这项工作是支持R01 DC 011582（AK）和RO1 DC011555（DJT）。

<ol><li>拉玻璃电极拉玻璃电极<ol><li> 拉出单桶电极使用单管玻璃毛细管用灯丝和拉小费测得的直径为约1-2微米，轴的长度为10-12毫米，和相应的约12兆欧姆（范围5-20兆欧姆）的电极电阻。在0.9％NaCl溶液。下电极的电阻会导致更多的背景活动从单个神经元中分离单一的活动，从而更加困难。对于拉动该电极，使用水平或垂直牵拉与任一加热丝或加热线圈。 </li><li> 拉出多管电极。由于更大的直径的多管玻璃和围绕整个多管受热均匀分布的需要，一个强大的牵拉与较大直径的加热线圈，不加热灯丝，是必要的。阿多管吸移管具有被插入在所述加热灯丝的中心与nO触点加热线圈。请注意，除了5桶移液器这里描述，3 - 的桶或7镜筒移液器是市售的替代品。拉出的玻璃吸移管前端的总直径约10微米，或更小。在下一步骤中的正确的直径的前端将被打破，因此，其准确的笔尖的大小比的整体形状的电极头中，这应该是长期的和相对薄的，在提拉过程中不太重要。请参阅图1C中的图像所需的电极头的形状。短而粗的电极形状（ 图1B），将导致大量的组织损伤非常长而细的电极形状（ 图1A）时提前进入大脑，而会弯曲，从而将难以打破的电极尖端到正确的直径（见步骤2）。 </li></ol></li><li>修改电极尖端修改电极秘诀。之前的两个电极可以粘在一起，它们必须被修改。该轴的单电极需要被弯曲之前，它可以连接到多管，使确保成品的小猪背面电极的结合轴是尽可能地薄。此外，的多管电极的前端具有用于离子电渗疗法，以确保低电阻的被折断。 <ol><li> 弯曲轴约20度的一个单一的筒电极。使用最小本生灯火焰可能。典型的“小”标准的实验室供应商创建本生灯火焰的大小，此应用程序是过于庞大。为了规避此问题，使用最小的商业本生灯和固定的注射器针（〜18号）的燃烧器的顶部，并使用牙科用粘固剂的密封连接。当操作时，火焰应该是很难看到的，约5毫米或更小的直径，约8毫米高。任何在房间内的空气流动将熄灭火焰，因此它是一个好主意，在操作的燃烧器封闭的房间，或使用风盾。通过火焰移动单桶电极弯曲约20度。目的有燃烧器的火焰熔化玻璃的过渡区，在远离电极尖端大约10毫米。为了避免熔化我们建议，该电极被保持在约45度的电极的前端，前端朝下，并通过火焰移动电极相对较快。 </li><li> 折断的多管电极的前端，为了确保视觉打破了电极尖端的控制，同时，使用显微镜的最低10x物镜和10倍目镜。插在眼的测量规模也将需要测量头的大小。将一块有机玻璃的显微镜这样的结束，可以看出，在大约三分之一的有机玻璃鉴于在显微镜领域的二分之一。有机玻璃片在我们的例子中，大约是25×70毫米和5毫米厚的和连接的，通过螺钉可固定到一个自定义在显微镜载物台的线程。重要的是，有一个设计，它允许为有机玻璃独立的滑动移动。将多管电极在一张床上的橡皮泥在玻片上，并插入幻灯片，包含电极显微镜载物台的幻灯片夹。使用显微镜载物台的XY机械手，轻轻地移动电极尖端对有机玻璃片，并通过显微镜目镜观察破的尖端。尝试干净突破多管的尖端直径约25-35微米的累积。丢弃移液管尖直径不均匀的休息断绝过大，或提示。我们放弃我们的多管电极由于不良尖形状的约30％。 </li></ol></li><li>组装背驮式电极装配背驮式电极。 <ol><li> 位置电极。删除步骤2.2从显微镜载物台的有机玻璃片。固定完成多管电极插入橡皮泥在玻片上，尖端指向略有上升。朝上将是重要的步骤3.2中，在两个电极的胶合。点上去的提示，导致胶水远离尖端，避免胶电极尖端。将弯曲单管电极，电极持有人定制的显微操纵器（ 图2）。使用可视化的指导意见，然后再微观指导，降低了单管的多管电极的电极上。直接进入的槽，其形成由5桶的布置的，其前端突出的多管尖端的前端由约5-10微米的单电极，应该降低。当降低单桶，密切观察]是在两电极之间形成的角度。对于最好的结果，在其中的提示点彼此分开，而是试图降低单电极到避免任何角度多完全平行或即使与它的端部第一次接触的多管，形成一个很轻微的“楔”的安排。由于单一的套筒尖端是非常灵活的，它会弯曲时，单个电极被降低一点进一步后的前端已达到多桶电极的顶表面，形成一个干净的复合尖端有少量的内置弹簧作用帮助的提示。但是，如果单管和多管电极之间的角度太陡（太多的楔形），弹簧的作用将是太高和向下弯曲的电极布置。 </li><li> 胶电极轴在一起。胶轴的两个电极一起使用氰基丙烯酸酯（强力胶）。将一个扁平的牙签和触摸电极组件用胶水下降到旁边的小滴一小滴胶水。在最远端的位置的提示开始，慢慢地将牙签用胶水降沿电极轴对电极尖端。使用太多胶水，或施胶吨的oo与接近的电极尖端将导致胶合电极开口，使所述电极的至少部分非功能。 </li><li> 用牙科水泥稳定关节。少量的牙科水泥和牙科丙烯酸混合在一个小的一次性塑料盘或权衡船，采用扁平的牙签。等待，直到水泥成为可模制的，并在两个电极之间的接头涂上少量稳定的联合（粉红色的图3中的材料）。允许约15分钟至干。 </li><li> 电极取出并妥善保管。小心地取出背驮式电极的微操作机器人的持有人，然后从载玻片上分离，并存储在防尘的容器中。 </li></ol></li><li>准备电极填充解决方案准备电极填充的解决方案。由于离子电渗疗法需要带电的分子，无论是在酸性或碱性的环境（通常是一个要溶解大多数代理商た约3-4的pH值，或约8-10的pH值，分别）。一些经常使用的化学物质，是在离子电渗疗法的列于表1。对于代理商而言，该表中未列出的，确定的pKa值，是否会更容易在酸性或碱性环境中使用的分子，使分子带电，并解散相应。为了达到最佳效果，混合所有的解决方案鲜的每日。 </li><li>填写并准备电极填写，并准备电极。使用电极前，请填写每个桶各自的药物，使用碳纤维28 - 34号针头的注射器针头式过滤器。填写4个外桶5桶配置的首选药物，该中心作为一个平衡的桶桶3M氯化钠。填写单每桶记录电极以及与3M氯化钠。添加染料的NaCl溶液中，如快速绿色或酚红将使它更容易看到的电极头在放置过程中的脑表面上的电极。将电极插入电极持有人的记录设置，所有的电线插入到合适的玻璃桶。使用从约1厘米的绝缘已被移除的尖端处的绝缘银线。应该有5根为多管电极（4药物桶和一个平衡的桶），再加上需要被插入到记录单管电极的放大器导线。 </li><li>打开电离子透入泵模块打开离子电渗泵模块和测试所有的酒桶。每一个泵模块的电极测试功能，将有助于确定如果电极筒功能。从桶的药物，以防止泄漏，在不使用时，保持在作为分子的电荷相反的极性的电压，需要施加。 </li></ol>

