This work was funded by the state program Investissements d&#8217;avenir managed by ANR (grant ANR-10-BINF-05 'Pherotaxis').

<html>该协议是基于市售的机器人（见材料表）和雄蛾（ 小地老虎 ）与他们的性信息素。然而，它可以适于与小的修改，其它昆虫物种，增味剂，和机器人。 1，昆虫<ol><li>用人工饲料，并在个别塑料杯，直到化蛹它们维持在23±1℃，如前面所述24 50±5％相对湿度: 小地老虎 HUFNAGEL（鳞翅目夜蛾科）的后幼虫。 </li><li>性别蛹，并分别保持成年男性从女性的塑料盒。给他们免费使用20％的蔗糖溶液。 </li><li>与男性进行实验。雄蛾是由他们的同种雌性发出的性信息素高度敏感。 A.中地老虎 ，主要的信息素组分， 顺 -7 -十二碳烯基乙酸酯（Z7-12:醋酸）是在天线上最活跃的化合物。 </li></ol>书名"&gt; 2。电<ol><li>从全虫准备录制东亚运动会，如下所述（ 图1A）。完整的触角优于切除的触角，因为他们表现出更长的寿命（见代表性的结果）。 </li><li>在10-20分钟浓漂白剂溶液氯化两银子电线浸泡和冲洗之后。这个过程可以防止从电极极化。它具有每当基线在实验过程中的漂移或当电极之间的偏移电压变得过大，由放大器进行补偿，以重复进行。 </li><li>使从火抛光的毛细血管与电极拉马玻璃电极。火焰抛光可以防止刮伤氯化银线与电极。 </li><li>麻醉雄性蛾和CO 2，并放置一个泡沫塑料块内具有从顶部的头部伸出。 </li><li>系绳昆虫的头部与脖子画家的磁带。 </li><li>将银线作为参比电极到颈部。 </li><li>在立体显微镜下，用固定在顶端和基部细条画家的磁带的天线之一。 </li><li>切出与手术剪天线的远端2-3段。 </li><li>定位用微操纵器天线的切口尖端附近的玻璃电极。 </li><li>切出用钳子将玻璃毛细管的末端，以获得直径小于天线的切尖稍大。 </li><li>带（以mM计）填充玻璃吸管6.4的KCl，340葡萄糖，10 HEPES，12的MgCl 2，1的CaCl 2，12氯化钠，pH值6.5。 </li><li>插入天线的切尖与显微操作的玻璃毛细管。 </li><li>打滑的银线作为记录电极插入玻璃毛细管的最大末端。 </li></ol> 3，硬体介面<ol><li>挂载整个制备， 即昆虫电极-微操作器上拧上所述机器人（ 图1B）的顶部的金属板。将电极连接到机器人，如下所述。 </li><li>根据以往的作品25-26，设计一个硬件接口，以适应东亚运动会的输出电压（为了1 mV在几MΩ）为适合机器人的扩展板的范围内。董事会接受0-5V模拟输入及以下-200 mV的负电压可能会造成严重的损害。按照步骤3.2.1至3.2.4设计界面与鹰。 <ol><li>使用稳压器78L10（①在图2A-2C）+12 V电池设计一个±5V电源。 </li><li>设计一个探头前置放大器（10X）的基础上的仪表放大器INA121（③在图2A-2C）。 </li><li>设计一个第二级放大器（25X）与噪声滤除（一阶高通0.1 Hz滤波器，二阶低通500 Hz滤波器，陷波50 Hz滤波器）基于四运算放大器LT1079（315;在图2A-2C）。 </li><li>设计一个信号调理阶段，与运算放大器LT1079和二极管1N4148计算（⑤在图2A-2C）。总增益为250和东亚运动会输出的范围是0-5V零是在2.5 V。 </li></ol></li><li>将电极连接到差分输入EAG（②在图2A-2C）。记录电极连接到INA121的反相输入端，以取得积极的EAGs。 </li><li>东亚运输出（⑥在图2A-2C）连接到机器人的扩展板的12个模拟输入。因为每个输入是顺序读取每毫秒，采样频率为1kHz。 </li></ol> 4，软件界面主线程包含一个图形用户界面（GUI），用于信号检测的各种功能，用于控制机器人的方法。 <ol><li>写一个GUI（ 图2D）中的Qt-C + + FO<html> R数据的可视化，数字滤波（20赫兹5 阶 Butterworth低通滤波器）和来自东亚运动会气味检测。后者可以通过两种方式进行:要么，或通过模拟的神经机制，允许快速和可靠的信息素检测A.解卷积东亚运动会用适当的过滤器（工程方法，第4.2节） 地老虎蛾（仿生方法，第4.3节）。 </li><li>反卷积滤波器。东亚运动会是由一个非线性级联27，它由一个静态非线性很好的描述<img alt="式（1）" fo:content-width="0.3in" src="/files/ftp_upload/51704/51704eq1.jpg" />和第一阶低通滤波器，指数脉冲函数<img alt="公式2" fo:content-width="1in" src="/files/ftp_upload/51704/51704eq2.jpg" />为<img alt="式（3）" fo:content-width="0.3in" src="/files/ftp_upload/51704/51704eq3.jpg" /> ， 见图3A。为了应对波动的臭气浓度uation 4"FO:内容宽度="0.3英寸"的src ="/ files/ftp_upload/51704/51704eq4.jpg"/&gt;中，第一阶段的输出由卷积积分给出<img alt="方程（5）" fo:content-width="1in" src="/files/ftp_upload/51704/51704eq5.jpg" /> 。