Name: a method for remotely silencing neural activity in rodents during discrete phases of learning
Uploaded: 2015-06-22T14:00:00.000+00:00
Duration: 9 min 22 s
Description: Watch this Scientific Journal Video about A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning at JoVE.com

我们感谢罗宾逊等人 12的作者为他们从本议定书部分派生的手稿贡献。

利用动物的奥伯林学院机构动物护理批准和使用委员会，并按照实验动物21与指南护理和使用。 1.准备输液病毒注:该协议使用BSL-1的预防措施。当采用BSL-2的预防措施，一次性白大褂，手套，鞋套，保护眼睛和防护口罩（N95型）是必需的。所有个人处理BSL-2化合物必须符合由当地公共卫生机构测试的微粒呼吸器。请参阅洛厄＆Majewska（2010）22对处理和储存病毒载体的其他详细信息。 <ol><li>当第一次使用，分装和储存未使用的病毒在20微升离心管，以避免反复冻融。 </li><li>通过去除任何不必要的对象和灭菌的表面用70％的乙醇制备的工作表面上。准备1用于净化废AAV 0％的漂白粉溶液。放置一个完整的漂白溶液和无菌的10微升注射器到工作表面的烧杯中。将离心管中设置了与碎冰容器的AAV。 </li><li>将离心管中的病毒变成一个标准的台虎钳。装载10微升注射器至少4微升的AAV。小心不要装载期间弯曲对微量离心管底部的注射器的尖端。确保没有气泡在4微升在注射器的AAV溶液。 </li><li>在含有10％的漂白剂的烧杯处置空病毒管。存储病毒的未使用的部分所指定的供应商。 </li><li>添加额外的10％的漂白粉到废液容器，并允许废物处置净化废液前坐了30分钟。 </li><li>在生物危害容器处理所有的防护设备和塑料废弃物的指示机构的指导方针。德科ntaminate中随附用10％漂白粉病毒接触的任何设备或表面。 </li></ol> 2.手术<ol><li>通过立体定位仪下，并在指定为专用的病毒处理区相邻的空间放置吸水纸板凳，准备手术区。 <ol><li>放置一个10％的漂白剂废物容器中的手术装置附近的专用的病毒处理区。 </li></ol></li><li>诱导麻醉一个稳定的水平，并准备大鼠进行手术。 <ol><li>将鼠到包含异氟醚气（1〜3％范围内）和氧（100％，在大约1升/分钟）的混合物中，直到有意识和缺乏目的运动总值的损失感应腔室。维持异氟烷麻醉整个手术过程中。 </li><li>经过6个深麻醉达到，刮老鼠从眼睛之间略有耳朵后面。 </li><li>将大鼠放回感应腔，用于一个另外1-3分钟。 </li><li>确保异氟烷麻醉系统连接到立体定位手术的鼻锥。打开对异氟烷油管旋塞阀开始异氟醚的stereotax流动。维持异氟烷和氧气在高于规定的电平。 </li><li>由固定到嘴咬栏和固定耳棒放置在立体定向仪的老鼠。 </li><li>敷眼润滑剂对眼睛和优碘到前切口手术部位。 </li></ol></li><li>使头骨的背表面皮肤的2.4厘米正中切口起约2毫米尾鳍的眼睛。收回的皮肤暴露颅骨。周期性地灌输在手术部位的边缘无菌的0.9％的无菌盐水以防止干燥组织。 </li><li>从头骨直到前囟和lambda清晰膜组织都清晰可见。 </li><li>确保头骨水平在测量背腹坐标前囟门和拉姆达。调整立体设备相应地，直到前囟和lambda背腹坐标差异不超过0.04毫米。 </li><li>此时，减少异氟烷气体之间2和2.5％。 </li><li>返回钻前囟门，重新调零坐标，然后钻移动到所需的内侧，外侧和前后坐标。 </li><li>钻一个孔在协调使用0.9毫米钻头，因为它会产生一个28克注射针筒适当大小的洞。 </li><li>设置输液泵以0.2微升/分的速率递送病毒。将注射器插入立体装置的输液臂和降低注射器成所需坐标。递送病毒的所需量（范围0.1〜0.8微升）。例如，递送0.8微升的AAV在4分钟。 注:开展试点研究，以确定理想的输液量和病毒在每个关心区域的典型传播。 <ol><li>后病毒的递送是共mplete离开注射器到位10分钟，以防止回流进入针道。慢慢提升注射器以约5毫米/分钟的速率。重复，直到所有的坐标都被注入AAV。 </li></ol></li><li>关闭使用手术钉和大衣搭配外用抗生素软膏伤口创面。 </li><li>遵循手术后的疼痛管理机构的指导方针。 </li><li>将老鼠与床上用品，盖和适当的标牌笼子里的老鼠恢复到动物设施。 <ol><li>的时间由机构指定的指导方针的金额（通常为48-72小时），如果在BSL-2注意事项工作，收集的床上用品。将被褥成生物危险废物的容器。 </li><li>所指定的为他们的安全处置规定进行处置所有AAV污染的材料。 </li></ol></li></ol> 3.行为仪器注:感觉预处理装置由一个标准的操作性条件反射湛误码率（12"长x 9.5"宽×11.5"高）用不锈钢格栅地板，2有机玻璃侧壁和2金属壁。 <ol><li>当在2赫兹闪过安装在铝幕墙作为光房子之一，作为视觉刺激2.8紫外线光。安装扬声器的室提供的听觉刺激（10秒1500赫兹，78分贝纯音和10秒78分贝白噪声）。 <ol><li>使用食品料斗提供45毫克的食物颗粒进入食品杯。内的食物杯中，红外光电检测在食品杯花之前，期间和刺激和食物奖励的介绍后的总时间。 </li><li>房子配备排风扇声音衰减柜室（22"宽x 22"高x 16"D）（〜68 dB为单位）。 </li><li>使用PC计算机与地中海Associates的软件来控制操作性室和获取数据。