我们感谢斯蒂芬施瓦茨和基督教Biobserve公司Gutzen赞助这篇文章中，相当多的技术援助。我们感谢与鱼护理援助奥利维亚奥乔亚，基督教考夫曼和扎卡里水芹，我们感谢墨西哥联邦政府权限收集鱼。我们要感谢格伦Vigus，弗雷德里克帕克，并在得克萨斯州A＆M可视化实验室雅典娜梅森和瑞安伊斯特林协助编写本出版物。经费是由美国得克萨斯州A＆M大学和美国国家科学基金会的IOS - 1045226。

1。设置硬件和查看器的系统。 该系统由一个测试舱两侧是两台监视器。 BIOBSERVE Viewer系统连接到一台摄像机记录的实时问题的运动。运动数据是通过交互式视频播放（IVP）单独的服务器上，这就决定了屏幕上的动画刺激的运动方案。 <ol><li>两个CRT显示器放置在两端用干净的水装满一个80升的水族馆;匹配监视器输出。 </li><li>将相机的开销捕捉水族馆众目睽睽之下;连接到BIOBSERVE视频卡。 </li><li>配置查看器的系统，以接收从相机跟踪信息。 </li><li>启用插件查看器，通过指定的网络IP地址发送动物的口鼻部，正文和尾部动画实时服务器的坐标。 </li><li>打开动画服务器。 </li></ol> 2。校准。 <ol><li>在动画的服务器，打开InteractiveDisplaySetup.txt。输入像素下的“监视器信息”屏幕宽度和屏幕的高度。在“PROGRAMTYPE”进入“校准”。保存文件的变化。 </li><li>双击IVP程序图标启动程序。 </li><li>在查看器的系统设置步骤2.3罐体外形尺寸范围的输入。这将确保查看器数据和交互式输出从初值之间的通信。 </li><li>由于鱼缸的两端均小于显示器，该软件允许用户使用键盘（宽度A和S，Z和X高度，箭头键位置）的移动可视面积（视口）刺激测试槽的两端对齐。观看动画的服务器上，粉红色的视是一个测试显示器和其他蓝色视口（图1）呼应。 </li><li>使用键盘上的Z和X，直到它匹配所需的刺激大小规模的雄性鱼都视口中的显示。 </li><li>移动显示框，让他们试验槽的边缘对齐。用户使用的Q或W键移动靠近或远离中心的鱼箱。 </li><li>程序提示时，输入实验信息。用户已经预装模型（在我们的演示，求爱男剑尾birchmanni，X malinche）之间进行选择的选项。用户还可以指定在每个阶段的每个鱼（非交互式或交互式），侧面和阶段的行为，每个刺激会出现，而且所需的大小，每个刺激（标准长度）。 </li><li>接下来的问题询问用户是否希望鱼动作要镜像，即水族馆两侧模型运动将完全相同。这仅仅是用来当鱼在动画模式。最后的问题是关于运动的背鳍。用户可以选择默认的背，这将使的背，只有提高在横向的求爱显示。如果背是不是设置为默认，用户可以决定是否该模型提出了它的背鳍，根据从监视器的主题距离。 </li></ol> 3.Starting IVP为队友选择测试。 <ol><li>打开InteractiveDisplaySetup.txt和设置“PROGRAMTYPE”liveTesting相同的文件名，包含实验数据，并设置“experimentName”。 </li><li>轻轻地放置在水族箱受鱼，并等待10分钟。 </li><li>启动浏览器2.0和IVP。 </li></ol> 4。材料的选择试验。 整体实验顺序如下1-4以往的研究，使用非交互式视频刺激。