解卷积是简单地通过该系统的逆获得的;就是说<img alt="公式6" fo:content-width="1in" src="/files/ftp_upload/51704/51704eq6.jpg" />在频域中。然后， <img alt="公式7" fo:content-width="1.2in" src="/files/ftp_upload/51704/51704eq7.jpg" />作为傅立叶脉冲响应的变换是<img alt="公式8" fo:content-width="1.2in" src="/files/ftp_upload/51704/51704eq8.jpg" /> 。对于信号的检测，按照步骤4.2.1至4.2.3。 <ol><li>在时域中执行去卷积处理<img alt="公式9" fo:content-width="1in" src="/files/ftp_upload/51704/51704eq9.jpg" />和<img alt="公式10" fo:content-width="1in" src="/files/ftp_upload/51704/51704eq10.jpg" /> ， 如图3B所示 。 </l我&gt; <li>近似​​非线性<img alt="公式11" fo:content-width="0.3in" src="/files/ftp_upload/51704/51704eq11.jpg" />由一个多项式函数。拟合多项式的参数和时间上的输入输出数据对恒定，以尽量减少与实际的均方误差<img alt="公式4" fo:content-width="0.3in" src="/files/ftp_upload/51704/51704eq4.jpg" />与重建<img alt="式（12）" fo:content-width="0.3in" src="/files/ftp_upload/51704/51704eq12.jpg" />臭气浓度。 </li><li>检测信息素命中时<img alt="式（12）" fo:content-width="0.3in" src="/files/ftp_upload/51704/51704eq12.jpg" />超过预定的阈值。 </li></ol></li><li>神经形态探测器。另一种方法来检测包含在模仿生物。 A.中地老虎蛾，从天线接收输入中枢神经元的信息素响应与激励-抑制13的刻板的点火模式。霍奇金 - 赫胥黎吨YPE有四个离子电流神经元模型<img alt="公式13" fo:content-width="0.3in" src="/files/ftp_upload/51704/51704eq13.jpg" /> （延迟整流钾电流，电压门控Na +和Ca 2 +电流，小电导钙依赖性钾电流）是以前开发的重现观察到的生理反应13。对于信号的检测，按照步骤4.3.1到4.3.3。 <ol><li>实现神经元模型的微分方程。以东亚运动会信号作为输入电流<img alt="公式14" fo:content-width="1.2in" src="/files/ftp_upload/51704/51704eq14.jpg" />在膜电势的演变<img alt="公式15" fo:content-width="1in" src="/files/ftp_upload/51704/51704eq15.jpg" /> 。使用膜电容C = 22.9 pF和由下式给出的泄漏电流<img alt="公式16" fo:content-width="1in" src="/files/ftp_upload/51704/51704eq16.jpg" />与电导G L = 0.011161μs和反转电位E L = -61.4 mV的。该离子电流由所述<img alt="公式17" fo:content-width="1.2in" src="/files/ftp_upload/51704/51704eq17.jpg" />同<img alt="公式18" fo:content-width="2in" src="/files/ftp_upload/51704/51704eq18.jpg" />由地方<img alt="公式19" fo:content-width="1.2in" src="/files/ftp_upload/51704/51704eq19.jpg" />五是非线性函数的详细信息，请参阅以前的工作13。 </li><li>通过集成一个4 阶龙格-库塔法微分方程和一个时间步长模拟实时与Sirene酒店的神经元模型<img alt="公式20" fo:content-width="0.1in" src="/files/ftp_upload/51704/51704eq20.jpg" /> = 0.01毫秒。运行测试秒杀V（T F）&gt; 0 mV和V（T F - <img alt="公式20" fo:content-width="0.1in" src="/files/ftp_upload/51704/51704eq20.jpg" /> ）&lt;0 mV的联机获取秒杀时间和峰峰间隔。 </li><li>检测每当突发激励的（3个连续峰峰间隔&lt;70毫秒）之后是抑制（放电脉冲时间间隔≥350毫秒）信息素命中， 见图3C。 </li></ol></li></ol>

