我们感谢瓦西莉奇 Chantzara 执行定时交配和生物资源单位的工作人员在伦敦大学学院眼科为老鼠饲养。这项研究得到了惠康信托调查员奖 095623/z/11/z 到 CR 的支持。

所有的动物工作都是按照英国内政部和当地的道德准则进行的。
1. 步骤和时间摘要
<ol><li>对交配的成年小鼠进行定时的自适应应答, 以获得胚胎日 (e) 9.5-e13.5 妊娠;需要 12-15 天。</li>
	<li>可选, 准备 5-溴-2-脱氧 (BrdU)/5-乙炔-2 '-脱氧 (教育) 解决方案和执行注入 (协议部分 2);需要1小时的前一天或前一天的胚胎隔离, 取决于所需的教育/BrdU 标签的长度。</li>
	<li>执行胚胎隔离和后脑解剖 (议定书3节);需要10分钟/胚。</li>
	<li>执行 wholemount 免疫荧光标记 (协议4节);需要3天。</li>
	<li>节使用 vibratome 和执行浮动部分免疫荧光标记 (协议节 4): 需要2天。</li>
	<li>节使用低温和执行免疫荧光标记的 cryosections (协议5节);需要2天。</li>
</ol>2. 用 BrdU 或教育注射怀孕雌性小鼠 (可选)
<ol><li>将无菌磷酸缓冲盐 (PBS) 中的 BrdU 或教育分解为10毫克/毫升和1毫克/毫升的浓度。 警告: BrdU 和教育是有毒的;穿戴适当的保护。</li>
	<li>称量怀孕的小鼠, 并计算 BrdU 或教育解决方案的体积, 应管理达到100毫克/千克 BrdU 或5毫克/千克教育。</li>
	<li>在采集胚胎前1小时或1天, 通过腹腔内路线注入 BrdU 或教育解决方案, 具体取决于所需的标签长度。 注: 标记为 1 h 可视后脑细胞 S 阶段。标记为1天的后脑 npc 的后裔。</li>
</ol>3. e9.5-e13.5 小鼠胚胎 Hindbrains 的解剖
<ol><li>安乐在规定的妊娠期 (如, 颈椎脱位) 中使用符合伦理要求的程序的定时怀孕雌性老鼠。使用锋利的剪刀, 切开腹腔和仔细切除子宫。将被切除的子宫放入含有20毫升冰冷 PBS 的60毫米的塑料碟中。 注意: 不需要无菌技术。</li>
	<li>使用解剖显微镜进行所有进一步的解剖。使用制表钳编号 5, 撕裂子宫肌壁暴露的胚胎, 释放每个胚胎通过切断脐带和删除卵黄囊。</li>
	<li>使用一个不育的巴斯德吸管与宽孔开口, 转移每个胚胎到一个干净的塑料盘与冰冷的 PBS。</li>
	<li>使用制表钳编号 55, 切断头部 (图 1E)。</li>
	<li>(可选) 保留一小部分组织 (如, ¼卵黄囊或大约2毫米尾尖) 进行基因组 DNA 分离和随后的基因分型。</li>
	<li>使用制表钳编号 55, 切除的组织包含后脑平行, 并立即低于后脑神经细胞, 以分离它从面部和前脑组织 (图 1F)。</li>
	<li>定位后脑和尾头组织背侧, 并确定 4th心室, 这是覆盖薄组织层 (顶板)。小心刺穿屋顶板使用钟表钳编号 55 (图 1G) 和剥离多余的组织钳, 移动 rostrally 沿中线上方的中脑, 然后尾在后后脑和脊髓 (图1G);后脑现在应该在开放的书籍准备 (图 1H) 中公开。</li>
	<li>准备 hindbrains wholemount immunolabeling (选项 1)。<ol>
<li>使用制表钳编号 55, 小心地戏弄了其余的头部间和任何脑膜连接到脑膜一侧的后脑使用钳 (图 1I)。取出中脑和脊髓组织 (图 1J) 只留下后脑组织 (图 1K)。 注意: 此步骤不应在 e9.5 或 e10.5 中遵循, 因为尝试这样做可能会损坏后脑神经细胞;此外, 去除脑膜是不必要的, 因为后脑神经细胞是足够薄, 在这个阶段, 以允许有效地渗透抗体 immunolabeling。然而, 这一步应该, 从 e11.5 开始, 当脑膜组织巩固, 以提高抗体渗透到后脑神经细胞。解剖是最简单的, 当在冰冷的 pbs (使用清洁 pbs 为每个后脑)。</li>
	</ol>
</li>
	<li>准备 hindbrains vibratome 和 cryosectioning (选项 2)<ol>
