这项工作得到了萨伏地基金会和治疗癫痫基金会的业务赠款和加拿大创新基金会 (共聚焦显微镜) 和 G H (旋转圆盘共焦显微镜) 的设备赠款的支持。急诊室从全宗 de 研究所Santé (FRQ S) 获得职业奖;临床医生-科学家奖) 并且从加拿大学院为健康研究 (卫生研究院;年轻研究员奖)。伦理是 FRQ 的资深学者。L. E 是 Steriade-萨伏基博士后培训奖的接受者, 从萨伏依基金会, 楚圣-贾斯汀基金会博士后培训奖和 FRQ 博士后培训奖, 与明星基金会合作。加拿大脑研究基金在加拿大卫生部的财政支持下, 通过加拿大大脑研究所, 使这个项目成为可能, 授予 L.E。

所有涉及动物的实验均由 Institutionnel 女佣 Pratiques avec Animaux de 研究所 (CIBPAR) 在楚圣贾斯汀研究中心批准, 并按照加拿大动物保育委员会指南进行实验动物的护理和使用 (Ed 2)。
此处描述的协议是在胚胎日 (e) 13.5 中对胚胎的电穿孔进行优化的, 此时在梅兰日兰派生的 ins 被主动生成之前, 在 CGE 派生的 ins 生产45,46的峰值之前。此外, 为了将电穿孔偏向 GABAergic, 我们使用在 ins 中有选择地表达的启动器 (例如, Dlx5/6启动器及其最小增强程序)47。
1. 电穿孔和脊髓切片培养液的制备
<ol><li>准备125毫升无菌培养基。<ol>
<li>测量125毫升的常规神经元特定培养基 (参见材料表, 用于配方) 在先前 UV 灭菌的生物安全柜中喷洒70% 乙醇的消毒瓶。添加2.25 毫升50x 无血清神经元特异补充剂, 1.75 毫升200mM 谷氨酰胺 (最终浓度0.5 毫米) 和6.25 毫升的热灭活马血清以前 aliquoted 在无菌条件下。彻底混合, 整除15毫升无菌锥形管, 并贮存在4摄氏度。 注: 制备培养基可贮存3周至4摄氏度。</li>
		<li>将 Botteinstein 的 N-2 配方48的100X 库存解决方案划分为150µL 整除数在无菌条件下, 并在20°c 处冻结直至使用。</li>
	</ol>
</li>
	<li>制备 1 L 无菌人工脑脊液 (ACSF)。<ol>
<li>在1升烧杯中测量800毫升蒸馏水。加入25.67 克蔗糖, 5.08 克氯化钠 (NaCl), 2.18 克碳酸氢钠 (NaHCO3), 1.80 克葡萄糖, 0.19 克氯化钾 (氯化钾), 0.15 克磷酸钙无水磷酸磷酸钠 (NaH2PO4), 1 毫升1M 库存 CaCl2. 2H2O 和2毫升1M 库存 MgSO47H2o 搅拌在室温下溶解。加蒸馏水达到总容积1升。</li>
		<li>使用0.22 µm 过滤器, 将溶液过滤成消毒后的生物安全柜中的无菌瓶, 贮存在4摄氏度至1月。</li>
	</ol>
</li>
	<li>在每次试验前, 在 ACSF 中准备4% 琼脂糖的新鲜溶液。<ol>
<li>测量25毫升以前制备的 ACSF 在50毫升无菌圆锥管和添加1克的低熔点琼脂糖。</li>
		<li>四十五年代在微波炉加热。为避免溢出, 每 3-4 秒加热时, 每次沸腾启动时, 打开管子让压力流出, 再关闭它, 并手动搅拌以混合琼脂糖。重复, 直到琼脂糖溶液是均匀的。保持琼脂糖溶液在42°c 在其余的实验, 以避免凝固。 注意: 温度升高会损伤脑组织。</li>
	</ol>
</li>
</ol>2. 注射用质粒的制备
<ol><li>拉玻璃微注射吸管<ol>
<li>用适当的参数设置微拉拔器, 在所提供的空间中确保玻璃毛细管的安全, 并确保其以灯丝为中心。</li>
