This work was supported by a BBSRC project grant to DMcK, DMcC and RGN (BB/K002775/1) and a PhD studentship [to JP, BB/J013854/1]. We gratefully acknowledge the fantastic advice on technical equipment and field techniques that we received throughout the project from Nozrat Mirzai (Bioelectronics Unit University of Glasgow), Ross McLeod and Matthew Clavey (ThermalVision Research) as well as SCENE for all their logistic support of the field work. We would also like to thank Debbie Kelton at Veracity for generously providing equipment used to power the thermal imaging camera in the field.

伦理声明: 这项工作涉及批准由英国信托鸟类和捕获协议引发的鸟温和的压力自由生活的小雀常规捕获方法进行了根据英国内政部的许可。 1.设置对于拍摄<ol><li>创建一个拍摄设置，其中鼓励鸟类到自己在相机（图1）的前方位置。鸟进入的建立通过孔在一个端壁，并具有获得食物接近相对的端壁。 </li><li>为了使其习惯鸟的设置，数周记录之前提供合适的食品（例如，花生仁）在送纸器。除了提供食物，留下的建立不受干扰。在此期间，将一个虚设三角架在送料器的前部，以允许鸟也，观察者到摄像机。 </li><li>上记录的天，减少availability在设置了由从给料器中除去所有剩余的食物，并与只用一个小孔在其中心上，通过该鸟类可以访问食物顶端包含在透明容器中的食物代替它花生仁。位置在笼子的在相对的入口孔端的中心这种食品容器。 </li><li>放置的已知发射率的黑色绝缘胶带小方向，在的框的网格的一个角自然珠被所以它出现在所有图像。将黑色绝缘带用温度记录仪，其中记录了黑色绝缘带的温度为0.1℃，每1秒的分辨率相连的热电偶。 </li><li>将热成像相机50厘米从箱阱的中心，使得摄像机视场中的所有盒的配合，和鸟类从透明容器馈送将被定位在聚焦相机的区域。将相机连接到一台笔记本电脑，将相机<html>在每秒（带有时间戳）平均7.5帧的记录影像，并且将图像发送到笔记本电脑的硬盘保存在那里。 </li><li>从设置了约20米 - 附加渔线的设置了旋转门，并推广到哪里实验者从视图中鸟隐蔽的位置，但设置了仍然可以观察到。 </li></ol> 2.拍摄鸟的响应轻度急性应激<ol><li>一旦鸟进入进给箱，留在不受干扰的框约 5秒鸟。 </li><li>拉钓线鸟度过之后，在框约 5秒以关闭馈送盒。注意使鸟仍处于框的远端，以便尽量减少受伤的鸟的风险。 </li><li>马上接近进给箱，站在一动不动背后的摄像头约3分钟。然后检索框中的小鸟，让它去。 </li></ol>从热像眼区域温度"&gt; 3。提取<ol><li>提取每个帧的最高温度。