AB衷心感谢美国国家科学基金会（1245680）根据本科教育科网络物理系统程序（1239243）和资金;和美国国防高级研究计划局（DARPA）为支持这项工作的早期阶段。这项工作的早期阶段通过AB公司阿米特教授拉尔的实验室在美国康奈尔大学进行。 AB感谢Ayesa辛哈和拉尔教授的实验指导和构思一代在那个阶段。 烟草天蛾 （林奈1763）是由生物系在杜克大学，北卡罗来纳州达勒姆，美国保持了殖民地获得的。飞蛾是5天之内羽化的使用。我们要感谢三角生物系统公司国际，尤其是大卫Juranas和Katy米莱为他们Neuroware系统其优良的技术援助和使用。我们还要在实验过程中要感谢威尔卡菲他的帮助。

注意:要按照协议要求的材料和试剂的来源，在"试剂"表下面提供。 1，准备印刷电路板（PCB）的记录电极连接注意:为了提供一个实用的实验程序，线电极被焊接到PCB上，以插入这些电极插入FFC（柔性扁平电缆）的连接器。 <ol><li>切一个0.5x5厘米2片覆铜板的。 </li><li>用细尖标记，绘制三个0.1x5cm 2矩形垫，作为蚀刻掩模图案。 </li><li>采用通风的地方或通风橱内的印刷电路板蚀刻液蚀刻暴露层压板。覆盖叠层体的切口的长度与非反应性的胶带约1厘米。填有刻度的烧杯中与至少百毫升PCB蚀刻剂和胶带的铜层压板切口到该带刻度的烧杯用胶带的内部。铜层压板cutou的一半吨应该浸没在PCB蚀刻剂。 </li><li>将烧杯20分钟的旋转平台上。 </li><li>从蚀刻剂中取出的切口，并将其放置于充满水10分钟的烧杯中。 </li><li>使用纸巾，应用异丙醇和删除的标记，露出非蚀刻铜垫。 </li><li>大约1厘米长的切断在印刷电路板成更小的正方形。 </li><li>用锋利的刀片，以3厘米长度每切两片涂覆，退火，不锈钢丝（0.11"涂覆，0.008"裸）。这些片的不锈钢丝是将要被插入到昆虫的胸部的有源电极。 </li><li>使用刀片，从每根电线的两端去掉4-5毫米的塑料涂层。建议使用显微镜。 </li><li>切10.7厘米一块绝缘的不锈钢丝，以创建一个尖端延伸的接地电极。轻轻地用刀片除去包衣或用焊接红外热融化在步骤1.9的执行。 </li><li>对于接地连接，切一块柔性（绞合或电感器）连接至4.5厘米长。 </li><li>焊接0.7 cm长的步骤1.10到1.11步准备的接地连接线制不锈钢。露出的不锈钢尖端应位于接地连接的末端。 </li><li>用胶带将制备的电极板牢固地用非反应性胶带的焊接工作区。使用磁带来掩盖所有的焊盘，但1-2毫米的板，其中的电极将被焊接。焊盘这个蒙面，无焊接结束时将被插入到在步骤4.1中所述的FFC连接。 </li><li>对准三个电极布线，使得每个的一端可被焊接到电极板的相应焊盘。适用于不锈钢磁通穿过电极焊盘，以便于焊接。 </li><li>焊接上的每个焊盘露出的电极。 </li><li>浸入电极在丙酮和异丙醇中进行10分钟每次清洁锡渣。使用超声波浴的提高了清洁性能。 </li></ol> 2，手术插入到烟草天蛾蛹注:昆虫将是最活跃的白天和黑夜之间的转换。因此，人工昼/夜循环应该被昆虫室中使用自动定时器插座成立。这些应该被设置为模拟7小时黑暗和17小时光照周期。 <ol><li>检查烟草天蛾蛹每天确定一个适当的插入时间。蛹是准备插入大约一天后，翅膀表现出黑斑。 </li><li>麻醉蛹，将它们放置在冰箱（4℃）大约为6个小时。 </li><li>准备插入工作区。工作区应包括异丙醇，锋利的镊子，刀片，和一个30 G注射针。作为一个选项，氰基丙烯酸酯粘合剂可用于增强电极的固定。</li><li>通过浸渍成或用异丙醇擦拭消毒针头，镊子，以及电极。 </li><li>从冰箱中取出蛹，并将其转移到工作空间。 </li><li>确定对应于所感兴趣的肌肉群的胸部的位置。在这个例子中，工作的重点是负责机翼上冲程运动的背腹肌肉。 </li><li>使用锋利的刀片，轻轻走过exocutical层划伤一个1x1 平方厘米的长方形。使用镊子，慢慢撕下这些作品。 </li><li> （可选）使用的真空，从胸部的暴露区域除去机翼的头发。 </li><li>慢慢地插入约5mm针头插入mesothorax所在的翅膀连接到胸部，以创建针对肌肉群的两个插入点。 </li><li>使用镊子，引导两个记录电极插入两个插入点。 </li><li> （可选）要加强机械耐久性，清洁电极周围的头发并慷慨应用氰基丙烯酸酯胶粘剂在每个插入点与线式施加的胸部。 </li><li>准备一个笼子出现适当的材料（粗糙，质感）覆盖的墙壁和天花板，使昆虫可以在出现攀升。也可使用穿孔纸箱或包装的纸张。 </li><li>准备一个坚强内固定棒具有约6厘米长和直径为2毫米。塑料搅拌器，棉签，或金属丝可用于该步骤。 </li><li>小心地伸出长鼻下方的孔滑动这根棍子。 </li><li>固定在轿厢表面上，使得蛹不能滚绕杆的两侧。定位蛹笼使得mesothorax朝上的内部。广泛的移动可能会损坏电极，损耗血淋巴，或使插入无用。 </li></ol> 3，插入接地电极进入烟草天蛾 注意:接地（参见ENCE）电极应该被插入到胸部，腹部或前端部分，以避免信号耦合。