		<li>按下拉按钮。</li>
		<li>小心地去除新做的微注射吸管从热拉器和地方在盒或清洁培养皿, 直到进一步使用, 以避免损坏的小费。</li>
	</ol>
</li>
	<li>建立生物安全柜与本实验所需的所有仪器 (见图 1A), 用70% 乙醇将生物安全柜中的所有仪器进行慷慨喷洒, 用 UV 光为 15-20 分钟消毒仪器和环境。</li>
	<li>在灭菌步骤中, 在冰上解冻质粒 (4 °c)。</li>
	<li>从库存溶液 (4 µg/µL) 到清洁1.5 毫升离心管中测量质粒的10µL。添加0.01% 的快速绿色 FCF。轻轻地混合, 旋转, 并保持在冰上, 直到使用。 注: 最大准备 DNA 应根据制造商的协议, 使用无内毒素的最重要的试剂盒准备。DNA 可以可溶性在 TE 缓冲器或无核酸酶 H 2 O 和用染料溶液制备质粒, 但不需要在无菌条件下进行.应 aliquoted 质粒从最大的准备, 以避免多个冻融循环。在使用前, Aliquoted 质粒应与染料混合, 2 小时以上, 不应解冻进一步使用。</li>
	<li>灭菌后, 准备纳米注射器如下。<ol>
<li>选择一个以前准备好的玻璃微注射吸管储存在一个干净的盒子或培养皿, 并使用小镊子削减的尖端的吸管在斜面的方式, 以达到约15µm 外径。 注: 这里给出的外径是一个近似的措施, 给用户一个想法, 我们在我们的实验中使用, 并已优化, 以方便穿刺的头骨的质粒注射, 而不损害大脑, 同时允许液体DNA 的加载和释放。外径可以通过观察玻璃微注射吸管并列在明亮的场显微镜下的测微刻度条上的切割尖端来测量。</li>
		<li>用注射器填充微的 unpulled 端的矿物油 (排出所有的空气)。</li>
		<li>按照制造商的说明, 在纳米喷油器中插入填充玻璃微。</li>
		<li>空 2/第三的玻璃微 (保持足够的油, 以防止空气进入)。</li>
	</ol>
</li>
	<li>在含有质粒/染料溶液的管中小心插入所制备的微, 并用质粒/染料溶液填充玻璃微。</li>
</ol>3. 从孕妇中收集小鼠胚胎
<ol><li>每日监测雌性繁殖, 以评估阴道堵塞情况, 最好是每天同一时间 (下午)。日 e0.5 对应于第一天, 当阴道插头被观察。 注意: 这里所描述的实验可以在野生型小鼠中进行。但是, 为了便于识别梅兰日兰并对所有 GABAergic (或特定的子集 (如梅兰日兰派生的 ins) 进行标记, 可以使用转基因动物 (例如: GAD67EGFP;Dlx5/6 的., 其中有一个有创报告的等位基因, 等等47,49)。在这种情况下, 注射的实验质粒应表达另一种荧光 (例如mCherry或TdTomato), 以允许可与未转染的 ins (绿色) 进行比较的转染 ins (黄色) 的可视化。</li>
	<li>在胚胎日 e13.5, 由颈部脱位牺牲雌性。 注: 在牺牲时给出的麻醉药可能影响迁移和生存50,51 , 应避免。</li>
	<li>按 C 节采集胚胎, 如下所示。<ol>
<li>用70% 乙醇慷慨喷洒女性腹部。用一双消毒钳拉腹部皮肤, 另一方面, 用消毒的手术剪刀从腹部切开皮肤。</li>
		<li>用第二对消毒钳和剪刀, 拉腹部筋膜, 并削减它, 而小心避免子宫。</li>
		<li>使用第三对消毒钳和剪刀, 拉子宫角, 并把他们从骨盆腔。将解剖后的子宫角放置在一个无菌的60毫米培养皿中, 用神经基培养基补充氨基酸、维他命和无机盐 (参见材料表以供商业使用的产品)。</li>
	</ol>
</li>
