We thank Shigeru Matsuyama for providing purified bombykol. This work was supported by the Japan Society for the Promotion of Science KAKENHI (grant numbers 22700197 and 24650090) and the Human Frontier Science Program (HFSP).

1.实验动物<ol><li>准备一个塑料盒，以保持公silkmoths的蛹（ 桑蚕 ），直到他们的羽化。在底部放纸巾和纸板件周围的框（ 图1A）的内壁。 注:纸板的作品是必要的成蛾，同时羽化（ 图1A）在扩大自己的翅膀举行。 </li><li>在框中放入雄silkmoth（ 家蚕铁道部 I）蛹，并让他们在一个孵化器，直到羽化16小时下:8小时光照:黑暗周期为25℃。 注:雌雄蛹可以通过在腹部（ 图1B）的标志性受到歧视。 </li><li>收集成年雄蛾羽化后并将其移动到一个新的框。 </li><li>保持成年蛾在培养箱16小时以下:8小时光照:黑暗周期和降低温度至15℃，实验前，以减少它们的活性。 </li></ol> 2.拴系Silkmoth <ol><li>附件制造的圈养（ 图2A） 注意:该附件包括在其前端的薄塑料片的带材的铜导线。这确保行走（ 图2B）期间胸廓的背腹运动。 <ol><li>制备薄塑料片的带材，2×40毫米（厚度:0.1 MM），并在中间折叠。 </li><li>折叠条附加到铜线用粘合剂的尖端。 </li><li>弯，其中一个silkmoth的胸部附着在折叠条带的前端。 </li></ol></li><li>在光照期为实验用成蛾（2-8天）。 注意:对信息素的敏感性强烈依赖于生物钟18。因为家蚕是一昼夜蛾，必须在光周期地进行实验。 </li><li>轻轻卸下DOR所有尺度采用了一块湿纸巾（或棉签）的SAL胸部（胸背板），并暴露胸背板（ 图2C）的角质层。 </li><li>在塑料上附着的带和暴露胸背板有小的平口螺丝刀表面上粘贴粘合剂，等待5-10分钟直至粘合剂不再粘。 注:粘合剂应不能触及机翼铰链或前翅tegulae（ 图2C）。 </li><li>粘接胸背板附件。 </li><li>请把它放在机器人的驾驶舱内前拴的飞蛾。持在支架上附着并把一张纸下的双腿休息的飞蛾。 </li></ol> 3.昆虫控制机器人<ol><li>设计基于以前的作品16,17,19昆虫控制机器人的硬件。 注意:昆虫控制机器人包括一个空气支撑的跑步机带的光学鼠标传感器capturE中的昆虫运动，定制基于AVR的微板用于处理和马达控制，和两个直流无刷电机（ 图3和4）。机器人可以在200毫秒的时间延迟内用96％的精度或更高的球转动的基础上运行。它也保证期间信息素跟踪行为16的最大前进速度的silkmoth的流动性（24.8毫米/秒）和角速度（96.3°/秒）。跑步机（ 图5A）和气味输送系统（ 图5B）的气流被设计用于板载蛾顺利走在球和通过两个天线获得的气味。跑步机的进气口和流路是从那些气味输送系统分开，以避免信息素的污染。 </li><li>设计基于以前的作品16板载微控制器的软件。 注:板载微控制器计算Ť他从用光学传感器测量的昆虫移动机器人的运动（旋转，ΔX;平移，Δÿ; 图6）。行驶距离（ΔL）和打开每机器人的单位时间的角度（Δθ）的每个轮子（左，ΔL L;右，ΔL R）的行进距离的基础上计算，如ΔL =（Δ L L +ΔL R）/ 2和Δθ=（ΔL L - ΔL R）/ D 轮 ，其中D 轮是两个轮（120 MM）之间的距离。 ΔL L，ΔL R中进一步描述为ΔL L =Δ 长 x，L +Δ 大号 Y，L，ΔL R =Δ 长 x，R +Δ 大号 Y，R，其中Δ 长 x，L &lt;/子&gt;，Δ 长 x，R是车轮上的左，右两侧通过Δ 点¯x控制的移动距离，和Δ 大号 Y，L，Δ 大号 Y，R是那些由Δý控制。理想的情况下，Δ 长 x，L和Δ 长 x，R被描述为Δ 长 x，L =-Δ 长 x，R = GΔ 的X（D 车轮 / D 球 ），和Δ 大号 Y，L，Δ 大号 ÿ中，R被描述为Δ 大号 Y，L =Δ 大号 Y，R = GΔy，其中G是马达增益和D 的球是球（50毫米）的直径。在实践中，电动机增益独立地由每个侧（左或右轮）和每个方向（向前或向后旋转），以便校准所述机器人运动设置。独立涨势进一步允许不对称马达旋转，以产生所述机器人的转动偏置设置（见步骤6.1）。 </li><li>与水以除去任何可能的嗅觉或视觉提示:;:洗白色发泡聚苯乙烯球的表面（50毫米质量直径约2克）。 注意:一个新的球的表面应以细砂纸，如P400，这确保对球腿的把手进行粗化处理。 </li><li>打开送风风扇，在9伏供应空气到跑步机和漂浮球（ 图5A）。观察球从杯底上浮约2毫米。 </li><li>使用螺钉，附加与蛾附件的铜丝（见步骤2），以在所述机器人的座舱夹具（参见图3插图）。确保中间腿的位置是在球（ 图7A）的中心。 </li><li>调整附件的垂直位置，以使蛾对将b正常走路所有。保持球在相同的高度之前和附加蛾（ 图7B）之后。 