<ol>
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J., Park, K. C., Hetling, J. R., Baker, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+discrimination+of+mixed+odor+strands+in+overlapping+plumes+using+an+insect-antenna-based+chemosensor+system.">Detection and discrimination of mixed odor strands in overlapping plumes using an insect-antenna-based chemosensor system.</a> J. Chem. Ecol. 35, 118-130 (2009).</li><li>Myrick, A. J., Park, K. C., Hetling, J. R., Baker, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+odor+discrimination+using+a+bioelectronic+sensor+array+based+on+the+insect+electroantennogram.">Real-time odor discrimination using a bioelectronic sensor array based on the insect electroantennogram.</a> Bioinspiration and Biomimetics. 3, (2008).</li><li>Vickers, N. J., Christensen, T. A., Baker, T. C., Hildebrand, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odour-plume+dynamics+influence+the+brain's+olfactory+code.">Odour-plume dynamics influence the brain's olfactory code.</a> Nature. 410, 466-470 (2001).</li><li>Vergassola, M., Villermaux, E., Shraiman, B. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Infotaxis'+as+a+strategy+for+searching+without+gradients.">'Infotaxis' as a strategy for searching without gradients.</a> Nature. 445, 406-409 (2007).</li><li>Martin-Moraud, E., Martinez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+and+robustness+of+robot+infotaxis+for+searching+in+dilute+conditions.">Effectiveness and robustness of robot infotaxis for searching in dilute conditions.</a> Frontiers in neurorobotics. , (2010).</li><li>Masson, J. -. 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Authors have nothing to disclose.

几乎是二十年前，神崎和他的同事们率先使用EAGs嗅觉29-30机器人的想法。他们的技术最初是基于对离天线。在这里，我们从完好触角记录，以改善制剂的敏感性和寿命。其他的研究31-32也注意到的全身准备在隔离天线的优越性。在我们的机器人的实验中，我们经历了一天之内稳定的录音。与此相反，EAGs上记录分离的天线有2小时的寿命（ 见图5）。 我们的东亚运动会-机器人平台的开发，主要以测试有关嗅觉编码和搜索策略，昆虫13生物学假说。类似昆虫的触角接收输入中枢神经元，我们连接的神经元模型对机器人真正蛾天线及进行信息素的检测是根据它的射击模式。检测和未检测次数分别为然后用来驱动朝向信息素的来源的机器人。这里考虑的反应检索策略的灵感来自于由一种性信息素吸引雄蛾的行为模式。它在实验室条件下（ 图6）表现不错，允许低排放源在一个比较大的搜索空间（初始距离（在我们的案例中10微克与10毫克的前期工作24信息素剂量）从2米源的定位与10厘米以前的实验20-21）。 这些机器人的实验应该被视为一个概念证明表明昆虫的触角适合机器人嗅觉搜索。虽然昆虫的触角是已知的有毒气体，毒品和爆炸物9-11反应，都需要与现实世界的应用程序应对几个扩展。首先，在更复杂的搜索方法34-36可以是更有效的在距离超过10μm时，在重新捕获时的羽毛变得不太可能。第二，它可能需要从不同的物种中，为了检测利益气味分子结合EAGs在生物电子鼻的配置14。第三，通过从相同的昆虫的两个天线的记录获得立体声感测功能可以证明是有益的有效性方面。两个传感器并联使用的可能确实增加方向性。第四，搜索策略的延伸集体机器人搜索37顷应该考虑实际应用，即使他们不是在飞蛾的情况下，生物相关。

