ESB的承认的美国国立卫生研究院主任的新的创新奖（DP2 OD002002 - 01），美国国立卫生研究院的挑战格兰特1RC1MH088182 - 01，美国国立卫生研究院大机会格兰特1RC2DE020919 - 01，美国国立卫生研究院1R01NS067199 - 01，美国国家科学基金会（0835878和0848804）的麦戈文研究所神网奖“计划的资助部，国防，NARSAD，Alfred P. Sloan基金会，杰里和玛吉伯内特，神经，麻省理工学院媒体实验室，贝尼塞基金会，和华莱士阁下科尔特基金会SFN创新研究奖。

1。构建立体定向钳<ol><li>立体钳，用于喷油器阵列连接到stereotax，喷油器，使立体手臂平行。 </li><li>每个实验室只需要一个钳，除非它是预期，一个以上的手术可能会同时发生。在这种情况下，相同数量的同时手术预期的最大数量。这可能也是可取作出额外的，第一的损害的情况下。 </li><li>首先，为了使立体钳，一个1.5毫米的外径（OD）钢套管切成2英寸长，一端是Dremeled直到持平。 </li><li>接下来，切出一小块（约0.5 × 0.5厘米）是印刷电路板原板，作为一个间隔使用。 </li><li>使用激光切割机，以确保作出的任何削减都垂直于材料表面，两个相同的矩形（1 / 2“× 3 / 8”）削减1 / 8“丙烯酸塑料布，每个矩形的两个圆孔矩形的对角第一洞的直径1.5毫米，只是大到足以容纳金属套管紧紧第二洞直径1 / 16“（ 图 1C ）。 </li><li>金属导管插入一个矩形1.5毫米的孔，​​然后通过第二个1.5毫米的孔。底端的套管与底部矩形底面对齐，形成一个曲棍球棒的形状。 </li><li>在保持两个矩形之间的紧密举行的间隔，这种结构是通过使用套管和1.5mm孔周围热胶胶结在一起，小心地避免胶合矩形的间隔。造型粘土可能会有所帮助，在此过程中举行的东西放在一起。 </li><li> 1-72螺丝胶水晾干后，插入从矩形的顶面的1 / 16洞。然后一个1-72六角螺母拧到1-72螺钉年底，并拧紧。螺丝和螺母，为共同举行钳。 </li><li>要重视六角螺母的底部矩形，少量的5分钟环氧底面被丢弃周围边的六角螺母，使环氧树脂进入六角螺母或螺钉的螺纹。任何造型粘土被删除，它是证实，垫片，可以牢固地拧紧螺丝举行到位。 </li></ol> 2。准备定制的喷油器阵列系统：汉密尔顿泵和stereotax <ol><li>喷油器阵列系统，可定制，以几乎所有在大脑中的坐标。 </li><li>用户首先定位坐标转换所需的注射部位在鼠标（或其他物种）脑图谱，stereotax适当的坐标（这里的X，Y，和Z坐标）。注射部位（ 图1a和1b 3） 此后称为 k。 </li><li> K的10μL的汉密尔顿注射器数量被放置在这一个从哈佛仪器如注射泵注入/退出。 </li><li>接下来，3英尺长的的聚乙烯管件牢固地连接到每个汉密尔顿注射器针头。 </li><li>对于每一块聚乙烯管材，一架F - 252厄普丘奇科学管套筒是在开放的结束下滑，并连接到一个P - 627管适配器，使用包括螺母和套圈，也从厄普丘奇科学。 </li></ol> 3。构建定制的喷油器阵列<ol><li>每个喷油器阵列是定制用户选择的坐标。然而，坐标，只能通过翻译和/或旋转不同的设置，相同的喷油器阵列可以使用。 </li><li>只考虑相对注射部位的X和Y坐标，K孔钻成的PCB厚度为1 / 32的原板“，具有相同的相对间距孔钻使用一个Modela小型轧机和一个0.011“麦克马斯特卡尔直径钻头。一定要使用的钻井速度缓慢（Z型高速），以避免破坏或穿出来的钻头。去除磨板前，一个矩形周围的小孔钻，用一个较大的钻头直径的1 / 32“，避免穿较小的一个小型轧机的代码可以生成容易使用MATLAB;。样本在以下文件中提供的代码。 <ol class="lroman"><li> generate.m - MATLAB代码生成所需的坐标Modela代码</li><li> holes_ex.rml - Modela代码钻井环模式进入原板（8孔） </li><li> outline_ex.rml - Modela钻孔孔周围的矩形的代码</li></ol></li><li> 245微米OD/100微米内径（ID），熔融石英毛细管，从Polymicro技术，切成K的3英寸件数。这包括个人的喷油器。一个一次性毛细管戳通过每个洞，扫清实际喷油器无堵塞碎片。喷油器，然后每孔中途插入，因此，他们紧紧彼此平行。喷油嘴epoxied向董事会，形成喷油器的阵列结构。剩下的就是正确的长度修剪喷油嘴。 </li><li>喷油器阵列连接到立体钳之间的丙烯酸矩形放在一个角落里的原板，然后通过收紧立体钳螺丝。下一步，钳的金属导管连接的stereotax，使用的stereotax附件机制。 </li><li>以下是在显微镜下完成。其中一个的喷油器（即，可以从侧面直边走近没有碰撞到任何其他的喷油器）外，尖延伸超出了原板的底部用剪刀剪开。注射皮质，这是适当的喷油器延长约超出原板表面5毫米。对于更深的注射，这个数目可以增加深度相应增加。用一个DREMEL工具提示是夷为平地。喷油器的提示也可以是地面作为一个额外的预防措施对堵塞角度，深入的精度并不那么重要。 </li><li>下一步，选择一个稳定的参考点的立体手臂范围内，喷油器的阵列移动，这样，夷为平地外的喷油器的提示，在这个参考点。 </li><li>选择第二个喷油器，以及与其相应的坐标。认为是喷油器之间的第一个和第二个坐标（Z方向）的相对高度差异。喷油器阵列是沿Z轴移动此相对距离。第二注射器是修剪和Dremeled正确的长度，使第二注射器的针尖现在平坦参考点的高度。喷油器阵列，可在X和Y方向移动，以方便与参考点的喷油器提示比较。 </li><li>这修剪的过程不断重复，剩下的喷油器。 </li></ol> 4。整个系统组装<ol><li>立体钳和定制的喷油器阵列，无论是已建成，这部分需要。 </li><li>每个喷油器的后端（尚未修剪后终点）插入一个蓝色的F - 240油管套管和使用的P - 235螺母和套圈的P - 200厄普丘奇科学，是连接到适配器已经连接螺纹聚乙烯管材的汉密尔顿泵。制造商的说明，可以详细咨询。 </li><li>使用27号针头，硅油注入，使整个系统是由汉密尔顿注射器填充注射器针尖的汉密尔顿注射器回，没有在所有的气泡。 </li><li>由于汉密尔顿注射器重新放置在汉密尔顿泵，硅油量最大可能保持在注射器。 </li><li>如果实验需要超过泵的设计（在这种情况下两个）的注射器，注射器可以间隔的塑料小件（如针帽），让他们互相平行，并确保所有件推泵相同程度。或者说，一个多机架升级套件可以购买从哈佛仪器举行一次10注射器。 </li></ol> 5。注射/手术<ol><li>一个平行的注射器注射的过程是非常类似的注射用一个吸管。 </li><li>至关重要的是，使用缓慢笔芯/注入率，在此过程中，因为快抽，可能把对大和小管之间的联合的压力。推荐的最大速率：2μL/分钟加载病毒，0.1μL/分钟注入病毒。 </li><li>一个麻醉鼠标放置在stereotax，并保持在整个实验的麻醉。 </li><li>需要准备的鼠标。例如，用手术刀，一个单一的削减是作出下来中线的皮肤，眼睛之间的两耳之间。拉回皮肤暴露​​颅骨，筋膜清除。一把拉住玻璃吸管连接的stereotax。耳棒的位置进行调整，直到前囟和lambda是一致的同一高度，使连接它们的线是平行于Y轴的stereotax。根据已计算出的坐标系，在其中注入坐标stereotax轴取向。前囟门的玻璃吸管提示脑立体定位归零，然后提示是略高于头骨定位在X和Y坐标的注射部位之一。 </li><li>使用牙钻，颅骨下面的提示仔细相比，直到仍然有一层极薄的骨。