注意:该附件的太低位置增加对蛾压力和引发向后行走来抵抗压力（ 图7C），而过高的位置会导致不稳定的步行和传感器的故障是由于在垂直位置的变化球（ 图7D）。要检查正常行走的行为，单膨化信息素的刺激被用来触发走在蛾（为信息素的刺激，见第4步）。请注意，由于以前接触过bombykol habituates silkmoths并降低其灵敏度（松山和神崎，未公布数据）测试刺激必须是最小的。 </li></ol> 4.气味源的制备 注:男家蚕是对同种雌性性信息素的主要成分敏感（bombykol:（E，Z）-10,12-hexadecadien -1-醇） <s达&gt; 20。实验设备与bombykol任何污染引起的气味追踪行为和影响蛾的响应。 <ol><li>在一张滤纸（约10mm×10mm）的的滴10微升溶解在正己烷（200毫微克/微升）的bombykol溶液。每一片滤纸bombykol的量为2000毫微克。 注意:要检查蛾的正常行走的行为，在此步骤中准备一个信息素的刺激盒。盒是一个玻璃巴斯德吸管用含有2000纳克bombykol之一一张滤纸。推球的喷着空气含bombykol。 </li></ol> 5.气味源定位实验<ol><li>打开一个拉出空气型风洞（1800×900×300毫米，长×宽×高; 图8）的风扇并设置风速至0.7米/秒。确保该温度超过20℃。 </li><li>设置臭味源（在p含bombykol风洞的上游滤纸）的IECE。 注意:羽宽度应在实验前，通过使用四氯化钛17,19来确认。 </li><li>打开机器人的微控制器板和建立通过蓝牙到PC的串行连接。 </li><li>发射被称为"生物信号"，它提供了在PC和机器人之间的界面的定制的Java程序。 注意:主窗口包括用于发送命令给机器人的按钮，用于显示输入和串行通信的输出，并且小箱配置参数文本窗口。随后的命令是通过点击相应的按钮，在这个程序中，除了视频捕捉发送。 </li><li>点击"关于设备"按钮，以确认通过指定的COM口机器人发送命令连接，并检查该消息是由机器人返回。 </li><li>点击"备忘录RY擦除"按钮，清除遗留在板载闪存先前的运动数据。 </li><li>点击"drivemode1"按钮发送默认马达收益的机器人。 注意:电机增益和昆虫运动和机器人的运动之间的时间延迟的操作应用此步骤之后（参见步骤6.1和6.3， 图9）。 </li><li>点击"不开车"按钮发送一个命令，直到实验开始固定机器人。 </li><li>把机器人在起始位置（从气味源600毫米下游），转动马达驱动板的开关。 </li><li>按下摄像机的录制按钮开始视频拍摄。 </li><li>点击"REC START"按钮，开始命令发送与板载闪存球旋转的同时记录来启动机器人。观察，该机器人开始移动并跟踪气味羽。 </li><li>点击"停止录像"和"不开车"按钮发送命令，如果机器人本地化气味源同时停止机器人的运动和记录。 </li><li>按下摄像机的录制按钮停止视频拍摄。 </li><li>下载通过串行连接从板载闪存到计算机记录运动数据。关闭程序。 </li></ol> 6.操纵昆虫控制机器人 注:各操作的定时图9中被指示。 <ol><li>马达收益操纵 注意:此操作改变机器人的平移和旋转速度。不对称马达收益产生转动偏置，其可用于研究昆虫如何补偿的偏置17。 <ol><li>限定通过编辑日为前向和每边17（ 图6B）的马达的反向旋转的旋转增益Ë配置文件命名为"param2.txt"使用文本编辑器。 </li><li>点击"设置参数2"读取软件程序的编辑过的配置文件。然后，点击"drivemode2"送操纵收益的机器人。 </li></ol></li><li>电动机输出的反转 注意:此操作提供类似于双边嗅觉输入的反转的状态（参见步骤6.4），并且可以用于研究双边嗅觉的意义。然而，电机的输出的反转也反转板载蛾的自感应视觉运动。倒自感应视觉输入的影响，可以通过与倒置嗅觉输入19进行比较来评价。 <ol><li>通过横渡控制电缆每个电机反转双边电机控制。 </li></ol></li><li>昆虫运动和机器人的运动之间的时间延迟的操纵。 注:此操作允许用在感觉运动处理机器人气味跟踪的时间段接受的调查。微控制器存储关于缓冲存储器的运动的数据，然后在指定的时间延迟后进行处理。注意，该机器人具有200毫秒的最大内部时间延迟;因此，实际的时间延迟预计将在指定的时间延迟加上200毫秒16,17。 <ol><li>在主窗口的一个小盒子输入一个数字（0-10），以指定从0-1,000毫秒，在100毫秒的步骤的时间延迟。 </li><li>点击"设置延时"按钮应用的时间延迟。 </li></ol></li><li>嗅觉输入的操纵。 注意:此操作可用于研究双边嗅觉输入的重要性。 silkmoths的浪涌方向施力的较高浓度侧22。 <ol><li>改变吸管尖端之间的间隙或反转它们的位置来改变在由每个天线获取的气味浓度差。 </li></ol></li><li>视觉输入的操作 注意:此操作是调查视觉输入的对气味的跟踪作用。 <ol><li>覆盖一个白皮书，分别闭塞板载蛾的水平和垂直视场的105°和90°，遮篷。 </li></ol></li></ol>

