我们感谢福米久保带人，托德蒂勒和HerwigBaier的的（UCSF /马克斯普朗克研究所）的行为实验和DPSS激光器成立的建议;的Heesoo金正日和Chiara CERRI MBL神经生物学课程协助厨师tdTomato实验的，PetronellaKettunen（哥德堡大学）最初的合作optogenetic实验BaljitKhakh，埃里克·哈德森，迈克·巴卡和约翰·米利根（UCLA）的技术咨询和钱永健（UCSD）的厨师tdTomato的建设。这项工作得到了NRSA奖（5F31NS064817）AMSP和赠款来自美国国家科学基金会（RIG：0819010）AS。

提前准备好以下。 1。准备光纤电缆<ol><li>创建光缆的存储单元，用于通过熔融玻璃巴斯德吸管的锥形颈部超过本生灯创建一个〜150°的角度。 </li><li>使用线切割或刀片，小心地剪光缆的成两部分。每一件作品都应该有一个结束与一个FC / PC适配器和一个露底。存储一块作为储备电缆。 </li><li>向下剥去光缆到包层，除去纤维外套和加强纤维（ 图1A），从5.0厘米的电缆的切割端。 </li><li>到预先准备的巴斯德吸管插入光缆。请确保电缆可以很容易地移入和移出的吸头。 </li><li>光缆突出的巴斯德吸管尖，小心地剪切和删除光纤包层玻璃纤维周围，2毫米的切口端面。 </li><li>使用金​​刚石笔/玻璃切割机，缺口的玻璃纤维和破晓FF到底要创造一个干净的切割/表面的纤维（ 图1B）。 </li><li>收回光缆巴斯德吸管存储。如果光纤末端被碎裂或断裂不均匀，重复步骤（1.6）。 </li><li>巴斯德吸移管的位置光缆在使用显微操作器（ 图1C）。 </li></ol>程序2-8描述用于注射一般适用于许多斑马鱼实验成胚的转基因的方法。这种方法的变化，所描述的其他视频朱庇特6，7，8，9，22也同样有效。 2。拔出注射针头<ol><li>使用针拔出器，拉一个逐渐变细尖成两个注射针头与高硼硅玻璃管材。拔轮器设置会有所不同。 （在我们使用一个萨特仪器针拔出器设置：P = 500，热= 720，拉出= 50，速度= 70，时间= 150）。每针拔出器是不同的，所以优化车夫设置时间pirically。对于更多的锥形针，增加热量和/或拉。对于不锥形针，增加时间和/或减少拉。 </li><li>将针在一个安全的容器（ 即培养皿中冷轧带，粘合面出）。 </li></ol> 3。倒入注塑模具<ol><li>熔体0.5 G琼脂糖在30毫升的胚胎/蓝水，直到琼脂糖完全溶解。 </li><li>倒入一个陪替氏培养皿的下半部。 </li><li>将安装井（ 图2）琼脂糖，小心地限制泡沫的形成孔周围的矩形模具。 </li><li>让琼脂巩固。 </li><li>取出模具，并填写蓝水/胚胎培养皿。 </li><li>竖直存放，充满洁净的水，在室温下当天使用或在4°C，以备将来使用。 </li></ol> 4。质粒DNA混合的注射<ol><li>质粒DNA稀释为1:10酚红在DDH 2 O的浓度为50纳克/微升为例如： <table border="1"><tbody><tr><td> 1.0毫升</td><td>质粒DNA（250毫微克/毫升） </td></tr><tr><td> 0.5毫升</td><td>酚红</td></tr><tr><td> 3.5毫升</td><td> DDH 2 O </td></tr></tbody></table></li><li> DNA混合物可在室温下保持，如果立即使用或储存在4℃下，数天。 </li></ol> 5。对繁殖这应该做的前一天晚上你打算做注射。 <ol><li>填写系统的水和雌雄鱼繁殖罐。如果注射不能尽快灯打开的设施，独立的男，女鱼分频器。 </li></ol> 6。准备注射剂（可以做到，而等待的胚胎） <ol><li>打开高压注射器台。确保系统设置为脉冲。开始PULSE DURATION设置为1。 </li><li>打开空气阀和调整高压注射器〜20磅。 </li><li>用解剖的范围内以最高的放大率，打破尖的针，用钳子或扑克创建1〜2微米的开口（ 图3，电影1）。 </li><li>填写针与DNA组合：1。配售的针，针尖侧，到DNA结构，并通过毛细管作用或2针填写。使用长距离尖到中移液1〜2μl的DNA混合直接进针。 </li><li>将已装好的针在一个安全的地方，直到准备好注入。 </li></ol> 7。收集胚胎<ol><li>设施后，灯已开启，删除分频器（如适用）。收集过滤器/小筛子的胚胎，并将它们传送到培养皿中，用新鲜的蓝/胚胎水。 </li></ol> 8。 1-2细胞阶段的胚胎注入<ol><li>将收获的胚胎用塑料吸管的注塑模具。 </li><li>用解剖显微镜，轻轻一推胚胎成井钳或钝扑克（ 图4，电影2）。 </li><li>将装载针到一个以上的胚胎显微操作位置。 </li><li>校准注射量以1级为单位调节的脉冲持续时间，直到达到所需的音量（NL）。这可以用千分尺幻灯片校准，中所描述的前一的JOVE视频8，22，但用于本实验的精度是没有必要的，因为范围广泛的进样体积将导致足够的表达。 </li><li>注1 NL DNA混合直接进入细胞，然后为所有的胚胎。 DNA也可以注入到蛋黄，但是这往往是低效率的（ 图5，电影3）。 </li><li>使用温和的流蓝/胚胎水从模具中取出注射的胚胎。 </li><li>商店在28.5°C注射的胚胎在黑暗中。 </li><li>定期删除未受精的，受损或死亡的胚胎。 </li><li>治疗胚胎无线网络日PTU 18-24 HPF之间，防止色素沉着。 </li></ol> 9。屏幕转基因表达<ol><li>手动dechorionate 24-48斑马鱼胚胎中使用产钳。 </li><li>用0.02％三卡因麻醉幼虫。 </li><li>使用荧光显微镜，确定幼虫的RB或三叉神经元表达，并将其传输到一个新的菜，新鲜的的PTU蓝色/胚胎水。胚胎与稀疏在容易识别细胞中的表达是最理想的，但个别神经元的激活与光纤电缆，以便有针对性地进行了广泛的表达强度是可以接受的。 </li><li>商店胚胎在28.5°C在黑暗中，直到所需的试验阶段。 </li></ol> 10。安装幼虫行为实验<ol><li> 1.5％的低熔化琼脂糖在DDH 2 O和存储在42°C加热块，以防止其凝固。 </li><li>使用玻璃巴斯德吸管，转移成桶的预先筛选的幼虫之一e为1.5％的低熔化琼脂糖尽可能少的水尽可能蓝/胚胎。 </li><li>转移到一个小培养皿下降的琼脂糖幼虫。 </li><li>在解剖显微镜下，位置幼虫背起来。 </li><li>当琼脂糖已经凝固，切断了琼脂糖用薄刀片（11刀），留下一块楔形的琼脂围绕整个幼虫。 </li><li>的蛋黄两侧的两条对角线切割照顾缺口的幼虫。 </li><li>填写周围的区域琼脂糖与胚胎/蓝水。 </li><li>拉琼脂糖远离躯干和尾幼虫（ 图6）。 </li></ol> 11。准备高速摄像机和成像软件<ol><li>高速摄像机安装到夹层范围。 </li><li>将相机连接到电脑上。 </li><li>打开电脑。 </li><li>开启高速摄像机。 </li><li>打开视频/图像处理软件（我们使用的AOS成像软件，将描述在这里使用的程序，但其他影像学检查软件同样是可以接受的）。 </li><li>调整相机设置（ 即 1000帧每秒（fps）的50％触发缓冲区或其他特别设置）。 </li><li>开始记录。 </li></ol> 12。激活单个神经元，用473 nm激光<ol><li>附加刺激，激光和光纤电缆。 </li><li>打开刺激。 </li><li>设置刺激的和最多5伏特，脉冲持续时间为5毫秒​​。 </li><li>根据制造商的说明，打开激光。 </li><li>使用在解剖显微镜与的厨师tdTomato表达（ 图6）的神经元的细胞体的附近的光纤电缆的前端定位。 </li><li>蓝色的光脉冲传递到激活感觉神经元。 </li><li>使用高速摄像机记录“行为设置为500或1000帧每秒。 </li><li>重复实验所需，等待1分钟之间，以避免习惯性的激活。 （我们每个神经元）录得最低的三个反应。 </li><li>对释放的幼虫，撬除了琼脂糖镊子，小心不要伤害动物。这种动物可以允许进一步发展和可重复的过程，在一个旧的阶段的特点开发的行为。高分辨率共焦成像的关联的行为与蜂窝结构，如下面描述的激活细胞的胚胎也可以重新安装。 </li><li>新鲜的蓝色水/胚胎，幼虫转移到培养板。我们使用24孔板的来跟踪个人幼虫。 </li></ol> 13。用激光共聚焦显微镜图像神经元（S） <ol><li> 0.02％三卡因的麻醉幼虫。 </li><li>安装幼虫，背了起来，在1.2％的低熔化琼脂糖或在背部模具。 </li><li>图片厨师tdTomato神经元与543 nm激光和适当的过滤器和目标。我们用20倍的目标。 </li><li>从琼脂糖凝胶中取出幼虫，并返回到个人与蓝/胚胎水。 </li><li>商店在28.5°C在黑暗中富rther分析。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

