我们感谢安吉拉Kralemann - 科勒为优秀的技术支持。本文所述的实验工作是在我们的实验室进行，由授予德国研究理事会（DFG）的CK和BK和BK的资助研究和教育的德国卫生部部（BMBF）的支持。

伦理声明这项研究进行了严格按照政府的动物和保健使用委员会，国家北莱茵 - 威斯特法伦州，（德国杜塞尔多夫）的LANUV的规定。所有的动物实验已根据许可证号8.87-51.04.20.09.317（LANUV，NRW）批准LANUV，杜塞尔多夫。所做的一切努力，尽量减少痛苦和需要研究的动物的数量。 1。动物护理及房屋<ol><li>在本文描述的协议中使用的所有动物应保持无特定病原体的条件下，由联邦欧洲实验动物科学协会（FELASA）所定义的。 </li><li>小鼠应保持在标准笼在温度和湿度控制（22℃）室温条件下昼夜（12小时亮/暗周期）根据与HEPA过滤空气。 </li><li>标准化的食物和水应提供自由采食 。&lt;/ LI&gt; <li>如果IκB/ tTA的和IκB/ -对照小鼠被使用时，对每个动物应该进行基于PCR的基因分型，如7,18说明。 </li><li>雄性动物具有小于四天年龄差异应该被使用，以减少个体差异。 </li><li> （可选）：对于NF-κB的激活实验，多西环素，必须在饮用水管理（2毫克/毫升与2.5％蔗糖）至少14天。 </li></ol> 2。空间格局分离 - 巴恩斯迷宫（SPS-BM） <ol><li>所有的行为测试应根据国际和当地的指引进行。 </li><li>重要！只使用雄性小鼠为六个月或以上的年龄。一个测试系列的动物之间的年龄差应不超过四天。测试必须由同一运营商为每个系列中进行。老鼠应该得到一个标准的饮食前测试，以进一步提高部分被作为驱动动力weet食物奖励。 </li><li>成立了一个白色的圆板由硬塑料制成（直径120mm厘米， 见图5A）在湿度和温度控制室内（22℃），照明用至少4×80 W和3×215 W霓虹灯荧光灯。重要！确保正确的照明使用，因为老鼠的动机进入食品屋部分由自己情绪的推动，以明亮，暴露的地方。 </li><li>设置在视频跟踪系统。相机应放在115厘米上面的板的中心（参见图5A）。 </li><li>附上五彩额外迷宫线索（EMC），以白色色布中的动物，从板的边缘大约100公分（ 见图5A）容易看见的位置。 </li><li>仔细清洁板具有快速消毒剂，可以从实验装置中移除任何气味。 </li><li>将7相同的黄色食物业（12厘米×7厘米×8厘米， 见图5A </STRONG&gt;）的白色车牌。该位置应明确标记（ 见图5A）。 </li><li>将甜的食物颗粒奖励（一季度的Kellogs单曲Froot循环/食品屋）上定义的位置所有的食物里面的房子（ 见图5A），只有一个定义的粮食房子是可以自由进出的动物（地点楼参见图5A ）。关闭非靶食品与房屋透明薄膜。 </li><li>试验前，进行习惯（一天开始任务之前）。使所有食品的房子可以自由进出，让小鼠自由探索迷宫和检索的食物颗粒奖励（10分钟/动物）。 </li><li>切换电脑和相机并启动视频跟踪系统的软件。 </li><li>开始录制。 </li><li>将动物在圆形板所定义的起点（ 图5A，S：起始位置），让老鼠来搜索目标食品的房子10分钟。 </li><li>申通快递P上的视频跟踪。 </li><li>重要！每次试验与快速消毒后清洁圆板和食品的房子。 </li><li>每天重复这些试验连续七天（每个动物）。 </li><li>使用适当的统计软件分析结果（延迟，覆盖距离和错误）。定义错误，接近了错误的食物及/或不输入和检索的食物颗粒奖励与适当的盒子接触。