注意:最高温度几乎总是从眼睛周围的区域内的记录，由periophthalmic环（图2）的暴露的皮肤有界，并且在下文称为眼区域的温度。 <ol><li>在热成像分析软件右键点击图片，"添加"一个新的剧情为图像最大。然后，右键单击该出口数据（眼部区域的温度和帧拍摄的时间）为CSV文件的情节。 </li></ol></li><li>从CSV文件中删除从帧，在此鸟的眼区是不可见的所有行。 </li><li>暗算时间最高气温在R 27，确定手动点在哪里时，瞬膜被拉扯了眼点低值outsid两个连续的读数（闪烁）和之间的温度峰值&gt; +0.2ºC体温鸟类28电子的范围 （即，当眼睛区域是可见的，但通过多帧运动模糊），然后从在步骤3.1生成的CSV数据文件中的相关行。 </li><li>测量环境温度。 <ol><li>从温度记录仪中的数据（时间和温度记录器）下载到计算机上，并出口到电子表格中。 </li><li>为了从热图像的环境温度，在热图像分析软件绘制一个正方形了，涵盖了温度记录仪探头黑色绝缘胶带，然后用鼠标右键单击在广场上，并​​选择方形的平均温度"添加新的阴谋"。 </li><li>然后，右键单击该地块，将数据（时间和IRT温度）导出为CSV文件。合并两个产生的温度时间序列进行温度记录仪和热图像匹配时间到一个电子表格。 </li></ol></li><li>正确的眼部区域temperatu再相对于环境温度。导出步骤3.4中创建成CSV文件中匹配时间的电子表格。到每个保留的眼区域温度增加温度记录器和热成像衍生温度值（记录器温度 - IRT温度）之间的差的同时进行测量。 </li><li>进行自动过滤以除去使用峰值搜索算法（见补充编码文件）自动提取数据中的最高的（因此最准确的）点不太准确低眼区域的温度值。 注意:峰值搜索算法重新组织的温度数据与用户定义的宽度（跨度）的载体，建议跨度= 3，提取的中心值的行其中数字任一侧较低， 即峰。 </li><li>用线性插值以关闭留下的空白由序列时没有峰萃取，当眼睛区域是不可见的。使用命令na.approx（动物园包v1.7-11在R 27），得到每秒单个温度值的每一个人。 </li><li>在CVS数据文件，0.2的值添加到每个眼睛区域的温度来校正的通过网格拍摄图像的影响。 注意:试验表明，当黑体，加热到近似平均〜41ºC鸟体温在其活性相28，被通过网状窗口具有相同的网目尺寸为在拍摄设置用于成像，记录，由温度在未由导线遮蔽区域的热成像照相机分别为0.2ºC低于没有居间网格得到的值（平均差异= -0.2ºC±0.085的SD，n = 30的黑体的温度范围= 41.6-42.5℃）。 注意:这特定于本研究中的条件的修正系数，并有可能随仪器制造，和类型和宽度的动物和摄像机之间的啮合，并且在每一种情况需要建立的藻ecific学习条件。 </li><li>从箱子被关闭之前选择的最高值记录到的最大眼区的温度，这将构成不受干扰鸟的基线眼部区域的温度。添加该基准眼区的温度作为一个新的列到CVS文件（基准线）。然后减去从每个保留最大的眼区域的温度值，产生在CVS数据文件中一个新的列中的基线值。 注意:这个新的价值现在快递鸟的应对轻度急性应激作为其原状的基准温度偏差。 </li><li>从之后的陷阱使用命令ggplot（以R 27包GGPLOT2 V1.0.0）被关闭小区内的所有个人的基本眼部区域温度最高眼部区域温度的偏差。生成使用选项mean_cl_boot（在R 27包GGPLOT2 V1.0.0）自举95％置信区间。有关详细信息，请参阅补充编码文件。 </l我&gt; </ol>

