This material is based on the works supported by Nanyang Assistant Professorship (NAP, M4080740), Agency for Science, Technology and Research (A*STAR) Public Sector Research Funding (PSF, M4070190), A*STAR-JST (The Japan Science and Technology Agency) joint grant (M4070198), and Singapore Ministry of Education (MOE2013-T2-2-049). The authors would like to thank Mr. Roger Tan Kay Chia, Prof. Low Kin Huat, Mr. Poon Kee Chun, Mr. Chew Hock See, Mr. Lam Kim Kheong and Dr. Mao Shixin at School of MAE for their support in setting up and maintaining the research facilities. The authors thank Prof. Michel Maharbiz (U.C. Berkeley) his advice and discussion, Prof. Kris Pister and his group (U.C. Berkeley) for their support in providing the GINA used in this study.

1.动物研究<ol><li>后个人Mecynorrhina torquata甲虫英寸（6厘米，将8g）在单独的塑料容器木质颗粒床上用品。 </li><li>每次喂糖甲虫果冻（12毫升）每3天杯。 </li><li>保持饲养室的温度和湿度在25℃和60％，分别为。 </li><li>植入细线电极之前测试每个甲虫的飞行能力。 <ol><li>轻轻扔甲虫到空气中。如果甲虫能飞的时间超过10秒，连续5个试验，得出这样的结论甲虫拥有定期航班的能力和使用它为后续飞行实验。夺回甲虫，关闭在房间里所有的灯，使其黑暗。这将导致甲虫终止飞行。 注:甲虫自发地开始飞走时释放到空气中。最好是在一个大的封闭室以进行飞行实验如在图1中（16×8×4米所示的<suP&gt; 3的一个动作捕捉空间12.5×8×4米3），作为飞行甲虫动作非常快（约3-5米/秒），并在空气中转动时绘制大弧。 </li></ol></li></ol> 2.植入电极<ol><li>通过将它放置在填充有CO 2的1分钟13,16,20-24塑料容器麻醉甲虫。 </li><li>通过在热水中浸渍10秒软化的牙模。放置一个木块上麻醉甲虫和软化的牙模固定它。牙科用蜡自然冷却并在几分钟之内固化。 </li><li>切割绝缘电线银（127微米裸内径，外径178微米与全氟烷涂时）到25毫米的长度，以用作细线电极植入。 </li><li>通过在每个导线的两端燃烧绝缘体露出裸露的银3毫米。 </li><li>用细尖的剪刀来创建SMA解剖甲虫的角质层的顶面在metepisternum（ 图2c）的约4×4mm的LL窗口。注意:再用柔软的褐色表皮暴露出来， 如图2c中 - 即该3AX肌肉位于软角质层下方。 </li><li>皮尔斯使用昆虫销（尺寸00）的两个孔中（ 图2d）之间2mm的距离暴露的棕色角质层两个孔。 </li><li>插入两个线电极（包括在步骤2.4制得一种活性和一个返回电极）小心穿过孔，并将它们植入到每个3AX肌肉以3毫米的深度。 </li><li>固定植入电极和举行的地方通过孔下降融化的蜂蜡，避免接触和短路。如果需要的话，通过用热烙铁的前端接触蜂蜡回流过角质层的蜂蜡。蜂蜡快速凝固并加强植入。 注意:要检查是否着床是正确的，甜菜的鞘翅乐可解除电刺激期间遵守3AX肌肉的运动。 </li></ol> 3.无线背包大会注:背包包括一个内置的无线电微控制器上的4层状FR-4板（1.6×1.6 平方厘米）的。背包是由锂聚合物微电池（3.7 V，350毫克，10毫安）驱动。背包包括电池的总质量为1.2±0.26 g的小于甲虫（10 g体重的30％）的有效负载容量。