These studies were funded by grants received from the NIH (MH082876, DA023988).

！注意：此协议涉及使用3B类激光器，并需要适当的培训和安全准则得到遵守。护目镜必须在任何时候运行激光器的时候穿，用对齐程序提出特别高的风险。接触激光提供商以确定眼镜，将对于给定的激光提供最大的衰减。如果有的话，报名参加的机构的激光安全培训课程。切勿操作激光没有适当的安全眼镜和培训。 1.激光装置的建立在适当情况下，在第1步骤被指定为（A）或（B）中，以单或双激光系统之间，分别区分。 <ol><li>安装并固定激光器的线路板。实验板是优良的热导体与作为散热器，以防止损坏内部激光元件长时间使用。 </li><li> （A）固定预连接拉斯呃到10“×12”面包板（或必须）使用¼-20“帽螺丝和垫圈（ 图2A）。如果面包板孔不与激光安装孔对齐，用小&#39;表夹“，以确保激光面包板上。 </li><li> （b）如果要使用的2激光器具有非常不同的光束的高度（&gt;约1厘米），用小4“×6”面包板创建平台激光器之一。这些附加电路板的主要大12“×18”采用¼-20“帽螺丝垫圈线路板，那么激光连接到使用盖螺丝或夹子表的小板， 如图2B。直接连接其他激光用螺丝帽或可变高度表夹在面包板上。 关键的一步：面包板，螺钉和光学元件可以购买英制或公制所以在购买物品的时候是一致的;默认此协议是帝王。 </li><li> （A）附加一个厚夹套平面裂开/物理接触（FC / PC）跳线到耦合器（称为成色帘线; 见图3），其物理地附接到激光（ 图2A）的前面。 </li><li> （B）的螺纹联接到3/4“光后，再环氧耦合器和使用JB佳柏，或类似的环氧柱的顶端之间的连接，以防止在使用过程中松动和错位。连接后的线路板（如线路板孔不会总是排队与光学元件，柱座，底座适配器所需的位置，并且夹紧叉是用来固定连接器的光学职位地方）。附加一个厚夹套接插线（耦合线）到耦合器的背面。 </li><li> （B） 插入第一个转向镜的蓝色激光进运动持有者，它使用的是¾“光学后连接到线路板。这个帖子附加到一个ADAP基地器和夹紧叉。定位夹紧叉并直接在蓝色激光的前支柱组件与反射镜成45°引导朝向分色镜的激光。用孔的格子图案的线路板作为一个粗略的定位导向。一旦粗略定位，用1/4“-20螺栓固定夹紧叉线路板（ 见图2B，C）。 </li><li> （B） 将分色镜成运动支架并将其连接到一个1“光后，直接固定到线路板。分色镜定位到最左侧的，和在同，蓝色激光镜像线。角的分色镜以45度角，使得蓝色光反射离开第一反射镜被反射到耦合器，然后拧紧螺钉的运动保持器附接至后（参见图2B，C）。 </li><li> （B）将第一转向镜黄色激光3/4“光学职。附加OPTICAL后到基本适配器和夹紧叉。定位夹紧叉，并直接在黄色激光和角镜的前面的后合奏以45°角，使得黄色光将朝向第二转向反射镜被导向。固定夹紧叉的地方与1/4“-20带帽螺丝和垫圈。 </li><li> （B）将第二转向镜黄色激光1“光学职。角反射镜，使得黄色光束从第一反射镜将通过分色并进入耦合器（ 图2C）被反射。直接固定岗位的线路板，拧紧固定螺丝，一旦镜角度的恰如其分。细镜调整将使用可调镜座在后面的步骤制备。 </li><li> （B）将中性密度滤镜轮到¾“光后和后放入连接到安装底座柱座。确保合奏的第一和本身之间的线路板COND黄色反光镜使用的是单一的¼“-20螺栓。这轮是用来调整的黄色激光到达耦合器中的功率。 提示：它们连接到基座前，单独收紧所有组件（如持有可调镜持有人的职位顶部的螺丝，并保持基本适配器的帖子底部的线程）。用小六角扳手的轴在所提供的通孔中的光职位以获得足够的扭矩。这将防止部件松动在使用过程中，因此需要调整。 </li><li>附加的FC / PC到FC / PC L型支架适配器的线路板。 </li><li>可选：直接固定1×2 50/50迷你立方体光纤分路器的线路板可同时在体内刺激两个或两个以上的动物。此外，手柄可被添加到辅助电路试验板组件的运动（如在图2A中看到的那样）。 </li></ol> 2.激光耦合（非接触式联轴器） 本节中涉及到双激光设置（ 图2B）。调整外黄激光路径之前对准内部的蓝色激光的路径。 ！注意：使用低光功率耦合（〜1毫瓦），以保证眼睛的安全。穿在激光和直到光强度被测量并且被认为是安全的防护眼镜到电源。 <ol><li>上设置激光的背面的开关，以“电流”（电流）和晶体管 - 晶体管逻辑模式（TTL）+为恒定的照明（相对于模拟模式）。确保在驾驶员的前方的电源按钮被设定为零。通过接通该驱动程序，然后再在激光键开启激光器。 </li><li>缓慢调节位于所述激光驱动器，使得〜1毫瓦的激光光被发射的前面的电源按钮。等待10 - 15分钟（或制造商指定的）激光预热。 </li><li>直接连接光缆测试仪的FRE耦合器尾纤电子端打开电缆测试仪（ 图3A）。调节耦合器的角度，使红色光束行进直背朝向分色镜的中心。红光从电缆测试器发射的光的光束路径是输入激光光将需要以跟随要被连接到激光器的准确路径。 </li><li>执行粗对准：使用横向和水平旋钮上的运动反射镜引导激光的光束进入耦合器。基座夹具可能需要被松开以略微重新定位反射镜和耦合器。运动坐骑应该还是有可用于进一步微调一些旅游。不要担心，如果没有蓝光在这个时候被排放出来的耦合器，连接跳线。 </li><li>放置一个单件半透明纸直接在二向色镜的前方，在二向色和耦合器之间。会有两个蓝色和ar编点对本文从激光和电缆测试仪，分别。使用的纸张是半透明足以同时从纸张的同一侧同时看到红色和蓝色斑点。 </li><li>进行微调，以第一转向反射镜（ 即，一个接近激光，而不是分色）通过仔细调节的横向和水平旋钮对准红点与蓝点的中心。 </li><li>移动纸张背面朝向耦合器，以便它是直接在耦合器的前面，并调整旋钮上的第二 （ 即，二向色）镜对准激光光束的红色光束。 </li><li>迭代步骤2.6＆2.7直至蓝色/黄色和红色光束的中心在两个位置被精确地对准（ 即，直到红色和蓝色光束是共线的）。 </li><li>卸下电缆测试仪从耦合线。激光光线现在应该从耦合器接插线的末端射出。 </li><li> Determi通过测量光功率从使用功率计成色接插线的光纤尖端发射ね耦合效率。使用在功率计上的光电二极管500毫瓦的设置和改变的波长设定（λ）为蓝色（473纳米）或取决于激光黄色（635 nm）的光谱的光被使用。 </li><li>放置光纤尖端沿垂直于光电二极管，以获得功率读数。比较光功率输入耦合到光功率从光纤末端射出。 &gt; 80％的耦合效率被认为是非常不错的。第二转向镜非常小的进一步调整，有时略有改善耦合。在一般情况下，当从耦合光纤的末端的波束图案是一个小的，紧，中央点（无环周围），耦合效率进入纤维芯是最佳的。 </li><li>重复步骤2.1 - 2.11黄激光耦合，除了使用两个导引反射镜为黄色激光（参见图2C）。没有做吨调整分色镜或蓝色激光的取向的位置将被丢失。 </li></ol> 3. 在体内光遗传学刺激确保涉及动物使用的任何程序都符合当地和国家的方针由相应的机构动物护理和使用委员会进行批准。 <ol><li>光纤设置（ 见图3B用于识别不同类型的接插线的下文）。激发一个单一的鼠标，使用直接连接到线路板的FC / FC L型托架适配器耦合接插线连接到一个厚夹套接插线（参见图4A）。刺激两种动物从一个激光器，使用1×2 50/50的微型立方体成色接插线连接两个厚夹套接插线（参见图4B）。为了刺激三个或更多的动物，附加连接器电源线使用1×2米的多模光纤分路器INI立方体已经连接到线路板（ 见图4C）。 </li><li>附加一个换向器/旋转接头的厚夹套接插线/纤维分离器的自由端。换向器是必不可少的，因为它们允许与啮齿动物的运动，从而防止转矩的积累在接插线的光纤的旋转。过多的扭矩可以捻帘线，导致断裂，而在测试期间与动物的自然运动干涉。 </li><li>附加动物跳线到换向器。 </li><li>附加的连接分离套筒动物跳线（ 图5）的自由金属套圈结束。不要强行套一路攀升套圈;离开〜0.5厘米套筒的外露因为这是连接到植入的光纤固定到动物（ 图6）。 关键的步骤：请购买包含分裂，使连接在胀紧套在植入光纤插芯的袖子，去除。过紧一个合适的能引起严重的创伤给动物如果从头骨试图断开从植入物的套筒，当植入物被驱逐出来。如果这确实发生，动物应该从研究中删除，并立即得到兽医照顾。同样地，利用第一次的新套筒之前，通过插拔的套圈，直到它与力的所需量的断开“打破它在&#39;。 提示：这是很容易折断的纤维，同时除去被紧紧附连到箍圈的套筒。为了避免这种情况， 推动套圈通过插入一个小木棒插入套管（标准棉签的手柄大小合适）的开口端。 </li><li>蓝色激光驱动器连接到使用BNC电缆脉冲发生器和打开脉冲发生器。 </li><li>穿戴合适的护目镜。上设置激光到“电流”和“TTL +”模式的背面的开关。确保塔吨对驾驶员的前面的电源按钮被设定在零andturn上的激光（打开第一驱动程序，然后该激光键）。 </li><li>调整激光的前面的电源按钮，使5 - 10毫瓦正在从动物跳线光纤尖端作为使用光功率计测量发射的光。 5 - 10毫瓦是一般准则-影响组织的一个给定的体积，应先于实验开始计算，如在Aravanis 等所需的具体功率强度3。 </li><li>切换蓝色激光来“模拟”模式在体内的刺激。注：黄色DPSS激光器在TTL +模式恒定的照度操作。等待10-15分钟的激光预热。 </li><li>轻轻抑制鼠标和连接分割套筒形式的动物用接插线的慢性植入纤维（参见图6）。确保两个光纤的端部使身体彼此接触。使用分割上的连接套筒作为窗口的可视化，两者之间的直接接触关键的一步：有时碎片可以收集关于动物的植入纤维的金属制套圈，干扰的正确连接。在这种情况下，使用乙醇擦拭，轻轻地清洁的动物的头套圈之前附件。切勿强行将连接套筒在套圈，因为这可能会导致严重创伤的动物。如果纤维端部之间的物理连接不能在清洗后进行，从研究中除去的动物。 提示：光泄漏可以发生在植入纤维和动物跳线之间的连接点。这种光的老鼠可视化可能会出现一个实验变乱10。热缩管可连接到接插线和滑过的连接点，以减少外来光。 </li><li>让鼠标恢复为以前的行为测试开始几分钟。 提示：Depending在行为测试到给药，最好是2，观察者只小鼠到连接和束缚方法 - 先有3天，根据需要的处理以连接该动物可能诱发应力，颠倒行为测试。 </li><li>将鼠标在行为测试设备，确保连接线是免费的碰壁的。从来没有刺激时留下的动物无人值守。即使使用换向器，跳线确实有一种倾向，在延长的时间段，以扭曲和与行为测试可能会干扰。 </li><li>使用脉冲发生器脉冲蓝色激光以预定的频率，这将激活选择的视蛋白。用于黄色激光用途：脉冲黄色激光与外部百叶窗或通过简单地阻挡的光束路径用不透明的，非反射性的，不易燃的对象。 </li></ol> 4.后在体内刺激的注意事项此部分不打算成为一个完整的原山坳但提供作为该应下在体内光遗传学刺激被认为是额外的程序指导。 <ol><li>在实验完成后，确认病毒和纤维铺放组织学的行为结果准确解释。根据机构指南安乐死的动物和灌注用冰冷的磷酸盐缓冲盐水（PBS）和4％（重量/体积）低聚甲醛在PBS中的动物。 </li><li>牢牢抓住外露的金属套圈用钳子止血或删除植入的光纤。在一个平稳，但迅速，运动拉起。它通过测量光输出在每个实验结束时，测试植入纤维的完整性是重要的。 </li><li>通过感兴趣区域切片之前48小时 - 在多聚甲醛后固定的大脑至少24。 （如果使用冷冻切片，切片之前孵育大脑中有30％的蔗糖溶液对数天）。使用standa进行免疫组化次协议用 ​​于检测适当视蛋白标记的荧光团， 即，绿色荧光蛋白（GFP），增强的黄色荧光蛋白（EYFP）或mCherry。 </li><li>查现场视蛋白的表达和纤维植入物的显微镜下目视确认病毒注射和植入物的基础上选择的坐标的合适的位置。 </li></ol>