<li>使用制表钳编号 55, 取出中脑和脊髓组织 (图 1J)。 注意: 不需要删除用于剖切的 hindbrains 的间和脑膜 (如图 1I所示)。</li>
	</ol>
</li>
	<li>使用巴斯德吸管将 hindbrains 从塑料盘转移到圆形底2毫升的管子, 在 pbs 中溶解4% 瓦/v 甲醛, 并在4° c 下用温和搅拌法固定2小时。 注意: 甲醛是有毒的;穿戴适当的保护。</li>
	<li>用 PBS 冲洗 hindbrains 3x 次。如果 immunolabeling 将在2-3 天内开始在4° c 或更换 pbs 与 50% methanol/50%pbs 在5分钟之前转移到100% 甲醇为更长的存贮在-20° c.
</li>
</ol>4. Wholemount 免疫荧光
<ol><li>如果需要, 水化 hindbrains 在 pbs (例如, 75% 甲醇, 50% 甲醇, 25% 甲醇) 在室温下连续5分钟, 然后转到 pbs, 以降低稀释的甲醇。 注: 需要用分级的甲醇来轻轻水化 hindbrains, 确保适当的组织保存。</li>
	<li>Permeabilize hindbrains 为30分钟, 在4° c 的 PBS 中含有0.1% 的海卫 X-100 (PBT) 与温和的鼓动。</li>
	<li>在4° c 下, 在含有10% 热灭活山羊血清的 PBT 中孵育 hindbrains, 并温和搅拌。 注意: 使用宿主物种中的血清来培养二次抗体。对于在山羊体内饲养的主要抗体, 在无血清蛋白块中孵育, 例如 PBS 中的5% 牛血清白蛋白或合适的商业替代品 (参见材料表)。这将减少非特异染色经常观察时使用的主要抗体在山羊饲养。</li>
	<li>在4° c 夜间孵育 hindbrains, 含有1% 热灭活血清和温和躁动的初级抗体 (例如, 兔抗磷组蛋白 H3 [pHH3] 稀释 1:400)。 注意: 对于山羊的主要抗体, 使用无血清 PBT。</li>
	<li>hindbrains 在4° c 5x 时用 PBT 清洗1小时。</li>
	<li>在4° c 的情况下, 在含有适当荧光共轭二级抗体 (例如, 山羊抗兔 Alexa 氟 488) 的 pbt 中孵育 hindbrains, 在1:200 的 pbt 中针对所用的主要抗体。让 hindbrains 在黑暗中从这里保护荧光免受漂白。 注意: 对于山羊的主要抗体, 使用抗山羊的二次抗体片段来减少非特异性染色。</li>
	<li>hindbrains 在4° c 5x 时用 PBT 清洗1小时。</li>
	<li>将 PBS 中的 hindbrains 在4% 的甲醛中进行15分钟的后缀, 用于长期保存抗体结合。在 PBS 中简单冲洗两次。</li>
	<li>用两层黑色电子胶带盖上玻璃显微镜, 并将小方块从层状胶带上剪下, 形成一个大到足以容纳一后脑的口袋。</li>
	<li>将每个后脑到一个口袋里, 用巴斯德吸管, 除去多余的液体, 然后在口袋里放上适当的 antifade 试剂, 然后用玻璃片, 以避免在片下捕捉气泡。密封的片和贴在幻灯片上薄薄一层指甲油。存储在4° c 的幻灯片在黑暗中, 直到图像获取 (协议部分 7)。</li>
</ol>5. Vibratome 切片和漂浮部分免疫荧光
<ol><li>如有需要, 水化 hindbrains 的甲醇, 如步骤4.1 所述。</li>
	<li>嵌入 hindbrains 在熔融3% 瓦特/v 琼脂糖在蒸馏水中制备。 注: 在嵌入前, 允许熔融琼脂糖冷却至近似 c, 以防热损伤后脑。</li>
	<li>使用 vibratome 将横向后脑剖面切割为70µm 的厚度。将每个刚切割的部分与一支画笔放入一个装有冰冷 PBS 的24井板的井中。</li>
	<li>按步骤 4.2-4.7 中的说明对浮动剖面进行标记, 但请按如下方式修改步骤: 在4° c 3x 处用 PBT 洗涤漂浮部分, 每15分钟, 减少二次抗体的潜伏期为 2 h, 并在 RT 的二级抗体中孵育。</li>
