Requirement for Hippocampal CA3 N M D A Receptors in Associative Memory Recall
Kazu Nakazawa,etc SCIENCE VOL 297 12 JULY 2002
Pattern completion, the ability to retrieve complete memories on the basis of incomplete sets of cues, is a crucial function of biological memory systems. The extensive recurrent connectivity of the CA3 area of hippocampus has led to suggestions that it might provide this function. We have tested this hypothesis by generating and analyzing a genetically engineered mouse strain in which the N-methyl-D-asparate N M D A receptor gene is ablated specifically in the CA3 pyramidal cells of adult mice. The mutant mice normally acquired and retrieved spatial reference memory in the Morris water maze, but they were impaired in retrieving this memory when presented with a fraction of the original cues. Similarly, hippocampal CA1 pyramidal cells in mutant mice displayed normal place-related activity in a full-cue environment but showed a reduction in activity upon partial cue removal. These results provide direct evidence for CA3 N M D A receptor involvement in associative memory recall。
基因转移的方法介绍，以鱼类为例 >
2．1 显微注射法
显微注射法是目前广泛使用、效果较好的一种鱼类基因导人方法，其主要 程序如下：(1)人工催情，分别收集鱼的卵子和精液，体外人工受精；(2)受精后3～5min，用0．25％的胰蛋白酶消耗以去除卵壳，将裸卵移入盛有 Holtfreter氏培养液的平面皿中；(3)将溶于ST(88mMNaCl、10mMTris—HCl，pH7．5)溶液中的外源基因装人玻璃微针， 在第一次卵裂前实施外源基因的显微注射手术。每卵注射1—2nL的DNA溶液，约含1×106拷贝的外源基因；(4)显微注射后，受体卵在 Holffreter氏溶液中培养发育。胚胎发育至原肠期后，将培养液逐渐用暴气的冷开水稀释；发育至心跳期后，将胚胎转移到暴气的冷开水中直至形成鱼 苗。在胚胎发育过程中，需精心管理，及时去除死胚胎。
上述方法主要适用于一些卵壳易被胰蛋白酶消化的淡水鲤科鱼类。对于一些冷水性的鲑鳟鱼类来 说，胰蛋白酶消化难以去除卵壳。因此发展了3种变通的显微注射方法。一是从受精孔将DNA溶液注入卵中，简称MP法；二是在虹鳟受精卵刚受精后卵壳尚未变 硬时直接注射，简称EL法；三是先用硬金属针在卵壳上打一个孔，再进行显微注射，简称LI法。
2．2 电脉冲法
电脉冲进行鱼类受精卵基因 转移的前两步与显微注射法相同。不同的是将胰酶消化后的裸卵与外源DNA溶液一同放人一特制的电脉冲处理槽中，然后施加一定强度的电脉冲。外源DN在电脉 冲处理下进入受精卵。这一方法的优点是操作比较简单，可同时处理大量的受精卵。缺点是导人无定向、效率较低，针对不同种鱼需要建立相应的电脉冲条件等。
外 源基因在电脉冲处理条件下进人受精卵的机制尚不十分清楚。在已成功的鱼类电脉冲基因转移报道中，使用的电压都较低(几百伏)。有人认为，在这样的电压下， 不足以使受精卵膜发生变态或产生小孔。因此，外源基因的进入机制也就不同于培养细胞，推测是在鱼类受精膜上天然存在一些小孔，在低电压下，外源基因就通过 这些小孔进入受精卵。
2．3精子携带法
精子在等渗液中先与外源基因保温，可将外源DNA片段带人精子细胞内，再与卵子受精，可将外源 DNA片段带人受精卵内。该方法简单、方便，依靠生理作用受精，对原核损害较小。此法已在鲤鱼、泥鳅[5l中试验成功，目前已有6种鱼的成功报道。其外源 基因的处理方法虽各有不同，但都得到分子杂交或PCR检测的阳性结果，阳性率在5％～38％。从总的实验结果来看，精子携带基因转移尚存在转基因阳性率 低、转移率不稳定现象。此外，精子携带外源基因的机制尚未明确，推测与精子表面吸附或精子细胞摄取有关。
2．4基因枪注射法
基因枪注射法 是通过把吸附DNA的金属微粒高速打人细胞内，使基因导入细胞内的方法。有人在泥鳅、斑马鱼、鲑鱼的3日胚上应用此法，结果70％的个体生存，一部分个体 导入了外源基因。此法不容易进行，但有短期可处理多个个体的优点，只是现在的报道太少，有待今后进一步的研究。
2．5 逆转录病毒感染法
此 类病毒是RNA病毒的一种，进入宿主后从RNA逆转录成DNA，并结合到宿主细胞的染色体上，这种纳入病毒基因的细胞染色体内就存在了病毒基因。如果把目 的基因组合到病毒染色体上，用改造的病毒侵染宿主细胞就有可能向细胞内导人外源基因17J。但转基因因病毒有严格的宿主特异性，而且鱼类转基因病毒的分析 非常落后，对于鱼类此法暂不太适用。
2．6组织注射法
有报道外源基因向体组织直接注射时，周围细胞把外源基因纳人，并且发现了那种外源基 因。有人把CAT(链霉素转移酶基因)注射到一些鱼的体侧肌肉上，从肌肉匀浆中检测出CAT活性”。另一方面，把含有合成黑色素的互补DAN质粒注射到一 些鱼的体侧肌肉上，使周围体表合成黑色素，体表黑化。这种方法能非常容易地把外源基因导人细胞内，所以对检测外源基因启动子非常有效。
2．7 卵母细胞的基因转移
选取、收集、准备发育到一定时期的卵母细胞，显微镜下检查胚泡，若卵可供注射，操纵注射管，扎进胚泡进行显微注射，即将外源基因准确地注入卵母细胞核中，离体培养卵细胞至成熟卵，然后与正常精于受精，使受精卵整合外源基因，发育成转基因鱼。
2，8基因打靶法
20 世纪90年代出现了新的外源基因导入技术，即基因剔除(gene knock out)和基因楔入(geneknockin)技术。基因剔除是基因打靶的(genetargeting)一种方法，类似于同源重组技术，指外源DNA与 受体细胞基因组合，这是一种先进的传染技术，克服了其它传染技术无法消除的盲目性和偶然性，具有整合位点确定、精确、转移基因频率较高等优势，但是基因剔 除技术不能产生核苷酸水平上的精确突变，新的挑战使条件性基因剔除(conditional gene knock out)应运而生，这是基因剔除技术的一个飞跃。它是指在某一特定的细胞类型或细胞发育的特定阶段剔除某一特定基因的技术，常采用打了就走(hit and runstrategy)、标记—交换(tag and exchange strategy)和Cre—loxP重组系统(Cre—lox P recombina-tion system)等新的转基因策略，这些新策略可对细胞中任一基因进行核苷酸水平上的精确突变。
基因楔入又称基 因置转技术(gene replace•menttechnique)，使双链的断裂点发生在同源序列的边缘或同源序列之外，转基因的结果是外源DNA取代内源靶序列，虽然基因 楔人技术刚刚起步，但已经展示了它在应用研究方面的广阔前景。以上介绍的几种基因打靶方法大大提高了外源基因的整合率，但是未能解决外源基因定点整合的问 题。小鼠ES细胞基因打靶技术可以获得对某一位点上基因进行改造的同合子个体。该技术首先把含有更改好的基因或基因的一部分插入到载体，并引入来自小鼠的 ES细胞系，ES细胞能进行组织培养，并能产生任何组织的细胞，细胞增殖一段时间后，筛选出发生同源重组的少数细胞进行克隆并扩增，然后用显微毛细管将细 胞注入小鼠早期胚胎，产生嵌合体。若该嵌合体在生殖细胞携带外源基因，则其后代中1／2为转基因个体，它们相互交配，后代中1／4为携带外源基因的同合 子，即为可用于研究的转基因个体。然而该技术的应用必须依赖于ES细胞，而ES细胞只能在小鼠上获得，故该技术只能局限在小鼠上展开。如何在大动物上做到 外源基因的定点整合是研究人员一直努力的方向。
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CNSNEURON是在神经科学领域有一定造诣的专家，对神经科学发展前沿有深入了解。这个帖子是前段时间CNSNEURON、gaalaa和我在MSN上聊天时，当我们提出让CNSNEURON写个专题的时候，没想到CNSNEURON很痛快的就答应了，而且不到一周的时间，就发了这个帖子。对此，我们十分感谢CNSNEURON老师！
里面的很多内容对我来说还很陌生，我现在正在仔细拜读，对此，我google了一下“基因敲除”，有这样一段话：
随着人类基因组草图的完成，生命科学的注意力开始向基因组计划的终极目标转移，即基因背后所隐藏的生物学功能以及由于其突变导致的病变机理。而基因敲除技术作为基因打靶技术中最新的技术之一，正是在此需求下诞生并被逐渐完善的一种极其有创意的新兴技术。
单单就基因敲除技术来说，这是一项近几年的新兴技术，国内还没有独立的实验室能够对于此项技术加以应用，而国外此项技术的运用开展的如火如荼，并且逐步的成熟起来。迄今为止至少有 2500 个基因做了基因敲除实验及后续的功能分析。然而，非常遗憾的是还没有一个基因的敲除是在国内独立完成的。