玻璃吸管可降低教育署和提出检查，被钻了孔，是在正确的位置为中心。使用30号针头，轻轻地拿起一个小片层的是正确的X和Y坐标，，因此，暴露硬脑膜。这个洞应该是大到足以容纳一个喷油器（0.25毫米宽）。请参阅图图1D。这样，小，0.25毫米宽孔是在颅骨，在X和Y坐标的对应每个注射部位。 </li><li>玻璃吸管被丢弃在锐器容器中，并采用脑立体定位夹具定制的喷油器阵列连接的stereotax。 </li><li>为了正确地设置了喷油器阵列的角度，两个外喷油器进行校准，以一个给定的参考点，如下选择。后喷油器之一，是与参考点相匹配，考虑相对的X，Y型注射部位之间的距离相对这个喷油器和另外一个。 stereotax轴调整，使整个喷油器阵列是在X和Y方向移动，这些相对距离。如果不是现在与参考点对齐第二注射器，金属套管松动和旋转。反复重复这一过程，直到喷油器阵列的角度正确。 </li><li>在灌装前与病毒的喷油器，喷油器与硅油填充，直到注射器2μL点（或更大）。这提供了一个缓冲地带，使任何气泡或堵塞的提示可以很容易地推油使用汉密尔顿泵，而不必硅油填充与整个系统从后面的注射器，描述以前，删除。 </li><li>一个封口膜无菌，放在头骨，和喷油嘴上的封口膜轻轻降低。对于有很大的不同深处，一个自定义铣削部分（如楼梯状物体）的坐标可以方便上落。 </li><li>下面的部分假设，1μL的病毒是在每个站点的加载（以下数量应缩小，如果需要小批量）。为了保证&gt; 1μL病毒注射在每个站点，1.5μL病毒吸管将在每个喷油器的尖端封口膜或楼梯。 </li><li>随着汉密尔顿泵的填充速度设置为1μL/分钟，1.2病毒液补充到每个喷油器。 </li><li>在囟最长的喷油器的提示是零，然后转移到的X和Y坐标，喷油器喷油器阵列。如果堵塞是一个问题，说如果涉及许多深目标：前插入大脑中的喷油器提示，甚至有时是最好启动泵注入，直到可以看到所有的喷油器提示出现病毒。这在喷油器提示删除任何气泡，并确保没有堵塞。在堵塞的情况下，汉密尔顿泵注入2μL/ min的速度在一个简短的脉搏，轻轻地清除堵塞。 </li><li>通过30表针的小孔，然后降低喷油器阵列，是正确的Z -深度。 </li><li> 1μL病毒注入到每个喷油器，在0.1μL/ min的。 </li><li>注射30分钟的单独留在家中。 </li><li>喷油器阵列后，慢慢地从大脑中提取，汉密尔顿泵注入0.1μL/ min的速率，以检查在每个喷油器堵塞。 </li><li>喷油嘴清洗注墨，注入2μL/ min的1.5μL乙醇。 </li><li>最后，喷油器加气硅油保持在每个汉密尔顿注射器2μl的缓冲地带。 </li><li>所有设备应进行消毒前和手术后，根据您的机构的生物安全和动物使用的协议。 </li></ol> 6。代表性的成果平行的喷油器阵列加快手术大约等于喷油器数量的一个因素，但不包括安装和恢复时间，虽然个别时间将取决于从业者的技能。 1微升注入，我们通常看到一个约直径1mm（ 图1E）的球体中的慢病毒表达。注射精度等尖端定位的变化，从审判开庭审理，约45微米（从针尖位置达到了预期的尖端位置的距离的标准偏差）。 <img src="/files/ftp_upload/1489/1489fig1.jpg" alt="图1" /> 图1：设计，实施和使用一个平行病毒喷油器阵列。一个平行的喷油器阵列系统，原理图，显示出三重喷油配置， 三同时注射 ，B，三重并行喷油阵列的照片图解在A 。 <st荣&gt; C， 从顶部的轮廓显示的立体钳，研发，技术效率，损害最小化，孔喷油器插入到大脑颅骨开放的例证：用牙钻下来的头骨，薄〜50微米的厚度，然后使用锋利的针，开了一个小的开颅手术的一角，E，荧光图像显示channelrhodopsin - 2（ChR2）GFP标记的细胞在小鼠皮层三重喷射器阵列的目标地区，在 B所示。