	<li>(可选) 在用步骤5.4 所描述的其他表位的抗体标记后, 根据制造商的说明检测教育+细胞核。在37° c 的黑暗中孵育反应鸡尾酒中的漂浮部分30分钟。在4° c 3x 时, 用 PBT 清洗后脑部分, 每次15分钟。</li>
	<li>(可选) 在标记为步骤5.4 中所述的其他表位后, 检测 BrdU 的+核如下。<ol>
<li>在黑暗中孵育 2 N 盐酸在37° c 的浮动剖面30分钟。</li>
		<li>在0.1 米硼酸钠缓冲液中孵育浮动剖面, 在黑暗中每两次在室温下为5分钟, 以中和盐酸。</li>
		<li>在4° c 的 PBS 中简单地洗涤漂浮的部分3x。</li>
		<li>如步骤5.4 所述, 对 BrdU 执行免疫荧光标记。</li>
	</ol>
</li>
	<li>在 PBS 的10µg/毫升 4 ', 6-diamidino-2-吲 (DAPI) 中孵育2分钟的漂浮部分, 在 counterstain 细胞核中进行。</li>
	<li>在 RT 中将4% 甲醛的浮动部分后缀为15分钟。</li>
	<li>在 PBS 中简要地洗涤浮动部分。使用画笔小心地将浮动剖面转移到玻璃显微镜幻灯片上。用吸印迹纸或纸巾把多余的 PBS 拖到这一节。</li>
	<li>在 PBS 中使用80% 的甘油, 用玻璃片慢慢盖住, 避免气泡。存储在4° c 的幻灯片在黑暗中, 直到图像采集 (步骤 7)。</li>
</ol>6. 低温切片和 Cryosection 免疫荧光标记
<ol><li>如有需要, 水化 hindbrains 的甲醇, 如步骤4.1 所述。</li>
	<li>孵育 hindbrains 在30% 瓦特/v 蔗糖在 PBS cryoprotect hindbrains 前冷冻。 注: Hindbrains 已准备好冻结时, 他们已经沉没到底部的管, 这通常采取≤ 2 h 为 e9.5-10.5 Hindbrains 和3-6 小时 e11.5-13.5 Hindbrains。</li>
	<li>在光学切削温度化合物 (oct) 中浸入 hindbrains, 通过将含有 hindbrains 的模具转移到戊冷却至-40 ° c 至-50 ° c 的干冰上, 快速冻结。 注: 冷冻后, 嵌入的 hindbrains 可贮存在-20 ° c 的短时和-80 度的长期, 直到切片。</li>
	<li>使用低温和转移到静电胶显微镜幻灯片, 将横向后脑剖面切割成厚度为10µm。 注: 后脑 cryosections 可储存在-20 ° c 的短期和-80 ° c 长期, 直到标签。</li>
	<li>在 RT 中孵育幻灯片15分钟至干燥部分到幻灯片。用 PBS 笔冲洗 cryosections, 以溶解 OCT 并在 cryosections 周围标记疏水屏障。重复步骤 5.4-5.8。</li>
	<li>重复步骤5.10 使用聚乙烯醇为基础的安装介质代替甘油。</li>
</ol>7. 图像采集
<ol><li>图像样本使用 epifluorescent 或共焦激光扫描显微镜, 配有适合于水介质安装的幻灯片和适合用于 immunolabeling 荧光的光学过滤器的透镜。</li>
	<li>要对整个后脑或后脑部分进行图像处理, 请使用镜头进行10x 放大 (例如,图 2B);要可视化 hindbrains 区域以进行量化, 请使用40X 放大倍数 (例如,图 2D), 并可视化单个单元格, 使用带有63x 放大倍数的镜头 (如,数字 2C)。</li>
</ol>8. 替代方法
<ol><li>在步骤3.8 之后, 质不固定 hindbrains 为流式细胞仪应用程序生成单个单元悬浮,15。</li>
	<li>下面的步骤 3.8, 质不固定的 hindbrains 提取 RNA 从单细胞悬浮为RT qPCR (例如, 16)。 注意: 确保所有的试剂和设备都保持无菌和无核糖核酸, 以防止 RNA 的降解。</li>
	<li>下面的步骤 3.8, 质不固定的 hindbrains 孤立 npc 和传播他们在体外作为球分析后脑 npc 行为17。 注意: 确保所有的试剂和设备保持不育, 以防止干细胞文化的细菌/真菌污染。</li>
</ol>