在空调的会议，收集PRESENTA期间用头花费在食品杯的总时间数据灰光刺激（5秒纪元）和呈现的食物奖励（5秒纪元）的过程中。在测试会话，收集上花费在食物杯中响应于听觉刺激的演示的总时间（10秒时期开始时的听觉刺激终止）数据。 </li></ol></li></ol>感官预处理范式4.概述<ol><li>从手术中恢复之后，逐渐限制食物的老鼠，以他们的自由喂养体重的85％。一个适当限食范式请教兽医人员。 注意:3相感官预处理范例示于图2。 </li><li>预处理会议（第1阶段;每日64分钟的培训课程跨连续4天进行的）: <ol><li>目前大鼠12混合试验是由交付听觉和视觉刺激。包括审间的间隔，每一个平均后4.5分钟（范围4.0〜5.0分钟）uditory演示。 </li><li> 6试验，目前音（预条件刺激）为10秒，随后立即闪烁光刺激的一个5秒的介绍。 </li><li>另外6项研究，提出了白噪声（配对的刺激）单独，持续10秒。 </li></ol></li><li>空调会议（第2阶段;跨连续5天每天进行64分钟的培训课程）: <ol><li>本大鼠8试验，它由输送视觉刺激（闪烁光刺激）为5秒的紧接着输送两个45毫克的食物颗粒。包括审间的间隔，每个审判后平均7分钟（范围6.0〜8.0分钟）。只偶尔需要超过5调理会话学习清淡的食物关联。 </li></ol></li><li>测试会议（第3阶段;一个78分钟的会话）: <ol><li>呈现大鼠12混合试验，它由输送单独的听觉刺激。包括AVE审间的间隔愤怒4.5分钟（范围4.0〜5.0分钟）每听演讲后。 </li><li> 6审判，呈现音刺激（预条件刺激）单独，持续10秒。 </li><li>另外6项研究，提出了白噪声（未成对刺激）单独，持续10秒。 </li></ol></li><li>通过完成学习与第二组接收白噪声作为预处理刺激和乐音作为未成刺激大鼠抵消刺激。 </li></ol> 5.药物和行为过程<ol><li>打开电源的行为的装置和装载MED-PC的代码为适当的行为会话。电线和程序行为装置，使得被安装在声音衰减机柜风扇打开时，接通电源上。 </li><li>以制备1mg / ml的溶液氯氮平N-氧化物（CNO）的，称重5.0毫克CNO的入15ml锥形管中并加入5毫升无菌0.9％萨利的无菌水或5毫升NE。涡旋直至溶液澄清。 <ol><li>如果CNO不溶解成溶液，或者如果CNO的更浓缩的溶液根据需要添加增溶剂，如DMSO 23。具体而言，以制备1mg / ml的溶液CNO的，用0.5％的DMSO，称量5.0毫克CNO，并添加25微升DMSO中至15ml锥形管中。轻弹轻轻确保内容保持在该管的基极。当溶液变得清晰，加入5毫升无菌水或5毫升无菌0.9％盐水中。可用的DREADD wiki资源第25页准备CNO的解决方案进一步的说明。 </li><li>它给予每天准备一个新的CNO的解决方案。 </li></ol></li><li>注入CNO腹膜内以1mg / kg的剂量约30分钟前行为测试。例如，注入一个200克大鼠用0.2ml的1mg / ml的CNO溶液。 CNO给药的剂量和时间过程基于先前发表的报告2,12被选定。<ol><li>之前注入到每个会话预处理CNO 30分钟（第一阶段，共4天注射），但事先管理该车辆的所有后续行为会话。 </li></ol></li><li> 30分钟后，将大鼠放入行为测试室，并启动了适当的行为会话的计算机代码。 </li><li>一旦该行为的任务完成后，立即从室的老鼠，并返回给大鼠动物殖民地。 </li></ol> 6.分析行为数据注:所有的行为会因变量是的时间量的老鼠的头部是食品杯内由食品杯红外光电中断的检测。 （在秒）的数据收集，并记录由计算机软件。 <ol><li>不进行分析了在预处理阶段产生的数据。 </li><li>对于空调的会议:分析每一个大鼠的一通过相容性陈述横跨5届行为的视觉刺激的过程中比较的食用杯行为学光食品协会。具体而言，计算5秒光划时代的每个8试验/会议期间在食品杯花费的平均时间（ 即，平均8试验每组是5秒的持续时间/大鼠）。比较平均控制值与实验值的每5日常会话（ 见图3A）。 <ol><li>分析老鼠的动机通过交付该食物奖励（5秒时代）跨越使用相同的计算上面指定的5秒光划时代的5届的行为过程中比较的食用杯的行为来获得食物奖励。这些值会比介绍的视觉刺激的期间获得始终如一更高（参见图3B）。 </li></ol></li><li>对于测试会话:计算判别比描述为响应PRES老鼠的食物一杯行为每一个听觉刺激entations。 <ol><li>具体而言，对于每只大鼠分为以下各6演示的感官刺激预处理（ 即，在音）通过在食品杯平均花费的时间总和在食品杯平均花费的时间在每次的6演示的感官刺激预处理（ 即刺激音）加在食品杯花费的总时间以下每6演示未成对刺激（ 即白噪声刺激）的平均水平。每个时期是10秒的持续时间。 注意:0.5或以上的判别得分表明大鼠学习固有的感官预处理范例的关联。 </li></ol></li></ol> 7.验证AAV安置和表达<ol><li>麻醉和灌注transcardially用4％多聚甲醛的作用。由于使用的荧光标记，不超过2小时的后固定时间最小imize损失抗原性，并保持了荧光信号。 </li><li>通过将它们插入含有对于至少2天，或直至大脑下沉到脑罐的底部将30ml在磷酸盐缓冲盐水（1×）20％的蔗糖溶液中的脑罐脱水灌注大脑。 </li><li>使用冷冻切片机滑动到整个感兴趣区域的整个rostrocaudal程度使冠状脑切片（20-40微米）。 </li><li>进行免疫组织化学针对标记受体或报告蛋白，以确定设计者受体和/或报道12的位置和表达。提供了DREADD wiki资源第25页上的免疫组织化学染色协议。 </li><li>安装使用100-200微升的水性封固剂的路段上的Superfrost加幻灯片和盖玻片。 </li><li>对脑组织进行荧光显微镜来验证AAV安置和蛋白表达12,24。</li>
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(45), (2010).</li><li>Cavaletti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+in+the+peripheral+nervous+system+of+systemically+administered+dimethylsulfoxide+in+the+rat:+a+neurophysiological+and+pathological.">Effect in the peripheral nervous system of systemically administered dimethylsulfoxide in the rat: a neurophysiological and pathological.</a> 119 (1-2), 1-2 (2000).</li><li>Parnaudeau, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mediodorsal+thalamus+hypofunction+impairs+flexible+goal-directed+behavior.">Mediodorsal thalamus hypofunction impairs flexible goal-directed behavior.</a> Biol. Psychiatry. 77 (5), 445-453 (2014).</li><li>wiki, D. R. E. A. D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2014).</li><li>Ferguson, S. M., Phillips, P. E. M., Roth, B. L., Wess, J., Neumaier, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-pathway+striatal+neurons+regulate+the+retention+of+decision-making+strategies.">Direct-pathway striatal neurons regulate the retention of decision-making strategies.</a> J. Neurosci. 33 (28), 11668-11676 (2013).</li><li>Cassatarro, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+pharmacogenetic+modulation+of+the+nucleus+accumbens+reduces+ethanol+consumption+in+a+limited+access+paradigm.">Reverse pharmacogenetic modulation of the nucleus accumbens reduces ethanol consumption in a limited access paradigm.</a> Neuropsychopharm. 39, 283-290 (2014).</li><li>Krashes, M. J., Shah, B. P., Koda, S., Lowell, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+versus+delayed+stimulation+of+feeding+by+the+endogenously+released+AgRP+neuron+mediators.">Rapid versus delayed stimulation of feeding by the endogenously released AgRP neuron mediators.</a> GABA, NPY and AgRP. Cell Metab. 18 (4), 588-595 (2014).</li><li>Gage, F. H., Bjorklund, A., Stenevi, U., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+correlates+of+compensatory+collateral+sprouting+by+aminergic+and+cholinergic+afferents+in+the+hippocampal+formation.">Functional correlates of compensatory collateral sprouting by aminergic and cholinergic afferents in the hippocampal formation.</a> Brain Res. 268 (1), 39-47 (1983).</li><li>Nelson, R. J., Young, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+in+mice+with+targeted+disruption+of+single+genes.">Behavior in mice with targeted disruption of single genes.</a> Neurosci. Biobehav. Rev. 22 (3), 453-462 (1998).</li></ol>

The authors declare that they have no competing financial interests.

这个协议描述了如何应用药理学方法（DREADD）调查如何特定脑区域有助于多相复杂的学习任务。随着跨学习阶段暂时和远程沉默的神经活动的离散的大脑区域的能力，这种方法相结合提供了一个平台，调查范围广泛的行为，包括学习更细致或屏蔽的形式。在这个协议中所描述的例子中，控制该表达在retrosplenial皮层（RSC）的设计者受体鼠和大鼠中的3相行为的任务12进行测试。任务的第一阶?...

行为神经科学领域内一个令人兴奋的挑战是确定复杂行为的神经基础。许多如永久性病变，通过套管植入大脑暂时失活和光遗传学技术已经被用来确定离散的大脑区域的贡献了复杂行为的子组件。虽然在学习这些方法告知我们的区域特殊性的理解，每种技术也不是没有限制的。具体地讲，永久病灶通常进行前行为测试，因此它们的作用是在整个范例的持续时间存在。插管研究涉及一个短期神经灭活剂（ 例如，河豚毒素）的呈现可以产生于脑组织实质损害，并且可以刚好在行为测试诱导应力的受试者。此外，通过插管灭活限于组织围绕的区域尖端的插管。最后，虽然光遗传学提供了一系列的灵活性可满足特定的大脑区域活动的时间控制，它是成本过高，技术要求高。 这些限制可以使用药理学方法（设计-受体独占激活的按设计药物，DREADDs）1,2有待克服。重要的是，而药物基因学的概念是复杂的，该技术的执行是简单的。类似于涉及输液毒素（ 例如，NMDA，鹅膏蕈氨酸）转换成离散的大脑区域，这种技术涉及输注腺相关病毒（AAV），其中包含的DNA片段进行修饰抑制G蛋白偶联型受体的传统立体手术方法（hM4Di;设计者受体）进入的标准实验室啮齿类感兴趣的区域（ 见图1）。所述病毒载体还含有荧光报道（mcitrine）。一旦合并到细胞中，设计者受体（和报告蛋白）的最大限度表达〜后3周输注和2-5小时由否则生物学惰性设计者药物的全身给药，氯氮平-N-氧化物可以被选择性地激活（CNO）1 3。因为实验者被赋予了超过在特定脑区域的神经活动的精确的，但遥控临时控制，药物遗传学结合特别好地与在多个阶段进行的行为范式。在这个例子中，retrosplenial皮质（RSC）的贡献对刺激刺激学习相比，其在巴甫洛夫学习的作用，然而办法这种组合非常适合于设法确定脑区如何具体向任意数量的问题复杂的行为。 此外，虽然在本协议中没有描述，病毒和转基因的方法可以用来实现细胞类型特异性DREADD表达式2。正如我固有在涉及药理学和/或其它类型的实验操作，仔细考虑的实验设计和随后的定量分析的行为范例是采用DREADD方法时需要。实验者新来的DREADD方法被称为当前DREADD 2技术的全面审查。 每一天，生物体了解新的刺激和事件以及它们彼此之间的关系。甚至在熟悉的环境，如家庭，一种是快速的以检测更改在刺激之间的关系，因为这些变化可能是预测有意义的事件。这种刺激的刺激（ 即，关系）学习涉及多种刺激的互联以及历来与海马，其中心位于内侧颞叶4内相关联。但是，海马并不存在，也没有采取行动，孤立;内和出皮层区域颞叶内侧的一边提供关键的感官信息的海马结构5-7。传统的永久损伤研究提供了令人信服的证据的一个数字皮层区域（ 例如，retrosplenial，postrhinal和内嗅皮质）中的海马依赖性学习的参与，但在其过程中的离散的阶段辨别一个特定区域的作用的能力是有限的学习8-10。 本协议测试的假设，即在RSC是必要的刺激-刺激的学习期间通过一个3相感官预处理范例11,12的单相沉默在RSC。简而言之，大鼠收到包含设计者受体和〜3周后施用设计者药物（CNO）30前行为测试开始分钟的AAV的输注。在本协议中，实验组大鼠在测试过程中的第一阶段（当刺激刺激李尔收到CNO宁发生），他们在接下来的2阶段测试接收车辆。为了控制CNO对行为的影响意外，注入老鼠与设计师受体（hM4Di），并注入一些车辆，而不是CNO。考虑到病毒输注和受体表达的一般效果，注入一个对照病毒不包含设计者受体和管理CNO。 许多AAV的不同血清型的用于输送遗传物质。研究涉及重组或合成的分子的当前的NIH指导方针主张的AAV（所有血清型）和重组的或合成的AAV构建体，其中所述转基因不编码任一种潜在的致瘤基因产物或毒素分子，并且产生在没有一辅助病毒，需要BSL-1的预防措施（附录B-1。风险组1（RG1）代理）13。可用14,15一些有关AAV结构，实用性和安全审查的。值得注意的是，虽然时，由于有关在啮齿动物可能生殖16,17和潜在的致癌机制18-20关注，一些机构要求使用的BSL-2的预防措施的AAV工作时。验证通过与地方研究会进行，疾病控制中心和美国国立卫生研究院准则研究使用病毒载体的基因操作在美国时涉及重组DNA分子13的监督委员会，在个别机构咨询，使用前适当的BSL。个人防护，调查培训，矢量遏制，净化，消毒处理的材料，以及注射后的动物住房需求是由这些原则规定。此外，参阅并遵循适当的机构动物护理和使用委员会的准则或同等机构的监督委员会的指导方针，以确保安全处理，管理和处置的AAV。

