Name: assessment of spontaneous alternation, novel object recognition and limb clasping in transgenic mouse models of amyloid-β and tau neuropathology
Uploaded: 2017-05-28T13:00:00.000+00:00
Duration: 10 min 2 s
Description: Watch this Scientific Journal Video about Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-Î² and Tau Neuropathology at JoVE.co

作者没有确认。

本出版物中详述的方法由Hilltop实验室动物机构动物护理和使用委员会（IACUC）审查，以确保适当的照顾，使用和人道对待动物，遵守适用的联邦，州和当地法律法规，例如作为联邦动物福利条例，或AWR（CFR 1985）和"人体护理和使用实验动物公共卫生服务政策"（PHS政策）（PHS，1996）。 1.所有行为评估总则<ol><li>在任何动物处理之前，使用新的笼子卡盖住现有的笼子卡，仅显示唯一的，盲人的动物标识符。 注意:在常规化合物/安慰剂给药期间处理小鼠的调查员不得处理小鼠进行行为评估。 </li><li>点亮或关闭头顶灯，调整照明，使得竞技场或迷宫地板的照明为30-35勒克斯。 </li><li> F或研究跨越几个星期，每周记录体重作为总体健康的间接测量。 注意:如果需要进行更健壮的身体检查，可以包括外套质量，姿势，步态和自发运动的额外的笼侧检查。 </li></ol> 2.调查小鼠处理小鼠<ol><li>在任何行为测试前两天，将老鼠习惯于处理。将笼子从笼架上取下，放在水平面上。 </li><li>从笼子上取下盖子。正如处理即将到来的行为测试时所处理的一样，处理鼠标。将鼠标放在家中笼子上的杯状手中。 </li><li>测量潜伏期从研究者的手跳回到家庭笼中。握住鼠标最多5秒。 注意:延迟≥2秒的小鼠被视为"习惯"。在第一次试验期间显示延迟&lt;2秒的小鼠经历2次额外的习惯那天的n个会议。 </li><li>让老鼠连续2天处理习惯。注意在第二天结束时没有习惯的任何鼠标。 </li></ol>通过测量Y型迷宫中的自发交替来评估空间工作记忆8 <ol><li>在初次使用之前，用无味的漂白杀菌擦拭物，70％EtOH，然后用dH 2 O 9彻底清洁Y-迷宫。明确将迷宫的手臂指定为"A"，"B"和"C"或其他相似的唯一标识符。 </li><li>在测试开始之前，设置数据采集系统或摄像机，并在迷宫中设置适当的鼠标跟踪。使用已知长度的标尺或其他物体的捕获视频图像校准迷宫中的距离。 注意:此过程中的行为方法将适用于各种数据采集系统，作者假设anyo执行此过程精通使用他们选择的数据采集系统。功率分析表明，β≤0.2需要每组10-15只小鼠的样本量。 </li><li>从机架上取下保持架，轻轻将其放置在靠近Y型迷宫的桌子上。将鼠标从其家中的笼子中取出，轻轻地将其放置在Y-迷宫的一个臂上，面向中心。让调查员远离迷宫，使鼠标看不到调查员。 </li><li>将鼠标放入迷宫后立即激活数据/视频采集系统。 </li><li>按播放并记录每只小鼠的自发行为10分钟。会话完成后，将鼠标轻轻放回家中的笼子并将笼子返回到机架。 </li><li>在每次会话之间彻底清洁迷宫，使用无味的漂白杀菌擦拭剂，70％EtOH，然后加入dH 2 O.重复步骤3.4，评估所有麦克风即</li><li>一旦所有小鼠完成了Y迷宫的探索，分析采集系统的数据或手动对会议视频进行评分。当鼠标的所有4只爪穿过中央区域的阈值并进入臂并且动物的鼻孔朝向手臂的端部时，发生臂进入。 注意:要分析的端点包括:迷宫中的总距离，每个臂（包括中央区域）中行进的总距离，每个臂（包括中央区域）的总时间，手臂总数，进入每个手臂的条目，以及输入的武器序列列表，以评估所做的更改次数。 </li><li>当三只连续的手臂条目中的每一只小鼠进入迷宫的不同臂时，发生自发的交替。然后用以下公式计算自发交替％。 <img alt="公式1" src="/files/ftp_upload/55523/55523eq1.jpg" /> 注意:例如，胳膊入门的顺序是:ABCCBABCABC，调查员总共得到6次自发的交替（按照ABC，CBA，ABC，BCA，CAB，ABC）。