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(47), (2011).</li><li>Sauer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis+in+the+neural+tube.">Mitosis in the neural tube.</a> The Journal of Comparative Neurology. 62 (2), 377-405 (1935).</li><li>Sessa, A., Mao, C. A., Hadjantonakis, A. K., Klein, W. H., Broccoli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tbr2+directs+conversion+of+radial+glia+into+basal+precursors+and+guides+neuronal+amplification+by+indirect+neurogenesis+in+the+developing+neocortex.">Tbr2 directs conversion of radial glia into basal precursors and guides neuronal amplification by indirect neurogenesis in the developing neocortex.</a> Neuron. 60 (1), 56-69 (2008).</li><li>Pilz, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification+of+progenitors+in+the+mammalian+telencephalon+includes+a+new+radial+glial+cell+type.">Amplification of progenitors in the mammalian telencephalon includes a new radial glial cell type.</a> Nat Commun. 4, 2125 (2013).</li><li>Fantin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+macrophages+act+as+cellular+chaperones+for+vascular+anastomosis+downstream+of+VEGF-mediated+endothelial+tip+cell+induction.">Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction.</a> Blood. 116 (5), 829-840 (2010).</li><li>Kayam, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+Pax6+in+the+segmental+organization+of+the+hindbrain.">A novel role for Pax6 in the segmental organization of the hindbrain.</a> Development. 140 (10), 2190-2202 (2013).</li><li>Tata, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+embryonic+neurogenesis+by+germinal+zone+vasculature.">Regulation of embryonic neurogenesis by germinal zone vasculature.</a> Proc Natl Acad Sci U S A. 113 (47), 13414-13419 (2016).</li></ol>