	<li>在无菌生物安全柜中, 使用两对细镊子 (每只手一只) 从胎盘中解剖胚胎, 用斩首将头颅分离。</li>
	<li>锥切的一个无菌3毫升塑料转移吸管的尖端, 吸头和转移他们在一个新的无菌60毫米培养皿分层的固化黑蜡和填充相同的神经基补充培养基, 如上所述。 注意: 这一步最小化污染物的转移 (老鼠头发, 血液)。黑色蜡用于在解剖过程中稳定头部。在这些过程中, 文化媒体不需要有氧。</li>
</ol>4. 梅兰日兰脑室内质粒注射液和体外电穿孔
注: 在以前准备的生物安全柜中, 必须在无菌条件下执行以下步骤。
<ol><li>将60毫米的培养皿分层为黑色蜡, 并在生物安全柜内的双筒望远镜下, 将被斩首的头颅置于神经基补充培养基中。</li>
	<li>稳定头部, 延髓部分面对右, 用细镊子与左手, 并使用纳米注射器在右手注入 1-2 µL 的质粒/染料溶液进入右侧脑室。 注: 通过将两种质粒混合在摩尔浓度上, 通过共 electroporating 一 shRNA 表达质粒, 进行共表达实验。</li>
	<li>Electroporate 注射的大脑。<ol>
<li>将磁头置于电极之间, 将负极定位背, 并平行于头部和正电极朝向头部的腹侧, 以梅兰日兰为目标。</li>
		<li>一旦电极定位良好, 在500毫秒间脉冲间隔内, 为50毫秒提供4平方脉冲 40 V。</li>
		<li>使用已置于生物安全柜中的镊子从电极上取出任何残余组织。 注意: 这些参数已针对我们实验中使用的 electroporator 进行了优化。建议用户在使用不同类型的 electroporator 时预先进行优化测试。</li>
		<li>对所有剩余的大脑重复步骤4.1 到4.3。 注意: 尽管本协议描述了一个大脑所需的操作, 但在 electroporating 每个大脑之前, 可以依次注入多达4个大脑, 从而提高了产量。当2种或更多不同的质粒在同一实验中 (例如控制或实验粒) 被注射 (允许窝比较) 时, 这种策略特别有利。此外, 通过将电极完全平行于脑表面, 可以同时注入和 electroporate 大脑两侧以增加产量。</li>
	</ol>
</li>
</ol>5. 脑解剖和脊髓切片培养
<ol><li>虽然仍然在生物安全柜的无菌环境中操作, 解剖大脑出头骨。<ol>
<li>将针头插入每只眼睛, 同时小心地避免大脑, 使头部在黑色蜡层上稳定下来。</li>
		<li>使用一对细镊子, 以保持颈部左侧和第二对细镊子撕裂皮肤从头骨, 从背部到前面。</li>
		<li>一方面用镊子将头部侧向, 另一方面, 用另一双镊子仔细地将头骨在脑干的水平上切开, 然后轻轻地将头骨向上拉。每镊子, 在矢状平面 (中线) 上切开头骨朝向前面, 然后侧向切开以解放头骨碎片。</li>
		<li>抬起脑干, 仔细切开脑膜和脑神经, 直到大脑完全脱离头骨。 注: 5.1 中描述的所有步骤都应在安全柜的严格无菌条件下进行。</li>
	</ol>
</li>
	<li>将大脑嵌入4% 低熔点琼脂糖切片。<ol>
<li>用上面准备的琼脂糖溶液填充35毫米培养皿 (保持液体在42摄氏度)。</li>
		<li>使用先前的割转吸管快速将转的大脑转移到琼脂糖填充皿中。把盘子放在室温下。</li>
		<li>用金属棍搅动琼脂糖, 使大脑保持在井中间 (防止下沉), 并将大脑定位在与盘子平行的 rostro 尾平面上。当琼脂糖开始凝固时, 不要搅拌, 以免脑损伤。</li>
		<li>用剃刀刀片切开围绕大脑的琼脂糖, 以形成一个长方形块, 在大脑周围留下 1-2 毫米的边缘。确保大脑的延髓部分垂直于块的前极限, 以利于后续切片步骤的方向。</li>