在这个专题中，CNSNEURON对基因敲除技术进行了比较系统的阐述，使我对此有了一个宏观了解，至少解开了基因敲除技术的神秘面纱，记得今年我们所有个博士在美国做完课题后，回国论文答辩时，她的试验中使用了基因敲除动物，当时真的很迷惑：怎么会把个体的特定器官/组织的基因剔除呢？
写了一点自己的感受，不知道各位战友的感受如何呢？我想我们其中肯定有人在做这方面的工作，对此，CNSNEURON和我们很乐意大家参与讨论，大家共同学习，共同提高！
谢谢lits的支持，也恭喜lits晋升版助！
即是同道中人，不分先后还是互称学友较好。两天后我会把条件性基因剔除技术原理和应用部分的图文附上。
非常感谢CNSNEURON学友对神经生物学版块的热心支持！！！
多谢楼主老师，期待后文！！！
感谢CNSNEURON 老师, 对条件性基因剔除我是门外汉, 希望通过此贴系统的学习!
现在 ,在国外的论文中"gene knock out"出现的频率越来越高, 虽然我对基因敲除的手段并不了解,但我知道利用基因敲除动物很大程度上使一些疾病或者药物机理的解释变的容易许多,所以我们有必要系统的了解和学习基因敲除这个先进的实验技术!
相信还有很多战友接触或或者精通基因敲除,希望你们积极的讨论,在讨论中共同进步,象我这样的门外汉也可以学到更多的东西
Transgenic vs. “knock-out”
Transgenic: an organism that has had DNA introduced into one or more of its cells artificially
“transgenic”: DNA is integrated in a random fashion by injecting it into the pronucleus of a fertilized ovum
Random (approx.. 10% disrupt an endogenous gene important for normal development)
multiple copies
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knock out protocol
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1 Cre/LoxP重组系统的发现 (文本部分摘自 组织特异性定点重组Ｃre/LoxP系统的研究概况 连正兴 李宁 吴常信)
噬菌体Ｐ1在大肠杆菌内处于溶源状态时是以单拷贝形式整合在宿主菌基因 组上的，它编码一个位点特异性重组系统。这个系统包含一个ＬoxP位点和一个称为Ｃre的基因，该系统负责将噬菌体整合到或脱离宿主菌基因组。其另一个主 要作用是降低基因组内外源基因的拷贝数，直至一个拷贝为止；同时还有助于在感染组织中使Ｐ1ＤＮＡ环化，以及ＤＮＡ复制后形成的二聚体的分离，保证噬菌体 子代在细胞分裂时只有一个Ｐ1的存在，但具有机制不详。
Ｃre重组酶以高效方式进行组织特异性、位点特异性和可遗传方式对基因组ＤＮＡ进 行重组。Cre和ＦＬＰ（也是一种重组酶，其作用的位点是ＦＲＴ）相似，不含有核定位信号，可能是通过融合或有丝分裂时核膜破裂进入核内，所重组的ＤＮＡ 长度一般为20kb，有时可达70kb以上。目前已经取得到了Cre蛋白，它在真核细胞内也可以发挥良好的ＤＮＡ重组作用。
2 Cre重组酶结构
Cre重组酶是整合酶家族的一员，它是从噬菌体Ｐ1中提取出来的。整合酶识别特定的核苷酸序列，通过ＤＮＡ和蛋白质的瞬间结合，发挥其重组作用。依据序 列同源性，来自细菌和酵母的重组酶可以分为整合酶家族和解离酶－转化酶家族。这两个家族采用两个截然不同的重组机制，但均是通过酪氨酸残基与靶ＤＮＡ共价 组合，借助一系列标定剪切位点裂解ＤＮＡ底物。
大于60个成员的重组酶家族成员间除4个用于催化反应的严格保守的残基外，其余的序列同源 性较差。这种非同源反映了重组酶在遗传重组时功能和形式的多样性。Cre活性区包含保守的催化三联体残基，精氨酸173、组氨酸289、精氨酸292，还 有保守的亲核酪氨酸324和色氨酸315。
Cre是一个38kD的蛋白质，调节LoxP位点的分子内（切除、反接）和分子间（整合）的特异性重组。Cre的作用与酵母中的ＦＬＰ重组酶作用想相似，但又有所不同。在体外状态下，无辅助因子、拓扑异构酶和ＤＮＡ不复制时也可发挥其生理作用。
Cre重组酶折叠成两个不同的区域，并通过一个较短的分隔区相连。Ｎ末端由20∽120个氨基酸组成的5个α螺旋，Ｃ、Ｄ、Ｅ形成反平行束；Ａ、Ｂ螺旋 垂直于三个反平行束。Ａ、Ｅ区负责4聚体的形成；Ｂ、Ｄ区分与ＬoxP DAN大沟半个回纹结构相连接；Ｃ末端Ｎ螺旋远离其他螺旋，有助于Cre亚基间接触。Cre的Ｃ端结构与λ和HPI整合酶的催化区较为相似，但2个与 LoxP结合的Cre分子间Ｃ末端构象出现较大的偏差，这种偏差说明只有一个亚基具有剪切活性。
3 Cre重组酶所作用的序列
Cre所作用的序列为中间有一个8bp非对称间隔区所分隔的回纹结构：LoxB、LoxR、Loxl和有两个13bp的反向重复序列的LoxP。
一个Cre分子结合一个重复序列或者二聚体结合两个反向重复序列，重组发生在8bp的分隔区内。此8bp的间隔区又负责位点的定向。LoxP有两个 13bp的反向重复序列，而LoxB有两个近10kp的反向重复序列。所有的Cre作用序列均具有一定的方向性。LoxP和LoxB的杂交可在两个方向上 进行，因此可分别形成两种LoxR与LoxL。LoxP与LoxB的重组效率较低，但LoxB是Ｐ1噬菌体整合到宿主菌基因组内的重要切入点。LoxP和 LoxL 的重组效率比LoxR与LoxB的重组效率高。Cre/LoxP系统的一个最为重要的特点是在两个LoxL位点间能发生重组，当两个LoxP反向时，使 LoxP间的外源基因反向连接；当同向时，Cre重组酶剪切LoxP间的外源片段，并保留后边一个LoxP位点。
4 作用方式
重组酶先和ＤＮＡ结合，但结合力较弱，当遇到LoxP位点时则结合力增加20倍以上；当有两个LoxP时结合力更高。在对体外表达出Cre蛋白的研究表明，Cre无核酸酶和拓扑异构酶活性。
其作用方式是将一端ＤＮＡ双链形成holliday结构的中间产物，然后与第二个双链交换，去掉中间产物发生重组。在Ｌox结构的两个回纹序列上各结合 一个Cre亚基，由于两个亚基作用的非一致性，由此造成Lox位点的非对称剪切，产生holliday结构，完成基因的插入或删除之作用。
Ｎ末端Ｅ区螺旋在裂解性Cre亚基中与ＤＮＡ骨架同向并和磷酸骨架形成静电结合。在非裂解性生长中，Ｃre亚基的Ｅ螺旋只处在邻近LoxP之间。Ｃ端主要与大小沟相接触，并与单个反向重复序列结合。
Cre和ＤＮＡ结合后形成对称结构。Cre中2个亚基只有一个活性中心，保证了只有一个反向重复处ＤＮＡ发生断裂，也只有一个Ｃ末端与邻近的亚基结合，短的Ｌ－Ｎ螺旋也是 Cre酶的主要活性中心。
一个活性区的氨基酸均来自于同一个亚基，这不同于ＦＬＰ重组酶共用一个活性区。Cre/LoxP的一个活性位点经过ＤＮＡ断裂后形成一个3＼磷酸酪氨酸，经过磷酸化酪氨酸的亲核攻击，使同一个短裂点的5＼端变成ＯＨ。同时由于构象变化和迁移使ＤＮＡ的方向发生改变。
在大肠杆菌内λLoxP2同向时有近100％在单个周期内发生重组，而λLoxP2反向时有 反接，40%∽50%重组是由于一次反接一次正接，两者机会均等所致。重组效率和Cre含量无直接联系。
重组既可在分子内发生，又可在分子间发生，但分子间效率比分子内效率低，并产生染色体易位。Cre重组酶不同于其他重组酶，对目的片段构象无太多要求， 也即与质粒结构（超螺旋、有缺口或线形）无关。现已经有了Cre重组酶的商品化产品，其作用效果和体内的一样。Cre重组酶对目的片段发生作用之后，使目 的片段产生两个分子，铰链在一起和游离式两种，但两者比率是等同的，这又不同于其他重组酶Ｔn3/re。 。
（缩略图，点击图片链接看原图）
1 内、外源基因失活或删除 借助组织特异性表达Cre基因，可以在发育的特定时期删除早期致死而在后期无法研究的基因；也可以用于删除在发育时过度表达的基因。
淋巴细胞抗原受体基因座位经常发生突变和基因重组以保证机体对外源抗原的免疫性。但为了保证基因组结构的稳定性，ＤＮＡ聚合酶β(Polyβ)基因的表 达产物参与ＤＮＡ的修复作用，如果使Polyβ基因失活则早期胚胎出现死亡，观察不到效果，然而采用可控重组技术则可以将该问题迎刃而解。
利用同源重组技术构建含有两个ＬoxP位点的Polyβ基因的转基因动物。研究发现插入的两个LoxP位点对Polyβ基因的表达和活性无任何不良影 响。选择具有LoxP-Polyβ-LoxP和无Polyβ的杂合体，然后与含有Ｔ细胞特异性表达Cre基因的转基因动物杂交，所产生的后代仅在Ｔ细胞内 产生缺失纯合体；其它组织和器官中无任何变化。
2 内外源基因的激活 转基因的主要目的是对转入基因进行准确、高效的操作。我们在结基因进行删除操作的目的是了解该基因的反向效应，但我们还需要了解转入基因开启后的特定效 应，也即是在特定时间或条件下对该基因进行开启，了解基因的正向效应。进行本操作之前首先制备在特定内源基因启动子和起始密码子之间插入一个带有两个同向 LoxP 位点的终止序列，将该基因失活的转基因动物。然后同特定组织内表达Cre基因的动物杂交，在该组织内将终止序列删除，使目的基因得以表达。
3 基因插入 经过同源重组技术，在基因组上人为构建一个LoxB位点；然后用连有外源基因的Cre/LoxP系统将外源基因借助LoxB位点整合到基因组上。此种重组是双向的，尽管整合效率较低，但是定点整合的一个重要手段。
4 染色体易位 在不同染色体上分别构建一个LoxP位点、经Cre重组酶的作用可以使得两条染色体上的LoxP位点重组，产生不同染色体上片段的交换。这种交换过程中可以产生一种含有两个着丝粒的一条染色体，进而产生染色体易位。
5 基因的条件性激活或删除 与类固醇激素受体的激素结合区相结合的融合蛋白，所加入的外源蛋白活性是配体依赖性的（特别是雌激素受体激素结合区），既当有配体存在时外源蛋白有活性， 无配体时则没有活性。现在已经研究的有癌蛋白、转录因子、丝/苏氨酸激酶等，当与配体结合时均呈现出高的活性。假如用雌激素受体构建含有Cre基因的融合 蛋白，所表达的融合蛋白在有雌激素作用时，Cre蛋白才具有生物活性，进而达到条件重组之目的。
