这项研究得到了国家卫生研究院 (K99MH104595) 和发育研究院校内研究计划的支持 T.J.P。 我们感谢 Gord Fishell, 在其实验室中最初建立了这种方法。

所有实验程序均按照国家卫生研究院的指导方针进行, 并经发育研究院动物护理和使用委员会 (ACUC) 批准。下面所述的协议利用Nkx2.1 的c++;Ai9+/- 幼崽收获梅兰日兰衍生 interneuron 前体, 但可以执行任何所需的荧光记者鼠标线。雄性和雌性早产后小鼠 (P0-P2) 被不分青红皂白地用于供体和宿主组织。
1. 解决方案准备
<ol><li>根据以下食谱 (单位毫米) 制备蔗糖人工脑脊液 (sACSF):87 氯化钠, 26 NaHCO3, 2.5 氯化钾, 1.25 NaH2PO4, 0.5 CaCl2, 7 氯化镁2, 10 葡萄糖, 75 蔗糖饱和 95% O2, 5% CO2在 pH 值 = 7.4。</li>
	<li>填充一个烧杯与350毫升纯 ddH2O 基于最终期望体积 (500 毫升的 sACSF 应该足够的1-2 垃圾), 添加一个磁搅拌杆, 并把烧杯放在搅拌板上。</li>
	<li>重量所有盐 (氯化钠, NaHCO3, 氯化钾, NaH2PO4, 葡萄糖, 蔗糖), 一个在时间和添加到水根据下表。金额可以根据最终所需的音量进行调整。<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>试剂</td>
				<td>分子量</td>
				<td>浓度 (毫米)</td>
				<td>Grams/500 毫升</td>
			</tr>
<tr>
<td>氯化钠</td>
				<td>58.44</td>
				<td>87</td>
				<td>2.54</td>
			</tr>
<tr>
<td>碳酸氢钠</td>
				<td>84.01</td>
				<td>26</td>
				<td>1.09</td>
			</tr>
<tr>
<td>氯化钾</td>
				<td>74.55</td>
				<td>2。5</td>
				<td>0.09</td>
			</tr>
<tr>
<td>磷酸磷酸钙钠</td>
				<td>119.98</td>
				<td>1.25</td>
				<td>0.08</td>
			</tr>
<tr>
<td>葡萄糖</td>
				<td>180.16</td>
				<td>10</td>
				<td>0。9</td>
			</tr>
<tr>
<td>蔗糖</td>
				<td>342。3</td>
				<td>75</td>
				<td>12.84</td>
			</tr>
</tbody></table>
</li>
	<li>气泡的解决方案与 95% O2, 5% CO2 (carboxygen) 至少20-30 分钟之前添加适当数量的 CaCl2 (0.25 毫升从1米解决方案500毫升 sACSF) 和氯化镁2 (3.5 毫升从1米解决方案500毫升 sACSF).</li>
	<li>使用标准 ph 表检查 ph 值。如果准备正确, 解决方案应该有 pH 值 = 7.4。</li>
	<li>在一个毕业的圆筒中, 用纯 ddH2O 来解决最终体积为500毫升的溶液。</li>
	<li>sACSF 可以提前1-2 天准备好。使用前, 放置在冰和气泡与 carboxygen 至少15分钟前开始的解剖, 并保持瓶 sACSF 冒泡整个解剖。</li>
</ol>2. 解剖准备
