这项工作是在药物滥用研究所（NIDA）授予R01 DA24040（DCC），科罗拉多大学的创新种子格兰特（DCC）和NIDA的培训资助T32 DA017637（MVB）支持的研究所。

1。病毒贮藏和准备<ol><li>为提供视蛋白基因的啮齿动物的大脑使用无法复制的AAV载体已被批准用于生物安全1级（BSL-1），并要求在美国政府刊物，在生物安全微生物和生物医学实验室，可在概述了适当的保护和处理程序在疾病控制中心网站（ <a href="http://www.cdc.gov/biosafety/publications/bmbl5/index.htm" target="_blank">http://www.cdc.gov/biosafety/publications/bmbl5/index.htm</a> ）。 </li><li>为了避免反复冻融，吸管病毒从制造商的小瓶成更小的分装在II级生物安全柜，存放于-80℃冰箱。 </li><li>此前立体定向注入，让等分在冰上融化，然后简要降速与台式离心机。 </li><li>慢慢回填一个汉密尔顿玻璃注射器和针头用少量的有机硅或矿物油。确保没有明显的气泡在注射器针筒。将注射器插入一个微量泵直接安装在泵的垂直立体定位臂（ 即立体机械手）。 </li><li>降低立体定位臂，直到针的尖端接触到病毒的等分试样管的底部。使用控制器的微量泵撤回病毒的所希望的体积（1.5微升的1微升注射）。 </li><li>所有的材料和表面，它们与病毒接触，应进行净化与消毒，如含氯漂白剂（10％）。 </li></ol> 2。手术和病毒注射液<ol><li>麻醉动物用氯胺酮（大鼠，125毫克/千克，IP） - 甲苯噻嗪（大鼠，10毫克/千克，IP）的鸡尾酒。为了衡量麻醉效果，检查到脚趾捏反射反应，并根据需要给予补充剂量的氯胺酮。使用标准的无菌手术方法，将动物变成立体定位仪（大卫科夫在剂量率仪）。 </li><li>通过对动物头骨手术小剪刀或手术刀上方的皮肤作正中切口。轻轻分开结缔组织及清洁用小刮刀骨颅骨的顶部。 </li><li>验证动物的头是水平的，这样的Z坐标为前囟和lambda是相等的。确定立体坐标从一个脑图谱如鼠脑立体坐标 （乔治·旅游Paxinos和查尔斯·沃森，科学出版社，2007）或小鼠大脑中的立体坐标 （基思·北京·富兰克林和乔治·旅游Paxinos，学术出版社的目标脑区，2007）。标记注入的预期部位与手术的笔。 </li><li>仔细薄颅骨比使用一个手持电钻目标区域并且当钻头到达头骨底部停止。使用一对额外的细镊子，取出以露出硬脑膜变薄的骨头。 </li><li>定位注射器针头在目标是一，降低针，直到它触及硬脑膜，用这点来计算的z坐标。非常缓慢地降低注射针入脑，直到正确的Z位置为止。在这个协议中，内侧前额叶皮质的前度区域（2.7毫米前到前囟，±0.5毫米横向中线和-2.2毫米从硬脑膜）或背侧下脚（-6.0毫米到前囟，±3.0毫米横向中线和-2.0毫米硬脑膜）是针对一个成年SD雄性大鼠（约275克）中。 </li><li>注射病毒以0.1微升/分钟的速率。注射完成后，等待10分钟，抽出针头，以避免病毒的倒流到大脑表面之前。 </li><li>用缝纫机缝合用针连接，以密封在皮肤和应用的抗生素与伤口。 </li><li>的ChR2和NpHR从AAV载体功能性表达的时间过程将取决于实验设计和病毒滴度变化。在这个实验中， 在体内 </eM&gt;录音被进行18-21天注射后。 </li></ol> 3。光传输和在 ChR2的或NpHR表达神经元在体内录音<ol><li>连接的钨电极（〜1-1.5MΩ，微探针）使用强力胶的玻璃毛细管（Fisher科学）。 </li><li>使用光纤剥线工具（Thorlabs公司）公开一个多模光纤（直径200微米的核心，Thorlabs公司）和清洁光纤头与乙醇的裸结束。很轻易得分光纤头与一个楔形钻石刀（Thorlabs公司），并小心地除去多余的纤维（ 如用细镊子）。光纤应在比分轻​​松切割。 </li><li>光纤的FC端连接到一个200毫瓦单个二极管激光（输出端口上的PC连接器<a href="http://www.lumiphy.com" target="_blank">www.Lumiphy.com</a> ）与所期望的波长。确保与激光器工作时，合适的保护眼罩戴在任何时候。 </li><li>测量L阿塞尔强度在光纤的使用光功率计（尖端<a href="http://www.lumiphy.com/" target="_blank">www.Lumiphy.com</a> ）。 在体内录音，光照强度少则20-100毫瓦/毫米2能可靠地唤起神经元活动的ChR2或NpHR介导的激活或沉默，分别。 </li><li>插入所述光纤插入毛细管。定位在纤维末端的钨电极的尖端约500微米以上而配合的薄缝合线两次在尖端附近的几个点，使得所述电极和所述纤维是直的。确保电极的尖端和最近的结之间的距离足够长，用于插入到目标区域。 </li><li>准备动物如步骤2.1和2.2以上。使用初始开颅手术为指导，以使一个新的干净的小窗口，在颅骨的定位optrode（ <a href="http://www.lumiphy.com/" target="_blank">www.Lumiphy.com</a> ）。使在D小切口市建局使用细针。 </li><li>通过头骨的窗口和进入大脑小心地将optrode用显微操作（成茂）的转区。然后推进optrode慢慢在10-50微米的步骤，直到一个光响应性细胞的出现。 </li><li>自定义软件的用户界面（NeuroLux临<a href="http://www.lumiphy.com/" target="_blank">www.Lumiphy.com</a> ）LabVIEW编写的（美国国家仪器）是用来控制单个二极管激光器和可视化和记录持续的神经活动。记录的信号被捕获与ExAmp-20K（Kation Scientific）的放大器的带通滤波（0.3-8千赫），和National Instruments的模拟到数字板（NI USB-6009）。该NeuroLux专业用户界面允许模拟输入的选择（选择两个通道的神经元和TTL信号）和数字输出。用户可以定义采样率（20千赫），基线期（预光），和后期的光周期。用户可以定义中的TTL激光stimulati上的脉冲宽度，频率和刺激期间（如果频率是20赫兹和刺激期间是500毫秒，10的激光脉冲与定义脉冲宽度交付）。用户也可以选择连续光传输（ 例如 ， 图1）。为重复的记录，用户还可以定义脉冲串的数目和列车之间的间隔。数据可以有或没有记录到磁盘被收购。 </li><li>以下记录，将动物麻醉，用氯胺酮/赛拉嗪混合物和以验证optrode安置和视蛋白表达心脏灌注生理盐水和多聚甲醛（4％）。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