这种插入可以在蛹发育的后期阶段或后的昆虫出现来完成。用于接地电极的窗口，必须在蛹期制备为任何一个蛹或成虫期的接地电极的插入。 <ol><li>对蛹期的插入:环绕有源电极剥离mesothoracic角质层后（参见步骤2.7），通过exocutical层划伤另一个矩形使用30 G皮下注射（约0.5x0.5厘米2）上的背腹部靠近胸部针。使用在第2节中描述的技术将接地电极到这个窗口。 </li><li>对于成年阶段接地电极插入:一旦昆虫已经出现，将其放置在冰箱中4℃，6〜24小时来固定。 剩下的步骤都是相同的两个蛹和成虫阶段插入。 </li><li>准备插入背板<html>化工作空间，包括异丙醇，尖锐的镊子，一个30 G注射针，氰基丙烯酸酯胶粘剂，一段导线的应用胶水，热cauterizer（可选），和一个牙科用蜡棒（可选）。 </li><li>定位插入点大约1-2厘米的距离沿后腹部的记录电极。 </li><li>慢慢地将针头刺穿腹部，并提供一个插入位点。 </li><li>用镊子小心地将接地电极插入插入位点和施加压力，直到它是3-4毫米深。握住电极的地方，并使用导线应用周围的插入位点胶。 </li><li> （可选）要提高机械强度，使用温度cauterizer，并在尖端收集蜡的小（2-3毫米）珠。将吸头接近插入位点和施加热，使得蜡围绕电极和牢固地保持在适当位置。 </li></ol> 4，准备适配器董事会ontent"&gt;注:适配器板需要通过FFC（柔性扁平电缆）连接器的电极板连接到无线录像探头对于这一点，类似于电极板一个板需要通过以下准备步骤1.1至1.7 。 <ol><li>焊接一个FFC连接到所制备的板的一端。 </li><li>焊接3 30 AWG（美国线规）接上电线的另一端​​三个垫片。 </li><li>焊接小三接头为下一阶段描述的三个焊盘上的适配板示波器读数。 </li><li>焊接的这些三根导线的另一端​​连接到探头连接器。 </li><li>固定探头电路板的悬浮框的顶部。 </li></ol> 5，用示波器（可选）预录注:为了评估该电极的可靠性和观察信号的信噪比，拴示波器记录可以部署WIREL之前获得ESS的记录系统。应该用于此在适配器板上的微型导线连接器。 <ol><li>将示波器连接到细胞外的神经录音放大器。设置放大器参数为1赫兹，20 kHz的低通截止频率，和100的增益的高通截止频率。 </li><li>每个在转接板上，女迷你导线连接器连接到放大器的输入通道。 </li><li>从笼子里植入电极板上卸下昆虫，当它处于活动状态（在它的黎明时间）。将一片纸巾昆虫在为它测量前休息。 </li><li>使用镊子，在转接板上滑动电极板插入FFC受体。观察一个平坦和低电压基准，当昆虫是休息和肌电图（EMG）的产生尖峰昆虫扇动它的翅膀。 注:请参阅第6章:观察昆虫飞行的无线录音系统representat香港专业教育学院示波器结果。 </li><li>根据需要调整示波器观察参数。捕获数据在示波器上并保存数据。 </li></ol> 6，观察昆虫飞行的无线录音系统注意:一个电磁悬浮平台可以为肌电信号的无线录音期间拴烟草天蛾飞行建成。该磁悬浮平台由设计来平衡一个圈养机制框架。悬浮允许的帧，因此，昆虫，在测试过程中偏航无束缚的电线的约束。该框架可以使用熔融沉积成型（FDM）的机器快速原型。磁铁需要被安装到该框架，以通过一系列的磁铁在基座平台被悬浮的底部。昆虫被连接到悬挂在框架的顶部的FFC连接。该磁悬浮平台位于这是构建加兹登LED的竞技场内克60板的5x7个别LED阵列组成。该系统是基于对苍蝇15，16，17开发的水果视觉刺激的环境中建立的方法。竞技场由微控制器允许模拟顺时针和逆时针旋转以及旋转速度的控制的控制。 <ol><li>由探头连接到适配器板连接器悬浮平台上设置无线记录系统。 </li><li>从笼中取出的昆虫，当它处于活动状态时它的黎明时间最好。 </li><li>使用镊子，小心将电极板插入磁悬浮框架，使昆虫被安装在牢固悬挂在FFC受体。 </li><li>将磁棒上的探头的磁性接近开关来激活无线数据传输。蓝色指示灯将亮起，表明探头处于活动状态。 </li><li>关掉的灯房间完全黑暗。一个红色的指示灯，可以用来添加灯光的房间。在电脑上打开遥测数据采集软件，并选择适当的预加载的配置文件，如果有的话。启动数据采集开始观看的信号。 </li><li>选择用于观察的EMG信号的无线记录系统上的相应的用户界面，以确保可靠的无线连接和电极的操作。 </li><li>打开所有的灯竞技场组成部分:直流稳压电源和微控制器。微控制器可以调整每个循环的光图案的微小的旋转，也可以控制光的旋转方向。 </li><li>竞技场内慢慢平衡磁悬浮平台。上面仔细地悬浮底座中心的框架，否则该帧将被迅速拉至地面可能伤及昆虫对齐。 </li><li>启动视频记录系统。 </li><li>选择该软件的相关记录片接口。指定录音时间和文件保存的目的地。选择合适的输出设置，以保存数据。单击开始按钮来启动该软件中记录会话。这将节省可以被导入到数值计算环境中的数据文件。 </li><li>观察的昆虫苍蝇在与该LED的移动对应的方向。反向LED的方向，并确认昆虫反转方向。随意执行此多次。 </li></ol>