没有一个作者有竞争利益或利益冲突。

本协议描述了如何使用小鼠胚胎后脑作为模型来研究发育性神经再生的机制。使用各种不同的 immunolabeling 方法, 后脑 npc 可以可视化和他们的数量量化的组织部分或跨器官 wholemounts。解剖和扁平解剖的方便, 确保了后脑可以被想象在一个 ' 开放的书籍 ' 准备收集信息的器官范围内的神经再生模式。
我们进一步表明, npc 的形态学和细胞周期相关的 npc 定位可以很容易地形象化的浮动或 cryosections 的后脑。这两种行为都可能被利用来定义新的祖群, 就像以前在哺乳动物端20中执行的那样。例如, 早期形成的Sox2+ neuroepithelia 和Pax6+顶端径向胶质细胞存在于后脑21、22中, 但后脑缺少Tbr2+基祖3.
这里描述的协议也可以通过荧光标记来观察特定 NPC 亚群的行为, 以便在固定组织中进行活体成像和/或血统追踪。这可以实现, 例如, 通过研究 hindbrains 的小鼠携带他莫昔芬诱导的Sox1-iCreERT2转基因和Rosa26tdTomato 记者23。
除了增强对小鼠神经形成的认识, 研究后脑可能阐明广泛相关的神经源机制, 共享的物种, 因为后脑是一个高度保守的大脑区域, 预计将更相似脊椎动物之间的物种比前脑。
由于后脑神经新生发生在一个相对地短时间窗口比前脑神经新生23, 重要的是要考虑是否需要比较适当的分期胚胎。因此, 在孤立其后脑之前, 通过计数和记录胚胎中节对的数量来避免实验偏差。后脑组织本身是脆弱的, 因此, 在将后脑组织从头部间和脑膜中分离时, 应小心处理镊子;因此, 在对有价值的胚胎进行解剖之前, 有两个 "练习跑" 可能是可取的。此外, hindbrains 应该从一个管转移到另一个使用宽口径的巴斯德吸管, 而不是用镊子, 以避免损害 (吸管的开放可以扩大通过削减尖端与清洁剪刀)。最后, 虽然不常见的, 教育/BrdU 纳入 S 阶段 npc 的程度可能是可变的, 特别是在短 (即, 1 h) 脉冲。为了改善标签, 确保教育/BrdU 在将溶液装入注射器并小心地注入腹腔之前, 正确溶解。不良的注射, 如那些由意外的皮下注射, 将导致失去或捕获的教育/BrdU 解决方案, 并防止它进入循环。

在胚胎哺乳动物发育过程中, npc 在扩张的神经细胞的心室 (VZ) 和脑室 (SVZ) 区中分裂, 在称为 "神经发生" 的过程中产生新的神经元。新神经元及其 NPC 前体的产生在前脑1中得到了广泛的研究, 而其他地区对此过程的了解较少。
前脑是一个复杂和错综复杂的折叠结构, 主要研究组织切片后的组织学方法, 这使得了解神经再生模式横跨整个器官的挑战。此外, 对前脑神经再生的研究也不一定是其他脑区或脊髓神经行为的预测。例如, 信号提示可能在不同的 CNS 区域引起不同的反应, 如在睫状神经营养因子和白血病抑制因子的情况下观察到, 促进侧节隆起的 npc 的自我更新, 但驱动脊髓祖细胞的分化2。此外, 一个子级的 npc, 填充发育前脑, 并大大促进大脑皮层的扩张1没有从后脑和脊髓3。相反地, 可以想象, 脊髓和后脑含有不存在于皮质中的其他 NPC 亚型。
小鼠胚胎后脑是哺乳动物大脑中进化最古老的区域, 产生小脑和脑干。尽管它在物种间的保护, 但相对较少的是已知的后脑神经再生, 包括 NPC 亚型或他们的规则。大多数的后脑研究都集中在小鼠的组织分割过程中, 由 Hox 基因4驱动, 以及后有丝分裂神经元的模式5。此外, 后脑已被用作研究发育性血管生成机制的模型6。
与鼠标后脑, 斑马鱼后脑已被广泛使用, 以跟踪 NPC 分化和沿袭进展脊椎动物模型生物体 (如,7,8)。小鸡后脑也被用来研究脊椎动物发育过程中的神经发生 (例如,9,10)。与斑马鱼后脑11相似, 小鸡后脑可以实时成像, 以研究 NPC 的行为和时间的调节12。活体成像的类似纵向观察目前在哺乳动物的生物体中是不可能的, 因为它们在子宫内发育.此外, 通过电穿孔等技术进行有针对性的操作可以很容易地应用于自由活斑马鱼胚胎或小鸡胚胎在蛋(例如,13), 但这些技术也更具挑战性的子宫内。
然而, 小鼠胚胎后脑非常适合于定义控制神经再生的分子和细胞机制。首先, 对老鼠后脑的分析, 在许多情况下, 提供的信息与人类的发展比通过研究低等脊椎动物所获得的更重要。此外, 还有大量的转基因小鼠菌株可用于对鼠 npc 的命运测绘, 或改变相关基因的本构或条件突变等位基因的调控机制。最后, 最近已经表明, 微注射心室后脑祖细胞前体允许至少一个简短的检查后脑鼻咽癌动力学14。然而, 目前对后脑 npc 在整个器官环境中的时空组织和行为的了解甚少。
在这里, 我们演示了一个简单和快速的方法, 使用后脑作为一个强大的模型, 分析哺乳动物鼻咽癌的行为在 wholemount 准备和组织切片。我们进一步提供协议使用 immunolabeling 研究不同的神经新生参数, 并进一步处理后脑样品的下游分子应用, 如定量逆转录酶 (qRT)-PCR。