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	<li>Chow, B., Han, X., Qian, X., Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-performance+halorhodopsin+variants+for+improved+genetically-targetable+optical+neural+silencing.">High-performance halorhodopsin variants for improved genetically-targetable optical neural silencing.</a> Frontiers in Systems Neuroscience. , (2009).</li><li>Bi, A., Cui, J., Ma, Y. P., Olshevskaya, E., Pu, M., Dizhoor, A. M., Pan, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+expression+of+a+microbial-type+rhodopsin+restores+visual+responses+in+mice+with+photoreceptor+degeneration.">Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration.</a> Neuron. 50, 23-33 (2006).</li><li>Han, X., Qian, X., Bernstein, J., Zhou, H., Franzesi, G., Stern, P., Bronson, R., Graybiel, A., Desimone, R., Boyden, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-Timescale+Optical+Control+of+Neural+Dynamics+in+the+Nonhuman+Primate+Brain.">Millisecond-Timescale Optical Control of Neural Dynamics in the Nonhuman Primate Brain.</a> Neuron. 62 (2), 191-198 (2009).</li><li>Zhang, F., Wang, L. P., Brauner, M., Liewald, J. F., Kay, K., Watzke, N., Wood, P. G., Bamberg, E., Nagel, G., Gottschalk, A., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+fast+optical+interrogation+of+neural+circuitry.">Multimodal fast optical interrogation of neural circuitry.</a> Nature. 446 (7136), 633-639 (2007).</li><li>Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale genetically targeted optical control of neural activity.</a> Nat Neurosci. 8 (9), 1263-1268 (2005).</li><li>Han, X., Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple-color+optical+activation,+silencing,+and+desynchronization+of+neural+activity,+with+single-spike+temporal+resolution.">Multiple-color optical activation, silencing, and desynchronization of neural activity, with single-spike temporal resolution.</a> PLoS ONE. 2, e299-e299 (2007).</li><li>Szobota, S., Gorostiza, P., Del Bene, F., Wyart, C., Fortin, D. L., Kolstad, K. D., Tulyathan, O., Volgraf, M., Numano, R., Aaron, H. L., Scott, E. K., Kramer, R. H., Flannery, J., Baier, H., Trauner, D., Isacoff, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+control+of+neuronal+activity+with+a+light-gated+glutamate+receptor.">Remote control of neuronal activity with a light-gated glutamate receptor.</a> Neuron. 54 (4), 535-545 (2007).</li><li>Lima, S. Q., Miesenbock, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+control+of+behavior+through+genetically+targeted+photostimulation+of+neurons.">Remote control of behavior through genetically targeted photostimulation of neurons.</a> Cell. 121 (1), 141-152 (2005).</li><li>Zhang, F., Wang, L. P., Boyden, E. S., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsin-2+and+optical+control+of+excitable+cells.">Channelrhodopsin-2 and optical control of excitable cells.</a> Nat. Methods. 3, 785-792 (2006).</li><li>Ishizuka, T., Kakuda, M., Araki, R., Yawo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+evaluation+of+photosensitivity+in+genetically+engineered+neurons+expressing+green+algae+light-gated+channels.">Kinetic evaluation of photosensitivity in genetically engineered neurons expressing green algae light-gated channels.</a> Neurosci Res. 54 (2), 85-94 (2006).</li><li>Luo, L., Callaway, E. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+neural+circuits.">Genetic dissection of neural circuits.</a> Neuron. 57 (5), 634-660 (2008).</li></ol>