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The authors have no conflicts of interest to report.

当前述激光调校和圈养策略是具有广泛的啮齿动物行为测试兼容。的确，各种行为测试已经使用以下，或伴随， 在体内光遗传学刺激，包括情绪行为任务，行为调节，学习和记忆范式，睡眠，觉醒，和食欲任务仅举几（参见聂等人。 6全面检讨）。光遗传学改变了传统的行为测试在多天的研究进行的方式，现在可以浓缩成一个会话中的行为相比，主体内“开”与“关”，在光不同的时期5。值得注意的是，行为装置包含门道，封闭室或其他障碍物可能必须进行修改，以适应系留纤维的通道。 所描述的网络共享策略许可证SI多个小鼠从单个激光multaneous刺激。高通量光遗传学行为测试，因此可以通过使用多个激光器和检测设备的实现。可以同时刺激动物的数量，但是，将被由最大光功率，可以在每根光纤前端来实现的限制。最大输出功率在光纤尖端取决于激光器的1）起始功率; 2）的耦合效率和3）数目光束分割的。对于一个100毫瓦的蓝色激光器以〜80％ ​​的耦合效率和最多4个光束分裂（如在图4C中示出），在光纤尖端的平均功率使用200微米芯，0.22 NA光纤接插线时5-10毫瓦之间的范围可以（NB期望从旋转接头的传输损耗为&lt;15％）。测量的光输出在光纤尖端是用于确定适当的光功率为视蛋白活化必需的，因为视蛋白在它们的光，因此，光功率密度的敏感性不同（毫瓦个 / mm 2）所需的活化11。例如，稳定的阶梯函数视蛋白（SSFO）充当一个光子累加器，因此需要用于激活非常少的光功率密度（&lt;8μW/毫米2）8。与此相比，传统的信道的视紫红质（的ChR2），需要一个最小的光引发动作电位的1毫瓦/毫米2 2。 表1提供了作为用于当前激活最常见视蛋白中需要已知最小光辐射能的快速参考使用。最后，必须考虑该光散射和吸收，因为它通过脑组织行进，使得更多的光功率所需的更深的大脑结构3。一个有用的在线资源提供的<a href="http://www.stanford.edu/group/dlab/cgi-bin/graph/chart.php">http://www.stanford.edu/group/dlab/cgi-bin/graph/chart.php</a> ，将计算的光强度在通过脑组织的不同深度通过考虑占该纤维芯的尺寸，数值孔径，光的波长使用，并且开始光功率在光纤尖端。对于这些计算背后的理论原则的很好的概述，请参阅Foutz 等 。 （2012）12。如何应用这些原理和计算，以实验设计实施例中演示Aravanis 等人。（2007）3和塔伊等人。（2012）13。前一个实验开始执行这些计算是至关重要的，以确保充分的光的辐照度为视蛋白活化。鉴于这些考虑，有利的是，购买供电更高的激光器，以确保有足够的功率输出。激光器100-200毫瓦之间的功率输出通常是足够的，以补偿小芯光纤，多纤维分裂，耦合效率低和传输损失7。如果使用高功率激光器，但是，必须小心，以避免神经损伤或热和光准可能发生的长期和/或高功率照明7天的文物。一个安全范围的体内实验是高达75毫瓦/毫米2。14 决定激光的购买类型可以是一件复杂的事情，因为有很多因素需要考虑。例如，直接二极管激光器提供更稳定的和可重复的脉冲式输出比二极管泵浦固态（DPSS）激光器，并随着时间的推移在实验室环境中更加可靠。在某些情况下，但是，直接二极管激光器可以发射一个较低光功率，〜0.1毫瓦，即使当命令电压为0V，由于以恒定的偏置电流被发送到二极管通过激光的控制电子设备。这个“自发的”发射具有更广谱比不从相同的激光发射的激光，所以可以通过安装在激光和光耦合器之间的窄的带通（或“清理”）过滤器（见零件清单）可以具体地降低。该过滤器还将激射时减少功率输出由〜50％，所以相应地购买供电更高的激光。应当指出的是黄色DPSS激光器是极其敏感的，并且可以运行不稳定并具有降低的寿命，如果迅速由脉冲发生器调制。黄激光功率的调整应该通过放置在光束路径（ 第1.7节 ），而操作激光TTL +模式下使用外部密度滤光片轮来完成。此外，购买绿色532纳米DPSS激光器是一种具有成本效益的替代方案，可以同时激活halorhodopsins和archaerhodopsins。 光纤的数值孔径（NA）是很重要的设计和购买光纤部件为激光组件设置时要考虑的问题。光纤的NA决定了可以被接受，并在纤维的端发出的光线的角度。如果需要更高NA的纤维配合到一个较低的NA纤维，将发生显著损失在那个接口，所以要一致无线是很重要的<html>个光纤的NA一次装夹中（或确保NA增大时沿着光路径）。纤维的NA的脑组织照射的体积上的效果是不那么重要，因为脑组织是高度散射，并且由于耦合来自激光源的光将趋向于“底部填充”高NA纤维;然而光纤带的0.22和0.37的NA是常用。同样地，从较大的芯到一个较小的芯光纤耦合也将导致在显著的损失，所以始终确保从激光源向动物植入进展时使用增加或等于芯直径。在一个一般的笔记，纤维端部应始终封在不使用时，以防止灰尘和颗粒积聚。这是一个好主意，定期清洁光纤末端和连接器（70％异丙醇效果很好），以确保最大的光输出功率，并开始每一天的实验，之前测试的光输出功率通过“虚拟植入”。 “&gt;在行为测试，当务之急是采取以控制病毒感染，外源蛋白的表达，可见光，并在动物的行为可能组织加热效果和伪像的影响的步骤，因此，适当的对照组应该包括动物转导的对照病毒（ 例如，绿色荧光蛋白，EYFP，mCherry）接收相同的轻刺激参数。实验验证是一个关键的最后步骤为用于分析的行为数据是完全依赖于适当的视蛋白和光纤放置在感兴趣的区域具体而言，在没有检测到免疫动物的信号，或在信号或光纤的位置并不在感兴趣的区域，然后行为数据为该动物应该从实验中删除​​。另外，有必要在测试光输出前两种手术植入，并再次验尸光纤头，以确保有足够的光功率的视蛋白的激活，在灵魂LS其中已经发生严重的光损耗通过光纤试验后（&gt; 30％）9，对于动物数据应被视为除去。去除标准应建立一个先验的 。最后，必须考虑以调节神经放电所需的脉冲频率，这将取决于大脑结构和神经元的子类型的有针对性。出版光遗传学光刺激参数为多个神经元的子类型，但是，以调节神经放电的能力应该被独立地通过在体内或脑切片电生理记录证实存在。 正如一个熟练的变激光的使用和激光调校改性，不同波长的组合可被拴到多个纤维在单个动物或递送向下同一纤维组合光遗传学8。多波长刺激会变得越来越重要，因为对红移的快速发展编辑channelrhodopsins 8，蓝移极化视蛋白15的工程，采用双稳态阶跃函数视蛋白8,16,17，并视蛋白具有独特的激活谱11的一般扩展列表。这种扩张的光遗传学工具箱将允许前所未有的控制多个神经子类型在国内和跨越大脑区域，以确定其在管理复杂的行为状态的作用。

