按照加拿大的动物保健和麦吉尔大学（McGill University）动物保健委员会理事会提出的指导原则和法规，对动物进行的实验。这项研究由加拿大卫生研究院（CIHR）授予KG的资金支持。没有利益冲突的声明。

1。动物房和驯化过程<ol><li>与同性窝，含有β的芯片的被褥和nestlet垫，线网格盖，以适应水的瓶子和食物标准的塑料笼，每笼5只，男性和女性都装CD-1小鼠。标准小鼠饲料和水在整个实验过程中的饲养笼可自由采食。 </li><li>维持小鼠的反向暗光的时间（灯在22:00至10:00）。 </li><li>允许小鼠使其恢复到IVSA检测室前一周的实验程序的开始。 </li></ol> 2。操作性培训<ol><li>所有的行为程序，包括的甜牛奶培训和静脉注射毒品自我管理会话进行使用操作性室测量15.9×14×12.7厘米，配备超灵敏的鼠标杆，斗杯，刺激灯和微量注射泵（医学Associ阿泰，圣奥尔本斯，VT，USA）。钱伯斯是连接到一台计算机使用MED Associates的智能CR接口和地中海 - PC软件编程加固的时间表和收集数据。 </li><li>天真的小鼠熟悉的操作性室的培训，以杠杆压使用了瓢杯加糖的牛奶奖励（0.1毫升）。操作性牛奶培训5-7天进行1小时的会议。完成一个固定的比例（FR）的日程安排上的主动杆的北斗杯和刺激光照明的演示文稿中。甜牛奶溶液中加入全脂牛奶（脂肪含量3.25％），蔗糖（10毫克/毫升，ACS试剂，Sigma-Aldrich公司公司圣路易斯MO，USA）。操作性培训甜奶溶液或液体食品增强剂是一种常用的技术，用于以方便操作性响应在小鼠1,2。 </li></ol> 3。在手术过程中使用的设备的制备热曲IRED设备 - 20，23和26号针，磨床，1毫升注射器，聚乙烯管，烙铁，肝素，抗生素和止痛剂。 <ol><li>准备一个剃了20号针头的针引导到颈静脉插入导管。针的轴的剃下来使用研磨机，以形成一个通道内的针引导导管进入静脉。针通道应仔细检查，可能已沉积的金属碎片。任何障碍物的通道，应该用细尖钳刮出来。 </li><li>适应两个1-cc的注射器用于冲洗和检查导管。准备两个注射器Tygon管12厘米的片，并将其附加在其一端固定到1毫升注射器至26号针头，和拉伸管子的另一端，23号针头。一个注射器0.9％无菌生理盐水，其他应充满肝素化的的替卡西林解决方案（33毫克替卡西林和肝素每10毫升的无菌盐水中的0.3毫克）。 </li><li>准备导管插管的上限。聚乙烯管伸过来的23号针，切1厘米的斜面。熔体的管子的开口端，以创建一个厚的密封。应熔化的油管，从而使整个插管帽是足够长的，以适应在导管插管，紧贴在密封的端部装配件的套管的端部。请注意，如果帽太长，存在这样的风险，它将被弯曲并其后在过程中的安装螺钉导管覆盖穿孔。因此，精心准备的导管帽的建议，以提供一个适合的套管。 </li><li>准备好所需的解决方案。所有试剂购自Sigma-Aldrich公司（圣路易斯MO，USA）。 <ol class="lalpha"><li>肝素化替卡西林导管冲洗溶液 - 溶解0.33克替卡西林（二钠盐）和0.003克的肝素在10ml的无菌生理盐水。 0.03毫升的溶液冲洗THRough每天导管。来自发展中国家的管理，以防止血液凝块和感染的抗生素溶液。 </li><li> amikicin（抗生素）用于皮下注射 - 单次皮下注射给予手术后的剂量为10毫克/公斤，以防止手术后感染。 </li><li>酮洛芬（止痛药）皮下注射 - 单次皮下注射手术后的剂量为5毫克/公斤，以管理任何手术后的疼痛。 </li></ol></li></ol> 4。导管植入手术所需的设备和试剂:异氟醚，无菌盐水，醇（70％），1和3毫升注射器填充用无菌生理盐水，1毫升，适于注射器，抗生素和镇痛的解决方案，在第3段中，鼠标的导管（CamCaths，剑桥，英国制备），导管盖（从的HRS科学，蒙特利尔，魁北克水晶帽），眼润滑剂，4厘米的塑料棒材，以提高静脉（这可以由一个塑料棉条），polysporin，无菌棉签，纱布，曲线和直线钳，动脉夹，精剪。 <img src="/files/ftp_upload/3739/3739step1.jpg" alt="第1步" fo:content-width="3in" fo:src="/files/ftp_upload/3739/3739step1highres.jpg"/><ol><li>使用标准的无菌操作技术，替补席上的手术，手术器械，导管手术前进行消毒。适当的消毒技术包括:蒸汽高压灭菌的手术器械和导管购自CamCaths。玻璃珠也可用于灭菌的外科器械上的提示。可以采用更细腻的导管或风险熔化的材料，环氧乙烷灭菌。啮齿动物手术的无菌技术的更详细的描述可以发现在所附的参考文献1,2。设置的长椅上，仪器及维持麻醉的头锥的照片说明。 </li><li>小鼠麻醉与异氟醚气体，并保持在麻醉状态下清除系统下使用呼吸管。