我们描述了一种单RB神经元在活的斑马鱼optogenetic激活。我们的方法采用了短暂的转基因表达的荧光标记的channelrhodopsin的变种，厨师tdTomato 13，在特定的躯体感觉神经元。这种方法可以很容易地适应用于其他幼虫斑马鱼的细胞群。 使用这种方法，我们一贯引起的行为反应，从34-48斑马鱼幼虫表达大厨tdTomato的。使用5毫秒的蓝色光脉冲，在5 V，我们能够激活单RB的神经元?...

促进或抑制神经细胞的兴奋与定义的波长的光的optogenetic方法的发展使我们能够研究不同人群的神经元的神经回路，控制行为，1，19，21的功能。这种技术通常被用于激活的神经元组，但它也可以被用来激活单个神经元的。斑马鱼幼虫，这些方法特别适合的，因为他们是半透明的，他们的神经系统迅速发展，并建立转基因动物是快速和常规。然而，重大的技术障碍必须克服，可靠地实现单神经元激活。 要优化程序optogenetic激活的单斑马鱼神经元，我们专注于躯体感觉神经元。斑马鱼幼虫检测各种体感刺激，使用两个群体神经元三叉神经元，支配头，及Rohon胡子（RB）的神经元，它支配身体的其他部位。每三叉和RB神经元投射的周轴突分支广泛在皮肤上，以检测的刺激和连接到下游的神经回路的中心轴突。动物早在21小时后受精（HPF）的响应触摸，表明已形成了相干的体感电路5星，18。幼体发育过程中至少有三叉神经和RB神经元突触上的Mauthner细胞激活经典的逃生反应，但越来越多的证据表明，有多种类型的体感神经元与不同的连接模式，可能引起变化的逃逸行为，4， 10，12，14，15，16，17。我们开发这种方法的动机是行为功能的不同类别的躯体感觉神经元的特征，但这种方法原则上可以用于研究的功能，几乎所有的神经元或神经元的LAR人口值斑马鱼。 道格拉斯等人以前描述了一种方法用于激活Channelrhodopsin-2表达的蓝色光的躯体感觉神经元，引出逃逸行为3。他们使用的方法ISL1基因的增强子元件驱动CHR 2-EYFP躯体感觉神经元中的表达。这种转基因，然而，据报道，以显示相对微弱的荧光，需要共同注射的第二记者，UAS :: GFP，允许细胞表达CHR 2-EYFP的可视化。这种方法被用来引出24-48斑马鱼的行为反应之间，但可能不会得到响应过去的72 HPF。因此，虽然此方法适用于研究神经回路在非常早期的幼体阶段（24-48 HPF），它是不足为特征的神经回路和行为反应幼虫，中老年时更是明显不同的行为反应和神经回路是比较成熟的。 我们试图该技术在提高灵敏度的特点幼虫RB神经元亚群的功能。为了提高表达，我们使用了一个特定的体感增强剂（CREST3）20驱动表达的LexA-VP16和LexA操纵的序列（4xLexAop）11拉伸，扩增的荧光标记的光激活的信道的表达。此配置扩增表达的通道，省去了用于共表达的第二记者和允许我们直接确定每个神经元中的信道的相对丰度。使用的LexA蛋白/ LexAop序列有额外的优点，允许我们引入转基因成斑马鱼记者使用GAL4/UAS系统的线。转基因瞬时表达，这造成了不同程度的表达，但通常是足够强大的可视化的单个神经元的胞体和轴突预测数天。为了优化灵敏度性点燃，我们使用的光激活通道厨师，channelrhodopsin的变体组成的一个嵌合体：channelopsin-1（Chop1）和channelopsin-2（Chop2），交叉点在螺旋环EF 13。此信道被激活为CHR 2在相同的波长，但需要较低的光强度进行激活，使得它比其他常用的途径，包括CHR 2更敏感。的厨师融合蛋白红色荧光蛋白，tdTomato，使我们能够筛选蛋白的表达没有激活的通道。作为光源，我们使用了一个二极管泵浦固态（DPSS）激光耦合到一个光纤电缆提供一个精确的，高功率的蓝色光脉冲到一个特定区域的幼虫。这使我们能够专注激光的单个神经元，省去了寻找罕见的转基因动物，在一个单一的神经元表达的通道。使用这种方法，我们能够激活单RB神经元，记录行为反应s的一个高速视频摄像机，和图像的激活的神经元，在高分辨率的激光共聚焦显微镜。