对于分组分析中使用双因素方差分析和Bonferroni事后检验。 </li><li> （可选）牺牲小鼠并分析如下所述的海马。 </li></ol> 3。 BrdU标记<ol><li>每天一次腹膜内注射50毫克/千克IP的BrdU 3天（微分和积分的分析）或200毫克/公斤的ip为单次注射（增殖的分析）。 </li><li>牺牲动物，解剖海马和下述准备40微米的部分。 </li><li> Denaturate的用2M HCl中10分钟的部分和孵育在10分钟的0.1M硼酸盐缓冲液。 </li><li>标注一在12系列从每个动物免疫组织化学40μm的切片（240微米外），如下所述使用针对的BrdU抗体。 </li><li>通过共聚焦显微镜分析量化整个颗粒细胞层和颗粒下层区域的rostrocaudal程度的标记的细胞。乘以12所得的数目由两个获得每海马和除法的BrdU标记细胞的估计总数量，就得到每个危险品标记细胞的总数量。 </li></ol> 4。去除大脑和冷冻切片的制备由未注动物<ol><li>请遵守当地批准的程序实施安乐死的动物。小鼠可直接安乐死颈椎脱位。 </li><li> （可选）可动物安乐死之前，根据当地和国际准则进行麻醉， 如腹腔麟蹄CTION为0.8 mL圣阿韦坦的33克鼠标（通过混合150与1.85毫升生理盐水或生理缓冲液由2.2毫克2,2,2 - 三溴乙醇1毫升乙醇异戊酯毫升原液新鲜的）。 </li><li>消毒的头部与聚维酮碘（10％聚维酮碘）浸泡过的纱布和棉签随后用纱布浸泡在70％乙醇中。 </li><li>采用合适的手术剪刀斩首动物和拉扯皮肤一边。 </li><li> （可选）固定器可应用于避免折返迟钝的皮肤。 </li><li>做中线切口在颅骨小心地将头骨碎片放在一边。 </li><li>小心使用适当的手术器械（ 如莫里亚勺）删除整个大脑。 </li><li>预冷加入25ml 2 -甲基丁烷在50ml的烧杯中（ 例如，肖特杜兰）到-30至-40℃，在干冰上。 注：对于这非常适合共聚焦激光扫描显微镜“厚”部分自由浮动的染色，取出大脑，洗3在缓冲液中，并储存在4℃下在磷酸盐缓冲的时间的30％蔗糖溶液的50ml管中，直到大脑已经下沉降到底部（通常过夜）。 </li><li>小心地将大脑一块Nescofilm（封口膜），并涵盖了充足的量TissueTek华侨城化合物，在-80℃直至使用2 - 甲基丁烷，店内冻结Nescofilm。 注意：对于在-20℃下储存在9％蔗糖，7毫摩尔MgCl 2，50 mM磷酸盐缓冲液，44％甘油长时间存储。 </li><li>切脑入10-12微米厚的部分适当的冷冻切片机。 注：对于厚，自由浮动的部分，大脑冻结在适当cryotome cryotome阶段（ 如赖克特荣，Frigomobil）和削减在-20°40微米的水平段至- 25℃。收集刀切片用细刷，并收集在缓冲区或保持存储解决方案（步骤4.9），在-20℃下长期贮存。 </li><li>小心地装上单显微镜载玻片两片。在我们电子商务的Superfrost UltraPlus型幻灯片强烈建议，最大限度地部分粘连。 </li><li>干燥载玻片5分钟，在室温下进行。幻灯片可以贮存于-80℃直至使用。 </li></ol> 5。从灌注动物部分的制备<ol><li>麻醉以上（步骤4.2）所描述的动物。 </li><li>仔细transcardially灌注动物用磷酸盐缓冲盐水（PBS）中含肝素（0.025 10g/100ml的PBS）和普鲁卡因（0.5 10g/100ml的PBS）中2〜4分钟，随后在PBS中的4％多聚甲醛10-15分钟。灌注是通过使用约1.2-1.4米或利用蠕动泵设置为大约15毫升/分钟的静水压力通过左心室和开口的右心房灌流最佳执行。