<ol><li>以下工具应消毒/蒸压: 至少一小锐锋利的剪刀, 2 精钳 (花柱 #5), 弯曲的细钳, 脑切片矩阵模具和几个刀片。一个小铲, 以消除大脑从头骨是可选的。</li>
	<li>在解剖显微镜附近建立工作站 (9 厘米和4厘米直径), 50 毫升锥形塑料管, 收集孤立的脑区和大口径塑料转移吸管, 全部保存在冰上。在冰上准备好50毫升的 carboxygenated sACSF 管。如果解剖一个以上的大脑区域, 确保正确和清楚地标记收集管和转移吸管, 以避免污染。</li>
	<li>有一个额外的冰桶, 冰麻醉的幼崽通过低温, 一个适当的危险废物袋处置尸体。</li>
	<li>为组织研磨准备火抛光玻璃巴斯吸管。使用本生燃烧器消毒和形状的吸管的尖端。准备几个吸管不同孔开口: 大 (~ 600 µm), 中等 (~ 300 µm) 和小 (100 µm)。通过使用水来测试流量, 以确保不同的研磨速度。每个单独的脑区都需要至少一个不同的吸管, 但如果堵塞或折断提示, 建议每种尺寸加几个。</li>
</ol>3. 移植制剂
<ol><li>使用电极拉拔器制备细尖玻璃 micropipettes。将笔尖折断或斜角, 使尖角点, 尖端直径为20-40 µm. 用显微镜观察小贴士, 确保没有灰尘或残留的玻璃微粒阻碍光圈。</li>
	<li>使用细针注射器, 用矿物油完全填充玻璃微。将微插入 Nanoject 设备 (或其他微注射装置)。为 Nanoject III, 设置以下节目: 容量 = 60 nL, 率 = 30, 周期 = 25, 延迟 = 1 s。</li>
	<li>将 Nanoject 固定在连接到磁基座的机械手上, 并调整机械手, 使 Nanoject 指向直线向下, 而不是沿 x 或 y 轴倾斜。</li>
	<li>将一些粘胶腻子 (P0-P2 幼崽的1-2 厘米) 贴在培养皿的顶部, 形成一个高的表面, 使幼崽的头部休息。</li>
	<li>剪切6-8 厘米长条纹的实验室胶带, 并在中间形成一个菱形孔。该胶带将用于稳定的头部的幼崽在过程中, 同时也伸展皮肤和允许容易可视化地标和目标区域。</li>
	<li>在注射站旁边准备一个加热垫, 然后在开始注射前打开 "低" 设置。用纸巾或尿布垫把它盖在垫子上。</li>
	<li>如果执行多个移植条件, 有一个小剪刀准备执行脚趾/尾部修剪标记小鼠接受不同的注射。</li>
</ol>4. P0-P2 鼠脑切除
<ol><li>在开始程序之前, 用冷冻的 carboxygenated sACSF 填充几个培养皿。也填写收集管 (s) 与〜25毫升 sACSF。</li>
	<li>把 P0-P2 的小狗裹在 parafilm 或手套里, 放在冰层下。设置一个定时器5分钟, 并启动它。之后, 删除小狗, 并检查它不响应捏 hindpaw。</li>
	<li>斩首的幼崽, 并收集在一个充满 sACSF 的培养皿头。沿中线切开皮肤, 露出头骨。</li>
	<li>在头骨的后脑/脊髓处找到开口, 并在颅骨的背部插入钳的一端。轻轻地抓住头骨在中线和 ' 剥离 ' 部分侧向暴露的大脑, 小心不损害大脑。</li>
	<li>当切除颅骨的背部和侧面部分时, 使用镊子或刮刀轻轻地将大脑从头骨中挖出, 然后在大脑的腹面下方挖, 尽量不破坏任何区域。把大脑放到另一个培养皿里, 里面装满了 carboxygenated sACSF。 