		<li>为每个大脑重复。 注意: 通过在不同高度设置每个大脑的同时, 通过形成单独的琼脂糖块, 可以一次切割超过一个大脑 (最大 3)。</li>
	</ol>
</li>
	<li>Vibratome 冠状部分和切片培养。<ol>
<li>解冻一整除 100X N-2 补充 (150 µL) 在冰上, 并添加到15毫升 aliquoted 培养基在无菌条件下。</li>
		<li>将750µL 培养基 (用 1X N2 补充) 输送到6井培养板的每一个井中。</li>
		<li>用弯曲的镊子, 安置一个细胞培养插入物 (30 毫米直径, 0.4 µm 孔大小, PTFE) 在每个中等填装的井。</li>
		<li>用连续含氧 ACSF 填充 vibratome 浴。冷却到4°c 与周围的冰浴, 或使用冷藏 vibratome。</li>
		<li>将 vibratome 速度设置为0.150 毫米/秒, 频率为80赫兹。</li>
		<li>将琼脂糖块粘附在 vibratome 平台上, 延髓边缘朝下, 面对用户的腹边边缘。</li>
		<li>切割冠状部分的大脑, 以获得250µm 厚的部分 (在4摄氏度)。</li>
		<li>与灭菌铲子, 收集 2-3 个部分包含梅兰日兰, 并把所有的大脑部分从一个动物在一个单一的30毫米膜插入, 而小心避免节之间的重叠。将插入物放置在6井培养板的一个井中 (如上所述), 其中包含750µL 的补充培养基。另外, 每节可以放置在一个单独的13毫米直径膜在一个12井培养板填充500µl 补充培养基。为每个井推荐的培养基数量允许大脑部分在不被淹没的情况下由培养基滋养。 注: 5.3.6 和5.3.7 中描述的步骤在完全不育条件下不进行, 除非 vibratome 可以在生物安全柜中进行灭菌和使用。因此, 必须小心地采取这些步骤, 以避免任何污染。适当的防护设备 (清洁口罩、手术手套和实验室大衣) 应佩戴在任何时候和身体部位, 甚至覆盖, 如头发, 脸部和手, 不应该通过文化板块 (有或没有培养基)。它还建议经常喷洒70% 乙醇的手套和铲子用于收集大脑部分。</li>
		<li>将文化板块放在通风不育的孵化器中, 温度为37摄氏度, 湿度为 60%, 5% CO2为48或 72 h。 注意: 这些潜伏期被优化为梅兰日兰的时间推移成像和固定切片共焦成像。每个实验设计都应预先测试最佳潜伏期。此外, 如果选择的孵化是72小时和下, 没有必要改变培养基。对于较长的潜伏期, 培养基应每 2-3 天改变一次。</li>
		<li>在理想的孵化时间后, 将感兴趣的部分转移到一个8腔的盖玻片中, 并添加 3-5 µL 培养基。将盖玻片放在环境室 (37 °c, 60% 湿度, 5%CO2) 上, 连接到一个倒置的旋转圆盘共焦, 配备了计算机辅助的采集软件来设置时间推移成像会话。 注: 或者, 节可以固定与4% 多聚甲醛 (隔夜在4摄氏度或2小时室温), 随后 immunostained 与不同的抗体, 以可视化的形态学特征的转在共焦显微镜。虽然 eGFP 和 mCherry 可以通过共聚焦显微镜可视化, 没有任何反染色程序, 我们建议对 GFP 和 mCherry 进行免疫组化, 以增强信号, 因为固定过程可以减少荧光, 减少检测胚胎神经元的较细成分, 如前导或尾随过程中较小的分支。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。此处所表达的意见不一定代表卫生部长或加拿大政府的意见。