光遗传学发生了革命性的系统级神经科学在其搜索的神经电路元件的驱动正常和疾病相关的行为状态。该光敏感微生物视蛋白1可以功能性地表达在哺乳动物细胞中的发现提供了平台，利用光来获得前所未有的控制神经活动的高空间和时间精度2。不同于传统的电生理学或药理学途径的神经活动的操纵，光遗传学允许特定类型的细胞内异质种群和在生理学相关的时间尺度的控制（基于遗传鉴定或空间投影）。随后引入神经 ​​光接口提供交付光的实用工具，动物行为3。这使得在苏醒做人啮齿类定义神经回路的实时调制以因果测试在相关的神经和精神疾病4-6行为状态的治理，这些神经回路的作用。光遗传学，因此，代表一个强大的工具用于引入兴趣调查大脑活动和行为或生理措施在动物模型之间的函数关系的任何实验室。 成功的设计是光遗传学实验和完成涉及的各个步骤和注意事项（ 见图1）。当前协议的目的是为个人提供的工具和组件，随着理论和实践知识，要清醒做人啮齿类动物进行光遗传学刺激。目前，有用于激活微生物视蛋白通道两种主要波长范围：在蓝色光谱（通常473纳米）和黄绿色光谱（通常532或591纳米）。两个激光器和发光二极管（LED），可以用作光源至deliver特定波长的光到脑组织。非相干光由LED发射，然而，这使得有效传输的光难以耦合成所需的体内啮齿动物的刺激小芯光纤时。决定适当的激光组件是一个关键的第一步和将取决于在实验室的预期用途的光遗传学。当前协议描述了两个基本的配置，不同之处它们易于组装和使用的：单个预耦合激光器和双激光系统（ 见图2）。被预连接制造商的单激光系统基本上都是现成的，去的到来与很少或几乎不需要的建立后，但有最小的最终用户定制的缺点。一种双激光系统使得递送两种不同波长下的相同的纤维。这将成为与组合光遗传学的出现越来越重要，由此不同的波长可用于激活/抑制distinc包含空间共定位吨的细胞类型。这也是与双稳态阶梯函数视蛋白，其中的光电流是发起并分别7,8-由蓝光和黄光终止使用是必不可少的。双激光系统也可定制的用户可以根据需要添加或删除的光路组件（ 例如，外窗，梁过滤器，馈线电源米）。由于它的多功能性，双激光设置，建议如果光遗传学将是在实验室中使用的持续的工具。激光器的耦合，但是，可以是一个挑战，因此快速，简便和可靠的联接机构在该协议提供的。注意，该协议细节的光学部件的组装，并利用被用于阶跃折射率多模光纤的优化具有200微米的芯和0.22的数值孔径（NA）的接插线和组件。不同的核心尺寸和NA可以购买，但是所有的组件应该在核心方面理想匹配大小和NA，以避免光损耗光纤连接点。另外，在光纤连接，光可以通过从一个比较小的一个较大的磁芯尺寸;和/或从一个较低的NA到更高的NA纤维，无需额外的损失。 提供束缚策略允许多个老鼠高通量行为测试的同时刺激。所提供的协议假定使用慢性植入纤维行为测试，但是可以修改为急性刺激协议。急性植入纤维是有利的，用于组合光遗传学刺激药理操纵，因为相同的套管可用于递送药物和光纤的同一位置的小费。使用长期植入的纤维是比较，但是，强烈建议对多个天行为测试，因为它减少了与纤维的重复插入和取出相关的组织损伤和在纤维的一致布置方​​面可以提高精度组织照明3。当与这里所描述的束缚结构结合时，行为可以可靠地跨多个天被记录。纤维植入后确实，可靠的透光报道9月这样的慢性刺激和行为测试范例可以，理论上，进行跨多个几天和几周。上的硬件组件更多的笔记已被添加到该协议，以允许读者选择中，将满足他们的个人需求，包括成本效益的替代品和产品，可以在内部进行的最佳产品。也提供了重要的提示，设置和实施过程中是有用的。