	  注意: 我们提出了两种不同的方法来解剖海马体、纹状体和皮质。第一个策略 (步骤 5.1-5.10) 对任何鼠标线都很有用, 而第二个策略 (步骤 6.1-6.7) 需要一个荧光记者鼠标, 以干净地将纹状体与苍白和其他大脑结构分开。如果不需要纹状体, 则忽略两种技术中的纹状特定部分。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57723/57723fig1.jpg"> 图 1: 脑解剖、技术 #1 的示意图和图像 在步骤 5.1-5.10 中描述的解剖技术。如果需要巴组织, 将 P1 大脑放在脑阵腹侧上。将剃刀刀片放入矩阵槽, 通过前脑, 通过纹状体获得冠状部分。从两个半球移除巴块, 重复所有包含海马前纹状体的部分。如果不需要巴组织, 只需 hemisect 大脑, 将半球内侧放在盘子中, 移除腹内侧脑结构 (丘脑、基底节等), 以暴露海马体。用镊子捏住海马, 然后翻转半球, 用镊子解剖出一大块皮质组织。垂直黑线通过示意图半球, 其中的削减将是删除巴部分。缩放栏 = 500μm。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57723/57723fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
5. 收获纹状体, 海马和皮质, 技术 #1
<ol><li>把整个大脑切片矩阵模具, 腹侧向上。将1刀片穿过大脑最前面的部分 (通过前嗅道)。</li>
	<li>插入另一剃刀刀片刚后到第一个产生0.5 毫米切片, 并放置一个额外的剃刀刀片后, 这一个。</li>
	<li>把这些切片转移到一个培养皿与 carboxygenated sACSF, 并将其余的大脑放在一个单独的菜与 sACSF。将巴切片转移到解剖范围以可视化纹状体。这些切片应包含很大一部分的前纹状体和缺乏海马。使用荧光解剖范围与Nkx2.1+/-;Ai9+/切片, 以区分弱巴 tdTomato 信号的强 tdTomato 信号的球苍白。</li>
	<li>有或没有荧光, 捏出的纹状体从两个半球上的所有部分和转移巴块, 正确标记50毫升管与 sACSF, 储存在冰上。</li>
	<li>用镊子或剃刀 Hemisect 大脑沿中线, 并放置半球, 使内侧表面朝上。</li>
	<li>用镊子掐掉腹侧脑组织 (丘脑、基底节等)。完成后, 海马体 (一种横跨前后轴沿背皮层的香肠状结构) 和大脑皮层的心室侧应清晰可见。</li>
	<li>移除海马体, 在前海马前插入镊子的尖端, 并通过移动钳向后和捏沿海马皮层边界轻轻地将海马体与皮层分开。将孤立的海马体转移到正确标记50毫升管与 sACSF, 储存在冰上。</li>
	<li>解剖皮层, 将 hemisected 皮质内侧向下。然后用镊子捏住皮层的最背、腹、前和后部, 留下一大块内侧体感皮层, 2 毫米 x 2 毫米. 翻转皮层, 使内侧侧向上和清洁任何多余的非皮质组织 (丘脑、基底节等)如果需要, 在内侧表面。转移皮层块, 以正确标记50毫升管与 sACSF, 储存在冰上。</li>
	<li>重复这个过程与另一个半球收集海马和皮层区域。</li>
	<li>对所有幼崽重复此过程, 确保在幼崽之间更换培养皿中的 sACSF, 以确保 sACSF 保持寒冷和新鲜。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57723/57723fig2.jpg"> 图 2: 脑解剖、技术 #2 的示意图和图像 在步骤 6.1-6.7 中描述的解剖技术。把大脑插到解剖盘背侧上。在剥去皮层后, 海马体和纹状体可见, 可以去除, 然后一节皮质可以像以前的技术所描述的那样被移除。根据转基因小鼠线, 纹状体可以清除, 以消除球苍白和其他组织。在Nkx2.1Cre;Ai9小鼠线, 球苍白有显着较高的密度番茄 + 细胞相比, 纹状体。刻度条 = 500 微米。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57723/57723fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