如今，动物，像狗经常用于涉及化学品泄漏，毒品和爆炸物的，因为其出色的嗅觉检测能力16本地化安全和安全应用。然而，他们表现出行为的变化，经过广泛的工作感到厌倦，并且需要频繁的再培训由于其性能随时 ​​间下降17。绕过这些限制的方法之一是通过嗅觉机器人来取代训练有素的狗。 尽管如此，跟踪气味和臭味的来源是在机器人技术的一个重大挑战。在动荡的环境中，气味羽的景观是非常庞杂且不稳定，以及由零星位于补丁4。即使在从源头抓起，短则数米的距离适中，检测成为零星的，只提供线索断断续续。此外，在检测的局部浓度梯度一般不指向源。鉴于盘信息和有限的本地信息ontinuous流量检测时作出如何导航朝着源的机器人？ 这是众所周知的昆虫，如雄蛾用化学通讯在长距离（几百米），成功地找到自己的伴侣。为此，他们采取一种刻板的行为18-20:他们迎风暴涨在检测气味的补丁并执行扩展的搜索称为铸造时的气味信息消失。此浪涌压铸策略是纯粹的反应， 即动作是由当前感知（检测和非检测事件）完全确定。然而，它的实施对嗅觉的机器人限制了在过去的成功，因为气味补丁的检测是由人工煤气传感器缓慢阻碍。 在大多数嗅觉机器人使用的金属氧化物传感器具有几十秒响应和恢复时间，以便它们通常过滤掉在动荡的羽状物21碰到的浓度波动。与此相反，昆虫化学感受器的响应时间要短得多， 例如 ，昆虫electroantennograms（EAGs）的上升时间小于50毫秒22。因此，通过使用昆虫EAGs，气味脉冲在几个赫兹23的频 ​​率解析。这个特性使得EAG传感器非常适合于天然羽毛气味长丝的检测。我们在这里介绍一个协议，用于在机器人允许使用高效激增嗅觉搜索嵌入昆虫EAGs和铸造的策略。

<table border="0" cellpadding="0" cellspacing="0" width="681">
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:179px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="85" rowspan="2" style="height:85px;width:216px;">Agrotis ipsilon&#160;</td>
			<td style="width:179px;">PISC&#160;</td>
			<td rowspan="2" style="width:119px;"></td>
			<td rowspan="2" style="width:167px;">moth</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:179px;">http://www-physiologie-insecte.versailles.inra.fr/indexenglish.php</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:216px;">Robot Khepera III&#160;</td>
			<td style="width:179px;">K-team&#160;</td>
			<td rowspan="2" style="width:119px;"></td>
			<td rowspan="2" style="width:167px;">Khe3Base + KorBotLE + KorWifi</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:179px;">www.k-team.com</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">KoreIOLE</td>
			<td style="width:179px;">K-team</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Input/output extension board</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:216px;">EAG-robot interface</td>
			<td style="width:179px;">LORIA&#160;</td>
			<td rowspan="2" style="width:119px;"></td>
			<td rowspan="2" style="width:167px;">Custom-made hardware and software</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:179px;">www.loria.fr</td>
		</tr>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:216px;">Sirene</td>
			<td rowspan="2" style="width:179px;">LORIA&#160;</td>
			<td rowspan="2" style="width:119px;"></td>
			<td rowspan="2" style="width:167px;">neuronal simulator sirene.gforge.inria.fr</td>
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		<tr height="21">
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:216px;">Eagle</td>
			<td rowspan="2" style="width:179px;">CadSoft www.cadsoftusa.com</td>
			<td rowspan="2" style="width:119px;"></td>
			<td rowspan="2" style="width:167px;">PCB design software</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Micromanipulator</td>
			<td style="width:179px;">Narishige / Bio-logic</td>
			<td style="width:119px;">UN-3C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Magnet base</td>
			<td style="width:179px;">Narishige/ Bio-logic</td>
			<td style="width:119px;">USM-6</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Adapter</td>
			<td style="width:179px;">Narishige/ Bio-logic</td>
			<td style="width:119px;">UX-6-6</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Rotule</td>
			<td style="width:179px;">Narishige/ Bio-logic</td>
			<td style="width:119px;">UPN-B</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Micro scisors</td>
			<td style="width:179px;">MORIA / Phymep</td>
			<td style="width:119px;">15371-92</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Stereo microscope Zeiss St&#233;mi 2000</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">B19961</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Light source 20W KL200</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">W41745</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Narishige PC-10 Na PC-1</td>
			<td style="width:179px;">Narishige</td>
			<td style="width:119px;">Narishige PC-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Capillaries Na PC-1</td>
			<td style="width:179px;">Fisher scientific</td>
			<td style="width:119px;">C01065</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Pheromone cis-7-Dodecenyl acetate(Z7-12:OAc)</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">259829</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pack of 3 pipettes&#160;</td>
			<td rowspan="2" style="width:179px;">Eppendorf</td>
			<td rowspan="2" style="width:119px;">4910000514</td>
			<td rowspan="2" style="width:167px;">For pheromone dilution and deposition on paper filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-20 &#181;l/ 50-200 &#181;l/ 100-1000 &#181;l&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Gas sensor TGS2620&#160;</td>
			<td style="width:179px;">Figaro www.figarosensor.com</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Optional, for comparison with EAG</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">electrode puller&#160;</td>
			<td style="width:179px;">Narishige&#160;</td>
			<td style="width:119px;">PC-10</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