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Neurophysiol. 80, 1686-1701 (1998).</li><li>Coleman, W. L., Fischl, M. J., Weimann, S. R., Burger, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+and+glycinergic+inhibition+modulate+monaural+auditory+response+properties+in+the+avian+superior+olivary+nucleus.">GABAergic and glycinergic inhibition modulate monaural auditory response properties in the avian superior olivary nucleus.</a> J. Neurophysiol. 105, 2405-2420 (2011).</li></ol>

没有利益冲突的声明。

我们描述了一种技术，它允许一个单一的神经元的微电路体内操纵，而在相同的时间，从而可以用于记录的实验操作过程中的神经元的响应。神经电路操纵通过突触的激动剂和拮抗剂的iontophoretical应用。过压喷射离子电渗疗法的主要优点是，离子电渗疗法不要求流体从所述电极到神经组织的物理运动，并因而不存在关注，通过所施加的压力或流体体积引起组织损伤。这种技术的主要限制是缺乏有关下列内容的信息的绝对药物在组织中的浓度，与受影响的组织体积。然而，由于喷射离子电渗疗法的药物制剂的量更小的和更精确的可控性比与压力喷射，从药物的应用的恢复通常是快得多的ð更完整的。在神经系统的感觉和其他一些微电泳已成功地被使用，并且很少或没有内在处理的脑区中最成功的应用。其原因是，喷出的药理活性剂中的一些内容可能从应用程序站点扩散到相邻的神经元，并也可以操纵的相邻神经元的响应特性。 单和多桶电极的单独制造的允许的组合，电极具有任意和无关的特性。拉出电极桶在一起并使用一些用于记录和一些用于离子电渗疗法的目的，会产生非常相似的性质与电极尖端，使得电极尖端是过大的单细胞记录，或过小的药物的应用。另外，有单管尖超出约20微米的多管电极尖端大大降低了噪音的录​​音，ð消除可能的干扰电流的影响，从保留或喷射电流的神经元发射3。 背驮式多管电极的第一个超过30年的前4-6，已经非常成功地用于解剖的神经回路7-18 19-29。因此，该方法本身并不新颖或独特的。然而，电极的制备和使用的特定细节已经被修改多年来，这里描述的指令集已被证明是特别容易和成功的，并没有被详细发表在其他文献中。特别地，弯曲的单管电极尖端允许最终小费的小猪背面电极相对苗条（ 图3），因此，允许以最小的损伤到大脑从深核录音;单筒突出在多管电极的电极删除几乎所有的CURREN吨的影响，这通常被认为是该技术的缺点3。这里提出的新的细节，如具有制造背叠电极时，电极尖端朝上在涂胶过程中和休息的多管电极的槽的单桶将确保高的成功率。该技术是相对容易的，通常可以在几天之内所掌握的新手。