6. 收获纹状体, 海马和皮质, 技术 #2
<ol><li>将大脑腹侧放在含有 sACSF 的 sylgard 涂层培养皿中, 然后通过小脑和前皮质或嗅球将其钉住。</li>
	<li>从一个半球开始, 使用弯曲钳轻轻地将后皮质与底层海马体和其他组织分开。轻轻地将去皮的皮质平放在培养皿上。对其他半球重复。海马和纹状体在暴露的大脑中应该清晰可见。</li>
	<li>用镊子捏住中线上的一个海马体, 侧剥海马体以去除。放置在收集瓶冰和重复与其他海马。</li>
	<li>对于纹状体, 轻轻地刮绕纹状的边缘, 从周围的组织松动。然后将纹状体从下面捏下来, 加入到冰上的收集管中。对其他纹状体重复。</li>
	<li>皮质切片可按步骤5.8 所述进行收割。</li>
	<li>如果使用转基因记者鼠标线 (例如, Nkx2.1;Ai9、 Lhx6-GFP等), 在荧光解剖范围内, 将纹状体转移到培养皿中 sACSF 和视图。使用镊子从纹状体中去除任何非巴组织 ( Nkx2.1;Ai9小鼠, 除去所有 ' 明亮 ' 组织, 因为球球苍白有更高的梅兰日兰派生, tdTomato + 细胞密度比纹状体)。对所有纹状体重复。</li>
	<li>对所有幼崽重复此过程, 确保在幼崽之间更换培养皿中的 sACSF, 以确保 sACSF 保持寒冷和新鲜。</li>
</ol>7. 生成单细胞 Dissociations
<ol><li>解剖完成后, 准备一个1毫克/毫升链霉蛋白酶溶液, 重10毫克链霉蛋白酶和溶解在10毫升 carboxygenated sACSF。</li>
	<li>将组织从50毫升的收集瓶转移到5毫升圆底管, 含有2毫升的链霉蛋白酶-sACSF 溶液。在室温下孵化组织20分钟, 在孵化过程中用手指摇动/弹管3-4 次以混合样品。</li>
	<li>在这一孵化过程中, 准备重组解决方案: 1% 个血清 (100 ul) + DNAse (1 ul 1:10,000 股票 DNAse) 在10毫升 carboxygenated sACSF。</li>
	<li>20分钟后, 小心移除链霉蛋白酶溶液, 不扰乱底部的组织, 并用1-2 毫升的重构溶液取代它 (总容积取决于组织的起始量)。</li>
	<li>机械分离组织通过研制过程的组织与先前准备的火抛光巴斯德吸管。从大口径 (~ 600 µm) 吸管开始, 吸入并排出组织溶液至少十次以分解组织。不要在解决方案中引入气泡。对中孔吸管和小孔吸管重复此过程。溶液应该是多云的, 并且在收集管中不应该有清晰的组织可见。</li>
	<li>吸管细胞裂解液通过50µm 过滤器成5毫升圆锥管, 以去除任何细胞团簇。继续这些单细胞解决方案流式细胞术 (或可能直接到细胞计数, 如果不需要排序)。</li>
</ol>8. 制备用于移植的纯化细胞溶液
<ol><li>在从流式细胞仪中接收单元解决方案后, 将溶液转移到一个 (或多个) 1.5 毫升圆锥管, 并在 4oC 上将细胞旋转500克, 5 分钟。离心后, 取出介质, 使20-40 µL 留在管中。重建剩余介质中的细胞 (如果需要多管, 则结合解决方案)。</li>
	<li>在一小块 parafilm, 混合2µL 细胞溶液 + 8 µL sACSF + 10 µL 的台盼蓝染色。吸管10µL 入 hemocytometer 与滑动盖子。</li>
