This work is supported in part by American Heart Association Scientist Development Grant and NARSAD Young Investigator Award to Yingwei Mao.

注：所有涉及动物的方案如下按照美国宾夕法尼亚州立大学的动物护理准则，IACUC＃44057 1.慢病毒生产注意：转染前一天，LentiX-293细胞应在80％汇合。 <ol><li>冲洗细胞用DMEM只是染前和孵育在10毫升的DMEM / 10％FBS中的与青霉素（100IU / ml）和链霉素（100微克/ ml）处理5分钟，在RT。 </li><li>在1ml DMEM中，并涡旋混合1分钟稀释的DNA和聚乙烯亚胺（PEI）（1毫克/毫升）中1：3的比例（3微升的PEI 1微克的DNA）。 DNA的量加入是依赖于DNA的浓度。在室温下孵育10分钟。 <ol><li>例如，使用20微克质粒DNA，以每皿60微升的PEI由10微克载体质粒，5微克psPax2 14质粒，5微克水泡性口炎病毒（VSV）的。 </li></ol></li><lI&gt;通过涡旋1分钟再次混合该DNA和PEI溶液。在室温下孵育10分钟。 </li><li>添加的DMEM（ 即 1毫升用于在100mm培养皿10毫升培养基）的总培养基的1 /第10卷。加入1毫升的DNA-PEI混合物一滴一滴放入盘中和周围摇动培养皿直到向培养基充分混合用DNA。 </li><li>孵育6小时，在室温，除去上清液。加入5毫升培养介质中。 </li><li>转染后48小时，在4℃在50ml管中收集病毒上清液和自旋在627×g离心5分钟。过滤30毫升病毒上清液通过0.45微米的注射器过滤器到超速离心管中。 </li><li>加入无菌水来平衡管，并覆盖管与小片封口膜。旋管在11249×g离心120分钟，在4℃。使用真空尖除去液体。 </li><li>加入100微升冷PBS管。轻轻摇动管在4°CO / N。 </li><li>重新悬浮的病毒，吸管吨他补充PBS在步骤1.8在颗粒的10倍，小心不要碰到沉淀的尖端。粒料不会被重悬浮，直到这是完整的。 </li><li>分装病毒以每管，闪光冻结10-20微升在液氮中，并储存在-80℃。 </li></ol> 2.立体定向注射 2.1）制备仪器<ol><li>放置在手术前的一对剪刀，钝端钳，持针器，解剖刀，棉签，并且在密封的包装用灭菌指示器布，并且高压釜中。此外，获得10％的聚维酮碘，70％的乙醇，可吸收缝线，加热垫，人工泪液，玻璃珠灭菌，钻头，钻头，2一次性注射器，注射针筒，电动剃须刀，镇痛药（酮洛芬），麻醉剂（阿佛丁），手套，并用注射泵立体定向装置。 </li><li>使1.25％阿佛丁解决方案新鲜的那一天，在过滤器无菌罩采用0.2＃181;米无菌注射器式滤器，并放入无菌血清小瓶。混合将2.5g 2,2,2-三溴醇到5毫升叔戊醇，然后溶解在200毫升水中。保持低于5.15的pH值</li></ol> 2.2）制备的鼠<ol><li>基于鼠标（375毫克/千克）的重量，施用阿佛丁15注入鼠标与阿佛丁经由腹膜内（IP）注射，然后将返回到它的笼子，直到完全麻醉。 </li><li>给通过IP注射（ketaprofen，5毫克/公斤），并放置在鼠标以防止干燥的眼中人工泪液的镇痛。 </li><li>确保鼠标完全睡着了捏他们的脚。如果鼠标响应捏脚，然后给予更多的麻醉剂（剂量50微升）。使用电动剃须刀刮胡子鼠标的头部。剃要被操作（通常是从刚耳朵后面，向口鼻部的顶部）的区域，和周边地区。 </li><li>将鼠标我NTO的立体定位仪。锁存门牙上的前夹，并降低夹具并拧紧，使下巴是完全安全的。 </li><li>插入耳棒进入耳道完全稳定的头部。要小心，不要插入耳酒吧太远，这可能损害内耳。一旦完成之后，将小鼠的体应当能够在不破坏头部的位置略微移动。 </li><li>放置在鼠标下的加热垫，以在整个手术过程调节小鼠的体温。 </li><li>彻底清洁手术区。用棉签，擦10％聚维酮碘的圆周运动，从手术部位的中间开始和向外移动。然后，用棉签擦70％的乙醇在以同样的方式的外科手术部位。重复两次。 </li></ol> 2.3）第一切口<ol><li>一旦鼠标坦然，打开无菌制袋设备。豆豉前更换手套纳克分析仪器。取出无菌布将仪器在布上。如果有任何乐器倒是一个未消毒的表面，用玻璃珠灭菌15秒消毒它。 </li><li>取手术刀在优势手和钝端镊子在非优势手。使用平末端镊子轻轻握鼠标的皮肤，并用手术刀做一个切口。开始切口约1.5厘米以上（朝向鼻子）的耳朵和延伸到耳朵下方约0.5公分。垂直延伸的切口按住鼠标的头部的中间，以暴露前囟（ 图1）。 </li><li>一旦前囟门可视化，采取无菌棉签轻轻取出任何血液覆盖在颅骨表面。使用两个棉尖端推皮肤向头部的侧面。 </li></ol> 2.4）设备安装<ol><li>之后的任何血液从头骨的表面去除，前囟门是清晰可见，准备注射器（CON中心定位的10 8 TU /毫升，量1微升）。在通风柜，填充与病毒的注射器被注入。确保没有泡沫在里面。将注射器中的立体定位仪，确保其完全固定。 </li><li>缓慢降低注射器，直到它是正确的颅骨的表面上方，使得无菌注射器针头的尖端被设置为前囟的交点与所述耳间线。设置此点作为零，并确定从该点的坐标。 </li><li>根据感兴趣的大脑区域，改变立体定向坐标。通过利用脑图谱4确定这些坐标。一旦坐标被确定，移动的注射器的针头，以匹配坐标。使用目标的坐标齿状回：M / L = +/- 1毫米，A / P = -1.82毫米。降低针向右颅骨上方形象化，其中该孔需要被钻出。 </li><li>提高注射器咯，拿钻，然后将双钻吨右目标钻探点以约45°角到所述颅骨上方。开始钻探，并保持，直到钻颅骨让位。当颅骨让位，在电阻的下降可以被检测到。要小心，不要钻到大脑，以免损坏皮质。 </li><li>以无菌棉签，擦去任何血液从孔。 </li><li>降低注射器，使尖端位于右对大脑（未头骨）的表面上。设置D / V坐标（深度）为0降低至所需深度的注射器，基于脑图谱坐标。为了达到齿状回，设置D / V至-1.79毫米。 </li><li>开始注入以0.2微升/分的速率。看，以确保注射器不超过所需的深度滑低。如果发生这种情况，再次轻轻抬起注射器至所需深度。 </li><li>注射后结束，等待约2分钟，以确保任何残留的病毒已被吸收。慢慢地将注射器用棉签去除任何丽从注射部位镑。 </li><li>重复另一半球。 </li></ol> 2.5）缝合<ol><li>从包装和抓地力取出消毒缝合针，并用持针针。面对针的尖锐边缘远离针夹持器。持在非优势手的钝端钳，并用它来夹住皮肤上的鼠标。先从优势手。 </li><li>轻轻通过皮肤推缝合并使用平末端镊子抓住皮肤上的切口的另一侧。通过直到大约0.75英寸仍然是拉动缝合材料。 </li><li>放掉针，并使用平末端钳子环绕针头夹持器一次缝线，然后，抓住与针保持器的0.75英寸剩余缝合，并通过拉缝线，使针和缝合针夹持器最终在头对面的来了，他们是在原来的一面。这应该形成一个结。 </li><li>重复此步骤三次以上，交替的头部侧的针保持器结束了每次上。 </li><li>切缝合线，并重复步骤2.5.1-2.5.3直到闭合切口。 </li></ol> 2.6）术后护理<ol><li>轻轻地取出来自小鼠的耳棒，以及由前夹删除其下颚。 </li><li>按住鼠标上的加热垫，直到它唤醒并四处移动它自己。监控鼠标每天，并保持和眼睛出疼痛的迹象，如不梳洗，进食或饮水。给予额外的镇痛认为必要时。 </li></ol> 3.打开现场测试 3.1）设置<ol><li>获得空方，塑料舞台尺寸为五十厘米乘50厘米，50厘米高的围墙（但可以稍大）。清洗之前用该试验开始时的70％的乙醇的竞技场。确保乙醇已放置鼠标进入竞技场之前完全干燥。 </li><li>广场上的舞台地板，并尽量保证照明设置，以尽量减少阴影和眩光。 </li><li>如果使用跟踪软件，开放领域的直接开销定位摄像头，约1.5 ft在开放的领域（远到足以完全涵盖整个球馆，但足够接近，来使鼠标的一个明确的说法）。 </li><li>设置区域中的框（30厘米×30cm的大致的区域）的中心16要设置一个区域，点击在侧面板区域1选项卡。 <ol><li>选择一个在上部面板形状的轮廓，和轮廓的中心区的周边交界。选择添加区域，然后单击中心区域内。使用该程序的提示，命名的区域（这将被称为“中心”在此过程中）。如果行为测试将进球了手，确保字段的底部被划分成10厘米×10平方厘米​​，使用磁带标记。 </li></ol></li><li>设置软件，使得设置是否正确，并且鼠标很容易VISI竹叶提取在屏幕上。通过调整机上的检测设置为与测试竞技场（照明）和鼠标的颜色相容做到这一点。 注：如果设置正确，系统只应跟踪鼠标，并没有任何阴影或尿液/粪便。具体的软件设置取决于舞台的灯光，和鼠标的颜色。 </li></ol> 3.2）的适应过程和测试<ol><li>移动实验小鼠进入房间的行为1小时测试前，以适应他们自己的环境。离开老鼠的笼子里的驯化。 </li><li>打开相机到磁带的行为的任务，然后将鼠标移向舞台的前排中使鼠标面壁。 </li><li>设置定时器为5分钟，步距舞台。如果可能的话，离开房间，以便不会意外影响鼠标。 </li><li>一旦5分钟都在涨，移开鼠标，并放置在自己的笼子里。 </li><li>使用70％的乙醇在进行下一鼠标之前彻底清理掉字段。确保乙醇已完全干燥。 </li></ol> 3.3）得分<ol><li>考虑鼠标作为该领域的中心被当所有四只爪子是中间30厘米×30cm的内部。 </li><li>量化时间的小鼠花费在中心的量。另外，确定使用跟踪软件（如Noldus动物行为轨迹分析），这个时候。 <ol><li>要做到这一点，去分析选项卡，然后在分析配置文件点击“动作”。删除当前设置的类别，然后在“中区”的位置选项卡下进行选择。选择中心区。下一步，选择“分析输出”（结果选项卡下）来查看结果。一个表格，列出所花的时间不动每次试验应该会出现。 </li></ol></li><li>否则，由专人得分OFT。开放的领域应该有郎德上OPE底部25 10厘米×10平方厘米n场（如步骤3.1.3概述）。量化时间的小鼠花费在中央，除了条目的数量进入竞技场的中心的数量。16 注：中心30厘米×30 CM区被认为是开放场的中心。鼠标被视为居住中心区域，如果所有四只爪子是30×30cm的区域内。 </li></ol> 4.强迫游泳实验注：允许最小的行为任务之间五天。 4.1）设置<ol><li>获得2升斗清晰，并填写22℃的水。放置在一个表中的水桶，并尽量保证照明设置，以尽量减少阴影和眩光。 </li><li>如果使用跟踪软件，直接过顶桶位置的摄像头。 </li><li>设置软件，使鼠标在屏幕上清晰可见。 </li></ol> 4.2）的适应过程和测试<ol><li>移动实验小鼠进入RO行为在测试之前OM 1小时，适应周围的环境。离开老鼠的笼子里的驯化。 </li><li>打开相机到磁带的行为的任务上。轻轻地，将鼠标在水的桶的中间。确保不要砸鼠标慢慢地放下鼠标，所以它的前脚先着水。请注意，以防止他们的头部淹没。 </li><li>设置定时器6分钟，并从行为区步距。 </li><li>一旦6分钟都在涨，移开鼠标从桶中，并放置在鼠标背部的笼子前，擦去多余的水分。 </li></ol> 4.3）得分<ol><li>如果使用跟踪软件，设置不动百分比为11％，由Noldus的建议（这应该是默认设置），以量化的时间与浮动游泳。 </li><li>为了量化使用的跟踪系统，转到分析选项卡，并根据分析型材点击运动。删除当前设置的类别，然后选择移动状态下，左侧面板（下个体行为）。设置不动到11％。 </li><li>选择可视化的轨道，结果选项卡下。选择分析输出（结果选项卡下）来查看结果。一个表格，列出所花的时间不动每次试验应该会出现。 </li><li>如果手得分，衡量鼠标花费游泳或登山，和大量的时间鼠标不动花费时间。此外，测量延迟到第一时间不动。参见图2中的结果部分。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetizing+mice.">Anesthetizing mice.</a> Methods Mol Biol. 18, 135-136 (1993).</li><li>Alachkar, A., Jiang, D., Harrison, M., Zhou, Y., Chen, G., Mao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+EJC+factor+RBM8a+regulates+anxiety+behaviors.">An EJC factor RBM8a regulates anxiety behaviors.</a> Current Molecular Medicine. 13 (6), 887-899 (2013).</li><li>Bernal, J., Baldwin, M., Gleason, T., Kuhlman, S., Moore, G., Talcott, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+rodent+survival+surgery.">Guidelines for rodent survival surgery.</a> Journal of Investigative Surgery. 22, 445-451 (2009).</li><li>Wahlsten, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+ready+for+testing.+Mouse+Behavioral+Testing:+How+to+use+mice+in+behavioral+neuroscience.">Getting ready for testing. Mouse Behavioral Testing: How to use mice in behavioral neuroscience.</a> Academic Press. , 133-141 (2011).</li><li>Kheirbek, M. A., 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=Differential+control+of+learning+and+anxiety+along+the+dorso-ventral+axis+of+the+dentate+gyrus.">Differential control of learning and anxiety along the dorso-ventral axis of the dentate gyrus.</a> Neuron. 6 (77), 955-968 (2013).</li><li>Snyder, J. S., Soumier, A., Brewer, M., Pickel, J., Cameron, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+hippocampal+neurogenesis+buffers+stress+responses+and+depressive+behaviors.">Adult hippocampal neurogenesis buffers stress responses and depressive behaviors.</a> Nature. 476 (7361), 458-461 (2011).</li><li>Sik-Lee, J., 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=Induction+of+neuronal+vascular+endothelial+growth+factor+expression+by+cAMP+in+the+dentate+gyrus+of+the+hippocampus+is+required+for+antidepressant-like+behaviors.">Induction of neuronal vascular endothelial growth factor expression by cAMP in the dentate gyrus of the hippocampus is required for antidepressant-like behaviors.</a> Journal of Neuroscience. 29 (26), 8493-8505 (2009).</li><li>Miller, D. G., Adam, M. A., Miller, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+by+retrovirus+vectors+occurs+only+in+cells+that+are+actively+replicating+at+the+time+of+infection.">Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection.</a> Molecular and Cellular Biology. 10 (8), 4239-4242 (1990).</li><li>Rubinson, D. A., 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=A+lentivirus-based+system+to+functionally+silence+genes+in+primary+mammalian+cells,+stem+cells+and+transgenic+mice+by+RNA+interference.">A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.</a> Nature Genetics. 33, 401-406 (2003).</li></ol>

The authors have nothing to disclose.

成功立体定向注射依靠三个因素：保持鼠标活着，设置正确的零点为坐标（针的针尖上囟的交点和耳间线），和右深度设置为零点针（尖端只要触摸的脑组织的外部）。小鼠的存活率是重要的。手术存活可以通过确保鼠标正确麻醉并接收足够的止痛来辅助。疼痛是已知的恢复差的主要原因手术后。确保鼠标充分整个手术麻醉（它应该不会响应脚箍缩），并给予正确的剂量止痛剂（基于鼠标的重量?...

鼠标已经被广泛地应用于神经生物学，因为它很容易操纵基因。基因敲除技术允许研究人员调查了每个遗传因素形状的鼠标行为。此外，CRE-loxP系统提供了用于组织-一个有价值的工具和细胞类型特异性基因敲除小鼠中，这使研究人员研究基因功能在不同的组织。1然而，在实践中，CRE启动子的表达模式是难以控制，到目前为止，许多老牌CRE司机都没有实现区域特异性表达。2,3 可替代地，立体定向注射是靶向成年小鼠的特定脑区的方法。通过注射遗传工程病毒表达的cDNA或shRNA，基因表达的区域特定调制可以实现。虽然各小鼠的脑大小而定，特定脑区域的位置可以通过立体定向coordi来确定纳茨从设置在地标在小鼠大脑的颅骨。最常用的地标是前囟，λ，和耳间线。使用从脑图谱获得的坐标，每个脑区的4的准确位置可以由前-后（A / P），内侧-外侧（M / L）进行标识，并且背腹（D / V）从轴前囟门/双耳线的交点。通常病毒注射到小鼠的大脑的标签带是红色或绿色荧光蛋白（RFP或GFP），以使注射可以通过荧光显微镜来确认。 小鼠的行为评估是对精神疾病的基础研究尤为必要。精神障碍的患者的症状通常涉及异常行为。一些这些人类行为是进化上保守的，并且可以直接模仿和在小鼠观察到。例如，抑郁症可以通过测量行为绝望来建模在小鼠。患有抑郁症的人经常恩觉得好像没什么他们将永远帮助，症状可最终导致自杀。在啮齿动物中，这可以通过使用强迫游泳测试（FST），其测量小鼠游泳与漂浮在水的池（视为放弃）的时间量被建模。这一模式是由抢救表型抗抑郁验证。这已收到抗抑郁药会花显著更少的时间比不动未处理对照小鼠7,8,9。另一个行为测试，在旷场测试（OFT）的目的是评估在小鼠中运动，并且另外可用于分析在小鼠中急表型。5,6-该试验是基于这样的前提，老鼠感到更安全当它们接近到墙壁以一种新颖的开放领域。野生型小鼠最终将探索新的环境，因为他们是好奇的动物。然而，花费更少的时间在该场的中心表示在小鼠的焦虑，如鼠标将无法克服初始升恐惧的一种新的环境所带来的。鼠标的焦虑，作为定量的时间在开放场的中心所花费的金额，可以比作临床焦虑在人类中，这是目前在许多精神疾病。 立体定向注射用行为范例的结合是一种新颖的方式来改变以靶向脑区域中的特定基因的表达。然后可确定调制基因表达对小鼠行为的作用。与此相反，以全脑敲除，这种方法，因为它仅针对特定的大脑区域是特别有用的。此外，立体定向注射通常在成年野生型小鼠进行的，因此，内源基因表达已被保持在整个发育阶段。该方法将避免变乱影响，如果在发育的胚胎或产后阶段是必需的生存的基因。一个主要的限制是，实验小鼠需要去通过量h的侵入性手术，其中小鼠的头骨必须打开。此外，基因调制程度是由病毒的滴度和效率决定的。病毒需要被注入到正确的区域使用立体定向坐标，这需要特殊的仪器。正确的注射部位的验证才能完成验尸。 这种方法已经被先前用于测试在各种神经疾病的特定基因的参与。例如，病毒介导的RNA干扰的钍基因（它允许多巴胺要合成）注射到黑质致密，和运动行为分析进行。10另一项研究中使用立体定向注射慢病毒沉默DISC1，以评估相对于鼠标行为精神分裂症。 DISC1击倒导致增加运动响应于新颖性（精神分裂症平行阳性症状），并在更大的动FST 11同样地，额外的研究发现，5-HT 1B的过度表达导致增加的探索行为在OFT，在使用这种方法的抗焦虑表型一致。12立体定向注射可以提供CRE病毒以诱导重组在CRE-loxP小鼠。这种方法被用来有选择地删除在杏仁核的Y2受体和终纹床核。在行为分析，这些小鼠被发现时，基因被在中央杏仁核删除的抗抑郁表型，但是不用当该基因在基底外侧杏仁核或终纹床核删除表型。13因此，这种技术为研究对动物行为的遗传效应的独特工具。

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applying stereotactic injection technique to study genetic effects on animal behaviors

立体定向注射是一种非常有用的技术，以提供高滴度慢病毒，以有针对性的大脑区域在小鼠。慢病毒可以过表达或在一相对集中的区域击倒的基因表达，但无脑组织显著损伤。恢复后，注入鼠标可以在各种行为的任务，如开放场测试（OFT）和强迫游泳实验（FST）进行测试。该OFT旨在通过测量时间，一个鼠标花费以一种新颖的开放场的中心的量，以评估运动和焦虑的表型的小鼠。更着急的鼠标将花费显著更少的时间在小说领域的中心与对照组相比。该FST通过量化的时间花在老鼠当动放入一桶水量评估了抗抑郁的表型。鼠标具有抗抑郁型将花费显著更少的时间不动相比，控制动物。此协议的目的是使用STEReotactic注射慢病毒与行为测试相结合，以评估因素是如何遗传调控动物的行为。

<html>准确的立体定位注射液在很大程度上依赖于设置正确的坐标。用于将病毒注射针的尖端应直接在囟的交点和耳间线（ 图1）被设置。是有帮助的使用立体显微镜来确定针是否被正确地放置。当通过显微镜看，针应该定位成使得如果病毒被注射，这将直接落在前囟的交点与所述耳间线。即，开口在注射器针应放置的交点的正上方。在这项研究中，表达抗RBM8a，在所述外显子连接复合?...

Watch this Scientific Journal Video about Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors at JoVE.com

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

立体定向注射慢病毒表达的cDNA或shRNA可在小鼠中的特定脑区调节基因表达。在这里，我们提出了一个协议，以立体定向注射结合行为的任务，如打开场测试（OFT）和强迫游泳试验（FST）。

应用立体定向注射技术在研究动物行为的遗传效应

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

applying-stereotactic-injection-technique-to-study-genetic-effects-on

Research

JoVE Journal

Behavior

14.0K 次观看.  The Pennsylvania State University. 立体定向注射慢病毒表达的cDNA或shRNA可在小鼠中的特定脑区调节基因表达。在这里，我们提出了一个协议，以立体定向注射结合行为的任务，如打开场测试（OFT）和强迫游泳试验（FST）。 

视频: Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

立体定向注射的microRNA表达慢病毒的小鼠海马CA1区和行为结果的评估

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

科研

行为学

A Within-subjects Experimental Protocol to Assess the Effects of Social Input on Infant EEG

Despite the importance of social interactions for infant brain development, little research has assessed functional neural activation while infants socially interact. Electroencephalography (EEG) power is an advantageous technique to assess infant functional neural activation. However, many studies record infant EEG only during one baseline condition. This protocol describes a paradigm that is designed to comprehensively assess infant EEG activity in both social and nonsocial contexts as well as tease apart how different types of social inputs differentially relate to infant EEG. The within-subjects paradigm includes four controlled conditions. In the nonsocial condition, infants view objects on computer screens. The joint attention condition involves an experimenter directing the infant's attention to pictures. The joint attention condition includes three types of social input: language, face-to-face interaction, and the presence of joint attention. Differences in infant EEG between the nonsocial and joint attention conditions could be due to any of these three types of input. Therefore, two additional conditions (one with language input while the experimenter is hidden behind a screen and one with face-to-face interaction) were included to assess the driving contextual factors in patterns of infant neural activation. Representative results demonstrate that infant EEG power varied by condition, both overall and differentially by brain region, supporting the functional nature of infant EEG power. This technique is advantageous in that it includes conditions that are clearly social or nonsocial and allows for examination of how specific types of social input relate to EEG power. This paradigm can be used to assess how individual differences in age, affect, socioeconomic status, and parent-infant interaction quality relate to the development of the social brain. Based on the demonstrated functional nature of infant EEG power, future studies should consider the role of EEG recording context and design conditions that are clearly social or nonsocial.

This novel protocol is designed to assess the neural bases of social interaction in infants. The paradigm is designed to tease apart how various social inputs such as language, joint attention, and face-to-face interaction relate to infant neural activation. Infant EEG power is recorded during both social and nonsocial conditions.

一个被试内实验协议，以评估对婴幼儿脑社会投入的影响

Despite the importance of social interactions for infant brain development, little research has assessed functional neural activation while infants ...

Testing Animal Anxiety in Rats: Effects of Open Arm Ledges and Closed Arm Wall Transparency in Elevated Plus Maze Test

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral science, conflicting outcomes are often provided from different laboratories. To identify reasons for the different outcomes, we previously focused on arm features, which differ between laboratories, most notably the presence/absence of ledges at the sides of open arms and the transparency/opaqueness of closed arm walls. In a previous report, we used a custom designed container to compare rat behavior on different combinations of open and closed arm designs under otherwise identical experimental conditions, and showed that differences in arm features interfere with experimental outcomes. In brief, open arm ledges significantly increased anxiety-like behavior in rats, while transparent arms may also have decreased this behavior. Furthermore, we verified a higher detection sensitivity of the effect of an anxiolytic drug in a combination of no-ledged open arm + opaque walled closed arm compared with a combination of ledged open arm + transparent walled closed arm. In this report, we introduce our protocol for the elevated plus maze test, together with discussion of the key results from the previous report and our experimental experience. We believe this report will provide useful information for researchers who have employed or who plan to use the elevated plus maze in their studies.

Rats' anxiety-like behavior tested by the elevated plus maze varies highly depending on the testing environment. Here we show how apparatus settings, especially arm structure, affect behavior on the maze, and we explain a reliable protocol to detect effects of anxiolytic drugs. This report will contribute to anxiety research.

大鼠动物焦虑症的实验研究: 开放臂壁和闭合臂墙透明度对高加迷宫试验的影响

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral ...

Assessment of Social Transmission of Food Preferences Behaviors

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy aging groups. For example, olfactory dysfunction in AD can present as impairment in olfactory recognition, emerging during early stages of the disease and worsening while the disease progresses. The social transmission of food preferences (STFP) task is based on a rudimentary form of communication between rodents concerning distant foods dependent on the transmission of olfactory cues. Healthy wild-type mice would prefer to eat a novel, flavored food that was previously cued by a conspecific, and this food preference would be hampered in transgenic AD mice, such as the APP/PS1 model. Indeed, a strong preference for the cued food in C57Bl6/J mice of 3 months of age was found, and this was reduced in 3 months old transgenic APP/PS1 mice. In summary, STFP task could be a powerful measure to be integrated in present subclinical detection assays of AD.

This paper presents a protocol for the investigation of social transmission of food preference in mice. The advantages and possible applications for this procedure, for instance, in detecting early changes in AD mouse models, are highlighted. To conclude, interpretation of the results in light of critical details are discussed.

食品偏好行为的社会传播评价

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy ...

Tickling, a Technique for Inducing Positive Affect When Handling Rats

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk for both the animals and the hands of their handlers, decreased animal welfare, and less valid research data. To minimize negative effects on experimental results and human-animal relationships, research animals are often habituated to being handled. However, the methods of habituation are highly variable and often of limited effectiveness. More potently, it is possible for humans to mimic aspects of the animals&#39; playful rough-and-tumble behavior during handling. When applied to laboratory rats in a systematic manner, this playful handling, referred to as tickling, consistently gives rise to positive behavioral responses. This article provides a detailed description of a standardized rat tickling technique. This method can contribute to future investigations into positive affective states in animals, make it easier to handle rats for common husbandry activities such as cage changing or medical/research procedures such as injection, and be implemented as a source of social enrichment. It is concluded that this method can be used to efficiently and practicably reduce rats&#39; fearfulness of humans and improve their welfare, as well as reliably model positive affective states.

This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.

痒, 一种诱发大鼠正向影响的技术

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk ...

Using a Classroom-Based Deese Roediger McDermott Paradigm to Assess the Effects of Imagery on False Memories

Associated word list procedures can elicit false memories in predictable ways by inducing associative processing, thus making it harder to monitor the accuracy of memories. The purpose of the method presented here was to induce false memories using lists of either semantically or phonologically related words and to assess the effects of imagery instructions on the recall and recognition of those false memories. To do this, we used a modified version of the Deese Roediger McDermott (DRM) paradigm. We adapted word lists from previous DRM studies to suit imagery procedures and created an automated presentation to present the word lists in classroom settings. We then recruited undergraduate classes and instructed some of the classes to create mental images of the list words as they were being presented, while instructing the other classes to simply remember the words. The automated presentation presented word lists to participants, one word at a time, alternating between phonologically and semantically related lists. Participants used paper-pencil recall packets to immediately recall list items, complete a distractor activity, and take a subsequent final recognition test. Often, participants immediately recalled and later recognized words that were related to the list items but were not actually presented; these are known as critical lures and indicate a false memory. The protocol detailed here describes a four-step procedure - list presentation, immediate recall, distractor phase, and final recognition - that can assess the effects of list type and imagery instruction within the DRM paradigm on memory. The automated nature of the list presentation provides the ability to systematically vary variables of interest, and the paper and pencil method of data collection affords an easily accessible method for collecting data in classroom settings. The protocol also offers options to modify the procedure to a more traditional DRM paradigm without imagery and/or list type manipulations. The use of this protocol can provide results relevant to both classroom learning and cognitive science principles.

The method presented here induced false memories using lists of related words and also assessed the effects of imagery instructions on the recall and recognition of those false memories. This protocol details a modified version of the Deese Roediger McDermott (DRM) paradigm.

利用基于课堂的 deese roediger mcdermott 范式评估意象对虚假记忆的影响

Associated word list procedures can elicit false memories in predictable ways by inducing associative processing, thus making it harder to monitor the ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

Assessing the Coherence of Parents&#039; Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

使用五分钟言语样本程序评估家长对孩子的简短叙述的一致性

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...

Applying an eMASS Customization Program as a Research Tool to Evaluate Consumer Benefits

As many scholars and practitioners study personalization and relationship marketing, it is important to provide personalization such as mass customization through marketing technology. The purpose of this study is to examine how to conduct consumer research using an online survey and analysis of data. This study examines consumers&#39;&#160;perceived benefits while customizing a product as well as emotional product attachment, attitudes toward a customization program, and loyalty intentions in the context of online retailing. In addition, this study investigates how consumer responses are different based on individual characteristics such as fashion innovativeness. An online survey company in South Korea recruited 290 female apparel shoppers who purchased apparel online. To enhance external validity, this study used an existing retail website with a well-established mass customization program. After completing the customization program, participants complete the online questionnaire. Structural equation modeling (SEM) and latent mean analyses (LMAs) are then performed for analyses. This study stresses the importance of testing measurement invariance for mean comparisons. Before the SEM and LMA, this study follows the hierarchy of invariance tests (configural invariance test, metric invariance test, and scalar invariance test), which are not considered by traditional approaches such as ANOVA. These statistical analyses provide applicability of the invariance test procedures and LMA to consumer behaviors. The conclusions of mean differences have integrity and validity because they are guided by a sophisticated statistical procedure to ensure measurement invariance.

Presented here is a protocol to examine consumer responses toward mass customization in the context of online retailing. The protocol details the online survey procedure and how to analyze data using structural equation modeling and group differences using latent mean analyses.

应用 eMASS 定制计划作为评估消费者权益的研究工具

As many scholars and practitioners study personalization and relationship marketing, it is important to provide personalization such as mass customization ...

An Experimental Paradigm for Measuring the Effects of Ageing on Sentence Processing

Previous studies have found that older adults have greater difficulties in processing syntactically complex sentences than younger adults. However, the exact regions where the difficulties arise have not been fully identified. In this study, a maze task was implemented to investigate how older adults and younger adults differentially processed two types of sentences with different levels of syntactic complexity, namely subject relative clauses and object relative clauses. Participants were asked to choose between two alternatives at each segment of the sentences. The task required participants to engage in a strictly incremental mode of processing. The reading times for each segment were recorded, allowing the quantification of the difficulty of sentence reading. The task allowed us to identify the exact locations of the processing difficulty and thus facilitate a more accurate assessment of the age-related decline in sentence processing. The results indicate that the effect of ageing was found mainly at the head nouns, but not at other regions of the sentences, a finding which suggests that the maze task is an effective method to identify the exact location of the ageing effect on sentence processing. The implications of this experimental paradigm for investigating the effect of ageing on sentence processing are discussed.

Here, we present a protocol to examine the age-related decline in sentence processing using a maze task that allowed us to localize the processing difficulty at each word of the sentences during reading.

测量衰老对句子处理影响的实验范式

Previous studies have found that older adults have greater difficulties in processing syntactically complex sentences than younger adults. However, the ...

The Use of Traditional Fear Tests to Evaluate Different Emotional Circuits in Cattle

Animal temperament is complex and has implications for productivity and economic profitability. Quantifying an animal&#39;s response to differing stimuli may facilitate breeding selections and identify animals that are better suited to specific management strategies. Multiple tests have been developed to evaluate cattle temperament (e.g., exit velocity, chute score, pen score, open field test, startle test, bovine zero maze), but each of these tests evaluates the animal&#39;s response to different stimuli (e.g., isolation, novel environment, startle, willingness to enter an enclosed area). Cattle temperament has been observed to be relatively stable over time. However, the evaluation of temperament has the potential to be influenced by current conditions, previous experiences, and observer bias. Many of these temperament tests have been improperly categorized as fear tests and have also been criticized for being subjective. This paper provides a framework for standardizing behavioral tests for cattle and suggests that these different evaluations assess different aspects of the animal&#39;s overall temperament.

Here, we present a protocol to conduct a variety of behavioral tests in cattle that have been designed to evaluate emotions. A battery of behavioral tests (open field test, startle test, bovine zero maze, exit velocity, pen score, and chute score) were conducted to evaluate different components of animal temperament.

使用传统恐惧测试来评估牛的不同情绪回路

Animal temperament is complex and has implications for productivity and economic profitability. Quantifying an animal&#39;s response to differing stimuli ...