眼睛的润滑剂（泪自然漆PM）被施加到双眼，以防止它们在手术过程中干燥。适当的手术切缘剃了对动物的背部和绕在脖子上。在任何切口，外科领域限制使用无菌单。 </li><li>为了制备用于插入到导管的心脏的右心房，多余的导管被切断1.2厘米从灯泡的导管。这是最佳的长度建立成年CD-1小鼠，约8周龄，体重20-25克。导管管材的长度可能需要稍微调整（通过试验和错误）的基础上的应变，大小和年龄的小鼠。在插入之前，含有无菌生理盐水的注射器（第3.2节）被连接到导管插管，该导管被刷新，并检查是否有泄漏。保持在整个手术过程中，这种注射器连接到导管。它将被用于冲洗导管，并绘制在步骤4.7中的回血。 </li><li>用70％酒精消毒后，2厘米长的midscapular切口是在背面的中间开始和结束只是颈部以下的，以容纳在导管的基。结缔组织应该用钳子被迫分开，下面的皮肤的导管基地，以腾出空间。 </li><li>配售的动物在它的后面，第二个浅1 - 2厘米对角线切口是由右锁骨向上的动物下颚的区域已经被擦洗后，用70％的酒精。将被发现的颈静脉表面的皮肤下的颈部。在准备插入导管，导管的基础上从油管被拉通过切口的背面，并通过油管皮肤下，刚刚超过右肩靠近颈静脉带来。的导管的端部是然后连接到的动脉夹，并放置在动物的侧保持到位。 </li><li>右颈静脉位于通过轻轻移开浅表从动物的脖子上的切口周围的结缔组织和脂肪组织。静脉周围的结缔组织，用弯钳破碎和静脉，然后用消毒过的塑料棒升高。松散开放的缝合线结围绕静脉的每一端，并穿过导管顶端的结，和环绕在缝合线休息解除夹紧在右肩。 </li><li>在插入之前，湿的20个计插入针和静脉用无菌生理盐水，以减少摩擦。针保持平行到静脉，和插入的底部附近的升高的静脉（ 注:约0.5厘米的针尖需要输入静脉）轻轻。用镊子针轴，向下滑动导管插入静脉。电阻表明，管我s结缔组织之内，而不是在静脉内。通过静脉推0.03毫升生理盐水，以确保没有泄漏。泄漏会表明静脉可刺穿或导管的位置需要调整。 注意:要检查管内静脉，试图制定一些血液，使用附带的盐水注射器。如果血液不能立即被制定，静脉或心脏壁可以阻断导管尖端，或没有被刺穿静脉;调整管，并再次尝试。针重新安置可能是必要的，如果血液中仍然不能被制定了。 </li><li>为了确保导管的位置，导管推灯泡插入点并取下针头。领带结底部，然后将导管冲洗第二个结绑在正上方的灯泡之前，对吧。再次测试，看是否可以得出血液和放松的结稍如果需要的话。塔克下导管的皮肤和缝合腹切口周围的动物的脖子上。应用Polysporin治愈快速使用无菌棉头涂药器，或任何其他抗生素软膏优选含有一定的镇痛到闭合切口。 </li><li>与动物在其腹部，放置导管基础的背部皮肤下内制备的切口。确保最低限度循环，隐藏在导管基地，以尽量减少动物的咀嚼和穿孔的机会，多余的管道。缝合切口两侧导管基地，并应用Polysporin效治疗快速用无菌棉棒敷贴。 </li><li>冲洗导管用0.03毫升的肝素替卡西林解决方案使用注射器管贴吧（3.2节）。盖上管与白色导管盖的塑料管帽和螺丝。在某些小鼠从手术是新鲜的，血液可能会泄漏的导管之前插管上限。重要的是要重新冲洗动物和rapidl的y替换插管帽前血的机会都流出来了。动物必须被刷新，每天都保持导管通畅。 </li><li>之间的动物后腿，用70％乙醇擦拭后的喷射区域，皮下注入镇痛酮洛芬一侧上为5毫克/公斤的剂量，并在剂量为10毫克/公斤的另一侧上的抗生素amikicin。 </li><li>麻醉后停止，动物可以恢复一个干净的笼子里，容易获得食物和水，5〜7天。小鼠被放置在一个恒温柜过夜，以防止手术后的低温。 </li></ol><img src="/files/ftp_upload/3739/3739step2.jpg" alt="第2步" fo:content-width="4.75in" fo:src="/files/ftp_upload/3739/3739step2highres.jpg"/> 5。行为测试 - 静脉自我管理<ol><li>在此之前的行为的测试导管的0.9％无菌生理盐水冲洗。然后将小鼠中的d操作性腔和连接到输液线和输液泵。活跃​​的杠杆印刷机导致一个3.2秒18μl反应药物输注加上刺激光照明。其次是由一个8秒的超时时间段，在此期间，刺激光停留在每一个杠杆压。 </li><li>继操作性会话，小鼠导管冲洗肝素替卡西林解决方案之前，回到自己的家乡笼。 </li><li>自我管理连续3个2小时的会议在各剂量组小鼠。剂量每只小鼠以随机的顺序，在下面的部分中所示。 </li><li>导管通畅每日评估确保生理盐水和抗生素的溶液通过导管可以被刷新。此外，氯胺酮/咪达唑仑测试如所附的参考文献5中描述的，可以进行。简言之，麻醉的迹象，如不动的5秒内的输液0.02-0.03毫升氯胺酮（15毫克/毫升），咪达唑仑（0.75毫克/毫升），咪达唑仑的专利导管5的证据。 </li></ol>

