我们感谢她的工作，在创建Virtools的三维示意图，在玛雅和埃里克孟森世界霍尔顿汤普森。研究由美国国立卫生研究院博士后F32 MH078471部分赞助NCH和NIDA的RO1 DA027802至九龙南线。潜水是由美国国家科学基金会资助的BCS - 0420632。

A.刺激和任务的设计 1。一般设计。 我们研究语境的影响，担心收购和记忆保持两天。这种设计是在啮齿类动物实验研究，为长期记忆的形成和现实世界的恐惧是在时空距离，从治疗和重新接触的经验中吸取的突发神经生物学的合并进程的帐户的同时。在杜克大学的沉浸式虚拟环境（潜水）称为完全沉浸式虚拟环境中，遇到了动态条件刺激（CS）（移动蛇和蜘蛛）和有条件介绍电气手腕刺激配对。采用差分恐惧制约程序使用皮肤电导反应（SCR）作为一种恐惧的依赖措施。在这里，我们展示了空调的恐惧和随后的存储器保留两天在26名健康男性和女性参加者，年龄18-30岁，在杜克大学的潜水测试。该协议被批准，根据杜克大学IRB标准。 2。参与者设立在潜水。 潜水是一个完全封闭的，六面，米× 3米× 3米，背投影虚拟现实（VR）的环境。潜水是坐落在一个专门建造的30英尺立方体跨学科的工程，医学和应用科学中心（控制室（VisRoom），图1）在杜克大学的。恐惧在潜水空调进行如上所述。 与会者坐在跟踪的3 - D眼镜头向前跳水面临的中心。这些体位约束，以避免头晕，帐户的高度可变性，背景和刺激之间的接触量的控制参与者都采取一个固定的“虚拟步行通过指定的环境在虚拟蛇和蜘蛛遇到的每一个学习阶段。参加者，并保证，是切实更新，根据参与者的情况下通过运动视觉显示。 3。歧视调节过程。 歧视程序被聘用，其中一个Visual CS演示部分钢筋（配筋率40％），合作终止的电刺激，无条件刺激（美国）在收购阶段。学员被分配到2个条件之一：虚拟蛇或虚拟蜘蛛的恐惧收购。与美国配对的强化刺激，简称为“CS +其他视觉刺激的”CS -“是明确作为控制未成。CS +和CS -随机分配和整个群体的平衡。 4。空调的刺激。 刺激是动态的蛇和蜘蛛，单独出现在中间，持续4秒的屏幕前的潜水中心。这种合作发生的蛇或蜘蛛的外观与听觉刺激信号，提醒参与者的环境中的新刺激（拨浪鼓或拍打声，分别）存在。广告蜘蛛与蛇的虚拟场景使用Maya动画软件创建和导入Virtools的软件（Virtool，行为公司的SA，巴黎，法国），在潜水观看。 5。无条件的刺激。 电刺激实验开始前进行了调整，根据每个主题的容忍程度，以促进集团的比较和消除混杂因素的影响整体觉醒水平的差异 ， 跨4组，5。每个参与者选择的刺激水平，他或她的看法“非常恼人的，但使用一个递增的楼梯程序不痛。电压最初被设置在30 V的水平低，在5 V的增量增加，直到与会者表示，他们的不引起疼痛耐受程度已达到transcutaneously管理，对参与者的主导手腕的正中神经刺激（200毫秒的持续时间在30-50赫兹）由双极表面刺激电极（21毫米电极间距：草Telefactor。担保由橡胶表带型号FE 10S2，西华威，RI）。电极导线连接通过同轴电缆通过射频滤波器，屏蔽和接地导线，以一个基层Telefactor的SD - 9刺激。，生理盐水为基础的凝胶（SIGMA凝胶：帕克实验室，新泽西州费尔菲尔德）被用来作为电解质导体（见图2），与会者被告知，所有脉冲将在相同的强度交付。 6。训练阶段。 这里描述的实验是在两会进行24小时的延迟。在杉杉吨的会议上，最初的习惯期4观看的3 - D完全沉浸在灰色的背景，但未经加固或培训或测试发生在虚拟世界中，每个CS型试验。