所有的程序，按照美国国立卫生研究院实验动物的指南和麻省理工学院的动物护理和使用生物安全委员会批准。

近年来，基因编码的光学增敏剂已启用神经元被激活， 并在体内沉默在时间上精确的方式，在简短的光脉冲响应（例如，1,4,5,6,7,8 ，11）。通过与神经元已认识到光在哺乳动物的大脑，是一个关键的方法，如慢病毒和腺相关病毒（AAV）的病毒，它可以提供从老鼠到猴子的动物大脑中的视蛋白编码基因，在安全和持久的方式（ 如 2,9,10） 。允许更快的周转时间比转基因的病毒，特别是生物，遗传模式生物如老鼠和猴子，和视蛋白可能使高表达水平，可能无法在转基因的情况下可能。在这里，我们展示了一个平行的喷油器阵列能够创造一个快速的时间表，“电路级转基因”，使整个三维大脑结构被病毒基因的目标在一个单一的手术步骤。喷油器阵列由一个或多个排量泵通过高压容错连接器，每个驱动器的注射器，其中每个饲料成聚酰亚胺/熔融硅毛细管。毛细血管的大小，然后插入到所需的位置，通过自定义铣立体定位板指定，从而使病毒或其他试剂被传递到大脑区域所需的设定。要使用的设备，医生首先填充流体子系统完全符合石油，回填与病毒的毛细血管，进入大脑中插入的设备，慢慢注入试剂（&lt;0.1μL/分钟）。 这项技术将使新品种的实验，如毫秒时间尺度（如海马）关闭complexly形结构过程中的行为在精确的时间，时间精确灭活双边冗余结构，可能会采取行动，种类繁多（如左，右杏仁核），和扰动的多个离散的大脑区域相驾驶两个相连的区域（例如，研究如何跨区域同步取决于每个区域内的活动后，或刺激输入到一个地区，而沉默的一个子集的目标，以了解其中的几个目标是调解这些投入的效果的关键）。像那些在灵长类动物的大脑，在我们最近表现出光的特定细胞类型的神经激活3，在行为上相关领域的活动的干扰，可能需要大，结构复杂的病毒标签。我们注意到，可用于几乎所有的有效载荷 - 注入药物，神经调质，神经递质，或者甚至细胞 - 在时间上精确地在大脑中复杂的3 - D模式，并行喷油器阵列。最后，从翻译的角度来看，它可以迅速，病人定制的基因治疗或药物输送装置，可迅速定制设计和制造，以满足个人的大脑形状，支持了各种病症的新疗法可能通过使用的，光控分子。 喷油器阵列设计要精确，在空间和体积。在X和Y方向，这是通过钻洞，非常准确地放置使用一种廉价的小型轧机，只是足够大，以适合的喷油器，喷油器，使彼此平行举行的孔，并在一个精确的位置。在Z方向，喷油器被修剪使用立体定位仪，允许立体定向手术本身的精度水平相当于。体积精密来自汉密尔顿泵的精度，以及接近零死体积连接器，从适应的高压液相色谱（HPLC）字段。喷油器是由熔融石英毛细管，这是强大的和足够的刚性，它保持压力下的精确形状和间距，没有替代品，如钢套管的壁厚较大。小的修改，可以很容易地适应各种实验并行的喷油器阵列。例如，如果需要体积更小的病毒或更细间距，可以聘用较小的毛细管，连同相应的较小的钻头。未来的设备可以利用微流体通道和泵，增加平行喷射器的数量，以减少大小（也许使这些设备装上自由移动的动物负责人）。