共有11个手臂入口，自发交替率为67％。 </li><li> 进行以下质量控制检查，以确保数据代表对自发交替的无偏估计。 <ol><li>执行Pearson的自发交替百分比与总行进距离和所进行的手臂数量的相关性。 注意:如果自发交替％与任一参数存在显着相关性，则由于超动力运动对明显认知终点的潜在影响，应进一步检查数据。 </li><li>使用单向方差分析测试分析进入每个手臂的条目数。 注意:如果这个分析是重要的，那么这将表明在环境中存在吸引小鼠到特定区域的线索在迷宫上。 </li></ol></li></ol> 4.通过测量新对象识别来评估中期识别记忆11,12,13 <ol><li>对于该测试的每个阶段，在初始使用之前，用无味的漂白杀菌擦拭剂，70％EtOH，然后用dH 2 O彻底清洁开放场地。 </li><li> 在暴露对象前一天，将老鼠习惯于野外野外。 <ol><li>在习惯开始之前，设置数据采集系统或摄像机，并确认迷宫中鼠标的正确跟踪。使用已知长度的标尺或其他物体的捕获视频图像来校准舞台上的距离。在软件中标记竞技场的角落，以允许评分位置偏差。 注意:此过程中的行为方法将适用于各种数据采集系统茎和作者假设执行此过程的任何人精通使用他们选择的数据采集系统。功率分析表明，β≤0.2需要每组15-20只小鼠的样本量。 </li><li>从机架上取下笼子，轻轻地放在靠近竞技场的桌子上。 </li><li>从家笼中取出鼠标，轻轻将鼠标放在竞技场的中心。将鼠标放入竞技场后立即打开跟踪软件和/或录像系统。 </li><li>允许老鼠自由探索竞技场30分钟。 注意:在此期间，调查员不会打扰小鼠。 </li><li>习惯性会话后，将小鼠放回自己的家笼内，用无味的漂白杀菌擦拭剂70％EtOH，然后用dH 2 O彻底清洗。 </li><li>从步骤4.2.2重复，直到所有的老鼠都习惯于竞技场。 </li><li>所有的老鼠都习惯了分享到舞台，分析视频。 注意:要分析的终点包括竞技场中的总距离和每个角落附近花费的时间。如果与小鼠模型相关，这些分析中包括刻板行为（ 即 ，肌阵挛角跳，盘旋等 ）。在竞技场的特定地区表现出偏袒的小鼠被排除在进一步的实验之外，因为这将影响对象探索。 注意:新颖的对象识别的第一阶段涉及熟悉一个对象的小鼠。在这里，新的对象识别过程的这一部分将被称为样本阶段。 </li><li>在采样阶段开始之前，将物体放置在竞技场中，并将其固定在地板上，并安装腻子，以使动物无法移动物体。将两个相同的物体对准一个墙壁和物体之间的足够距离的特定墙壁，以便老鼠可以从所有角度自由探索物体LES。 </li><li>设置数据采集系统或摄像机，并确认迷宫中鼠标和对象的正确跟踪。使用已知长度的标尺或其他物体的捕获的视频图像校准竞技场中的距离。 </li><li>在软件中标记竞技场的角落，以允许评分位置偏差。在软件中标记对象并分别跟踪每个对象的探索行为（ 即 "对象A"和"对象B"）。 </li><li>从机架上取下机架，轻轻将其放在靠近舞台的桌子上。 </li><li>从家笼中取出鼠标，轻轻将其放入竞技场的中心，面对物体。 </li><li>让鼠标自由探索物体15分钟。在此期间不要打扰老鼠。 </li><li>在会话结束时，将鼠标轻轻放回家中的笼子中。用70％EtOH和dH 2 O清洁竞技场和物体。将这些物品放回e竞技场</li><li>重复步骤4.2.11，直到所有小鼠熟悉一个对象。 </li><li>一旦所有的老鼠熟悉一个对象，分析视频。 注意:一旦符合以下条件，对象探索将被计数:鼠标面向对象，鼻子在对象的2厘米以内，动物身体的中点距离对象超过2厘米，并且以前的标准已经达到至少1秒。另外，如果一只动物已经满足了勘探标准，但是表现为10秒以上的不动，那么这个探索性的回合就被认为是完成的。 </li><li>计算每个鼠标的对象偏差得分如下。 <img alt="公式2" src="/files/ftp_upload/55523/55523eq2.jpg" /> 注意:显示目标偏差得分低于20％或高于80％的小鼠被排除在进一步的实验之外。 </li></ol></li><li> 新的对象识别的最后阶段涉及评估探索行为指向环境中的一个新颖和熟悉的对象，这里被称为测试阶段。此阶段在样品相完成2-3小时后进行。 <ol><li>在测试阶段会话开始之前，将对象放入竞技场，并将其固定在地板上，以使动物无法移动对象。 <ol><li>将物体放在相对于样品阶段13的竞技场中的相同位置。 </li><li>平衡基因型和治疗组中新颖和熟悉的对象的相对位置。 </li><li>确保墙壁和物体之间有足够的距离，以便老鼠可以从各个角度自由探索物体。 </li></ol></li><li>设置数据采集系统和/或摄像机。确认在迷宫中正确追踪小鼠和物体。使用已知长度的标尺或其他物体的捕获视频图像校准竞技场中的距离。 </li><li>在软体中标记竞技场的角落是允许得分的位置偏见。用软件标记对象，并对每个对象（ 即 "小说"和"熟悉"）追踪探索性行为。 </li><li>从机架上取下机架，轻轻将其放在靠近舞台的桌子上。 </li><li>轻轻地将动物放在竞技场的中心，面对物体。记录小鼠自由探索物体10分钟。 </li><li>在测试结束时，将小鼠从竞技场中取出，将小鼠放回家中。每个会话之后，彻底清洁竞技场和物体，含有无味的漂白杀菌擦拭剂，70％EtOH和dH 2 O。 </li><li>从步骤4.4.3重复，直到所有动物进行了评估。 </li><li>一旦对所有小鼠进行物体探查，就分析视频。 注意:满足以下条件后，对象探索进行计数:鼠标面向对象，鼻子位于对象的2厘米以内，中点动物的身体距离物体超过2厘米，并且以前的标准已经达到至少1秒。另外，如果一只动物已经满足了勘探标准，但是表现为10秒以上的不动，那么这个探索性的回合就被认为是完成的。 </li><li>通过比较探索小说与熟悉对象的时间来评估新颖的对象识别。文献中通常报道了三种方法。 <ol><li>分析使用重复测量测试探索新颖和熟悉的对象的原始时间。当基因型和/或治疗不影响总的探索时间时，最好使用这种方法。 </li><li>使用以下公式计算新颖性偏好: <img alt="公式3" src="/files/ftp_upload/55523/55523eq3.jpg" /> 注意:这提供了探索新对象相对于探索对象的总时间所花费的时间百分比。价值范围从0％（没有探索新物体）到100％（仅探索小说object），值为50％，表示探索小说和熟悉对象的时间相等。 </li><li>计算辨别指数11 ，使用下式: <img alt="公式4" src="/files/ftp_upload/55523/55523eq4.jpg" /> 注意:这产生了相对于探索对象的总时间来探索小说和熟悉对象的时间差异。值范围从-1（只有熟悉的对象的探索）到+1（仅探索新颖的对象，值为0表示等待时间花费在探索小说和熟悉的对象上。 </li></ol></li><li>从动物运动或其他刻板印象中删除不参加测试的动物，从考虑11 。 注意:用于去除的标准必须客观和先验确定小鼠模型（ 即 ，总探查时间的第 5百分位数，&gt; 100平均值测试期间转弯角度或呈现肌阵挛角跳跃的第 50百分位数时间）。 </li></ol></li></ol> 5.用肢体夹住评估小鼠皮质脊髓功能14 <ol><li>视频记录整个会话。使用便携式手持设备（ 即 ，智能手机或等效物体）录制视频。 注意:功率分析表明，β≤0.2需要每组10-15只小鼠的样本量。 </li><li>将家用笼子从机架上取下并放在桌子上。在下一步之前记录视频中的动物ID。 </li><li>轻轻地将鼠标从笼子中取出并由尾巴悬挂5-10秒。视频必须在暂停时记录动物的后爪和前爪。 </li><li>拍摄至少5秒的视频后，将鼠标放回家中的笼子，然后将笼子放回机架。 </li><li>清理桌子。从步骤5.2重复，直到所有的小鼠都被记录<html>天。 </li><li> 评分肢体从小尾巴悬挂的小鼠的视频上按0-4的比例（见表1的评分描述）。检查暂停的小鼠的视频，然后根据以下标准分配分数。 <ol><li>没有肢体紧握。正常逃生扩展。一个后肢显示不完全的显示和流动性的丧失。脚趾表现正常。 </li><li>两肢后肢表现不均匀，流动性下降。脚趾表现正常。 </li><li>两个后肢都表现出蜷缩的脚趾和不动。 </li><li>前肢和后肢表现出扣合，交叉，卷曲的脚趾和不动。 </li></ol></li><li> 所有小鼠均由2名独立研究者评分。任何一个2分的差异超过1分的鼠标再次被打破。 <ol><li>不同的得分平均。 </li></ol></li></ol>p_upload / 55523 / 55523table1.jpg"/&gt; 表1:肢体分离得分的描述。