通过同源重组制备在视黄醇基因两侧含有两 个同向LoxP位点的转基因动物；用Cre基因替换雌激素受体的ＤＮＡ结合区，制备成另一个可组织特异性表达Cre雌激素配体结合区融合蛋白的转基因动 物，两者杂交后。通过使用雌激素，Cre通过两个LoxP位点间重组，使得视黄醇Ｘ受体基因被删除，这样经过使用雌激素便可达到基因重组之目的。为了避免 体内雌激素的干扰，可将雌激素受体基因的配体结合区做一定程度的改变，使其只能和某些雌激素衍生物相结合，使条件重组的特异性更强。
（缩略图，点击图片链接看原图）
以N M D A受体方面的研究进展为例进一步了解KO 和CKO：
1) NR1 global knockout
Last updated: 9th December 2004
generated by homolgous recombination in ES cells
NR1 expression lost thoughout the brain
NR2B expression reduced
complete loss of the N M D A -induced rise in intracellular calcium
complete loss of formation of whisker-related patterns ('barrels') in the brainstem
lethal within a day of birth (10 hours, cf NR2B global knockout)
rescued by ectopic expression of the NR1-1a splice variant
viability is dependent on level of NR1-1a expression
extent of somatosensory map formation dependent on expression level of NR1-1a
migration of neocortical neurons is normal - possible compensatory mechanisms
3-5 fold increase in cell death in the ventrobasal nucleus of the thalamus (V, beginning at day of birth
significant reduction in the size of the VB starting before birth (E17.5)
2)NR1 global knockout
generated by homolgous recombination in ES cells
express only 5-10% of normal levels of NR1
develop to normal size and weight
show increased motor activity during habituation to new environment
increased stereotypic movements
social behaviour deficits - lower levels of social investigation and increased escape behaviour
abnormal sexual function
behavioural and locomotor deficits are reduced following treatment with clozapine
behavioural deficits are related to schizophrenia
3)NR1 conditional knockout - hippocampal CA1 deletion
Last updated: 9th December 2004
generated by cre-loxP recombination
loss of NR1 expression restricted to CA1 region of the hippocampus
NR1 expression lost two weeks post natal
mice are viable and develop normally
short-term potentiation (STP), long-term potentiation (LTP) or long-term depression (LTD) cannot be induced in CA1 neurons
LTP can be induced in the dentate gyrus
deficient in spatial memory (hidden platform in Morris water maze)
decreased specificity of individual CA1 neuronal place fields
deficit in the co-ordinated firing of pairs of neurones tuned to similar place fields
deficit in relational memory - the ability to associate odour pairs with rewards where a single odour leads to a reward when associated with a specific second odour
Olfactory perception and the ability to associate specific odours with specific rewards is unimpaired
Trafficking of NR2 subunits is disrupted in the CA1 region with NR2A and NR2B being retained in somatic ER
a further mutant has been produced in which the knockout of NR1 expression in hippocampal CA1 neurones is placed under tetracyclin control (Tet-OFF)
this mutant shows a deficit in both spatial and contextual fear momory consolidation when NR1 expression is switched off by treatment with doxycyclin (dox) soon after the initial learning
no effect on memory consolidation is seen when NR1 expression in maintained for 7 days following initial learning
disruption of remote contextual and cued fear memories by prolonged NR1 downregulation (30 days) 6 months after initial learning
no effect of short NR1 down-regulation (7 days) on remote contextual and cued fear memories
a lack of LTP in entorhinal neurones following dox treatment for 5 days in 8-11 month old mice
4)NR1 conditional knockout - hippocampal CA3 deletion
Last updated: 18th February 2003
generated by cre-loxP recombination (cre expression driven by the regulatory sequences of the KA-1 gene)
loss of NR1 expression is restricted to CA3 region of the hippocampus
mice are viable and develop normally
NR1 deletion in pyramidal cells begins at five weeks post natal and reaches near completion at eighteen weeks post natal
NR1 expression in CA1 pyramidal cells and general hippocampal architecture is normal
N M D A R-dependent LTP in C/A-CA3 synapses is essentially absent, but N M D A R-independent LTP in MF-CA3 synapses is normal
LTP in SC-CA1 synapses is normal
spatial memory is normal (cued hidden platform in Morris water maze)
deficit in pattern completion, assessed by removing three out of four cues (partial cue conditions) in the hidden platform test (less time spent at the recalled platform site).