<table><tbody><tr><td>Male, Long Evans Rats, 55-60 days old</td><td>Hilltop Lab Animals Inc</td><td></td><td></td></tr><tr><td>rAAV8/hSyn-HA-hM4D(Gi)-IRES-mCitrine</td><td>Virus Vector Core</td><td></td><td>Caution: This is a BSL-1 compound</td></tr><tr><td>rAAV8/hSyn-GFP</td><td>Virus Vector Core</td><td></td><td>Caution: This is a BSL-1 compound</td></tr><tr><td>Clozapine-N-oxide</td><td>R&amp;amp;D Systems</td><td>4936-10</td><td>Designer drug</td></tr><tr><td>Rat Cage lid (polycarbonate)</td><td>Alternative Design&amp;nbsp;</td><td>FT 8XL-PC</td><td>Used to cover animal cages 48-72 hr post infusion</td></tr><tr><td>Filter paper (replacement)</td><td>Alternative Design</td><td>FP-R-1018XAD</td><td>Filter paper that goes with cage lids</td></tr><tr><td>Table top vise</td><td>JETS</td><td>2201-265</td><td>For holding microscentrifuge tubes containing AAV in the hood</td></tr><tr><td>Biohazard trash bags</td><td>Staples</td><td>113444</td><td>Medline biohazard liners</td></tr><tr><td>Biohazard trash can</td><td>Amazon.com</td><td></td><td>United Solutions 34 gallon rectangular wheeled trashcan with hook and lock handle</td></tr><tr><td>Isoflurane, 100 ml</td><td>Patterson Veterinary Supply Inc.</td><td>07-890-8540</td><td>Anesthetic</td></tr><tr><td>Dual small animal stereotaxic with digital display readout console</td><td>David Kopf</td><td>Model 942</td><td>Surgical equipment</td></tr><tr><td>Non-rupture ear bars, set of 2 (rat)</td><td>David Kopf</td><td>Model 955</td><td>Surgical equipment</td></tr><tr><td>Anesthesia mask (rat)</td><td>David Kopf</td><td>Model 906</td><td>Surgical equipment</td></tr><tr><td>High speed stereotaxic drill includes table top motor controller, foot pedal, handpiece, stereotaxic handpiece holder</td><td>David Kopf</td><td>Model 1474</td><td>Surgical equipment</td></tr><tr><td>Microdrill burrs, 0.9 mm</td><td>Fine Science Tools Inc</td><td>19007-09</td><td>Surgical supply</td></tr><tr><td>Automated syringe pump with micro4 controller</td><td>David Kopf</td><td>Model UMP3-1</td><td>Surgical equipment</td></tr><tr><td>Pro-animal detachable ceramic blade clipper kit</td><td>Ahdis</td><td>21420</td><td>Surgical supply</td></tr><tr><td>Betadine skin cleanser</td><td>Perdue Products L.P</td><td>67618-149-04</td><td>Surgical supply</td></tr><tr><td>Triple antiobiotic ointment</td><td>Medline Supply</td><td>53329-087-01</td><td>Surgical supply</td></tr><tr><td>Puralube vet ointment</td><td>Only Veterinary Supply</td><td>17033-211-38</td><td>Surgical supply</td></tr><tr><td>Dino-lite</td><td>Microscope</td><td>AD7013MTL</td><td>An alternative to the traditional