<ol>
	<li>Elder, G. A., Gama Sosa, M. A., De Gasperi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mouse+models+of+Alzheimer's+disease.">Transgenic mouse models of Alzheimer's disease.</a> Mt Sinai J Med. 77 (1), 69-81 (2010).</li><li>Onos, K. D., Sukoff Rizzo, S. J., Howell, G. R., Sasner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+more+predictive+genetic+mouse+models+of+Alzheimer's+disease.">Toward more predictive genetic mouse models of Alzheimer's disease.</a> Brain Res Bull. 122, 1-11 (2016).</li><li>Webster, S. J., Bachstetter, A. D., Nelson, P. T., Schmitt, F. A., Van Eldik, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+mice+to+model+Alzheimer's+dementia:+an+overview+of+the+clinical+disease+and+the+preclinical+behavioral+changes+in+10+mouse+models.">Using mice to model Alzheimer's dementia: an overview of the clinical disease and the preclinical behavioral changes in 10 mouse models.</a> Front Genet. 5, 88 (2014).</li><li>Rodgers, S. P., Born, H. A., Das, P., Jankowsky, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+APP+expression+during+postnatal+development+causes+persistent+locomotor+hyperactivity+in+the+adult.">Transgenic APP expression during postnatal development causes persistent locomotor hyperactivity in the adult.</a> Mol Neurodegener. 7, 28 (2012).</li><li>Hsiao, K., 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=Correlative+memory+deficits,+Abeta+elevation,+and+amyloid+plaques+in+transgenic+mice.">Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice.</a> Science. 274 (5284), 99-102 (1996).</li><li>Santacruz, K., 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=Tau+suppression+in+a+neurodegenerative+mouse+model+improves+memory+function.">Tau suppression in a neurodegenerative mouse model improves memory function.</a> Science. 309 (5733), 476-481 (2005).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> What's wrong with my mouse? : behavioral phenotyping of transgenic and knockout mice. 2nd edn. , (2007).</li><li>Hughes, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+value+of+spontaneous+alternation+behavior+(SAB)+as+a+test+of+retention+in+pharmacological+investigations+of+memory.">The value of spontaneous alternation behavior (SAB) as a test of retention in pharmacological investigations of memory.</a> Neurosci Biobehav Rev. 28 (5), 497-505 (2004).</li><li>Rutala, W. A., Weber, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guideline+for+disinfection+and+sterilization+in+healthcare+facilities.">Guideline for disinfection and sterilization in healthcare facilities.</a> Centers for Disease Control. , (2008).</li><li>Wes, P. D., 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=Tau+overexpression+impacts+a+neuroinflammation+gene+expression+network+perturbed+in+Alzheimer's+disease.">Tau overexpression impacts a neuroinflammation gene expression network perturbed in Alzheimer's disease.</a> PLoS One. 9 (8), 106050 (2014).</li><li>Ennaceur, A., Delacour, 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+new+one-trial+test+for+neurobiological+studies+of+memory+in+rats.+1:+Behavioral+data.">A new one-trial test for neurobiological studies of memory in rats. 1: Behavioral data.</a> Behav Brain Res. 31 (1), 47-59 (1988).</li><li>Taglialatela, G., Hogan, D., Zhang, W. R., Dineley, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermediate-+and+long-term+recognition+memory+deficits+in+Tg2576+mice+are+reversed+with+acute+calcineurin+inhibition.">Intermediate- and long-term recognition memory deficits in Tg2576 mice are reversed with acute calcineurin inhibition.</a> Behav Brain Res. 200 (1), 95-99 (2009).</li><li>DeVito, L. M., Eichenbaum, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+contributions+of+the+hippocampus+and+medial+prefrontal+cortex+to+the+"what-where-when"+components+of+episodic-like+memory+in+mice.">Distinct contributions of the hippocampus and medial prefrontal cortex to the "what-where-when" components of episodic-like memory in mice.</a> Behav Brain Res. 215 (2), 318-325 (2010).</li><li>Lalonde, R., Strazielle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+regions+and+genes+affecting+limb-clasping+responses.">Brain regions and genes affecting limb-clasping responses.</a> Brain Res Rev. 67 (1-2), 252-259 (2011).</li><li>King, D. L., Arendash, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+characterization+of+the+Tg2576+transgenic+model+of+Alzheimer's+disease+through+19+months.">Behavioral characterization of the Tg2576 transgenic model of Alzheimer's disease through 19 months.</a> Physiol Behav. 75 (5), 627-642 (2002).</li><li>Lalonde, R., Lewis, T. L., Strazielle, C., Kim, H., Fukuchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+expressing+the+betaAPP695SWE+mutation:+effects+on+exploratory+activity,+anxiety,+and+motor+coordination.">Transgenic mice expressing the betaAPP695SWE mutation: effects on exploratory activity, anxiety, and motor coordination.</a> Brain Res. 977 (1), 38-45 (2003).</li><li>Lewis, 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=Neurofibrillary+tangles,+amyotrophy+and+progressive+motor+disturbance+in+mice+expressing+mutant+(P301L)+tau+protein.">Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein.</a> Nat Genet. 25 (4), 402-405 (2000).</li><li>Spittaels, K., 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=Prominent+axonopathy+in+the+brain+and+spinal+cord+of+transgenic+mice+overexpressing+four-repeat+human+tau+protein.">Prominent axonopathy in the brain and spinal cord of transgenic mice overexpressing four-repeat human tau protein.</a> Am J Pathol. 155 (6), 2153-2165 (1999).</li><li>Terwel, D., 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=Changed+conformation+of+mutant+Tau-P301L+underlies+the+moribund+tauopathy,+absent+in+progressive,+nonlethal+axonopathy+of+Tau-4R/2N+transgenic+mice.">Changed conformation of mutant Tau-P301L underlies the moribund tauopathy, absent in progressive, nonlethal axonopathy of Tau-4R/2N transgenic mice.</a> J Biol Chem. 280 (5), 3963-3973 (2005).</li><li>Merrow, M., Spoelstra, K., Roenneberg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+cycle:+daily+rhythms+from+behaviour+to+genes.">The circadian cycle: daily rhythms from behaviour to genes.</a> EMBO Rep. 6 (10), 930-935 (2005).</li><li>Smarr, B. L., Jennings, K. J., Driscoll, J. R., Kriegsfeld, L. 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+time+to+remember:+the+role+of+circadian+clocks+in+learning+and+memory.">A time to remember: the role of circadian clocks in learning and memory.</a> Behav Neurosci. 128 (3), 283-303 (2014).</li><li>Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+memory+storage:+a+dialogue+between+genes+and+synapses.">The molecular biology of memory storage: a dialogue between genes and synapses.</a> Science. 294 (5544), 1030-1038 (2001).</li><li>Stough, S., Shobe, J. L., Carew, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermediate-term+processes+in+memory+formation.">Intermediate-term processes in memory formation.</a> Curr Opin Neurobiol. 16 (6), 672-678 (2006).</li><li>Stevens, L. M., Brown, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+and+working+memory+deficits+in+the+3xTg-AD+mouse+between+2+and+15-months+of+age:+a+cross-sectional+study.">Reference and working memory deficits in the 3xTg-AD mouse between 2 and 15-months of age: a cross-sectional study.</a> Behav Brain Res. 278, 496-505 (2015).</li><li>Yue, X., 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=Brain+estrogen+deficiency+accelerates+Abeta+plaque+formation+in+an+Alzheimer's+disease+animal+model.">Brain estrogen deficiency accelerates Abeta plaque formation in an Alzheimer's disease animal model.</a> Proc Natl Acad Sci U S A. 102 (52), 19198-19203 (2005).</li><li>McAllister, C., 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=Genetic+targeting+aromatase+in+male+amyloid+precursor+protein+transgenic+mice+down-regulates+beta-secretase+(BACE1)+and+prevents+Alzheimer-like+pathology+and+cognitive+impairment.">Genetic targeting aromatase in male amyloid precursor protein transgenic mice down-regulates beta-secretase (BACE1) and prevents Alzheimer-like pathology and cognitive impairment.</a> J Neurosci. 30 (21), 7326-7334 (2010).</li><li>Blaney, C. E., Gunn, R. K., Stover, K. R., Brown, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+genotype+influences+behavioral+development+of+3xTg-AD+mouse+pups.">Maternal genotype influences behavioral development of 3xTg-AD mouse pups.</a> Behav Brain Res. , 40-48 (2013).</li></ol>