这个阶段允许驯化在潜水和减少定向条件刺激的反应的实验环境。 16紧随习惯相后，担心收购相混合每个CS类型，其中CS是16 CS +试验单独和5加强审判。大约24小时后，发生记忆保持和灭绝培训测试。这个阶段包括16每个CS类型的试验，美国没有，在一个虚拟的背景下，无论是相同或不同的恐惧收购范围内（跨与会者平衡）的。一个上下文是室内环境（室内配有家具的公寓，上下文）和其他方面的室外环境（邻里现场，上下文乙）。受试者被随机分配到实验组，其中1和2天的背景下介绍的顺序确定。他们被分配到相同的上下文条件（AA或BB）或上下文移条件（AB或BA）。路径长度和课程与虚拟世界之间的一致性，以及对象/在不同的环境刺激的数量和位置。 7。实验参数。 审间的间隔时间为14 ± 2秒。 CSS的序列是伪，受约束，不超过2试验相同的CS（状态焦虑和认知的预期，以避免混淆归纳）连续发生。局部加固的CS +（40％）用于延迟迅速灭绝，通常发生在人类参与者100％的CS + 加固 6,7 。此外，局部加固提供了一个更现实的空调应急厌恶的事件并不总是发生后担心刺激的程度。 8。任务说明。 每个实验阶段之前，学员们了解以下设计特点：他们在虚拟环境中，会遇到动画蛇和蜘蛛，沿固定路径的虚拟步行路程，他们将通过环境引导;他们可能会收到电刺激手腕在整个研究过程中的任何时间来调理前的水平。他们奉命直接面对，并出席在屏幕的正面中心提出的蛇和蜘蛛的图像，并提醒说，他们没有任何超过他们通过自己的运动世界或电刺激的发生的控制。他们还被告知，他们可以随时终止，而无需对他们的刑罚的研究。 B.心理生理测量 1。数据收集。 可控硅被用作依赖恐惧措施，如前面所述4，6。可控硅录得通过心理生理监测系统（BIOPAC系统，圣巴巴拉，CA）。可控硅监测从银 - 氯化银电极魔术贴绑带连接到非惯用手的第一和第二位的中间指骨的磁盘。生理盐水为基础的凝胶（西格玛凝胶）被用作导电的电解质。受试者指示，以保持他们的手仍然在SCR记录电极，以避免运动文物。信息达到BIOPAC生理记录系统，这是位于外的中控室的潜水。 BIOPAC系统同步运行Virtools的软件的计算机的刺激呈现。图1说明了在潜水的参与者，沉浸在背景A.控制计算机（Virtools与脚本生成）设立BIOPAC（SCR）和电刺激技术，如图2所示。 皮肤电导在200 Hz采样，放大，存储使用AcqKnowledge软件（BIOPAC系统，圣巴巴拉，CA）的离线分析。 Virtools的软件控制刺激的演示，并触发通过国家仪器DIO - 24数据采集卡（德克萨斯州奥斯汀​​市）的震荡发生器。记录的波形的低通滤波使用布莱克曼窗口（截止频率为31赫兹）和3点多，连续的数据平滑。皮肤电导响应振幅时间锁定到每个CS和美国的相对发病到刺激前的基线，推导出一个依赖条件和无条件的恐惧措施，分别为4-6，8。列入数据分析，建立了以下标准：延迟= 1 - 4秒，持续时间= 0.5 - 5秒，最小幅度= 0.02微西门子（μS）。答复不符合这些标准的得分为零。 2。 Analys可控硅。 由于可控硅数据通常是向零倾斜，数据平方根转化之前，统计分析，以达到一个正常的分布。 “早”和“晚”每个阶段的试验块，作为学习跨越时间通常在每个学习阶段不同，从每个CS类型（虚拟蛇或蜘蛛）的数据倒塌。反复措施方差分析分析被用来计算空调皮肤电导反应组的差异作为一个学习为主体内的变量相与CS型（晚采集功能（CS + CS -），早期或晚期灭绝（CS +，CS - ）和上下文之间的可变数据，每次试验的条件的响应值除以每个参与者自己的最大的美国的反应，以手腕的刺激（对任何审判）考虑到个体差异归转让（相同或Shift）。