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Authors have no conflict of interest in this study.

有许多影响在后续步骤中的协议的记录数据的功能的记录电极的外科插入过程中几个关键步骤。记录电极应插入蛹1天参展翼点在其背侧之后。如果这个时间之后两天或两天以上进行插入，昆虫的组织将没有足够的时间来开发和周围稳定插入电极。这可能导致在成年阶段的植入电极和不可靠的记录的动作。 不插入记录电极成蛹飞行肌在超过5毫米的深度是重要的。否则，血淋巴...

插入电极，即使附有电子系统，以昆虫为遥测记录应用，一直是在自然飞行1，了解神经系统如何作用的主要方式。昆虫中安装或植入人工系统已经构成涉及到扰乱昆虫的自然飞行的潜在许多挑战。浅表附件或对成年昆虫人工平台外科插入是不可靠的而引起的身体引起的惯性和应力的力量插入的设备的可能的转移。表面附着或手术插入电极也容易出现由昆虫作为一种异物被拒绝。此外，植入操作需要周围的外骨骼去除鳞及桩。厚厚的角质层也需要穿透手术神经支配，可能导致抵押品组织损伤，从而与昆虫的自然飞行干扰。所有的t灰褐色的因素可以使手术或表面植入手术一个具有挑战性的和微妙的任务。为了缓解这些问题涉及外部连接的控制和传感系统，以昆虫，一种新颖的方法，涉及变质的增长将在本文中加以说明。 holometabolic昆虫的变态发育开始于幼虫（若虫或）改造成一个成年人与一个中间蛹期（ 图1）。变态过程涉及广泛的组织重新编程，包括变性后重塑。这种转型变成一个陆地幼虫到成虫演示几个复杂的行为2,3。 极端连体手术后昆虫的生存已经证明，其中在早期变质阶段4,5分别进行了手术。在这些手术中，发展​​组织发生喧嚣编手术伤口被修复在较短的持续时间。根据这些意见，一种新技术已经开发，其中变质增生的早期阶段（图1）中进行导电电极的植入。这使得对昆虫6生物力学安全依恋。高度可靠的接口也确保与昆虫的神经和神经肌肉系统7。这种技术被称为"早期变形记插入技术"（发射）8。 整个组织系统的重建之后，在蛹插入结构出现的成虫。飞行的肌肉群组成，以65％的总胸椎体量，因此，对于EMIT程序9比较方便的目标。在基本振翅的变化飞行供电dorsolongitudinal（DL）的形态和背腹（DV）肌肉造成机翼articulat离子的几何形状来产生升力10。因此，DL和dv肌肉功能协调已经下飞行神经生理学的一个活跃的研究课题。在电子编程的视觉环境圈养昆虫一直是研究的复杂运动器官行为11,12的神经生理学的最常用方法。发光二极管面板组成的圆柱形领域已被用于这些虚拟现实环境中，飞虫被拴在中间，其运动由动态地更新周围的全景视觉显示了模拟。在较小的昆虫，如果蝇的情况下，栓系是通过将一个金属销的昆虫的背侧胸部和放置针下的永久磁铁13,14来实现。此方法只允许运动反应的量化，通过视觉观察用高速摄影机，没有任何电生理分析。此外，该甲基OD一直是低效的，暂停烟草天蛾的较大和较重的车身。为了解决这个问题，我们从磁磁悬浮帧，其中重量轻帧，重视他们的底部磁铁是通过电磁力悬浮受惠。当与市售神经放大器和LED阵列相结合，这提供了一个平台来控制飞行，电机输出和记录烟草天蛾的相关电。

<table border="0" cellpadding="0" cellspacing="0" width="579">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Coated stainless steel wire</td>
			<td style="width:113px;">A-M Systems</td>
			<td style="width:137px;">791900</td>
			<td style="width:135px;">0.008&#8217;&#8217; bare, 0.011&#8217;&#8217; coated, annealed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Flexible electrode wire</td>
			<td style="width:113px;"></td>
			<td style="width:137px;"></td>
			<td style="width:135px;">Litz or inductor wire can be used.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Surface-mount FFC connector</td>
			<td style="width:113px;">Hirose Connector</td>
			<td style="width:137px;">FH28E-20S-0.5SH(05)</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Tweezers</td>
			<td style="width:113px;">Grobet USA</td>
			<td style="width:137px;">N/A</td>
			<td style="width:135px;">Clean with 70% alcohol before use on the insect.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Kim-Wipes</td>
			<td style="width:113px;">Kimberly-Clark Worldwide</td>
			<td style="width:137px;">34155</td>
			<td style="width:135px;">Any size delicate-wipe tissues can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Teflon tape</td>
			<td style="width:113px;">N/A</td>
			<td style="width:137px;">N/A</td>
			<td style="width:135px;">5 mm width Teflon tape.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">Hypodermic Needle</td>
			<td style="width:113px;">Becton Dickinson &#38; Co.</td>
			<td style="width:137px;">30511</td>
			<td style="width:135px;">20-30 gauge hypodermic needle can be used. Video showed 30 gauge.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">Rigid Fixation Stick</td>
			<td style="width:113px;">N/A</td>
			<td style="width:137px;">N/A</td>
			<td style="width:135px;">Variety of materials can be used (e.g. coffee stirrers)</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:195px;">Insect Emergence Cage</td>
			<td style="width:113px;">N/A</td>
			<td style="width:137px;">N/A</td>
			<td style="width:135px;">Plastic pet cage lined with packing paper or similar padding. Ventilation holes are needed.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">Thermal Cauterizer</td>