JM Levenson由Proclara Biosciences，Inc.雇用.Miedel，J.Partton，A.Miedel和E.Miedel由Hilltop Laboratory Animals雇用。

技术对现有方法的重要意义 该方法设计用于筛选化合物在β-淀粉样变性和tau蛋白病的转基因小鼠模型中的体内活性。这里使用的分期方法确保检测与AD 3相关的认知领域中的有效化合物。此外，此处详细介绍的方法使用具有明确定义的端点的行为测试，易于实施的质量控制检查，可以以适度的吞吐量格式运行，并且几乎不需要调查员的干预。这些特征导致在动物和跨群体中表现出良好的再现性的测定法，其导致低的体内和分析间差异和效应大小（2≤f≤6），其足够强大以支持药物发现环境中的行为分析。 Pr。中的关键步骤otocol 用于AD药物发现的许多小鼠模型表现出与高度焦虑和侵略相一致的行为。这使得处理习惯对于执行此处描述的任何行为测试至关重要。由于这些测试依赖于无动力的行为，由于过度活跃和焦虑或侵略性的小鼠，研究者的粗暴处理可能会显着影响性能。焦虑增加可能导致执行任务失败，降低了测试的整体力量。此外，竞技场的光线水平对于促进每次测试所需的自发运动至关重要。明亮的光线往往会增加焦虑，抑制啮齿动物的运动，因此应注意调整环境光线水平至30-35勒克斯。 该过程的另一个重要方面是最小化强烈的环境线索，这将妨碍动物执行任务的能力。清洁竞技场和运动之间的对象是必不可少的，因为小鼠被吸引到环境中的新鲜气味。未能彻底清洁竞技场和物体可能会导致鼠标的自发活动倾斜，并掩盖真正的认知表现。调查员也应尽量减少在执行这些程序时使用个人卫生用品和古龙水/香水。最后，啮齿动物在许多公开行为20中表现出强大的昼夜和昼夜变化，包括学习和记忆21 。因此，为了尽可能减少由于基础行为和认知表现的昼夜节律引起的差异，所有的测试都应该在同一时间进行队列和研究。 此外，具体地关于新颖的对象识别，样本和测试阶段之间的延迟间隔以及对象在环境中的选择和放置是关键参数。内存存在3种不同的形式:短期记忆ory（STM），中期记忆（ITM）和长期记忆（LTM） 22,23 。将样本和测试阶段之间的间隔从分钟（STM）更改为小时（ITM）或天数（LTM）将会更改程序12测试的内存类型。此外，在运行新颖的对象识别测试之前，应该在小鼠的测试队列中筛选许多对象，用于潜在的探索偏倚。在评估新颖的对象识别时，不能使用对测试队列过于吸引或排斥的对象。理想情况下，在测试中使用的所有对象，当放置在竞技场中时，将从幼稚的小鼠中引出相同的探索时间。物体的测试和优化不足可以显着降低新物体识别的力量。 修改和故障排除 有几个因素可能会增加这里描述的认知测试中的表观变异性。 AD的许多小鼠模型表现出超动力运动3 ，其可以掩盖或改变作为认知终点测量的行为。此外，越来越多的证据表明， 24,25,26甚至母亲基因型27可以影响AD小鼠模型中神经病理学和认知表型的发展和进展。意外的变化或未能实施行为任务可能是由于这些因素中的任何一个。当首次实施特定行为测试时，结果应始终按性别，年龄和母亲基因型分类。此外，应始终执行本程序中概述的质量检查，以确保多动症或其他定型行为不会干扰认知终点的量化。 信封熨烫也可以影响啮齿动物的自发探索行为。对研究人员无法察觉的气味或声音可能会吸引或排斥小鼠，从而偏离依赖于自发行为的认知测试结果。当初始建立Y迷宫或新颖的物体识别时，执行控制措施以确保在物体和/或环境的勘探中没有位置偏差是至关重要的。如果观察到位置偏差，调查员必须彻底审查环境，并可能调整照明，竞技场位置，测试室相对于设施中其他房间的位置（ 即 ，不靠近高流量区域或重型设备）和竞技场清洁程序。 测试环境的习惯是在新颖的对象识别测试中实现最佳性能的关键。例如，总勘探时间较短可能是由于习惯不适。作为proc的替代方法本文概述的处理（第2节）和竞技场（第4.2节）习惯，习惯处理和测试环境可以每天3,5分钟的时间进行连续2天。 技术的局限性 与任何程序一样，这些行为测试有局限性。已经使用这些程序，因为它们测试各种皮层区域和海马的功能。如果小鼠模型在这些测试探测到的脑区域中没有显示功能缺陷，那么这些技术将不会有用。此外，我们选择了探索短期记忆的认知测试。如果化合物临床前评估的作用机制不会影响短期记忆，则应相应修改这些程序（ 即增加样本 - 测试相间隔以测试长期记忆）。最后，这些测试使用没有动机的行为。因此，如果鼠标模型是exces活跃的活动或显示其他刻板行为，阻止对环境的探索，那么这些程序可能不是最佳的。作为替代方案，可以使用Tg2576或其他β-淀粉样变性小鼠模型的恐惧调理，或者用于rTg4510或其他小鼠模型的tau蛋白病3的空间水迷宫。 未来的应用 一旦这些程序已经在实验室中成功采用，可以进行几项修改或扩展，以评估额外的认知和功能运动措施。例如，改变新颖的对象识别任务以确定鼠标是否可以识别对象13的位置的改变。或者，不是使用对象，可以使用其他鼠标并实施社会识别测试。关于肢体夹紧和运动功能，可以用线悬挂和/或抓力测试来补充该测试。测试在这种方法中详细描述形成一个坚实的基础来筛选在 AD的翻译小鼠模型中具有体内功效的化合物，并且可以以许多方式进行调整或修饰以最好地询问特定的小鼠模型或满足独特药物发现程序的需要。