在响应和排除非应答者（个人谁显示很少或根本没有可控硅）有关数据在图3差可控硅分数可视化。被减去反应的CS的CS +整个审判块学习的指标计算。根据这一措施，零差异分数反映没有学习，而在零以上的差异分数反映学习的恐惧响应。然而，统计学上确定保留的情境恐惧，如在图3所示学生吨的测试是可控硅值计算，以CS +和CS -上下文操作的功能（同样的背景下，与作为一个群体间的分析上下文移位）在第2天的生物灭绝初期。 C.硬件系统描述杜克大学的潜水系统是基于预计的虚拟现实“洞穴” 设计 9 。潜水系统是3 × 3米× 3米的房间里，所有6个“墙”（4墙壁，天花板和地板）显示背投立体电脑图像。每个墙，DLP投影机（视数字幻影S +2 K时，在1056x1056 @ 110 10赫兹），这又是一个专门提供电脑（Windows XP双核心2.0 GHz的与NVIDIA Quadro 3000FX - G的显卡）控制。一面墙上滑开，让流入和流出的潜水的访问。 6使计算机控制由主控计算机，通信，跟踪系统（Intersense IS - 900 11），控制音响系统，并发送一个脉冲电击系统通过并行端口。参与者的头部和手部位置跟踪系统提供三维位置和方向信息。主动立体视觉是通过液晶快门眼镜（CrystalEyes 3月12日 ）。 7电脑（6使计算机和主计算机）是同步的帧图像的边界上，通过同步锁相（G同步）NVIDIA显卡的能力。 D.软件简介由两个不同的虚拟世界，通过它的参与者都对导游的恐惧条件和保留本实验测试上下文。虚拟世界为蓝本，使用3D建模包玛雅 13 。导航是有限的，以一个固定的路径，是在所有的虚拟世界相同。沿着这条道路的运动是通过Virtools 的 14个软件系统控制。 Virtools是一个游戏引擎，主要设计用于桌面或基于Web的经验。通过Virtools的VRPack延伸，预计虚拟世界中潜水。 Virtools的通信，通过虚拟现实外设网络跟踪系统（VRPN 15），一个开源库。 VRPN寄存器参与者头部和手的位置和方向以及按下按钮信息。 Virtools的使用头部跟踪信息，以正确的观点呈现参与者的三维场景。 <img src="/files/ftp_upload/1993/1993fig1.jpg" alt="图1" /> 图1。控制室（VisRoom）和一个人观看虚拟场景的参与者潜水立方体示意图。 <img src="/files/ftp_upload/1993/1993fig2.jpg" alt="图2" /> 图2。图与左手上的皮肤电导测量的滋补和阶段性的反应刺激电极的参与者。电刺激电极上的右手腕。 BIOPAC生理数据收集通过笔记本电脑上的应答软件。代码发送通过Virtools的软件生成虚拟现实技术在潜水预计脚本的台式电脑OSC。 <img src="/files/ftp_upload/1993/1993fig3.jpg" alt="图3" /> 图3。比较恐惧的收购和潜水和实验室的灭绝。差分皮肤电导反应（SCR）的+ / - 扫描电镜，空调和24小时后测试，在劳动的参与者atory或虚拟现实（潜水）。图文说明相当于恐惧收购潜水和实验室研究的参与者和灭绝。潜水产量相同的上下文（N = 12）的测试，第2天更强大的恐惧记忆保留相移上下文测量SCR到CS +，在我们的潜水参与者，而不是在我们的实验室参与者，* P = 0.05（N = 14） 。

