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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Guide for the Care and Use of Laboratory Animals.</a> National Academy Press. , (1996).</li><li>Lowery, R. L., Majewska, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+injection+of+adeno-associated+viral+vectors.">Intracranial injection of adeno-associated viral vectors.</a> J. Vis. Exp. (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. 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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 border="0" cellpadding="0" cellspacing="0" >
	
	
	<tbody>
		<tr >
			<td >Male, Long Evans Rats, 55-60 d</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:&#160; This is a BSL-1 compound</td>
		</tr>
		<tr >
			<td >rAAV8/hSyn-GFP</td>
			<td >Virus Vector Core</td>
			<td ></td>
			<td>Caution:&#160; This is a BSL-1 compound</td>
		</tr>
		<tr >
			<td >Clozapine-N-Oxide</td>
			<td >R&#38;D Systems</td>
			<td >4936-10</td>
			<td>Designer Drug</td>
		</tr>
		<tr >
			<td >Rat Cage lid (Polycarbonate)</td>
			<td >Alternative Design&#160;</td>
			<td >FT 8XL-PC</td>
			<td>Used to cover animal cages 48-72 hours post infusion</td>
		</tr>
		<tr >
			<td >Filer Paper (Replacement)</td>
			<td >Alternative Design&#160;</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 >Medline Biohazard liners&#160;</td>
			<td >Staples</td>
			<td >113444</td>
			<td>Biohazard Trash Bags</td>
		</tr>
		<tr >
			<td >United Solutions 34 gallon rectangular wheeled trashcan with hook and lock handle&#160;</td>
			<td >Amazon.com</td>
			<td ></td>
			<td>Biohazard Trash Can</td>
		</tr>
		<tr >
			<td >Isoflurane, 100 mL</td>
			<td >Patterson Veterinary Supply Inc.&#160;</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&#160;</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&#160; 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&#160;</td>
			<td >Microscope</td>
			<td >AD7013MTL</td>
			<td>An alternative to the traditional disection scope</td>
		</tr>
		<tr >
			<td >Dino-lite Rigid TableTop Boom Stand</td>
			<td >Microscope</td>
			<td >MS36B</td>
			<td>Surgical equipment</td>
		</tr>
		<tr >
			<td >28 Gauge 10uL Syringe</td>
			<td >Hamilton&#160;</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&#39;Wx22&#34;Hx16&#34;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&#160;</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&#160;</td>
			<td >Med Associates, Inc</td>
			<td >ENV-203M-45</td>
			<td>Behavioral equipment</td>
		</tr>
		<tr >
			<td >Pellet Recetacle, 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&#160;</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&#34; 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:&#160; carcinogen, weigh in hood</td>
		</tr>
		<tr >
			<td >Rabbit Monoclonal antibody (HA-Tag)</td>
			<td >Cell Signaling Technologies&#160;</td>
			<td >3724S</td>
			<td>Histology reagent</td>
		</tr>
		<tr >
			<td >XP Rabbit monoclonal antibody (GFP)</td>
			<td >Cell Signaling Technologies&#160;</td>
			<td >2956S</td>
			<td>Histology reagent</td>
		</tr>
		<tr >
			<td >Anti-Rabbit IgG</td>
			<td >Cell Signaling Technologies&#160;</td>
			<td >4412S</td>
			<td>Histology supplies</td>
		</tr>
		<tr >
			<td >Superfrost Plus slides&#160;</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?...

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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 ...

a-method-for-remotely-silencing-neural-activity-rodents-during

Research

JoVE Journal

Behavior

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

Meal Duration as a Measure of Orofacial Nociceptive Responses in Rodents

A lengthening in meal duration can be used to measure an increase in orofacial mechanical hyperalgesia having similarities to the guarding behavior of humans with orofacial pain. To measure meal duration unrestrained rats are continuously kept in sound attenuated, computerized feeding modules for days to weeks to record feeding behavior. These sound-attenuated chambers are equipped with chow pellet dispensers. The dispenser has a pellet trough with a photobeam placed at the bottom of the trough and when a rodent removes a pellet from the feeder trough this beam is no longer blocked, signaling the computer to drop another pellet. The computer records the date and time when the pellets were taken from the trough and from this data the experimenter can calculate the meal parameters. When calculating meal parameters a meal was defined based on previous work and was set at 10 min (in other words when the animal does not eat for 10 min that would be the end of the animal&#39;s meal) also the minimum meal size was set at 3 pellets. The meal duration, meal number, food intake, meal size and inter-meal interval can then be calculated by the software for any time period that the operator desires. Of the feeding parameters that can be calculated meal duration has been shown to be a continuous noninvasive biological marker of orofacial nociception in male rats and mice and female rats. Meal duration measurements are quantitative, require no training or animal manipulation, require cortical participation, and do not compete with other experimentally induced behaviors. These factors distinguish this assay from other operant or reflex methods for recording orofacial nociception.