The authors have nothing to disclose.

用于通过silkmoth机器人的成功控制的最重要的点是让蛾行走顺畅的空气支撑球和稳定地测量球的旋转。因此，圈养的silkmoth并在适当的位置将其安装在球在此协议中的关键步骤。蛾到附件或球上的蛾不适当定位不当粘附将导致在其上不自然的压力，这扰乱它的正常行走行为和/或引起该光学传感器的故障来测量球的旋转。粗糙化的聚苯乙烯球也很重要，以防止蛾打滑。系留蛾的响应的移动来刺激气?...

Autonomous robots capable of finding an odor source can be important for the safety and security of society. They can be used for the detection of disaster victims, of drugs or explosive materials at an airport, and of hazardous material spills or leaks in the environment. At present, we rely entirely on well-trained animals (e.g., dogs) for these tasks, and robotic odor source localization has been strongly expected to relieve the workload of these animals. Finding an odor source is a challenging task for robots because odorants are distributed intermittently in an atmosphere1; therefore, continuous sampling of the odor concentration gradient is not always possible. Thus, a search strategy using intermittent odor cues is necessary for the achievement of robotic odor source localization2-4.
Odor source localization is essential for organism survival and includes tasks such as finding food, mating partners, and sites for oviposition. To overcome the difficulty in tracking patchy distributed odorants, organisms have evolved various behavioral strategies consisting of two fundamental behaviors: moving upstream during odor reception and cross-stream during cessation of odor reception5,6. These reactive strategies have been well-documented in insects and further combined with other modalities, such as wind direction and vision5-8. The insect behavioral models have also been useful examples for robotics3,9-11, in which behavioral algorithms or neural circuit models are implemented into mobile robots for the evaluation of odor source localization abilities10,12-15. From biomimetic perspectives, this "bottom-up" approach is certainly a fundamental way to develop biomimetic robots. However, the bottom-up approach is not a shortcut to obtaining a useful search strategy, because biological analyses are still ongoing, and the modeling of the sensory-motor systems behind insect behaviors has not been completed. Therefore, it is still unknown how such a biological system actually works as a controller of a robotic platform.
In this article, we demonstrate the protocol of a straightforward "top-down" approach to develop an odor-tracking mobile robot controlled by a biological system16,17. The robot is controlled by a real insect and can be regarded as a prototype of future insect-mimetic robots. In the robot's cockpit, a tethered adult male silkmoth (Bombyx mori) walked on an air-supported ball in response to the female sex pheromone, which was delivered to each antenna through air suction tubes. The ball rotations caused by the walking of the onboard moth were measured by an optical sensor and were translated into the movement of the two-wheeled robot. The advantage of this "hybrid" approach is that experimenters can investigate how the insect sensory-motor system works on the robotic platform where a pilot insect is in a closed loop between the robot and a real odor circumstance. The manipulation of the robotic hardware alters the closed loop; therefore, the insect-controlled robot is a useful platform for both engineers and biologists. For engineering, the robot represents the first steps of applying a biological model to meet the requirements for robotic tasks. For biology, the robot is an experimental platform for studying sensory-motor control under a closed loop.