using insect electroantennogram sensors on autonomous robots for olfactory searches

机器人设计，在危险的工业设施1或爆炸的痕迹在地雷场2面对同样的问题，跟踪化学品泄漏昆虫觅食或寻找配偶3:嗅觉搜索是通过湍流输送4的物理限制。风传气味的浓度景观是不连续的，由零星位于补丁。一个先决条件嗅觉搜索的是间歇性的气味补丁检测。由于其高速度和灵敏度5-6，昆虫的嗅觉器官提供检测一个独特的机会。昆虫天线已被用于在过去检测不仅性信息素7，而且化学物质有关的人， 例如 ，挥发性化合物从癌细胞8或有毒的和非法物质9-11发出。我们在这里描述的协议使用昆虫触角上的自主机器人的D存在概念的跟踪气味的羽毛其来源证明。嗅觉神经元的全球反应被记录在原位 electroantennograms（EAGs）的形式。我们的实验设计的基础上，全虫准备，允许在一个工作日内稳定的录音。相比较而言，在切除触角EAGs有2小时的寿命。自定义硬件/软件接口是东亚运电极与机器人之间展开。测量系统可以解决个别气味补丁高达10赫兹，这超过了人工化学传感器12的时间刻度。 EAG传感器嗅觉检索数据的效率被进一步证实在驱动机器人朝向信息素的来源。通过使用相同的嗅觉刺激和传感器在现实的动物，我们的机器人平台为测试有关嗅觉编码和搜索策略13的生物学假说的直接手段。它也可能被证明有利于通过检测的权益外的气味从不同的昆虫种类结合EAGs在生物电子鼻配置14或使用纳米气体传感器，模仿昆虫触角15。