individual CA1 neuronal place fields are normal under full-cue conditions, although complex burst spike frequency and complex spike index of CA1 pyramidal cells are reduced
co-ordinated firing of pairs of CA1 neurones tuned to similar place fields is normal under full cue conditions
deficit in burst spike frequency, place field size and co-ordinated firing of CA1 neurones under partial cue conditions
4)NR1 conditional knockout - cortical deletion
Last updated: 21st February 2003
generated by cre-loxP recombination (cre expression driven by the regulatory sequences of the homeobox gene, Emx1)
mice are viable but develop more slowly than control littermates (70% of the body weight of littermates at P7)
loss of NR1 expression is restricted to excitatory neurones in cortical regions, including the somatosensory barrel cortex and the hippocampus at P7
absence of N M D A receptor-mediated excitation in the barrel cortex; AMPA receptor-mediated excitation is normal
whisker development and neural pattern in the brain-stem, dorsal column nuclei and the ventrobasal thalamus in normal
small and indistict barrels patterns formed in the barrel cortex, patches due to the bundles of thalamocortical axons
barrel cortex cell numbers are unaltered, but no barrel boundaries are formed (uniform granule cell distribution)
patterns related to sinus hairs and digits are mostly absent
thalamocortical axons (TCA) undergo normal structural plasticity in response to whisker lesion
dendtritic fields of spiny stellate cells do not orient towards TCA terminal patches, but radiate in all directions
dendritic fields of spiny stellate cells show profuse branching and increased spine density
TCA terminal branching is poorly developed
thus, cortical N M D A R activation is necessary in the transfer of periphery-related patterns to the cortex
5)NR1 N598 Mutants
Last updated: 18th February 2003
multiple lines generated replacing asparagine 598 (N598) with glutamine (Q) or arginine (R)
generated by homologous recombination in ES cells
expression of mutant alleles is 'silenced' by presence of a 'floxed' neomycin resistance sequence (neo)
neo gene removed either in vitro (Cre recombinase expressed in ES cells) or in vivo (silent mutant mice crossed with Cre-deleter mice)
expression of pure mutant NR1 (either N598Q or N598R) is lethal within 1 hour of birth (resipratory deficits, lack of suckling response; cf global NR1 knockout)
silencing of either mutation (through retention of the neo sequence) converts the phenotype to that of the NR1 knockout (lethal within 10 hours of birth)
heterozygotes for either mutation exhibit increased mortality but more severe for N598R mutation
heterozygotes expressing silenced mutations are phenotypically indistinguishable from wild-type mice
homozygotic N598Q mice show a four-fold reduction in calcium permeability
homozygotic N598Q mice show incomplete magnesium block (and thus increased ion flux) at resting membrane potentials;
homozygotic N598Q mice show a strong, voltage-dependent calcium block that is dominant over usual magnesium block at depolarising potentials
N M D A receptor complexes with both native and N598Q mutant subunits show functional characteristics of native receptors
N M D A receptor-dependent LTP in CA1 hippocampal neurons is normal in N598Q heterozygotes
N598Q heterozygotes show impaired maternal behaviour
N M D A receptor currents are undetectable in hemizygotic N598R mice (only one (mutant) allele is expressed)
N598R mutant subunit is functionally dominant over native NR1 subunits within mixed receptors
heterozygotic N598R mutant mice show normal somatosensory map formation in the brain stem
further mutants utilizing the Tet-off expression system linked to the alpha-CAMKII promoter controlled expression of the transgene in a normal background
expression of the transgene in the absence of doxycycline is highest in the forebrain and not detectable in the cerebellum or brainstem
transgene expression in mice in which the mother did not receive doxycycline during pregnancy was lethal within 22-28 days
transgene expression in mice in which the mother received doxycline up to birth was generally reduced and was completely absent in the hippocampus
mice in which transgene expression was suppressed during embryonic development and at up to P10 were phenotypically normal
Calcium permeability is impaired in 69% of olfactory bulb granule cells
N M D A R2 Subunit Transgenic Mice
The expression of all four N M D A receptor subunits NR2A-D have been modified. In addition to the generation of knockout mice, in which the expression of the subunits have been lost, both NR2B and NR2D have been over-expressed. Mutanat receptor subunits with C-terminal deletions of NR2A-C have also been expressed, replacing the native subunits. Studies such as these have demonstrated that correct expression of the NR2B subunit is essential to neonatal survival and somatosensory map formation (cf NR1). Deletion studies have shown that the C-terminus of the NR2B subunit is important in the synaptic targetting of the N M D A receptor complex. In addition to this, over-expression of either NR2B or NR2D results in N M D A receptors with characteristics found in juvenile animals. It is interesting that both these receptor subunits undergo developmental down-regulation - NR2B is expressed at higher levels in juvenile animals while NR2D is expressed primarily during embryonic development. In behavioural tests, over-expression of NR2B appears to enhace the ability to retain information leading to better long-term memory whereas over-expression of NR2D retards the development of induced epileptic seizures, suggesting a role for this receptor subunit in the development of epilepsy.