<table border="0" cellpadding="0" cellspacing="0" width="961">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:267px;">Male adult silkmoth 
			&#160;(Bombyx mori)</td>
			<td style="width:159px;"></td>
			<td style="width:171px;"></td>
			<td style="width:365px;">Rear from eggs, or purchase as pupae.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Incubator</td>
			<td style="width:159px;">Panasonic</td>
			<td style="width:171px;">MIR-254</td>
			<td style="width:365px;">Store pupae or adult silkmoths at a constant temperature, 238 L.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Plastic box</td>
			<td style="width:159px;">Sunplatec</td>
			<td style="width:171px;">O-3</td>
			<td style="width:365px;">Store pupae or adult silkmoths, 299 &#215; 224 &#215; 62 mm L &#215; W &#215; H.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Copper wire</td>
			<td style="width:159px;"></td>
			<td style="width:171px;"></td>
			<td style="width:365px;">2-mm diameter for the attachment. Any rigid bar can be used as an alternative for making the attachment to tether a silkmoth.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Plastic sheet</td>
			<td style="width:159px;">Kokuyo</td>
			<td style="width:171px;">VF-1420N</td>
			<td style="width:365px;">Sold as overhead projector film with thickness of 0.1 mm. Use at the tip of the attachment.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Forceps</td>
			<td style="width:159px;">As one</td>
			<td style="width:171px;">5SA</td>
			<td style="width:365px;">Remove scales on the thorax.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Adhesive</td>
			<td style="width:159px;">Konishi</td>
			<td style="width:171px;">G17</td>
			<td style="width:365px;">Bond a silkmoth to the attachment.</td>
		</tr>
		<tr height="93">
			<td height="93" style="height:93px;width:267px;">Insect-controlled robot</td>
			<td style="width:159px;">Custom</td>
			<td style="width:171px;"></td>
			<td style="width:365px;">Bearing an air-supported treadmill, an optical sensor, custom-built AVR-based microcontroller boards, and two DC brushless motors. It is powered by 8 &#215; AA and 3 &#215; 006P batteries.</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:267px;">Microcontroller</td>
			<td style="width:159px;">Atmel</td>
			<td style="width:171px;">ATMEGA8</td>
			<td style="width:365px;">A component of the insect-controlled robot.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">DC blower</td>
			<td style="width:159px;">Nidec</td>
			<td style="width:171px;">A34342-55</td>
			<td style="width:365px;">A component of the insect-controlled robot for floating a ball in an air-supported treadmill.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">DC fan</td>
			<td style="width:159px;">Minebea</td>
			<td style="width:171px;">1606KL-04W-B50</td>
			<td style="width:365px;">A component of the insect-controlled robot for suctioning air containing an odor.</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:267px;">Optical mouse sensor</td>
			<td style="width:159px;">Agilent technologies</td>
			<td style="width:171px;">HDNS-2000</td>
			<td style="width:365px;">A component of the insect-controlled robot, obtained from an optical mouse (M-GUWSRSV, Elecom, Japan).</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Brushless motor</td>
			<td style="width:159px;">Maxon</td>
			<td style="width:171px;">EC-45</td>
			<td style="width:365px;">A component of the insect-controlled robot for driving a wheel.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">White polystyrene ball</td>
			<td style="width:159px;"></td>
			<td style="width:171px;"></td>
			<td style="width:365px;">A component of the insect-controlled robot. Diameter 50 mm, mass approximately 2 g.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Bombykol: 
			&#160;(E,Z)-10,12-hexadecadien-1-ol</td>
			<td style="width:159px;">Shin-Etsu chemical</td>
			<td style="width:171px;"></td>
			<td style="width:365px;">Custom synthesis.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">n-hexane</td>
			<td style="width:159px;">Wako</td>
			<td style="width:171px;">085-00416</td>
			<td style="width:365px;">Solvent for bombykol.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Wind tunnel</td>
			<td style="width:159px;">Custom</td>
			<td style="width:171px;"></td>
			<td style="width:365px;">Pulling-air type, sized 1800 &#215; 900 &#215; 300 mm L &#215; W &#215; H.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:267px;">BioSignal program</td>
			<td style="width:159px;">Custom</td>
			<td style="width:171px;"></td>
			<td style="width:365px;">A program to establish serial communication between the insect-controlled robot and a PC via Bluetooth. Used for sending commands to start/stop the robot or configuring its motor properties.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Camcorder</td>
			<td style="width:159px;">Sony</td>
			<td style="width:171px;">HDR-XR520V</td>
			<td style="width:365px;">Capture robot movements.</td>
		</tr>
	</tbody>
</table>