			<td style="width:113px;">Advanced Meditech International</td>
			<td style="width:137px;">CH-HI CT2103 (tip)</td>
			<td style="width:135px;">Optional equipment used for application of dental wax.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">Dental Wax</td>
			<td style="width:113px;">Orthomechanics LC., Broken Arrow, Oklahoma</td>
			<td style="width:137px;">N/A</td>
			<td style="width:135px;">Optional material used for stabilizing the electrodes on the insect.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:195px;">Magnetic Levitation Platform</td>
			<td style="width:113px;">N/A</td>
			<td style="width:137px;">N/A</td>
			<td style="width:135px;">Custom designed frame fabricated in-house with 3D prototyping</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:195px;">CA40 Instant Adhesive</td>
			<td style="width:113px;">3M</td>
			<td style="width:137px;">62-3803-0330-5</td>
			<td style="width:135px;">Avoid skin contact. Use gloves when handling.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">70% Isopropyl alcohol</td>
			<td style="width:113px;">store brand</td>
			<td style="width:137px;"></td>
			<td style="width:135px;">Commercially available from many suppliers.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:195px;">PCB Etchant</td>
			<td style="width:113px;">RadioShack</td>
			<td style="width:137px;">276-1535</td>
			<td style="width:135px;">Toxic if swallowed or ingested, skin irritant&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;"></td>
			<td style="width:113px;"></td>
			<td style="width:137px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EQUIPMENT:</td>
			<td style="width:113px;"></td>
			<td style="width:137px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Name of Reagent/Material</td>
			<td style="width:113px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="231">
			<td height="231" style="height:231px;width:195px;">TBSI Neuroware Software</td>
			<td style="width:113px;">Triangle Biosystems International</td>
			<td style="width:137px;">N/A</td>
			<td style="width:135px;">NeuroWare v1.4. Software for importing .nex files obtained at Technologies, N. NeuroExplorer Code and Scripts. (2012).at http://www.neuroexplorer.com/code.html</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">TBSI Wireless Recording System</td>
			<td style="width:113px;">Triangle Biosystems International</td>
			<td style="width:137px;">W5 FI</td>
			<td style="width:135px;">USB Base station, headstage unit, charger</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">16 Channel Amplifier A-C Amplifier</td>
			<td style="width:113px;">A-M Systems&#160;</td>
			<td style="width:137px;">950000</td>
			<td style="width:135px;">Model 3500 (110 V)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Oscilloscope&#160;</td>
			<td style="width:113px;">Agilent Technologies&#160;</td>
			<td style="width:137px;">DSO1014A</td>
			<td style="width:135px;">Oscilloscope, 100 MHz, 4 channel</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:195px;">Microscope</td>
			<td style="width:113px;">N/A</td>
			<td style="width:137px;">N/A</td>
			<td style="width:135px;">5x magnification microscope to assist visualization during electronics construction.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Ultrasonic Cleaner</td>
			<td style="width:113px;">ColeParmer</td>
			<td style="width:137px;">EW-08848-10</td>
			<td style="width:135px;">Ultrasonic Cleaner with Timer,&#160;</td>
		</tr>
	</tbody>
</table>