1)NR2A global knockout
Last updated:
generated by homolgous recombination in ES cells
NR2A expression lost thoughout the brain
overall neuroanatomy normal
reduced N M D A receptor-mediated component of synaptic transmission in hippocampus (CA1)
reduced induction of LTP in hippocampus (CA1; stratum radiatum and stratum oriens) and cerebellum
reduced synaptic transmission and LTP induction in associational input to hippocampal CA3 neurons (stratum radiatum)
no effect on synaptic transmission or LTP in fimbrial inputs (stratum oriens) to same cells (cf NR2B knockout)
LTP deficits are overcome by giving a stronger tetanus
mice show deficits in Morris water maze learning, eyeblink response and associative learning
complete C-terminal deletion produces similar effects on LTP
2)NR2B global knockout
Last updated: 20th February 2003
generated by homolgous recombination in ES cells
subunit is essential for neonatal survival (cf NR1 global knockout), but overall neuroanatomy is normal
complete loss of whisker-related patterns ('barrels') in the brainstem
LTD cannot be induced in CA1 hippocampal neurons
LTP in fimbrial input to CA3 neurons is severely impaired in heterozygotes (cf NR2A knockout)
heterozygotes show increased acoustic startle response (ASR) and a slight increase in prepulse inhibition of ASR
no effect on ASR seen with homozygous NR2A,C or D kcockouts
3)NR2 C-terminal deletion knock-ins
Last updated: 21st February 2003
generated by homolgous recombination in ES cells
NR2B
deletion of the entire C-terminus is lethal prenatally (Sprengel et al,1998)
deletion of two-thirds of the C-terminus results in mice that are viable up to postnatal day three
disrupted barrel formation in the brainstem
LTP can be induced in some CA1 hippocampal neurons (cf NR2B global knockout)
reduced synaptic expression of NR2B subunit but the number of synapses in normal
in embryonic neocortical neurons from homozygous mice, N M D A receptor-mediated EPSCs show a reduced peak amplitude and mean open probability
severe reduction in the fraction of N M D A receptrs that are synaptically localised
synaptic localization recovers with increasing time in culture as receptors containing NR2A are expressed
NR2A/C
marked reduction in N M D A receptor-mediated EPSC at the cerebellar mossy-fibre-granule cell relay
no effect of neurotransmitter release
no effect on N M D A receptor development or expression pattern
reduced peak open probability in synaptic N M D A channels
impaired N M D A receptor-dependent LTP
4)NR2B knock-in
generated by pronuclear injection
NR2B subunit is overexpressed by linkage to the CaMKII promoter, producing overexpression in the forebrain
overall neuroanatomy and development is normal
N M D A receptor-mediated synaptic currents are similar to those in juvenile mice (slow kinetics) from 18 days post natal (Note: NR2B undergoes a developmental down-regulation - cf NR2D knock-in)
LTP induced by tetani in the 10-100 Hz range is enhanced in CA1 neurons
LTD and paired pulse facilitation is normal
better long term visual recognition memory that can be further enhanced by rearing animals in an enriched environmemt, but no effect on plasticity at the synpatic level in the visual cortex
stronger freezing responses in contextual and cued fear conditioning
enhanced ability to dissociate fear responses from contextual and tonal cues
faster spatial learning in Morris water maze
no further increases in contextual and cued fear conditioning responses or fear extinction when raised in an enriched environment
enhanced N M D A receptor mediated fEPSP in anterior cingulate cortex and insular cortex (areas involved in processing of pain-related information)
no effect on fEPSPs or any transgene expression found in dorsal horn
selective increase in c-fos expression in response to tissue injury/inflammation in areas with enhanced NR2B expression
enhanced display of a withdrawal response to a previously non-noxious stimulus
enhanced odour memory function
5)NR2C global knockouts
Last updated:
two strains generated by homolgous recombination in ES cells
first strain shows reduced N M D A receptor-mediated component of cerebellar granule cell EPSC
second strain shows a higher EPSC peak amplitude but shorter decay time than in wild-type mice
Low conductance channels are also lost (channels with conductance below 37 pS)
no motor impairments have been seen with single NR2C gene knockouts
mice with combined gene deletions of NR2A and NR2C can only manage simple co-ordination tasks
6)NR2D global knockout
Last updated: 20th February 2003
generated by homolgous recombination in ES cells
minimal effects - NR2D is expressed at it's highest levels during embryonic and early postnatal stages of development
neuroanatomy is normal
motor and anxiety responses are normal
spontaneous activity in the open field is reduced
expression of NR2A and B subunits is normal
specific binding of MK-801 to the N M D A receptor in adults is reduced
uptake of calcium through in response to pharmacological activation of the N M D A receptor is reduced but is unaffected by high K+ stimulation in adults
altered monoaminergic neuronal function (dopaminergic, setotoninergic, nor-adrenergic)
reduced locomotion and rearing behaviour
mice may have a reduced susceptibility to stress and/or reduced psychological anxiety (elevated maze, light-dark box and forced swimming tests)
7)NR2D global knock-in
Last updated:
generated by pronuclear injection
NR2D subunit is overexpressed by linkage to the CaMKII promoter
overall neuroanatomy and barrel formation is normal
selective expression of NR2D in cortex, hippocampus and striatum reaching maximum by 8 weeks (adult)
lower amplitude and slower kinetics of evoked currents in hippocampal CA1 cells from adult mice (cf NR2B subunit knockin)
impaired LTD in juvenile mice in hippocampal CA1 cells ; no effect on LTP
impaired LTP in adult mice in hippocampal CA1 cells; LTD not induced in either mutant or wild-type mice
spatial memory is normal (Morris water maze) but mutants are less motile and show reduced exploratory behaviour in the open field
supression of kindling epileptogenesis (retarded development of motor seizure and afterdischarge duration)
no effect on pre-established epileptic seizures
N M D A R3 Subunit Knockout Mice
The N M D A receptor subunit NR3A is a recently characterised subunit (also known as c-1 and N M D A R-L) that is expressed primarily during brain development and has also been shown to exist in complexes with NR1 and NR2 subunits. Little is yet known about the function of this subunit, but from work with the only knockout mouse described, it appears to be a regulatory subunit involved in dendritic spine development.
1) NR3A global knockout
Last updated:
generated by homolgous recombination in ES cells
no obvious behavioural abnormalities
normal levels of other N M D A receptor subuhnits
three-fold increase in N M D A -induced current density in acutely dissociated cerebrocortical neurons
three to four-fold increase in dendritic spine density on both basal and apical dendrites of cortical neurons from layers IV and V in P19 mice
increased size of spine head and increased length of spine neck
no effects seen in cerebellar purkinje cells, which do not express the NR3A subunit
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已保存，多谢楼主老师！
Very exciting to see such a high-level Journal Club is presented here.
Thank you!
我也做过一个类似的，现将部分内容呈上。
割成四份了。周末前可以下载的。
文字说明在幻灯下面有。
>
已保存，谢谢smallcatter!
CLUB里重点在讨论：讨论文中的方法，试验设计，数据、图表、文字结构和表达，结论的详实，引述是否恰当，值得借鉴的地方......。希望大家能广开言论。
几个相关概念：
学习和记忆
学习: 获取外界信息，形成新的行为、习惯的神经过程。
记忆: 将获取的信息或新的行为习惯进行储存和读出的神经过程。
这两个过程是互相联系的。
学习的两种形式
1． 非联合型学习 (nonassociative learning)： 也称简单学习。所谓“简单”或“非联合”，是指在学习过程中引起反应的刺激是单一的，该刺激不需要和其它刺激相联系或联合。
（2）敏感化：是指在较强的伤害性刺激后，机体对原先弱刺激引起的反应明显增强的过程。强刺激和弱刺激不需要在时间上的结合，故又称为假性条件化。
2．联合型学习（associative learning）：所谓“联合”，是指在学习过程中需要两种不同的刺激（或行为与某种刺激）按照一定次序进行配对，由此在脑内形成相互联系。
（2） 操作性条件反射（operate conditioning）：也称为工具性条件反射。
学习和记忆的机制
* 记忆痕迹贮存部位：用损毁个别脑区来探查记忆痕迹贮存部位的实验表明，学习和记忆是中枢神经系统许多神经元共同活动的结果，似乎不可能定位于某个特定的脑区。然而，人们也发现某些脑区损伤后造成的记忆障碍程度较其它脑区严重。例如，海马和颞叶联合皮层的损伤，将会引起短时性记忆向长时性记忆的转移发生障碍。
为了进一步揭示大脑高级功能的奥秘，人们从神经生理、神经生化和神经解剖多方面对学习和记忆的机制进行了广泛的研究。
1． 神经生理学机制
① 神经元活动的后作用（刺激停止后，神经活动仍能持续短暂时间）：感觉性记忆的机制可能属于这一类；
② 环路式联系：进行的连续活动可能是第一级记忆的基础，如海马环路的活动与第一级记忆的形成有关，其反复活动又促进了第一级记忆向第二级记忆转移；
③ 突触的可塑性改变：是长时性记忆的一个重要基础。这方面的知识主要来自于对习惯化、敏感化和长时程增强等突触传递现象的研究。习惯化和敏感化的突触机制最早是在对低等海洋动物海兔缩鳃反射的研究中得以阐明的。
习惯化的发生机制：是由于感觉神经元轴突末梢释放的兴奋性递质减少，导致与其构成突触的运动神经元和中间神经元上突触后电位减小、甚至消失的缘故。Ca2+内流减少和可动员的突触小泡数量减少被认为是突触前膜递质释放减少的原因。
敏感化的发生机制：是突触传递效能的增强。电刺激海兔引起敏感化过程中，有中间神经元终止于缩鳃反射的感觉神经元末梢，通过G蛋白和第二信使的作用，使该感觉神经元突触前末梢兴奋时产生的动作电位时程延长，Ca2+内流增多，从而使其释放递质的数量增加。
长时程增强现象及机制：在哺乳类动物中，长时程增强被视为学习和记忆的重要机制。
a. 长时程增强1923年，Bliss等人首先在麻醉兔上发现，用一串高频率、大强度的电刺激作用于某些脑区的传入纤维后，原先单个刺激诱发的兴奋性突触后电位的幅度将增大，并且该易化现象能持续数小时以上。这种单突触诱发反应的长时程易化现象被称为长时程增强(Long term potentiation, LTP)。
b. LTP的产生机制: 与***A受体的作用特点有关。
双重门控: ***A受体是一种独特的双重门控通道，既受递质（Glu）门控也受电压门控，只有当膜去极化达到一定程度后，才能移开堵塞通道的Mg2+，使结合了Glu的***A通道打开; 在正常低频突触传递时，突触前膜释放的Glu可同时作用于突触后膜上***A和非***A受体，但只能激活非***A受体出现较弱的去极化反应；强直刺激可使突触后膜的去极化达到一定程度，使Mg2+对***A通道的阻塞效应解除，从而打开***A通道，出现Ca2+内流，导致LTP的产生。
实验表明，动物的记忆能力与其LTP反应强度有显著相关性，影响LTP的因素或药物也能影响学习和记忆过程。因此，LTP的发现是学习和记忆乃至神经科学史上的一个里程碑。
突触的可塑性
概念：突触传递在某些情况下可发生功能性增强或减弱，此即突触的可塑性。
表现形式：强直后增强；习惯化；敏感化；长时程增强和长时程减弱。
意义：突触可塑性与脑的学习和记忆功能有关
该文的试验方法：
Materials and Methods
Generation of CA3-NR1 knockout mice. We isolated an approximately 120-kb-long bacterial artificial chromosome (BAC) clone BAC1, that contained a part of the genomic sequence of mouse KA-1, one of the kainate receptor subunits, by screening a C57BL/6 (B6) BAC library (Genome Systems) with a probe composed of the 5' untranslated region of a mouse KA-1 cDNA clone (S1). The BAC1 clone contained the genomic DNA sequence of the putative promoter, exon 1 (~338 bp) and exon 2 (45 bp), but not exon 3 (105 bp) carrying the translation initiation codon. The entire BAC1-DNA fragment was coinjected into fertilized B6 mouse eggs with a DNA construct carrying both the minimal heat shock promoter (from pIND plasmid, Invitrogen), and the Cre cDNA with nuclear localization signal (pBS317, provided by B. Sauer). Out of eight founder lines in which both Cre and BAC1 sequences were cointegrated into the genome, the line referred to as G32-4 was crossed with the Rosa26 reporter line (S2), or the "floxed" ***A receptor subunit-1 (fNR1) mouse line, which we reported earlier (S3) and subsequently backcrossed to B6 eight times. All procedures relating to animal care and treatment conformed to the Institutional and NIH guidelines.
Immunocytochemistry. Mice were perfused transcardially with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (P. For X-gal staining, the brains were removed and post fixed in the same fixative at 4°C for 30 min. Coronal sections (50 m thick) were cut on a Vibratome and collected in PB. Sections were then incubated in 0.1 M PB containing 0.01% SDS, 0.02% NP-40, 2 mM MgCl2 at 4°C for 15 min followed by -galactosidase reaction in 1 PBS pH 8.0 containing 0.5 mg/ml X-gal, 5 mM K4Fe(CN)6/3H2O, 5 mM K3Fe(CN)6, 2 mM MgCl2 at 37°C for 24 hours. Section were post fixed in 10% formalin for at least 2 hours, and counterstained with Nuclear Fast Red (Poly Scientific). For immunofluorescent staining in Fig. 1, the brains were post fixed in 4% paraformaldehyde overnight, and 50- m-thick Vibratome sections were prepared. Sections were then incubated at 4°C overnight with gentle shaking in primary antibody [anti-Cre polyclonal antibody (pAb), 1/4000, BABCO; anti-GAD67 pAb, 1/1500, Chemicon; anti-calretinin pAb, 1/4000, Swant] with 0.05% Tween20 following pre-incubation with 10% normal goat serum at room temperature for 2 hours. The sections were then incubated with a biotinylated anti-rabbit IgG (1/200; Vector) at room temperature for 2 hours, and then with streptavidin-biotin/horseradish peroxidase complex (Vector) for 30 min. Cre-IR was visualized by 5-min treatment with fluorescein isothiocyanate (FITC)-tyramide at room temperature. GAD67-IR and calretinin-IR were visualized by a 5-min treatment with the Cy3-tyramide, according to the manufacture's protocol (NEN). For double immunofluorescence staining, brain sections were further incubated with anti -galactosidase (1/2000, Cappel) at 4°C for overnight. After rinsing, the sections were incubated in Alexa488-conjugated anti-rabbit IgG (1/2000, Molecular Probes) at room temperature for 2 hours. Fluorescent images were captured with a confocal laser scanning microscope (BioRad). For immunoperoxidase staining in Fig. 2, 5- m-thick paraffin sections were prepared as described (S4). Briefly, prior to primary antibody incubation, sections were subjected to pepsin pretreatment (1 mg/ml in 0.2 N HCl) at 37°C for 10 min. After blocking with normal goat serum, sections were incubated overnight with anti-NR1 pAb (GluR 1 COOH-terminus, 0.5-1.0 g/ml), anti-PSD-95 pAb (0.5 g/ml), anti-GluR1 pAb (GluR 1 COOH-terminus, 0.5 g/ml), or anti-calbindinD28k pAb (1/10,000) followed by incubations with biotinylated anti-rabbit IgG for 1 hour and streptavidin-peroxidase complex for 30 min (Vector). Immunoreaction was visualized with 3,3'-diaminobenzidine, and photographs were taken by AX-50 light microscope (Olympus).
In situ hybridization of NR1 mRNA. Fresh-frozen brain sections (14 m in thickness) were prepared in the parasagittal plane with a cryostat, and mounted onto pre-coated glass slides. Sections were post fixed with 4% paraformaldehyde in PBS for 15 min, and treated with 10 g/ml proteinase K at 37°C for 30 min followed by 0.2 M HCl for 10 min. After rinsing, sections were further incubated in 0.25% acetic anhydride and 0.1 M triethanolamine for 10 min to avoid non-specific binding of the probe. Following dehydration with ethanol, hybridization was performed at 55°C for 18 hours in a hybridization buffer containing 50% formamide. For detection of the mouse NR1 mRNAs, a complementary RNA (cRNA) probe, derived from the AvrII-SphI 0.4-kb antisense DNA fragment of rat NR1 cDNA containing from exon 13 to exon 16 (S5), was labeled with [33P]UTP (5 105 cpm), and added to the hybridization buffer. The brain sections were serially washed at 55°C with a set of SSC buffers of decreasing strength, the final strength being 0.2 and then treated with RNase A (12.5 g/ml) at 37°C for 30 min. The sections were exposed to hyper-beta max (Amersham) for 2 days and were dipped in Kodak NTB3 nuclear emulsion followed by exposure to X-ray film for 3-4 weeks.
Slice electrophysiology. Transverse hippocampal slices (300-350 m thick) were prepared from 18- to 28-week-old male homozygous fNR1 mice (control) and CA3-NR1 KO (mutant) mice as described previously (S6). In addition, we found that the viability of surface neurons was noticeably improved by cutting slices with freshly broken glass blades (Ralph Glass Knife Maker; Ted Pella, Inc.) placed in a Vibratome blade holder. Individual slices were transferred to a recording chamber and submerged in oxygenated (95% O2 to 5% CO2) bathing solution maintained at 30°-31°C and containing (in mM): 124 NaCl, 2.5 KCl, 25 NaHCO3, 2-4 MgCl2, 2-5 CaCl2, 10 dextrose. Whole-cell patch recordings were made from visually identified CA3 and CA1 pyramidal cells with an SEC 05L amplifier (Adams and List Associates) in bridge or discontinuous voltage-clamp modes. Recording electrodes were either filled with (in mM) 120 K-gluconate, 20 KCl, 10 Hepes, 2 MgCl2, 4 ATP (disodium salt), 0.3 Tris-GTP and 7 phosphocreatine phosphocreatine (osmolarity 280-290, pH 7.3, KOH), or 107 gluconic acid, 107 CsOH, 5 QX-314-Cl, 10 BAPTA, 0.2 EGTA, 5 TEA, 20 Hepes, 0.7 NaCl, 4 MgATP, 0.3 NaGTP (osmolarity 280-290, pH 7.3 CsOH). In most experiments, biocytin was added to the patch electrode solution for cellular staining and subsequent morphological confirmation. Although not systematically analyzed, no obvious differences in recorded-cell morphology were observed between control and mutant animals. Synaptic responses were evoked in either CA3, CA1, or dentate gyrus with single stimulation or brief bursts of stimulation (5 pulses at 50-100 Hz) delivered to one of three afferent pathways (Fig. 3A): medial perforant path input to the dentate gyrus (MPP-DG), recurrent commissural/associational (C/A-CA3), or Schaffer collateral input to CA1 (SC-CA1). ***A currents were characterized by their slow time course, insensitivity to DNQX and sensitivity to 2-amino-5-phosphonopentanoic acid (APV). Where specified, ***A currents were isolated pharmacologically by adding the AMPA/kainate antagonist, 6-cyano-7-dinitroquinoxalline-2,3-dione (DNQX, 40 M) and the GABAB antagonist, CGP 35348 (50-100 M) to the bath solution. To ensure that ***A currents were fully activated, extracellular Mg2+ was removed and glycine (1-10 M) added. In some experiments ***A currents were recorded by depolarizing the cells to a variety of membrane potentials after filling the cells with cesium. Synaptic currents were evoked by electrical stimulation with a. glass or metal bipolar stimulating electrode placed in one of three afferent pathways: DG molecular layer (~ 50 m from stratum granulosum) to activate medial perforant path axons, (ii) the CA3 stratum radiatum (~200 m from stratum pyramidale) to activate C/A axons, (iii) CA3 stratum lucidum (~50 m from the cell body layer and ~100 m toward the granule cell layer in the mossy fiber track) to activate the mossy fiber pathway, and (iv) CA1 stratum radiatum (~200 m from stratum pyramidale) to activate Schaffer collaterals. ***A currents were evoked with single pulses or a brief burst of stimulation (5-pulse bursts at 50 or 100 Hz). The presence of a synaptically evoked ***A current was confirmed by blocking the response with the ***A antagonist APV (50-100 M). For LTP experiments, 4 mM MgCl2 and GABAA blockers (10 M bicuculline and 10 M picrotoxin) were added to the bathing solution. To induce LTP, three long trains of stimulation (100 pulses at 100 Hz; 1 train per 20 sec) were delivered to either MF-CA3, C/A-CA3, or SC-CA1 synaptic pathways. In addition, a 1-s depolarizing pulse (100-500 pA) was given during the stimulation trains in an effort to elicit an equivalent number of action potentials for each of the synaptic pathways during the train.
Morris water maze. The Morris water maze tasks were carried out with male mice 18 to 24 weeks of age. All the experiments were conducted by operators who were blind to the genotypes of the mice used. The mice were kept in a temperature-controlled room on a constant 12-hour light/dark cycle. The experiments were conducted at approximately the same time each day. The facility was in a square dark room and consisted of a circular pool (160 cm diameter, 60 cm deep at the edge) filled with water at 20°-22°C that was completely covered with floating polypropylene beads (Hanna resin distribution). The pool was encircled by black curtains (90 cm from the pool periphery) on which four large illuminated objects that served as extramaze cues were hung. An overhead infrared-sensitive CCD camera tracked the movement of mice at 30 Hz and sent the feed to a VCR and a computerized tracking system. The entrance and exit for an experimenter were at NW and NE, respectively. The escape latency (platform search time), path length (the distance traveled to reach the platform), swimming velocity and wall hugging (the time spent in an 18 cm-wide ring area bordering the pool wall, a measure of the degree of the animal's thigmotaxic behavior) for each trial were measured. For the hidden platform task, the mice were trained to find a hidden circular platform (10 cm in diameter and set 1.5 cm below the surface of water) that was placed at the center (46.8 cm from the center of the pool) of a radial quadrant, which is defined as the largest circle inscribed in a quadrant. In order to balance the effects of the environment, the mice of each genotype were divided into four groups and each group of mice were trained for a platform located in a distinct quadrant, NE, SE, SW, or NW. The data from the four groups of mice, except obvious "floater" mice (less than 5% of the mice tested), were then combined. The training was carried out in blocks of four trials per day (an inter-trial interval of about 30 min) for 12 days. During each block of trials, the mice were released from four pseudorandomly assigned start locations (N, S, E, and W) and allowed to swim for 90 s. If a mouse did not find a platform within 90 s, it was manually guided to the platform and allowed to rest on the platform for 15 s. Probe trials were performed on days 2, 7, 13, 14, and 15 under various conditions (see the text). In probe trials, the mice were released at the center of pool with a head direction that was pseudorandomly assigned, and were allowed to swim for 90 s in the absence of the platform. We measured the time (in seconds) the mice spent in the area, which corresponded exactly to the area occupied by the platform during the training session (absolute platform occupancy). We also measured the time mice spent in each of the four radial quadrants (the largest circles inscribed in each quadrant) and calculated the percent of time they spent in the target radial quadrant relative to the total time spent in the four radial quadrants (relative radial quadrant occupancy). For the partial-cue probe trial (the fourth probe trial, P4) conducted on day 14, only one of the two cues that were located more distant from the platform during the training session was kept and the other three cues were removed. Among the eight possible combinations of the remaining extramaze cue and platform position (NE, SE, SW, or NW), mice of each genotype were tested in six different combinations in order to balance the effects of the environment. The data from the six groups of mice were then combined. To evaluate the memory recall capability of each animal, we defined a relative recall index (RRI) as the ratio of the target platform occupancy of the fourth (P4) or fifth (P5) probe trial to that of the third (P3) probe trial for each animal that exhibited significant target platform occupancy (more than 0.23 sec) in P3. The values were then averaged over mice of a particular genotype.