<table border="0" cellpadding="0" cellspacing="0" width="763">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Round-bottomed reagent tube, 2.0 ml (Safe-Lock)</td>
			<td style="width:97px;">VWR</td>
			<td style="width:119px;">211-2120</td>
			<td style="width:291px;">Also available from other commercial suppliers</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Plastic cell culture dish, 60 mm</td>
			<td style="width:97px;">Thermo Fisher</td>
			<td style="width:119px;">150288</td>
			<td style="width:291px;">Also available from other commercial suppliers</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Cell culture plates, 12-well</td>
			<td style="width:97px;">Thermo Fisher</td>
			<td style="width:119px;">150628</td>
			<td style="width:291px;">Also available from other commercial suppliers</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Pasteur pipettes</td>
			<td style="width:97px;">Copan</td>
			<td style="width:119px;">200C</td>
			<td style="width:291px;">Also available from other commercial suppliers</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Dumont Watchmaker forceps, no. 5</td>
			<td style="width:97px;">FST</td>
			<td style="width:119px;">91150-20</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Dumont Watchmaker forceps, no. 55</td>
			<td style="width:97px;">FST</td>
			<td>11295-51</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">29G needle/syringe</td>
			<td style="width:97px;">BD</td>
			<td>BD Micro-Fine +1ml</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-phospho-histone H3 primary antibody</td>
			<td style="width:97px;">Millipore</td>
			<td>06-570</td>
			<td style="width:291px;">Goat, dilution 1:400</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Anti-BrdU primary antibody</td>
			<td style="width:97px;">Abcam</td>
			<td>ab6326</td>
			<td style="width:291px;">Rat, dilution 1:400</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-Ki67 primary antibody</td>
			<td style="width:97px;">BD Biosciences</td>
			<td>550609</td>
			<td style="width:291px;">Mouse, dilution 1:400</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:257px;">Anti-RC2 primary antibody</td>
			<td style="width:97px;">Developmental Studies Hybridoma Bank</td>
			<td>RC2</td>
			<td style="width:291px;">Mouse (IgM), dilution 1:400</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Alexa Fluor 488-conjugated goat anti rabbit secondary antibody</td>
			<td style="width:97px;">Thermo Fisher</td>
			<td style="width:119px;">A11029</td>
			<td style="width:291px;">Dilution 1:200</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Alexa Fluor 594-conjugated goat anti rabbit secondary antibody</td>
			<td style="width:97px;">Thermo Fisher</td>
			<td style="width:119px;">A11037</td>
			<td style="width:291px;">Dilution 1:200</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Alexa Fluor 488-conjugated goat anti mouse IgM secondary antibody</td>
			<td style="width:97px;">Thermo Fisher</td>
			<td style="width:119px;">A21042</td>
			<td style="width:291px;">Dilution 1:200</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Alexa Fluor 488-conjugated goat anti mouse secondary antibody</td>
			<td style="width:97px;">Thermo Fisher</td>
			<td style="width:119px;">A11001</td>
			<td style="width:291px;">Dilution 1:200</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Alexa Fluor 594-conjugated goat anti rat secondary antibody</td>
			<td style="width:97px;">Thermo Fisher</td>
			<td style="width:119px;">A11007</td>
			<td style="width:291px;">Dilution 1:200</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:119px;">D9542</td>
			<td style="width:291px;">Use at 10 &#956;g per mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">5-ethynyl-2&#8217;-deoxyuridine (EdU)</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:119px;">900584</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">5-bromo-2&#180;-deoxyuridine (BrdU)</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:119px;">B5002</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Click-iT EdU Alexa Fluor 594 Imaging Kit</td>
			<td style="width:97px;">Thermo Fisher</td>
			<td style="width:119px;">C10086</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Heat-inactivated goat serum</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:119px;">G9023</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Bovine serum albumin</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:119px;">A7906</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">DAKO protein-block serum free</td>
			<td style="width:97px;">Agilent</td>
			<td style="width:119px;">X0909</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Triton X-100</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:119px;">T8787</td>
			<td style="width:291px;">Also available from other commercial suppliers</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Phosphate buffer saline</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:119px;">P4417</td>
			<td style="width:291px;">Also available from other commercial suppliers</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Paraformaldehyde</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:119px;">P6148</td>
			<td style="width:291px;">Also available from other commercial suppliers</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Sodium tetraborate</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:119px;">B9876</td>
			<td style="width:291px;">Also available from other commercial suppliers</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Sucrose</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:119px;">S0389</td>
			<td style="width:291px;">Also available from other commercial suppliers</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Agarose</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:119px;">A9539</td>
			<td style="width:291px;">Also available from other commercial suppliers</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Super PAP pen liquid blocker</td>
			<td style="width:97px;">Ted Pella, Inc.</td>
			<td style="width:119px;">22309</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">SlowFade Antifade Kit</td>
			<td style="width:97px;">Thermo Fisher</td>
			<td style="width:119px;">S-2828</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Glycerol</td>
			<td style="width:97px;">Fisher Scientific</td>
			<td style="width:119px;">10337700</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Mowiol</td>
			<td style="width:97px;">Millipore</td>
			<td style="width:119px;">475904</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">OCT</td>
			<td style="width:97px;">Scigen</td>
			<td style="width:119px;">4583</td>
			<td style="width:291px;">Also available from other commercial suppliers</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Isopentane</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:119px;">M32631</td>
			<td style="width:291px;">Also available from other commercial suppliers</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Methanol</td>
			<td style="width:97px;">Thermo Fisher</td>
			<td style="width:119px;">10675112</td>
			<td style="width:291px;">Also available from other commercial suppliers</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Hydrochloric acid</td>
			<td style="width:97px;">Thermo Fisher</td>
			<td style="width:119px;">10316380</td>
			<td style="width:291px;">Also available from other commercial suppliers</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Microscope slides</td>
			<td style="width:97px;">VWR</td>
			<td style="width:119px;">631-0912</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Superfrost microscope slides</td>
			<td style="width:97px;">VWR</td>
			<td style="width:119px;">631-0108</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Thermometer</td>
			<td style="width:97px;">VWR</td>
			<td style="width:119px;">620-0858</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Cover glass</td>
			<td style="width:97px;">VWR</td>
			<td style="width:119px;">631-0137</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Stereo Microscope, Leica MZ16</td>
			<td style="width:97px;">Leica</td>
			<td style="width:119px;">not applicable</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Confocal laser scanning microscope&#160; LSM710</td>
			<td style="width:97px;">Zeiss</td>
			<td style="width:119px;">not applicable</td>
			<td style="width:291px;"></td>
		</tr>
	</tbody>
</table>