early metamorphic insertion technology for insect flight behavior monitoring

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on insects during their metamorphic development. Here, the implants are fused within the structure and function of the neuromuscular system as a result of metamorphic tissue remaking. The implants emerge with the insect where the development of tissue around the electronics during pupal development results in a bioelectrically and biomechanically enhanced tissue interface. This relatively more reliable and stable interface would be beneficial for many researchers exploring the neural basis of the insect locomotion with alleviated traumatic effects caused during adult stage insertions. In this article, we implant our electrodes into the indirect flight muscles of Manduca sexta. Located in the dorsal-thorax, these main flight powering dorsoventral and dorsolongitudinal muscles actuate the wings and supply the mechanical power for up and down strokes. Relative contraction of these two muscle groups has been under investigation to explore how the yaw maneuver is neurophysiologically coordinated. To characterize the flight dynamics, insects are often tethered with wires and their flight is recorded with digital cameras. We also developed a novel way to tether Manduca sexta on a magnetically levitating frame where the insect is connected to a commercially available wireless neural amplifier. This set up can be used to limit the degree of freedom to yawing &#8220;only&#8221; while transmitting the related electromyography signals from dorsoventral and dorsolongitudinal muscle groups.

整体EMIT过程的示意图示于图1中 ，示出了在天蛾的变质周期和相应的电极插入步骤的主要阶段。电极插入应该在后期蛹期羽化前4-7天进行。这使得肌纤维开发围绕电极和固定在植入物中的昆虫。 一个完成后期蛹期的插入已经插入两个活性电极和接地电极，其中的典型结果示于图2。 已完成的成年阶段插入已经插入两个活性电极和?...

Watch this Scientific Journal Video about Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring at JoVE.com

Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring

We present a novel surgical procedure to implant electrodes in Manduca sexta during its early metamorphic stages. This technique allows mechanically stable and electrically reliable coupling with the neuromuscular tissue to study flight neurophysiology dynamics. We also present a novel magnetic levitation platform for tethered studies of insect yaw.

早期变质插入技术昆虫飞行行为监控

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on ...

early-metamorphic-insertion-technology-for-insect-flight-behavior

Universidad de Cartagena UDEC

Research

JoVE Journal

Behavior

14.5K 次观看.  North Carolina State University. We present a novel surgical procedure to implant electrodes in Manduca sexta during its early metamorphic stages. This technique allows mechanically stable and electrically reliable coupling with the neuromuscular tissue to study flight neurophysiology dynamics. We also present a novel magnetic levitation platform for tethered studies of insect yaw.