<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> 物料 </td></tr><tr><td>玻璃巴斯德吸管</td><td>费舍尔</td><td> 1367820B </td><td>或同等学历（直径10-15毫米） </td></tr><tr><td> 200微米光纤</td><td> Thorlabs公司</td><td> AFS200/220Y-CUSTOM </td><td>跳线，长度：3米，完A：B：FC / PC，FC / PC，结束外套：FT030 </td></tr><tr><td> 50微米光纤</td><td> Thorlabs公司</td><td> AFS50/125Y-CUSTOM </td><td>跳线，长度：3米，完A：B：FC / PC，FC / PC，结束外套：FT030 </td></tr><tr><td>可调节剥线工具</td><td> Thorlabs公司</td><td> AFS900 </td><td>或三孔剥线工具（FTS4） </td></tr><tr><td>钻石楔形隶</td><td> Thorlabs公司</td><td> S90W </td><td></td></tr><tr><td>烈焰红唇/褐色微管牵引机</td><td>萨特仪器</td><td> P-97 </td><td>或同等学历</td></tr><tr><td>高硼硅玻璃管材用灯丝</td><td>萨特仪器</td><td> BF-100-78-10 </td><td></td></tr><tr><td>注塑模具</td><td> N / A </td><td> N / A </td><td>图5 </td></tr><tr><td> 1.5 mL离心管</td><td>任何</td><td>任何</td><td></td></tr><tr><td>陪替氏培养皿（100x15毫米） </td><td>任何</td><td>任何</td><td></td></tr><tr><td>陪替氏培养皿（60x15毫米） </td><td>任何</td><td>任何</td><td></td></tr><tr><td>高压注射器</td><td> ASI </td><td> MPPI-3 </td><td>或同等学历</td></tr><tr><td>微操作机器人及金属架</td><td> Narashige </td><td> M152 </td><td>或同等学历</td></tr><tr><td>一次性塑料移液管</td><td> Fisherbrand </td><td> 13-711-7 </td><td>或同等学历</td></tr><tr><td>扑克（针座，昆虫针） </td><td>精细的科学工具， </td><td> 26018-17和26000-70 </td><td>或同等学历</td></tr><tr><td>镊子</td><td>精细的科学工具， </td><td> 11255-20 </td><td>或同等学历</td></tr><tr><td> Microloader吸头</td><td> Eppendorf公司</td><td> 9300001007 </td><td></td></tr><tr><td> 28.5°C孵化器</td><td>任何</td><td>任何</td><td></td></tr><tr><td> 42°C加热块</td><td>任何</td><td>任何</td><td></td></tr><tr><td>非无菌手术刀片＃11 </td><td>精细的科学工具， </td><td> 10011-00 </td><td>或同等学历</td></tr><tr><td>解剖小号应付</td><td>蔡司</td><td> STEMI </td><td>或同等学历</td></tr><tr><td>荧光解剖范围与标准过滤器</td><td>任何</td><td>任何</td><td>或同等学历</td></tr><tr><td>共聚焦显微镜</td><td>蔡司</td><td> LSM 510或710 </td><td>或相当于激光器的GFP和RFP，10倍，20倍和40倍的目标</td></tr><tr><td>高速摄像机</td><td> AOS技术公司</td><td> X-PRI（130025-10） </td><td>或同等学历</td></tr><tr><td>便携式激光波长473 nm </td><td>晶体激光器</td><td> CL-473-050 </td><td>或更高的功率，与TTL选项</td></tr><tr><td> S48刺激</td><td>天文杂志，草仪器事业部</td><td> S48K </td><td>或同等学历</td></tr><tr><td> FC / PC FC / PC配合套筒</td><td> Thorlabs公司</td><td> ADAFC1 </td><td>五月NE为光缆连接</td></tr><tr><td>激光防护眼镜</td><td> Thorlabs公司</td><td> LG10 </td><td>或同等学历</td></tr><tr><td> 24培养板</td><td>杰纳西</td><td> 25-102 </td><td>或同等学历</td></tr><tr><td>单抑郁症幻灯片</td><td>费舍尔</td><td> S175201 </td><td>或同等学历</td></tr><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td>即时海洋</td><td>水生生态系统</td><td> IS50 </td><td></td></tr><tr><td>亚甲基蓝</td><td>费舍尔</td><td> S71325 </td><td></td></tr><tr><td>酚红</td><td>西格玛</td><td> P4758 </td><td></td></tr><tr><td>琼脂糖</td><td> EMD </td><td> 2125 </td><td>或同等学历</td></tr><tr><td>低熔化琼脂糖</td><td>西格玛</td><td> A9045 </td> &lt;TD&gt;或同等学历</td></tr><tr><td> PTU </td><td>西格玛</td><td> P7629 </td><td></td></tr><tr><td>三卡因</td><td>西格玛</td><td> A5040 </td><td></td></tr><tr><td>蓝色的水/胚胎</td><td></td><td></td><td> 10 L DDH 2 O 0.6克即溶海洋 6滴亚甲基蓝</td></tr><tr><td>酚红</td><td></td><td></td><td> （5毫克/毫升在0.2M氯化钾） </td></tr><tr><td> 100X PTU </td><td></td><td></td><td> 0.150克PTU 50 ML DDH 2 O 溶解在70°C，常常动摇 分装并储存于-20°C </td></tr><tr><td> 1个PTU </td><td></td><td></td><td> 1毫升100倍PTU 99毫升蓝/鱼水</td></tr><tr><td>三卡因原液</td><td></td><td></td><td> 400毫克三卡因 97.9 DDH 2 O </td></tr><tr><td> 〜2.1毫升1M​​ Tris，pH9.0 </td><td></td><td></td> &lt;TD&gt;调整pH值7.0〜 商店在4°C或-20°C用于长期存储</td></tr></tbody></table>