在本文中, 我们提供了一个可靠的方法来执行前子宫内电穿孔的小鼠梅兰日兰在 e13.5 和脊髓培养胚胎脑切片。虽然体外方法 (如博伊登室化验) 相对容易执行, 可用于评估不同基因和蛋白质在不受其他因素干扰的情况下的具体作用, 但它们排除了在关于方向性和迁移路径的迁移动态25。梅兰日兰外植体提供了一种有用的方法来研究在迁移过程中发生的动态骨架变化, 正?...

皮质 GABAergic 中间神经元 (INs) 不同于它们的生化特性、生理特性和连通性, 它们在成熟网络中调解不同的功能 1, 2, 3 ,4,5。通过对1、2进行了广泛研究的遗传级联, 对皮质 INs 的不同亚型进行了严格的规范。大多数 (70%) 皮质 GABAergic 的起源于祖细胞的内侧节隆起 (梅兰日兰), 腹部定位胚胎结构, 并必须跨越较长的距离迁移到皮质板1,2,6. 当皮质锥体细胞从心室区 (VZ) 向皮质板块沿径向胶质组织支架进行径向迁移时, 不依附于这种支架的 INs 的切向迁移需要多种内在和外部提示, 吸引迁移神经元朝向皮质板块, 同时引导他们远离非皮层结构2,7,8。在退出细胞周期后, 在梅兰日兰的 VZ 中表达的化疗排斥信号会使 INs 被排斥在梅兰日兰中, 这会触发向皮质板块9,10的切向迁移。迁移 INs 避免纹状体与不同的排斥线索11 , 并在到达皮质板块后, 他们从一个切线切换到径向迁移模式, 并达到他们的最终层流位置, 部分是响应从金字塔的线索单元格12和其他蜂窝群13。对于其他神经元种群, INs 的迁移涉及各种动态形态学变化, 以允许神经元的实际运动。这种所谓的神经元运动的特点是重复循环三个连续步骤: 一个领先的进程的伸长, 核的主动顺运动 (nucleokinesis), 并撤回的尾随进程14。在迁移过程中, 通过许多内在和外在的线索来调节引导进程的分支和主动重塑, 以正确的方向指导 INs, 同时确定迁移的方向和速度14,15 ,16。
在迁移中调节皮质的决定因素近年来得到了广泛的研究1,2,7,17,18,19,20,一些分子行为者的中断被假定导致神经发育紊乱, 如小儿难治性癫痫或自闭症频谱紊乱1,2,21,22,23,24. 因此, 开发了各种体外和体内方法, 以极大地提高我们研究这个动态过程的能力, 如前所述25。体外方法, 包括博伊登室法和条纹选择法, 提供了评估神经元迁移过程中特定基因或蛋白质的需求和细胞自主影响的最快和最可重现的手段,没有其他因素的影响25。当与实时成像8、26、27结合使用时, 这些检测尤其有用。通过这些技术, INs 可以很容易地从 e13.5 梅兰日兰中提取出来, 并通过酶和机械分离被分离, 然后通过不同的信号通路和指导线索进行研究, 如前所示8,28.然而, 这些化验发生在一个人工细胞外基质中缺乏三维组织结构, 这可能会改变神经细胞的行为和细胞的性质, 可能影响细胞迁移和/或生存25。为了规避这些限制,前体梅兰日兰外植体已被开发为一种替代工具, 用于量化迁移过程中发生的动态形态学变化以及诸如速度和方向14等参数, 29。生成梅兰日兰外植体相对简单, 在其他地方也被广泛描述为30。它需要在有吸引力或令人反感的提示25的情况下, 将梅兰日兰的小提取物涂在混合皮质细胞的单层上, 或者在胶和胶原蛋白的混合物中, 尽管后者是可选的31。梅兰日兰外植体允许高分辨率成像的疏生标记细胞, 简化了细胞内过程的研究, 如骨架重塑在领先的过程分支, 如前所示32,33 、34和本研究中。梅兰日兰外植体已成功地用于评估在2D 环境中迁移过程中的动态骨架变化, 例如在特定的药理或趋化操作之后 (参见, 例如, Tielens et 201633).然而, 通过这种方法, INs 在一个人工矩阵中迁移, 这可能改变行为和实验结果的再现性和意义。
相比之下,在子宫内电穿孔能够在其本土环境中对 INs 进行遗传操作, 是一种广泛使用的方法, 可以快速有效地评估基因功能的增益和丢失的影响, 同时规避成本高昂且耗时的挖空和敲入策略25,35。在子宫内电穿孔可能会偏向于祖细胞, 通过使用单元类型特定的促进器, 并将电极定位到丘脑结构, 包括梅兰日兰36。此外,在子宫内电穿孔允许在 1-2 天内及时表达实验性构造, 与使用病毒载体25构建表达式所需的 7-10 天相比。然而, 在祖细胞中的在子宫内电穿孔往往是低产量。的确, 虽然位于背心室区的锥体细胞祖体可以用在子宫内电穿孔中有效地转染, 但针对更多的腹部定位结构, 如梅兰日兰, 在技术上更具挑战性,特别是在小 e13.5 胚胎中, 胚胎致死率的高速度进一步降低了实验产量25。
为了规避与体外梅兰日兰植块实验和体内在子宫内的的一些技术局限性电穿孔,前体脊髓切片培养已经开发8,37, 38,39。大脑脊髓切片区域性提供了模拟体内条件的优势, 同时它比生成动物模型25更便宜, 而且耗时。事实上, 这些准备工作可以方便地访问梅兰日兰, 同时还可以通过特定的 INs 可视化, 并可与焦电穿孔结合, 调查在更具生理环境中迁移的 ins 中的特定分子通路8,39,40,41. 因此, 我们优化了脊髓文化的方法38, 我们结合前子宫电穿孔和时间推移共焦成像, 进一步评估的形态学和动态过程中发生在梅兰日兰的切向迁移过程中。本议定书是根据使用了前子宫或子宫内脑电穿孔和脊髓切片培养的其他人进行调整和优化的, 以研究锥体细胞的迁移42,43和皮质 INs36,39,44。具体来说, 小鼠胚胎被斩首, 梅兰日兰是转的前体在脑室注射实验质粒后, 允许更有效和精确的靶向梅兰日兰祖细胞比什么可以达到在子宫内电穿孔。然后将大脑提取并切片成全脑冠状切片, 可以培养几天, 从而允许连续跟踪和成像转染的 INs。这种方法通常标签为每脑切片 5-20 个切向迁移的 INs, 尽量减少达到统计学意义所需的实验迭代次数, 同时标记足够稀疏的神经元种群, 以确保容易分离单个神经元进行重建和精细形态学评估。此外, 与梅兰日兰外植体相比, 脊髓文化确保迁徙的 INs 接触到更自然的环境, 包括局部分泌的趋化因子和丘脑传入的投入。因此, 这种方法非常适合于量化转染的 INs 所采用的方向性和迁徙路径, 同时提供足够的解剖细节, 以便能够刻画出更精细的动态过程, 如领先的过程分支,nucleokinesis 和尾随过程撤回。