视频: Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

背景和线索恐惧制约试验小鼠中使用视频分析系统

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

科研

行为学

Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing locomotive impairment in animal models of neuromuscular disease and efficacy of therapeutic drugs that may improve locomotion and/or muscle function. The open field activity measurement provides a different measure than muscle strength, which is commonly assessed by grip strength measurements. It can also show how drugs may affect other body systems as well when used with additional outcome measures. In addition, measures such as total distance traveled mirror the 6 min walk test, a clinical trial outcome measure. However, open field activity monitoring is also associated with significant challenges: Open field activity measurements vary according to animal strain, age, sex, and circadian rhythm. In addition, room temperature, humidity, lighting, noise, and even odor can affect assessment outcomes. Overall, this manuscript provides a well-tested and standardized open field activity SOP for preclinical trials in animal models of neuromuscular diseases. We provide a discussion of important considerations, typical results, data analysis, and detail the strengths and weaknesses of open field testing. In addition, we provide recommendations for optimal study design when using open field activity in a preclinical trial.

Open field activity levels are used to assess locomotive and behavioral activity levels. This protocol provides a well-designed, standardized protocol to use in preclinical trials for neuromuscular disorders.

行为和运动测量使用一个开放的领域活动监控系统的骨骼肌疾病

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing ...

Examining Recall Memory in Infancy and Early Childhood Using the Elicited Imitation Paradigm

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is commonly assessed using verbal report paradigms in adults, studying the development of this ability in preverbal infants and children proved challenging. Over the past 30 years, researchers have developed a non-verbal means of assessing recall memory known as the elicited or deferred imitation paradigm. In one variant of the procedure, participants are presented with novel three-dimensional stimuli for a brief baseline period before a researcher demonstrates a series of actions that culminate in an end- or goal-state. The participant is allowed to imitate the demonstrated actions immediately, after a delay, or both. Recall performance is then compared to baseline or to performance on novel control sequences presented at the same session; memory can be assessed for the individual target actions and the order in which they were completed. This procedure is an accepted analogue to the verbal report techniques used with adults, and it has served to establish a solid foundation of the nature of recall memory in infancy and early childhood. In addition, the elicited or deferred imitation procedure has been modified and adapted to answer questions relevant to other aspects of cognitive functioning. The broad utility and application of imitation paradigms is discussed, along with limitations of the approach and directions for future research.

The elicited imitation procedure was established to examine the development of recall memory in infancy and early childhood. This procedure has been widely used to establish a solid foundation of the nature of recall memory in infancy and early childhood.

使用仿引起范式检查召回内存在婴幼儿时期

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is ...

Protocol for Data Collection and Analysis Applied to Automated Facial Expression Analysis Technology and Temporal Analysis for Sensory Evaluation

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis (AFEA) software. Additionally, we demonstrate a method for extracting relevant emotional data output and plotting the emotional response of a population over a specified time frame. By time pairing each participant's treatment response to a control stimulus (baseline), the overall emotional response over time and across multiple participants can be quantified. AFEA is a prospective analytical tool for assessing unbiased response to food and beverages. At present, most research has mainly focused on beverages. Methodologies and analyses have not yet been standardized for the application of AFEA to beverages and foods; however, a consistent standard methodology is needed. Optimizing video capture procedures and resulting video quality aids in a successful collection of emotional response to foods. Furthermore, the methodology of data analysis is novel for extracting the pertinent data relevant to the emotional response. The combinations of video capture optimization and data analysis will aid in standardizing the protocol for automated facial expression analysis and interpretation of emotional response data.

A protocol for capturing and statistically analyzing emotional response of a population to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis software is described.

协议进行数据采集和分析应用到自动人脸表情分析技术和时序分析感官评价

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial ...

Large Volume, Behaviorally-relevant Illumination for Optogenetics in Non-human Primates

This protocol describes a large-volume illuminator, which was developed for optogenetic manipulations in the non-human primate brain. The illuminator is a modified plastic optical fiber with etched tip, such that the light emitting surface area is &#62; 100x that of a conventional fiber. In addition to describing the construction of the large-volume illuminator, this protocol details the quality-control calibration used to ensure even light distribution. Further, this protocol describes techniques for inserting and removing the large volume illuminator. Both superficial and deep structures may be illuminated. This large volume illuminator does not need to be physically coupled to an electrode, and because the illuminator is made of plastic, not glass, it will simply bend in circumstances when traditional optical fibers would shatter. Because this illuminator delivers light over behaviorally-relevant tissue volumes (&#8776; 10 mm3) with no greater penetration damage than a conventional optical fiber, it facilitates behavioral studies using optogenetics in non-human primates.

