我们感谢D. Johnson和K. Doelling协助数据收集。我们也感谢M.-H.实验的协议，孟菲尔斯，J.勒社，Y. NIV和，M. MILAD建议。这项研究是由詹姆斯·麦克唐纳基金会和美国国立卫生研究院（NIH）资助R21 MH072279（EAP），国立卫生研究院资助R37 MH038774，P50 MH058911，RO1 MH046516和K05 MH067048（JEL），博士后奖学金NSERC，CIHR和AHFMR （M.-HM）和富布赖特和布拉瓦尼克奖（DS）。

概观在本协议中描述的研究中，我们进行了连续三天，三个实验组与主体之间的设计。第1天，所有受试者经历恐惧，他们学会区分两个线索，一个预测的冲击，另一种是不。第2天，所有受试者进行灭绝培训，但不同的方面会发生什么事先：两组收到提醒的提示，重新启动前的恐惧记忆消除训练，而第三组没有收到这样的提示。一组接收的提醒提示10分钟前灭绝，因此，他们经历灭绝，，而恐惧记忆reconsolidating。另一组接收的提醒提示6小时前灭绝，因此他们进行灭绝后，这的窗口大概已经关闭。第三组没有收到提醒，提示前接受灭绝，因此他们接受标准的灭绝培训。第3天，所有受试者ŕE打开另一个灭绝会话。他们的恐惧时，他们首先看到的第3天空调线索表明是否仍有恐惧记忆。所有这三个天进行相同的实验设置，这是一个声音衰减，温度受控室中。空调的刺激呈现在计算机屏幕上的彩色方块。诱导和测量用温和的电击和皮肤电导的恐惧，分别使用BIOPAC系统模块（ 见表1）。使用的台式计算机运行的AcqKnowledge的软件程序，允许同时使用一台单独的计算机记录和监控SCR运行，目前的实验脚本的主题。研究对象接受所有三个阶段大约在同一时间的一天。 1。恐惧的评估与皮肤电反应（SCR） - 电极的应用及录音<ol><li>理想情况下，试验室应保持温暖（我们的房间某处中外N 70和75度），以促进可靠和可测量的SCR。请清洁使用棉条蘸蒸馏水主体的右侧或左侧指尖，我们不建议磨料cleansors，因为他们可以干的皮肤，破坏出汗的反应。确保的指尖完全干燥前电极的应用程序。 </li><li>高度导电的电解质凝胶的两个电极（这里我们使用“Signa的凝胶”帕克实验室公司的，和电极连接到一个MP100数据采集系统由Biopac系统）。允许凝胶小幅上扬以上的SCR电极，并确保在凝胶中有没有气泡，因为这可以interefere与SCR信号。 </li><li>电极连接到第一和秒的手指的手之间的第一和秒指骨，是更加明显的皮肤电信号。确保电极尼龙搭扣带是否牢固，并要求受测者报告他们是否在他们的指尖感觉不到任何脉冲。这种脉冲印度阿泰的带子已经被应用得紧紧的，可以interefere与SCR信号。 </li><li>一旦皮肤电导电极被固定，开始录制皮肤电导样品约5分钟，以测试被检者的皮肤电信号。在这里，我们使用的是AcqKnowledge 3.9软件（从系统公司BIOPAC），并记录SCR波形上一个MAC台式电脑。我们使用的采样速率为200Hz，作为慢作为SCR，但是更高的速率，可以使用适当的水平的信号。提出实验的脚本和对象的视线从电脑记录的屏幕应该是独立的。确定的主题，具有强大的SCR反应，指示他们采取和保持深深吸了一口气，这应该大幅上涨的SCR（这种反应的一个例子见图1）。 </li></ol> 2。恐惧轻微电击刺激 - 电极感应与应用程序和工作程序<ol><li>在手腕上，OPPO网站的手与SCR电极，附加的刺激吧。在这里，我们使用条形码的电极（产品代码EL350系统公司从BIOPAC），连接到SD9方波脉冲刺激与珠电导凝胶应用到每个（由草技术，萎缩医学，Inc。产品集团）所述电极头。 </li><li>的冲击触发的刺激中在工作过程中，应拔下。将酒吧在手腕内侧，掌​​长肌腱两侧的固定用尼龙搭扣表带，以保持它在实验过程中，但，确保表带是不是太紧张。 </li><li>通知的主题，他们将获得手动交付的冲击。开始在一个较低的水平（我们建议20 V），和工作的水平，他们的决定是不舒服，但不痛。我们建议的震动持续时间设定为200毫秒，50个脉冲/秒，在过去的恐惧条件反射的研究21,24数组的使用一个标准的水平，但其他人有使用时间略有不同，导致同样的感觉25,26。 </li><li>手动触发的冲击，在20伏的开始。持续触发单个的冲击，逐渐在5V递增，直到力度越来越大的主题表示的冲击不舒服，但不痛。应该有一个大的SCR的冲击（参见图2的一个例子的冲击的响应）。记录的最后的冲击水平，并保持这个实验的其余部分的电压电平。 </li></ol> 3。第1天 - 收购<ol><li>受直接在屏幕上在任何时候都保持自己的眼睛，找到一个舒适的位置，将不需要运动，自然呼吸，因为突发性的呼吸变化可能会干扰SCR。应该被放置在一个放松的姿势，手腕上的表/台前面主题的怀抱一把舒服的椅子上，双臂。请告诉主体，他们将看到屏幕上的视觉刺激并不定期收到冲击。指示受监视他们所看到的刺激和冲击接收时之间的关系。 </li><li>将触发回的刺激和改变触发AcqKnowledge设置从OFF到外部。的主题是准备开始后，将电压恢复到记录的水平，并开始都AcqKnowledge的文件和实验脚本。在这里，我们从心理学的软件工具，公司使用E-Prime软件2.0版</li><li>收购会议期间，一个条件刺激（在这里，一个黄色正方形）的合作终止与厌恶的结果（在这里，轻度休克）的一个子集（38％）的试验，我们参考，这是我们的CS +。其他条件刺激（在这里，一个蓝色的方块）永远不会与美国配对;我们指这是我们的CS-。我们使用局部加固，以减缓快速灭绝，因为以前的工作发现，100％的配筋结果一次学习的主题实现之间的关系CS +和美国已改变21。一个局部加固范例也让我们以限制我们的分析，以非增强CS +试验，从而不包括CS +相关可控硅，是潜在的污染的生理反应震荡（见每个CS型空调和无条件的SCR的例子的图3）。 </li><li> （CSS）是空调的刺激，持续4秒。有10-12秒的随机变量间试间隔（ITI），在此期间，受试者看到一个黑色的屏幕与白色的固定十字中心。每个会话开始时的屏幕显示的实验即将开始（READY *）和终止的屏幕表示，它们已经成功地完成了实验的一部分（*您已经完成了这部分的实验中*）。在收购过程中，总数量的10个非增强型试验CS +试验10 CS-试验，除了6 CS +的试验，加强了休克。 </li><li>产生两种不同的伪RANDO整个实验，例如，没有试验型（CS +，CS-或CS + US）重复两次以上mized订单。在每一个订单，平衡的CSS的颜色。这将导致在各组内个人之间的分配是随机的4个数量级。我们建议包括至少出现一次连续的钢筋CS +美国临床试验，以防止被摄物体假设他们永远不会接收consecutives冲击的试订单的例子（ 见表2）。 </li></ol> 4。第2天 - 活化和消光培训主题细分： 所有受试者，随机分为已证明有足够的收购从第1天的恐惧（见下面的排除标准），分为三组： 第1组（重新启动10分钟）：10分钟后，检索的再固结窗口内接收灭绝培训 组2（激活6小时）：接收灭绝培训后6小时内检索的再固结窗口外 第3组（没有激活）：接收标准灭绝的训练没有任何检索<ol><li>抵达后，每个主题应当按照步骤1 - 2.2，具有相同的震撼级的第1天（没有工作）。有没有新的指令。告诉舒舒服服地坐在注意刺激，就像前一天的主题。 </li><li>在重新组（10分钟，6小时）进行主题单一，无筋介绍CS +（无冲击）后的10分钟休息时间。在没有激活的主题进行设置程序，然后直接去突破没有重新启动会话。中场休息时，停止在SCR的AcqKnowledge记录，将刺激电压零和直接当事人的注意​​，有一个单独的屏幕预先选定的电视节目（ 例如 ，在这里，我们使用了一个随机选择的10分钟部分的“辛普森一家”的插曲）。受试者观看10分钟，而所有电极保持连接。 </li><li>中场休息后，重新启动，10 min组进行与灭绝训练。主题的回报灭绝训练6小时后重新-6小时组。对于无活化受试者，半立即进行消光的培训，和半返回6小时后进行训练。这组控制的提醒提示，以及不同时间的流逝，在每个提醒激活和灭绝培训。此外，由于激活，6小时组的受试者可能发生的灭绝，无论是在下午或晚上（以便有充裕的时间结束后重新启动）它是重要的运行在上午的10分钟激活和不激活组的科目，下午和晚上，允许甚至学科分布在每一天的时间。 </li><li>期间ING灭绝，10无筋CS +试验和CS-11试验总数为每个激活组。由于CS-不重新启动会话过程中，添加一个额外的演示的灭绝会，让所有的CSS相同数量的第2天的时间内。没有激活组得到11未增强演示的每一个CS。 </li><li>在灭绝会议，恢复AcqKnowledge记录档案和，刺激电压恢复到设定的水平。指导的主题，参加实验的消光脚本正在等待READY *屏幕上的屏幕。前一天的方向往往是有用的提醒。恢复灭绝的培训讲稿，收到重复的主题介绍两个CSS无冲击（ 见表2）。 </li></ol> 5。第3天 - 自发恐惧回收试验<ol><li>如果这个问题表现出足够的灭绝学习（见排除标准以下），他们应该返回到实验室24小时后灭绝会话。到达后，它们应该被设置为每步骤1和2.2;电压被设定为相同的水平收购会话。提醒的方向前几天的主题。 </li><li>每个主题将进行重新灭绝的会议，使他们再次收到10个未增强的演示，以评估恐惧反应的程度，恢复时间的推移，由于这两个CSS（ 见表2）。第3天的第一次审判应该是一个额外的CS-（ 见表3），并在会议开始时的定向反应被忽略。 （同样可以做第2天开始，然而，因为只有后期采集的数据是用来评估灭绝的学习（见下面的排除标准），并在最后的分析中，这一建议并不重要，第2天）。第3天的下一个试验应CS +和CS之间的平衡。 SPontaneous恢复测量中减去SCR过程中的第一个CS +试用期间重新灭绝的最后一个CS +试验过程中灭绝。 </li><li>在结束的会议的主题是简要介绍，并支付$ 40出发前。 </li></ol> 6。前处理及分SCR数据<ol><li>离线数据分析AcqKnowledge软件。在这里，我们设定三个参数来衡量我们的数据图表：t（时间），δ-T（延迟）和PP（峰-峰值）。这些测量记录所选的SCR的时间点，延迟时间和峰值振幅。开始一个新的杂志图来记录你的测量。通波形，通过低通滤波器，以减少高频率数据，通过选择变换→数字滤波→FIR→低通（我们使用的默认设置在确认软件指南“的：截止频率= 25; 32系数;过滤器整个波）。平滑数据，以减少噪声的选择性婷变换→平滑→变换整个波（我们使用的默认设置提供的应答软件指南“的：平均价值平滑;平滑系数= 3的样品;过滤器整个波;然而，更高的采样率或增加噪声，平滑因子可以是增加）。 </li><li>选择一个皮肤电反应在每个试验基地的最大波形（微西门子，微秒），开始在0.5〜4.5秒窗口下面的刺激发病高峰差异。发病的SCR必须在此窗口内开始有资格作为一个刺激的反应，但可能需要更长的时间达到峰值。 SCR可能被记录，直到他们达到峰值，但是，响应5秒后，峰值不考虑特定的刺激和应该被忽视。这也正是为什么钢筋试验不应记录：一个SCR可能被污染，仍然是在试验结束时达到峰值的冲击和后续增加SCR是n催产素有关的CS本身。 </li><li>一个响应峰值为0.02μs的标准应利用;，反应低于这个标准通常被视为噪音，并编码为“0”。进球后，所有非增强型试验应划分为每个主题，各自受美国的平均响应。这是源自整个收购会议，并占主体的冲击反应的个体差异，所有冲击试验的平均SCR。最后，所有的试验应转化为减少偏态的平方根。 </li></ol>定时 第1天 制备方法：由5-7分钟采集脚本：约9分钟总计：15分钟 第2天 制备方法：由5-7分钟重新激活脚本：≈30秒休息：10分钟总计：20分钟 第3天 制备方法：由5-7分钟再消光脚本：约6.5分钟总计：15分钟 7。每个组的代表结果受试者在第1天（收购），应证明差SCR的CSS，那就是更高的CS +与CS-（ 见图4，收购，个别科目的一个例子）。结束第2天（重新启动/熄火），这种不同的反应减弱，使得不再有CS +和CS-（参见图4，消光，个别科目的一个例子）之间的差异。当检查第3天的自然恢复，我们希望看到的缺乏恐惧恢复激活10 min组因为这组收到灭绝培训的再固结窗口内，从而使原来的内存的跟踪，reconsolidate与新的安全性信息，并防止恢复的恐惧反应（参见图4，面板顶部，从个别科目的一个例子）。