的技术的广泛可用于在啮齿类动物基因靶向微生物视蛋白基因离散的大脑区域。病毒基因传递提供了一种相对便宜和快速的方法来调解的ChR2和NpHR的表达与细胞类型特异性。 AAV载体系统是使用共同的选择中，由于高的生产滴度的存储，其缺乏致病性，并且其产生的长期基因表达的能力10中保持稳定光遗传学实验。许多视蛋白的结构与细胞类型特异性启动子是市售的各种载体从核心设施，如宾夕法尼亚大学（AAV血清型的<a href="http://www.med.upenn.edu/gtp/vectorcore" target="_blank">http://www.med.upenn.edu/gtp/vectorcore</a> ）或大学北卡罗莱纳州教堂山分校（ <a href="http://genetherapy.unc.edu" target="_blank">http://genetherapy.unc.edu</a> ）。一个缺点AAV技术是有限的封装能力（〜4.7 kb的），其中放置一个限制，可以用于细胞特异性靶向的转基因盒的大小。作为替代，慢病毒载体，其中有一个较大的包装能力，都能够容纳较大的启动子序列的控制下靶向视蛋白。 使用一个optrode的允许可靠的检测电生理信号与时间上精确的光传输相结合。如上所述，然而，充分注意是必要的，它的构造。因为在切割的光纤很脆弱，捆扎缝合线得太紧会引起纤维断裂。即使optrode可用于多个记录时，电极和光纤应清洗（或手动切割的光纤裸露端的情况下）插入之前，因为传送的光的电极和质量的阻抗将降低重复使用。 在元素ctrophysiological记录是很重要的，该optrode被缓慢推进。这里使用的optrode拥有约350微米的厚度（钨电极：125微米;光纤芯：200微米）和快速的降低有可能导致脑组织的运动。光致文物也可能被认为是特别录制和光传输调校11-12。尽管我们没有发现光致工件在我们的实验条件下，转导的脑区域之外的记录有可能被用作用于光的工件的控制。在记录过程中的另一个考虑是所需要的光强度用于观察变化的ChR2-或NpHR-表达的神经元，特别是对长持续时间的记录。强激光强度和长激光照射可能导致的组织损伤和/或减少响应于后续的输送轻12-13。对行为的实验中使用的控制病毒载体需要eliminatin克由于热量从光纤传送的任何影响。许多市售的光遗传学构建体的存在，其中视蛋白基因序列已被删除互补控制结构。 但应注意的是，设计的ChR2与改善的性能和动力学的变体已与其他沉默视蛋白可能更适合于特定的实验问题14-15开发的。例如， 通过靶向诱变建立了一个新的ChR2变体，称为“CHETA”，可以在频率驱动高保真神经扣球（高达至少200赫兹）高于原来的ChR2 14。 体内的光传输和同步记录的神经元反应的组合是在基因靶向细胞群和相应的时间锁定行为事件建立图形化的活动之间的因果关系的关键一步。的程序和公顷这里列出rdware提供一个简单的方法来实现光遗传学体内的头固定在录音啮齿动物。