	<li>用标准的实验室手计数计数器 (死细胞将是蓝色的染料) 计算 hemocytometer 角落 4 x 4 平方网格中每一个活细胞的数量, 平均这4计数。将此数字乘以100以确定每个µL 的单元格数。</li>
	<li>如果需要, 调整音量, 使细胞在重构溶液中浓度为1000万细胞/µL。最终浓度取决于细胞的总数量和所需的移植数量。把细胞放在冰上移植</li>
</ol>9. 移植到 P0-2 幼崽
<ol><li>把一只小狗裹在一些 parafilm 或手套里, 放在冰层下。设置一个计时器5分钟, 并启动它。之后, 删除小狗, 并检查它不响应捏 hindpaw。</li>
	<li>当小狗在冰上, 混合细胞溶液通过吹打它几次, 以确保细胞悬浮在加载之前很好混合 (混合细胞之前, 加载微为每一个注射)。然后清空矿物油的微, 完全用细胞悬浮液填满。</li>
	<li>将被麻醉的小狗放在培养皿上, 头躺在粘合剂腻子上, 使头部的顶端相对平坦。把带钻石孔的带子放在幼崽头上, 拉紧它, 以确保皮肤伸展, 头部牢固, 但老鼠舒适, 呼吸良好。通过钻石孔可以看到 Lambda。</li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57723/57723fig3.jpg"> 图 3: 移植的示意图和图像 (A)注塑装置的图片。注意, lambda 是通过幼犬的头骨清晰可见的, 应该用于微的零化。刻度条 = 1 英寸。(B)描述注射程序以确定海马体目标的示意图。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57723/57723fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="4"><li>在 Nanoject 下移动安全的小狗并降低微, 以便微的尖端直接位于 lambda 之上。在机械手上记录 x y 坐标。</li>
	<li>调整机械手旋钮, 将微移动到所需的坐标沿 mediolateral 和前后轴的头部: S1 皮质→1.0 毫米前部和1.0 毫米侧, 海马→ 0.75-1. 0 毫米前和 1.0-1.2 毫米侧, 纹状体→2.0-2.2 毫米前部和1.5 毫米侧向。坐标可以稍微调整以补偿幼崽的年龄 (例如, P0 与 P2) 或小鼠的应变 (例如, CD1 鼠大于和 C57/B6 P2)。</li>
	<li>降低微, 直到它形成一个小凹在皮肤上。然后坚定地转动 z 轴机械手旋钮, 但轻轻地驱动微通过皮肤和头骨进入大脑。当微进入大脑时, 头骨上的压力将被释放, 凹陷将消失。</li>
	<li>稍微收回微, 直到尖端被一锥形皮肤包围, 形状像一个帐篷。读取沿 z 轴的坐标: 这将是 z 轴零点。</li>
	<li>降低微到所需的深度: 皮质→ 0.75-1. 0 毫米, 海马→ 1.2-1.5 毫米, 纹状体→ 2.0-2.5 毫米. 深度可以稍微调整以补偿幼崽的年龄或应变。</li>
	<li>使用上面描述的 Nanoject III 程序注射细胞悬浮液。注射程序完成后, 在缓慢缩回微之前等待15-20 秒, 以尽量减少注入部位泄漏的解决方案。</li>
	<li>如果执行双侧注射, 重新零微以上的 lambda 和移动到适当的坐标在另一个半球。另外, 你可以通过移动第一个注射的微1毫米前, 使两个注射进入同一半球的皮层。</li>
	<li>移植完成后, 取出胶带, 将幼崽转移到加热垫上。如有必要, 通过尾部或脚趾修剪标记小狗。一旦小狗库藏了红色, 并移动, 把它放回笼子里与母亲。</li>
</ol>