我们将期待以看到实质性复苏的担心，但是，在CS +（不CS-）的激活，6小时组，收到的灭绝培训外再固结窗口中（请参阅图4，中间面板中，从个人的一个例子主体）以及无活化组接受标准灭绝培训（请参阅图4，底部面板，从一个个体对象的一个例子）。 <img alt="图1" src="/files/ftp_upload/3893/3893fig1.jpg" /> 图1实施例的SCR成立期间电极。黑色，垂直箭头表示在该点受指示采取深深吸了一口气，导致在明显增加，SCR。 <a href="http://www.jove.com/files/ftp_upload/3893/3893fig1large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/3893/3893fig2.jpg" /> 图2。SCR在冲击工作程序中的示例。黑色的垂直箭头表示休克管理点。灰色箭头指示的峰值的皮肤电导上升的冲击。 <a href="http://www.jove.com/files/ftp_upload/3893/3893fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/3893/3893fig3.jpg" /> 图3。条件反应（CR）的发病CS +其次较大的无条件反应（UR）交付的冲击（美国）;随后的审讯是，一个CS-。灰色箭头指示的CS起点和终点，黑色箭头印度吃了美国提供的。 <a href="http://www.jove.com/files/ftp_upload/3893/3893fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" src="/files/ftp_upload/3893/3893fig4.jpg" /> 图4。在一个单独的问题从每个实验条件下（顶部面板：激活10分钟，中间面板：激活6小时;底板：没有激活）的收购，消光及自然恢复。 <img alt="表1" src="/files/ftp_upload/3893/3893table1.jpg" /> 表1。表相关产品和设备。 <img alt="表2" src="/files/ftp_upload/3893/3893table2.jpg" /> 表2。彭兰霞协议和时间表。 <img alt="表3" src="/files/ftp_upload/3893/3893table3.jpg" /> 试订单，每个阶段的一个例子见表3。实验。注意一个额外CS-应加入2.2天（消光）都提醒组以帐户为重新CS +。由于在会议开始时的定向反应的第一个CS-试验第3天（以灰色背景表示）被忽略。 实验设置设置包括PC上运行的E-贷，Mac上运行的承认，的MP100 BIOPAC系统，和一个箱体震荡。行代码被编入E-贷脚本的沟通与确认软件和箱体震荡。这使得E-贷发送信号，承认汉语表事件起始和信号的箱体震荡，引发休克管理。从E-素数的输出通过一个打印机端口发送到BIOPAC的系统，该系统依次发送的事件信号的Mac记录生理应答设置与BIOPAC系统通过USB连接，以及休克框的（见表1产品UCT信息）。 排除标准主题应排除在完成所有的实验会话或从数据分析，如果他们表现出以下任何一种： <ul><li>不显示检测出可靠的SCR - 应用和测试皮肤电反应时，受试者可以显示平SCR。如果发生这种情况，建议等待额外的5分钟的反应可能不会立即出现，或者检查是否有气泡与电导凝胶中（在这种情况下，电极应被除去，清洁和重新填充）。即使这些故障排除策略的主题有平面SCR反应，应获豁免的实验。 </li><li>否则，以展示收购的恐惧反应（第1天）：差分SCR的CS +和CS在年底的收购会话（意味着下半年的采集试验），是在错误的方向（ 即 CS-&gt; CS +）或太小concl的的ude学习发生（ 即 ，不同的是小于0.1微秒）。 </li><li>未能证明灭绝的恐惧反应（第2天）：差动SCR到CS +和CS-灭绝会议（最后一次试验），是大于0.1微秒的末尾。 </li></ul>