在时间上精确的方式中的神经回路激活或沉默特定的细胞类型的能力是如何理解的神经回路处理不同类型的信息相关的情感和认知的关键。在完好的神经活性的实验控制已聘请loss-/gain-of-function工具（ 例如 ，电刺激，药物调节，损伤），不提供的，用于控制神经元的特定人群，无论是在时间或空间尺度选择性要求。直接应对这些技术挑战，基因可编码感光工具的开发和应用，使​​神经科学家在明确界定的行为事件来控制选择单元格类型的电活动。许多这些光敏蛋白是微生物来源的，具有光门控阳离子通道，channelrhodopsin-2（CHR 2）1，和光驱动氯泵，halorhodop罪（NpHR）2,3，在广泛使用。 光遗传学的主要优点是在异构的大脑区域的能力，基因靶标特异性细胞群和技术已成功应用于多个模式生物（无脊椎动物到灵长类动物）4-6。许多光遗传学转基因动物已生成和是市售的7,8，但是建立转基因系可以是劳动密集型的，成本高昂。在这里，我们描述了协议的的ChR2和NpHR基因下使用重组腺相关病毒（AAV）载体大鼠前度皮质和背下托的CaMKIIα启动病毒介导的递送。内的前脑，CaMKIIα表达是独家谷氨酸锥体神经元9。 AAV是常用的基础研究，由于其相对容易地生产和缺乏致病性的，以及在强和PERSI已实现了与这些载体支架10的转基因表达。此外，我们概述的步骤和硬件麻醉头部固定大鼠同时光传输和记录。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Syringe </td>
 <td>Hamilton</td>
 <td>7653-01</td>
 <td>10 &mu;l</td>
 </tr>
 <tr>
 <td>Removable Needle</td>
 <td>Hamilton</td>
 <td>7803-03</td>
 <td>31 gauge, beveled tip</td>
 </tr>
 <tr>
 <td>Microinjector Pump</td>
 <td>World Precision Instruments</td>
 <td>UMP3</td>
 <td></td>
 </tr>
 <tr>
 <td>Pump Controller</td>
 <td>World Precision Instruments</td>
 <td>SYS-MICRO4</td>
 <td></td>
 </tr>
 <tr>
 <td>Silicone Oil</td>
 <td>Alfa Aesar</td>
 <td>A12728</td>
 <td></td>
 </tr>
 <tr>
 <td>Laser Protective Eyeware</td>
 <td>Kentek </td>
 <td>KMT-4501</td>
 <td></td>
 </tr>
 <tr>
 <td>Multimode Optical Fiber</td>
 <td>Thorlabs</td>
 <td>BFL37-200</td>
 <td>200 &mu;m diameter core, 0.37 NA</td>
 </tr>
 <tr>
 <td>Fiber Stripping Tool</td>
 <td>Thorlabs</td>
 <td>T12S21</td>
 <td>for 200 &mu;m diameter core multimode fiber</td>
 </tr>
 <tr>
 <td>Optical Power Meter</td>
 <td>Lumiphy LLC</td>
 <td><a href="http://www.lumiphy.com/" target="_blank">www.lumiphy.com</a></td>
 <td></td>
 </tr>
 <tr>
 <td>Photodetector</td>
 <td>Newport</td>
 <td>818-SL/DB</td>
 <td></td>
 </tr>
 <tr>
 <td>Blue Laser (445-473 nm, 100-200 mW)</td>
 <td>Lumiphy LLC</td>
 <td><a href="http://www.lumiphy.com" target="_blank">www.lumiphy.com</a></td>
 <td>coupled to a 200 &mu;m multimode fiber with FC/PC adapter </td>
 </tr>
 <tr>
 <td>Green Laser (532 nm, 100-200 mW)</td>
 <td>Lumiphy LLC</td>
 <td><a href="http://www.lumiphy.com" target="_blank">www.lumiphy.com</a></td>
 <td>coupled to a 200 &mu;m multimode fiber with FC/PC adapter </td>
 </tr>
 <tr>
 <td>Tungsten/Fiber Optrode</td>
 <td>Lumiphy LLC</td>
 <td><a href="http://www.lumiphy.com/" target="_blank">www.lumiphy.com</a></td>
 <td>Lumitrode</td>
 </tr>
 <tr>
 <td>Glass Capillary Tube</td>
 <td>Fisher Scientific</td>
 <td>22-362-566</td>
 <td></td>
 </tr>
 <tr>
 <td>Hydraulic Micromanipulator</td>
 <td>Narishige</td>
 <td>MO-22</td>
 <td></td>
 </tr>
 <tr>
 <td>Amplifier</td>
 <td>Kation Scientific</td>
 <td>ExAmp-20K</td>
 <td></td>
 </tr>
 <tr>
 <td>Data Acquistion Device</td>
 <td>National Instruments</td>
 <td>NI USB-6009</td>
 <td></td>
 </tr>
 <tr>
 <td>Rodent Head Restraint for Recording</td>
 <td>Lumiphy LLC</td>
 <td><a href="http://www.lumiphy.com/" target="_blank">www.lumiphy.com</a></td>
 <td></td>
 </tr>
 <tr>
 <td>Small Animal Stereotaxic Unit</td>
 <td>David Kopf Instruments</td>
 <td>Model 963</td>
 <td></td>
 </tr>
 </tbody>
</table>