the mouse hindbrain as a model for studying embryonic neurogenesis

小鼠胚胎前脑是研究哺乳动物神经发生发育过程中最常用的系统。然而, 高度折叠前脑神经细胞不适于 wholemount 分析, 以检查器官的神经再生模式。此外, 确定前脑神经再生的机制不一定是大脑其他部位神经再生的预测;例如, 由于前脑特定的祖类型的存在。小鼠后脑提供了一个研究胚胎神经再生的替代模型, 可 wholemount 分析, 以及组织切片观察神经祖细胞的时空分布和行为。此外, 它很容易被解剖的其他下游应用, 如细胞分离或分子生物学分析。由于老鼠后脑可以很容易地分析在大量的细胞谱系报告和突变的小鼠菌株已成为可用, 它提供了一个强大的模型, 研究细胞和分子机制的发展神经性在哺乳动物有机体.在这里, 我们提出了一个简单和快速的方法, 使用小鼠胚胎后脑分析哺乳动物神经祖细胞 (NPC) 行为的 wholemount 制剂和组织切片。

本节说明了在通过 wholemount 和组织切片分析研究小鼠胚胎后脑神经发生时可以得到的结果。
我们表明, wholemount immunolabeling 的 microdissected 后脑与抗体的有丝分裂标记 pHH3 可视化划分 npc 在 VZ (图 2B - D)。我们以高放大率显示 pHH3+ npc, 以突出不同的有丝分裂阶段 (图 2C)。我们已经说明, 这种标记方法适合在后脑发育的几个连续阶段进行, 以观察该器官的 NPC 有丝分裂的时间过程 (图 2D)。
我们表明, 成像横向 immunolabeled vibratome 部分后脑 1 h 后, 教育注射, 形象化的分裂方向的有丝分裂 npc (图 3B), 假, interkinetic 核迁移模式的循环祖18 (图 3B, d) 和整体 VZ 结构 (图 3B - d)。请注意, 有丝分裂 pHH3+ npc 只存在于心室表面, 而不是更多的基部 (图 3C), 它对比了前脑19中更有承诺的 npc 的基底划分模式。
我们还说明了如何循环 npc 和他们的分化后代可以标记为 BrdU 或教育, 以评估 npc 谱系进展 (图 4)。BrdU 注射 BrdU 和 Ki67 后的小鼠后脑1天的横向 cryosections 的 immunolabeling 演示在神经细胞中循环 npc 的数量和位置 (图 4A, B), 据此,可以通过计算所有 BrdU+单元格 (图 4A, C) 中 Ki67+ BrdU+单元格的百分比来定义自更新 npc。
最后, 我们展示了一个例子的 immunolabeling 后脑 vibratome 部分 RC2, 一个抗原在神经特异性中间灯丝巢, 以可视化 NPC endfeet (图 5B) 和进程 (图 5C)。Vibratome 部分, 而不是薄 cryosections, 允许改进的观察高度分支 npc 和整体上皮结构。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56793/56793fig1.jpg"> 图 1: 从 e11.5 小鼠胚胎解剖后脑的关键步骤.(A - D)后脑显微解剖的图解描述。(A) 沿红色虚线移除尾头组织。(B) 屋顶板被刺穿, 然后沿中线 (垂直红线) 的侧和尾侧方向撕裂, 然后侧面显示后脑神经细胞。(C) 在两个结构之间插入镊子后, 神经细胞从脑膜中被撬开, 中脑和脊髓组织通过沿红色虚线的镊子切断组织而被移除。(D) 此过程生成一个扁平后脑。(E-K)后脑显微切割过程中关键阶段的图像捕获。(E) 胚胎通过沿虚线被切断而被斩首。(F) 后脑和 4th心室, 由顶部板围住, 通过沿虚线切断并与头部分离。(G) 4th心室在一个小孔被刺穿到顶板 (星号) 之前朝向向上。rostrally 和尾沿中线 (箭头) 仔细剥开组织, 以揭露后脑。后脑被安置脑膜边下来和左折叠入一个开放书准备 (H; 黑色箭头表示后脑神经组织)。如果 wholemount immunolabeling 的后脑是必需的, 脑膜膜被用镊子轻轻地从周围的脑膜膜中窥探后脑神经细胞 (I)。多余的中脑 (mb) 和脊髓 (sc) 组织被移除 ( J中的虚线) 以隔离后脑 (K)。缩放栏: 500 µm (E), 300 µm (F K)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56793/56793fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56793/56793fig2.jpg"> 图 2: Wholemount immunolabeling 可用于在后脑中量化 NPC 分裂.(A) 示意图, 说明在后脑心室层中的有丝分裂 npc 的en 面成像。心室后脑侧;P, 脑膜后脑侧。(B) 共焦平铺扫描 z 堆栈 e10.5 后脑以下 wholemount immunolabeling 为有丝分裂标记 pHH3 (红色)。白色框表示在第二个面板 (D) 中显示的区域的放大倍数。(C) 前后期 (白箭头) 和后期 (黑箭头) 有丝分裂图在有丝分裂 npc 的总队列中是可区分的. (D) wholemount pHH3 标记的时间过程, 从 e9.5-13.5。缩放条形图: 500 µm in (B);20µm in (C);100µm in (D)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56793/56793fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56793/56793fig3.jpg"> 图 3: 在侧后脑的生发区内循环 npc.(A) 示意图, 分别描述有丝分裂 (绿色) 和 s-相 (红色) npc 在后脑心室表面和脑室区的位置。通过后脑的虚拟剖面在右侧显示更高的放大倍数。黑色箭头指示了向分化带退出细胞周期的 NPC 后代的迁移方向。(B) 从接受1小时脉冲的胚胎中浮 e11.0 后脑部分的共焦 z 叠加;immunolabeling 的 pHH3 和教育被用来可视化有丝分裂 (绿色) 和 s-相 (红色) npc, 分别。后脑表面用虚线表示;心室后脑侧;P, 脑膜后脑侧。白盒显示的区域在放大倍数中显示;箭头指鼻咽癌在后期的分裂平面, 相对于心室表面。(C) 仅显示 pHH3 immunolabeling 的单通道。有丝分裂 npc 用青色箭头表示;缺乏基本的分裂被表明由Δ. (D) 单通道只显示教育标签。由于 interkinetic 的核迁移, 教育+ npc 采用了假分布。可以测量 npc 从心室表面移出的距离, 如有代表性的橙色线所示。缩放条形图: 100 µm in (B -d);10µm 在嵌入中 (B)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56793/56793fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56793/56793fig4.jpg"> 图 4: 将 BrdU 与 Ki67 标记结合以确定后脑的 NPC 自我更新能力.(a) 在1天 BrdU 脉冲后为 Ki67 (绿色) 和 BrdU (红色) 标记后, e10.5 后脑 cryosection 的共焦 z 堆栈。双阳性细胞在心室区是 npc, 已纳入 BrdU, 并仍在通过细胞周期, 如 Ki67 标签所示。在总 BrdU+总体中, 双标记细胞的比例代表自我更新 npc 的百分比. (B, C) 单通道显示 Ki67 (b) 和 BrdU (c) immunolabeling。缺少 Ki67 并因此可能退出单元格周期的 BrdU+单元格由 (a,C) 中的青色箭头表示。后脑表面用虚线表示;心室后脑侧;P, 脑膜后脑侧。缩放栏:50 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56793/56793fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56793/56793fig5.jpg"> 图 5: 巢 immunolabeling 可视 NPC 进程和 endfeet.(A - C)免疫荧光 RC2, 它承认一个表位的神经中间灯丝巢, 说明 NPC 的形态学横跨后脑神经细胞。(a) 从 e11.5 后脑跟随 RC2 immunolabeling 的浮动剖面的共焦 z 栈;在 (b、 b; 顶端/心室区域) 和 (c、 c; 基底区域) 中, 盒装区域的放大倍数显示。后脑表面用虚线表示;心室后脑侧;P, 脑膜后脑侧。(B, C)(A) 中盒装区域的共焦 z 栈;单个光学剖面分别显示在 (B, C) 中。在心室表面固定的 NPC 顶端 endfoot 用箭头表示 (B ")。单一和 fasciculated 的 NPC 进程分别用白色和黑色箭头表示 (C ")。缩放条形图: 100 µm, (A);25µm (B, C)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56793/56793fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis at JoVE.com