背包被预编程成接收无线通信，并且有两个输出通道。 <ol><li>使用双面胶带清洁前胸背板表面（去除角质层蜡层）。然后，附上甲虫搭配了一块双面胶带的前胸背板的背包。 </li><li>植入电极的端部连接至背包的输出。 </li><li>环绕微电池的反光带，产生一个标记FO- [R动作捕捉摄像头检测。 </li><li>附加微电池使用一块双面胶带，使反光带可以通过运动捕获相机被检测的背包的顶部。 </li></ol> 4.无线控制系统注意:在此情况下，术语无线控制系统包括用于在遥控器的接收器，膝上型计算机，以运行定制飞行控制软件，基站，背包，以及动作捕捉系统。 <ol><li>经由USB端口连接的遥控器的膝上型计算机的基站和接收器。 </li><li>切换动作捕捉系统，它通过一个以太网端口连接到膝上型计算机。 </li><li>挥动魔杖校准（由运动捕捉系统的供应商公司提供的）完全覆盖运动捕捉空间进行体积校准。 <ol><li>从笔记本电脑的桌面上打开动作捕捉软件。点击和DR公司选择"资源"面板的"系统"菜单上的所有摄像头。 </li><li>点击"3D透视"菜单，选择"相机"切换到摄像机视图。单击"工具"面板上的"照相机"选项卡上显示校准设置。点击"开始"，在"创建相机面具"菜单上，以消除来自摄像机的噪音，然后选择"停止"噪音以蓝色掩盖了。 </li><li>单击并从"魔杖"菜单和"照相机"选项卡上的"L型框架"菜单中选择"5标记棒＆L型框架"。将"棒数"到2500，点击"开始"的"校准相机"菜单上，并在整个动作捕捉空间波校准魔杖。当魔杖计数达到2,500校准过程将停止。 </li><li>重复校准过程，如果图像错误（在"工具"面板中的"摄像机"选项卡的底部）高于0.3˚F或任何摄像头。校准后，放在地上的魔杖的动作捕捉空间的中间，然后点击"设置音量起源"菜单上的"开始"设置动作捕捉空间的原点。 </li></ol></li><li>使用虚拟测试记录由在运动捕获空间的用户挥手的标记的运动路径，并确认该标记是否被检测和跟踪检查动作捕捉系统的覆盖范围。如果标记检测过程中经常丢失，重复体积校准，直到假人测试成功。 <ol><li>点击"捕捉"选项卡上的"工具"面板，然后通过整个动作捕捉空间挥舞采样标记来记录它的轨迹前的"捕捉"菜单上的"开始"。 </li><li>录制完毕后，点击"运行重构流水线"来重建标记的位置，并检查录音质量。 </li></ol></li><li>连接为MicroB的端子attery（附接到背包在步骤3.4），以背包的电源引脚。 </li><li>测试笔记本电脑和使用自飞行控制软件背包之间的无线通信。在软件点击"开始"命令，检查显示连接状态。 </li></ol> 5.免费飞行实验<ol><li>开展飞行测量竞技场16×8×4米3的自由飞行实验。 </li><li>输入相应的参数，以飞行控制软件（电压，脉冲宽度，频率，和刺激持续时间）。注意:对于示范，我们固定的电压3伏，脉冲宽度为3毫秒，而刺激的持续时间为1秒，变化后的频率为60至100赫兹。 <ol><li>在软件的屏幕，类型3 3 V在"电压"中，1000为1000毫秒的"刺激持续时间"中，3为在"脉冲宽度"框3毫秒，而在赫兹的期望的频率的"频率"框Øn中的命令窗口。 </li></ol></li><li>松开背包式甲虫到空气中允许它飞行舞台上自由飞翔。手动触发刺激时，甲虫进入动作捕捉空间。按遥控器上的相应命令按钮（左或右），以刺激对甲虫的左侧或右侧的目标肌肉。 注意:一旦该按钮被按下，在笔记本电脑上运行的飞行控制软件生成的命令，并将其发送到背包。背包然后输出该电刺激到感兴趣的肌肉（左或右侧）。 </li><li>观察甲虫的实时反应的刺激时，重建使用三维绘图软件中的数据。 <ol><li>选择记录在"甲壳虫显示"窗口的数据表的试验之一，并单击"导出熊猫"该试验的数据复制到文件夹的分析和运行3D图形模块。 </li><li>对按"N"键盘刺激信号与记录的轨迹结合。按我以显示与突出显示的刺激时期甲壳虫的轨迹。 </li></ol></li></ol>

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The authors declare that there are no conflicts of interest.