女性测试水族馆的两端具有两种不同的显示器上的刺激。女性行为Biobserve Viewer系统实时跟踪。响应实验是协会的时间，所需的时间，女性10厘米内的一台监视器或其他花费。协会的时间是自动计算的Viewer。 <ol><li>视频刺激显示在审判中的科目，包括两个20分钟的阶段。一个阶段包括4个5分钟的环节： <ol><li>第一部分acclimatizes测试罐五分钟的女性，在两个监视器上显示的单色屏幕。 </li><li>在第二部分中，有两种不同的视频刺激，显示女性，左边的监视器上和其他五分钟的权利。 </li><li>紧随视频刺激显示，单色屏幕又是五分钟的两个监视器上显示。 </li><li>在第四和最后一节，提出了从第二部分的刺激，但每个刺激的位置切换​​。这提供了一个侧的偏置内女性的控制。 </li></ol></li><li>美国证券交易委员会第二个阶段重复同样的模式，与同时提出了不同的刺激。 </li><li>额外的试验运行，返回到步骤2，系统地呈现不同的侧面和秩序。 </li></ol> 5。代表性的成果。 我们评估了交互式和非交互式动画拉拢男性比较，女性的反应交互播放的效果。非交互式的刺激进行求爱刺激屏幕上，独立的女性行为，因为在以往的研究 1-4 。 互动刺激追踪在屏幕上的女性的水平位置。在操作模拟鱼或受鱼的位置定义为连接线的质心和吻中点。指导男性行为的三个规则如下，其中X方向是指坦克的长度，和Z方向是指坦克的宽度（图2）： 规则1：模拟男性始终遵循在屏幕上的问题，跟踪她在Z方向。 规则2：模拟男性的背鳍是它正在执行一个横向求爱显示时只提出。 规则3：模拟的男剑尾鱼将只执行一个横向求爱显示它正在显示对女性的总时间的50％。横向求爱是引发男性在四分之一的男性在Z方向的雌鱼体长的鱼。横向求爱是独立的女性是在X方向的监控密切。 互动刺激密切跟踪女性的实时位置（图3）。 使用非交互式刺激4,5以前的工作表明，女性的 X birchmanni强烈的首选自己种的视觉信号。 IVP创建非交互式的刺激，从而引发同种交配偏好（T = 1.923，N = 9，P = 0.046）同样有效。当女性被测试模拟互动的同种和heterospecific男性立即之前或之后在同一审判，然而，他们没有表现出偏好（图4）。 <img src="/files/ftp_upload/2374/2374fig1.jpg" alt="图1" /> 图1。显示器校准设置。 <img src="/files/ftp_upload/2374/2374fig2.jpg" alt="图2" /> 图2。坦克和显示器安装示意图，用来描述位置指示轴。 <img src="/files/ftp_upload/2374/2374fig3.jpg" alt="图3" /> 图3。随着时间的推移动画的典范和代表女性主体的水平位置。 <img src="/files/ftp_upload/2374/2374fig4.jpg" alt="图4" /> 图4。协会与交互式和非交互式的同种和heterospecific男性刺激女性的时间（S ）。每个小区创建原型从5分钟的位置输出记录。在这些地块中，时间的推移在垂直Y方向从0到5分钟。 <img src="/files/ftp_upload/2374/2374fig5.jpg" alt="图5" /> 图5。代表协会的实时数据和跟踪两个互动的刺激。两个五分钟的两名女性细分。