disection scope</td></tr><tr><td>Dino-lite rigid table-top boom stand</td><td>Microscope</td><td>MS36B</td><td>Surgical equipment</td></tr><tr><td>28 G 10 &amp;mu;l syringe</td><td>Hamilton</td><td>80308-701SN</td><td>Surgical equipment</td></tr><tr><td>Extra tall MDF sound attenuating cubicle</td><td>Med Associates, Inc</td><td>ENV-018MD</td><td>22&#039; W x 22&quot; H x 16&quot; D</td></tr><tr><td>Extra tall modular test chamber</td><td>Med Associates, Inc</td><td>ENV-007</td><td>Behavioral equipment</td></tr><tr><td>Stainless steel grid floor</td><td>Med Associates, Inc</td><td>ENV-005</td><td>Behavioral equipment</td></tr><tr><td>House light</td><td>Med Associates, Inc</td><td>ENV-215M</td><td>Used as the house light and stimulus light</td></tr><tr><td>Modular pellet dispenser</td><td>Med Associates, Inc</td><td>ENV-203M-45</td><td>Behavioral equipment</td></tr><tr><td>Pellet receptacle, cup type</td><td>Med Associates, Inc</td><td>ENV-200R1M</td><td>Behavioral equipment</td></tr><tr><td>Head entry detector for rat</td><td>Med Associates, Inc</td><td>ENV-254-CB</td><td>Behavioral equipment</td></tr><tr><td>Dustless precision food pellets, 45 mg</td><td>Bio-Serv</td><td>F0165</td><td>Behavioral supply</td></tr><tr><td>Cage speaker for rat chamber</td><td>Med Associates, Inc</td><td>ENV-224AM</td><td>Behavioral equipment</td></tr><tr><td>Programmable audio generator</td><td>Med Associates, Inc</td><td>ANL-926</td><td>Behavioral equipment</td></tr><tr><td>Smart ctrl 8 input/16 output package</td><td>Med Associates, Inc</td><td>DIG-716P2</td><td>Behavioral equipment</td></tr><tr><td>Large table-top cabinet and power supply</td><td>Med Associates, Inc</td><td>SG-6510D</td><td>Behavioral equipment</td></tr><tr><td>PCI interface package</td><td>Med Associates, Inc</td><td>DIG-700P2-R2</td><td>Behavioral equipment</td></tr><tr><td>MED Intel core computer pkg with X Pro 19&quot; monitor</td><td>Med Associates, Inc</td><td>COM-103V</td><td>Behavioral equipment</td></tr><tr><td>Paraformaldehyde (grannular), 1 kg</td><td>Electron Microsopy Sciences</td><td>19210</td><td>Hazard: carcinogen, weigh in hood</td></tr><tr><td>Rabbit monoclonal antibody (HA-Tag)</td><td>Cell Signaling Technologies</td><td>3724S</td><td>Histology reagent</td></tr><tr><td>XP Rabbit monoclonal antibody (GFP)</td><td>Cell Signaling Technologies</td><td>2956S</td><td>Histology reagent</td></tr><tr><td>Anti-rabbit IgG</td><td>Cell Signaling Technologies</td><td>4412S</td><td>Histology supplies</td></tr><tr><td>Superfrost plus slides</td><td>VWR international</td><td>483111-703</td><td>Histology supplies</td></tr></tbody></table>