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作者没有什么可透露的。

该协议的一个关键方面是最大限度地提高细胞的生存能力。确保组织和细胞始终处于冰冷 carboxygenated sACSF 是促进细胞存活所必需的。这需要一个有效的解剖和离解策略, 以尽量减少细胞在不同的解决方案和大脑环境之外的时间长短。根据被解剖和移植的大脑区域的数量, 在解剖和/或移植步骤中有一个合作伙伴帮助减少实验的长度是有益的。为了保证幼犬的健康和生存, 在移植过程中尽量减少冰麻?...

适当的皮质功能需要平衡兴奋投射神经元和抑制 GABAergic 中间神经元, 一个极异质的人群, 具有明显的形态, 电生理特性, 连通性和神经化学标记。异常发育和功能的中间神经元 (和特定的 interneuron 子群) 已与病理精神障碍, 如精神分裂症, 自闭症和癫痫1,2,3。此外, 许多与这些脑部疾病相关的基因在年轻的中间神经元4中有强烈的丰富。因此, 需要更多地了解调节 interneuron 命运确定和成熟的机制, 以了解许多脑部疾病的正常发展和潜在病因。
前脑中间神经元主要来自两个瞬时胚胎结构, 内侧和尾节显赫人士 (梅兰日兰和 CGE 分别)。这些分裂后细胞 (interneuron 前体) 然后经历一个长期的切向迁移阶段, 分散在整个前脑, 在那里他们集成到广泛的电路。梅兰日兰衍生中间神经元由三个主要的非重叠的, neurochemically 定义的子群: 快速峰值 parvalbumin (PV+) 中间神经元, 非快速峰值生长抑素 (SST+) 中间神经元, 和晚峰值神经元一氧化物合酶 (nNOS+) 中间神经元, 构成海马 neurogliaform 和常春藤细胞。许多实验室已经确定了梅兰日兰中的几种机制, 将初始命运决定调整为 PV+或 SST + 中间神经元, 包括格局来说的空间梯度、interneuron 前体的出生日期和神经源性分裂的模式。5,6,7,8,9,10. 有人建议, 中间神经元最初将其区分为 "主要阶级", 然后逐渐成熟为 "决定性阶级", 因为它们与环境11互动。最近的证据表明, 一些成熟的 interneuron 亚型可能是遗传的, 因为这些细胞成为分裂后在节显赫人士, 表明早期定义的内在基因程序可能发挥比以前更大的作用赞赏12,13。然而, 如何将内在遗传程序与环境线索相互作用来驱动分化为不同的 interneuron 亚型的关键问题仍在很大程度上未被探索。
许多研究已经将胚胎梅兰日兰细胞直接移植到了不同的脑区, 以协商一致的结果, 移植细胞成熟和释放 GABA 一般抑制局部内源电路14,15, 16,17,18,19。这些有希望的观察已经产生了极大的兴趣, 使用人类诱导多潜能干细胞 (hIPSC) 衍生中间神经元治疗各种脑疾病。然而, 很少的这些研究评估, 如果这些嫁接细胞成熟的预期类型的成熟中间神经元, 一个关键组成部分, 当你思考的翻译方法。
为解决环境对 interneuron 分化和成熟的影响, 设计了一种将未成熟 interneuron 前体移植到新的脑环境中的策略, 以检查嫁接中间神经元是否采用宿主的特征。环境或保留来自捐助者环境的特征20。梅兰日兰移植不适合解决这个问题, 因为梅兰日兰含有混合种群的 interneuron 和 GABAergic 投射细胞分散在许多脑区21。如果不知道这些梅兰日兰细胞将迁移到哪里, 就无法充分评估这些移植如何受大脑环境的影响。通过在产后早期 timepoints 收割 interneuron 前体, 通过获得完成迁移并到达目标脑区的未成熟细胞来规避这个问题, 但与环境的交互作用最小。通过专注于不同脑区之间差异表达的中间神经元的特定特征, 可以确定宿主环境如何改变 interneuron 属性。本议定书概述的一般方法应适用于任何想研究年轻神经元在新环境中受到挑战时如何行为的调查人员。

<table><tbody><tr><td>Sodium chloride</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S6297</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma</td><td>S0751</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C5080</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma</td><td>M2670</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G7528</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S7903</td><td></td></tr><tr><td>Brain Matrices</td><td>Roboz</td><td>SA-2165</td><td>Only needed if harvesting striatum</td></tr><tr><td>Fine point Dumont Forceps</td><td>Roboz</td><td>RS-4978</td><td></td></tr><tr><td>Microdissecting scissors</td><td>Roboz</td><td>RS-5940</td><td></td></tr><tr><td>Razor blades</td><td>ThermoFisher</td><td>12-640</td><td></td></tr><tr><td>Pasteur pipettes</td><td>ThermoFisher</td><td>1367820C</td><td></td></tr><tr><td>Nanoject III</td><td>Drummond</td><td>3-000-207</td><td></td></tr><tr><td>Manual Manipulator w/ stand</td><td>World Precision Instruments&amp;nbsp;</td><td>M3301R/M10</td><td></td></tr><tr><td>5 ml round bottom plastic tubes</td><td>ThermoFisher</td><td>149591A</td><td></td></tr><tr><td>60 mm Petri dishes</td><td>ThermoFisher</td><td>12556001</td><td></td></tr><tr><td>100 mm Petri dishes</td><td>ThermoFisher</td><td>12565100</td><td></td></tr><tr><td>Pronase</td><td>Sigma</td><td>10165921001</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ThermoFisher</td><td>16140063</td><td></td></tr><tr><td>DNase I</td><td>Sigma</td><td>4716728001</td><td></td></tr><tr><td>Celltrics 50um filters</td><td>Sysmex</td><td>04-0042327</td><td></td></tr><tr><td>Trypan blue</td><td>ThermoFisher</td><td>15-250-061</td><td></td></tr><tr><td>Hemocytometer</td><td>ThermoFisher</td><td>02-671-6</td><td></td></tr></tbody></table>