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dremel Tool</td>
<td>Tool</td>
<td>Dremel</td>
<td>3956-02</td>
<td>&nbsp;</td>
<tr>
<td>Laser Cutter</td>
<td>Tool</td>
<td>Universal Laser Systems</td>
<td>VLS2.30</td>
<td>&nbsp;</td>
<tr>
<td>Hot glue gun</td>
<td>Tool</td>
<td>Stanley Bostitch</td>
<td>GR20</td>
<td>&nbsp;</td>
<tr>
<td>Injection/withdrawal syringe pump (Hamilton pump)</td>
<td>Tool</td>
<td>Harvard Apparatus</td>
<td>702001</td>
<td>&nbsp;</td>
<tr>
<td>10 &mu;l Hamilton syringes</td>
<td>Tool</td>
<td>Hamilton Company</td>
<td>701N</td>
<td>Need one per injection site</td>
</tr>
<tr>
<td>Mouse stereotax</td>
<td>Tool</td>
<td>Stoelting</td>
<td>51725D</td>
<td>&nbsp;</td>
<tr>
<td>Modela mini-mill</td>
<td>Tool</td>
<td>Roland</td>
<td>MDX-15</td>
<td>&nbsp;</td>
<tr>
<td>0.011" diameter drill bit for mini mill</td>
<td>Tool</td>
<td>Mcmaster-Carr</td>
<td>8915A12</td>
<td>&nbsp;</td>
<tr>
<td>1/32" diameter drill bit For mini-mill</td>
<td>Tool</td>
<td>McMaster-Carr</td>
<td>8848A35</td>
<td>&nbsp;</td>
<tr>
<td>High speed dental drill</td>
<td>Tool</td>
<td>Lynx</td>
<td>333</td>
<td>&nbsp;</td>
<tr>
<td>Dental drill accessories</td>
<td>Tool</td>
<td>Pearson</td>
<td>F 35-08-25 F 35-07-10 P 86-02-38</td>
<td>&nbsp;</td>
<tr>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td>1.5mm outer diameter (OD) stainless steel cannula</td>
<td>Material</td>
<td>Small-Parts</td>
<td>HTX-15R</td>
<td>&nbsp;</td>
<tr>
<td>1-72 binding slotted machine screw</td>
<td>Material</td>
<td>Small-Parts</td>
<td>MX-0172B</td>
<td>&nbsp;</td>
<tr>
<td>1-72 hex nut</td>
<td>Material</td>
<td>Small-Parts</td>
<td>HNX-0172</td>
<td>&nbsp;</td>
<tr>
<td>PCB proto-board, 1/32" thick</td>
<td>Material</td>
<td>Digi-Key</td>
<td>PC57-T-ND</td>
<td>&nbsp;</td>
<tr>
<td>Acrylic Sheet, 1/8" thick</td>
<td>Material</td>
<td>Mcmaster-Carr</td>
<td>8560K239</td>
<td>&nbsp;</td>
<tr>
<td>HPLC connectors</td>
<td>Material</td>
<td>Upchurch Scientific</td>
<td>F-252, P-627, P-200, P-235, F-240 (some of these can be bought in 10-packs; simply add an &#x2018;x&#x2019; to the end of the part number)</td>
<td>Need one per injection per site, except F-252, P-627, and P-235, which can be reused</td>
</tr>
<tr>
<td>Fused silica capillary tubing, OD: 245 &mu;m, ID: 100 &mu;m</td>
<td>Material</td>
<td>Polymicro Technologies</td>
<td>2000022-10M</td>
<td>&nbsp;</td>
<tr>
<td>5-minute general purpose epoxy</td>
<td>Material</td>
<td>Permatex</td>
<td>84101</td>
<td>5-minute general purpose epoxy</td>
</tr>
<tr>
<td>Polyethylene tubing .066 x .095 inch</td>
<td>Material</td>
<td>VWR</td>
<td>63018-827</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

scalable fluidic injector arrays for viral targeting of intact 3-d brain circuits

我们了解神经回路 - 他们如何调解，对...有感觉，思想，情感，和行动，以及它们是如何在神经和精神疾病的损坏计算 - 将极大地促进由技术，用于快速定位的基因复杂的3 - 三维神经电路，使快速创作的“电路级的转基因。”我们最近开发的光敏感蛋白编码病毒能够认识到毫秒的时间刻度激活特定的细胞类型和完整的大脑沉默的方法。我们在座的喷油器阵列内提供的病毒（或其他液体）几十个定义点的三维结构的大脑的设计和实施（<strong&gt;图。 1A，1B</strong&gt;）。喷油器阵列由一个或多个排量泵通过高压容错连接器，每个驱动器的注射器，其中每个饲料成聚酰亚胺/熔融硅毛细管。毛细血管的大小，然后插入到所需的位置，通过自定义铣立体定位板指定，从而使病毒或其他试剂被传递到大脑区域所需的设定。要使用的设备，医生首先填充流体子系统完全符合石油，回填与病毒的毛细血管，进入大脑中插入的设备，慢慢注入试剂（&lt;0.1微升/分钟）。喷油器阵列的并行性，有利于快速，准确，稳健和整个神经回路与光增敏剂，如病毒有效载荷的标签，使定义的大脑回路的光激活和沉默。除了与其他技术，如光传递到大脑区域所需设置的光纤阵列，，我们希望创造一个工具箱，使因果完整的大脑的神经功能系统的探测。这项技术不仅可以开放电路集中在哺乳动物神经科学等系统的方法，并促进在非人类灵长类动物，如大动物的大脑区域的标签，但也可能开辟细胞特异的光学控制假肢的一个临床平移路径，其精度可以使改善顽固性脑部疾病的治疗。最后，这里描述的这种设备可方便精确定时的流体提供的其他有效载荷，例如干细胞和药理代理人，在一个易于用户定制的时尚，三维结构。