<ol>
	<li>Rocha, B. A., Ator, R., Emmett-Oglesby, M. W., Hen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Intravenous Cocaine Self-Administration in Mice Lacking 5-HT1B Receptors. Pharmacology Biochemistry and Behavior. 57 (3), 407-412 (1997).</li><li>Zapata, A., Chefer, V. I., Ator, R., Shippenberg, T. S., Rocha, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+sensitization+and+enhanced+dopamine+response+in+the+nucleus+accumbens+after+intravenous+cocaine+self-administration+in+mice.">Behavioural sensitization and enhanced dopamine response in the nucleus accumbens after intravenous cocaine self-administration in mice.</a> European Journal of Neuroscience. 17 (3), 590-596 (2003).</li><li>Pritchett-Corning, K. R., Mulder, G. B., Luo, Y., White, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+Rodent+Surgery+for+the+New+Surgeon.">Principles of Rodent Surgery for the New Surgeon.</a> J. Vis. Exp. (47), e2586 (2011).</li><li>Hoogstraten-Miller, S. L., Brown, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Techniques in Aseptic Rodent Surgery Current Protocols in Immunology. , 1.12-1.14 (2001).</li><li>Thomsen, M., Caine, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+Intravenous+Drug+Self-Administration+in+Rats+and+Mice.">Chronic Intravenous Drug Self-Administration in Rats and Mice.</a> Current Protocols in Neuroscience. . 9, 20.1-20.40 (2005).</li><li>Gill, K. J., Boyle, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confirmation+of+quantitative+trait+loci+for+cocaine+induced+activation+in+the+AcB/BcA+series+of+recombinant+congenic+strains.">Confirmation of quantitative trait loci for cocaine induced activation in the AcB/BcA series of recombinant congenic strains.</a> Pharmacogenetics. 13, 329-338 (2003).</li><li>Gill, K. J., Boyle, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+influences+on+drug-induced+psychomotor+activation+in+mice.">Genetic influences on drug-induced psychomotor activation in mice.</a> Genes, Brain &#38; Behavior. 7, 859-868 (2008).</li><li>Boyle, A. E., Gill, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+AXB/BXA+recombinant+inbred+lines+of+mice+to+the+locomotor+activating+effects+of+cocaine:+A+QTL+analysis.">Sensitivity of AXB/BXA recombinant inbred lines of mice to the locomotor activating effects of cocaine: A QTL analysis.</a> Pharmacogenetics. 11 (3), 255-264 (2001).</li><li>Boyle, A. E., Gill, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+verification+of+previously+identified+QTLs+for+cocaine-induced+activation+using+a+panel+of+B6.A+chromosome+substitution+strains+(CSS)+and+A/JxC57Bl/6J+F2+mice.">A verification of previously identified QTLs for cocaine-induced activation using a panel of B6.A chromosome substitution strains (CSS) and A/JxC57Bl/6J F2 mice.</a> Psychopharmacology. 207 (2), 325-3234 (2009).</li><li>Thomsen, M., Caine, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychomotor+stimulant+effects+of+cocaine+in+rats+and+15+mouse+strains.">Psychomotor stimulant effects of cocaine in rats and 15 mouse strains.</a> Experimental and Clinical Psychopharmacology. 19 (5), 321-341 (2011).</li><li>Thomsen, M., Caine, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+drug+self-administration+in+mice:+practical+considerations.">Intravenous drug self-administration in mice: practical considerations.</a> Behavior genetics. 37 (1), 101-118 (2007).</li></ol>