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1。结果相当于内会担心收购和跨群体灭绝被发现（图3）。这些数据表明，可靠，翔实的恐惧空调的研究可以在一个完全身临其境的环境能力的制约和执行。此外，我们也表现出强劲的担心同样的情况下保留在潜水（1和​​第2天在相同的情况下，相对于那些经验丰富的上下文转移的参与者）参与者上下文相关的恐惧记忆。保留的恐惧是在比16匹配范式传统的实验室观察（见图3）潜水强。身临其境的VR的设置，还可以检查和操纵丰富的上下文环境，不像在现实多式联运上下文操作是很难完成的的实验室环境，探测人类的陈述性记忆过程。最后，可以很容易地被移植的VR世界功能磁共振成像，采用立体VR的护目镜期间进行编码或检索恐惧收购，灭绝，复发脑活化分析（fMRI）技术结合使用。这种方法可以利用桥的恐惧和焦虑的啮齿类动物和临床研究结果。 2。控制在虚拟现实的背景和刺激曝光。 一个实验用的VR与利用的主要问题是它的实力。具体来说，完全沉浸VR提供的复杂性，困惑，和对现实世界的的自由。例如，在现实生活中，创伤受害者的经验，在数量不明的时间范围内的厌恶刺激。上下文曝光，具体功能和其他感官输入出席也未知的，或者不confirmable。同样的道理，如果我们允许参与者自由探索的虚拟环境，我们将不能够考虑到上下文或刺激的曝光时间或持续时间。例如，一位与会者可步行速度非常快，错过3 4 CS +演示。另一个可能只探讨在虚拟公寓的一个房间。同样，如果不刺激呈现在屏幕的中心，那里的目光是定向之前，要启动指定，与会者将避免或错过CS演示。这些潜在的混淆，我们的解决方案是采取上一个层次，每个环境的速度，将允许一个特定的刺激间隔（ISI）和刺激持续时间的导游的参与者。然后我们就可以从特定时间点和所有参与者的具体位置（例如，美国的CS +和CS -刺激的反应）中提取的可比可控硅数据。在作出这个决定包括寻找路径的形状，长度和运动速度，不会引起恶心或本体不协调的参与者后，遇到的困难和尚未觉得合适的一个新的环境中通过模仿自然下地。 3。执行标准的恐惧空调参数的VR系统。 为了模拟现实条件刺激蛇和蜘蛛的设计后，野生动物图像。首次在Maya建模，计算机图形三维建模和动画软件包，然后导入到VR系统的蛇和蜘蛛。我们这样做，因为Virtools是一个虚拟现实制作系统，而不是一个建模应用程序。因此，最好使用，运行一个虚拟现实系统，并添加场景的互动和导航。具体来说，每个CS类型的四个不同的动画Maya中创建的（例如，盘绕的蛇，蜘蛛在地板上运行，蛇张开嘴向前冲刺），然后到Virtools的进口。 从Maya导入Virtools的动态蛇和蜘蛛模型前，在Virtools的创建路径周围环境的参与者，指导顺利循环的方式，允许超过32个条件刺激演示过程中在恐惧的环境采样空调。是我们的三个虚拟世界的每一个相同的路径的形状。路径的建立是为了每个刺激呈现四个秒停止，刺激间隔为11 + / - 4秒，在此期间，主题是缓慢移动（指导）通过环境。此间隔确定从我们先前的担心空调的实验，在实验室中8，16，因为它允许恢复皮肤电导反应之间的刺激演讲。刺激，然后放置在时序参数指定点的道路上。这个安装程序创建特定的刺激和上下文连词（如滑行的餐桌周围的沙发腿走路，一只蜘蛛，蛇），以后可以探测外显记忆。通过脚本的伪随机刺激出场。所有的刺激演示出现在中间屏幕前，以防止从搜索刺激参与者。这为我们提供了一个刺激的曝光时间的控制量，并定义的上下文位置。正向的看法的一个限制是，它并没有身临其境的系统（例如，蛇能不能进入房间，从后面的参与者）的全部功能的优势。此外，刺激小心放置蛇和蜘蛛各地的参与者的位置，使从来没有侵占后，参与者的个人空间边界框之外。 4。查看点和头部跟踪。 潜水的角度，从坐姿，使参与者有正确的前进面临的角度。这种控制在参与者之间的高度的变化，并尽量减少运动伪影，对我们的生理录音。指示参加者面对尽可能少移动，这也是与会者控制，并因此保持一致的刺激和参与者之间的接触范围内。与会者佩戴3D眼镜，以确保他们查看现场，用正确的观点，我们选择打开一个头部跟踪装置。如果头部跟踪尚未当选，头部运动到左边或右边不会正确闭塞如何出现在世人面前的对象（例如，对象将出现在屏幕潜水一心想为与会者走过）。当选为头部跟踪，我们可以肯定的环境中的功能保留其正常的比例和潜水六个墙壁上，得出正确的实验时间。 5。数据收集。 16刺激呈现在我们的恐惧空调8的标准实验室的版本 ，控制简报软件包编程的计算机上运行脚本。为了保持实验室和虚拟环境之间的一致性，我们进口我们的标准代码格式恐惧收购和灭绝的脚本在控制室的控制计算机主机潜水立方体（见图1）。并行端口代码，发送的数字代码生成列表信号不同的事件，如蛇，蜘蛛，和电刺激onsets介绍。在我们的设计中，Virtools的发送一个开放式的音效控制消息（OSC 17）OSC / UDP消息到一个自定义的C + +程序集的并行端口值。我们的C + +程序使用OSCpack 18库。 BIOPAC的数字输入是连接到计算机的并行端口。 SCR数据收集从BIOPAC通过并行端口的笔记本电脑上，然后归，并计算到CS / CS和美国onsets在特定的参数（详见上文）。除了渲染场景和控制导航，Virtools是用来记录用户事件（按键）。总之，在一个实验中，消息是从主计算机发送BIOPAC系统通过并行端口。由于Virtools的不能与并行端口上的计算机进行通信，直接一个小的C + +程序侦听从Virtools的OSC消息，然后传送到并行端口。