<table border="0" cellpadding="0" cellspacing="0" width="798">
	<tbody>
		<tr height="20">
			<td colspan="5" height="20" style="height:20px;width:800px;">1. Laser Set-up</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:304px;">Name of Equipment</td>
			<td style="width:59px;">Company</td>
			<td style="width:65px;">Quantity</td>
			<td style="width:127px;">Catalog Number</td>
			<td style="width:245px;">Comments</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:304px;">100mW 473nm or 488nm Diode Laser System , &#60;2% Stability</td>
			<td style="width:59px;">Omicron</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">Luxx/Phoxx 473/488-100</td>
			<td style="width:245px;">Optional accessory includes a remote control box with key switch and LED Display</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:304px;">100mW 594nm DPSS laser</td>
			<td style="width:59px;">Colbolt</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">0594-04-01-0100-300</td>
			<td style="width:245px;">04-01 series yellow laser; sensitive to back-reflection from fibers</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:304px;">200mW 532nm DPSS laser; 5% power stability</td>
			<td style="width:59px;">Shanghai Lasers</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">GL532T3-200</td>
			<td style="width:245px;">Cost-effective alternative to yellow DPSS laser for activation of halorhodopsins and archaerhodopsins</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:304px;">Non-contact style laser to multimode fiber coupler&#160;</td>
			<td style="width:59px;">OZ Optics</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">HPUC-23-400/700-M-20AC-11</td>
			<td style="width:245px;">For use with dual laser set-up; Specs: 33mm OD for 400-700nm; FC receptacle, f=20mm lens with post mount laser head adapter #11</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:304px;">Aluminum breadboard, 12&#34; x 18&#34; x 1/2&#34;, 1/4&#34;-20 Threaded</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">MB1213</td>
			<td style="width:245px;">For dual laser system</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:304px;">Aluminum breadboard, 10&#34; x 12&#34; x 1/2&#34;. 1/4&#34;-20 Threaded</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">MB1012</td>
			<td style="width:245px;">For single laser system</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:304px;">Aluminum breadboard, 4&#34; x 6&#34; x 1/2&#34;, 1/4&#34;-20 Threaded</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">2</td>
			<td style="width:127px;">MB4</td>
			<td style="width:245px;">For blue laser; dual laser system</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">Compact variable height clamp, 1/4&#34;-20 Tapped</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">4</td>
			<td style="width:127px;">CL3</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">3/4&#34; stainless post</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">TR075</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">1&#34; stainless post</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">4</td>
			<td style="width:127px;">TR1</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:304px;">Post holder with spring-loaded hex-locking thumbscrew</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">2</td>
			<td style="width:127px;">PH1</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">Pedestal Base Adapter</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">3</td>
			<td style="width:127px;">BE1</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">Small Clamping Fork</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">3</td>
			<td style="width:127px;">CF125</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:304px;">Kinematic mount for 1&#34; optics with visible laser quality mirror</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">3</td>
			<td style="width:127px;">KM100-E02</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">Neutral filter density wheel</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">NDC-50C-2M</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">1&#34; Longpass dichroic mirror 50%</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">DMLP505</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">Kinematic mount for 1&#34; optics&#160;</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">KM100</td>
			<td style="width:245px;">For dichroic mirror</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">20-piece hex wrench kit with stand</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">TC2</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">1/4&#34;-20 cap screw and hardware kit</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">HW-KIT2</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">Mounting base 1&#34; x 2.3&#34;x3/8&#34;</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">BA1S</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">FC/PC to FC/PC L-Bracket mating sleeve</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">2</td>
			<td style="width:127px;">ADAFCB1</td>
			<td style="width:245px;">Dual FC/PC L-bracket also available</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">Breadboard lifting handles</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">3</td>
			<td style="width:127px;">BBH1</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:304px;">&#216;1&#34; Bandpass Filter, CWL = 450 &#177; 2 nm, FWHM = 10 &#177; 2 nm</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">FB450-10</td>
			<td style="width:245px;">For use with diode lasers that spontaneously emit</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;"></td>