<ol>
	<li>Pavlov, I. P., Anrep, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Reflexes. , (1927).</li><li>LeDoux, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emotion+circuits+in+the+brain.">Emotion circuits in the brain.</a> Annual Review of Neuroscience. 23, 155-184 (2000).</li><li>Myers, K. M., Davis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+neural+analysis+of+extinction.">Behavioral and neural analysis of extinction.</a> Neuron. 36, 567-584 (2002).</li><li>Bouton, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context,+ambiguity,+and+unlearning+:+sources+of+relapse+after+behavioral+extinction.">Context, ambiguity, and unlearning : sources of relapse after behavioral extinction.</a> Biological Psychiatry. 52, 976-986 (2002).</li><li>Nader, K., Schafe, G. E., LeDoux, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fear+memories+require+protein+synthesis+in+the+amygdala+for+reconsolidation+after+retrieval.">Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval.</a> Nature. 406, 722-726 (2000).</li><li>Nader, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+traces+unbound.">Memory traces unbound.</a> Trends in Neuroscience. 26, 65-72 (2003).</li><li>Alberini, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+memory+stabilization:+are+consolidation+and+reconsolidation+similar+or+distinct+processes.">Mechanisms of memory stabilization: are consolidation and reconsolidation similar or distinct processes.</a> Trends in Neuroscience. 28, 51-56 (2005).</li><li>Sara, S. J., Hars, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+memory+of+consolidation.">In memory of consolidation.</a> Learning and Memory. 13, 515-521 (2006).</li><li>Dudai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconsolidation:+the+advantage+of+being+refocused.">Reconsolidation: the advantage of being refocused.</a> Current Opinions in Neurobiology. 16, 174-178 (2006).</li><li>Schiller, D., Monfils, M. -. H., Raio, C. M., Johnson, D. C., LeDoux, J. E., Phelps, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 463, 49-54 (2010).</li><li>Monfils, M. -. H., Cowansage, K. K., Klann, E., LeDoux, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extinction-reconsolidation+boundaries:+Key+to+persistent+attenuation+of+fear+memories.">Extinction-reconsolidation boundaries: Key to persistent attenuation of fear memories.</a> Science. 324, 951-955 (2009).</li><li>Brunet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+post-+retrieval+propranololon+psychophysiologic+responding+during+subsequent+script-driven+traumatic+imagery+in+post-traumatic+stress+disorder.">Effect of post- retrieval propranololon psychophysiologic responding during subsequent script-driven traumatic imagery in post-traumatic stress disorder.</a> Journal of Psychiatric Research. 42, 503-506 (2008).</li><li>Kindt, M., Soeter, M., Vervliet, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+extinction:+erasing+human+fear+responses+and+preventing+the+return+of+fear.">Beyond extinction: erasing human fear responses and preventing the return of fear.</a> Nature Neuroscience. 12, 256-258 (2009).</li><li>Schiller, D., Phelps, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+reconsolidation+occur+in+humans.">Does reconsolidation occur in humans.</a> Frontiers in Behavioral Neuroscience. 5, 24 (2011).</li><li>Duvarci, S., Nader, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+fear+memory+reconsolidation.">Characterization of fear memory reconsolidation.