<ol>
	<li>Fisher, H. S., Mascuch, S., Rosenthal, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivariate+male+traits+misalign+with+multivariate+female+preferences+in+the+swordtail+fish,+Xiphophorus+birchmanni.">Multivariate male traits misalign with multivariate female preferences in the swordtail fish, Xiphophorus birchmanni.</a> Anim. Behav. 78, 265-269 (2009).</li><li>Fisher, H. S., Rosenthal, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hungry+females+show+stronger+mating+preferences.">Hungry females show stronger mating preferences.</a> Behavioral Ecology. 17, 979-981 (2006).</li><li>Fisher, H. S., Rosenthal, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+swordtails+court+with+an+audience+in+mind.">Male swordtails court with an audience in mind.</a> Biology Letters. 3, 5-7 (2007).</li><li>Wong, B. B. M., Rosenthal, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Female+disdain+for+swords+in+a+swordtail+fish.">Female disdain for swords in a swordtail fish.</a> American Naturalist. 167, 136-140 (2006).</li><li>Fisher, H. S., Wong, B. B. M., Rosenthal, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+the+chemical+environment+disrupts+communication+in+a+freshwater+fish.">Alteration of the chemical environment disrupts communication in a freshwater fish.</a> Proceedings of the Royal Society B-Biological Sciences. 273, 1187-1193 (2006).</li><li>Rosenthal, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+considerations+and+techniques+for+constructing+video+stimuli.">Design considerations and techniques for constructing video stimuli.</a> Acta Ethol. 3, 49-54 (2000).</li><li>Turnell, E. R., Mann, K. D., Rosenthal, G. G., Gerlach, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mate+choice+in+zebrafish+(Danio+rerio)+analyzed+with+video-stimulus+techniques.">Mate choice in zebrafish (Danio rerio) analyzed with video-stimulus techniques.</a> Biol. Bull. 205, 225-226 (2003).</li><li>Rosenthal, G. G., Ryan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assortative+preferences+for+stripes+in+danios.">Assortative preferences for stripes in danios.</a> Animal Behaviour. 70, 1063-1066 (2005).</li><li>Reynolds, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flocks,+herds+and+schools:+A+distributed+behavioral+model.">Flocks, herds and schools: A distributed behavioral model.</a> , 256 (1985).</li><li>Reynolds, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steering+Behaviors+For+Autonomous+Characters.">Steering Behaviors For Autonomous Characters.</a> , (1999).</li><li>Hoare, D. J., Couzin, I. D., Godin, J. -. G. J., Krause, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context-dependent+group+size+choice+in+fish.">Context-dependent group size choice in fish.</a> Animal Behaviour. 65, 663-669 (2004).</li><li>Hoare, D. J. &. a. m. p. ;. a. m. p., Krause, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+organisation,+shoal+structure+and+information+transfer.">Social organisation, shoal structure and information transfer.</a> Fish and Fisheries. 4, 269-279 (2003).</li></ol>

