Mohammed R. Milad is supported by NIMH grant R01 MH097880 and institutional funds from the Department of Psychiatry at MGH.

注：在本协议中的所有程序已经批准了小组委员会研究动物护理，作为该机构动物护理和使用委员会（IACUC）在马萨诸塞州总医院，并符合卫生准则的国家机构。 1.动物住房和处理程序<ol><li>抵达后，房屋成年SD雌性大鼠约8-10周龄（体重200-225克），在3-4英寸的塑料箱用木片床上用品组。维持它们在饮食中随意获得食物和水，并在12小时的光/暗周期。离开老鼠适应的住房条件和动物设施至少1周。 注：本稿件所有过程都是在光周期中进行。 </li><li>在此之后的适应期，处理所有动物的每个2-3天5分钟。正确地拿起大鼠一次一个（由躯干，而不是由尾支持），追究他们的安全，并在此期间抚摸。处理出现更多的强调，神经质，或焦虑的时间较长的动物。这样做是为了尽量减少处理压力，这可能会影响本协议的下列步骤。 注：天数和时间处理的动物可以在不同的实验室之间变化量;然而，这些都应该是相同的实验室内的实验之间保持一致。 </li></ol> 2.监测发情周期：阴道拭子拖尾和染色过程<ol><li>使0.9％盐溶液（溶解在蒸馏水氯化钠）。滋润在此溶液中的棉签。印迹使用纸巾，以避免过饱和，这可能导致样品收集或细胞的损失不足的小费。抱着尾巴向​​上远离阴道口，轻轻插入棉花尖端插入阴道运河和打滚的墙壁捕捉宽松阴道细胞。 </li><li>如果动物小便的婴儿床吨尖，更换一个新的，因为尿液污染将难以相鉴别。要格外小心，因为在此过程中不造成困扰的动物，因为接触应力破坏发情周期。当抽汲，旨在实现一个快速的插拔，是不是太深，以减少额外的刺激子宫颈，可引起假孕。 注：假孕可确定为无环发情周期通常持续约12天， 即发情间期25个连续天。 </li><li>小心地取出敷贴尖端和滚动它到一个预先标记的显微镜载玻片。避免按下太难为幻灯片上过多的液体可以相鉴别困难，一旦干燥。 注：一张幻灯片可以包含多个天的每只动物，尽管这可以根据个人的喜好来完成。 </li><li>确保样品转移到幻灯片。采集样品SEVER在一个人的滑动连续两天，以便能够更好地跟踪和识别每日发情期的变化。 注：如果可能的话，它也是理想的，如果人们采集样品保持相同的所有动物。 </li><li>一旦幻灯片是干的，开始染色协议相鉴别。使用染色试剂盒（见表材料），浸泡载玻片10倍，在固定液（浅蓝色），在染色溶液的10倍（粉红色）和5倍于染液溶液（紫色）。冲洗轻轻滑动，在流水下。让幻灯片完全风干显微镜下观察之前。 注：一般4-5天的长发情周期包括（按顺序）：发情期（E），发情后期（M），动情（D），和发情前期（P）26。发情的特点是蓝色，角化细胞，动情后期由白细胞和角化和有核细胞的组合，间情期是相似于细胞类型的存在，以动情后期除了它们是非常稀疏，动情前期由紫染色的聚集体的有核细胞（ 图1）。有各种染色方法，除了上述的那些，这是同样有效的用于阴道涂片细胞学检查。 </li><li>为了监测准确前实验阶段收集的样品在至少两个完整的周期（〜8-10天）。是同一天的样品所用的时间是一致的。每日贯穿并直到实验结束收集阴道涂片样品。 </li></ol> 3.预曝光<ol><li>在实验前的启动，将动物预暴露于调节腔室允许动物适应环境之前的任何行为测试的上下文中（调理室）。 <ol><li>预暴露的女性的调理室（位于声音衰减框）与房子灯每天20-30分钟3天之前的任何实验。它们是预先暴露于作为其腔室为研究分配的框。彻底清洁T字会话和动物之间他盒（墙壁和托盘），以去除异味，因为他们可以影响人的行为和发情周期。 </li></ol></li></ol> 4.第1天：习惯化/恐惧条件<ol><li>一旦动物已被证明是在其发情周期的发情期，开始为期3天的实验的习惯化/调理相。 <ol><li>取预音在实验中的每一天冷冻前到第一音调条件刺激（CS）的演示的基线测量。通过收购冻结百分之几十的CS发病前第一CS-单独开庭审理时的刺激，缺席的时间间隔内（由试验的持续时间，再乘以100花不动秒来计算的）做到这一点。 注意：可以用这些措施来评估，并与习惯，空调，灭绝和召回过程中比较基准冻结水平。 </li><li>要在发情后期阶段进行消退训练，调理老鼠发情期的最后一天。拭子并确定动情期（步骤2）的每一天开始的实验方案（调节/消光/调用）之前。由于发情期可能会持续更长的时间超过一天，使用周期的历史来预测阶段的长度来确定当动物准备开始训练。 </li></ol></li><li>在调理室中，对足部电击格栅地板连接到电刺激发生器。使用0.5-0.6 mA的刺激电平作为条件刺激（US）。使用4 kHz的80 dB音调作为条件刺激（CS）。 注意：这些参数可能跨越实验室变化，并且可以被修改为最佳调节。 <ol><li>如果冷冻分析程序需要视觉线索（相对于时间），共激活的LED光与色调。定位在光上的操作性室的外部，所述视频帧内的。在每次试验的开始，启动LED灯，并保持照明，直到日结束Ë审判。 注意：此光不被动物可见和仅作为一个标记为起始和所记录的视频文件内的试验结束（将用于测量冷冻行为）。 </li></ol></li><li>把动物进入空调室的习惯5 CS单机试验。紧随其后的习惯，应执行恐惧条件的7成对CS-美国选拔赛。确保CS音调持续30秒和coterminates与0.5秒美国休克。运行试验中的所有会话（习惯，调节，消光，并召回）具有可变intertrial间隔平均3分钟。 </li><li>在调理会议结束，返回动物到其家笼中，并给动物设施，直到第二天。 </li></ol> 5.第2天：准备雌二醇<ol><li>拭子并确定发情阶段所有接受该协议的第1天动物。制备雌二醇（也称为E2，17β-雌二醇，或β-雌二醇）用于15微克/千克的剂量皮下注射使用芝麻油或0.9％盐水作为溶媒。 </li><li>为了计算雌二醇的量为注射准备，使用所有的动物，这是测量和记录，当他们被空调有关此协议的第1天的体重。 </li><li>进行计算，以规范每鼠雌二醇（ 即 0.2ml）中的管理音量。因为雌二醇难以溶解，由于其不溶于水，并且需要大量的溶剂为小剂量时，通过混合雌二醇在芝麻油过热直至溶解制成原液。该溶液然后可以通过0.22微米过滤器以除去杂质。例如，为了实现15微克/千克的剂量在每只动物0.2注射体积添加的3000微克雌二醇/1毫升芝麻油原液芝麻油总所需体积计算的容积。 注：另外，用其他形式的雌激素的塔<html>吨是水溶性的和更容易溶解。 </li></ol> 6.第2天：雌二醇管理/消退训练<ol><li>管理雌二醇皮下。抬起肩胛骨之间的皮肤松弛的脖子形成平缓的捏和将注射器针头插入一个由皮肤折叠形成的三角形。 </li><li>下面雌二醇皮下注射30分钟后，再次将动物的调理室，并开始消退训练。在这种灭绝会议，进行20 CS单机试验，每个试验包括一个30秒，4千赫，80分贝色调，以可变intertrial间隔平均为3分钟介绍。 注：雌二醇对召回的影响时，雌二醇之​​前灭绝培训或灭绝后立即给予发生。效果没有观察4小时后的消光，因此建议注入在时间上接近的训练会话15的开头或结尾。 </li></ol>。乐“&gt; 7天3：灭绝召回<ol><li>灭绝训练后24小时，棉签上的天完成1和2本协议的动物。 </li><li>将动物进入分配给他们的空调室（同日1和2）。通过提供3 CS单机试验（灭绝召回三次试验），其中只包含3.80分贝与intertrial间隔类似给药灭绝培训20次试验CS单机试验按步骤6.2中所述开始灭绝召回会话。 </li><li>在召回会话完成后，返回的动物，以他​​们家的笼子。 </li></ol> 8.数据分析<ol><li>通过视频采集行为数据，并使用计算机软件对其进行分析。 注：冻结的分数也可以通过实验者是盲目的药物治疗的CS表示每次试验过程中定时冻结发作手工计算，表现为时间的百分比花的基调期间冻结。结冰由完全缺乏运动与在30秒CS-美国选拔赛异常呼吸定义。由花30秒（试验持续时间）乘以100冻结（动）秒除以计算百分比冻结。 </li></ol>

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B., 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=Strategies+and+methods+for+research+on+sex+differences+in+brain+and+behavior.">Strategies and methods for research on sex differences in brain and behavior.</a> Endocrinology. 146, 1650-1673 (2005).</li><li>Westwood, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+female+rat+reproductive+cycle:+a+practical+histological+guide+to+staging.">The female rat reproductive cycle: a practical histological guide to staging.</a> Toxicologic Pathology. 36 (3), 375-384 (2008).</li><li>Hurn, P. D., Macrae, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+as+a+Neuroprotectant+in+Stroke.">Estrogen as a Neuroprotectant in Stroke.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 20, 631-652 (2000).</li><li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> Journal of Pharmacology and Pharmacotherapeutics. 1 (2), 87-93 (2010).</li><li>Gillies, G. E., McArthur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+actions+in+the+brain+and+the+basis+for+differential+action+in+men+and+women:+a+case+for+sex-specific+medicines.">Estrogen actions in the brain and the basis for differential action in men and women: a case for sex-specific medicines.</a> Pharmacological Reviews. 62 (2), 155-198 (2010).</li><li>Tolin, D. F., Foa, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+trauma+and+posttraumatic+stress+disorder:+a+quantitative+review+of+25+years+of+research.">Sex differences in trauma and posttraumatic stress disorder: a quantitative review of 25 years of research.</a> Psychological Bulletin. 132, 959-992 (2006).</li></ol>

这个手稿的作者有没有竞争经济利益或其他利益冲突的披露。

恐惧灭绝已被广泛研究在雄性大鼠调处下各种操作识别和评估条件性恐惧灭绝的神经机制。相对而言，很少有研究探讨雌性大鼠或性腺激素的恐惧消退的作用。具体研究雌激素对恐惧消退的影响，自然循环雌性大鼠受到了为期三天的行为范式。这个过程包括习惯/调理，灭绝，灭绝和召回阶段。使用此协议，它已经证明，雌性大鼠低雌激素发情后期阶段表现出较高的冻结（或受损灭绝召回）相比，...

固有的性别差异在人类和啮齿类动物在不同的认知行为和学习的范式观察。例如，据报道，妇女一般有较强的口头和注重细节的能力，而男性有更好的空间能力1-3。这些性别差异可能是部分原因是由于性腺激素的影响。高雌激素水平提高，妇女更好的工作性能，但恶化的任务，男人通常做的很好4-6性能。虽然这方面的证据是引人注目的，有限的能力，以在人类研究中完全控制实验环境使得它以确定是否对行为这些效果可以特异性归因于激素难。动物研究，与此相反，允许完全控制的情况。 虽然恐惧消退的基本神经机制已经确定，并很好的研究，男性，目前还不清楚是否这些系统是在女性或他们如何在整个发情周期变化一样。恐惧空调和灭绝被广泛应用于行为范式在啮齿动物和人类进行相关的焦虑症的研究。鉴于妇女对焦虑症的风险较高，以及较高的症状的持续时间和严重程度7-13，这是至关重要的，包括女性在这些研究中。女性在这项研究中代表性不足可能是由于发情期的监测和会计的性激素的行为影响的挑战。实验室是做检查的女性中，往往没有报告或方法的研究，没有说明这方面遇到的问题。 新出现的证据在啮齿类动物表明，在恐惧中灭绝的性别差异是由性腺激素14-18调制。在恐惧条件，动物训练害怕一个特定的刺激。一些无筋介绍后的刺激下，动物学会不害怕线索，一个叫灭绝的过程。如何做好动物学习和巩固学习这个任务可以给灭绝训练一段时间后的消光回忆测试过程中应遵循的记忆。期间灭绝召回少恐惧的表情显示出良好的消记忆的巩固。从我们实验室最近的研究结果表明，雌激素能调节恐惧消退记忆的巩固和提高灭绝召回。具体来说，雌性大鼠被扑灭在发情前期，发情周期的阶段期间，循环雌激素水平的高峰，显示出增强记忆灭绝保留。与此相反，雌被熄灭在低雌动情后期阶段显示相对差的消光召回，它可以与外源性雌二醇给药之前或消退训练15,16后，立即得到改善。灭绝记忆巩固的神经机制（包括UDING性腺激素在女性中的作用）不清晰。 在动物实验中，激素的作用，可通过微创手术切除的程序，如阉割去势，并进行调查。随后，从手术后恢复，性腺激素往往外生一种行为的任务19的性能在操纵。这种方法提供了关于性激素关键信息和，因为它允许对性腺激素的良好控制的操作（在定时和剂量计）20-23是有用的。这种方法，然而，没有评估在整个发情周期发生也不它们代表“正常”的动物，从而限制了平移潜在对人的研究的天然存在的波动的影响。它已被充分证明了雌性激素水平峰值和下降的发情周期的特定阶段，和在循环内的雌激素受体的表达的变化，和卵巢切除后24。因此，有必要进行研究，女性用完整性腺和缩小的实验设计，以可靠地研究了高和低雌激素状态可能对女性在其整个生命周期的影响。 该协议的重点是雌激素对参与恐惧消退的神经生物学系统的作用。它描述了如何：1）认真监测发情周期，2）准备有效剂量雌激素的全身用药，和3）遵循的行为模式，包括恐惧空调，灭绝，并记得在自然循环雌性大鼠。此协议可与其它药理学和操纵细胞和分子的工具进行修改，以帮助研究，以更好地理解，在条件性恐惧观察消光特性的性别差异。注意，以下描述的过程是那些我们实验室内使用，且存在许多的这些过程的变化i的n中的文献。

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			<td height="22" style="height:22px;width:120px;">Name</td>
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			<td style="width:199px;">Comments</td>
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			<td height="43" style="height:43px;width:120px;">Fear conditioning chamber</td>
			<td style="width:159px;">Coulbourn Instruments</td>
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			<td style="width:199px;"></td>
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			<td height="22" style="height:22px;width:120px;">Graphic State</td>
			<td style="width:159px;">Coulbourn Instruments</td>
			<td style="width:135px;"></td>
			<td style="width:199px;"></td>
		</tr>
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			<td height="43" style="height:43px;width:120px;">Sound-attenuating box</td>
			<td style="width:159px;">Med Associates, Inc.</td>
			<td style="width:135px;">NIR-022MD</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">Estradiol</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:135px;">E1024</td>
			<td style="width:199px;">In sesame oil for subcutaneous injection</td>
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			<td height="22" style="height:22px;width:120px;">Sesame oil</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:135px;">S3547-250ML</td>
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			<td height="22" style="height:22px;width:120px;">Freezescan</td>
			<td style="width:159px;">Cleversys, Inc.</td>
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			<td height="43" style="height:43px;width:120px;">Dip quick stain</td>
			<td style="width:159px;">Jorgensen Laboratories, Inc.</td>
			<td style="width:135px;">J0322A1, J0322A2, J0322A3</td>
			<td style="width:199px;"></td>
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			<td height="43" style="height:43px;width:120px;">Cotton-tipped applicators</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:135px;">23-400-114</td>
			<td style="width:199px;">6-inch, sterile</td>
		</tr>
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			<td height="22" style="height:22px;width:120px;">0.9% saline</td>
			<td style="width:159px;">LabChem, Inc.</td>
			<td style="width:135px;">LC23460-2</td>
			<td style="width:199px;">Sodium chloride w/v</td>
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			<td height="43" style="height:43px;width:120px;">Selectfrost microscope slides</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:135px;">12-550-003</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:120px;">Virex II 256</td>
			<td style="width:159px;">Diversey, Inc.</td>
			<td style="width:135px;">5019317</td>
			<td style="width:199px;">Dilute 1:256 with water</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:120px;">Luer-Lok Tip 1ml Syringes</td>
			<td style="width:159px;">Becton Dickinson</td>
			<td style="width:135px;">309628</td>
			<td style="width:199px;"></td>
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		<tr height="64">
			<td height="64" style="height:64px;width:120px;">Hypodermic disposable needles</td>
			<td style="width:159px;">Exelint International, Co.</td>
			<td style="width:135px;">26402</td>
			<td style="width:199px;">26-gauge</td>
		</tr>
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protocol for studying extinction of conditioned fear in naturally cycling female rats

的条件性恐惧灭绝已被广泛研究在雄性啮齿动物。最近，已经有越来越多的研究表明，对某些行为的任务和应对行为的神经机制是在女性和男性不同。在调查研究使用雌性可以表示由于性腺激素在其动情周期的变化的一个挑战。本协议描述既定程序，在调查中雌性大鼠恐惧消退记忆的巩固雌激素的作用是有用的。发情周期和外源性雌激素的管理之前灭绝培训阶段可以在24小时后影响灭绝召回。这里描述的发情相鉴别阴道擦拭技术辅助检查和处理的自然循环性腺激素。使用这个基本啮齿动物模型可进一步划定由雌激素能调节中灭绝的恐惧记忆的机制女性。

在这种恐惧灭绝召回的协议，冻结百分比是衡量恐惧的指标。动物熄灭很好，保留了灭绝培训的内存表现出较低的恐惧在行为测试期间灭绝召回的最后一天。雄性和雌性大鼠不显著在条件性恐惧表达的调节，消光期间不同，并且召回相（ 图2）。然而，性别差异变得明显，当动物如高和低雌激素发情期组分别进行了分析。 ，在发情周期的低雌激素发情后期阶段进行消退训练雌鼠不记?...

Watch this Scientific Journal Video about Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats at JoVE.com

Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

协议在骑自行车自然雌性大鼠学习条件性恐惧灭绝

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that ...

protocol-for-studying-extinction-conditioned-fear-naturally-cycling

Research

JoVE Journal

Behavior

13.1K 次观看.  Massachusetts General Hospital. Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

视频: Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

立体定向显微注射病毒载体表达Cre重组酶研究的靶基因的作用在可卡因条件性位置偏爱

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

科研

行为学

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

一个过程来研究延长食品的限制对海洛因寻求戒断大鼠的影响

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

背景和线索恐惧制约试验小鼠中使用视频分析系统

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.

In the following experiment we describe a protocol for trace fear conditioning in mice. This type of associative memory includes a trace period that separates the neutral stimulus and the unconditioned stimulus.

跟踪恐惧条件小鼠

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

一个过程来观察大鼠巴甫洛夫空调酒精寻求行为的背景下引发的重建

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

A Treatment Package without Escape Extinction to Address Food Selectivity

Feeding difficulties and feeding disorders are a commonly occurring problem for young children, particularly children with developmental delays including autism. Behavior analytic interventions for the treatment of feeding difficulties oftentimes include escape extinction as a primary component of treatment. The use of escape extinction, while effective, may be problematic as it is also associated with the emergence of challenging behavior (e.g., extinction burst). Such challenging behavior may be an acceptable side effect in treatment cases where feeding problems are severe and chronic (e.g., failure to thrive). However, in more acute cases (e.g., selective eating), the negative side effect may be unwarranted and undesired. More recent research on the behavioral treatment of food selectivity has begun to evaluate treatments for feeding difficulties that do not include escape extinction (e.g., demand fading, behavioral momentum), with some success. However, research to date reveals individual differences in responsiveness to such treatments and no clear preferable treatment has emerged. This manuscript describes a multi-component treatment package that includes shaping, sequential presentation and simultaneous presentation, for the treatment of food selectivity in four young children with developmental delays. This treatment package extends the literature on the behavioral treatment for food selectivity and offers a multi-component treatment protocol that may be clinically applicable across a range of treatment scenarios and settings.

Feeding difficulties are a common problem for children with developmental disorders, including autism, and behavioral interventions often include escape extinction. Recent research has begun to evaluate treatments that do not include escape extinction. This manuscript describes a multicomponent treatment package that does not use escape extinction to treat feeding difficulties.

一个疗程装没有逃脱灭绝解决粮食选择性

Feeding difficulties and feeding disorders are a commonly occurring problem for young children, particularly children with developmental delays including ...

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 ...

Pavlovian Conditioned Approach Training in Rats

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the attribution of incentive salience, or the transformation of neutral stimuli into attractive, &#34;wanted&#39; stimuli capable of motivating behavior. Pavlovian conditioned approach (PCA) develops after the response-independent presentation of a conditioned stimulus (CS; e.g., a lever) that predicts the delivery of an unconditioned stimulus (US; e.g., a food pellet) and can be used to measure incentive salience. During training, three patterns of conditioned responses (CRs) can develop: sign-tracking behavior (CS-directed CR), goal-tracking behavior (US-directed CR), and an intermediate response (both CRs). Sign-trackers attribute incentive salience to reward-related cues and are more vulnerable to cue-induced reinstatement of drug-seeking as well as other addiction-related behaviors, making PCA a potentially valuable procedure for studying addiction vulnerability. Here, we describe materials and methods used to elicit PCA behavior from rats as well as analyze and interpret PCA behavior in individual experiments.

Here, we present a protocol to elicit Pavlovian conditioned approach behavior in rats. This procedure can be used to measure individual differences in the tendency to approach and attribute incentive salience to reward-related cues and investigate addiction vulnerability.

大鼠条件性巴甫洛夫训练方法

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the ...

Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders such as post-traumatic stress disorder (PTSD). To understand the biological mechanisms of fear dysregulation in PTSD, it is important to start with a valid animal model of the disorder. This protocol describes the methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of PTSD, in both rats and mice. SEFL was developed to recapitulate critical aspects of PTSD, including long-term sensitization of fear learning caused by an acute stressor. SEFL uses aspects of Pavlovian fear conditioning but produces a distinct and robust sensitized fear response far greater than normal conditional fear responses. The trauma procedure involves placing a rodent in a conditioning chamber and administering 15 unsignaled shocks randomly distributed over 90 minutes (for rat experiments; for mouse experiments, 10 unsignaled shocks randomly distributed over 60 minutes are used). On day 2, rodents are placed in a novel conditioning context where they receive a single shock; then, on day 3 they are placed back in the same context as on day 2 and tested for changes in freezing levels. Rodents that previously received the trauma display enhanced levels of freezing on the test day compared to those that received no shocks on the first day. Thus, with this model, a single highly stressful experience (the trauma) produces extreme fear of the stimuli associated with the traumatic event.

Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents.

压力增强恐惧学习--创伤后应激障碍的健壮啮齿动物模型

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders ...

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

一种用于大鼠渴望培养的条件场所偏好协议

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...