Erratum: Human Fear Conditioning Conducted in Full Immersion 3-Dimensional Virtual Reality

human fear conditioning conducted in full immersion 3-dimensional virtual reality

恐惧条件是广泛使用在非人类的动物研究的范式调查潜在的恐惧和焦虑的神经机制。在人类进行调节研究的一个重大挑战的能力，以强烈的操纵或模拟的环境背景与调节情绪的行为关联。在这方面，虚拟现实（VR）技术是一种很有前途的工具。然而，以适应这种技术，以满足实验的限制，需要特殊的住宿。在这里，我们完全沉浸6片面VR环境和目前的恐惧空调数据进行恐惧条件时，涉及的方法论问题。 在现实世界中，创伤性事件的发生在复杂的环境中，是由许多线索，参与我们所有的感官方式。例如，环境配置的线索，不仅包括视觉元素，听觉，嗅觉甚至触觉。在恐惧制约动物的啮齿类动物研究中，完全沉浸在一个背景下，是丰富新颖的视觉，触觉和嗅觉线索。然而，标准的实验室测试，在人类的恐惧空调通常在一个平面或二维电脑屏幕前的不伦不类的房间进行，不复制现实世界的经验的复杂性。另一方面，旨在减少（灭火）的恐惧和预防焦虑症复发的临床研究的一个主要限制是，治疗后，参与者都获得了害怕失控，很大程度上是未知的背景下发生的。因此，实验者都没有关于曝光时间，刺激的真实性质，与环境相关的背景线索1的信息。在此信息的情况下，它可以提示和上下文相关的一个担心是难以真正熄灭。虚拟现实环境中解决这些问题提供了对现实世界的的复杂性，并在同一时间允许实验者约束恐惧空调和灭绝参数收益率的实证数据，可以提出更好的治疗方案和/或分析机理的假说。 为了测试的恐惧制约可能是丰富的编码和上下文相关完全身临其境的环境中进行的假设，我们开发了独特的虚拟现实3 - D上下文中，参加者有经验的虚拟蛇或蜘蛛的恐惧空调。听觉线索的合作，以进一步唤起定向反应和2科目的“存在”的感觉与CS发生。皮肤电导反应担任恐惧收购，内存保留和灭绝的依赖措施。