<table border="1"><tbody><tr><td> 产品名称 </td><td> 生产厂家 </td><td> 评论 </td><td> 猫。 ＃ </td></tr><tr><td>本生灯</td><td></td><td> ：VWR </td><td> 17928-027 </td></tr><tr><td> 双组分 “冷固化”牙科材料牙科水泥： </td><td>联合口服ITE牙科制造有限公司</td><td> ：AM系统公司</td><td> 525000 </td></tr><tr><td> 双组分的牙科水泥：义齿材料的交联液体化合物</td><td>联合口服ITE牙科制造有限公司</td><td> ：AM系统公司</td><td> 525000 </td></tr><tr><td>液体胶</td><td>汉高</td><td> 从 ：乐泰瞬间胶</td><td> 01-06849 </td></tr><tr><td>微电离子透入元：Neurophore BH-2 </td><td>哈佛设备</td><td> 可从 ：哈佛仪器</td><td> 65-0200和65-0203 </td></tr><tr><td>绝缘银线</td><td> AM-系统</td><td> ：AM-系统</td><td> 785500 </td></tr><tr><td>水平拉马</td><td>蔡茨DMZ，通用拉马</td><td> 可从 ：自动化科学</td><td> NA </td></tr><tr><td> 微机械手部分组成：电极持有人</td><td> WPI </td><td> ：WPI </td><td> M3301EH </td></tr><tr><td> 微机械手部分：线性阶段</td><td>新港423系列</td><td> 可从 ：纽波特</td><td> 423 </td></tr><tr><td> 微机械手的作品：旋转舞台</td><td>新港RSP-2 </td><td> 可从 ：新港</td><td> RSP-2 </td></tr><tr><td> 微机械手的作品：Z翻译</td><td>新港433系列</td><td> 可从 ：纽波特</td><td> 433 </td></tr><tr><td> 微机械手的作品：角度90°组装支架Z和XY轴</td><td>新港360-90 </td><td> 可从 ：纽波特</td><td> 360-90 </td></tr><tr><td> 微操纵件：X平移/线性阶段</td><td>新港423系列</td><td> 可从 ：纽波特</td><td> 423 </td></tr><tr><td> 微机械手的作品：y平移/线性阶段</td><td>新港423 </td><td> 可从 ：系列新港</td><td> 423 </td></tr><tr><td> 显微镜 </td><td>莱茨Laborlux 11 </td><td></td><td></td></tr><tR&gt; <td> 显微镜：目标</td><td>莱茨韦茨拉尔10倍，NA 0.25 </td><td></td><td> 519760 </td></tr><tr><td> 显微镜：eypieces </td><td>莱茨韦茨拉尔，Periplan 10x/18 </td><td></td><td> 519748 </td></tr><tr><td> 显微镜：第一阶段</td><td>莱茨韦茨拉尔</td><td></td><td> 513544 </td></tr><tr><td>多管毛细管</td><td> N / A </td><td> 来自 ：AM系统， </td><td> 612000 </td></tr><tr><td> Sinlge桶毛细管（GC 150F-10） </td><td>哈佛设备</td><td> 可从 ：哈佛仪器</td><td> 30-0057 </td></tr><tr><td>立式拉拔器</td><td>成茂PE-2型</td><td></td><td></td></tr><tr><td></td><td></td><td>自定义元素的微操纵器（标记为光b魏利于图1） </td><td></td></tr><tr><td>钢板</td><td></td><td></td><td></td></tr><tr><td>倾斜基地</td><td></td><td></td><td></td></tr><tr><td>附件电极支架</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td> 表2。程序中所使用的所有设备和用品的制造商和项目编号。 </td><td></td></tr></tbody></table>

manufacturing and using piggy-back multibarrel electrodes for in vivo pharmacological manipulations of neural responses

从单个神经元在体内的录音，让调查员审查的神经元发射性能，例如，在对感官刺激的反应。神经元通常会收到多个传入和/或传出兴奋性和抑制性输入，相互融合，并最终测得的响应特性的神经元的神经整合这些投入驱动。为了研究在神经系统中的信息处理，它是必要理解的一个神经细胞或神经系统的各种输入，这些输入的特定属性。一个强大的和技术上相对简单的方法来评估一个给定的神经元接收的职能作用，一定的投入是动态的和可逆的抑制或消除这些投入，并测量这种操作所造成的神经元的输出的变化。这可以通过药理学改变神经元的直接环境与背叠多管ELECTR赞歌。这些电极由一个单一的筒记录电极和多管可携带多达4个不同的突触的激动剂或拮抗剂的药物电极。药物可以用于离子电渗疗法在所希望的时间，在实验过程中，允许时间控制送货和可逆的重新配置的突触输入。因此，药物的微环境是一个功能强大，无与伦比的操作方法来测试特定的假设有关的神经回路的功能。 在这里，我们描述，如何背驮式电极的制造，以及它们是如何在体内实验过程中使用的。背驮式系统允许研究者结合了单管的任意属性（电阻，针尖大小，形状等）的记录电极与多管药物电极。这是一个标准的多电极，所有的桶都或多或少类似的形状和性能的主要优势。多管Ë首次推出了超过lectrodes 40年前的1-3，经历了一些设计上的改进2,3，直到背驮式在20世纪80年代4,5。在这里，我们提出了一系列重要的改进，使脑深部渗透在动物体内制剂由于比较薄电极轴，使损害最小的完整的背驮式电极在实验室中生产。此外，这些电极，其特征在于由低噪声录音，并具有低电阻桶所需的药物非常有效的离子电渗疗法的药物。