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The authors declare that they have no competing financial interests.

本文的目的是描述了必要的方法来测量响应于急性应激改变一个自由生活的动物的皮肤温度。这项研究表明，与野生鸟类的急性应激反应有关的皮肤温度急剧变化可以被捕获的非侵入性使用IRT。此过程涉及到五个重要阶段（1）设计相应的字段使用高度便携式热成像系统设置的，（2）测量基准温度，（3）应用温和的压力源，涉及捕捉的鸟在设置起来， （4） 事后自动化的眼区域温度和（5）校准萃取温度数据的采样。这里介绍的方法适用于野生蓝山雀捕获。代表基线眼区域的温度可能会被捕获在低至5秒。在现场记录一个鸟所需的估计时间是大约1小时，用以处理所需的一小时3分钟热的视频序列。 在收集动物的皮肤温度好的数据需要高质量的热图像。热成像仪的区别仅在于从根本上从可见光摄像机在它们检测出的电磁辐射的波长而言，因而许多适用于摄影的概念（例如，视场，场的深度）也适用于热成像。如由野生动物摄影师通常实践，该动物的访问可以预见，其中天然（例如，巢位点）或人工（例如，馈线），可用于收集的热图像的任何位置。吸引动物相机而不是试图跟着他们，虽然它们的栖息地已经允许我们设置摄像头提前，尽快收集干扰最小的高品质的图像作为动物映入眼帘的优势。然而，使用这种方法，这是至关重要的，以检查或考虑的影响测量应力或皮肤温度的具体情况。例如，在这里，鸟衡量一个饲养环境，食物是用来积极引诱蓝山雀进入菜单。研究鸡表明，急性积极经验，如食物奖励的一种联想学习试验还可以降低眼气温 29的预期和消费。因此，进入这个特殊的设立个体的"基线水平"可以通过这一行为与奖赏之间的关联影响。这种可能性值得进一步研究，但如果存在，预期会提高从基线水平的下降已经观察到。此外，生热发生食物的消化，但不期望食物期间基线测量所消耗的具体效果的3分钟捕集周期30内显著升高温度。 这种限制认为，测量所有动物在同样的情况下也是这种方法的巨大力量。代表结果表明，有一个大的变化的个体之间的眼区域温度的急性应激涂布之前（基线眼区温度）。在基线眼温度的变化（变化= 3.9％的系数），可能是部分原因是由于测量误差或个体之间的差异的真实写照。期间IRT的相机记录的黑色绝缘带的温度在5秒时，磁带的温度不发生变化，变化的平均系数为0.26％（范围为0.08％至0.59％）。虽然测量误差很可能是在移动的鸟较大它仍然显示，大多数这种变异在基线眼区域的温度确实个人之间。相对较小的测量误差对移动鸟的概念在基线测量进一步支持了眼睛区域的温度高重复性超过20秒（ <stroNG&gt;图3）。通过控制的情况下，我们可以保证所有的鸟都从事相同的活动。我们还可以最大限度地减少在测定时，以在皮肤温度变化的个体之间，例如，通过确保局部性从混杂入射辐射保护环境的贡献，将，热成像，导致动物的表面温度过高估计。这可以通过在阴凉处或通过策略性地放置的色调，包括阻断红外线波长最透明的塑料的工作来实现。这里介绍的协议，显示了一个简单的设置，可以很容易地应用到越冬蓝山雀的情况。蓝山雀的拍摄，而在与避免阳光直接照射了坚实的屋顶进给箱。这种设置可能也适用于定期来人工饲养在一年中的某些时候许多其他雀形目。蓝山雀是比较小的动物，它们需要被拍摄特写镜头捕捉细节的良好水平。与摄影相机，越来越接近的主题 - 无论是在实际的距离，放大倍率方面 - 显着减少景深。有鸟尽可能大的视场需要从相机的最小焦距，而在这种情况下，为500毫米，并减少景深至40毫米拍摄它们。因此，在拍摄设置的所有特征需要被设计，以鼓励动物内的所用的特定相机的锐聚焦的相对窄的区中显示他们的头部。 响应于一个完善的急性轻度应激的动物，我们预期在血流模式的快速变化，从经由交感神经介导的血管收缩外围向芯，减少皮肤温度17。眼眶周围区域被选为该规定的身体，这是绝缘的，是阿索的唯一地区ciated用小血管中，RETE ophthalmicum能影响热损失从眼睛31中的富混合体网络。热videoing技术的快速帧速率是能够显示在大约2℃眼区温度下降在10秒，随后通过后续的温度上升到内基线的在3分钟内0.5℃。虽然在应对轻度应激周边散热一直在约1分钟的间隔17,26，32以前记录的，该技术在这里表明，在温度最高降幅可能会非常迅速。凭借较低的时间分辨率的这种影响的程度可能会错过。比较在不同个体的应激反应的差异时，这可能是重要的。重新升温迅速"战斗或逃跑"反应后，小鸟没有恢复到基线水平鸟依然在箱体内表明禽流保持生理应激。