需要注意, 例如从注入的站点到标记的目标的距离, 这取决于实验中使用的模型动物种类3,5,18. 总而言之, 一个成功的追踪研究取决于整个实验过程中的每一步。
在本研究中, 我们提供了一个完善的协议, 以证明荧光 BDA 染色法是获得高质量的神经标记的有效途径, 它可以同时与其他神经标记结合使用荧光组织化学或免疫化学。通过对传统的 bda 染色与民建联程序的比较, 可以更容易地分析 bda 标签的精细结构, 并在共聚焦激光扫描显微镜下, 利用目前的荧光技术, 将其与其它神经元区别开来。方法。

高分子量 BDA (1万分子量), 一种高度灵敏的示踪剂, 已用于在中枢神经系统中追踪神经通路20年以上的1。虽然 bda 的使用是一种常见的神经道追踪技术, 但在动物中, bda 标签的质量可能受到各种因素的影响, 如1,2,3。最近的研究表明, BDA 标签的优化结构不仅与适当的注射后生存时间有关, 而且与染色方法4相关。到目前为止, 传统的亲和-生物素-过氧化物酶复合物 (ABC), 链亲和素异硫氰酸酯, 和 streptavidin-AF594 染色方法, 以揭示 BDA 标签在以前的研究2, 3, 4,5。相比之下, BDA 的荧光染色可以很容易地进行。
为了推广高分子量 BDA 的应用, 本研究引入了一种精细化的协议。在将 bda 注射液注入大鼠脑丘脑的 VPM 后, 采用常规 ABC 染色法和双荧光染色技术对 bda 标签进行了分析, 观察了 bda 标签的相关性和基本streptavidin-AF594 和荧光尼斯尔组织化学或 PV 免疫化学的皮质靶上的神经元素或中间神经元。通过 VPM 与原发性体感皮层 (S1)6、7、8之间的相互神经通路, 我们集中观察了在丘脑-皮质预测轴突和 corticothalamic 中的 BDA 标记。投射的细胞 somas 在 S1。通过这一过程, 我们希望不仅提供一个详细的协议, 以获得高品质的神经标记的高分子量 bda, 而且还有一个完善的协议, 结合荧光 BDA 标签和其他荧光神经标记与组织化学或免疫化学。这种方法在共聚焦激光扫描显微镜下研究局部神经回路及其化学特性是比较可取的。

<table><tbody><tr><td>Biotinylated dextran amine (BDA)</td><td>Molecular Probes</td><td>D1956</td><td>10,000 molecular weight</td></tr><tr><td>Streptavidin-Alexa Fluor 594</td><td>Molecular Probes</td><td>S32356</td><td>Protect from light</td></tr><tr><td>500/525 green fluorescent Nissl stain</td><td>Molecular Probes</td><td>N21480</td><td>Protect from light</td></tr><tr><td>Brain stereotaxis instrument</td><td>Narishige</td><td>SR-50</td><td></td></tr><tr><td>Freezing microtome</td><td>Thermo</td><td>Microm International GmbH</td><td></td></tr><tr><td>Confocal imaging</td><td>Olympus</td><td>FV1200</td><td></td></tr><tr><td>system</td><td></td><td></td><td></td></tr><tr><td>Micro Drill</td><td>Saeyang Microtech</td><td>Marathon-N7</td><td></td></tr><tr><td>Sprague Dawley</td><td>Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences</td><td>SCKX (JUN) 2012-004</td><td></td></tr><tr><td>Vectastain ABC Kit</td><td>Vector Laboratories</td><td>PK-4000</td><td></td></tr><tr><td>superfrost plus microscope slides</td><td>Thermo</td><td>#4951PLUS-001</td><td>25x75x1mm</td></tr><tr><td>Photoshop and Illustration</td><td>Adobe</td><td>CS5</td><td></td></tr></tbody></table>

improving the application of high molecular weight biotinylated dextran amine for thalamocortical projection tracing in the rat