<table>
	<tbody>
		<tr>
			<td>Neurobasal Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>21103049</td>
			<td>Commercially available neuron-specific culture medium. Complete formulation available on this website: https://www.thermofisher.com/ca/en/home/technical-resources/media-formulation.251.html</td>
		</tr>
		<tr>
			<td>B-27 serum-free supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>17504044</td>
			<td>50X Serum-free neuron specific supplement</td>
		</tr>
		<tr>
			<td>15 mL sterile centrifuge tubes</td>
			<td>Sarstedt</td>
			<td>62.554.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s (1X) L-15 Medium (+ L-Glutamine)</td>
			<td>ThermoFisher Scientific</td>
			<td>11415064</td>
			<td>Commercially available neural-based culture medium supplemented with amino acids, vitamins and inorganic salts. Complete formulation available on the distributor&#39;s website&#160;</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum, heat inactivated</td>
			<td>Millipore-Sigma</td>
			<td>H1138-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurocell supplement N-2 100X</td>
			<td>Wisent</td>
			<td>305-016</td>
			<td>Botteinstein&#39;s N-2 Formulation</td>
		</tr>
		<tr>
			<td>VWR Square PETG Media Bottles 125 mL</td>
			<td>VWR</td>
			<td>89132-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II Type A Biosafety Cabinet</td>
			<td>Nuaire</td>
			<td>NU-540</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>BioShop</td>
			<td>SUC700.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>BioShop</td>
			<td>SOD001.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>ThermoFisher Scientific</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr>
			<td>D+ glucose</td>
			<td>Millipore-Sigma</td>
			<td>G7528-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>ThermoFisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic anhydrous</td>
			<td>BioShop</td>
			<td>SPM400.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>BiosShop</td>
			<td>MAG522</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>BioShop</td>
			<td>AGA002.500</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile centrifuge tubes</td>
			<td>Sarstedt</td>
			<td>62.547.004</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL centrifuge tubes</td>
			<td>Sarstedt</td>
			<td>72.690.001</td>
			<td></td>
		</tr>
		<tr>
			<td>P-97 Flaming/Brown Micropipette puller</td>
			<td>Sutter Instruments Co.</td>
			<td>Model P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>0.4 mm I.D. x 75 mm Capillary Tube</td>
			<td>Drummond scientific</td>
			<td>1-000-800/12</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>E193</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Becton Dickinson &#38; Co</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil (heavy)</td>
			<td>Rougier Pharma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>WPI Swiss Tweezers #5</td>
			<td>World Precision Instruments</td>
			<td>504511</td>
			<td>11 cm, straight, 0.06x0.07mm tips, antimagnetic. You will need 2 of these.</td>
		</tr>
		<tr>
			<td>WPI Swiss Tweezers #7</td>
			<td>World Precision Instruments</td>
			<td>504504</td>
			<td>11.5 cm, 0.18x0.2mm, curved tips</td>
		</tr>
		<tr>
			<td>HTC Tweezers</td>
			<td>World Precision Instruments</td>
			<td>504617</td>
			<td>11 cm, Straight, flat</td>
		</tr>
		<tr>
			<td>Operating scissors</td>
			<td>World Precision Instruments</td>
			<td>501225</td>
			<td>16 cm, Sharp/sharp, straight. You will need 3 of these.</td>
		</tr>
		<tr>
			<td>Dressing Forceps</td>
			<td>World Precision Instruments</td>
			<td>501217</td>
			<td>12.5 cm, straight, serrated</td>
		</tr>
		<tr>
			<td>Iris Forceps</td>
			<td>World Precision Instruments</td>
			<td>504478</td>
			<td>10.2 cm, full curve, serrated</td>
		</tr>
		<tr>
			<td>DeBakey Tissue Forceps</td>
			<td>World Precision Instruments</td>
			<td>501996</td>
			<td>15 cm, 45&#176; angle, Delicate Jaw, 1.5mm wide</td>
		</tr>
		<tr>
			<td>Fisherbran Microspatula with rounded ends</td>
			<td>FisherScientific</td>
			<td>21-401-5</td>
			<td>You will need 2 of these.</td>
		</tr>
		<tr>
			<td>Nanoject II Auto-Nanoliter Injector</td>
			<td>Drummond scientific</td>
			<td>3-000-204</td>
			<td></td>
		</tr>
		<tr>
			<td>TC Dish 60, Standard</td>
			<td>Sarstedt</td>
			<td>83.3901</td>
			<td>60-mm dish</td>
		</tr>
		<tr>
			<td>Tissue culture dish</td>
			<td>Sarstedt</td>
			<td>83.1800</td>
			<td>35-mm dish</td>
		</tr>
		<tr>
			<td>Black Wax</td>
			<td>FisherScientific</td>
			<td>S17432</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes&#160;</td>
			<td>Ultident</td>
			<td>170-CTB700-212</td>
			<td>3 mL, small bulb</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Leica Biosystems</td>
			<td>Leica M80</td>
			<td>In replacement to our stereomicroscope which has been discontinued by the manufacturer (StereoMaster from FisherScientific)</td>
		</tr>
		<tr>
			<td>Electro Square Porator</td>
			<td>BTX Harvard Apparatus</td>
			<td>ECM 830</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezertrodes, Plattinum Plated, 3mm</td>
			<td>BTX Harvard Apparatus</td>
			<td>45-0487</td>
			<td></td>
		</tr>
		<tr>
			<td>25G 1 1/2</td>
			<td>Becton Dickinson &#38; Co</td>
			<td>305127</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica VT1000S Vibrating blade microtome</td>
			<td>Leica Biosystems</td>
			<td>VT1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>GEM, Single edge razor blade</td>
			<td>Electron Microscopy Sciences</td>
			<td>71952-10</td>
			<td>Remove the blunt end before inserting in the blade designated space of the vibratome</td>
		</tr>
		<tr>
			<td>&#181;-Slide 8 well</td>
			<td>Ibidi</td>
			<td>80827</td>
			<td>Pack of 15</td>
		</tr>
		<tr>
			<td>Millicell cell culture insert</td>
			<td>Millipore-Sigma</td>
			<td>PICM0RG50</td>
			<td>30 mm, hydrophilic PTFE, 0.4 &#181;m pore, pack of 50.&#160;</td>
		</tr>
		<tr>
			<td>Leica DMi6000 microscope</td>
			<td>Leica Microsystems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disk confocal head Ultraview Vox</td>
			<td>Perkin Elmer</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Volocity 6.0 acquisition software</td>
			<td>Improvision/Perkin Elmer</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>LiveCell Stage top incubation system</td>
			<td>Pathology devices</td>
			<td>LC30030</td>
			<td>Provides Temperature, CO2 and humidity control.&#160;</td>
		</tr>
		<tr>
			<td>SP8 confocal microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mCherry-Lifeact-7</td>
			<td>Addgene</td>
			<td>54491</td>
			<td>Gift from Michael Davidson</td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Millipore-Sigma</td>
			<td>F7258-25G</td>
			<td>25G bottle, certified by the Biological Stain Commission</td>
		</tr>
	</tbody>
</table>

ex utero electroporation and organotypic slice cultures of embryonic mouse brains for live-imaging of migrating gabaergic interneurons

GABAergic 中间神经元 (INs) 是神经网络的重要组成部分, 它驱动着认知和行为。用于填充皮层的 INs 在腹侧前脑 (包括从内侧和尾部节显赫人士 (梅兰日兰、CGE)) 向背皮质板块迁移, 以响应各种内在和外在线索。多年来, 已经制定了不同的方法来基因操作特定的途径, 并调查它们如何调节在迁移过程中适当的动态骨架变化。在子宫内电穿孔广泛用于研究基因抑制或过度表达在亚型中的作用, 同时评估对形态学和最终位置的影响。然而, 虽然这种方法很容易用于修改径向迁移的锥体细胞, 但它在技术上更具挑战性, 当靶向 INs.在子宫内电穿孔产生低产量, 因为幼崽的存活率降低时电穿孔是在 e14.5 之前进行的, 就像研究梅兰日兰的习惯一样。在另一种方法中, 梅兰日兰外植体为梅兰日兰提供了方便的途径, 并促进了基因修饰的 INs 的成像。然而, 在这些外植体中, INs 迁移成一个人工基质, 缺乏内源性指导线索和丘脑输入。这促使我们优化一种方法, 使 INs 可以在更自然的环境中迁移, 同时规避在子宫内方法中的技术挑战。本文描述了胚胎小鼠脑中的前宫内电穿孔与脊髓切片培养的结合, 以方便地跟踪、图像和重建基因改造的 INs 沿着自然路径迁徙, 以应对内源性线索。这种方法既可以量化的动态方面的迁移与时间推移共焦成像, 以及详细分析各种形态学参数使用神经元重建的固定 immunolabeled 组织。