鸟人释放d从实验装置的约3分钟任意选择的分界点后，自由生活的动物克制的时期减少。然而，在未来，试验可能需要更长以记录完整的温度响应，直到动物的眼睛区域温度恢复到基线水平，如果后勤可能的。 这涉及到一系列的前后应激现在被应用后，采集到的图像的新协议允许响应急性应激的动态的详细研究，以及动物个体之间的多个时间点的比较。重复个人对不同季节或环境条件在该试验可以允许解缠结上基线或后急性应激皮肤温度环境的影响，作为一个可能的途径为慢性压力评估为好。 。 鸟的皮肤温度不仅取决于代谢产热和血流量也对从太阳辐射，风速和润湿25的热交换。这妨碍了精确的热录音风雨交加的条件。记录的热成像仪的眼区温度可在任一时间被低估（负误差）或高估（正误差）。大量正误差将需要能量输入，但是这是可以避免的，在馈送盒的鸟从太阳被屏蔽。正误差的另一个来源包括当鸟眨了眨眼。偶尔鸟简要地拉动其瞬膜在眼睛区域和，因为它是内部存储的温度更接近于身体的核心，闪烁给出了一个异常高的温度读数时，它覆盖眼球。这是，然而，很容易地检测为它留下了非常标记签名和受影响的帧可以被删除。主要的原因在我们的记录负误差为运动模糊。任何运动也迅速被捕获莎尔帘布层由照相机的帧速率混淆从与周围冷区（运动模糊）的图像的小眼暖区域中捕获的数据，从而在眼区温度的低估。除去正误差后因而这使得从眼部区域测得的最大温度最精确的测量，并使用峰值函数命令的自动过滤除去最大眼区域的温度的可能性较小较低的值。 此外，作为红外线辐射被水蒸汽吸收，表面温度记录将由环境的相对湿度的影响。这可以通过输入空气温度，相对湿度，距离物体插入分析热图像分析软件进行核算。然而，更精确和有效的方法（如在此进行的）是包括已知的温度和发射率的基准体的视野内。在我们的情况下，这是的黑色绝缘胶带的正方形为0.97的辐射率，这大约相当于天然外皮33。这允许通过使用测得的绝缘带的实际和热成像衍生温度值之间的差校正眼区域的温度。黑体的表面温度可以被用来在整个测量周期连续地校准图像。 由于在开发的技术的进步，在热感摄像机，他们现在可以部署为小型轻量的相机能够收集每秒许多帧在延长的时间周期。虽然热成像是在禽类研究25一种广泛使用的技术中，尺寸和费用的热成像系统的限制了其在野外使用。在这项研究中，系统是高度可移植的，大约花费。 £6,000，并能提供高分辨率热成像视频非侵入捕捉精确的TE从自由生活的动物mperature数据，而无需处理鸟的需要。记录每秒多个帧允许的高分辨率时间序列外围温度的测量，并因此提供探索急性应激反应的动态的可能性。这是很难实现与由可以在一段时间内采取的样本数的限制的常规的糖皮质激素的样品。这里，我们已经萃取1秒的时间间隔，这是足以证明眼区域温度响应于急性应激（捕捉）时间序列的眼区域的温度，但较高的时间分辨率也将是可能的。的大量图像的积累，但是，需要的自动化中的从图像信息中提取一定程度。该协议描述了一个简单的半自动化的进程来提取每个图像的最高温度。我们能够做到这一点作为感兴趣的区域中，眼区域选择性N，总是在图像中最温暖的地方。分析可能是更成问题，如果感兴趣的区域是更复杂的，并且可能需要定制设计的图案识别软件（例如，34）。在这项研究中一些手动过滤物所需的情况下，眼区域温度为过度或估计但这些都容易地检测，但当然进一步自动化是可取的。应力引起的周围冷却的由热成像测量提供了一个有价值的除了应力等生理措施和该技术的非侵入性的方面是涉及圈养野生动物进一步的研究非常有利的。 捕获完整的应激反应，红外热显​​然有着巨大的潜力，作为压力评估的工具。成为一种非侵入性的替代既定激素和核心温度测定法虽然，这将是必要的交叉验证之中，这些措施并确定皮肤温度显示了相同的比例与应激强度， 即皮肤温度响应的程度上可以反映应激的实力。未来的研究还应解决皮肤温度是否捕捉慢性压力。虽然SIH响应于急性应激预计是短暂的，经常暴露于身体或心理应激源急性可以生成核心体温35,36慢性升高。无论是持续的血管收缩有助于这一核心海拔一直没有明确的测试。然而，在人类慢性压力有关的疾病患者相关研究做表明降低手指温度37。最后一个属性探讨的是价:区分正从消极事件的能力。激素测定分不清价5，有出现，反映的激励水平，而不是压力特别糖皮质激素水平。研究梳理脾气ATURE暴露在厌恶和积极刺激国内鸡建议皮肤温度可能同样类似于一般的觉醒22,29。然而，在人类中，不同的情绪状态引发皮肤温度区域的具体变化，如眶周变暖和脸颊冷却时吓了一跳38，但笑39时整体下降皮肤温度。不同地区之间的比较还可能揭示的情绪状态。使用本文所提出的建议，将有可能解决所有这些问题，并验证皮肤温度应力的一种非侵入性标记物。