在这个实验中，甘氨酸受体拮抗剂士的宁盐酸盐离子电渗疗法的应用。阻断甘氨酸抑制发射的神经元通常会增加。 图4显示了样本数据从他们的反应正弦声音刺激强度的增加，传递到动物的耳朵的听觉神经元。这种类型的一个实验被称为为神经元的放电率与强度函数。大声导致穗率较高（黑线）的声音。在本实验中使用的的初始离子导入电流为15 nA的。排出电流后，电流接通和稳定在新的水平（深蓝色曲线）的变化率强度功能，逐步增加至30，45，和60 NA（橙色，绿色和淡蓝色的曲线，分别）。在每一种情况下，在相同的范围内的声音强度中的神经元的响应后，记录的变化在dischaRGE率强度的功能，新的喷射电流的稳定。最合适的电流，在这个例子中使用的喷射是45 nA至60 nA的，因为这些水平的电流不再改变不同的神经元的反应。这一结果表明，在45 nA电流，该神经元的所有甘氨酸受体已被封锁士的宁盐酸盐。喷射电流和释放甚至更多的马钱子碱没有任何进一步的增加导致的神经元的放电速率级别的功能的进一步的变化。的协议完成后，关闭喷射电流。回到基线恢复的神经反应实现了约25分钟后（红线）。这可能需要，这取决于药物的类型和量的喷出，几秒钟和几十分钟之间。 <table border="1"><tbody><tr><td> 药物 </td><td> 浓度 </td><td> 的溶液的pH值 </td><td> 溶剂 </td><td> 公司 </td><td> 猫。 ＃ </td><td> 典型的保持电流 </td><td> 典型的顶出电流 </td></tr><tr><td> GABA </td><td> 500毫米</td><td> 3.5-4.0 </td><td> DH 2 O </td><td>西格玛</td><td> A-2129 </td><td> -15 nA的</td><td> +5 nA到100 nA的</td></tr><tr><td> 甘氨酸 </td><td> 100毫米</td><td> 3.5-4.0 </td><td> DH 2 O </td><td>西格玛</td><td> G-7126 </td><td> -15 nA的</td><td> +5 nA到100 nA的</td></tr><tr><td> 荷包牡丹碱甲碘化物 </td><td> 10毫</td><td> 3.0 </td><td>在卫生署2 O 0.165 M氯化钠</td><td>西格玛</td><td> B-6889 </td><td> -15 nA的</td><td> +5 nA至60 nA的</td></tr><tr><td> 盐酸士的宁 </td><td> 10毫</td><td> 3.0 </td><td>在卫生署2 O 0.165 M氯化钠</td><td>西格玛</td><td> S-8753 </td><td> -15 nA的</td><td> +5 nA至80 nA的</td></tr><tr><td> L-谷氨酸 </td><td> 500毫米</td><td> 8 </td><td> DH 2 O </td><td>西格玛</td><td> G-1251 </td><td> 30 nA的</td><td> -10 nA至-150 nA的</td></tr><tr><td> L-天冬氨酸 </td><td> 500毫米</td><td> 8 </td><td> DH 2 O </td><td>西格玛</td><td> A-8949 </td><td> 30 nA的</td><td> -10 nA至-150 nA的</td></tr><tr><td> 红藻氨酸 </td><td> 1毫米</td><td> 9 </td><td> DH 2 O </td><td>西格玛</td><td> K-0250 </td><td> 30 nA的</td><td> 10nA至-100 nA的</td></tr></tbody></table> 表1。常用药物溶解和浓度的pH值。该表列出了最常用的突触使用的离子电渗疗法的激动剂和拮抗剂。上市的pH值环境需要分化吨的账户<html>灰褐色剂，和在不同药物之间的有效性的可变性在所建议的浓度占。 <img alt="图1" src="/files/ftp_upload/4358/4358fig1.jpg" /> 图1。三个多管移液管尖的长度不同。答：这5桶电极的尖端已经拉得长而薄的。需要注意的是秘诀是弯曲的，而且很软。这种类型的前端很难突破到所需的直径。 B：该电极的前端是太短和粗短。当推进到更深的脑区中，该电极将导致不必要的脑损伤，由于这样的事实，所述电极相对变后的前端只有几毫米厚的。 C：与正确的拉小费的电极的一个例子。虽然是长而细，尖端是坚定的，可以很容易出问题所需的尖端直径。 gure 2“SRC =”/ files/ftp_upload/4358/4358fig2.jpg“/&gt; 图2。制图电极操纵器组件操纵器组件一起使用，用显微镜来组装的背叠电极。资料标记为灰色的是市售的产品，并在表2中列出。标记为蓝色的定制在我们的机构的机加工车间加工。它们是1）1/4英寸的钢板尺寸43x26厘米有孔纽波特阶段423根据提供的纽波特孔图案钻了进去; 2），允许装配在任意角度倾斜的倾斜阶段3）连接器，安装电极支架顶端的翻译阶段。 <img alt="图3" src="/files/ftp_upload/4358/4358fig3.jpg" /> 图3。照片的样本背驮式电极 。完成后的5桶单桶的记录EL电极组装在一起ectrode。注意长轴约7mm允许的脑深部的记录。 <img alt="图4" src="/files/ftp_upload/4358/4358fig4.jpg" /> 图4。弹射电流滴定。该图显示了速度记录从一个单一的听觉神经元的功能，而动物的耳朵色调不同强度的刺激强度。大声的声音往往引起点火率较高。前神经元的速度，强度功能药物的应用，显示出穗率最低（黑线）。更高的喷射电流的阻碍日益甘氨酸受体的神经元，导致点火率逐步提高。在此神经元的最佳喷射电流为45〜60 nA的。这些喷射电流，实现了完全堵塞的所有神经元的甘氨酸受体。实验协议完成后，终止的离子电渗疗法是与神经元被允许恢复。完全恢复时的恢复率 - 强度函数实现与初始预药物恢复功能匹配。转载许可克鲁格等人 ，1995年从美国生理学会，。