在这一节中, 我们提供了具有代表性的结果后, 在前子宫内电穿孔的控制质粒, 或一个实验质粒靶向感兴趣的基因, 在 e13.5 小鼠胚胎梅兰日兰后脊髓切片培养孵化37°c 为48小时 (用于时间推移成像) 或 72 h (用于固定和免疫组化标记) (请参见图 1B用于示意图协议)。还说明了从梅兰日兰外植体迁移的 INs 的代表性示例 (参见图 1C?...

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Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

在这里, 我们提供了一个低成本和可靠的方法来产生转脑脊髓切片培养的小鼠胚胎适合共焦显微镜和实时成像技术。

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

ex-utero-electroporation-organotypic-slice-cultures-embryonic-mouse

Research

JoVE Journal

Neuroscience

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

9.9K 次观看.  Centre de recherche du CHU Sainte-Justine. 在这里, 我们提供了一个低成本和可靠的方法来产生转脑脊髓切片培养的小鼠胚胎适合共焦显微镜和实时成像技术。

视频: Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

科研

神经科学

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

在子宫内电穿孔:鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

胚胎腹侧中脑器官型片文化:一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

浦肯野细胞树突形态的器官切片培养分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

扩展胚胎及成体神经干细胞<em&gt;在子宫内</em电穿孔或病毒立体定向注射

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

皮质的interneurons的亚型选择性电穿孔

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

神经血管重塑的分析Entorhino，海马脑切片培养

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em&gt;在Utero</em&gt;电穿孔

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...