最佳灌注导致苍白的大脑，没有红色的血管是可见的。 </li><li>解剖和后固定于4％多聚甲醛的大脑，在4℃放置24小时。 </li><li>低温保护大脑在PBS中30％蔗糖，在4℃下至少24小时。 </li><li>冻结在cryotome舞台的大脑。 </li><li>使用适当的cryotome制备40μm的由整个前脑横截面。 </li><li>幻灯片可以在-20℃直至使用被存储在冷冻保护剂溶液（0.1M磷酸盐缓冲液，50％甘油，0.14％的MgCl 2，8.6％的蔗糖）。 </li></ol> 6。未注动物的脑切片的免疫组化<ol><li>后固定冷冻切片中，如上​​所述，使用-20℃下的冷甲醇10分钟制备。 </li><li>用含0.3％的Triton-X（从哪个被用于提高二次抗体的物种）中过夜，4℃5％正常血清阻断</li><li>冲洗3次用1×PBS，并与第一抗体孵育过夜，在4℃下</li><li>冲洗3次用1×PBS，孵育在室温下的二抗一小时。 </li><li>冲洗3次用1×PBS </li><li>凑nterstain的部分使用SYTOX绿，或DAPI（或替代的DNA染料）的DNA。 </li><li>冲洗3次用1×PBS </li><li>嵌入在水性介质中嵌入的部分。 </li><li>让包埋剂聚合至少48小时。 </li></ol> 7。灌注动物的脑切片的免疫组化<ol><li>如步骤6.2中所述，随后孵育含有0.3％的Triton-X（自由浮动的）在4℃过夜固定脑切片中第一抗体溶液稀释在PBS块</li><li>漂洗三次，用1×PBS，孵育在稀释于PBS中含有0.3％的Triton-X（自由浮动）在室温下搅拌3小时的二级抗体溶液。 </li><li>冲洗3次用1×PBS </li><li>染液的部分使用SYTOX绿色或DAPI（或替代的DNA染料）的DNA。 </li><li>漂洗三次，用1×PBS </li><li>嵌入在水性介质中嵌入的部分。 </li><li>让包埋剂聚合一吨至少48小时。 </li></ol> 8。的苔藓纤维预测调查<ol><li>牺牲动物，准备上述40微米的冠状切片，用抗体神经丝染色米海马节</li><li>扫描部分在使用共聚焦激光扫描显微镜可视化苔藓纤维和核的适当波长。启动扫描在低倍镜下。 </li><li>使用低倍图像方向和目标的海马苔藓纤维的预测。 </li><li>扫描部分（Z-切片），在高分辨率和高放大倍数。 </li><li>通过分析染色的NF-M可视化的苔藓纤维的外观形态和连通性。 </li><li> （可选）分析海马叶片的尺寸和体积。使用DNA信号的测量。 </li></ol> 9。氟翡翠C含量（神经细胞死亡） <ol><li>牺牲动物，解剖hippocAMPUS，并准备上述12微米的部分。 </li><li>让该部分干燥30分钟，在室温下进行。 </li><li>固定在4％PFA的部分为40分钟，在室温下进行。 </li><li>简述双蒸2 O洗部3X </li><li>孵育0.06％的高锰酸钾（ 高锰酸钾 ）的部分为10分钟连续下，轻轻摇动。 </li><li>洗部3X用双蒸2 O。 </li><li>孵育切片用20分钟，在室温下0.002％氟玉C溶液。 </li><li> （可选）对于同时核染色，0.002％氟翡翠C溶液可补充10微克/毫升的DAPI。 </li><li>简述双蒸2 O洗部3X </li><li>让该部分干燥30分钟，在室温下进行。 </li><li>孵育二甲苯的部分（1分钟） </li><li>采用二甲苯为基础的安装介质（DPX）安装部分。 </li></ol>

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作者宣称，他们有没有利益冲突。

成年神经发生，并通过经多西环素在神经元抑制NF-κB的，其激活后其操纵的可能性，提供了调查，​​在成人大脑，以及为神经元去和重新生成的新生神经元引人入胜系统。该系统的优点在于，NF-κB信号通路抑制神经元不仅导致改变神经细胞死亡，祖细胞的增殖和迁移，以及严重的结构和解剖学上的变化，而且在学习的明显缺陷。重要的是，IκB/ tTA的动物的表型可逆转实验和多西环素给药后获救?...

本体论上，海马是已知的最古老的大脑解剖结构之一。它负责不同的复杂的任务，如在长期记忆，空间定位，并形成各自的存储器中，并固结的调节枢转功能。在解剖学上，海马由锥体细胞层（ 层锥体 ）包括大角Ammonis（CA1，CA2，CA3，CA4和）区和齿状回（ 齿状回 ），其中包含的颗粒细胞和一些神经祖细胞内的颗粒下层区。的颗粒细胞经所谓苔藓纤维（颗粒细胞的轴突）突出朝向CA3区。 直到最后世纪末，成年哺乳动物大脑被认为是静态的器官缺乏细胞可塑性和神经发生。然而，在过去的二十年中，越来越多的证据量清楚地表明了成年神经发生在至少2脑区，脑室下区（SVZ）和海马的颗粒下层区域。 我们以前的研究，并与其他团体，都表明，转录因子NF-κB的是成年神经发生的关键分子调节剂之一，其放松管制导致严重的结构缺陷，海马和认知障碍1-6。 NF-κB是五DNA结合亚基二聚体的不同组合组成的诱导转录因子的通用名称：P50，P52，C-REL，RelB基因和P65（的RelA），后三家的反式激活域。于大脑，在细胞质中发现的最丰富的形式是P50和P65，这是由卡巴B（IκB）蛋白的抑制剂保持在非活性形式的异源二聚体。 学习和直接操纵的NF-κB驱动的神经，我们用转基因小鼠模型，以使所有的NF-κBsubun的简单的抑制作用它的，特别是在脑7（ 见图1）。为了这个目的，我们杂交以下转基因小鼠系，IκB/ - 和-/tTA。采用NF-κB抑制剂IκBa（超阻遏IκBa-AA1）的反式显性负突变体产生的8号线-转基因IκB/。相反，野生型IκBα，IκBα-AA1具有两个丝氨酸残基突变为丙氨酸（V32和V36），这阻碍了该抑制剂的磷酸化和随后的蛋白酶体降解。对IκBa-AA1-转基因的前脑神经元特异性表达，IκB/ - 小鼠杂交小鼠窝藏钙 - 钙调蛋白依赖性蛋白激酶IIα（CAMKIIα）的启动子，可以通过四环素反式激活物（TTA）被驱动9。 P65基因敲除小鼠的胚胎致死表型，由于大量肝细胞凋亡10，所以这里显示的方法提供了一个优雅的方法为调查的NF-κB在产后和成年神经的作用。 经典的行为学测试，以空间学习和记忆研究20世纪80年代由理查德·莫里斯，测试被称为Morris水迷宫（MWM）11被描述。在这个开放的场水迷宫，动物学会从不透明水中逃脱到基于方向和额外迷宫的线索隐藏的平台。 MWM的干变体是所谓的巴恩斯迷宫（BM）12。本次测试采用了圆形板布置在板的边缘20圆孔，用一个定义孔作为转义框，视觉多余的迷宫线索方向。这两种实验范式依靠飞行行为引起的啮齿动物单曲厌恶水，或打开，明亮的照明空间。这两项测试让空间方位的调查，以及相关的内存性能。虽然海马起着空间记忆形成的一般和重要作用，海马Ř参与egions不同，根据不同的应用测试。在BM测试的内存源于enthorinal皮层（EC）和位于海马CA1区域无危险品13-16的贡献锥体神经元之间的神经元活动。特别是，经典的BM主要是通过突触颞ammonic途径从欧盟三CA1至欧盟五，重要的是，DG关键是参与所谓的空间模式识别17，这意味着不仅是处理依赖于导航视觉和空间信息，但也差不多陈述或记忆转化为不同的，不重叠的陈述。这个任务需要的功能的三突触回路从欧盟二DG可以CA3到CA1和EC六，不能在BM 15进行测试。 为应对这些挑战，我们设计了一套SPS-BM作为一个行为测试，具体测试齿状回依赖的认知表现在合作ntrol动物，并在IκB/ tTA的超阻抑物模型下的NF-κB的抑制。重要的是，与此相反的MWM或骨髓，在SPS-BM可以揭示从神经发生减值产生微妙的行为缺陷。因为空间图案分离是严格依赖于EC II和DG和CA3和CA1和EC VI之间的功能性电路，该测试是内中的神经发生电位变化，苔藓纤维通路的修改或组织稳态的变化高度敏感DG。 技术上，设定了我们的试验是基于研究Clelland 等，其中的空间间隔图案是用一个木制的8臂旋臂迷宫（RAM）19进行测试。在我们修改设置，八臂，7个相同的黄色食物房屋所取代。总之，这里显示的方法，包括doublecortin表达（DCX +）海马，苔藓纤维突起，神经元细胞内德细胞分析ATH特别是SPS-BM这里提出，可应用到的结合具有在成年神经发生的影响的转基因其他小鼠模型的研究。进一步的应用可能包括药物制剂的研究和测量它们对DG和空间格局分离的影响。