Watch this Scientific Journal Video about Human Fear Conditioning Conducted in Full Immersion 3-Dimensional Virtual Reality at JoVE.com

Human Fear Conditioning Conducted in Full Immersion 3-Dimensional Virtual Reality

古典恐惧制约范式被改编为人类完全沉浸式虚拟现实设置参与者。皮肤电导反应在两个不同的虚拟环境的动态虚拟蛇和蜘蛛（条件刺激）使用歧视范式，条件恐惧，提示和上下文记忆保持，和灭绝。

人类的恐惧充分浸泡三维虚拟现实中进行的空调

Fear conditioning is a widely used paradigm in non-human animal research to investigate the neural mechanisms underlying fear and anxiety. A major ...

human-fear-conditioning-conducted-full-immersion-3-dimensional

Research

JoVE Journal

Neuroscience

20.8K 次观看.  Duke University. 古典恐惧制约范式被改编为人类完全沉浸式虚拟现实设置参与者。皮肤电导反应在两个不同的虚拟环境的动态虚拟蛇和蜘蛛（条件刺激）使用歧视范式，条件恐惧，提示和上下文记忆保持，和灭绝。

视频: Human Fear Conditioning Conducted in Full Immersion 3-Dimensional Virtual Reality

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16.
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.

在蜜蜂的长鼻扩展响应的经典条件反射行为药理学（蜜蜂）

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

科研

神经科学

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Organotypic Culture of Full-thickness Adult Porcine Retina

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human retina and is therefore a valuable species for studying mechanisms of human retinal injury and degenerative disease. Here we describe a cost-effective technique for organotypic culture of adult porcine retina isolated from eyes obtained from an abattoir. After removing the anterior segment, a trephine blade was used to create multiple neural retina-Bruch's membrane-RPE-choroid-sclera explants from the posterior segment of adult porcine eyes. A piece of sterile filter paper was used to lift the neural retina off from each explant. The filter paper-retina complex was cultured (photoreceptor side up) atop an insert, which was held away from the bottom of the culture dish by a custom-made stand. The stand allows for good circulation of the culture medium to both sides of the retina. Overall, this procedure is simple, reproducible, and permits preservation of native retinal structure for at least seven days, making it a useful model for a variety of morphological, pharmacological, and biochemical studies on mammalian retina.

Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.

器官型培养的全厚成年猪视网膜

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

在脑片恐惧电路体外光遗传学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

使用的跟踪经典条件反射，评估胎儿酒精谱系障碍大鼠模型海马功能障碍

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

果蝇调理作为学习和记忆的措施

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the regulation of cerebral blood flow (CBF) is incompletely understood, especially at the capillary level. Two-photon microscopy is a powerful tool widely used to study CBF and its regulation. Currently, this field is limited by the lack of in vivo two-photon microscopy studies examining (1) CBF responses in three-dimensions, (2) conducted vascular responses, and (3) localized interventions within the vascular network. Here, we describe a 3D in vivo method using two-photon microscopy to study conducted vascular responses elicited by&#160;local ejection of ATP with a glass micro-pipette. Our method uses fast and repetitive hyperstack two-photon imaging providing precise diameter measurements by maximal intensity projection of the obtained images. Furthermore, we show that this method can also be used to study 3D astrocytic calcium responses. We also discuss the advantages and limitations of glass micro-pipette insertion and two-photon hyperstack imaging.

We present an optimized local ejection procedure using a glass micro-pipette and a fast two-photon hyperstack imaging method, which allows precise measurement of capillary diameter changes and investigation of its regulation in three dimensions.

在 Vivo 三维双光子显微镜中,利用玻璃微移器研究本地 ATP 喷射的血管反应

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the ...

A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction of high-resolution imaged stacks to reveal ultrastructural patterns that could not be resolved using 2D images. Indeed, the latter might lead to a misinterpretation of morphologies, like in the case of mitochondria; the use of 3D models is, therefore, more and more common and applied to the formulation of morphology-based functional hypotheses. To date, the use of 3D models generated from light or electron image stacks makes qualitative, visual assessments, as well as quantification, more convenient to be performed directly in 3D. As these models are often extremely complex, a virtual reality environment is also important to be set up to overcome occlusion and to take full advantage of the 3D structure. Here, a step-by-step guide from image segmentation to reconstruction and analysis is described in detail.