insect-controlled robot: a mobile robot platform to evaluate the odor-tracking capability of an insect

Robotic odor source localization has been a challenging area and one to which biological knowledge has been expected to contribute, as finding odor sources is an essential task for organism survival. Insects are well-studied organisms with regard to odor tracking, and their behavioral strategies have been applied to mobile robots for evaluation. This "bottom-up" approach is a fundamental way to develop biomimetic robots; however, the biological analyses and the modeling of behavioral mechanisms are still ongoing. Therefore, it is still unknown how such a biological system actually works as the controller of a robotic platform. To answer this question, we have developed an insect-controlled robot in which a male adult silkmoth (Bombyx mori) drives a robot car in response to odor stimuli; this can be regarded as a prototype of a future insect-mimetic robot. In the cockpit of the robot, a tethered silkmoth walked on an air-supported ball and an optical sensor measured the ball rotations. These rotations were translated into the movement of the two-wheeled robot. The advantage of this "hybrid" approach is that experimenters can manipulate any parameter of the robot, which enables the evaluation of the odor-tracking capability of insects and provides useful suggestions for robotic odor-tracking. Furthermore, these manipulations are non-invasive ways to alter the sensory-motor relationship of a pilot insect and will be a useful technique for understanding adaptive behaviors.

我们在座的一个气味源的成功国产化所需的昆虫控制机器人的基本特征。机器人和silkmoths，气味递送系统的有效性，并准确双边嗅觉和视觉输入的重要性之间的比较被检查。 气味跟踪行为自如行走蛾和昆虫控制机器人之间的比较示于图10A和B.在相同气味的情况下，无论是行走蛾和机器人拿下100％的成?...

Watch this Scientific Journal Video about Insect-controlled Robot: A Mobile Robot Platform to Evaluate the Odor-tracking Capability of an Insect at JoVE.com

Insect-controlled Robot: A Mobile Robot Platform to Evaluate the Odor-tracking Capability of an Insect

本地化气味源的能力是必要的昆虫存活和预计适用于人工气味跟踪。昆虫控制机器人由一个实际silkmoth驱动，使我们可以通过一个机器人平台，以评估昆虫气味跟踪能力。

昆虫控制的机器人:移动机器人平台来评估昆虫的气味跟踪能力

Robotic odor source localization has been a challenging area and one to which biological knowledge has been expected to contribute, as finding odor ...

insect-controlled-robot-mobile-robot-platform-to-evaluate-odor

Research

JoVE Journal

Neuroscience

14.6K 次观看.  The University of Tokyo. 本地化气味源的能力是必要的昆虫存活和预计适用于人工气味跟踪。昆虫控制机器人由一个实际silkmoth驱动，使我们可以通过一个机器人平台，以评估昆虫气味跟踪能力。 