<table border="0" cellpadding="0" cellspacing="0" width="872">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Moria MC17 Perforated Spoon&#160;</td>
			<td style="width:127px;">FST</td>
			<td style="width:132px;">10370-18</td>
			<td style="width:167px;">removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting microscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi SV8</td>
			<td>removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical scissors&#160;</td>
			<td style="width:127px;">FST</td>
			<td>14084-08</td>
			<td>removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical scissors&#160;</td>
			<td style="width:127px;">FST</td>
			<td>14381-43</td>
			<td>removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #5 forceps</td>
			<td style="width:127px;">FST</td>
			<td>11254-20</td>
			<td>removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">SuperFrost Slides</td>
			<td>Carl Roth&#160;</td>
			<td align="right">1879</td>
			<td>slides for immunohistochemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Paraformaldehyde</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>P6148</td>
			<td>fixative</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">TissueTek OCT compound</td>
			<td>Sakura Finetek</td>
			<td style="width:132px;">1004200018</td>
			<td>embedding of the brains</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:447px;">Normal Goat Serum</td>
			<td style="width:127px;">Jackson Immunolabs</td>
			<td style="width:132px;">005-000-001</td>
			<td>blocking in IHC</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:447px;">Normal Rabbit Serum</td>
			<td style="width:127px;">Jackson Immunolabs</td>
			<td style="width:132px;">011-000-001</td>
			<td>blocking in IHC</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Normal Donkey Serum</td>
			<td style="width:127px;">Jackson Immunolabs</td>
			<td style="width:132px;">017-000-001</td>
			<td>blocking in IHC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Neurofilament-M antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>2H3</td>
			<td>IHC, Dilution 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-doublecortin&#160;antibody</td>
			<td>sc-8066</td>
			<td>Santa Cruz</td>
			<td>IHC, Dilution 1:800</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">anti-GFP antibody</td>
			<td>ab290</td>
			<td>abcam</td>
			<td>IHC, Dilution 1:2000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">anti-BrdU antibody</td>
			<td>OBT0030G</td>
			<td>Accurate Chemicals</td>
			<td>IHC, Dilution 1:2000&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Fluoro Jade-C</td>
			<td>FJ-C</td>
			<td>HistoChem</td>
			<td>Determination of neuronal cell death</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Betadine</td>
			<td style="width:127px;">&#160;MUNDIPHARMA</td>
			<td>D08AG02&#160;</td>
			<td>disinfectant</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cryomicrotome</td>
			<td>Leica</td>
			<td>CM1900</td>
			<td>preparation of brain slices</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Heparin sodium salt</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:132px;">H3393</td>
			<td>perfusion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">circular plate made from hard-plastic (diameter 120 cm)</td>
			<td style="width:127px;">lab made</td>
			<td style="width:132px;">none</td>
			<td>plate for SPS-BM, diameter 120cm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buraton rapid disinfectant&#160;</td>
			<td style="width:127px;">Sch&#252;lke &#38; Mayr</td>
			<td style="width:132px;">113 911</td>
			<td>disinfectant</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">video-tracking system TSE VideoMot 2 with Software Package VideoMot2</td>
			<td>tse systems</td>
			<td style="width:132px;">302050-SW-KIT</td>
			<td>tracking and analysis of SPS-BM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T8787</td>
			<td>permeabilisation/IHC</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:447px;">cryotome</td>
			<td style="width:127px;">Reichert&#160; Jung/Leica</td>
			<td style="width:132px;">Frigomobil 1206</td>
			<td>preparation of 40&#181;m brain slices</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Mowiol 4-88</td>
			<td style="width:127px;">Carl Roth&#160;</td>
			<td>Art.-Nr. 0713</td>
			<td>embedding of the slides</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">SYTOX green</td>
			<td style="width:127px;">Invitrogen</td>
			<td>S7020</td>
			<td>Nuclear staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Food pellets (Kellog`s Froot Loops)</td>
			<td>Kellog`s</td>
			<td style="width:132px;"></td>
			<td>SPS-BM</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:447px;">Prism, Version 3.0</td>
			<td style="width:127px;">Graph Pad Software, San Diego, USA&#160;</td>
			<td style="width:132px;"></td>
			<td>Statistical evaluation of SPS-BM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Zen 2008 or Zen 2011 Software</td>
			<td style="width:127px;">Carl Zeiss</td>
			<td style="width:132px;"></td>
			<td>Software (Confocal microscope)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">D.P.X</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:132px;">317616</td>
			<td>mounting medium for Fluoro Jade C staining</td>
		</tr>
	</tbody>
</table>