A lengthening in meal duration represents orofacial nociceptive behavior in rodents similar to the guarding behavior of humans with orofacial pain. Eating is a behavior that requires no training or animal manipulation, requires cortical participation, and is not competing with other experimentally induced behaviors, distinguishing this assay from alternative reflex or operant measurements.

膳食持续时间作为衡量啮齿动物的奥体非感知反应的指标

A lengthening in meal duration can be used to measure an increase in orofacial mechanical hyperalgesia having similarities to the guarding behavior of ...

科研

行为学

Automated Visual Cognitive Tasks for Recording Neural Activity Using a Floor Projection Maze

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have developed visual cognitive tasks for rats using the Floor Projection Maze1,2&#160;that are optimized for visual abilities of rats permitting stronger comparisons of experimental findings with other species.
In order to investigate neural correlates of learning and memory, we have integrated electrophysiological recordings into fully automated cognitive tasks on the Floor Projection Maze1,2. Behavioral software interfaced with an animal tracking system allows monitoring of the animal's behavior with precise control of image presentation and reward contingencies for better trained animals. Integration with an in vivo electrophysiological recording system enables examination of behavioral correlates of neural activity at selected epochs of a given cognitive task.
We describe protocols for a model system that combines automated visual presentation of information to rodents and intracranial reward with electrophysiological approaches. Our model system offers a sophisticated set of tools as a framework for other cognitive tasks to better isolate and identify specific mechanisms contributing to particular cognitive processes.

We describe protocols for training rats for chronic electrophysiological recordings in fully automated cognitive tasks on a Floor Projection Maze.

使用地面投影迷宫自动化视觉认知任务记录神经活动

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have ...

Measuring Neural and Behavioral Activity During Ongoing Computerized Social Interactions: An Examination of Event-Related Brain Potentials

Social exclusion is a complex social phenomenon with powerful negative consequences. Given the impact of social exclusion on mental and emotional health, an understanding of how perceptions of social exclusion develop over the course of a social interaction is important for advancing treatments aimed at lessening the harmful costs of being excluded. To date, most scientific examinations of social exclusion have looked at exclusion after a social interaction has been completed. While this has been very helpful in developing an understanding of what happens to a person following exclusion, it has not helped to clarify the moment-to-moment dynamics of the process of social exclusion. Accordingly, the current protocol was developed to obtain an improved understanding of social exclusion by examining the patterns of event-related brain activation that are present during social interactions. This protocol allows greater precision and sensitivity in detailing the social processes that lead people to feel as though they have been excluded from a social interaction. Importantly, the current protocol can be adapted to include research projects that vary the nature of exclusionary social interactions by altering how frequently participants are included, how long the periods of exclusion will last in each interaction, and when exclusion will take place during the social interactions. Further, the current protocol can be used to examine variables and constructs beyond those related to social exclusion. This capability to address a variety of applications across psychology by obtaining both neural and behavioral data during ongoing social interactions suggests the present protocol could be at the core of a developing area of scientific inquiry related to social interactions.

Research on social exclusion has grown tremendously in recent years. As the field expands, it is imperative to develop sophisticated methodologies allowing for the simultaneous measurement of neural and behavioral outcomes during social exclusion. This protocol utilizes event-related brain potentials to record ongoing neural activity during computerized social interactions.

测量神经和行为活动期间持续计算机化的社会互动：对事件相关电位的考察

Social exclusion is a complex social phenomenon with powerful negative consequences. Given the impact of social exclusion on mental and emotional health, ...

The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their behavioral flexibility and required memory duration, the number of goals and pathways, and also the overall task complexity. A confounding feature in many of these tasks is the lack of control over the strategy employed by the rodents to reach the goal, e.g., allocentric (declarative-like) or egocentric (procedural) based strategies. The double-H maze is a novel water-escape memory task that addresses this issue, by allowing the experimenter to direct the type of strategy learned during the training period. The double-H maze is a transparent device, which consists of a central alleyway with three arms protruding on both sides, along with an escape platform submerged at the extremity of one of these arms.
Rats can be trained using an allocentric strategy by alternating the start position in the maze in an unpredictable manner (see protocol 1; &#167;4.7), thus requiring them to learn the location of the platform based on the available allothetic cues. Alternatively, an egocentric learning strategy (protocol 2; &#167;4.8) can be employed by releasing the rats from the same position during each trial, until they learn the procedural pattern required to reach the goal. This task has been proven to allow for the formation of stable memory traces.
Memory can be probed following the training period in a misleading probe trial, in which the starting position for the rats alternates. Following an egocentric learning paradigm, rats typically resort to an allocentric-based strategy, but only when their initial view on the extra-maze cues differs markedly from their original position. This task is ideally suited to explore the effects of drugs/perturbations on allocentric/egocentric memory performance, as well as the interactions between these two memory systems.

The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

双-H迷宫：一个强大的行为测试学习和记忆的鼠害

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their ...

Quantifying Learning in Young Infants: Tracking Leg Actions During a Discovery-learning Task

Task-specific actions emerge from spontaneous movement during infancy. It has been proposed that task-specific actions emerge through a discovery-learning process. Here a method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process. This discovery-learning task uses an infant activated mobile that rotates and plays music based on specified leg action of infants. Supine infants activate the mobile by moving their feet vertically across a virtual threshold. This paradigm is unique in that as infants independently discover that their leg actions activate the mobile, the infants&#8217; leg movements are tracked using a motion capture system allowing for the quantification of the learning process. Specifically, learning is quantified in terms of the duration of mobile activation, the position variance of the end effectors (feet) that activate the mobile, changes in hip-knee coordination patterns, and changes in hip and knee muscle torque. This information describes infant exploration and exploitation at the interplay of person and environmental constraints that support task-specific action. Subsequent research using this method can investigate how specific impairments of different populations of infants at risk for movement disorders influence the discovery-learning process for task-specific action.

A method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process.

量化学习婴幼儿：跟踪腿操作期间发现，学习任务

Task-specific actions emerge from spontaneous movement during infancy. It has been proposed that task-specific actions emerge through a discovery-learning ...

Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for quantifying locomotor deficits in rodents is to measure minute disturbances of unconstrained gait overground (e.g., manual BBB score or automated CatWalk). However, cortical inputs are not required for the generation of basic locomotion produced by the spinal central pattern generator (CPG). Thus, unconstrained walking tasks test locomotor deficits due to motor cortical impairment only indirectly. In this study, we propose a novel, precise foot-placement locomotor task that evaluates cortical inputs to the spinal CPG. An instrumented peg-way was used to impose symmetrical and asymmetrical locomotor tasks mimicking lateralized movement deficits. We demonstrate that shifts from equidistant inter-stride lengths of 20% produce changes in the forelimb stance phase characteristics during locomotion with preferred stride length. Furthermore, we propose that the asymmetric walkway allows for measurements of behavioral outcomes produced by cortical control signals. These measures are relevant for the assessment of impairment after cortical damage.

Here, we present a protocol to quantify precise stepping in rodents. Cortical and the spinal central pattern generator signals are required for precise foot-placement during obstructed locomotion. We report here the novel constrained walking task that directly examines precise stepping behavior.

非对称走道：一种新的行为检测方法的研究不对称步态

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for ...

Using the Activity-based Anorexia Rodent Model to Study the Neurobiological Basis of Anorexia Nervosa

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A challenging illness to treat, due to the lack of understanding of the underlying neurobiology, AN has the highest mortality rate among psychiatric illnesses. To address this need, neuroscientists are using an animal model to study how neural circuits may contribute toward vulnerability to AN and may be affected by AN. Activity-based anorexia (ABA) is a bio-behavioral phenomenon described in rodents that models the key symptoms of anorexia nervosa. When rodents with free access to voluntary exercise on a running wheel experience food restriction, they become hyperactive &#8211; running more than animals with free access to food. Here, we describe the procedures by which ABA is induced in adolescent female C57BL/6 mice. On postnatal day 36 (P36), the animal is housed with access to voluntary exercise on a running wheel. After 4 days of acclimation to the running wheel, on P40, all food is removed from the cage. For the next 3 days, food is returned to the cage (allowing animals free food access) for 2 hr daily. After the fourth day of food restriction, free access to food is returned and the running wheel is removed from the cage to allow the animals to recover. Continuous multi-day analysis of running wheel activity shows that mice become hyperactive within 24 hr following the onset of food restriction. The mice run even during the limited time during which they have access to food. Additionally, the circadian pattern of wheel running becomes disrupted by the experience of food restriction. We have been able to correlate neurobiological changes with various aspects of the animals&#8217; wheel running behavior to implicate particular brain regions and neurochemical changes with resilience and vulnerability to food-restriction induced hyperactivity.

Here we present a protocol to induce activity-based anorexia (ABA) in female adolescent mice. ABA is a condition of hyperactivity evoked by imposing food restriction on rodents with access to a running wheel. This phenomenon is being used as a model to study the underlying neurobiology of anorexia nervosa.

使用基于活动的厌食症啮齿动物模型来研究神经性厌食症的神经生物学基础

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A ...

A Novel Experimental and Analytical Approach to the Multimodal Neural Decoding of Intent During Social Interaction in Freely-behaving Human Infants

Understanding typical and atypical development remains one of the fundamental questions in developmental human neuroscience. Traditionally, experimental paradigms and analysis tools have been limited to constrained laboratory tasks and contexts due to technical limitations imposed by the available set of measuring and analysis techniques and the age of the subjects. These limitations severely limit the study of developmental neural dynamics and associated neural networks engaged in cognition, perception and action in infants performing &#8220;in action and in context&#8221;. This protocol presents a novel approach to study infants and young children as they freely organize their own behavior, and its consequences in a complex, partly unpredictable and highly dynamic environment. The proposed methodology integrates synchronized high-density active scalp electroencephalography (EEG), inertial measurement units (IMUs), video recording and behavioral analysis to capture brain activity and movement non-invasively in freely-behaving infants. This setup allows for the study of neural network dynamics in the developing brain, in action and context, as these networks are recruited during goal-oriented, exploration and social interaction tasks.

This protocol presents a novel methodology for the neural decoding of intent from freely-behaving infants during unscripted social interaction with an actor. Neural activity is acquired using non-invasive high-density active scalp electroencephalography (EEG). Kinematic data is collected with inertial measurement units and supplemented with synchronized video recording.

在自由，表现人类婴儿一种新的实验和分析方法意向的多模态神经解码期间社会互动

Understanding typical and atypical development remains one of the fundamental questions in developmental human neuroscience. Traditionally, experimental ...

Feeding Experimentation Device (FED): Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents

Food intake measurements are essential for many research studies. Here, we provide a detailed description of a novel solution for measuring food intake in mice: the Feeding Experimentation Device (FED). FED is an open-source system that was designed to facilitate flexibility in food intake studies. Due to its compact and battery powered design, FED can be placed within standard home cages or other experimental equipment. Food intake measurements can also be synchronized with other equipment in real-time via FED's transistor-transistor logic (TTL) digital output, or in post-acquisition processing as FED timestamps every event with a real-time clock. When in use, a food pellet sits within FED's food well where it is monitored via an infrared beam. When the pellet is removed by the mouse, FED logs the timestamp onto its internal secure digital (SD) card and dispenses another pellet. FED can run for up to 5 days before it is necessary to charge the battery and refill the pellet hopper, minimizing human interference in data collection. Assembly of FED requires minimal engineering background, and off-the-shelf materials and electronics were prioritized in its construction. We also provide scripts for analysis of food intake and meal patterns. Finally, FED is open-source and all design and construction files are online, to facilitate modifications and improvements by other researchers.

Feeding Experimentation Device FED: Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents

Feeding Experimentation Device (FED) is an open-source device for measuring food intake in mice. FED can also synchronize food intake measurements with other techniques via a real-time digital output. Here, we provide a step-by-step tutorial for the construction, validation, and usage of FED.

饲养实验装置（FED）：建筑和验​​证的开源设备的在鼠害测量摄食

Food intake measurements are essential for many research studies. Here, we provide a detailed description of a novel solution for measuring food intake in ...

A Computational Method to Quantify Fly Circadian Activity

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. Fundamental mechanisms that underlie this temporal control are widely studied using the fruit fly Drosophila melanogaster as a model organism. In flies, the clock is typically studied by analyzing multiday locomotor recording. Such a recording shows a complex bimodal pattern with two peaks of activity: a morning peak that happens around dawn, and an evening peak that happens around dusk. These two peaks together form a waveform that is very different from sinusoidal oscillations observed in clock genes, suggesting that mechanisms in addition to the clock have profound effects in producing the observed patterns in behavioral data. Here we provide instructions on using a recently developed computational method that mathematically describes temporal patterns in fly activity. The method fits activity data with a model waveform that consists of four exponential terms and nine independent parameters that fully describe the shape and size of the morning and evening peaks of activity. The extracted parameters can help elucidate the kinetic mechanisms of substrates that underlie the commonly observed bimodal activity patterns in fly locomotor rhythms.

A method to quantify the main temporal features seen in fly circadian locomotor rhythms is presented. The quantification is achieved by fitting fly activity with a multi-parametric model waveform. The model parameters describe the shape and size of the morning and evening peaks of daily activity.