a method for high fidelity optogenetic control of individual pyramidal neurons in vivo

光遗传学方法已经成为一个强大的工具，阐明神经回路活动的基础在广泛种类的多样化行为。微生物来源的光遗传学工具由光敏膜蛋白质，是能够激活（ 例如 ，channelrhodopsin-2，CHR 2）或沉默（ 例如 ，halorhodopsin，NpHR）以上行为上相关的时间尺度神经活动ingenetically定义的细胞类型。我们首先证明的腺相关病毒介导的交付的ChR2和NpHR转基因的背下托和大鼠前额叶皮层的前度区域的简单方法。因为的ChR2和NpHR的基因靶向，我们描述了使用这种技术来控制神经元（ 即锥体神经元）的嵌入式异构组织高时间精度特定人群的电活动。我们这里所描述的硬件，软件定制的用户界面，和程序，以便从转导的锥体神经元同步光递送和电记录在麻醉的体内制备。这些光响应性工具提供了这个机会识别不同类型的细胞，以信息处理和行为的因果贡献。

图1描述了用于同时记录神经元的活动，并控制光脉冲参数（ 如脉冲频率，脉冲宽度，刺激期）定制软件（NeuroLux专业版）在LabVIEW环境下开发的（美国国家仪器公司）的屏幕截图。该软件程序发送的命令信号，以产生TTL脉冲在单个二极管激光器控制一个数字采集装置。此外，该软件程序显示和存储记录的神经元的活动，并发表TTL信号。这些信号是通过在确定的采样率（ 例如 ，20千赫）的采集装置数字化。记录的信号被保存为一个二进制文件中的二维双精度数组。 图2显示了自定义optrode由连接到光纤的钨电极。的光纤是用薄的缝合线在3固定在电极点。如果需要的话，超级胶可用于附着它们。然而，避免永久地固定光纤的电极，因为虽然电极可以通过在纤维被重复使用几次，光传输将降低用重复使用，而且应该被切割和清洗或与每一个插入到大脑取代。 图3展示了荧光增强的黄色荧光蛋白的图像的左侧面板，显示实际的ChR2和NpHR表达。这些照片显示了在大鼠背下托代表NpHR表达（ 图3A）和前度皮质的ChR2表达（ 图3B）。病毒表达被限制主要是为了背下托和前度皮质（ 如一些荧光在齿状回观测到（ 图3A，左））。也观察到电极（薄轨道，箭头）和光纤（粗跟踪，箭头）的曲目。 图4的S =“jove_content”&gt;左面板显示的ChR2诱导大鼠皮质前度在时间上精确的尖峰。 图4A是CHR 2 -触发的动作电位响应于10毫秒的蓝色光脉冲，20赫兹递送的示例跟踪对大鼠前度皮质。光栅图 （ 图4B）示出了在6代表神经元的ChR2诱导的活化。所有记录的神经元表现出光诱发扣球与完美的保真度。在对比的ChR2，NpHR快速和可逆地压制在体内对大鼠皮质前度（ 图4的右侧）的自发活动。 图4C是示例跟踪显示该前度皮质表达NpHR下的连续532nm的光照（10次） CaMKllα促进消除体内自发单单位活动。需要注意的是前度锥体细胞活性的完整的沉默是时间锁定在10秒连续光传送。低呃光栅图 （ 图4D）示出了在6代表神经元NpHR诱导的沉默。 在体内的录音是典型的成年Sprague-Dawley大鼠获得的，其中的微生物视蛋白基因的AAV-介导的递送发生前18-21天。 图5示出了鼠前度锥体神经元的重复的ChR2刺激的结果。蓝色的光脉冲（10毫秒）交付在20赫兹，持续5秒（100个脉冲总数）。光诱发尖峰电压的痕迹2小时录音会议（61次重复总）期间多次获得每2分钟。使用这种重复刺激协议即使当，ChR2的诱导稳定和健壮扣球响应于所述光传送。 图6和图7显示NpHR引起的光抑制和大鼠subicular神经元活动的ChR2的驱动光敏化的结果。既然是这样的前度皮质，NpHR和ChR2的能介导的光诱导时间锁定的抑制和活化的视蛋白转导subicular神经元具有高再现性。 <img alt="图1" src="/files/ftp_upload/50291/50291fig1.jpg" /> 图1。截图同时进行光传输和电生理记录的NeuroLux Pro软件界面 。图为跟踪显示大鼠前度锥体细胞的响应10秒的连续532 nm的光传输的自发活动NpHR诱导的沉默。 <a href="https://www.jove.com/files/ftp_upload/50291/50291fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/50291/50291fig2.jpg" /> 图2。定制optrode高功率形象 。的光纤被插入到连接到钨电极，并使用缝合线固定到电极的玻璃毛细管。的中心到中心的距离本月与EEN电极尖端与纤维末端大约为500微米。 <img alt="图3" src="/files/ftp_upload/50291/50291fig3.jpg" /> 图3。病毒表达和optrode位置 。 （左）的照片显示在背下托代表NpHR表达式（A）和前度皮层（B）的ChR2表达。 （右）示意图，它表示每个照片的拍摄位置。在每张照片的箭头和箭头表示的钨电极的位置和光纤，分别为。 SUB：下托，DG：齿状回，PL：前度皮质<a href="https://www.jove.com/files/ftp_upload/50291/50291fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" src="/files/ftp_upload/50291/50291fig4.jpg" /> 图4， 在体内从的ChR2或NpHR转导的大鼠prelimb电生理记录IC皮层。 （ 一 ）ChR2的触发动作电位响应蓝色（473 nm）的光脉冲（10毫秒，蓝条）的20赫兹交付示例跟踪。 （二）光栅图，显示在六个代表神经元的ChR2引起的尖峰。每单位活性被绘制为一个点。 （ 三）自发活动期间连续绿光（532 nm）的光照射（10秒，绿巴）NpHR诱导抑制的例子痕迹。 （四）光栅图，显示在六个代表神经元NpHR诱导的沉默。每个单元活动被绘制成一个点。 <a href="https://www.jove.com/files/ftp_upload/50291/50291fig4large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图5" src="/files/ftp_upload/50291/50291fig5.jpg" /> 图5。 在体内大鼠前度锥体神经元的重复20赫兹的ChR2驱动的扣球 。 （A）电压的光诱发扣球的痕迹在时间点0，30，60，90购入，录音开始后120分钟。 （二）光栅图，显示所有的61次重复的光诱导活化的（2分钟试验间隔，120分钟总）。每个单元活动被绘制成一个点。 <a href="https://www.jove.com/files/ftp_upload/50291/50291fig5large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图6" src="/files/ftp_upload/50291/50291fig6.jpg" /> 图6。 在体内从NpHR转导的大鼠背下托电生理记录 。 （ 一 ）范例跟踪显示，表达NpHR背下托的10秒连续532 nm的光照（绿色条）消除自发活动。两个记录的单位从上面的跟踪（B）的平均波形。振幅的阈值被用于识别两种不同的神经元。 （三）光栅图，显示5重复S这些单位NpHR诱导沉默。每单位活性被绘制为一个点。 <img alt="图7" src="/files/ftp_upload/50291/50291fig7.jpg" /> 图7。 在体内大鼠背subicular神经元重复20赫兹CR2驱动的扣球 。 （A）电压的光诱发的时间点0，30，扣球收购的痕迹60，90，录音开始后120分钟。插图：该小区的典型爆发活动。 （二）光栅图，显示所有的61次重复的光诱导活化的（2分钟试验间隔，120分钟总）。每个单元活动被绘制成一个点。 <a href="https://www.jove.com/files/ftp_upload/50291/50291fig7large.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo at JoVE.com

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

神经科学的工具，结合遗传学和光学的最新发展，被称为“光遗传学”，使过度的神经回路活动控制与空间和时间分辨率的一个前所未有的高度。在这里，我们提供了一个协议，用于在体内录音与集成光遗传学操作的前额叶皮层和subicular锥体神经元的基因定义的子集。

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 ...

a-method-for-high-fidelity-optogenetic-control-individual-pyramidal

Research

JoVE Journal

Neuroscience

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

18.9K 次观看.  University of Colorado Boulder. 神经科学的工具，结合遗传学和光学的最新发展，被称为“光遗传学”，使过度的神经回路活动控制与空间和时间分辨率的一个前所未有的高度。在这里，我们提供了一个协议，用于在体内录音与集成光遗传学操作的前额叶皮层和subicular锥体神经元的基因定义的子集。 

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

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

科研

神经科学

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 ...

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 ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

在对小鼠坐骨神经体内的电测量

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

在脑片恐惧电路体外光遗传学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

的简化方法超低密度，长期主要海马神经元文化

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

比率钙显像对单个神经元的行为线虫线虫

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.