这项工作是由慷慨的加拿大自然科学和工程研究理事会（NSERC）和健康研究加拿大学院（CIHR）的支持。

此过程符合渥太华动物护理委员会大学的规定。没有利益冲突的声明。请参阅材料与试剂的表的设备和下列材料的品牌和型号。自书面的Spike2和MATLAB脚本和样本数据均在补充文件中提供。 1。水族箱和隔离室设置<ol><li> 反吸振地板 。通过堆叠橡胶垫，吸音泡沫塑料，船用胶合板面板，聚氨酯泡沫垫从底部到顶部（ 图1A）构造一个抗振动面（2.1米×2.1米）。打下胶合板面板上有四个木螺栓（5厘米×10厘米），以支持水族箱的边缘。 </li><li> 地板暖气 。莱电屏蔽加热元件在热梯度泡沫填充（ 见图1D底部）。覆盖有金属的加热元件LIC网电气屏蔽。 </li><li> 空箱 。使用1.3厘米厚的钢化玻璃面板，L形的铝制框架和水族馆级硅（ 见图1A）构造一个宽而浅的水族箱（1.8米×1.8米×30厘米）。覆盖所述槽的底面具有一大张白色背景上提供高成像对比度（见方案3）。 </li><li>通过做压克力板（哑光白0.64厘米厚）的墙壁安装（22.5厘米高）除以水族箱到中央舞台（直径1.5米）与四个角车厢（ 见图1B）。 <ol><li>弯曲4压克力板（22.5厘米×102.7厘米）通过加热创建四个弧形墙段，并使用硅填缝从四个角车厢分开中央的舞台将它们连接到罐底。保留20厘米的空间的弯曲部分之间的门安装。 </li><li>单独的邻近车厢的角落通过安装四个双墙无线日15厘米差距，提供额外的电气隔离和场所作水下传感器，如一个水听器。 </li></ol></li><li>装配4电动门，角室和中央舞台之间安装它们。 <ol><li>组装4门框， 如图1C所示 。每个门框创建六口井（0.64厘米深），嵌入尼龙橡子螺母（0.64厘米直径的螺纹），并用环氧树脂固定。 </li><li>切四门板由丙烯酸和橡胶板，并在丙烯酸和橡胶板的锁定机构建立六个孔（0.64厘米直径的）。加入用硅胶嵌缝丙烯酸和橡胶板。 </li><li>安装铰链的丙烯酸加盟门板与门框。 </li><li>摩摇摆伺服电机武器，并在门框顶部安装它们（ 见图1C）。使回路用电缆扎带的摆臂链接到门板。 </li><li>放置在GA的门组件PS弧形墙段之间创建，并使用硅填缝固定。 </li><li>所有的伺服电机连接到伺服控制器，并将其连接到通过一个活动的USB延长线的电源和一台电脑。使用与伺服控制器提供的控制软件测试的大门。 </li><li>硅胶变硬后，通过锁定所有的门用尼龙螺丝和灌装一室在同一时间检查水密性。 </li></ol></li><li> 隔离室 。构建一个隔离室包围的水族馆和阻断光线，声音和电气噪声外部来源（ 见图1D）。 <ol><li>提出三点墙板（2米×2米×5厘米）和四门板（1.9米×0.95米×5厘米）。对于每一个面板，加入铝嵌条（5厘米×2.5厘米）来创建一个矩形框，以及铆钉白色塑料波纹面板上的铝框。填写声学玻璃纤维棚板的面板，并关闭带有黑色塑料波纹板。</li><li>在抗震动地板安装三个墙板，并安装钢琴铰链可以加入四大门板上的墙板。 </li><li>环绕的隔离室中铝网格，和地面网格各方创造一个法拉第笼。 </li></ol></li><li> 湿度控制 。安装低噪音排气扇（ 图1F顶部），以除去多余的湿气积聚的加热。将排气扇最少2米距离的录制现场，并安装隔离室和排气风扇之间的风道。 </li><li>定期监测和维护鱼缸水和​​动物的条件。 <ol><li>保持恒定的水质条件为10厘米深，100μS/ cm的电导率和pH值7.0，加入水或盐溶液（请参考努森33的配方）。加入一包碎珊瑚，如果pH值低于6.5。 </li><li>安装立式鱼缸过滤器可从浅水操作进行清洗和曝气的目的（ 图1F底部）。断开过滤器和带他们出去了中央舞台的过程中记录会话。 </li><li>通过用弹性部件安装在其中吸盘提供在槽的底部活黄粉虫。避免自由浮动的猎物，如blackworms以防止录音过程中的杂散猎物的不受控制的喂养。 </li></ol></li></ol> 2。排爆跟踪<ol><li> 电极安装 。装配在中央舞台上的弧形墙8石墨电极，和空间他们平分。 <ol><li>得到画线（15厘米长;火星碳2毫米型HB​​）和剃除引线的外涂层。 </li><li>切同轴电缆（RG-174）的8个10厘米的段，环绕石墨棒的一端的电缆芯，并应用热缩管在他们的强大和稳定的电气连接。附加BNC插座连接器上的相对端（ 图2A左）。&lt;/ LI&gt; <li>放置在墙壁上的电极被录音，并应用在电极表面上的细条状胶带从硅胶保护。涂上硅填缝永久保持电极和硅变硬（ 图2A右）前取出所有磁带。 </li></ol></li><li>建造8的电缆组件，通过测量距离从每个电极到放大器单元，和在长度切割同轴电缆（RG-54）。连接BNC接头连接器的电缆的两端。 </li><li>使用电缆组件来连接所有电极到放大器单元。通过配对两个90°取向电极通过连接到法拉第笼差分放大（ 见图2B），和地面所有的同轴屏蔽线。 </li><li>设置为低于所述信号饱和极限的放大器的增益，并应用一个带通滤波器（200 Hz至5千赫）来去除噪声。数字化的四个记录电极对40 KS /秒。 </li><li> 在线信号处理。该指令对Spike2的软件写的，参数设置为Gymnotus SP进行了优化。 （ 见图2C的总结）。 <ol><li>加直流删除所有记录通道的过程（τ= 0.1秒）。 </li><li>添加精馏过程的所有录像通道。 </li><li>通过总结所有四个记录通道创建一个虚拟通道。 </li><li>通过增加一个有效值提取每EOD脉冲单峰信封（根均方， <img alt="" fo:content-width="40px" src="/files/ftp_upload/50962/50962_clip_image002.gif" width="70px" /> ）过程（τ= 0.25毫秒）到虚拟信道，用于产生每EOD周期单峰明确地确定脉冲定时。 </li><li>从虚拟信道建立realmark信道和记录时间和峰值振幅的值，设定适当的阈值来捕获所有的EOD脉冲的Wi后thout缺少一个脉冲，同时避免误报。 </li><li>由realmark通道的通道显示选项设置为瞬时频率模式监测瞬时排爆率的实时性。 </li><li>监视鱼类运动实时通过复制realmark信道，并且显示选项设置为一个波形模式。 </li><li>通过创建从realmark通道（0.01秒的采样周期） 的虚拟信道从EOD振幅斜率的RMS量化的活性水平，并加斜率（τ= 0.25毫秒）和均方根（τ= 0.5毫秒）的过程。 </li><li>导出realmark通道中的Spike2软件MATLAB的格式。 </li></ol></li></ol> 3。同步视频跟踪<ol><li>创建一个背景场景。 <ol><li>隐藏蒙上覆盖有磨砂的白色台面膜在水面上反射的任何对象。 </li><li>安装哑光白塑料瓦楞板15厘米以下的天花板以隐藏摄像机和通风口。 </li><li>在打印校准相机一大张白纸网格图案，打下它的油箱下方，以提供高对比度的背景。 </li></ol></li><li>安装光源。 <ol><li>获得红外LED灯，并删除内置风扇来减少噪音。驱动LED与放置法拉第笼外的电流稳压直流电源。 </li><li>安装红外灯在黑暗中灯成像，和白光LED灯驱动昼夜光周期中的试水产品。直接所有光源向天花板，实现间接和均匀照明（ 图3A）。 </li><li>通过驱动白光调节昼夜光周期的LED灯带定时控制开关（ 例如 12小时on/12小时关闭）。 </li></ol></li><li>安装一个摄像头水族箱的正上方。 <ol><li>得到近红外感光摄像头，或移除IR阻塞滤清器r除以断裂的薄有色玻璃的片材在所述透镜组件的背面。确保可视角度是足够宽，图像整体的中央舞台。 </li><li>使在天花板面板的中间一个小的观察孔，并且将相机放置在孔的正上方。 </li><li>镜头周围安装一个白色的环控卫，如果光源产生的眩光。 </li></ol></li><li>做一个时间同步的视频录制。 <ol><li>放置一个红外灯在四个罐的角落之一，产生时间同步脉冲（1毫秒的持续时间，10秒内）。加的负载限流电阻（1KΩ）呈串联，以及来自数字转换器硬件的数字输出端口驱动IR LED。 </li><li>使用捆绑在一起的相机，如果可用的视频录制软件。选择最高的录音质量（ 如无损压缩），支持最高分辨率。 </li><li>开始排爆记录之前，立即开始录像，停止录像I​​MMEdiately后，排爆记录。 </li><li>在记录之后，由第一和由信号数字转换器和视频记录捕获的最后的光脉冲之间线性内插变换图像帧编号，以数字化的时间单位。 </li></ol></li><li>自动图像跟踪 该指令用于在MATLAB 图像处理工具箱编写的，利用它的功能。自定义MATLAB脚本设有这次提交的自动图像跟踪。 <ol><li> 导入视频 。导入录像文件直接使用MATLAB工作空间"Videoreader。读 "功能。 </li><li>通过组合两个图像帧创建合成的背景图像。替换为从另一个帧的相同区域的未占用的图像（参见图3B）所占用的动物的图像区域。 </li><li>指定的图像区域通过绘制围绕中心舞台上的圆形面具排除一个跟踪REA之外（ 图3B底部），以及由一个常数（R INT）乘以设定最低门槛，强度差。例如，设置RINT = 0.85将抑制强度波动15％=（1 - R int）的背景下。 </li><li> 图像相减 。减去背景图像的图像帧（IM = K）（IM = 0），得到差分图像（=ΔIMK）。使用无符号整数数值精度来存储图像灰度值非负整数。 </li><li>段通过施加从graythresh函数所确定的强度的阈值的差分图像。使用bwmorph函数清洁二进制图像，并选择使用regionprops函数计算所有的blob区域后，相应的动物最大的blob。 </li><li>确定的质心和主要取向的x是通过应用regionprops功能，旋转图像，使其与x轴的长轴的最大BLOB的。分割图像的头和尾部分的质心（ 图3D顶部）。 </li><li>确定头部的长轴，并且旋转整个图像，使其与x轴（ 图3D左下方）。适合包围盒周围的头和尾部分平行于它们的长轴使用regionprops功能。 </li><li>确定在左，中和的包围盒（绿点在图3D底部）右侧垂直边缘的中间y坐标的Blob的，并将其分配到五个特征点（头尖，中间头，中体，中尾，尾巴尖）。 </li><li>种植集中在其前一帧确定动物的心一帧图像后处理连续帧。 </li><li>手动分配头方向的第一帧，并使用一个点积betwe连接从两个连续帧的方向矢量，以自动确定的头部方向。检查的结果，并手动翻转头部方向，如果不正确地分配。 </li></ol></li><li>通过连接头部的技巧绘制的动物运动轨迹，并顺利通过中位数和平均滤波器（N = 3），如果它有一个紧张的外观。叠加的轨迹与背景图像，并使用5的特征点（参见图2E）插鱼中线。 </li><li>通过重采样的瞬时排爆率（100 Hz采样率）和平均（0.0625秒的时间窗口）计算平均排爆率在每个图像捕获时间。剧情从时间匹配的排爆率决定的伪色的轨迹，并叠加的背景图片（ 见图2F）。 </li></ol>

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我们的技术意义。综上所述，我们首先描述一个大水族箱的建设和隔离室观察世界经济论坛所产生的自发的探索行为。接下来，我们展示了记录和跟踪EOD率和从无节制的鱼使用多个电极对的运动状态进行实时的技术。最后，我们描述了在时间同步的方式，通过水的红外视频记录技术，图像跟踪算法来测量身体的位置和姿势。作为实验的准备，世界经济论坛提供了通过展示容易量化的排爆率...

背景 。对动物行为的定量实验（ 如被迫的选择，避免震动，T-迷宫等 ）通常用于调查有关的感觉运动技能，学习和记忆的形成特定的假设。然而，这些限制性实验错过很多自然动物行为的丰富性，并有可能导致行为的潜在的神经基础的过于简单的模型。因此，更多的自然条件下的实验是由我们可以探索更充分地一个物种的行为习惯的重要补充。涉及自由移动的动物实验，但必须满足独特的技术挑战，如运动诱发的记录文物。不像刺激诱发反应，自发产生的探索行为是无法预测的，因此实验对象必须不断监测和跟踪在一段较长的时间。具体的研究问题的CAN为最好由精心挑选的生物体和可用的技术工具解决。例如，如基因编码的钙传感器1和光遗传学2光学记录和刺激技术已成功地应用于自由移动的遗传模式生物3-5。另外，小型化神经遥测系统可以记录和刺激自由活动小动物6,7。 电鱼 。世界经济论坛的物种产生电子琴放电（EODS），这让他们感觉到他们的周围环境或在更远的距离进行通信。 EODS的时间模式不同的条件，例如自我运动8,9，感官刺激10,11，和社会交往12,13下有所不同。脉冲型WEF物种产生离散的脉冲串，而不是其产生连续的准正弦波波型物种。一般情况下，脉冲型品种表现出铁道部Ë可变排爆率相比，波浪型的物种，以及动物的排爆率密切地反映他们的感官环境10,14的新奇内容。脉冲型物种可以立即在一个脉冲周期中响应缩短脉冲间的时间间隔（IPI），以一种新颖的感官扰 ​​动（新颖响应10,11,14）。这些鱼的不断电的行为可以通过从外部来源不受控制的感官刺激的扰动，以及不同类型的刺激，如振动，声，电，光被称为触发新奇的反应。因此，特别注意必须采取在一个长期观察的自由游动的世界经济论坛，以阻止或减弱外界感官刺激。以这种方式，改变EOD速率和运动轨迹可以被指定属于由实验者呈现的刺激。 水族箱和隔离室 ，因此， 我们放置的振动吸收材料的多层üNDER大型水族箱（2.1米×2.1米×0.3米），并包围了罐隔热的外壳挡住光，电噪声，声音和热通量的外部来源。排爆速率取决于环境温度15,16，因此水温严格调控在一个热带范围内（25±1℃）南美世界经济论坛的物种。我们构建了一个大而浅（10厘米水深）水箱，观察世界经济论坛的空间探索行为主要局限于在两个维度（ 图1A）。罐被分隔成中央舞台观察空间的行为，以及四个角室分别容纳个别鱼（ 图1B）。每个隔间建防水，以防止个人之间的电气连通。动物进入中央舞台从外面由四个电动门被控制。门被放置在车厢之间，他们成为水密锁定时由尼龙翼螺母。无金属部件被用于水下自WEF反应灵敏金属。 排爆记录 。在刻板的方式通过激活单（在Mormyrids）或多个空间分布的电动器官（在Gymnotiforms）17,18产生EODS。在排爆率的时间调制可以揭示更高层次的神经活动，因为延髓起搏器接收到来自更高的脑区，如间脑prepacemaker细胞核，从而接收来自前脑轴索19直接推算神经的投入。然而，在EOD定时必须小心地从原始波形记录中提取，而不是由动物的运动诱发的扭曲偏置。由WEF产生的电场可以近似为一个偶极子，因此EOD脉冲振幅在记录电极依赖于动物和电极8,20之间的相对距离和方向。动物的自我MOVEM已废除改变相对几何关系的动物并在电极之间，从而变动引起的EOD的振幅在不同的电极，以随时间变化的易失性地（参见图2B中君等人 8）。此外，自变动，改变记录的EOD的波形的形状，因为来自不同组的电动机构的相对贡献取决于它们的位置沿所述主体的长度和它们的局部曲率由尾部的弯曲引入。在EOD幅度和形状的运动引起的失真会导致不准确和不可靠的排爆定时测量。我们克服这些问题，通过空间平​​均记录在不同位置的多个EOD的波形，并通过添加一个包络提取滤波器，以精确地确定从自由游动WEF的EOD定时。此外，我们的技术还测量EOD振幅，这表明动物是否是休息或积极地移动的基础上，EOD的变化振幅随着时间的推移（ 见图2E和2F）。我们记录了差分放大信号从记录电极对，以减少共模噪声。因为在不规则的时间间隔产生的脉冲排爆，排爆事件的时间序列具有可变采样率。在EOD时间序列可以通过内插，如果需要通过所选择的分析工具被转换为一个恒定的采样速率。 视频录制 。虽然排爆记录可以监控动物的总变动活动，录像允许的动物的身体位置和姿势的直接测量。近红外（NIR）照明（λ= 800〜900 nm）的允许泰然自若目测的自由游泳的鱼21,22，因为WEFs是最活跃的在黑暗中，他们的眼睛是不近红外光谱23,24敏感。大多数数码成像传感器（ 如 CMOS或CCD），可以捕捉近红外光谱与wavelengt800-900纳米之间h不等，消除红外（IR）阻断滤波器25后。某些高端消费级网络摄像头提供了高清晰度，宽视角和良好的低光灵敏度，它可以产生的图像质量相当于或优于专业级红外摄像机可在更大的成本。此外，某些消费级网络摄像头捆绑在一起的录音软件，允许延长记录时间由没有质量损失压缩视频。大多数专业级摄像机与数字化信号的视频之间调整的时机提供时间同步TTL脉冲输出或TTL触发脉冲输入26，不过这个功能一般是没有的消费级网络摄像头。然而，视频记录和一个信号数字转换器之间的定时可以准确地匹配通过同时拍摄周期性地闪烁红外相机和信号数字化的LED。初始和最后的IR脉冲的定时可以用一个的两个时间刻度标记物，用于将所述视频帧号到信号数字化时间单元，反之亦然。 灯光及背景 。通过图像捕捉水可因光线反射在水面技术上的挑战。水面可以作为一面镜子来反映一个视觉场景上面的水，和模糊的视觉特征的水下，因此上面的水现场必须提供无特色，以防止视线干扰。以图像整体的水族馆，照相机需要被放置在水的上方，以及它应该在一个小的观察孔顶棚背后被隐藏，以防止其在水面上的反射。此外，水面可以产生刺眼和不均匀的照明，如果光源不正确投影。间接照明可实现均匀的亮度在整个水族箱通过瞄准光源朝向天花板，这样，天花板和周围沃尔LS可以反映和到达水面之前弥漫的光线。选择一个红外照明相匹配的相机（ 如 850纳米峰值波长）的光谱响应。来自光源的电噪声可以通过使用LED灯，并把它们的DC电源的法拉第笼的外侧被最小化。放置一个白色的背景油箱下方，因为鱼的对比以及在一个白色的背景在近红外波长。同样，隔离室的内表面上使用的亚光白色可提供均匀，明亮的背景照明。 视频跟踪 。视频录制完成后，自动图像跟踪算法可以测量一段时间内动物的身体位置和姿势。该视频跟踪可以通过现成使用的软件（观点或Ethovision），或用户可编程软件（基于OpenCV或MATLAB 图像处理工具箱 ）自动执行。由于图像跟踪的第一步，一个有效的跟踪区域需要通过绘制几何形状外排除的区域（屏蔽操作）来定义。接着，动物的图像需要从背景中通​​过从含有该动物的图像中减去背景图像来进行分离。该减影图像是通过将强度阈值，从而使得质心和所述定向轴线可以从二值形态学运算来计算转换为二进制格式。在Gymnotiforms 27-29和30-32 Mormyrids的electroreceptor密度最高的头部区域附近，因此在任何时刻的头部位置表示最高感官敏锐度的位置。头部和尾部的位置可以通过将图像旋转和边界框的操作被自动地确定。头部和尾部末端可以彼此通过手动定义它们在第一帧中，并通过跟踪它们的位置从比较两个连续帧区别开来。

<table border="0" cellpadding="0" cellspacing="0" width="1020">
	<colgroup>
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	<tbody>
		<tr height="21">
 <td></td>
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 <td></td>
			<td height="21" style="height:21px;width:216px;">[Aquarium construction]</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrically shielded floor heater</td>
			<td style="width:277px;">ThermoSoft Corp., IL, USA</td>
			<td style="width:241px;">ThermoTile</td>
			<td>www.thermosoft.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tempered glass panel</td>
			<td style="width:277px;">generic</td>
			<td style="width:241px;"> </td>
			<td>.5 inch thick, used for the aquarium construction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aquarium grade silicone</td>
			<td style="width:277px;">generic</td>
			<td style="width:241px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acrylic sheet</td>
			<td style="width:277px;">generic</td>
			<td style="width:241px;"> </td>
			<td style="width:287px;">.25 inch thick, matt white</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Natural rubber sheet</td>
			<td style="width:277px;">generic</td>
			<td style="width:241px;"> </td>
			<td>.25 inch thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Servomotor</td>
			<td style="width:277px;">HITECHRCD Inc., Korea</td>
			<td style="width:241px;">HS-325HB, 180deg rotation</td>
			<td>www.servocity.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Servomotor arm mount</td>
			<td style="width:277px;">HITECHRCD Inc., Korea</td>
			<td style="width:241px;">56362 Large Spline</td>
			<td>www.servocity.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Servomotor controller (6 chan.)</td>
			<td style="width:277px;">sparkfun.com</td>
			<td style="width:241px;">ROB-09664 </td>
			<td style="width:287px;">Micro Maestro 6-channel USB Servo Controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Active USB extension cable</td>
			<td style="width:277px;">C2G</td>
			<td style="width:241px;">38990</td>
			<td style="width:287px;">12m USB 2.0 A Male to A Female 4-Port Active Extension Cable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Exhaust fan</td>
			<td style="width:277px;">Nutone</td>
			<td style="width:241px;">ILFK120</td>
			<td>www.homedepot.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vertical aquarium filter</td>
			<td style="width:277px;">Tetra, Germany</td>
			<td>Whisper Internal Power Filter - 40i</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Crushed coral</td>
			<td style="width:277px;"> </td>
			<td></td>
			<td>Used to increase the pH of the tank water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"> </td>
			<td style="width:277px;"> </td>
			<td></td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">[EOD recording setup]</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Graphite Electrodes</td>
			<td style="width:277px;">Staedtler, Germany</td>
			<td>Mars Carbon 2-mm type HB</td>
			<td>Shave the outer coating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Physiological Amplifier/Filter</td>
			<td style="width:277px;">Intronix, Canada</td>
			<td style="width:241px;">2015F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coaxial Cable</td>
			<td style="width:277px;">generic</td>
			<td style="width:241px;">RG174</td>
			<td>For electrodes assembly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coaxial Cable</td>
			<td style="width:277px;">generic</td>
			<td style="width:241px;">RG54</td>
			<td>For wiring use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BNC jack connector for RG-174</td>
			<td style="width:277px;">Amphenol Connex</td>
			<td style="width:241px;">112160</td>
			<td>For electrodes assembly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BNC plug connector for RG-54</td>
			<td style="width:277px;">Amphenol Connex</td>
			<td style="width:241px;">112116</td>
			<td>For wiring use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Signal digitizer hardware</td>
			<td style="width:277px;">Cambridge Electronic Design, UK</td>
			<td style="width:241px;">Power MKII 1401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Signal digitizer software</td>
			<td style="width:277px;">Cambridge Electronic Design, UK</td>
			<td style="width:241px;">Spike 2. ver 7</td>
			<td></td>
		</tr>
		<tr height="21">
 <td></td>
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			<td height="21" style="height:21px;width:216px;">[Visual tracking setup]</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">White LED light</td>
			<td style="width:277px;">IKEA, Sweden</td>
			<td style="width:241px;">DIODER 201.194.18</td>
			<td>www.ikea.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Infrared LED light (850 nm)</td>
			<td style="width:277px;">Scene Electronics, China</td>
			<td style="width:241px;">S8100-60-B/C-IR</td>
			<td>Remove built-in fan</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">USB webcam</td>
			<td style="width:277px;">Logitech Inc., CA, USA</td>
			<td style="width:241px;">C910</td>
			<td>Remove Infrared blocking filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Motorized camera</td>
			<td style="width:277px;">Logitech Inc., CA, USA</td>
			<td style="width:241px;">Quickcam Orbit</td>
			<td>Remove Infrared blocking filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Video recording software</td>
			<td style="width:277px;">Logitech Inc., CA, USA</td>
			<td>Logitech Quickcam Software</td>
			<td>Download from www.logitech.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Matlab</td>
			<td style="width:277px;">Mathworks, MA, USA</td>
			<td style="width:241px;">2012a</td>
			<td>Image processing toolbox</td>
		</tr>
	</tbody>
</table>