生产这种视频文章Biobserve研究赞助。

前动物行为的交互式视频播放方法依靠人工操作，提供响应的主体行为的线索。有了IVP，我们创建了一个程序，适用于以规则为基础的互动响应，对主体行为的实时自动数据。我们简单介绍一下创建下面的程序中所涉及的步骤。 第一步是创建数字化X 的男性典范birchmanni和X 。 malinche。我们花了6以往的研究方法类似。我们创建了一个基于真正的 X的照片上的纹理建模的三维网格birchmanni和X 。 malinche。为了捕捉真正的鱼，用来模拟鱼的形状相同的照片，逼真的纹理被用作鱼的纹理。一个平面贴图应用到自己的紫外线的坐标对齐与照片纹理的UV贴图。其次，数字鱼网状必须像一个真正的鱼变形。而要做到这一点，创建一个虚拟的骨骼是身体和鳍和“剥皮”网格。关节旋转时，使网格变形蒙皮过程。 第二，我们添加了数字鱼的议案。六关键的动作，使得男性的剑尾鱼的动画。三个动作被用来代表不同的速度，鱼会游泳。其他三个乐章，其余仍鱼，转弯，或参展横向的求爱显示。由于雄性能够按照提高或降低其背鳍，无论男性还是女性接收机目前的 3，我们从外侧求爱运动解耦的背鳍。背鳍键，这样它可以循环过程中提高或降低在任何时候。共有二十四个动画周期使用。每个周期的开始和截至鱼在同一姿势，使动画周期可以很容易地交融在一起。二十四动画周期对位7,8所需的议案，从现场的开销视频， 拉拢男性 X。 birchmanni。 第三，我们启用了交互性。我们使用的Biobserve查看器的系统，实时跟踪的吻，身体和女剑尾尾的位置和传输实时IVP方案信息。这样做是单独为每个监视器上的每个求爱男。男性动画跟着受鱼的位置。我们仿照下面的使用雷诺抵达转向行为9,10，允许男性遵循女性和减速，因为它接近女性。 要计算出在每个时间步的男剑尾鱼的位置，该系统提供与当前位置的女性，这使得程序来计算的力量，推动男性。首先，目标偏移向量计算，减去从雄鱼雌鱼的位置的位置。二雄鱼雌鱼的距离是由目标偏移矢量的幅度。第三，雄鱼所需的速度是由除以一个常数的减速值的距离。这使雄鱼放慢，因为它接近雌鱼。最后减去所需的速度从目前男性的速度计算，所需的加速度。 由于动画是在60赫兹的离散视频帧渲染，计算每个离散的时间步长，在0.016秒的时间间隔。最大速度设置为10厘米/小号这些实验的价值。如果新速度的幅度大于最大速度，速度设置为最大。 对于这个特殊的模拟，互动雄鱼背鳍提出50％的时间，只有在求偶相互作用。横向的求爱行为是引发男性的刺激时，在0.25 Z尺寸的女剑尾鱼的体长。 我们很惊讶，交互取消conspecifics女性的偏爱，尽管事实上，非交互式的动画引起了强烈的偏好和女性度过了大部分时间与关联的互动刺激。一种可能性是，可以重写用于评估男性的视觉线索，如剑和背鳍，密切关注女性。此外，女性可能不太可能在拉拢男性失去兴趣，因此不大可能样品不论是个人（图5）。 然而，我们的结果表明，视频游戏技术的工作原理，即软件驱动，响应用户输入的规则为基础的药物可以成功地应用于互动式播放，在研究动物的行为。这种类型的以规则为基础的的互动式播放证明对研究变浅和集体运动11,12有用。在操纵一个虚拟的典范变浅使用规则的能力，特别是应该让我们见识到动物使用的进程，使变浅决定。

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<td>C# program using Microsoft&#x2019;s XNA Game Studio 2.0</td>
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<td>BIOBSERVE Viewer 2</td>
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<td>Dell 15&#x201D; CRT monitor (2)</td>
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<td>20 X 20 X 80 cm Plexiglas testing aquarium</td>
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automated interactive video playback for studies of animal communication

视频播放是一个广泛使用的技术，为控制动物传播的视觉信号的处理和介绍。特别是，参数为基础的电脑动画，提供了机会，独立操纵任意数量的行为，形态，或光谱特征逼真，动物在屏幕上移动图像的背景下。然而，常规播放的一个主要限制是视觉刺激，缺乏互动能力与活的动物。从视频游戏技术的借贷，我们已经创建了一个自动化，视频播放，从视频跟踪系统的实时信号控制动画的交互系统。女剑尾鱼， 剑尾birchmanni进行材料的选择试验，我们证明了这种方法。女性被赋予了一个献殷勤的男同种和水族馆两侧的一个求爱男heterospecific（X. malinche）之间的同步选择。虚拟男性刺激编程跟踪水平位置的女性，在野外求偶的男性。伴侣的选择试验，对野生捕捞的X 。 birchmanni女性被用来验证原型的能力，有效地生成一个逼真的视觉刺激。

Watch this Scientific Journal Video about Automated Interactive Video Playback for Studies of Animal Communication at JoVE.com

Automated Interactive Video Playback for Studies of Animal Communication

视频播放是一种广泛使用的技术在动物行为。我们创建和评估程序，适用于回应，主体行为的实时自动数据的规则为基础的，三维电脑动画的交互播放。

自动化研究动物交流的交互式视频播放

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

automated-interactive-video-playback-for-studies-animal

Research

JoVE Journal

Neuroscience

13.2K 次观看.  Texas A&M University (TAMU). 视频播放是一种广泛使用的技术在动物行为。我们创建和评估程序，适用于回应，主体行为的实时自动数据的规则为基础的，三维电脑动画的交互播放。