</a> Journal of Neuroscience. 24, 9269-9275 (2004).</li><li>Walker, M. P., Brakefield, T., Hobson, J. A., Stickgold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociable+stages+of+human+memory+consolidation+and+reconsolidation.">Dissociable stages of human memory consolidation and reconsolidation.</a> Nature. 425, 616-620 (2003).</li><li>Hupbach, A., Gomez, R., Hardt, O., Nadel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconsolidation+of+episodic+memories:+A+subtle+reminder+triggers+integration+of+new+information.">Reconsolidation of episodic memories: A subtle reminder triggers integration of new information.</a> Learning &#38; Memory. 14, 1-7 (2007).</li><li>Forcato, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconsolidation+of+declarative+memory+in+humans.">Reconsolidation of declarative memory in humans.</a> Learning &#38; Memory. 14, 295-303 (2007).</li><li>McNally, G. P., Johanses, J. P., Blair, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placing+prediction+into+the+fear+circuit.">Placing prediction into the fear circuit.</a> Trends in Cognitive Neuroscience. 34, 283-292 (2011).</li><li>Herry, C., Ferraguti, F., Singewald, N., Letzkus, J. J., Ehrlich, I., Luthi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuits+of+fear+extinction.">Neural circuits of fear extinction.</a> European Journal of Neuroscience. 31, 599-612 (2010).</li><li>Phelps, E. A., Delgado, M. R., Nearing, K. I., LeDoux, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extinction+learning+in+humans:+Role+of+the+amygdala+and+vmPFC.">Extinction learning in humans: Role of the amygdala and vmPFC.</a> Neuron. 43, 897-905 (2004).</li><li>Milad, M. R., Quirk, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+in+the+medial+prefrontal+cortex+signal+memory+for+fear+extinction.">Neurons in the medial prefrontal cortex signal memory for fear extinction.</a> Nature. 420, 70-74 (2002).</li><li>Schiller, D., Levy, I., Niv, Y., LeDoux, J. E., Phelps, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Fear+to+Safety+and+Back:+Reversal+of+Fear+in+the+Human+Brain.">From Fear to Safety and Back: Reversal of Fear in the Human Brain.</a> Journal of Neuroscience. 28, 11517-11525 (2008).</li><li>Schiller, D., Delgado, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlapping+neural+systems+mediating+extinction,+reversal+and+regulation+of+fear.">Overlapping neural systems mediating extinction, reversal and regulation of fear.</a> Trends in Cognitive Neuroscience. 14, 268-2627 (2010).</li><li>Esteves, F., Parra, C., Dimberg, U., Ohman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonconscious+associative+learning:+Pavlovian+conditioning+of+skin+conductance+responses+to+masked+fear-relevant+facial+stimuli.">Nonconscious associative learning: Pavlovian conditioning of skin conductance responses to masked fear-relevant facial stimuli.</a> Psychophysiology. 31, 375-385 (1994).</li><li>Knight, D. C., Cheng, D. T., Smith, C. N., Stein, E. A., Helmstetter, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+Substrates+Mediating+Human+Delay+and+Trace+Fear+Conditioning.">Neural Substrates Mediating Human Delay and Trace Fear Conditioning.</a> The Journal of Neuroscience. 24, 218-228 (2004).</li><li>Venables, P. H., Christie, M. J., Martin, I., Venables, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+in+psychophysiology.">Techniques in psychophysiology.</a> Electrodermal activity. , 3-67 (1980).</li></ol>

没有利益冲突的声明。

我们这里介绍的协议，可以创建一个简单的恐惧厌恶的结果搭配一个中性刺激的响应。一天后，我们恢复了恐惧记忆，以触发其再巩固。在这个阶段，我们诱导干扰的形式灭绝训练。要验证灭绝会影响再巩固我们使用了两个对照组。一组接受没有提醒试验引发的再固结灭绝，而另一组经历了提醒的试验，然后进行灭绝的再固结窗口外。然后，我们测量了，在第二​​天，无论存储器自发收回或是?...

extinction training during the reconsolidation window prevents recovery of fear

恐惧是不适应的，持续时长后，情况已变得安全。因此，关键是开发一种方法，坚持防止返回的恐惧。巴甫洛夫恐惧调节范式通常是创建一个受控，新的恐惧在实验室协会。配对后无害的刺激（条件刺激，CS）与厌恶的结果（无条件刺激，美国），我们就可以引起恐惧反应（条件反射，或CR）呈现的刺激1,2。担心的是一旦收购，它可以减少使用灭绝训练，条件刺激重复出现，直到怕是没有厌恶的结果不再对3。这种抑制作用的学习创造一个新的，安全的CS表示，竞争的表达与原来的恐惧记忆4。虽然的灭绝是有效抑制恐惧，它不是永久性的。恐惧可以自发地追随着时间的推移。暴露于压力或返回的背景下，最初的学习也可能导致恐惧再度出现3,4。 我们的协议针对的的再固结窗口，修改情感记忆更持久的方式解决了过渡性质的灭绝。充足的证据表明，重新激活合并存储器，它返回到一个不稳定的状态，在此期间，存储器是再次易受干扰5-9。这一机会之窗打开后不久，激活和关闭约6小时后5,11,16，尽管这可能取决于的实力和存储器15岁。使新的信息纳入原有的记忆痕迹，内存可能会被更新，，因为它reconsolidates 10,11。涉及非人类的动物研究已经成功地阻止恐惧记忆的表达，通过引入药理内再操作整合的窗口，但是，大多数剂是对人体有毒的或使用时，在人类研究中12-14显示模棱两可的效果。我们的协议解决了这些难题提供了一个有效的，但非侵入性的，操纵行为，是对人类安全的。 提示的恐惧记忆检索的灭绝之前，我们基本上是触发再巩固过程，使新的安全信息（ 例如 ，消光）被纳入恐惧记忆仍然容易受到干扰。最近的一项研究在大鼠采用这种行为的操作已成功阻止恐惧记忆，用这些时间参数11。进一步的研究表明，在人类引入新的信息后，以前综合电机16，短暂的17，或声明的18记忆的检索干扰与原来的记忆痕迹14。下面我们概述了一种新的亲tocol用来阻止人类的恐惧恢复。

Watch this Scientific Journal Video about Extinction Training During the Reconsolidation Window Prevents Recovery of Fear at JoVE.com

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

条件性恐惧可以减少通过抑制过程的灭绝，但可以重新浮现根据条件，如通过时间或接触应力。我们的协议防止恐惧复苏过程中引入灭绝的的再固结窗口（重新存储阶段的恢复了记忆），提出了一种新的方式。

灭绝培训在彭兰霞窗口无法还原的恐惧

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents ...

extinction-training-during-reconsolidation-window-prevents-recovery

Research

JoVE Journal

Neuroscience

24.9K 次观看.  Mt. Sinai School of Medicine. 条件性恐惧可以减少通过抑制过程的灭绝，但可以重新浮现根据条件，如通过时间或接触应力。我们的协议防止恐惧复苏过程中引入灭绝的的再固结窗口（重新存储阶段的恢复了记忆），提出了一种新的方式。

视频: Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

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

Fear conditioning is a widely used paradigm in non-human animal research to investigate the neural mechanisms underlying fear and anxiety. A major challenge in conducting conditioning studies in humans is the ability to strongly manipulate or simulate the environmental contexts that are associated with conditioned emotional behaviors. In this regard, virtual reality (VR) technology is a promising tool. Yet, adapting this technology to meet experimental constraints requires special accommodations. Here we address the methodological issues involved when conducting fear conditioning in a fully immersive 6-sided VR environment and present fear conditioning data.
In the real world, traumatic events occur in complex environments that are made up of many cues, engaging all of our sensory modalities. For example, cues that form the environmental configuration include not only visual elements, but aural, olfactory, and even tactile. In rodent studies of fear conditioning animals are fully immersed in a context that is rich with novel visual, tactile and olfactory cues. However, standard laboratory tests of fear conditioning in humans are typically conducted in a nondescript room in front of a flat or 2D computer screen and do not replicate the complexity of real world experiences. On the other hand, a major limitation of clinical studies aimed at reducing (extinguishing) fear and preventing relapse in anxiety disorders is that treatment occurs after participants have acquired a fear in an uncontrolled and largely unknown context. Thus the experimenters are left without information about the duration of exposure, the true nature of the stimulus, and associated background cues in the environment1. In the absence of this information it can be difficult to truly extinguish a fear that is both cue and context-dependent. Virtual reality environments address these issues by providing the complexity of the real world, and at the same time allowing experimenters to constrain fear conditioning and extinction parameters to yield empirical data that can suggest better treatment options and/or analyze mechanistic hypotheses.
In order to test the hypothesis that fear conditioning may be richly encoded and context specific when conducted in a fully immersive environment, we developed distinct virtual reality 3-D contexts in which participants experienced fear conditioning to virtual snakes or spiders. Auditory cues co-occurred with the CS in order to further evoke orienting responses and a feeling of "presence" in subjects 2 . Skin conductance response served as the dependent measure of fear acquisition, memory retention and extinction.