			<td style="width:59px;"></td>
			<td style="width:65px;"></td>
			<td style="width:127px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="20">
			<td colspan="5" height="20" style="height:20px;width:800px;">2. Laser Coupling</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:304px;">Name of Equipment</td>
			<td style="width:59px;">Company</td>
			<td style="width:65px;">Quantity</td>
			<td style="width:127px;">Catalog Number</td>
			<td style="width:245px;">Comments</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:304px;">! Laser protective eyewear</td>
			<td style="width:59px;">Various</td>
			<td style="width:65px;">One for every user at each wavelength</td>
			<td style="width:127px;"></td>
			<td style="width:245px;">! Consult with laser provider to ensure proper selection of eyewear that will provide maximal light attenuation for the purchased laser&#160;</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">Fiber optic cable tester</td>
			<td style="width:59px;">Eclipse</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">902-186N</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">One-step fiber connector cleaner</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">FBC1</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:304px;">Coupler patch cord (0.75 meter)</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">0.75m 200um core, 0.22NA, FC/PC connectors multimode fibers</td>
			<td style="width:245px;">For dual laser system</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:304px;">Coupler patch cord (0.5 meter)</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">0.5m 200um core, 0.22NA, FC/PC connectors, multimode&#160;</td>
			<td style="width:245px;">For single laser system</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">Doric mini cube</td>
			<td style="width:59px;">DORIC</td>
			<td style="width:65px;">2</td>
			<td style="width:127px;">DMC_1x2_FC-2FC</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">Compact power and energy meter console</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">PM100D</td>
			<td style="width:245px;">Digital 4&#34; LCD</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:304px;">C-series slim power sensor 5-500mW</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">S130C&#160;</td>
			<td style="width:245px;">Multiple detectors types are available; check with vendor</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;"></td>
			<td style="width:59px;"></td>
			<td style="width:65px;"></td>
			<td style="width:127px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="20">
			<td colspan="5" height="20" style="height:20px;width:800px;">3. In vivo Optogenetic Stimulation</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:304px;">Name of Equipment</td>
			<td style="width:59px;">Company</td>
			<td style="width:65px;">Quantity</td>
			<td style="width:127px;">Catalog Number</td>
			<td style="width:245px;">Comments</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:304px;">Multimode fiber splitters</td>
			<td style="width:59px;">FONT Canada</td>
			<td style="width:65px;">2</td>
			<td style="width:127px;">Large core fiber optic 1 X 2 splitter, 50/50 ratio, FC connectors, ruggedized&#160;</td>
			<td style="width:245px;">Length, core size and numerical aperture can be specified when ordering; cost-effective smaller core sizes available</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">Arbitrary waveform function generator (2 channel)</td>
			<td style="width:59px;">Rigol</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">DG1022</td>
			<td style="width:245px;">Can control up to 2 lasers at once</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:304px;">Fiber optic rotary joint (commutator)</td>
			<td style="width:59px;">DORIC*</td>
			<td style="width:65px;">6 to 8</td>
			<td style="width:127px;">FRJ_1X1_FC-FC</td>
			<td style="width:245px;">*Also available through Thorlabs and Prizmatix</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:304px;">Animal patch cords (Custom Mono Fiberoptic Cannula with 10mm ferrules, FC/PC connector)</td>
			<td style="width:59px;">DORIC</td>
			<td style="width:65px;">8</td>
			<td style="width:127px;">MFP_200/240/900-0.22_2m_FC-MF2.5</td>
			<td style="width:245px;">Length, core size and numerical aperture can be specified when ordering; alternatively, these can be made custom made in-house (see Sparta et al. 2012)9.</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:304px;">PFP ceramic split sleeve, 2.5mm ID, 11.40mm length (25/pkg)</td>
			<td style="width:59px;">Precision fiber Products</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">SM-CS1140S</td>
			<td style="width:245px;">Used for attaching implanted fiber optic on animal to a light-delivering fiber patch cord with flat cleeve (FC) end</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">Clear dust caps for &#216;2.5 mm ferrules (25/pkg)&#160;</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">1</td>
			<td style="width:127px;">CAPF</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:304px;">Metal cap for FC/PC and FC/APC mating sleeves&#160;</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">2</td>
			<td style="width:127px;">CAPF1</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:304px;">Thick-jacketed patch cords (custom order)</td>
			<td style="width:59px;">Thorlabs</td>
			<td style="width:65px;">4</td>
			<td style="width:127px;">200um core, 0.22NA, FC/PC connectors multimode fibers</td>
			<td style="width:245px;">Length, core size, and numerical aperture can be specified when ordering&#160;</td>
		</tr>
	</tbody>
</table>