Watch this Scientific Journal Video about Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses at JoVE.com

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

离子导入过程中细胞外的神经激动剂和拮抗剂<em在体内</em&gt;录音是一个功能强大的方式来操作一个神经细胞的微环境。最轻松地完成这些操作可以通过背驮式多管电极。在这里，我们描述了如何制造和使用它们在听录音。

生产和使用背驮式多管电极<em在体内</em&gt;药理手法的神经反应

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. ...

manufacturing-using-piggy-back-multibarrel-electrodes-for-vivo

Research

JoVE Journal

Neuroscience

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

10.4K 次观看.  University of Colorado Medical Campus. 离子导入过程中细胞外的神经激动剂和拮抗剂

视频: Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

一个慢性双光子薄颅骨窗口技术在体内 Neuroinflammation模型小鼠小胶质细胞成像

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

科研

神经科学

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

睾酮和社会决策，结合行为内分泌和实验经济学：

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

用于记录单机活动和Electrocorticographic信号的慢性神经电极植入手术

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

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

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

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

记录神经反应和注射示踪剂一个清醒小鼠的制备

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

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics.&#160;This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters.&#160;This article develops a protocol for in vitro and in vivo testing of neural recording electrodes.&#160;Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed.&#160;This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models.&#160;The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio.&#160;This protocol can easily be adapted to test other electrode materials and designs.&#160;The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators.&#160;The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.

Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes.

对于系统电化学神经记录电极和电生理评价方法

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.