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

小鼠后脑作为研究胚胎神经再生的模型

本文说明了如何将小鼠胚胎后脑作为研究整个器官和组织切片中发育性神经再生的模型。

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

the-mouse-hindbrain-as-a-model-for-studying-embryonic-neurogenesis

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.9K 次观看.  UCL Institute of Ophthalmology. 本文说明了如何将小鼠胚胎后脑作为研究整个器官和组织切片中发育性神经再生的模型。

视频: The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

在斑马鱼后脑细胞标记和成像

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

科研

神经科学

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

The most insidious aspect of drug addiction is the high propensity for relapse. Animal models of relapse, known as reinstatement procedures, have been used extensively to study the neurobiology and phenomenology of relapse to drug use. Although procedural variations have emerged over the past several decades, the most conventional reinstatement procedures are based on the drug self-administration (SA) model. In this model, an animal is trained to perform an operant response to obtain drug. Subsequently, the behavior is extinguished by withholding response-contingent reinforcement. Reinstatement of drug seeking is then triggered by a discrete event, such as an injection of the training drug, re-exposure to drug-associated cues, or exposure to a stressor 1.
Reinstatement procedures were originally developed to study the ability of acute non-contingent exposure to the training drug to reinstate drug seeking in rats and monkeys 1, 2. Reinstatement procedures have since been modified to study the role of environmental stimuli, including drug-associated cues and exposure to various forms of stress, in relapse to drug seeking 1, 3, 4.
Over the past 15 years, a major focus of the reinstatement literature has been on the role of stress in drug relapse. One of the most commonly used forms of stress for studying this relationship is acute exposures to mild, intermittent, electric footshocks. The ability of footshock stress to induce reinstatement of drug seeking was originally demonstrated by Shaham and colleagues (1995) in rats with a history of intravenous heroin SA5. Subsequently, the effect was generalized to rats with histories of intravenous cocaine, methamphetamine, and nicotine SA, as well as oral ethanol SA 3, 6.
Although footshock-induced reinstatement of drug seeking can be achieved reliably and robustly, it is an effect that tends to be sensitive to certain parametrical variables. These include the arrangement of extinction and reinstatement test sessions, the intensity and duration of footshock stress, and the presence of drug-associated cues during extinction and testing for reinstatement. Here we present a protocol for footshock-induced reinstatement of cocaine seeking that we have used with consistent success to study the relationship between stress and cocaine seeking.

Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.

在实验室老鼠研究求可卡因电击诱导恢复原状的程序

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

基于凝集素的分离和培养的小鼠胚胎运动神经元

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

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

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

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

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

方法研究抗精神病药物的作用机制在<em&gt;秀丽隐杆线虫</em

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

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 ...

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

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

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

角膜微囊含量:血管生成在小鼠眼模型

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

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering the molecular signals that cooperatively guide neuronal migration in the embryonic brain is therefore important to understand how the complex neural networks form which later support postnatal life. Facial branchiomotor (FBM) neurons in the mouse embryo hindbrain migrate from rhombomere (r) 4 caudally to form the paired facial nuclei in the r6-derived region of the hindbrain. Here we provide a detailed protocol for wholemount ex vivo culture of mouse embryo hindbrains suitable to investigate the signaling pathways that regulate FBM migration. In this method, hindbrains of E11.5 mouse embryos are dissected and cultured in an open book preparation on cell culture inserts for 24 hr. During this time, FBM neurons migrate caudally towards r6 and can be exposed to function-blocking antibodies and small molecules in the culture media or heparin beads loaded with recombinant proteins to examine roles for signaling pathways implicated in guiding neuronal migration.

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the neural tube, but migrate to reach appropriate targets. Facial branchiomotor (FBM) neurons are a useful model to study neuronal migration. This protocol describes the wholemount ex vivo culture of mouse embryo hindbrains to investigate mechanisms that regulate FBM migration.

鼠标后脑<em&gt;前体内</em&gt;企业文化来研究面部Branchiomotor神经元迁移

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

一<em&gt;在体外</em&gt;成年小鼠肌肉，神经准备留学肌肉传入放电的放电特性

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...