视频: Automated Interactive Video Playback for Studies of Animal Communication

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

器官型小脑文化：细胞凋亡的挑战和检测

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

科研

神经科学

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

闪光光解笼状化合物在嗅感觉神经元的纤毛

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to investigate the effects of locally manipulated neurotransmitter and signaling pathways in awake, behaving animals. Moreover, the stereotaxic implantation of electrodes for electrophysiological stimulation and recording studies has been instrumental to our current understanding of neuroplasticity and brain networks in behaving animals. Ever-increasing knowledge about optimizing surgical techniques in rodents1-4, public awareness concerning animal welfare issues and stringent legislation (e.g., the 2010 European Union Directive on the use of laboratory animals5) prompted us to refine these surgical procedures, particularly with respect to implementing new procedures for oxygen supplementation and the continuous monitoring of blood oxygenation and heart rate levels during the surgery as well as introducing a standardized protocol for post-surgical care. Our observations indicate that these modifications resulted in an increased survival rate and an improvement in the general condition of the animals after surgery (e.g. less weight loss and a more active animal). This video presentation will show the general procedures involved in this type of stereotaxic surgery with special attention to our several modifications. We will illustrate these surgical procedures in rats, but it is also possible to perform this type of surgery in mice or other small laboratory animals by using special adaptors for the stereotaxic apparatus6.

Stereotaxic surgery on rodents allows for targeted administration of drugs or electrical stimulation and recordings in awake, behaving animals. In this video presentation we will demonstrate recent procedural refinements to this long-standing procedure that successfully improved survival rate and reduced post-surgical weight loss.

啮齿动物的立体定向手术和动物福利成果改进行为神经科学

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to ...

Whole Animal Perfusion Fixation for Rodents

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate 5,7. Often, changes in response to hypoxia begin before the tissue can be preserved 12. The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures 3. It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.

Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain.

整体动物灌注固定鼠害

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small ...

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser.&#160;Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible.&#160;The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

在鼠背耳蜗核的听觉通路的光遗传学刺激直接可视化

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

Assessment and Communication for People with Disorders of Consciousness

In this experiment, we demonstrate a suite of hybrid Brain-Computer Interface (BCI)-based paradigms that are designed for two applications: assessing the level of consciousness of people unable to provide motor response and, in a second stage, establishing a communication channel for these people that enables them to answer questions with either &#39;yes&#39; or &#39;no&#39;. The suite of paradigms is designed to test basic responses in the first step and to continue to more comprehensive tasks if the first tests are successful. The latter tasks require more cognitive functions, but they could provide communication, which is not possible with the basic tests. All assessment tests produce accuracy plots that show whether the algorithms were able to detect the patient&#39;s brain&#39;s response to the given tasks. If the accuracy level is beyond the significance level, we assume that the subject understood the task and was able to follow the sequence of commands presented via earphones to the subject. The tasks require users to concentrate on certain stimuli or to imagine moving either the left or right hand. All tasks are designed around the assumption that the user is unable to use the visual modality, and thus, all stimuli presented to the user (including instructions, cues, and feedback) are auditory or tactile.

With this experiment, one might be able to detect consciousness in people with disorders of consciousness. Furthermore, the approach can create a simple communication channel that enables people to give simple YES/NO answers to questions.

有意识障碍者的评估与沟通

In this experiment, we demonstrate a suite of hybrid Brain-Computer Interface (BCI)-based paradigms that are designed for two applications: assessing the ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

用双光子激光消融神经种群及其对斑马鱼幼虫钙成像和行为记录的评价

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

微粒相关α synuclein 聚合体对原代神经元的外源性管理--纤维形成的强大细胞模型

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

体外楔形切片准备模拟在Vivvo神经元电路连接

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...