A protocol to build a tissue penetrating illuminator for delivering light over large volumes with minimal diameter is presented.

非人类灵长类动物遗传学的大体积、行为相关光照

This protocol describes a large-volume illuminator, which was developed for optogenetic manipulations in the non-human primate brain. The illuminator is a ...

An Objective and Child-friendly Assessment of Arm Function by Using a 3-D Sensor

Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment of muscle functions is an essential and important, although challenging, prerequisite for successful clinical trials. Current clinical rating scales restrain the movement abnormalities to certain predefined coarse-grained individual items. The Kinect 3-D sensor has emerged as a low-cost and portable motion sensing technology used to capture and track people&#39;s movement in many medical and research fields. A novel approach using this 3-D sensor was developed and a game-like test was designed to objectively measure the upper limb function of patients with SMA. The prototype test targeted joint movement capability. While sitting in a virtual scene, the patient was instructed to extend, flex, and lift the whole arm in order to reach and place some objects. Both kinematic and spatiotemporal characteristics of upper limb movement were extracted and analyzed, e.g., elbow extension and flexion angles, hand velocity, and acceleration. The first study included a small cohort of 18 ambulant SMA patients and 19 age- and gender-matched healthy controls. A comprehensive analysis of arm movement was achieved; however, no significant difference between the groups were found due to the mismatch of patient&#39;s capability and the test difficulty. Based on this experience, a second version of the test consisting of a modified version of the first game with increased difficulties and a second game targeting muscle endurance were designed and implemented. The new test has not been conducted in any patient groups yet. Our work has demonstrated the potential capability of the 3-D sensor in assessing such muscle function and suggested an objective approach to complement the clinical rating scales.

An objective measure of muscle functions is challenging especially in children. Based on a commercially available digital 3-D sensor, a child-friendly gaming test was developed to assess upper limb function for clinical trials.

基于3维传感器的 Arm 功能的客观和儿童友好评估

Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment ...

Key Elements of Photo Attraction Bioassay for Insect Studies or Monitoring Programs

Optimized visual attractants will increase insect trapping efficiency by using the target insect&#39;s innate behaviors (positive photo-taxis) as a means to lure the insect into a population control or monitoring trap. Light emitting diodes (LEDs) have created customizable lighting options with specific wavelengths (colors), intensities, and bandwidths, all of which can be customized to the target insects. Photo-attraction behavioral bioassays can use LEDs to optimize the attractive color(s) for an insect species down to specific life history stages or behaviors (mating, feeding, or seeking shelter). Researchers must then confirm the bioassay results in the field and understand the limited attractive distance of the visual attractants.
The cloverleaf bioassay arena is a flexible method to assess photo attraction while also assessing a range of natural insect behaviors such as escape and feeding responses. The arena can be used for terrestrial or aerial insect experiments, as well as diurnal, and nocturnal insects. Data collection techniques with the arena are videotaping, counting contact with the lights, or physically collecting the insects as they are attracted towards the lights. The assay accounts for insects that make no-choice and the arenas can be single (noncompetitive) color or multiple (competitive) colors. The cloverleaf design causes insects with strong thigmotaxis to return to the center of the arena where they can view all the options in a competitive LED tests. The cloverleaf arena presented here has been used with mosquitoes, bed bugs, Hessian fly, house flies, biting midges, red flour beetles, and psocids. Bioassays are used to develop accurate and effective insect traps to guide the development and optimization of insect traps used to monitor pest population fluctuations for disease vector risk assessments, the introduction of invasive species, and/or be used for population suppression.

Photo-attraction bioassay arenas are used to determine the optimal light color(s) to maximize insect attraction; however bioassays and methods are specific to target insect behaviors and habitats. Customizable equipment and modifications are explained for nocturnal or diurnal and terrestrial or aerial insects.

昆虫研究或监测项目照片吸引力生物测定的关键要素

Optimized visual attractants will increase insect trapping efficiency by using the target insect&#39;s innate behaviors (positive photo-taxis) as a means ...