long-term behavioral tracking of freely swimming weakly electric fish

长期行为跟踪可以捕获和量化自然动物的行为，包括那些不经常发生。行为，如勘探和社会交往能得到最好的研究通过观察奔放，自由活动的动物。弱电鱼（WEF）的显示屏很容易观察到探索性和社会行为所散发电子琴放电（EOD）。在这里，我们描述了三个有效的技术来同步测量排爆，身体姿势，以及一个自由游动的世界经济论坛为长时间的姿势。首先，我们描述了一个实验箱的结构的隔室旨在阻止感官刺激如光，声音和振动的外部源的内部。水族馆分配，以容纳四个测试样本，并自动闸门远程控制动物的访问中央舞台。其次，我们从自由游泳世界经济论坛描述准确，可靠的实时排爆定时测量方法。引起动物的身体运动的信号失真是通过空间平​​均和瞬时处理阶段修正。第三，我们描述了一个水下近红外成像的设置来观察泰然自若夜行性动物的行为。红外光脉冲被用来在一个较长的记录时间同步的视频和生理信号之间的定时。我们的自动跟踪软件测量动物的身体位置和姿势可靠地在水上的场景。在组合中，这些技术可实现长期观察的可靠和精确的方式可自由游动弱电鱼自发行为。我们相信，我们的方法可以通过探索性或社会行为与他们的生理信号同样适用于其他水生动物的研究。

排爆跟踪结果从不同的电极对所记录的排爆波形变化的幅度和形状从他们独特的位置和方向（ 图2C顶部）如预期。使用多个电极对确保强劲的信号接收在水箱内的所有可能的位置和世界经济论坛的方向。该包络波形（ 图2C底，绿色曲线）总是包含每个排爆周期的单峰，其中担任一个可靠的时间标记，精确测定脉冲间的时间间隔和瞬时排爆率（IPI = -1?...

Watch this Scientific Journal Video about Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish at JoVE.com

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

我们描述了一组技术研究的自由游动弱电鱼在较长的时间内，通过同步测量动物的电子琴放电时间，体位和姿势既准确，可靠地在一个专门设计的水族箱内感官自发行为隔离室。

自由游泳弱电鱼的长期行为跟踪

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

long-term-behavioral-tracking-of-freely-swimming-weakly-electric-fish

Research

JoVE Journal

Neuroscience

12.5K 次观看.  University of Ottawa. 我们描述了一组技术研究的自由游动弱电鱼在较长的时间内，通过同步测量动物的电子琴放电时间，体位和姿势既准确，可靠地在一个专门设计的水族箱内感官自发行为隔离室。 

视频: Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

线虫跟踪和行为测量

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

科研

神经科学

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

长期的嗅觉适应性的分子读数<em&gt; C.线虫</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

在两个神经元处理阶段在做人蜜蜂同时长期录音

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

老鼠耳蜗外植体的长期时间推移成像

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

琼脂糖微室的长期钙成像<em&gt;秀丽隐杆线虫</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction. However, these responses may be subtle, and measurement can be challenging. Using juvenile white sturgeon (Acipenser transmontanus) with copper exposures, this paper illustrates techniques used for quantifying behavioral responses using computer assisted video and digital image analysis. In previous studies severe impairments in swimming behavior were observed among early life stage white sturgeon during acute and chronic exposures to copper. Sturgeon behavior was rapidly impaired and to the extent that survival in the field would be jeopardized, as fish would be swept downstream, or readily captured by predators. The objectives of this investigation were to illustrate protocols to quantify swimming activity during a series of acute copper exposures to determine time to effect during early lifestage development, and to understand the significance of these responses relative to survival of these vulnerable early lifestage fish. With mortality being on a time continuum, determining when copper first affects swimming ability helps us to understand the implications for population level effects. The techniques used are readily adaptable to experimental designs with other organisms and stressors.

Measuring the impacts of environmental contaminants on fish behavior is often subjective and challenging particularly when dealing with sublethal endpoints. We describe methods including video technology to quantify swimming behavior of early life stage white sturgeon (Acipenser transmontanus) during and after 96 hr acute exposures to various concentrations of copper.

使用计算机辅助视频和数字图像分析量化鱼游泳行为应对水铜的急性接触

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction.  ...

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

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

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

的简化方法超低密度，长期主要海马神经元文化

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

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

一种新的行为分析调查个人的味觉，可自由移动的熊蜂（<em&gt;欧洲熊蜂</em&gt;）

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

无神经的一代和长期维持<em&gt;水润</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

Noninvasive EEG Recordings from Freely Moving Piglets

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

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

自由移动仔猪的无创脑电图记录

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