没有利益冲突的声明。

滥用药物的动物模型是特别有用的理解与毒品有关的行为的遗传基础。例如，小鼠与不同基因图谱显示，遗传差异在6-8可卡因的敏感性，并帮助识别潜在的候选基因介导的表型变异观察9。相当大的成功，静脉导管本文中所描述的程序已被用于检查药物IVSA在不同品系的小鼠以及不同遗传背景的小鼠10。 这个视频中显示的程序，突出重要的因素集中在以下导管插入手术，以获得可靠的静脉自我管理数据。首先，导管在右心房的位置是至关重要的，为了防止导管失败印迹血栓。在手术过程中，它是重要的，以确保导管到底是畅通的，而不是闭塞的心脏或静脉组织。其次，需要每日导管冲洗操作性会话之前和之后，以防止堵塞。最后，导管插管，必须不断与插管帽和晶体涵盖当动物是在他们的家笼覆盖，以防止碎片的条目。罚款26号针头，但是每天肝素抗生素溶液冲洗是必需的，特别是当动物没有测试过IVSA的轻微堵塞的导管插管可能会脱落。 要执行，镇痛和麻醉的动物的生存手术，无菌操作技术，良好的知识是必要的。虽然这个视频不能取代适当的外科手术培训，它可以用来为指导，研究人员希望获得这个范例所必需的技术。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>替卡西林二钠</td><td> Sigma-Aldrich公司</td><td> T5639-1G </td><td> 1克瓶</td></tr><tr><td>眼泪博纳自然PM（眼睛的润滑剂） </td><td>爱尔康</td><td> N / A </td><td> 3.5克管 此眼的润滑剂可以发现在大多数药店。可以使用任何眼科眼润滑剂。 </td></tr><tr><td>可卡因</td><td> Medisca Pharmaceutique </td><td> 0723-06 </td><td> 1克瓶</td></tr><tr><td>氯胺酮</td><td> Medisca Pharmaceutique </td><td> 1754年至1704年</td><td> 25克瓶</td></tr><tr><td>咪达唑仑</td><td> Medisca Pharmaceutique </td><td> 2519-06 </td><td> 1克瓶</td></tr><tr><td>高效率空气RIN钠盐</td><td> Sigma-Aldrich公司</td><td> H4784-250MG </td><td> 250克瓶</td></tr><tr><td>酮洛芬</td><td> Sigma-Aldrich公司</td><td> K1751-1G </td><td> 1克瓶</td></tr><tr><td> Amikicin硫酸盐</td><td> Sigma-Aldrich公司</td><td> A2324-5G </td><td> 5克瓶</td></tr><tr><td>蔗糖ACS试剂</td><td> Sigma-Aldrich公司</td><td> S5016-500G </td><td> 500克瓶</td></tr><tr><td>标准鼠标导管软网</td><td> CamCaths </td><td> MIVSA/20/26/BC-1S </td><td>管规格:26SWG; 0.451; 0.254 </td></tr><tr><td>水晶帽装配</td><td> HRS科学</td><td> 313CAC </td><td>这是白色的导管帽。 </td></tr><tr><td>聚乙烯微口径管材</td><td>托马斯科学</td><td> 9561S41 </td><td>管规格（缸径×外径×壁厚英寸）:0.01所述0.03所述0.01 </td></tr>
 </tbody>
</table>