应力是在生物学的中心概念，描述一个生物体的响应于环境挑战试图恢复动态平衡1,2-应急。在压力下，血糖，脂肪酸和氨基酸的水平，心脏，呼吸率和代谢率都增加，血液从外围向芯器官2被转向。此通用的生理变化的图案素数的动物，以便能够迅速地和自适应地社会和生理挑战的阵列响应。虽然认识和了解强调的是在两个纯洁的心脏和应用动物研究，评估奔放的动物的压力仍然是一个挑战。 应力的一种广泛使用的生理标记物是增加循环糖皮质激素，诸如皮质醇和皮质酮1,2-水平。这种方法的一大优点是，它们的浓度增加我Ñ​​比例应激强度，从而允许定量应力。但是，糖皮质激素都没有"应力"激素本身 ，但能量存储 2个的动员。作为这样糖皮质激素水平也更改与节能要求，一天，年龄和生殖状态3,4的时间，以及响应于明显阳性的情况下，如配合机会5。糖皮质激素水平，因此，必须慎重和一个上下文中解释。测量急性应激糖皮质激素反应也具有一定的局限性。急性应激引发的最初增加糖皮质激素水平，随后返回到基线水平1,2个动态响应。糖皮质激素的样品通常是侵入性获得，通过血液取样，这本身就是一个应激，因而可能会影响测量的糖皮质激素水平6。而且糖皮质激素水平只能在一个或很少的时间p内进行测量oints，这可能不是捕获的变化的峰值水平和定时或响应的持续时间，限制了我们通过个体内的动态应力响应的时间来检查变化的能力。激素采样的非侵入性的方法，如从粪便7，毛发或羽毛8，测量平均糖皮质激素水平天或数月的在较长的时间尺度，虽然有用研究慢性压力，并不适用于急性应激的研究。因为即使是最好的已建立的方法只提供一个有限的角度上的变化在急性应激之间的个体有必要在生理应力测量的替代方法。 作为应激反应涉及多个生理效应有其他候选人可能表明应力。其中，交感神经介导的血管收缩通道血液从外围向芯体。此浓度的血液，因此，热，以及各种形式的应激性产热S，温暖的铁心 9。这样，核心变暖，称为应激诱导的体温过高（SIH）也被用作急性应激的药物研究10一个标记。 SIH通常在提出一种急性应激10的10-15分钟的核心体温0.5-1.5℃。它是一个捕捉和处理的动物的一个普遍适用的实验应激可以提高核心体温的范围哺乳动物和鸟类物种11的相对有案可稽现象- 17。重要的是，相关SIH与其他已建立的指标的压力，如心脏速率18，糖皮质激素水平19和行为20,21。而像糖皮质激素水平，核心温度已经线性相关的应力水平在某些物种22。然而，与血液采样，核心温度的测量是侵入性本身，因此需要在插入Ò探针10的ř植入。基线核心温度时，逐步应力感生处理或探针插入必须重复23每次增加。在温度记录设备的最新发展，允许访问数据远程可以提供一种解决方案，至少对于大型动物24。 内生成SIH相同的机制，但是，在于还应力的另一个潜在标记:末梢血管收缩是移动的血液，并用其加热，在芯同时冷却皮肤17。不像核心温度，皮肤温度可以测量完全非侵入性，由红外热成像（IRT）。 IRT将来自一个物体的表面发射到温度25的红外辐射。作为一个动物的表面通常有一些裸露的补丁从皮肤温度可以直接派生，它需要获得自然外露的皮肤（ 如面积吨左右他的眼睛）或修剪毛皮或羽毛创建一个光秃秃的补丁。到皮肤和动物的一个合适的露出面积提供的访问可以保持在视摄像机的领域，IRT照相机可以用于远程收集连续皮肤温度测量，可能允许的完整的温度响应于被拍摄下来，和个人之间的比较。尽管已在该表面鸡所示温度响应急性应激26，本研究的新颖之处在于它测量野生动物的表面体温在比以前的研究更精细的时间分辨率，也表明了预期皮肤温度下降可以在该领域，其中的温度，湿度和天气是可变来检测。本文的目的是描述了必要的方法来测量使用热成像无约束动物的皮肤温度。我们使用捕捉诱发轻度急性应激在自由生活的蓝山雀（Cyanistes青色，林奈1758 </EM&gt;）和描述如何捕获合适的图像，分析图像和处理数据，以产生温度响应曲线相对于时间，展示响应于轻度应激的鸟类的皮肤温度的显著冷却。这可以提供评估野生和圈养动物有用了广泛的研究人员从生态学家，生物学家和生理学家动物福利研究人员强调一种有价值的替代方法。 此项研究是在17日期间日 2013年12月，与4 日 2014年1月在落叶栎（ 栎属）林苏格兰中心的生态与自然环境（场景），格拉斯哥大学东海岸的洛蒙德湖，西环苏格兰（56.13°N，4.13°W）。该协议涉及三个主要步骤:（1）设置在其下以捕获热图像从自由测距动物合适的条件下，（2）applyi纳克急性应激同时服用的热视频，和（3）从该随后可用于表征动物的反应，急性应激的热图像中提取和处理数据。在我们的例子中，我们吸引了自由生活的雀形目，蓝山雀到饲养箱（拍摄设置），其中热图像拍摄条件进行了优化，并应用于捕获的压力通过远程关闭进料箱时，鸟在里面。热图像捕获和数据提取的描述特定于我们所使用的设备，并且可以热成像系统之间变化。数据处理是使用开源软件描述。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Thermal Imaging Camera</td>
			<td>FLIR</td>
			<td>A65 (f=25mm)</td>
			<td>Compact (106 &#215; 40 &#215; 43 mm) &#38; low cost</td>
		</tr>
		<tr>
			<td>Thermal Image Analysis Software</td>
			<td>FLIR</td>
			<td>ResearchIR v3.4</td>
			<td>Allows high speed thermal video recording and thermal pattern analysis</td>
		</tr>
		<tr>
			<td>Temperature logger</td>
			<td>Gemini Data Loggers</td>
			<td>Tinytag Talk 2 TK-4023-PK</td>
			<td>Monitors from -40 to +125&#176;C using accompanying temperature probe</td>
		</tr>
	</tbody>
</table>