The described pipeline is designed for the segmentation of electron microscopy datasets larger than gigabytes, to extract whole-cell morphologies. Once the cells are reconstructed in 3D, customized software designed around individual needs can be used to perform a qualitative and quantitative analysis directly in 3D, also using virtual reality to overcome view occlusion.

胶质细胞和神经细胞的三维重建与虚拟现实分析方法

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...

Simultaneous Application of Transcranial Direct Current Stimulation during Virtual Reality Exposure

Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation that changes the likelihood of neuronal firing through modulation of neural resting membranes. Compared to other techniques, tDCS is relatively safe, cost-effective, and can be administered while individuals are engaged in controlled, specific cognitive processes. This latter point is important as tDCS may predominantly affect intrinsically active neural regions. In an effort to test tDCS as a potential treatment for psychiatric illness, the protocol described here outlines a novel procedure that allows the simultaneous application of tDCS during exposure to trauma-related cues using virtual reality (tDCS+VR) for veterans with posttraumatic stress disorder (NCT03372460). In this double-blind protocol, participants are assigned to either receive 2 mA tDCS, or sham stimulation, for 25 minutes while passively watching three 8-minute standardized virtual reality drives through Iraq or Afghanistan, with virtual reality events increasing in intensity during each drive. Participants undergo six sessions of tDCS+VR over the course of 2-3 weeks, and psychophysiology (skin conductance reactivity) is measured throughout each session. This allows testing for within and between session changes in hyperarousal to virtual reality events and adjunctive effects of tDCS. Stimulation is delivered through a built-in rechargeable battery-driven tDCS device using a 1 (anode) x 1 (cathode) unilateral electrode set-up. Each electrode is placed in a 3 x 3 cm (current density 2.22 A/m2) reusable sponge pocket saturated with 0.9% normal saline. Sponges with electrodes are attached to the participant&#8217;s skull using a rubber headband with the electrodes placed such that they target regions within the ventromedial prefrontal cortex. The virtual reality headset is placed over the tDCS montage in such a way as to avoid electrode interference.

This manuscript outlines a novel protocol to allow the simultaneous application of transcranial direct current stimulation during exposure to warzone trauma-related cues using virtual reality for veterans with posttraumatic stress disorder.

虚拟现实曝光期间颅外直接电流刺激的同步应用

Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation that changes the likelihood of neuronal firing through ...

Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish

Zebrafish (Danio rerio) are an excellent model to investigate the effects of chronic hyperglycemia, a hallmark of Type II Diabetes Mellitus (T2DM). This alternate immersion protocol is a noninvasive, step-wise method of inducing hyperglycemia for up to eight weeks. Adult zebrafish are alternately exposed to sugar (glucose) and water for 24 hours each. The zebrafish begin treatment in a 1% glucose solution for 2 weeks, then a 2% solution for 2 weeks, and finally a 3% solution for the remaining 4 weeks. Compared to water-treated (stress) and mannitol-treated (osmotic) controls, glucose-treated zebrafish have significantly higher blood sugar levels. The glucose-treated zebrafish show blood sugar levels of 3-times that of controls, suggesting that after both four and eight weeks hyperglycemia can be achieved. Sustained hyperglycemia was associated with increased Glial Fibrillary Acidic Protein (GFAP) and increased nuclear factor Kappa B (NF-kB)&#160;levels in retina and decreased physiological responses, as well as cognitive deficits suggesting this protocol can be used to model disease complications.

This protocol noninvasively induces hyperglycemia in zebrafish for up to 8 weeks. Using this protocol, an in-depth study of the adverse effects of hyperglycemia can be made.

交替浸入葡萄糖中，在斑马鱼中产生长期血糖超血症

Zebrafish (Danio rerio) are an excellent model to investigate the effects of chronic hyperglycemia, a hallmark of Type II Diabetes Mellitus (T2DM). This ...