的协议如上所述首先用信息素的短20毫秒脉冲（剂量1微克和10微克）直接膨化的天线上进行测试。 图4A示出在响应信息素脉冲EAGs。它们是正的，因为在记录电极连接到该放大器的反相输入，如在步骤3.3中描述。正如所指出的功率谱，测量系统能够解析信息素脉冲高达10赫兹。为了比较，我们还测试了可商购的气体传感器。在TGS2620是制造的溶剂蒸气的检测金属氧化物传感器。虽然传感器呈现出高灵敏度的乙醇，这是无法跟随变化的浓度（参见图4B中的虚线）。问题来自传感器外壳。在TGS2620商品化，一顶帽子，有一个防爆不锈钢纱布。响应时间慢，因为，在实践中，它需要一个一定时间扩散穿过纱布和到达金属氧化物表面上的气体。恢复也慢，因为它需要时间来清洁感应器时，气体被困在帽内。因此我们去掉了帽和该变形改善了动力学显著（参见图4B中的纯曲线）。不过，有东亚运动会和TGS2620（10赫兹对1赫兹）之间的一个因素10。这种比较是定性的还是作为第一阶段和TGS2620没有在相同的条件下进行测试。 然后，我们评估了稳定过我们的全虫制剂（N = 12飞蛾）相比，切除天线​​（N = 7天线）的时间。东亚运动会是为响应信息素的刺激（持续时间500毫秒，剂量为1微克）定期记录。原EAGs（单位为mV）转换为相对EAGs（在时间t = 0获得初始值的百分比）。 图5显示了我们整个昆虫准备的非常好的稳定性aration在一个工作日。与此相反，EAGs上记录分离的触角迅速下降随着时间的推移，这样的信号下降到一半其初始值之后只有1.5小时。这一次的依赖性是由一个指数衰减有2小时的寿命很好的描述。 最后，我们测试了东亚运动会的机器人平板要搜索的气味源（信息素化合物，Z7-12:醋酸）的能力，利用活性搜索策略（ 图6A）。搜索策略的信息素检测与螺旋铸在没有检测28每一次结合了上风激增。信息素的存在下，从第一阶段检测到由神经形态检测器，如在步骤4.3中描述。第一阶段的两个例子，在搜索过程中记录示于图6B。如果没有气味源，东亚运仍然在零附近（ 即 2.5 V）与极少数或没有检测。该机器人进行螺旋铸件，一般离开搜索空间BEF矿石到达目标位置（在试验中92％，N = 26的试验， 图6C右）。与气味源（ 图6C左），东亚运动会呈现活性（检测）的交织在一起的静默期（无检测）爆发。螺旋铸件主要发生在羽轮廓（ 图6C左边，红色线），似乎是搬迁羽中心线时，气味是失去了一个有效的策略。在这种情况下，源通常发现（成功率= 96％，N = 44试验）。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51704/51704fig1highres.jpg" width="500" /> 图1:全虫东亚运动会筹备和机器人设置A）的触角电位（EAG）是由全虫准备（详见说明书），B）记录的准备安装在抢OT。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51704/51704fig1highres.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51704/51704fig2highres.jpg" width="500" /> 图2。硬件-软件界面。 A）鹰的硬件原理图。该电路由六个部分（详见说明书）。它允许过滤（频带0.1-500赫兹，缺口在50 Hz），放大（总增益250X），并在0-5范围内五，B信号调理）鹰布局，可以显示铜的线（顶部是红色，底部蓝色）和孔（绿色）。 三）印刷电路板（PCB），显示离散元素写在Qt的C + +实现数据可视化（红色曲线= EAG输入，绿色曲线D）图形用户界面（GUI）=神经元模型输出），滤波器设计和信号检测。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51704/51704fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51704/51704fig3highres.jpg" width="500" /> 图3:信号检测从东亚运动会。 A）触角电位（EAG）模型。东亚运动会是由一个非线性级联27，它由一个静态非线性其次是第一阶低通滤波器，指数脉冲函数的建模<img alt="公式21" fo:content-width=".2in" src="/files/ftp_upload/51704/51704eq21.jpg" /> 。在第一阶段的输出由卷积积分与给定的<img alt="公式22" fo:content-width="2in" src="/files/ftp_upload/51704/51704eq22.jpg" /> B）工程的办法。去卷积滤波器 -呃写道: <img alt="公式23" fo:content-width="1in" src="/files/ftp_upload/51704/51704eq23.jpg" />和<img alt="公式24" fo:content-width="1in" src="/files/ftp_upload/51704/51704eq24.jpg" /> ，请见文中说明。气味的遭遇（点击）检测时<img alt="式（12）" src="/files/ftp_upload/51704/51704eq12.jpg" />超过预定阈值。C）仿生的方法。用5内部的电流（泄漏，K +，钠离子 ， 钙离子和SK）甲霍奇金-赫胥黎类型的神经元模型来再现激励-抑制（EI）所观察到的点火模式实验观察到13。对信号的检测，对EAG信号被用作输入电流和命中检测每当突发激励的后面是抑制在烧成活性。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51704/51704fig4highres.jpg" width="500" /> &lt;<html> BR /&gt; 图4。东亚运动会的响应时间。 A）第一阶段的录音响应于20毫秒的脉冲信息素（剂量1微克和10微克），以不同的速率（1，2，4，6，8和10个脉冲/秒）的发送。归一化的第一阶段的功率谱示出了用于脉冲在1和10赫兹（剂量1微克和10微克）的刺激。东亚运解决个别脉冲高达10赫兹B）录音从气体传感器TGS2620响应乙醇（浓度波动）。的虚线和滑动曲线是具有和不具有盖子的传感器响应，分别。与盖的传感器具有几十秒的响应时间，因此不能遵循在气体浓度的波动。无盖的TGS2620解决个别波动可达1赫兹。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51704/51704fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 总是"&gt; <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51704/51704fig5highres.jpg" width="500" /> 图5。东亚运动会的稳定性（全虫准备与切除天线 ​​）。东亚运动会期间，8小时内为3.2小时的切除天线​​（N = 7天线）的全虫制剂（N = 12飞蛾），并每20分钟记录每隔一小时。该图显示了相对EAGs（在时间t = 0得到的初始值的百分比）。对于切除触角的时间依赖性以及拟合指数衰减2小时（1.5小时半衰期）的一生。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51704/51704fig6highres.jpg" width="500" /> 图6。机器人实验A）浪涌压铸策略结合在上风激增用螺旋铸造的气味在其不存在存在28 B）的搜索过程中，当机器人正在移动（有和没有气味）。C）机器人轨迹与气味（N = 44的试验）和无臭味（正记录的典型EAG = 26试验）。红色虚线表示，其中90％的所有检测的试验期间发生的羽轮廓。实验条件:搜索空间= 4米×2.5M，机器人的速度= 5.6厘米/秒，目标= 10微克信息素沉积在过滤纸和更换每2试验中，机器人的起始位置= 2米的目标，风速= 0.9± 0.2米/秒的目标位置。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51704/51704fig6highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches at JoVE.com

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

在自主机器人以昆虫触角电位传感器的嗅觉搜索

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects ...

using-insect-electroantennogram-sensors-on-autonomous-robots-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

22.9K 次观看.  Centre National de la Recherche Scientifique (CNRS). We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

视频: Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

科研

神经科学

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

隔离啮齿类动物或人类的鼻嗅干细胞

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

从使用吸吸液管技术的嗅觉受体神经元的气味引起的反应记录

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

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 ...

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

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

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

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

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

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

Designing and Implementing Nervous System Simulations on LEGO Robots

We present a method to use the commercially available LEGO Mindstorms NXT robotics platform to test systems level neuroscience hypotheses. The first step of the method is to develop a nervous system simulation of specific reflexive behaviors of an appropriate model organism; here we use the American Lobster. Exteroceptive reflexes mediated by decussating (crossing) neural connections can explain an animal's taxis towards or away from a stimulus as described by Braitenberg and are particularly well suited for investigation using the NXT platform.1 The nervous system simulation is programmed using LabVIEW software on the LEGO Mindstorms platform. Once the nervous system is tuned properly, behavioral experiments are run on the robot and on the animal under identical environmental conditions. By controlling the sensory milieu experienced by the specimens, differences in behavioral outputs can be observed. These differences may point to specific deficiencies in the nervous system model and serve to inform the iteration of the model for the particular behavior under study. This method allows for the experimental manipulation of electronic nervous systems and serves as a way to explore neuroscience hypotheses specifically regarding the neurophysiological basis of simple innate reflexive behaviors. The LEGO Mindstorms NXT kit provides an affordable and efficient platform on which to test preliminary biomimetic robot control schemes. The approach is also well suited for the high school classroom to serve as the foundation for a hands-on inquiry-based biorobotics curriculum.

An approach to neural network modeling on the LEGO Mindstorms robotics platform is presented. The method provides a simulation tool for invertebrate neuroscience research in both the research lab and the classroom. This technique enables the investigation of biomimetic robot control principles.

LEGO机器人神经系统仿真设计和实现

We present a method to use the commercially available LEGO Mindstorms NXT robotics platform to test systems level neuroscience hypotheses. The first step ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

嗅觉测定的神经退行性疾病的小鼠模型

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

成像膜电位与基因编码荧光电压传感器的两种类型

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

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.

The capability to localize an odor source is necessary for insect survival and is expected to be applicable to artificial odor-tracking. The insect-controlled robot is driven by an actual silkmoth and enables us to evaluate the odor-tracking capability of insects through a robotic platform.

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 ...