optogenetic activation of zebrafish somatosensory neurons using chef-tdtomato

幼虫斑马鱼正在成为一个模型，用于描述简单的神经回路的发育和功能的。由于体外受精，快速发展，半透明，斑马鱼特别适合optogenetic方法来研究神经回路的功能。在这种方法中，光敏感的离子通道是在特定的神经元中表达，使实验者来激活或抑制它们的意愿，从而评估其贡献的特定行为。应用这些方法斑马鱼幼虫的概念很简单，但需要优化的技术细节。在这里，我们展示了一个程序，表达一个channelrhodopsin的变种在仔鱼斑马鱼的体感神经元，光活化的单个细胞，并记录产生的行为。引入了一些修改先前建立的方法，这种方法可以用来引发的行为反应，从单一的神经元激活到受精后至少4天（DPF）。具体来说，我们创建了一个转基因，用躯体感觉神经元增强，CREST3，推动标签channelrhodopsin变种，厨师tdTomato的表达。注射这种转基因成1 - 细胞期胚胎中的结果，在镶嵌在躯体感觉神经元中的表达，可以用激光共聚焦显微镜成像。照明确定在这些动物中的细胞从473 nm的DPSS激光器的光，引导通过光纤电缆，引出行为，可以用高速摄像机记录，并进行定量分析。这种技术可以适于研究行为引起通过激活任何斑马鱼神经元。与遗传或药物扰动的这种方法相结合，将是一个有力的方式来研究电路的形成和功能。

<img src="/files/ftp_upload/50184/50184fig1.jpg" alt="图1"/> 图1。光缆成立。（A）层的光纤电缆。 （B）的剥离的光纤电缆在巴斯德吸管。 （C）的光纤电缆在巴斯德吸管定位使用显微操作。 <img src="/files/ftp_upload/50184/50184fig2.jpg" alt="图2"/> 图2。注塑模具的模板。 <p class="jove_content" fo:keep-together.wit...

Watch this Scientific Journal Video about Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato at JoVE.com

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

optogenetic技术已使人们有可能研究特定的神经元的行为的贡献。我们描述了一个激活单躯体感觉神经元表达channelrhodopsin变种（厨师）二极管泵浦固态（DPSS）激光器和记录引起的行为与一个高速摄像机的斑马鱼幼虫的方法。

Optogenetic激活斑马鱼的体感神经元使用厨师tdTomato的

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

optogenetic-activation-zebrafish-somatosensory-neurons-using-chef

Research

JoVE Journal

Neuroscience

14.7K 次观看.  University of California, Los Angeles. optogenetic技术已使人们有可能研究特定的神经元的行为的贡献。我们描述了一个激活单躯体感觉神经元表达channelrhodopsin变种（厨师）二极管泵浦固态（DPSS）激光器和记录引起的行为与一个高速摄像机的斑马鱼幼虫的方法。

视频: Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

针对嗅球神经元采用组合在体内电穿孔和GAL4基于增强陷阱斑马鱼线

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

科研

神经科学

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.

使用光刺激神经元的负担得起的LED阵列

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

单个锥体神经元的高保真光遗传学控制方法<em&gt;在体内</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

听觉神经的光遗传学刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Somatosensory Event-related Potentials from Orofacial Skin Stretch Stimulation

Cortical processing associated with orofacial somatosensory function in speech has received limited experimental attention due to the difficulty of providing precise and controlled stimulation. This article introduces a technique for recording somatosensory event-related potentials (ERP) that uses a novel mechanical stimulation method involving skin deformation using a robotic device. Controlled deformation of the facial skin is used to modulate kinesthetic inputs through excitation of cutaneous mechanoreceptors. By combining somatosensory stimulation with electroencephalographic recording, somatosensory evoked responses can be successfully measured at the level of the cortex. Somatosensory stimulation can be combined with the stimulation of other sensory modalities to assess multisensory interactions. For speech, orofacial stimulation is combined with speech sound stimulation to assess the contribution of multi-sensory processing including the effects of timing differences. The ability to precisely control orofacial somatosensory stimulation during speech perception and speech production with ERP recording is an important tool that provides new insight into the neural organization and neural representations for speech.

This paper introduces a method for obtaining somatosensory event-related potentials following orofacial skin stretch stimulation. The current method can be used to evaluate the contribution of somatosensory afferents to both speech production and speech perception.

从口面皮肤牵拉刺激体感事件相关电位

Cortical processing associated with orofacial somatosensory function in speech has received limited experimental attention due to the difficulty of ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

M&amp;#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

激光诱导的斑马鱼视网膜变性及再生模型中的 m ü ller 胶质细胞活化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

跟踪果蝇幼虫行为对嗅神经元 Optogenetic 刺激的反应

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...