阿尔茨海默病（AD）是进行性神经变性疾病，导致认知衰退衰弱，影响全球约4400万人。目前尚无AD治疗可以进行疾病修复，强调迫切需要临床前发现这种疾病的新型治疗策略。已经创建了许多不同的转基因小鼠模型，其重述了AD1,2的各个方面，包括在患者中破坏的认知结构域的缺陷3 。这些小鼠模型代表有助于在体内有效筛选的工具。 当评估化合物潜在的体内功效时，必须采取分阶段的方法，筛选适当的认知领域的功效，还可以监测可能影响特定终点的行为ssess认知AD的许多转基因小鼠模型表现出多动症和其他可能干扰特定认知测试的行为，并禁止其用于药物筛选4 。此外，对于在药物筛选环境中实施的方法，所使用的特定测试应至少保持适度的产量，具有明确定义的终点，以及调查人员最少干预的程序。使用这些标准，可以实施行为屏幕，其显示复合筛选所需的再现性，低的测定内和测定间差异以及效果尺寸。这里详细描述了我们用于筛选有效减轻β-淀粉样变性和tau蛋白病转基因小鼠模型中存在的认知和运动表型的化合物的方法5,6 。所描述的方法适用于l中报告的常用行为范例迭代7 ，具有特定的优化和质量控制检查，以便它们可以用于与AD相关的转基因小鼠模型。该协议可用于各种数据采集和分析系统，并假设调查员具有相关软件的工作知识。

<table><tbody><tr><td>Topscan Lite-High Throughput</td><td>Cleversys</td><td></td><td>Automated behavioral analysis. Includes cameras and video acquisition system, laptop.</td></tr><tr><td>ObjectScan</td><td>Cleversys</td><td></td><td>Software module for accurate object exploration quantification</td></tr><tr><td>Open field for mouse</td><td>Cleversys</td><td>CSI-OF-M</td><td>Arena for novel object recognition</td></tr><tr><td>Y-maze for mouse</td><td>Custom</td><td></td><td>Arms: 30 cm long, 10 cm wide, 20 cm high walls, placed 120 deg apart.</td></tr><tr><td>Camera mount for open field</td><td>Custom</td><td>Custom</td><td>76 cm tall, 115 cm wide, cameras mounted @ 30 cm in from either side.&amp;nbsp; Two mounts, each covers two boxes.</td></tr><tr><td>Camera mount for Y-maze</td><td>Custom</td><td>Custom</td><td>76 cm tall, 115 cm wide, cameras mounted @ 30cm in from either side.&amp;nbsp; One mount covers two mazes.</td></tr><tr><td>Marbles</td><td>Inperial Toy</td><td>8565</td><td>Standard (15.5 mm Dia) glass marbles.</td></tr><tr><td>Dice</td><td>Cardinal Industries</td><td>770</td><td>Standard (0.650 inch) white dice with black dots.</td></tr><tr><td>LOCTITE Fun-Tak</td><td>Henkel</td><td>B018A3AG0W</td><td>Standard blue sticky tak</td></tr><tr><td>EtOH</td><td>Nexeo Solutions</td><td>82452</td><td>100% Ethanol Diluted to 70% using distilled Water</td></tr><tr><td>dH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Tulpenhocken Spring Water Co.</td><td>-</td><td>PA D.E.P. #31, NJ D.O.H. #0049, NYSHD Cert. #320</td></tr><tr><td>Paper towels</td><td>Procter &amp;amp; Gamble</td><td>B019DM86LA</td><td>Bounty, White</td></tr><tr><td>Handheld video camera</td><td>Apple, Inc.</td><td>MKV92LL/A</td><td>Acquisition of Limb clasping video, Iphone 6S Plus (or functional equivalent).</td></tr><tr><td>Gloves</td><td>SafePOINT, L.L.C.</td><td>GL640-2</td><td>Standard, Powder free Latex Gloves, Medium</td></tr><tr><td>Light meter</td><td>Dr. Meter</td><td>LX1330B</td><td>Lighting @ the bottom of Open Field= 35 LUX, Lighting @ bottom of Y-Maze= 32 LUX</td></tr><tr><td>Bleach germicidal wipes</td><td>Clorox</td><td></td><td>Sterilization of equipment during &amp;amp; after use</td></tr></tbody></table>

assessment of spontaneous alternation, novel object recognition and limb clasping in transgenic mouse models of amyloid-&#946; and tau neuropathology