视频: Insect-controlled Robot: A Mobile Robot Platform to Evaluate the Odor-tracking Capability of an Insect

An Experimental Platform to Study the Closed-loop Performance of Brain-machine Interfaces

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine interface to assess the robustness of different control-laws applied to a closed-loop image stabilization task. Taking advantage of the well-characterized fly visuomotor pathway we record the electrical activity from an identified, motion-sensitive neuron, H1, to control the yaw rotation of a two-wheeled robot. The robot is equipped with 2 high-speed video cameras providing visual motion input to a fly placed in front of 2 CRT computer monitors. The activity of the H1 neuron indicates the direction and relative speed of the robot's rotation. The neural activity is filtered and fed back into the steering system of the robot by means of proportional and proportional/adaptive control. Our goal is to test and optimize the performance of various control laws under closed-loop conditions for a broader application also in other brain machine interfaces.

We use a closed-loop fly-machine interface to investigate general principles in neuronal control.

研究脑机接口的闭环性能的实验平台

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine ...

科研

神经科学

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

用气相色谱 - 多单元的录音（GCMR）昆虫触角叶嗅觉挥发物的鉴定

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular recording has historically been a common technique used to study sensitivities of individual photoreceptor cells to different light stimuli that is still being used today. However, there remains a dearth of detailed methodology in the literature for researchers wishing to replicate intracellular recording experiments in the eye. Here we present the insect as a model for examining eye physiology more generally. Insect photoreceptor cells are located near the surface of the eye and are therefore easy to reach, and many of the mechanisms involved in vision are conserved across animal phyla. We describe the basic procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly, with the goal of making this technique more accessible to researchers with little prior experience in electrophysiology. We introduce the basic equipment needed, how to prepare a live butterfly for recording, how to insert a glass microelectrode into a single cell, and finally the recording procedure itself. We also explain the basic analysis of raw response data for determining spectral sensitivity of individual cell types. Although our protocol focuses on determining spectral sensitivity, other stimuli (e.g., polarized light) and variations of the method are applicable to this setup.

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

The electrophysiological technique of intracellular recording is demonstrated and used to determine spectral sensitivities of single photoreceptor cells in the compound eye of a butterfly.

光感受器细胞光谱灵敏度在从昆虫模型决定<em&gt;在体内</em&gt;细胞内记录

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

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.

高接触密度的制作，平面接口神经电极用于记录和刺激的应用

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 &#181;L or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 &#181;L can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

一种改进的麻醉小鼠脑脊液采集方法

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

基于改进多平台法的大鼠中心疲劳模型

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

小鼠脊髓损伤模型中内源性神经干细胞活化的干细胞检测

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

小鼠蛛网膜下腔出血模型脑血管痉挛量化的容积法

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Functional MRI in Conjunction with a Novel MRI-compatible Hand-induced Robotic Device to Evaluate Rehabilitation of Individuals Recovering from Hand Grip Deficits

Functional magnetic resonance imaging (fMRI) is a non-invasive magnetic resonance imaging technique that images brain activation in vivo, using endogenous deoxyhemoglobin as an endogenous contrast agent to detect changes in blood-level-dependent oxygenation (BOLD effect). We combined fMRI with a novel robotic device (MR-compatible hand-induced robotic device [MR_CHIROD]) so that a person in the scanner can execute a controlled motor task, hand-squeezing, which is a very important hand movement to study in neurological motor disease. We employed parallel imaging (generalized auto-calibrating partially parallel acquisitions [GRAPPA]), which allowed higher spatial resolution resulting in increased sensitivity to BOLD. The combination of fMRI with the hand-induced robotic device allowed precise control and monitoring of the task that was executed while a participant was in the scanner; this may prove to be of utility in rehabilitation of hand motor function in patients recovering from neurological deficits (e.g., stroke). Here we outline the protocol for using the current prototype of the MR_CHIROD during an fMRI scan.

We performed functional MRI using a novel MRI-compatible hand-induced robotic device to evaluate its utility for monitoring hand motor function in individuals recovering from neurological deficits.

功能 MRI 与新型 MRI 兼容手感应机器人设备结合，以评估从手柄缺陷中恢复的个人的康复情况

Functional magnetic resonance imaging (fMRI) is a non-invasive magnetic resonance imaging technique that images brain activation in vivo, using endogenous ...