a method for remotely silencing neural activity in rodents during discrete phases of learning

这个协议描述如何暂时和远程沉默神经元活动中的离散的大脑区域，而动物从事学习和记忆任务。该方法结合了药物遗传学（设计 - 受体 - 独占激活的逐设计-用药）与行为范式（感官预处理），其被设计不同形式的学习的区别开来。具体地，病毒介导的递送是用来表达基因修饰抑制G-蛋白偶联受体（设计器受体）插入在啮齿动物的离散大脑区域。三周后，当设计师受体表达水平是高的，药理剂（在设计药物）的全身给药30分钟之前，一个特定的行为会话。该药具有设计师受体的亲和力，从而导致抑制表达设计师受体神经元，但在其他生物惰性。该大脑区域保持沉默2-5小时（depen鼎上的剂量和给药途径）。在行为模式完成后，脑组织被评估正确的位置和受体的表达。这种方法是用于确定个体的大脑区域，以行为的特定组件的贡献，并且可以跨越任何数量的行为范例的使用是特别有用的。

行为结果在实验结束后，将区域专用临时灭活的有效性应该被定量和定性评估。本实施例涉及一个3相行为范式（感官预处理），其中CNO施用以减弱在预处理会话中在RSC神经活动来检验这一假设在RSC是必要的协会之间中性刺激12的形成。重要的是，实验者不局限于行为范式或本文描述的药物遗传学方法可以加上最多行为范例实验设计。 而分析通常不?...

Watch this Scientific Journal Video about A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning at JoVE.com

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

为远程沉默的神经活动在鼠害的方法在学习独立的阶段

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and ...

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14.6K 次观看.  Oberlin College. This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

视频: A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

Long-term Potentiation of Perforant Pathway-dentate Gyrus Synapse in Freely Behaving Mice

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential cellular mechanism underlying learning and information storage, have long been used to elucidate the physiology of various neuronal circuits in the hippocampus, amygdala, and other limbic and cortical structures. With this in mind, transgenic mouse models of neurological diseases represent useful platforms to conduct long-term&#160;potentiation (LTP) studies to develop a greater understanding of the role of genes in normal and abnormal synaptic communication in neuronal networks involved in learning, emotion and information processing. This article describes methodologies for reliably inducing LTP in the freely behaving mouse. These methodologies can be used in studies of transgenic and knockout freely&#160;behaving mouse models of neurodegenerative diseases.

Transgenic and knockout mouse models of neurological diseases are useful for studying the role of genes in normal and abnormal neurophysiology. This article describes methodologies which can be used to study long-term potentiation, a cellular mechanism which may underlie learning and memory, in transgenic and knockout freely&#160;behaving mouse models of neuropathology.

穿通通路 - 齿状回突触在自由活动的小鼠长时程增强

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential ...