高分子量生物素化葡聚糖胺 (BDA) 已被用作高度敏感的神经解剖学示踪剂数十年。由于其标签的质量受到各种因素的影响, 在这里, 我们提供了一个完善的协议, 以应用高分子量的 BDA 研究最佳神经标记在中枢神经系统。经立体定向注入内侧核 (VPM) 的大鼠丘脑通过一个微妙的玻璃吸管, bda 染色的荧光链亲和素 Alexa (AF) 594 和复染荧光尼斯尔染色 AF500/525。在绿色尼斯尔染色的背景下, 在体感皮层显示了红色的 BDA 标记, 包括神经元细胞体和轴突末端。此外, 对 bda 和钙结合蛋白 parvalbumin (pv) 进行了双荧光染色, 观察了 bda 标记与中间神经元在皮质靶上的相关性, 为研究局部神经电路及其化学特性。因此, 这种精制方法不仅适用于通过丘脑和大脑皮层之间的相互神经通路对高品质的神经标记进行可视化, 而且还可以同时证明其他具有荧光组织化学或免疫化学的神经标记。

10天后, BDA 注入 VPM 的生存是足够的, 以产生强烈的神经标记, 在相应的皮质区域与注射方 (图 2)。BDA 的常规 ABC 和荧光染色程序都揭示了 S1 上类似的神经标记模式, 包括 anterogradely 标记的丘脑-皮质轴突和 retrogradely 标记 corticothalamic 神经元(图 2C, D).
对于 anterogradely 标记的轴突, 它们从2层到6层, 在4层上具有较高的密度, 在桶区观察到典型的丘脑-皮质轴突类型(图 2C, D).在更高的放大倍数视图中, BDA 标签清楚地显示在轴突主干、分支、络和小静脉曲张 (图 3A) 中。在被标记的丘脑-皮质轴突将显示, retrogradely 被标记的皮层锥体神经元被显示, 并且他们的细胞身体分布在层数 5/6, 但他们的顶端树突延伸到2层(图 2C, D).BDA 标签不仅显示在神经细胞体和树突, 而且在树突状棘, 出现如高尔基样的分辨率 (图 3B)。
此外, 还进行了双荧光染色, 以观察与 streptavidin-AF594 和光伏免疫化学在皮质靶上的 BDA 标记和中间神经元的相关性。在大脑皮层目标的 BDA 标签周围, PV 阳性神经元从2层分布到6层, 高度集中于4层 (图 4A)。没有与 bda 标记的 pv 阳性神经元, 但是, 在光伏阳性细胞体表面周围发现了 bda 标记的轴突端子, 在 bda 标记的皮质锥体神经元周围有 pv-正轴突端子 (图 4B, C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55938/55938fig1.jpg"> 图1。在本实验中使用的关键手术步骤和主要仪器的照片。 A: 立体定向设备。B: 牙科钻头。C: 外科工具 (手术刀、microforceps 和等等) D: 装有玻璃微的微型注射器。E: 将鼠头置于立体定向装置中。F: 清理外科站点。G: 确认 Bregma 点。H: 钻头骨。I: 暴露大脑皮层。J: 将 10% BDA 加载到微注射器中。K: 将注射器装入用于微注射的装置中。L: 调整微泵的速度。M: 将玻璃微插入 VPM。N: 用无菌螺纹缝合伤口。O: 灌注在引擎盖中的老鼠。P: 解剖出大脑。Q: 将大脑分成三块与大脑矩阵。R: 在冻结阶段滑动切片系统上切割大脑。S: 在6井盘中有序地收集大脑部分。T: 在显微镜幻灯片上装入节。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55938/55938fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55938/55938fig2.jpg"> 图2。在体感皮层, 注射部位和典型的神经标记具有高分子量的 BDA。 : 在立体定向地图集上确定注射点。B: 具有代表性的显微照片在绿色尼斯尔染色的背景下, 显示 VPM 腹侧内侧核的 BDA (red) 注射部位。C: 荧光 BDA 标记的显微照片, 包括5/6 层4和 corticothalamic 神经元中的丘脑-皮质轴突。D: 显微照片常规的 bda 标签与标准的亲和-生物素过氧化物酶程序, 显示了类似的神经标记模式荧光 BDA 标签。VPL, 丘脑腹后外侧核。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55938/55938fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55938/55938fig3.jpg"> 图3。在绿色尼斯尔染色的背景下, 高分子量的神经标记具有较高的质量。 一个: 更高的放大视图的 anterogradely 标记轴突乔木 (红色) 详细。B: 详细显示 retrogradely 标记的神经元细胞体、树突和树突状刺 (红色) 的高分辨率照片。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55938/55938fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55938/55938fig4.jpg"> 图4。中间神经元与钙结合蛋白 PV 阳性在皮质靶上的相关性。 : 在体感皮层中, BDA 标记 (红色) 和 PV 阳性中间神经元 (绿色) 的分布。B: 在4层中, 对 BDA 标记的轴突和 PV 正中间神经元分布的放大视图更高。C: 在5/6 层中, BDA 标记的皮质锥体细胞 (红色) 和 PV 阳性中间神经元 (绿色) 的高分辨率照片。所有样品均复染 DAPI (蓝色)。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55938/55938fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充视频文件:补充视频简要介绍了外科手术, VPM 的注射, 灌注和切片, 以及结果。在名为 "iv" 的视频文件中。典型结果表明: 三维共焦激光扫描显微术和分析系统结果。高分辨率动画显示 retrogradely 标记的神经细胞体, 树突和树突状刺 (红色) 详细。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55938/Supplemental_Videos.zip">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat at JoVE.com