Classical fear conditioning paradigm was adapted for human participants in a fully immersive virtual reality setting. Using a discrimination paradigm, conditioned fear, cue and context memory retention, and extinction was measured with skin conductance response to dynamic virtual snakes and spiders (the conditioned stimuli) in two distinct virtual contexts.

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 ...

科研

神经科学

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

活体鼠脑微循环显微镜使用一个封闭的颅窗口

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Investigating the Neural Mechanisms of Aware and Unaware Fear Memory with fMRI

Pavlovian fear conditioning is often used in combination with functional magnetic resonance imaging (fMRI) in humans to investigate the neural substrates of associative learning 1-5. In these studies, it is important to provide behavioral evidence of conditioning to verify that differences in brain activity are learning-related and correlated with human behavior. 
Fear conditioning studies often monitor autonomic responses (e.g. skin conductance response; SCR) as an index of learning and memory 6-8. In addition, other behavioral measures can provide valuable information about the learning process and/or other cognitive functions that influence conditioning. For example, the impact unconditioned stimulus (UCS) expectancies have on the expression of the conditioned response (CR) and unconditioned response (UCR) has been a topic of interest in several recent studies 9-14. SCR and UCS expectancy measures have recently been used in conjunction with fMRI to investigate the neural substrates of aware and unaware fear learning and memory processes 15. Although these cognitive processes can be evaluated to some degree following the conditioning session, post-conditioning assessments cannot measure expectations on a trial-to-trial basis and are susceptible to interference and forgetting, as well as other factors that may distort results 16,17 .
Monitoring autonomic and behavioral responses simultaneously with fMRI provides a mechanism by which the neural substrates that mediate complex relationships between cognitive processes and behavioral/autonomic responses can be assessed. However, monitoring autonomic and behavioral responses in the MRI environment poses a number of practical problems. Specifically, 1) standard behavioral and physiological monitoring equipment is constructed of ferrous material that cannot be safely used near the MRI scanner, 2) when this equipment is placed outside of the MRI scanning chamber, the cables projecting to the subject can carry RF noise that produces artifacts in brain images, 3) artifacts can be produced within the skin conductance signal by switching gradients during scanning, 4) the fMRI signal produced by the motor demands of behavioral responses may need to be distinguished from activity related to the cognitive processes of interest. Each of these issues can be resolved with modifications to the setup of physiological monitoring equipment and additional data analysis procedures. Here we present a methodology to simultaneously monitor autonomic and behavioral responses during fMRI, and demonstrate the use of these methods to investigate aware and unaware memory processes during fear conditioning.

A methodology to investigate the neural mechanisms that support aware and unaware memory processes during fear conditioning is described. This method monitors blood oxygen level dependent (BOLD) functional magnetic resonance imaging, skin conductance response, and unconditioned stimulus expectancy during Pavlovian fear conditioning to assess the neural correlates of distinct memory processes.

调查的意识到，不知道用功能磁共振成像的恐惧记忆的神经机制

Pavlovian fear conditioning is often used in combination with functional magnetic resonance imaging (fMRI) in humans to investigate the neural substrates ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

抛光和钢筋，颅骨变薄窗口，为长期的小鼠脑成像

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

通过圆窗膜病毒介导的基因传递给鼠标内耳手术方法

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

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 ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

实时 fMRI 神经训练管理协议

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with neurodegenerative diseases or injuries, but also in the normal brain. Here, we describe a newly-established micromechanical system to rapidly, reliably, and reversibly induce axonal varicosities, allowing us to understand the mechanisms governing varicosity formation and heterogeneous protein composition. This system represents a novel means to evaluate the effects of compression and shear stress on different subcellular compartments of neurons, different from other in vitro systems that mainly focus on the effect of stretching. Importantly, owing to the unique features of our system, we recently made a novel discovery showing that the application of pressurized fluid can rapidly and reversibly induce axonal varicosities through the activation of a transient receptor potential channel. Our biomechanical system can be utilized conveniently in combination with drug perfusion, live cell imaging, calcium imaging, and patch clamp recording. Therefore, this method can be adopted for studying mechanosensitive ion channels, axonal transport regulation, axonal cytoskeleton dynamics, calcium signaling, and morphological changes related to traumatic brain injury.

This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

中枢神经元轴突静脉曲张形成和恢复的 Microbiomechanical 系统研究

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...