在这里，我们描述了一种分阶段的行为测试方法，可用于筛选在β淀粉样变性和tau蛋白病的转基因小鼠模型中对体内功效的认知和功能运动行为的化合物的筛选。范式包括Y迷宫中自发交替的测试，新颖的对象识别和肢体扣紧。选择这些测试是因为:1）询问认知或运动领域的功能以及与人类疾病状态相关的相关神经电路，2）具有明确界定的终点3）可以轻松实施质量控制检查4）中等吞吐量格式，以及5）调查员几乎不需要干预。这些方法设计用于寻找筛选化合物用于短期和工作记忆任务或与阿尔茨海默病小鼠模型相关的功能运动行为的研究者。这里描述的方法使用行为测试年龄不同的大脑区域包括海马和各种皮层区域。要求认知测试专门评估由单个脑区介导的认知的研究者可以使用这些技术补充其他行为测试。

老化的Tg2576小鼠表现出在Y-迷宫15,16内发生的自发变化的强大缺陷，可以使用本文详述的方法复制表型（ 图1A ）。虽然在这些小鼠中观察到增加的臂进入的趋势（ 图1B ），但是在该小鼠系列中观察到的多动症并不影响自发的交替率（ 图1C ）。相比之下，老化的rTg4510小鼠在放置在Y型迷宫中似乎表现出增加的自发交替（ 图1D ）。这是由于极度多动（ 图1E ）和定型10 ，这显着干扰了自发交替的测量（ 图1F ）。最初在这项任务中评估小鼠时，确保手臂输入和/或行驶距离不是至关重要的与自发交替率显着相关。 在评估新颖的对象识别之前，小鼠习惯于进行测试的竞技场。在习惯中，可以评估与小鼠模型相关的多动症（ 图2A ）和其他定型行为。在样本阶段，单独测量每个对象的检测是至关重要的，因此在进一步的评估中可以排除在探索行为中显示出显着偏差的小鼠（ 图2B ，空心圆圈）。通过比较一个熟悉和新颖的对象的探索来评估新的对象识别，并以三种不同的方式进行分析。如果总体探索时间在基因型和/或治疗组之间是可比较的，则可以使用探索每个对象的原始时间和适当的重复测量测试来确定新物体r是否存在差异识别（ 图2C ）。如果特定的小鼠品系在总探索时间上表现出差异，则可以使用新颖性偏好（ 图2D ）或鉴别指数（ 图2E ）来评估新颖的对象识别。 肢体扣紧是量化皮质脊髓功能缺陷的功能运动测试。在几种转基因tau小鼠模型6,17,18,19中观察到肢体扣紧，其不是认知措施，并且概括了晚期AD患者中观察到的一些功能性运动障碍。由尾巴悬挂小鼠引起逃生反应（ 图3A ，"0"）。根据他们的严重程度，从0-4开始，打破后肢和伸展脚趾的能力下降得分（ 图3A）。使用本文概述的程序，可以观察到在rTg4510小鼠中显着的肢体夹紧（ 图3B ）。 <img alt="图1" src="/files/ftp_upload/55523/55523fig1.jpg" /> 图1:Y迷宫中的自发交替。 （ A ）当放置在Y型迷宫中时，小鼠采用失调搜索策略，导致探索模式，每三臂进入一次，每只手臂只进行一次探索。老化的Tg2576小鼠表现出自发交替的显着缺陷。使用该方法中概述的程序，在用专有化合物处理后观察到自发交替的显着恢复。使用单因素方差分析数据，并使用Dunnett检验进行与Tg-PBS的事后比较。 ** p &lt;0.01。误差条表示SEM。 （ B ）任何一个手臂条目的数量没有显着差异在本实验中监测的组。使用单因素方差分析测试数据。误差条表示SEM。 （ C ）自发交替与所进行的手臂数量之间无相关性，表明自发运动活动中的任何差异不影响自发交替的量化。使用Pearson相关分析进行相关性检验。 （ D ）当置于Y型迷宫中时，相对于同窝鼠WT小鼠，rTg4510小鼠（6个月）似乎显示出显着更多的自发交替。通过单因素方差分析数据，并使用Dunnett检验进行与Tg-PBS的事后比较。 ** p &lt;0.01。误差条表示SEM。 （ E ）rTg4510小鼠由于极端的高动力运动而显着增加了手臂入口。通过单因素方差分析数据，并使用Dunnett检验进行与Tg-PBS的事后比较。 *** p &lt;0.001。错误栏表示SE（ F ）自发交替行为与手臂条目显着相关，表明超动力运动表型掩盖了真实的自发交替。使用Pearson相关分析进行相关性检验（r = 0.7，p &lt;0.0001）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55523/55523fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55523/55523fig2.jpg" /> 图2:新对象识别。 （ A ）竞技场习惯允许测量与特定小鼠模型相关的自发运动和其他定型行为。在这里，老年Tg2576小鼠（22个月）相对于同窝鼠WT小鼠显示出显着更多的自发运动。数据通过单因素方差分析进行分析，并使用Dunnett&#39;的测试。 ** p &lt;0.01。误差条表示SEM。 （ B ）在采样阶段，分别追踪两个相同物体的探测。从测试阶段排除显示对探测两个物体（空心圆）之一的大偏差的小鼠。 （ C - E ）通过测量一个新颖和熟悉的对象的探索来评估新的对象识别。使用（ C ）原始探索时间，（ D ）新颖性偏好或（ E ）辨别指数来评估新的对象识别。使用重复测量的双向ANOVA分析C组中的数据，并使用Sidak检验进行成对比较。使用单因素方差分析进行面板DE中的数据，并使用Dunnett检验进行与Tg-Veh的事后比较。 * p &lt;0.05，** p &lt;0.01。误差条表示SEM。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55523/55523fig2large.jpg" target="_blank">请点击</a>这里可以查看这个数字的较大版本。 <img alt="图3" src="/files/ftp_upload/55523/55523fig3.jpg" /> 图3:肢体扣紧。 （ A ）如表1所示，表现出各种程度的肢体紧握的小鼠的代表性图像。（ B ）rTg4510小鼠（6个月）相对于同窝小鼠WT小鼠显示出显着的肢体扣紧，使用这些方法评分。使用单因素方差分析数据，并使用Dunnett检验进行与Tg-PBS的事后比较。 *** p &lt;0.001，**** p &lt;0.0001。误差条表示SEM。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55523/55523fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-Î² and Tau Neuropathology at JoVE.co