methods for intravenous self administration in a mouse model

动物模型已经发展到学习的增强作用的药物，包括静脉-的管理（IVSA）范例。使用IVSA范式来研究药物如可卡因滥用的补强性的优点包括这样的事实，该药物是自我给药，而不是实验者给药，钢筋的时间表可以被改变，和精确的测量的数量药物以及消耗能够得到静脉注射液的定时和模式。此外，静脉给药，避免了首过代谢或味道有关的潜在的混淆，并产生血液和脑组织的药物浓度迅速增加。中列出此视频，静脉自我管理，恕不另行限食或药物训练前，小心导管放置在手术过程中和细致的日常导管冲洗和维护。在此列出实验程序本文包括动物住房和驯化方法的描述，使用甜牛奶解决方案的操作性培训，导管植入手术。

<img src="/files/ftp_upload/3739/3739fig1.jpg" alt="图1" /> 图1。响应静脉注射毒品的自我管理的模式会有所不同的药物，剂量范围内，小鼠品系采用。所提出的数字显示可卡因自我管理数据导管插入手术成功使用在视频中描述的步骤。图中显示的平均（±SEM）可卡因输液收入和平均（±SEM）可卡因消费量（毫克/公斤体重），在一系列的可卡因剂量以随机的顺序上FR1的加固进度。横坐标:自我管理的每次输注药物的剂量。左边的纵坐标总数:输液2小时操作性会议期间所赚取。右边的纵坐标:在2小时测试会话毫克/公斤的可卡因总量的摄入量。所有13个导管保持专利的研究的持续时间（4周）。一个单因素方差分析进行剂量透露，话筒e的管理可卡因的剂量依赖的方式[F（1,12）= 42.8，P &lt;0.05]。在剂量反应曲线[F（3,36）= 29.6，P &lt;0.05]，尽管减少了杆压在高剂量的可卡因消费在增加。每个数据点代表平均3测试会在每个的可卡因剂量（±SEM）收集的CD-1小鼠（N = 13 /剂量，男性和女性的结合）。使用2-way ANOVA分析，以确保两个杠杆之间的鉴别，小鼠的活性（药物增强）与非活动的杠杆响应跨越的剂量响应曲线的比较。对于CD1小鼠，分析揭示了偏好主动杆[F（1,12）= 10.255，p &lt;0.05】在整个的剂量 - 反应曲线（未示出的数据在这里）。

Watch this Scientific Journal Video about Methods for Intravenous Self Administration in a Mouse Model at JoVE.com

Methods for Intravenous Self Administration in a Mouse Model

的静脉-的管理（IVSA）的范式被认为是研究在啮齿类动物中滥用药物的补强性的黄金标准。这手稿列出了实验步骤和手术技术必要获得可靠的IVSA的数据。特别突出，细致的导管植入术和维护。

在小鼠模型中静脉自我管理的方法

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

methods-for-intravenous-self-administration-in-a-mouse-model

Nanyang Technological University

Research

JoVE Journal

Neuroscience

53.0K 次观看.  McGill University Health Centre. 的静脉-的管理（IVSA）的范式被认为是研究在啮齿类动物中滥用药物的补强性的黄金标准。这手稿列出了实验步骤和手术技术必要获得可靠的IVSA的数据。特别突出，细致的导管植入术和维护。

视频: Methods for Intravenous Self Administration in a Mouse Model

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

在大鼠的药物自我管理的评估超声发声

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

科研

神经科学

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

微透析的乙醇操作性乙醇的自我管理和乙醇的测定气相色谱

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

方法研究抗精神病药物的作用机制在<em&gt;秀丽隐杆线虫</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

角膜微囊含量:血管生成在小鼠眼模型

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

在Wholemount大鼠视网膜免疫组化和钙成像方法

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

方法在小鼠肾上腺嗜铬细胞细胞贴附电容测量

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

实时 fMRI 神经训练管理协议

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

腺相关病毒对大鼠的静脉管理方法和技巧及中枢神经系统转导的评价

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

FITC 标记白蛋白静脉滴注对神经退行性疾病模型血脑屏障通透性的评价

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...