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

在这里, 我们提出了一个完善的协议, 以有效地揭示生物素化葡聚糖胺 (BDA) 标签的荧光染色方法通过一个相互的神经通路。它适用于分析 BDA 标签的精细结构, 并在共焦激光扫描显微镜下将其与其他神经元进行区分。

高分子量生物素化葡聚糖胺在大鼠丘脑-皮质投影示踪中的应用

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of ...

improving-application-high-molecular-weight-biotinylated-dextran

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

8.9K 次观看.  China Academy of Chinese Medical Sciences. 在这里, 我们提出了一个完善的协议, 以有效地揭示生物素化葡聚糖胺 (BDA) 标签的荧光染色方法通过一个相互的神经通路。它适用于分析 BDA 标签的精细结构, 并在共焦激光扫描显微镜下将其与其他神经元进行区分。

视频: Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain4. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats7-9 to mouse10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

记录神经反应和注射示踪剂一个清醒小鼠的制备

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

科研

神经科学

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 ...

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

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

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, ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

禽流感胚胎听神经听觉神经纤维的选择性跟踪

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)1. PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site2,3. Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)4,5&#160;.&#160;This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3&#39;-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites6-11.

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

从外围目标跨突触跟踪与伪狂犬病病毒其次是霍乱毒素和生物素葡聚糖胺双标

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

方法以使对新生儿鼠害的腹头骨开颅手术

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive functions including decision making, sensory-guided behavior or memory. To understand how such intricate neuronal networks perform such tasks, a crucial step is to determine the function (or electrical activity) of individual cell types within the network, preferentially when the animal is performing a relevant cognitive task. Additionally, it is equally important to determine the anatomical structure of the network and the morphological architecture of the individual neurons to allow reverse engineering the cortical network. Technical breakthroughs available today allow recording cellular activity in awake, behaving animals with the valuable option of post&#160;hoc identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loose-patch) configuration using conventional patch pipettes. The juxtasomal recording configuration is relatively stable and applicable across behavioral conditions, including anesthetized, sedated, awake head-fixed, and even in the freely moving animal. Thus, this method allows linking cell-type specific action potential spiking during animal behavior to reconstruction of the individual neurons and ultimately, the entire cortical microcircuit. In this video manuscript, we show how individual neurons in the juxtasomal configuration can be labeled with biocytin in the urethane-anaesthetized rat for post&#160;hoc identification and morphological reconstruction.

To understand the structure of neuronal networks, functional and morphological characterization of individual neurons is a necessity. Here, we demonstrate juxtasomal biocytin labeling, which allows electrophysiological recordings in the extracellular configuration, yet maintaining the ability to intracellularly label the neuron for post&#160;hoc reconstruction of dendritic and axonal architecture.

Juxtasomal生物胞素标记来研究单个皮层神经元的结构与功能的关系

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

在Wholemount大鼠视网膜免疫组化和钙成像方法

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

激光制导神经跟踪在脑外植体

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

在河马区文特尔区谷氨酸终端的 Vivo 解剖和功能跟踪组合

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...