A Wind Tunnel for Odor Mediated Insect Behavioural Assays

Olfaction is the most important sensory mechanism by which many insects interact with their environment and a wind tunnel is an excellent tool to study insect chemical ecology. Insects can locate point sources in a three-dimensional environment through the sensory interaction and sophisticated behavior. The quantification of this behavior is a key element in the development of new tools for pest control and decision support. A wind tunnel with a suitable flight section with laminar air flow, visual cues for in-flight feedback and a variety of options for the application of odors can be used to measure complex behaviour which subsequently may allow the identification of attractive or repellent odors, insect flight characteristics, visual-odor interactions and interactions between attractants and odors lingering as background odors in the environment. A wind tunnel holds the advantage of studying the odor mediated behavioural repertoire of an insect in a laboratory setting. Behavioural measures in a controlled setting provide the link between the insect physiology and field application. A wind tunnel must be a flexible tool and should easily support the changes to setup and hardware to fit different research questions. The major disadvantage to the wind tunnel setup described here, is the clean odor background which necessitates special attention when developing a synthetic volatile blend for field application.

Here, we describe the construction and use of a wind tunnel for odor mediated behavioural assays with insects. The wind tunnel design facilitates the release of odor sources by several methods, with and without visual stimuli. Wind tunnel experiments are important methods to identify behaviorally active volatile chemicals.

气味介导的昆虫行为检测风洞

Olfaction is the most important sensory mechanism by which many insects interact with their environment and a wind tunnel is an excellent tool to study ...

Using Flight Mills to Measure Flight Propensity and Performance of Western Corn Rootworm, Diabrotica virgifera virgifera (LeConte)

The western corn rootworm, Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), is an economically important pest of corn in the northern United States. Some populations have developed resistance to management strategies including transgenic corn that produces insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt). Knowledge of western corn rootworm dispersal is of critical importance for models of resistance evolution, spread, and mitigation. Flight behavior of an insect, especially over a long distance, is inherently difficult to observe and characterize. Flight mills provide a means to directly test developmental and physiological impacts and consequences of flight in the laboratory that cannot be obtained in field studies. In this study, flight mills were used to measure the timing of flight activity, total number of flights, and the distance, duration, and speed of flights taken by female rootworms during a 22-h test period. Sixteen flight mills were housed in an environmental chamber with programmable lighting, temperature, and humidity control. The flight mill described is of a typical design, where a flight arm is free to rotate about a central pivot. Rotation is caused by flight of an insect tethered to one end of the flight arm, and each rotation is recorded by a sensor with a time-stamp. Raw data are compiled by software, which are subsequently processed to provide summary statistics for flight parameters of interest. The most difficult task for any flight mill study is attachment of the tether to the insect with an adhesive, and the method used must be tailored to each species. The attachment must be strong enough to hold the insect in a rigid orientation and to prevent detachment during movement, while not interfering with natural wing motion during flight. The attachment process requires dexterity, finesse, and speed, making video footage of the process for rootworms of value.

Using Flight Mills to Measure Flight Propensity and Performance of Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

Flight mills are important tools for comparing how age, sex, mating status, temperature, or various other factors may influence an insect&#8217;s flight behavior. Here we describe protocols to tether and measure the flight propensity and performance of western corn rootworm under different treatments.

使用飞行磨坊测量西部玉米根虫的飞行倾向和性能，二溴二恶十二药病毒病毒（LeConte）

The western corn rootworm, Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), is an economically important pest of corn in the northern ...

A Novel Pavlovian Fear Conditioning Paradigm to Study Freezing and Flight Behavior

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat are characteristic of various anxiety disorders, which are the most prevalent form of mental illness in the United States. Discovering the neurobiological mechanisms responsible for defensive behaviors will aid in the development of novel therapeutic interventions. Pavlovian fear conditioning is a widely used laboratory paradigm to study fear-related learning and memory. A major limitation of traditional Pavlovian fear conditioning paradigms is that freezing is the only defensive behavior monitored. We recently developed a modified Pavlovian fear conditioning paradigm that allows us to study both conditioned freezing and flight (also known as escape) behavior within individual subjects. This model employs higher intensity footshocks and a greater number of pairings between the conditioned stimulus and unconditioned stimulus. Additionally, this conditioned flight paradigm utilizes serial presentation of pure tone and white noise auditory stimuli as the conditioned stimulus. Following conditioning in this paradigm, mice exhibit freezing behavior in response to the tone stimulus, and flight responses during the white noise. This conditioning model can be applied to the study of rapid and flexible transitions between behavioral responses necessary for survival.

Defensive behavioral responses are contingent upon threat intensity, proximity, and context of exposure. Based on these factors, we developed a classical conditioning paradigm that elicits clear transitions between conditioned freezing and flight behavior within individual subjects. This model is crucial for the understanding the pathologies involved in anxiety, panic, and post-traumatic stress disorders.

研究冻结和飞行行为的新型帕夫洛夫恐惧调理范式

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat ...