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. 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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。

<table><tbody><tr><td>Stereotactic Apparatus item 51725</td><td>Stoelting co.</td><td>51725</td><td></td></tr><tr><td>Quintessential stereotaxic pump&amp;nbsp;</td><td>Stoelting co.</td><td>53311</td><td></td></tr><tr><td>Injection Styringe, 65 RN</td><td>Hamilton</td><td>7633-01</td><td></td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D5796</td><td></td></tr><tr><td>PEI&amp;nbsp;</td><td>Polysciences Inc.</td><td>23966</td><td></td></tr><tr><td>Scissors</td><td>Fine Surgical Tools</td><td>14084-08</td><td></td></tr><tr><td>Blunt end forceps</td><td>Fine Surgical Tools</td><td>11002-12</td><td></td></tr><tr><td>Needle holder&amp;nbsp;</td><td>Fine Surgical Tools</td><td>12001-13</td><td></td></tr><tr><td>Drill</td><td>Ram Products Inc</td><td>Microtorque control box, Tech2000 handpiece</td><td>with pedal</td></tr><tr><td>Glass bead sterilizer</td><td>Inotech&amp;nbsp;</td><td>IS-400</td><td></td></tr><tr><td>Absorbable sutures&amp;nbsp;</td><td>Unify</td><td>M-K518r19</td><td>Absorbable, reverse cutting</td></tr><tr><td>Cotton swabs</td><td>VWR</td><td>89031-270</td><td></td></tr><tr><td>Heating pad</td><td>Gaymar</td><td>T-pump TP-500 PN11184-000</td><td></td></tr><tr><td>Artificial tears</td><td>Rugby</td><td>NDC 0536-6550-91</td><td></td></tr><tr><td>Disposable syringe</td><td>BD Syringe</td><td>309623</td><td></td></tr><tr><td>Cloth</td><td></td><td></td><td></td></tr><tr><td>Gloves&amp;nbsp;</td><td>Ansell</td><td>Senseitouch #7823</td><td></td></tr><tr><td>Avertin or other anesthetic</td><td>see recipe citation</td><td></td><td></td></tr><tr><td>Ketoprofen or other analgesic</td><td>see veternarian&amp;nbsp;</td><td></td><td></td></tr><tr><td>Tracking software</td><td>Noldus</td><td>Ethovision XT</td><td></td></tr><tr><td>Tracking Camera</td><td>Noldus</td><td>Media Recorder</td><td></td></tr></tbody></table>

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Behavior

14.1K 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

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 ...

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

啮齿动物脑显微注射学习动机行为的分子基板

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

通过颅内注射和磁共振成像对大脑转移进行建模

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

癌症研究

Establishment of Orthotopic Patient-derived Xenograft Models for Brain Tumors using a Stereotaxic Device

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of neuro-oncology. One of the most widely used techniques is the orthotopic intracranial injection, a method that allows investigating of tumor growth, invasion, and dissemination within a controlled setting. This technique involves transplanting tumor cells from a specific patient region into the corresponding anatomical site in an animal. By doing so, these orthotopic brain tumor models offer a unique advantage, as they more accurately replicate cancer's biological behavior and its interactions with the brain environment seen in human patients. This makes them especially valuable for preclinical therapeutic testing, where a close resemblance to the clinical scenario is essential for evaluating potential treatments. This protocol shares experiences in developing patient-derived xenograft (PDX) models for pediatric brain tumors, including diffuse midline glioma (DMG), glioblastoma (GBM), medulloblastoma, and ependymoma. This method delineates the procedure for conducting intracranial stereotaxic injections in mice, ensuring the correct targeting of the injection site within the brain. Additionally, we describe the post-procedural monitoring system employed to detect signs of successful tumor engraftment. Following tumor injection, a rigorous monitoring system is implemented to observe the animals for any signs of neurological impairment, behavioral changes, and/or weight loss, which are common indicators of tumor progression. This system allows for timely intervention and provides critical data regarding the tumor's growth dynamics. By refining these models and protocols, we aim to enhance the reliability and translational potential of preclinical studies, contributing to the development of more effective treatments for pediatric CNS tumors.

The development of orthotopic pediatric brain tumor models requires meticulous precision, using a stereotaxic device to implant cancer cells precisely. The methodology presented here outlines the steps involved in preparing brain tumor cells, performing intracranial injections, and implementing a post-operative monitoring system to assess brain tumor engraftment.

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

新生小鼠脑巴细胞基因调控的立体定向手术

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

神经科学

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

在Vivo脑内立体注射中，用于小鼠脑切片中远距离输入的光遗传学刺激

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

Adaptable Angled Stereotactic Approach for Versatile Neuroscience Techniques

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain regions still presents a challenge, particularly when targeting brain structures along the midline. These challenges include avoiding of the superior sagittal sinus and third ventricle and the ability to consistently target selective and discrete brain nuclei. In addition, more advanced neuroscience techniques (e.g., optogenetics, fiber photometry, and two-photon imaging) rely on targeted implantation of significant hardware to the brain, and spatial limitations are a common hindrance. Presented here is a modifiable protocol for stereotactic targeting of rodent brain structures using an angled coronal approach. It can be adapted to 1) mouse or rat models, 2) various neuroscience techniques, and 3) multiple brain regions. As a representative example, it includes the calculation of stereotactic coordinates for targeting of the mouse hypothalamic ventromedial nucleus (VMN) for an optogenetic inhibition experiment. This procedure begins with the bilateral microinjection of an adeno-associated virus (AAV) encoding a light-sensitive chloride channel (SwiChR++) to a Cre-dependent mouse model, followed by the angled bilateral implantation of fiberoptic cannulae. Using this approach, findings show that activation of a subset of VMN neurons is required for intact glucose counterregulatory responses to insulin-induced hypoglycemia.

Described here is a stereotactic procedure that can target challenging and difficult-to-reach brain regions (due to spatial limitations) using an angled coronal approach. This protocol is adaptable to both mouse and rat models and can be applied to diverse neuroscientific applications, including cannula implantation and microinjections of viral constructs.

多功能神经科学技术的适应性角度立体定向方法

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain ...

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

We describe how to successfully inject solutions into specific brain areas of rodents using a stereotaxic frame. This survival surgery is a well-established method used to mimic various aspects of Parkinson's disease.

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in ...

优化小鼠立体定向显微注射的准确性

Preliminary Validation of Stereotaxic Injection Coordinates via Cryosectioning

Stereotaxic injection of a specific brain region constitutes a fundamental experimental technique in basic neuroscience. Researchers commonly base their choice of stereotaxic injection parameters on mouse brain atlases or published materials that employed various populations/ages of mice and different stereotaxic equipment, necessitating further validation of the stereotaxic coordinate parameters. The efficacy of calcium imaging, chemogenetic, and optogenetic manipulations relies on the precise expression of reporter genes within the region of interest, often requiring several weeks of effort. Thus, it is a time-consuming task if the coordinates of the target brain region are not verified in advance. Using an appropriate dye instead of a virus and implementing cryosectioning, researchers can observe the injection site immediately following dye administration. This facilitates timely adjustments to coordinate parameters in cases where discrepancies exist between the actual injection site and the theoretical position. Such adjustments significantly enhance the accuracy of viral expression within the target region in subsequent experiments.

作者聚焦:通过立体定向注射和经济高效的染料加速小鼠认知障碍研究

The present protocol describes a practical strategy to expedite the verification step of stereotaxic injection coordinates before conducting viral tracing using dyes and frozen sections.

通过冷冻切片初步验证立体定向注射坐标

Stereotaxic injection of a specific brain region constitutes a fundamental experimental technique in basic neuroscience. Researchers commonly base their ...