In vivo multielectrode recording. All recordings were carried out by operators who were blind to the genotype of the mice. Male mice (18-24 weeks of age) were implanted with a microdrive array consisting of six independently adjustable tetrodes (Stereotaxic coordinates from bregma: 2.0 mm lateral; 1.8 mm posterior). Recording sessions generally consisted of one or two "Run" epochs (20-30 min each) bracketed by "Sleep" sessions in which the animal rested quietly on a small platform outside of the behavioral environment. "Run" sessions were conducted in a low walled open field arena (50-cm in diameter) placed near the center of a rectangular black-curtained chamber (1.8 m 2.0 m). Diffuse room lighting was provided by low intensity spotlights focused onto four salient visual cues located on each of the walls of the recording chamber. As animals randomly explored the open field arena, extracellular action potentials were recorded while the animal's position was tracked using a pair of infrared diodes placed 3 cm above the animals head. Following data acquisition, action potentials were assigned to individual cells based on a spike's relative amplitudes across the four recording wires of a tetrode (S7). In order to be included for analysis, isolated cells had to satisfy three criteriai) fire a minimum of 100 spikes during the "Run" session, (ii) have a mean firing rate greater than 0.1 Hz, and (iii) have less than 0.5 % of the cells spikes fall within a 1 ms refractory period. For cue removal experiments cells also had to satisfy the following conditions: the waveform profiles of isolated cells had to remain stable across the 2-hour delay between recording sessions, (ii) cells had to fire at least 150 spikes in the first environment and (iii) cells had to have an initial place field size greater than 10 pixels.
Place cell analysis. All isolated cells were divided into two subclasses based on waveform and firing characteristics. Putative pyramidal cells were defined as cells with relatively broad waveforms (peak to trough width > 300 s) and a strong tendency to produce complex spike bursts (CSI > 3%) whereas putative interneurons had relatively narrow waveforms (peak to trough width < 240 s), and few if any complex spike bursts (CSI < 3%). When comparing the cellular properties between mutant and control animals, analysis was performed separately for these two classes of cells. To determine the consequence of CA3-NR1 disruption on the firing of CA1 cells, we measured several electrophysiological properties of hippocampal activity. First, we measured the bursting tendencies of hippocampal pyramidal cells using two measures: Complex spike index (CSI)-defined as the percentage of spikes with first lag interspike intervals that fall between 2 and 15 ms and whose second spike is smaller than the first and (ii) burst spike frequency-the ratio of number of spikes involved in a burst relative to the total number of spikes produced by a cell. As a measure of the intrinsic properties of pyramidal cells we measured both spike width (peak to trough width), and the degree of amplitude attenuation within a three-spike burst (S8). We also used two measures to assess the spatial tuning of individual place cells: place field size, which is defined as the number of pixels in which a cell's mean firing rate exceeded 1 Hz and (ii) integrated firing rate, which is defined as the sum of average firing rates across all pixels, a measure that is less sensitive to noise than place field size. For these experiments, the coordinated firing of overlapping place fields was assessed by calculating the firing rate covariance coefficient of all cell pairs within 9 pixels of each other using a bin size of 200 ms (S9). Finally, in order to quantify relative changes in place field properties for cells recorded across experimental conditions, we calculated, for each cell, a relative change index (RCI) (defined as the difference between the cell's firing between two conditions divided by the sum of the cell's firing across the two conditions. Note that RCI does not correspond to simple percentage change). An RCI value of -0.17 is equivalent to a 30% reduction.
技术：
分子生物学：NR1 KO,
形态学：免疫组化、原位杂交
电生理：patch clamp, 在体多电极纪录
行为试验：莫氏水迷宮学习，place cell (定位细胞)分析
Fig 1 G32-4 mice 的Cre 免疫染色及Cre/loxP 重组分布.主要分布在CA3
Fig 2 CA3-NR1 KO mice 中 NR1 mRNA, NR1 protein, and GAD67 CR 的分布.
Fig 3 CA3-NR1 KO mice 的C/A-CA3回路中 N M D A R 功能缺失 ，导致该回路的LTP缺失（FIG4）
Fig 5 CA3-NR1 KO mice回想记忆受损
FIG 6 CA3-NR1 KO mice CA1定位细胞 活性降低
文中最重要的图是Fig 4、5
KO/CKO的优点我就不提了，大家能在文中体会得到。
KO/KI技术能够回答例如N M D A在记忆中的特定功能（CKO 特定时间、特定组织），但突触可塑性中N M D A的精细调节如在突触后膜N M D A内部化、膜上的迁移、以及不同配体形成不同敏感性N M D A的功能等等。
（缩略图，点击图片链接看原图）
谢谢楼主这么好的文章。我就是对分子生物不感冒，一定要静下心把它看完！
谢谢楼主这么好的文章。我就是对分子生物不感冒，一定要静下心把它看完！
楼主在做这个吗，我正在做这方面的东西，我现在用的cre-ERT2,启动子用在神经干系胞里特定表达的，比如，nestin, camkinase等，基因吗就不好说了，呵呵，老鼠是已经flox好了的。
但这个需要很多bac 的工作，有机会可以聊聊啊。
正需要这方面的资料，可能下一步就要做相关的实验了，谢谢CNSNEURON，smallcatter能不能将那个幻灯在上传一次，我刚到这个版块，没有下到，谢谢！！！！或发到我Email：garywang05@yahoo.com
这么现在cko-1.rar下载后说文件错误不能打开，可能是上传的时间太久了吧，CNSNEURON，能不能再传一次，十分感谢您
请大虾帮忙翻译一下，谢谢先！
1.To determine whether the degree of respiratory coupling in the vasculature is involved in vascular disease, we generated mice with doxycycline-inducible expression of UCP1 restricted to aortic smooth muscle cells. Transgenic mice carrying the reverse tetracycline transactivator (rtTA) driven by the 2441 SM22a promoter (SM22-rtTA mice) were mated with mice transgenic for a tetracycline responsive element (TRE)-UCP1 minigene(TRE-Ucp1 mice), yielding SM22-TRE-Ucp1 mice.
2.There was no doxycycline-dependent induction of Ucp1 gene expression in the aortae of SM22-rtTA mice (lacking a TRE target) or TRE-Ucp1 mice(lacking an antibiotic-inducible transactivator; data not shown).
Gary2006 wrote:
这么现在cko-1.rar下载后说文件错误不能打开，可能是上传的时间太久了吧，CNSNEURON，能不能再传一次，十分感谢您
请pm CNSNEURON战友，他很热心的！
“然而，非常遗憾的是还没有一个基因的敲除是在国内独立完成的。”
这句话在2004年是正确的。但2005年，我就亲眼看着targeting and conditional targeting 在国内取得成功。（上海南方模式动物中心王铸钢研究员、费俭研究员，上海赛达生物技术研究中心郭礼和研究员。）
Useful information relevant to my present project.
Now I am constructing the vector for calcium channel alpha1A knockout mouse. I think Prof. CNSNEURON must be a big deal on neuroscience. My boss is also a very famouse neuroscientist on the world. As a beginner on this exciting field, I am suffering many problems. Do you mind if I pick your brains since you are so knowledgeable on this field. I am looking forward to discuss with you some day.
Useful information relevant to my present project.
Now I am constructing the vector for generation of calcium channel alpha1A knockout mouse. I think Prof. CNSNEURON must be a big deal on neuroscience. My boss is also a very famouse neuroscientist on the world. As a beginner on this exciting field, I am suffering many problems. Do you mind if I pick your brains since you are so knowledgeable on this field. I am looking forward to discuss with you some day.
太好了，茅塞顿开，谢谢提供美图得老师
对不起各位，最近比较忙没有上来回帖。
请需要全文的战友下载 >
也欢迎新来的各位多多发言。