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.

与体内光遗传学刺激获得的行为的结果是完全依赖的神经电路上被定位的，所用的动物模型，和调制参数。对于当前的示范目的酪氨酸羟化酶，多巴胺神经元在腹侧被盖区，或VTA，::的Cre小鼠转导稳定阶跃函数视蛋白（SSFO）8，或控制病毒（EYFP），和纤维的植入物是长期植入。利用TH ::酶Cre转基因小鼠可确保视蛋白表达仅限于在VTA TH +细胞（多巴胺）。 图7描述了使用当前述激光设置为多个小鼠的同时进行刺激获得代表的行为的结果。这里，小鼠系留，并在使用不同的激光的同时刺激（3只小鼠/激光器如在图4C中 ），并记录运动行为1小时。在VTA多巴胺神经元的反复刺激导致多动表型在整个刺激的持续时间持续。在自发的行为没有变化主要出现在EYFP小鼠（见视频1）。以下行为测试，免疫组织化学进行验证准确病毒靶向VTA多巴胺神经元和纤维铺放目视确认（参见图7）。 <img alt="图1" fo:content-="" src="/files/ftp_upload/51483/51483fig1highres.jpg" /> 体内光遗传学刺激图1.实验步骤。有设计和执行在体内光遗传学刺激时，参与4的一般步骤。这个协议具体细节涉及光的输送从激光光源到深部脑结构中的做人啮齿动物的步骤，并包括：1）激光系统组件和光耦合;对于C 2）网络共享策略onnecting高通量行为测试多种动物到光源和3）提供的指导方针，以确认所述靶向策略光递送 - 一个步骤是对数据的解释是必不可少的。注意：虽然该协议并不专用于慢性植入纤维束缚的目的，它建议，当与行为测试组合光遗传学刺激假定。看到这两个翁和Arenkiel，2012 18和斯巴达等 ，2012 9内部生产和慢性光纤植入。实线=涵盖在本协议中的步骤。 <img alt="图2" fo:content-="" src="/files/ftp_upload/51483/51483fig2highres.jpg" /> 用于体内刺激光遗传学图2激光系统：（A），用于在体内刺激的单激光系统。这种激光是pH值<html> ysically预耦合由制造商，需要很少的终端用户设置。（B）中的双激光系统。两个激光器耦合到一个单一的纤维，通过使用其作用引导每个光束路径成非接触式的联接器反射镜。这是最通用的设置作为光学部件可以被删除或根据需要而呈现更有效的激光耦合方面挑战的加入。（C）的（B）中示出的双激光系统的原理图激光器和镜的指示放置与相应的激光光束路径（箭头）所描绘。这里，分色镜的“D”是用于偏转光的蓝色波长而经由发送黄色波长的成色剂“C”，并进入所连接的耦合器的接插线。 B =蓝色激光; C =非接触式连接器; D =分色镜; FW =滤光轮; M =镜子; Y =黄色激光。获得=“_空白”&gt;点击此处查看该图的放大版本。 <img alt="图3" fo:content-="" src="/files/ftp_upload/51483/51483fig3highres.jpg" /> 图中的非物理耦合协议，用于3.（A）电缆测试仪底部：电缆测试仪直接连接到一个跳线。插入物示出了测试器到电缆的连接点（B）的跳线称为整个协议从外到内：多模光纤分路器，黑夹套动物接插线，用白色的氧化锆开口套筒附着到平面解理（FC）的端部，厚夹套接插线（也被称为“联接线”）。厚夹套跳线涂有聚氯乙烯（PVC）管，用于提供额外的保护。这些电缆的行业标准颜色代码用于不同类型的纤维，其中，橙=多模光纤之间进行区分。动物跳线是<html>更薄夹套以允许在行为测试的灵活性动物的运动。需要注意的是防尘帽放置在FC / PC结束时，电缆在不使用。 <a href="https://www.jove.com/files/ftp_upload/51483/51483fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" fo:content-="" src="/files/ftp_upload/51483/51483fig4highres.jpg" /> 图4.网络共享策略， 在体内光遗传学（A）一个单一的动物，（B）两种动物的刺激; （C）的三个或四个动物可能的结构并不限于上述所示的-多重配置是可能的，通过适配器，光纤分路器，和分支跳线的独特的组合是可商购或由客户订单。注意：接插线和纤维分离器含有在两端FC / PC连接器（只有一端被描绘）。ww.jove.com/files/ftp_upload/51483/51483fig4large.jpg“目标=”_空白“&gt;点击此处查看该图的放大版本。 <img alt="图5" fo:content-="" src="/files/ftp_upload/51483/51483fig5highres.jpg" /> 图5。一个跳线来使用分离式套管植入光纤正确和不正确（红色的X）连接。 （左面板）一种氧化锆分割套筒用于接插线连接到可植入的光纤（这里未示出固定到动物）的套箍。箭头指向的接插线和可植入光纤之间的连接点。比较（右面板），其中的接插线和可植入光纤之间存在间隙，由于通过连接套筒的裂开可视化。注意漏光可能发生的不正确的连接（右下图）。 U上的底部插入PPER左侧面板描述中个别组件。从顶部插入底部：多利安植入的光纤套管，氧化锆白分袖，黑色护套动物跳线平克利夫（FC）结束（ 图3B描绘充满跳线）。在所有面板，请注意，连接套筒不与接插线的FC端平齐。留下约0.5厘米的超挂起连接植入光纤贴于动物。 <a href="https://www.jove.com/files/ftp_upload/51483/51483fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" fo:content-="" src="/files/ftp_upload/51483/51483fig6highres.jpg" /> 图6.侧（左）和额叶（右）图的鼠标与植入光纤连接到接插线，用于连接套筒分割以帮助观察的接插线吨的正确连接澳植入光纤的套圈。连接点由红色虚线框突出，也描绘在上插入。 <a href="https://www.jove.com/files/ftp_upload/51483/51483fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" fo:content-="" src="/files/ftp_upload/51483/51483fig7highres.jpg" /> 图7.代表性的结果。 （左） 在体内光遗传学刺激行为读数。可以使用所描述的激光设置和束缚协议来获得的行为的例子。转导的任一步骤，函数视蛋白（AAV5-DIO-SSFO-EYFP - 运动活性在腹侧被盖区（VTA）中的酪氨酸羟化酶（TH）::的Cre小鼠（8 /组N = 7）的光遗传学刺激记录）或控制病毒（AAV5-DIO-EYFP）在VTA。三只小鼠的组分别同时拴到单个激光所描绘在图4C和具有447或473纳米的光有5秒脉冲刺激一次交付每15分钟。双向重复测量ANOVA揭示了一个显著组×时间交 ​​互作用（F = 3,39 15.27，P &lt;0.0001），时间显著的主效应（F = 3,39 4.67，P = 0.007），即光遗传学刺激增加自发活动只有在SSFO小鼠（邦费罗尼事后P &lt;0.0001，相对于T = 0 - 15时斌）导致与EYFP小鼠（组的主要作用相比自发活动的整体增加：F = 1.39 10.69，P = 0.0061;邦费罗尼事后P &lt;0.01在t = 15 - 30和P &lt;0.001在t = 30 - 45和t = 45 - 60）。