植入过程是重要的，因为它影响了实验的可靠性。电极应在3毫米或取决于甲虫（避免与附近的肌肉接触）的大小更小的深度插入到肌肉。如果电极触摸附近的肌肉，可能由于附近的肌肉的收缩发生不期望的马达动作和行为。两个电极应该很好对齐，以确保不会发生短路。当熔融和使用烙铁回流蜂蜡，该实验者有要小心和焊料尽快，由于肌肉可以通过用高温下长时间接触被燃烧，导致肌肉的故障。虽然除去角质层需要访问3AX肌肉，插入和密封过程花费不到一分钟，并设法尽量减少对肌肉损伤。昆虫被送回的实验之后的饲养室和可为生存长达3个月以上（其使用寿命结束）。为了保持甲虫，甲虫应投喂，并允许每20个连续试验后休息3〜4小时尽可能多的连续（40〜50）的飞行试验后，昆虫会变得疲劳，可能无法打开的良好表现它的翅膀。 作为用于自由飞行的实验中，对于动作捕捉系统体积校准是必要的，因为它影响轨迹跟踪精度。它填补了摄像机的视图充分校准棒的波具有小于0.3的所有的摄像机的图像误差，以保持运动跟踪系统的准确度是重要的。此外，标记的表面应洁净，或三维运动捕捉系统可能会经常想念标记。校准后，假人的测试应该通过挥动电池在规定的容积逆向反光胶带包裹检查动作捕捉系统的覆盖范围进行。为了测试运动跟踪精度，我们测量的两个标记在飞行舞台上移动的距离。所述标记物固定在一个硬纸板为200毫米到彼此的距离。董事会在整个飞行竞技场搬到获得两个标志的各种职务。的标准偏差，然后计算为1.3毫米（N = 3000）。 自由飞行测试设施（ 图1和4）让我们跟踪飞行昆虫的位置（X，Y和Z）与时间戳一起。由于只有单个标记附着到甲虫和3D运动捕捉系统只检测该标记，甲虫被视为一个粒子或一个质点。这样，从飞行甲虫数据具有的位置信息，但缺乏取向。因此，从甲虫的位置数据运动学分析仅提供沿X，Y上的平移速度和加速度，以及未经角速度或角加速度沿Z轴有关偏航，俯仰和翻滚轴旋转。固定在甲虫多个标记（诸如图6中所示的一个），必须使用对于3D运动捕捉系统治疗飞翔昆虫作为刚体和记录旋转和平移的数据。然而，实验者必须注意到这些标记到一个飞甲虫的动力学的贡献，因为标记是一个不小片胶带的，但需要足够大，以通过与最小的跟踪损耗照相机系统进行检测。这样的安排与多个标记的附件可能显著增加其质量和惯性25的时刻。此外，飞行竞技场的大小可设置为尽可能大的运动跟踪系统的覆盖范围内，以减少约束甲虫的自由飞行的行为。在本文中，飞行舞台的大小定义了基于运动捕捉系统（12.5×8×4米3）的最大覆盖范围。 例如 ，对对侧转7和3AX肌肉为同侧转13 basalar肌肉。此外，昆虫的神经系统的某些部分可以诱导各种反应。视叶刺激可诱发飞行萌生7，而天线的刺激可以在行走的昆虫12诱发对侧转动。此外，我们可以从一种电刺激器的肌电录音机其自然行为3,26过程中记录的昆虫的活动改变背包的功能。 甲虫的自由飞行的刺激有助于揭示和确认由enabli的3AX肌肉的自然功能的昆虫自由空气中移动的瞬时反应纳克的意见。这些信息是不是拴条件下11,13,27-30可用。昆虫的行为被拴系的条件下约束并可能自由飞行从不同的，可能导致的昆虫的行为的不正确的理解。因此，使用这种技术自由飞行的刺激是用于确认从拴系实验得出的假设一个强有力的工具。此外，昆虫机混合系统优于当前人工中的机车的能力和功耗13,17,31,32而言扑机器人。 作为它们继承活昆虫的复杂和灵活的结构和机车能力，降低了制造工艺的生产时间昆虫机混合系统可以代替在未来人工机器人。各种机车功能可以帮助昆虫机的混合动力系统操作更有效地约束的空间，包括步行和飞行， 如相结合，在救援任务。此外，昆虫机混合系统可以潜在地用作农业昆虫控制的工具，因为它可能能够融入天然昆虫菌落并有助于控制其活动。