科研

行为学

A Fully Automated Rodent Conditioning Protocol for Sensorimotor Integration and Cognitive Control Experiments

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. Higher cognitive functions that require precise control over sensorimotor responses such as decision-making and attentional modulation, however, are typically assessed in nonhuman primates. Despite the richness of primate behavior that allows multiple variants of these functions to be studied, the rodent model remains an attractive, cost-effective alternative to primate models. Furthermore, the ability to fully automate operant conditioning in rodents adds unique advantages over the labor intensive training of nonhuman primates while studying a broad range of these complex functions.
Here, we introduce a protocol for operantly conditioning rats on performing working memory tasks. During critical epochs of the task, the protocol ensures that the animal's overt movement is minimized by requiring the animal to 'fixate' until a Go cue is delivered, akin to nonhuman primate experimental design. A simple two alternative forced choice task is implemented to demonstrate the performance. We discuss the application of this paradigm to other tasks.

A fully automated protocol for rodent operant conditioning is proposed. The protocol relies on precise temporal control of behavioral events to investigate the extent to which this control influences neural activity underlying sensorimotor integration and cognitive control experiments.

为感觉运动整合与认知控制实验完全自动化的啮齿动物调理协议

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. ...

Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the necessary equipment is now available by several commercial companies. This protocol provides a detailed description of a two-way shuttle-box avoidance learning paradigm in rodents (here Mongolian gerbils; Meriones unguiculatus) in combination with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical electrophysiological in vivo recordings. The detailed protocol is applicable to study multiple aspects of learning behavior and perception in different rodent species.
Site-specific ICMS of auditory cortical circuits as conditioned stimuli here is used as a tool to test the perceptual relevance of specific afferent, efferent and intracortical connections. Distinct activation patterns can be evoked by using different stimulation electrode arrays for local, layer-dependent ICMS or distant ICMS sites. Utilizing behavioral signal detection analysis it can be determined which stimulation strategy is most effective for eliciting a behaviorally detectable and salient signal. Further, parallel multichannel-recordings using different electrode designs (surface electrodes, depth electrodes, etc.) allow for investigating neuronal observables over the time course of such learning processes. It will be discussed how changes of the behavioral design can increase the cognitive complexity (e.g. detection, discrimination, reversal learning).

Shuttle-box avoidance learning is well-established in behavioral neuroscience. This protocol describes how shuttle-box learning in rodents can be combined with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical in vivo recordings as a tool to study multiple aspects of learning and perception.

联合穿梭盒子训练与电的Cortex记录和刺激作为一种工具来研究感知和学习

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the ...

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

迷走神经刺激作为一种工具来诱导可塑性相关途径灭绝学习

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

一个多认知功能测试，并记录在啮齿动物的神经元活动的完全自动化和高度灵活的系统

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

神经科学

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman&#160;et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons&#39; action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

自由移动鸽子穿越三个阶段学习记录单个神经元&#39;动作电位

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

同时电生理记录和抑制剂在啮齿动物大脑微注射

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.

分层膜片钳技术在学习诱导可塑性分析中的应用

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and deafferentation of neuronal groups, which are both invasive methods that permanently impair the integrity of the target area. These methods are accompanied by collateral effects that can affect the analysis of acute and temporal regulatory mechanisms. The stereotaxic implantation of guide cannulas aimed at specific brain regions, followed by a recovery period, allows researchers to microinject different drugs after the disappearance of the undesired effects of the surgery. Tetrodotoxin has been used to determine the roles of several brain areas in diverse physiological processes because it transiently inhibits the sodium-dependent action potentials, thus blocking all neural activity in the target region. This protocol combines this method with strategies for the assessment of the estrous cycle and ovulation to reveal the role of discrete brain regions in the regulation of ovulation at particular times of any given stage of the estrous cycle. Awake and unrestrained rats (Rattus norvegicus) were used to avoid the blocking effects that anesthetics and stress hormones exert on ovulation. This protocol can be easily adapted to other species, brain targets and pharmacological agents to study different physiological processes. Future improvements to this method include the design of a microinjection system using glass capillaries of small diameter instead of guide cannulas. This will reduce the amount of tissue damaged during the implantation and decrease the spread of the infused drugs outside the target area.

This protocol describes the construction of a low-cost microinjection system, its stereotaxic implantation into deep-brain structures and the procedure for timed microinjections of tetrodotoxin in awake and unrestrained rats. The goal is to reveal the participation of hypothalamic structures in the regulation of ovulation by inhibiting their neural activity.

为远程沉默的神经活动在鼠害的方法在学习独立的阶段

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