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

这里描述的是一种分阶段的行为筛选方法，可用于筛选在β淀粉样变性和tau蛋白病的转基因小鼠模型中对体内功效的认知功能运动行为进行筛选的化合物。这些方法被优化以筛选化合物用于短期和工作记忆任务中的活动。

自发交替评估，淀粉样蛋白β和脑神经病理学转基因小鼠模型中的新物体识别和肢体紧扣

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

assessment-spontaneous-alternation-novel-object-recognition-limb

Universidad San Sebastian

Research

JoVE Journal

Medicine

26.5K 次观看.  Hilltop Laboratory Animals. 这里描述的是一种分阶段的行为筛选方法，可用于筛选在β淀粉样变性和tau蛋白病的转基因小鼠模型中对体内功效的认知功能运动行为进行筛选的化合物。这些方法被优化以筛选化合物用于短期和工作记忆任务中的活动。 

视频: Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-β and Tau Neuropathology

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer&#8217;s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer&#8217;s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

Morris水迷宫实验:优化小鼠品系和测试环境

 The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, ...

科研

行为学

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

在阿尔茨海默氏症小鼠模型神经炎斑检测

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

神经科学

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

一个串行收集脑脊液从池鼠标麦格纳技术

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

生物学

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

嗅觉测定的神经退行性疾病的小鼠模型

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

正常小鼠β淀粉样肽侧脑室注射敏锐地诱导老年痴呆样认知缺陷

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer's Disease with Stroke

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk factor for AD, stroke has also been implicated as a risk factor and an exacerbating factor. The co-morbidity of stroke and AD results in worsened stroke-related motor control and AD-related cognitive deficits when compared to each condition alone. To model the combined condition of stroke and AD, a novel transgenic rat model of AD, with a mutated form of amyloid precursor protein (a key protein involved in the development of AD) incorporated into its DNA, is given a small unilateral striatal stroke.
For a model with the combination of both stroke and AD, behavioral tests that assess stroke-related motor control, locomotion and AD-related cognitive function must be implemented. The cylinder task involves a cost-efficient, multipurpose apparatus that assesses spontaneous forelimb motor use. In this task, a rat is placed in a cylindrical apparatus, where the rat will spontaneously rear and contact the wall of the cylinder with its forelimbs. These contacts are considered forelimb motor use and quantified during video analysis after testing. Another cost-efficient motor task implemented is the beam-walk task, which assesses forelimb control, hindlimb control and locomotion. This task involves a rat walking across a wooden beam allowing for the assessment of limb motor control through analysis of forelimb slips, hindlimb slips and falls. Assessment of learning and memory is completed with Morris water maze for this behavioral paradigm. The protocol starts with spatial learning, whereby the rat locates a stationary hidden platform. After spatial learning, the platform is removed and both short-term and long-term spatial reference memory is assessed. All three of these tasks are sensitive to behavioral differences and completed within 28 days for this model, making this paradigm time-efficient and cost-efficient.

To investigate the co-morbid Alzheimer&#39;s disease (AD) and stroke condition in a novel model, three behavior tasks are described that assess both motor control and cognitive behaviors. These tasks include the beam-walk task, cylinder task and Morris water maze.

汽车和海马依赖空间学习和记忆参考评估与中风阿尔茨海默氏病的转基因大鼠模型

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk ...

医学

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

使用基于酶的生物传感器来衡量进补和阶段性谷氨酸在阿尔茨海默氏症小鼠模型

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

一种新的用于全中枢神经系统光透明快速生成的无源清除方法

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

正常小鼠和亨廷顿小鼠急性脑切片中谷氨酸释放和服用的单突触指标

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...