thermal imaging to study stress non-invasively in unrestrained birds

压力，在生物学的中心概念，描述了一套紧急应对的挑战。在其他的反应，压力导致在血流量的改变，其导致的血液净流入到关键器官和增加核心温度。这种应激诱导的体温过高是用来评估应力。但是，衡量核心温度是侵入性。作为血流被重定向到芯，主体的周边可以冷却。本文介绍了采用红外热像，其中外围体温测量非侵入野生蓝山雀协议（Cyanistes青色 ）。在现场，我们创建了一个设置使用诱饵盒使鸟儿在镜头前一个理想的位置。该相机采用原状鸟的短热录像施加温和的压力源（关闭对话框，因此捕捉鸟）之前，和鸟的回应被困记录。眼区域的裸露皮肤是最温暖的地区的形象。这允许自动提取从每个图像帧中的最大眼区域的温度，然后进一步的步骤的手动数据过滤除去误差的最常见的来源（运动模糊，闪烁）。这个协议提供用细的时间分辨率，使我们能够研究应激反应非侵入性的动力学的时间序列眼区域的温度。进一步的工作需要证实了该方法的有效性，以评估应力，例如，调查的眼区域温度响应是否是正比于压力源的强度。如果能得到证实，它将提供压力评估的一个有价值的替代方法，在动物和将有利于大范围的研究人员从生态学家，生物学家，生理学家动物福利研究人员。

