早期变质插入技术昆虫飞行行为监控
Summary
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.
Abstract
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 “only” while transmitting the related electromyography signals from dorsoventral and dorsolongitudinal muscle groups.
Introduction
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。
Protocol
Representative Results
Discussion
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…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Coated stainless steel wire | A-M Systems | 791900 | 0.008’’ bare, 0.011’’ coated, annealed |
| Flexible electrode wire | Litz or inductor wire can be used. | ||
| Surface-mount FFC connector | Hirose Connector | FH28E-20S-0.5SH(05) | |
| Tweezers | Grobet USA | N/A | Clean with 70% alcohol before use on the insect. |
| Kim-Wipes | Kimberly-Clark Worldwide | 34155 | Any size delicate-wipe tissues can be used. |
| Teflon tape | N/A | N/A | 5 mm width Teflon tape. |
| Hypodermic Needle | Becton Dickinson & Co. | 30511 | 20-30 gauge hypodermic needle can be used. Video showed 30 gauge. |
| Rigid Fixation Stick | N/A | N/A | Variety of materials can be used (e.g. coffee stirrers) |
| Insect Emergence Cage | N/A | N/A | Plastic pet cage lined with packing paper or similar padding. Ventilation holes are needed. |
| Thermal Cauterizer | Advanced Meditech International | CH-HI CT2103 (tip) | Optional equipment used for application of dental wax. |
| Dental Wax | Orthomechanics LC., Broken Arrow, Oklahoma | N/A | Optional material used for stabilizing the electrodes on the insect. |
| Magnetic Levitation Platform | N/A | N/A | Custom designed frame fabricated in-house with 3D prototyping |
| CA40 Instant Adhesive | 3M | 62-3803-0330-5 | Avoid skin contact. Use gloves when handling. |
| 70% Isopropyl alcohol | store brand | Commercially available from many suppliers. | |
| PCB Etchant | RadioShack | 276-1535 | Toxic if swallowed or ingested, skin irritant |
| EQUIPMENT: | |||
| Name of Reagent/Material | Company | Catalog Number | Comments |
| TBSI Neuroware Software | Triangle Biosystems International | N/A | NeuroWare v1.4. Software for importing .nex files obtained at Technologies, N. NeuroExplorer Code and Scripts. (2012).at http://www.neuroexplorer.com/code.html |
| TBSI Wireless Recording System | Triangle Biosystems International | W5 FI | USB Base station, headstage unit, charger |
| 16 Channel Amplifier A-C Amplifier | A-M Systems | 950000 | Model 3500 (110 V) |
| Oscilloscope | Agilent Technologies | DSO1014A | Oscilloscope, 100 MHz, 4 channel |
| Microscope | N/A | N/A | 5x magnification microscope to assist visualization during electronics construction. |
| Ultrasonic Cleaner | ColeParmer | EW-08848-10 | Ultrasonic Cleaner with Timer, |
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