An insect-machine hybrid system, often referred to as a cyborg insect or biobot, is the fusion of a living insect platform with a miniature mounted electronic device. The electronic device, which is wirelessly commanded by a remote user, outputs an electrical signal to electrically stimulate neuromuscular sites in the insect via implanted wire electrodes to induce user desired motor actions and behaviors. In the early stages of this research field, researchers were limited to conducting wireless recording of the muscular action of an insect, using simple analog circuits comprised of surface-mounted components1-3. The development of system-on-a-chip technology with radio frequency functionality enabled not only the wireless recording of neuromuscular signals but also the electrical stimulation of the neuromuscular sites in living insects. At present, a built-in radio microcontroller is small enough to be mounted on living insects without causing any obstructions to their locomotion4-13.
The development of the built-in radio microcontroller allows researchers to determine electrical stimulation protocols to induce desired motor actions to control the locomotion of the insect of interest. On the ground, researchers have demonstrated walking control by stimulating the neuromuscular sites of cockroaches4,12,14, spiders15, and beetles16,17. In the air, the initiation and cessation of flight were achieved using different methods such as the stimulation of the optic lobes (the massive neural cluster of a compound eye) in beetles7,9 and brain sub-regions in bees18, whereas turning control has been demonstrated by stimulating the antennae muscles and nervous system of the abdomens in moths11,19 and the flight muscles of beetles7,9,13. In most cases, a built-in radio microcontroller was integrated on a custom-designed printed circuit board to produce a miniature wireless stimulator (backpack), which was mounted on the insect of interest. This allows wireless electrical stimulation to be applied to a freely walking or flying insect. Such a microcontroller-mounted insect is what is referred to as an insect-machine hybrid system. 
This study describes the experimental protocols for building an insect-machine hybrid system, wherein a living beetle is employed as the insect platform, and instructs on how to operate the robot and test its flight control systems. The third axillary sclerite (3Ax) muscle was chosen as the muscle of interest for electrical stimulation and demonstration of left or right turning control13. A pair of thin silver wire electrodes was implanted in both the left and right 3Ax muscles. Moreover, a backpack was mounted on the living beetle. The other ends of the wire electrode were connected to the output pins of the microcontroller. The backpack was small enough for the beetle to carry in flight. Thus, this allows an experimentalist to remotely stimulate the muscle of interest of an insect in free flight and investigate its reactions to the stimulations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mecynorrhina torquata beetle</td>
			<td>Kingdom of Beetle Taiwan</td>
			<td></td>
			<td>10 g, 8 cm, pay load capacity is 30% of the body mass 
			Aproval of importing and using by Agri-Food and Veterinary Authority of Singapore (AVA; HS code: 01069000, product code: ALV002).</td>
		</tr>
		<tr>
			<td>Wireless backpack stimulator</td>
			<td>Custom</td>
			<td></td>
			<td>TI CC2431 micocontroler 
			The board is custom made based on the GINA board from Prof. Kris Pister&#8217;s lab. The layout of GINA board can be found at&#160;&#160;&#160; https://openwsn.atlassian.net/wiki/display/OW/GINA</td>
		</tr>
		<tr>
			<td>Wii Remote control</td>
			<td>Nintendo</td>
			<td></td>
			<td>Bluetooth remote control to send the command to the operator laptop</td>
		</tr>
		<tr>
			<td>BeetleCommander v1.8</td>
			<td>Custom. Maharbiz group at UC Berkeley and Sato group at NTU</td>
			<td></td>
			<td>Establish the wireless communication of the backpack and the operator laptop. Configure the stimulus parameters and log the positional data. Visualize the flight data.</td>
		</tr>
		<tr>
			<td>GINA base station</td>
			<td>Kris Pister group at UC Berkeley</td>
			<td></td>
			<td>TI MSP430F2618 and AT86RF231</td>
		</tr>
		<tr>
			<td>Motion capture system</td>
			<td>VICON</td>
			<td>T160</td>
			<td>8 cameras for a flight arena of 12.5 x 8 x 4 m</td>
		</tr>
		<tr>
			<td>Motion capture system</td>
			<td>VICON</td>
			<td>T40s</td>
			<td>12 cameras for a flight arena of 12.5 x 8 x 4 m</td>
		</tr>
		<tr>
			<td>Micro battery</td>
			<td>Fullriver</td>
			<td>&#160;201013HS10C&#160;</td>
			<td>3.7V, 10 mAh</td>
		</tr>
		<tr>
			<td>Retro reflective tape</td>
			<td>Reflexite</td>
			<td>V92-1549-010150</td>
			<td>V92 reflective tape, silver color</td>
		</tr>
		<tr>
			<td>PFA-Insulated Silver Wire&#160;</td>
			<td>A-M systems</td>
			<td>786000</td>
			<td>127 &#181;m bare, 177.8 &#181;m coated, 3 mm bare silver flame exposed at tips</td>
		</tr>
		<tr>
			<td>SMT Micro Header&#160;</td>
			<td>SAMTEC</td>
			<td>FTSH-110-01-L-DV</td>
			<td>0.3 x 6 mm, bend to make a 3 mm long slider to secure the electrode into the PCB header.</td>
		</tr>
		<tr>
			<td>Beeswax</td>
			<td></td>
			<td></td>
			<td>Secure the electrodes</td>
		</tr>
		<tr>
			<td>Dental Wax</td>
			<td>Vertex</td>
			<td></td>
			<td>Immobilize the beetle</td>
		</tr>
		<tr>
			<td>Insect pin</td>
			<td>ROBOZ</td>
			<td>RS-6082-30</td>
			<td>Size&#160; 00; 0.3mm Rod diameter; 0.03 mm tip width; 38 mm Length&#160; 
			Make electrode guiding holes on cuticle</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>DUMONT</td>
			<td>RS-5015</td>
			<td>Pattern #5; .05 X .01mm Tip Size; 110mm Length 
			Dissecting and implantation</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>ROBOZ</td>
			<td>RS-5620</td>
			<td>Vannas Micro Dissecting Spring Scissors; Straight; 3mm Cutting Edge; 0.1mm Tip Width; 3&#34; Overall Length&#160; 
			Dissecting and implantation</td>
		</tr>
		<tr>
			<td>Potable soldering iron</td>
			<td>DAIYO</td>
			<td>DS241</td>
			<td>Reflow beeswax</td>
		</tr>
		<tr>
			<td>Hotplate&#160;</td>
			<td>CORNING</td>
			<td>PC-400D</td>
			<td>Melting beeswax and dental wax</td>
		</tr>
		<tr>
			<td>Flourescent lamp</td>
			<td>Philips</td>
			<td>TL5 14W</td>
			<td>Light the entire flight arena with 30 panels (60 x 60 cm2). Each panel has 3 lamps. 
			14 W, 549 mm x 17 mm&#160;</td>
		</tr>
	</tbody>
</table>

insect-machine hybrid system: remote radio control of a freely flying beetle (mercynorrhina torquata)

启用无线电数字电子设备的兴起，促使利用小型无线神经肌肉录像机和刺激为研究飞行昆虫的行为。借助该技术使用本协议中所述活虫平台昆虫机混合动力系统的开发。此外，该协议提出的系统结构和用于在不受限制的昆虫评估飞行肌的功能自由飞行的实验程序。为了演示，我们有针对性的第三腋骨片（3AX）肌肉来控制和实现飞行甲虫向左或向右转向。薄银线电极植入上的甲虫两侧的肌肉3AX。这些被连接到一个无线背包的输出（ 即神经肌肉电刺激器）装上甲虫的前胸。肌肉是由交替的刺激侧（左或右），或者改变stimulatio在自由飞行刺激N频点。甲虫转向同侧当肌肉受到刺激并展出，以增加频率的召。植入过程和三维运动捕获相机系统的体积校准需要小心进行，以避免损坏肌肉和丢失标记的轨道，分别。该方法是非常有益的，研究昆虫飞行，因为它有助于揭示感兴趣的飞行肌的功能在自由飞行。

电极植入手术方式如图2薄银线电极植入通过刺穿上的肌肉（ 图2d - E）软角质层小孔甲虫的肌肉3AX。此软角质层刚好在basalar肌肉的apodema上面除去metepisternum的前部后（ 图2d - 三 ）。然后在电极用蜂蜡（ 图2F）固定。 图3示出了使用一个完整的甲虫构造昆虫机的混合动力系统中的步骤。 图2和图 3b示出用于植入金属细线（刺激电极）到感兴趣的肌肉（例如，方法，在图2中 ，3AX肌肉在此研究中使用），并安装在甲虫的前胸背板的背包。电线的自由端分别插入在跳线连接器中的孔上的背包，其被电连接到集成在背包（ 图3c）的微控制器的输入/输出管脚。最后，一​​个微电池被安装和微电池的电源线被连接到在跳线连接器中的孔，导致微控制器的接地端和正电源端子。 无线控制系统在图4中，当用户按下遥控器上的控制器（ 图4c）的命令按钮，在膝上型计算机（ 图4d）的飞行控制软件示出产生并经由基站无线地发送命令到背包站（ 图4b）。运动捕获系统（ 图4e）检测的甜菜的位置（X，Y和Z）乐和时间戳标记它。然后，该数据被馈送到膝上型电脑，和飞行控制软件同步与刺激信号的数据。 代表转弯控制的结果示于图5中 。在3AX肌肉的活化被认为引起同侧13的翼拍幅减少，从而导致执行在自由飞行中的同侧转弯的甲虫。所述3AX肌肉的电刺激显示出类似的效果作为甲虫转向同侧当左或右3AX肌肉被刺激13。甲虫的旋转速度评级为刺激频率的函数。 <img alt="图1" src="/files/ftp_upload/54260/54260fig1.jpg" /> 图1:免费飞行的舞台安排在自由飞翔的舞台被安排进两个部分:控制空间（3.5×8×4米3）用于建立注入试剂盒（显微镜和清扫工具）和控制室（计算机，无线基站，和摄像机控制器），而运动捕捉空间（ 12.5×8×4米3）上覆盖20的近红外摄像机来记录甲虫的位置（X，Y和Z）。飞行竞技场装备有30采光板（60×60 平方厘米，48 W），使其亮如在实验过程中日间的条件。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54260/54260fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54260/54260fig2.jpg" /> 图2:程序电极植入甲虫被麻醉并固定有牙科蜡为植入过程的木块上。 （ 一 - &lt;STRONG&gt; c）一个小窗口是在甲虫访问3AX肌肉metepisternum打开。 （d）利用昆虫销，两孔为2毫米的距离进行穿孔上承载3AX肌肉内角质层。 （e）有电极插入通过这些孔的肌肉和用镊子保持就位，以确保尖端之间不发生串扰。 。（F - G），然后将电极固定在用蜂蜡甲虫<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54260/54260fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54260/54260fig3.jpg" /> 图 3: 使用程序一个完整的甲虫产生昆虫机的混合动力系统 （A）的利息为生的甲虫肌肉植入机智HA对银线电极。 （b）与蜂蜡固定电极之后，我们安装在使用双面胶带甲虫的前胸背板的背包。 （c）该电极的自由端插入到背包的输出，并与micropin头固定。 （d）一个微电池，其上覆盖着复古反光带，安装在使用双面胶带，并连接到背包的电源引脚，背包<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54260/54260fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54260/54260fig4.jpg" /> 图 4: 为自由飞行实验无线系统中的无线系统由（a）一种电子人甲虫，（b）一种无线基站，（ 三的</s仲&gt;）的远程，（d）与蓝牙接收器的工作笔记本插上，和（e）三维运动捕捉系统。当用户按下遥控器上的命令按钮，在笔记本电脑定制的飞行控制软件，通过它通过USB接口插入笔记本电脑基站发送的刺激命令无线传输到电子人甲虫。一旦背包接收命令，它产生刺激肌肉的电刺激信号。同时，运动捕捉系统记录了甲虫的三维坐标和将它们送至笔记本电脑与刺激数据同步。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54260/54260fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54260/54260fig5.jpg" /> 图5:beetl的行为È由于在自由飞行的3AX肌肉的电刺激（a）中的甲虫转向同侧时向左或向右3AX肌肉受到刺激，及回转运动被分级为刺激频率的函数。 （ 二 ）当左或右3AX肌肉在序列被刺激的飞行甲虫的锯齿形路径。刺激参数为为3 V的幅度，3毫秒的脉冲宽度和60-100赫兹的频率。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54260/54260fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54260/54260fig6.jpg" /> 图6: 建议的标记套用于使用（ 一个 ）三个标记跟踪甲虫配置的三维方向（滚动，俯仰和偏转），（b）四。标记，以及（c）五种标志物。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54260/54260fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata at JoVE.com

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata

This protocol describes the process of constructing an insect-machine hybrid system and carrying out wireless electrical stimulation of the flight muscles required to control the turning motion of a flying insect.

昆虫机的混合动力系统:一个自由飞翔的甲壳虫无线电远程控制（<em&gt; Mercynorrhina torquata</em&gt;）

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight ...

insect-machine-hybrid-system-remote-radio-control-freely-flying

Nanyang Technological University

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Neuroscience

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle (Mercynorrhina torquata)

12.1K 次观看.  Nanyang Technological University. This protocol describes the process of constructing an insect-machine hybrid system and carrying out wireless electrical stimulation of the flight muscles required to control the turning motion of a flying insect.

视频: Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

本地化的蛋白质三维神经干细胞培养:一个混合的可视化方法

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

科研

神经科学

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

自由游泳弱电鱼的长期行为跟踪

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

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

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

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

的简化方法超低密度，长期主要海马神经元文化

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

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

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

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

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

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

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

约束和自由飞行，蜜蜂的神经药理学操纵，<em&gt;蜜蜂</em

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

使用新颖的方法在自由移动的新生大鼠中记录脑电图

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

自由移动仔猪的无创脑电图记录

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

自由生育小鼠的长期感觉冲突

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, conventional microdrive designs often only allow use of one type of electrode to record from single or multiple regions, limiting the yield of single-unit or depth profile recordings. It also often limits the ability to combine electrode recordings with optogenetic tools to target pathway and/or cell type specific activity. Presented here is a hybrid microdrive array for freely moving mice to optimize yield and a description of its fabrication and reuse of the microdrive array. The current design employs nine tetrodes and one opto-silicon probe implanted in two different brain areas simultaneously in freely moving mice. The tetrodes and the opto-silicon probe are independently adjustable along the dorsoventral axis in the brain to maximize the yield of unit and oscillatory activities. This microdrive array also incorporates a set-up for light, mediating optogenetic manipulation to investigate the regional- or cell type-specific responses and functions of long-range neural circuits. In addition, the opto-silicon probe can be safely recovered and reused after each experiment. Because the microdrive array consists of 3D-printed parts, the design of microdrives can be easily modified to accommodate various settings. First described is the design of the microdrive array and how to attach the optical fiber to a silicon probe for optogenetics experiments, followed by fabrication of the tetrode bundle and implantation of the array into a mouse brain. The recording of local field potentials and unit spiking combined with optogenetic stimulation also demonstrate feasibility of the microdrive array system in freely moving mice.

This protocol describes the construction of a hybrid microdrive array that allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe in two brain regions in freely moving mice. Also demonstrated is a method for safely recovering and reusing the opto-silicon probe for multiple purposes.

混合微驱系统,带可回收的光电探头和Tetrode,用于自由移动小鼠的双位点高密度记录

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

自由游泳鱼中可移动的特龙对神经元的无线电生理记录

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...

昆虫机的混合动力系统:一个自由飞翔的甲壳虫无线电远程控制（

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