结果从9自由生活的蓝色山雀呈现，说明可从热成像来获得和证实应力在鸟瞰区域温度预测的信号可以以游离测距动物中检测到的信息。每只鸟被拍摄的平均时间为禁区前5.1±0.9秒（9例）&#39;门被关闭了。这允许不受干扰鸟瞰区域温度（基准温度）的计算，这是所有后续措施可以被引用。在20秒的热视频原状蓝山雀到达和陷阱内饲养的剪辑进行的试验（N = 9鸟类用于轻度应激的应用程序的有所不同），R的相关性&gt; 0.7被发现的最大的存在前5秒期间眼区域温度记录，最大记录在第一10，15和20秒（图3）。这被解释为表示最大VA略记录在最初几秒钟后陷阱条目为代表的长期的水平，并在紧接盒闭合前述大约5秒的平均眼区域的温度被用作基线值。蓝山雀在此示例中有38.4±0.5°C（平均值±SE）的平均基线眼部区域的温度和范围为35.8至39.9℃（N = 9）。一旦鸟喂食5秒以允许基线眼区域温度的计算中，箱门被关闭由实验者。所有的鸟注意到盒子的关闭就证明了他们试图逃跑航班。 在陷阱闭合，眼区域温度迅速下降，并〜10秒（图4）之后达到最小值眼区域温度〜1.3℃，低于基线的温度。 图4示出了基于最大心眼复合曲线所有的九个人的区域温度平均每一秒。由于定时和的眼睛区域的温度下降的幅度个体之间变化的复合曲线掩模温度响应的真实程度。计算每个个体的平均温降分别为2.0±0.2℃，并经过9.4±2.8秒（平均值±SE）到达最低点。从那以后，眼部区域温度逐渐向基准眼部区域温度恢复在接下来的2-3分钟，但没有被审判结束完全达到基线。 <img alt="图1" src="/files/ftp_upload/53184/53184fig1.jpg" /> 图1.拍摄舞台的设置在现场的目的是要吸引鸟的照相机视其中轻度急性应激可以应用的领域，使用一个馈送盒。在设定了包括的25 14厘米，16.5厘米高箱胶合板建成，具有镀锌铁丝网与光圈SIZ前面板电子商务1 2.5公分。网格允许红外辐射通过，使得能够拍摄鸟类，而内部的菜单。丝网被选择，如玻璃和大多数塑料不传输红外辐射。毫米直径60的孔切成左端壁以允许鸟进入设置和获取食物放置在盒子的右端。的黑色绝缘胶带（标记黑色箭头）两个正方形之间夹着一个温度记录器探头连接至前面板以记录一个基准温度的右上角。轻度急性应激通过关闭框施加到鸟内。拉动附连到旋转门在进入孔的钓线允许实验者当鸟是内关闭该盒。热成像相机的陷阱前面设置记录事件的整个序列。 <a href="https://www.jove.com/files/ftp_upload/53184/53184fig1large.jpg" target="_blank">请点击此处查看大图Ø</a>F该数字。 <img alt="图2" src="/files/ftp_upload/53184/53184fig2.jpg" /> 一个蓝山雀图2.热像，大部分雀形目的身体既受羽毛绝缘（或由腿秤在较小程度上），但是眼睛周围的皮肤暴露并且辐射在正常情况下最多热量，并且是包围冷却器包膜或背景。这是由黄色（最高温度）眼睛周围的显示在该热图像，橙色，红色，紫色和蓝色表示冷却器和更低的温度。 <a href="https://www.jove.com/files/ftp_upload/53184/53184fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53184/53184fig3.jpg" /> 基准温度测量Ø图3.重复性版本不同时期的时间内记录的第10，15和20秒的最高值0.5度记录在第1-8秒"的最大值平均"的剪辑和最高温度之间的相关系数夹子（分别由红，蓝，绿线表示）。所有的剪辑为20秒的持续时间（N = 9）。在使用更大的数据集的初步分析，一些"的最大值来"的计算从仅有1值，并给出了相同的值在所有的比较RHO。从分析中删除了这些片段，只有那些从超过3次测量，计算办法离开。 <a href="https://www.jove.com/files/ftp_upload/53184/53184fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53184/53184fig4.jpg" /> 图4.响应眼部区域的温度来捕获的，有一个非常清晰和明显的诱捕事件信号的眼区的温度随时间的变化反映出来。为了比较跨越该变化在其基准温度，残留的眼区域温度的个体，如实际温度之差与那个个体的基线温度，标绘在垂直轴上。这是相对于时间绘制与收集设置为0的闭合和鸟从陷阱3分钟后取出。红线显示的平均剩余温度和灰带代表了95％的置信区间。 <a href="https://www.jove.com/files/ftp_upload/53184/53184fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds at JoVE.com

Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

有必要对压力的非侵入性的评估。本文介绍使用热成像检测野生蓝山雀眼区的温度为轻型急性应激一个显著响应一个简单的协议。

热成像学习压力非侵入性的无拘无束的小鸟

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood ...

thermal-imaging-to-study-stress-non-invasively-in-unrestrained-birds

Research

JoVE Journal

Neuroscience

13.3K 次观看.  University of Glasgow. 有必要对压力的非侵入性的评估。本文介绍使用热成像检测野生蓝山雀眼区的温度为轻型急性应激一个显著响应一个简单的协议。 

视频: Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

糖尿病大鼠的热痛觉阈值的快速测定

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

局部和全局的方法评估热痛觉<em&gt;果蝇</em&gt;幼虫

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

膜下丘脑腹内侧神经元的电位染料成像从成年小鼠为研究葡萄糖传感

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

鼠神经母细胞核转染到研究神经细胞迁移<em&gt;在体外</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

New Methods to Study Gustatory Coding

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons (GRNs) detect tastant molecules, they encode information about the identity and concentration of the tastant as patterns of electrical activity that then propagate to follower neurons in the brain. These patterns constitute internal representations of the tastant, which then allow the animal to select actions and form memories. The use of relatively simple animal models has been a powerful tool to study basic principles in sensory coding. Here, we propose three new methods to study gustatory coding using the moth Manduca sexta. First, we present a dissection procedure for exposing the maxillary nerves and the subesophageal zone (SEZ), allowing recording of the activity of GRNs from their axons. Second, we describe the use of extracellular electrodes to record the activity of multiple GRNs by placing tetrode wires directly into the maxillary nerve. Third, we present a new system for delivering and monitoring, with high temporal precision, pulses of different tastants. These methods allow the characterization of neuronal responses in vivo directly from GRNs before, during and after tastants are delivered. We provide examples of voltage traces recorded from multiple GRNs, and present an example of how a spike sorting technique can be applied to the data to identify the responses of individual neurons. Finally, to validate our recording approach, we compare extracellular recordings obtained from GRNs with tetrodes to intracellular recordings obtained with sharp glass electrodes.

We present three new methods to study gustatory coding. Using a simple animal, the moth Manduca sexta (Manduca), we describe a dissection protocol, the use of extracellular tetrodes to record the activity of multiple gustatory receptor neurons, and a system for delivering and monitoring precisely timed pulses of tastants.

研究风味编码的新方法

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

温度梯度法测定果蝇幼虫的热偏好

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

结合行为和脑电图研究正念冥想对情景记忆的影响

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. Imaging studies are widely used to investigate their function and plasticity in physiological and pathological conditions. Because of their size and structure, localization studies of proteins require high-resolution imaging techniques. In this protocol, we describe a procedure to study in primary neurons the co-localization of target proteins with synaptic markers at a super-resolution level using structured illumination microscopy (SIM). SIM is a patterned-light illumination technique that doubles the spatial resolution of wide-field microscopy, reaching a detail of around 100 nm. The protocol indicates the required controls and settings for robust co-localization studies and an overview of the statistical methods to analyze the imaging data properly.

This protocol shows how to employ super-resolution microscopy to study protein co-localization in primary neuronal cultures.

超分辨率成像研究原发神经元中蛋白质和突触标记的共同定位

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. ...

热成像学习压力非侵入性的无拘无束的小鸟

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