Watch this Scientific Journal Video about Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits at JoVE.com

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

控制和分析的神经回路<em&gt;在体内</em&gt;将有助于为提供所需的3维的大脑区域的病毒和其他试剂技术。我们展示定制流体喷射器阵列制造，并交付维拉利编码的光学增敏剂，使光学操纵复杂的大脑回路。

可伸缩流控喷油器阵列病毒完整的3 - D的大脑回路为目标

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

scalable-fluidic-injector-arrays-for-viral-targeting-intact-3-d-brain

Research

JoVE Journal

Neuroscience

14.2K 次观看.  Massachusetts Institute of Technology. 控制和分析的神经回路在体内将有助于为提供所需的3维的大脑区域的病毒和其他试剂技术。我们展示定制流体喷射器阵列制造，并交付维拉利编码的光学增敏剂，使光学操纵复杂的大脑回路。

视频: Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an injecting needle produces during its trajectory is a major concern especially in the mouse brain for not only the brain is small but also sometimes multiple injections are needed.  We show here a method to produce glass capillary needles with a 50-&mu;m lumen which significantly reduces the brain damage and allows a precise targeting into the rodent brain. This method allows a delivery of small volumes (from 20 to 100 nl), reduces bleeding risks, and minimizes passive diffusion of drugs into the brain parenchyma. By using different size of capillary glass tubes, or changing the needle lumen, several types of substances and cells can be injected. Microinjections with a glass capillary tube represent a significant improvement in injection techniques and deep brain targeting with minimal collateral damage in small rodents.

In this article, we show a method to make glass capillary needles with a 50-&mu;m lumen. This technique significantly reduces the brain damage, minimizes passive diffusion of drugs and allows a precise targeting into the rodent brain.

定向脑深部结构与配送药品，病毒载体，或细胞移植Microinjections

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an ...

科研

神经科学

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.
Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

脑植入的微型器件及周围组织的完整的组织学特性研究

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

在完整的成年斑马鱼大脑电生理记录

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 &#181;m) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Using this protocol, we were able to image 160 &#181;m thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron.

神经元的3-D成像和分析感染<em&gt;在体内</em&gt;与<em&gt;弓形虫</em

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

修改一个Colliculo  - 丘脑鼠脑切片，收纳室部件的3-D打印和多尺度光学成像

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

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 ...

Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not represent the underlying cytoarchitectural and functional complexity of the affected individuals carrying suspected disease variants. Conventional 2-D models remain incomplete representations of in vivo-like structures and do not adequately capture the complexity of the brain. Thus, there is an emerging need for more 3-D hiPSC-based models that can better recapitulate the cellular interactions and functions seen in an in vivo system.
Here we report a protocol to develop a 3-D system from undifferentiated hiPSCs based on the serum free embryoid body (SFEB). This 3-D model mirrors aspects of a developing ventralized neocortex and allows for studies into functions integral to living neural cells and intact tissue such as migration, connectivity, communication, and maturation. Specifically, we demonstrate that the SFEBs using our protocol can be interrogated using physiologically relevant and high-content cell based assays such as calcium imaging, and multi-electrode array (MEA) recordings without cryosectioning. In the case of MEA recordings, we demonstrate that SFEBs increase both spike activity and network-level bursting activity during long-term culturing. This SFEB protocol provides a robust and scalable system for the study of developing network formation in a 3-D model that captures aspects of early cortical development.

Here we report a protocol to develop a three-dimensional (3-D) system from human induced-pluripotent stem cells (hiPSCs) called the serum free embryoid body (SFEB). This 3-D model can be used like an organotypic slice culture to model human cortical development and for the physiological interrogation of developing neural circuits.

开发HiPSC衍生的无血清胚胎体用于使用生理相关测定的3-D干细胞培养物的询问

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

生成基于3-D胶原蛋白的水凝胶,分析神经系统发育过程中的Axonal生长和行为

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

多纤维光测量记录自由移动动物的神经活动

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...