methods for the modulation and analysis of nf-&#954;b-dependent adult neurogenesis

海马起着举足轻重的作用，在情节记忆的形成和巩固，并在空间定位。从历史上看，成年海马一直被看作是哺乳动物大脑的一个非常静态的解剖区域。然而，最近的研究结果已经表明，在海马的齿状回是巨大的可塑性在成人的区域，不仅包括修改现有的神经元回路的，但也成年神经发生。此可塑性被复杂的转录调控网络，其中，所述转录因子NF-κB中起着突出的作用。研究和操纵成年神经发生，转基因小鼠模型的NF-κB活性的前脑特异性神经元的抑制可以使用。 在这项研究中，方法是用于对NF-κB依赖的神经发生的分析，包括其结构方面，神经元凋亡和祖细胞的增殖和认知意义，whic描述h的通过齿状回（DG）相关的行为测试，空间格局分离 - 巴恩斯迷宫（SPS-BM）的特别评估。在SPS-BM协议可以简单地适用于与设计，以评估特定基因对成年海马神经发生的影响等转基因动物模型的使用。此外，SPS-BM可以在其他实验设置旨在调查和处理DG-依赖的学习，例如，使用药剂使用。

杂交育种的IκB/ -以及tTA的转基因小鼠系导致NF-κB活性的海马条件抑制。 调查IκBα-AA1-转基因的双转基因小鼠（ 图1A）的表达，大脑分离，cryosectioned和使用抗体抗GFP（绿色荧光蛋白）染色。共聚焦激光扫描显微镜观察发现高表达在CA1和CA3区的转基因，并在危险品（ 图1B）。 该IκBα-AA1-转基因表达类型-2B祖细胞?...

Watch this Scientific Journal Video about Methods for the Modulation and Analysis of NF-ÎºB-dependent Adult Neurogenesis at JoVE.com

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

方法的NF-κB依赖型成年海马神经发生的操作和分析的描述。详细的协议提出了一个齿状回依赖性行为测试（称为空间格局分离巴恩斯迷宫）的认知结果小鼠的调查。这项技术也将有助于使在其他实验设置的调查。

方法对NF-κB依赖的成年神经发生的调制与分析

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

methods-for-modulation-analysis-nf-b-dependent-adult

Research

JoVE Journal

Neuroscience

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

14.2K 次观看.  University of Bielefeld. 方法的NF-κB依赖型成年海马神经发生的操作和分析的描述。详细的协议提出了一个齿状回依赖性行为测试（称为空间格局分离巴恩斯迷宫）的认知结果小鼠的调查。这项技术也将有助于使在其他实验设置的调查。 

视频: Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

成年鼠脑铸造三维成像和形态分析

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

科研

神经科学

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

成人下丘脑神经发生局灶性放射性抑制功能审讯

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

斑马鱼成人端脑的一刀，受伤：一个方法调查脊椎动物脑部神经发​​生与再生

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

免疫组化和多标签与同一宿主物种抗体研究成人神经发生

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

评估主要神经再生<em&gt;爪</em&gt;胚胎使用免疫染色

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

分析突触调制<em&gt;果蝇</em&gt;曝光时间延长后，光光感受器

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

小鼠后脑作为研究胚胎神经再生的模型

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

果蝇幼虫和成年脑的代谢分析

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

成人脊髓细胞核在大规模平行单核 RNA 测序中的分离

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...