纤维规格：200微米的核心，0.22 NA。光辐照= 6 - 66毫瓦/毫米2，相应于0.1光纤尖端的距离- 0.6毫米的病毒注射部位以5毫瓦的光功率在光纤尖端之前圈养射出。误差棒代表平均值的标准误差。 EYFP与 SSFO：** P &lt;0.01; *** P &lt;0.001;时间效应：#### P &lt;0.0001（右）组织学的病毒和光纤布置确认。上获得的Leica TCS SP5扫描激光显微镜共聚焦荧光图像被用于可视化纤维铺放（虚线）和病毒介导的表达（绿色）在小鼠腹侧被盖区以下的体内光遗传学刺激。多巴胺神经元（TH +）是出现在蓝色。 <a href="https://www.jove.com/files/ftp_upload/51483/51483fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 视频1. 在体内的光遗传学刺激：肺热期间TH :: Cre重组酶的小鼠使用SSFO VTA刺激<a href="https://www.jove.com/files/ftp_upload/51483/JOVE_video_1.mov" target="_blank">请点击此处观看该视频。</a> <table border="0" cellpadding="0" cells起搏=“0”&gt; <tbody><tr><td colspan="5">要求表1.光辐照度，以激活常用的视蛋白。 </td></tr><tr><td rowspan="2">视蛋白变体</td><td rowspan="2"> λ </td><td rowspan="2">功率密度（/毫米2） </td><td></td><td rowspan="2">性能</td></tr><tr><td>开/关动力学</td></tr><tr><td colspan="5">光激发：快熔channelrhodopsins </td></tr><tr><td> 2的ChR2 </td><td> 470 </td><td> 1 - 为5mW </td><td> 1.21 / 12毫秒</td><td>火灾高达40赫兹</td></tr><tr><td> Cheta酒店19 </td><td> 490 </td><td> 5毫瓦</td><td> 0.86 / 8.5毫秒</td><td>火灾高达200赫兹</td></tr><tr><td>首席20 </td><td> 450 </td><td> 1.65毫瓦</td><td> 1.62 / 12毫秒</td><td>的ChR2非脱敏形式</td></tr><tr><td rowspan="2"> C1V 18 </td><td rowspan="2"> 540 - 630 </td><td> 8毫瓦（540nm处） </td><td> 5/34毫秒54为0nm </td><td rowspan="2">红移</td></tr><tr><td> 3.2兆瓦（630） </td><td> 67毫秒（上）在630 </td></tr><tr><td colspan="5">光激发：慢作用通道视紫红质</td></tr><tr><td>稳定步骤函数视蛋白（SFO）8 </td><td>五百九分之四百七十〇 </td><td> 8μW（435-460） </td><td> 20毫秒/ 29分钟</td><td>新的SFO变体;再打开状态。通过470纳米开业，由590纳米关闭</td></tr><tr><td colspan="4">光抑制</td><td></td></tr><tr><td rowspan="2"> eNpHR3.0 21 </td><td rowspan="2"> 560 - 630 </td><td rowspan="2"> 3 - 5毫瓦</td><td rowspan="2"> 2.5毫秒/ &lt;10毫秒</td><td> 30分钟22持续抑制一定的光* </td></tr><tr><td></td></tr><tr><td> ArchT3.0 11，23 </td><td> 520 - 560 </td><td> 1 - 5毫瓦</td><td> 2 / &lt;10毫秒</td><td>更敏感比eNpHR3.0 &lt;光电流较大/ TD&gt; </tr><tr><td colspan="5">该表被提供作为唯一的导向;需要神经调制特定的光辐照度应独立证实。 </td></tr><tr><td colspan="5">实验验证是重要的，以验证该视蛋白，靶向策略，和光刺激参数调节在预定的方式5神经放电。 </td></tr><tr><td colspan="5">功率密度（毫瓦/毫米2），是指光照射在脑组织的给定区域的功率，并且不指光功率从光纤前端射出。 </td></tr><tr><td colspan="5"> *经常使用的最低光照强度可能，尤其是长时间强光刺激。 </td></tr></tbody></table> 要求表1.光辐照度，以激活常用的视蛋白。 缩略语 AAV =腺相关病毒 DPSS =二极管泵浦固态<br/&gt; EYFP =增强黄色荧光蛋白 FC / PC =平板切割/身体接触 GFP =绿色荧光蛋白 PBS =磷酸盐缓冲盐水 PVC =聚氯乙烯 MW =毫瓦 NA =数值孔径 SSFO =稳定的阶跃函数视蛋白 TH =酪氨酸羟化酶 TTL =晶体管 - 晶体管逻辑 V =电压 VTA =腹侧被盖区

Watch this Scientific Journal Video about In vivo Optogenetic Stimulation of the Rodent Central Nervous System at JoVE.com

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 ...

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Research

JoVE Journal

Neuroscience

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

59.0K 次观看.  University of Pittsburgh Medical Center. 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.

视频: In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

全身和局部给药治疗的中枢神经系统疾病的啮齿动物模型

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

科研

神经科学

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

视神经横断：一个成人在中枢神经系统的神经元凋亡模型

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

调查中枢神经系统修复中受伤范式<em&gt;果蝇</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

在中枢神经系统Translatome由多核糖体分馏体内拷问

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

鼠标中枢神经系统的光透明使用被动CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...