homochronic transplantation of interneuron precursors into early postnatal mouse brains

神经元的命运决定和成熟需要一个复杂的相互作用的基因程序和环境信号。然而, 解析的作用, 内在的和外在的机制, 调节这种分化过程是一个难题, 所有发展 neurobiologists。这个问题被放大的 GABAergic 中间神经元, 一个令人难以置信的异构细胞群体出生的瞬态胚胎结构和经历一个长期的迁移阶段, 分散在整个前脑。为了探索不同的大脑环境如何影响 interneuron 的命运和成熟, 我们制定了一个协议, 以收获荧光标记未成熟的 interneuron 前体从特定脑区的新生小鼠 (P0-P2)。在这个年龄, interneuron 迁移几乎完成, 这些细胞居住在他们的最后休息环境与相对较少的突触整合。通过流式细胞仪收集单细胞溶液后, 这些 interneuron 前体被移植到 P0-P2 wildtype 产后幼崽中。通过执行伦 (如皮质对皮质) 或异位 (如皮质对海马) 移植, 你可以评估在新的大脑环境中如何挑战未成熟中间神经元影响他们的命运, 成熟和电路整合。大脑可以在成年小鼠身上收获, 并对移植细胞进行广泛的 posthoc 分析, 包括免疫组化、电生理和转录分析。这一一般方法为调查人员提供了一种策略, 用于分析不同的大脑环境如何影响神经元发育的许多方面, 并确定特定的神经元特征是否主要由硬质遗传程序驱动或环境暗示。

该协议展示了如何从产后早期大脑中采集特定的脑区 (图 1-2), 收集 interneuron 前体的单细胞 dissociations, 并将这些细胞移植到不同的脑区产后幼崽 (图 3)。在 posthoc 分析中, 接受 interneuron 前体移植的大脑在 P30-35 之间采集细胞形态学、神经化学标志物和电生理特性的特征。这些类型的化验通常是在正常小鼠的 P21-P30 之?...

Watch this Scientific Journal Video about Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains at JoVE.com

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

挑战新脑区的年轻神经元, 可以揭示环境如何造型神经元的命运和成熟的重要洞察力。本议定书描述了从特定脑区获取 interneuron 前体的程序, 并将其移植到产后幼崽的大脑中 homotopically 或 heterotopically。

Interneuron 前体 Homochronic 移植早期小鼠脑的研究

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

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Neuroscience

7.8K 次观看.  Norwegian University of Science and Technology. 挑战新脑区的年轻神经元, 可以揭示环境如何造型神经元的命运和成熟的重要洞察力。本议定书描述了从特定脑区获取 interneuron 前体的程序, 并将其移植到产后幼崽的大脑中 homotopically 或 heterotopically。

视频: Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

工程神经前体宫内移植,用于Vivo神经结构研究

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

科研

遗传学

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

新生儿脑室下区电穿孔

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

神经科学

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 ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

小鼠胚胎干细胞向皮质 Interneuron 前体细胞的分化

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

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 ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

将化学遗传工程皮质内神经内生前生移植到早期产后小鼠大脑

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

We describe the intraparenchymal transplantation of human neural progenitor cells transduced with a dual reporter vector expressing luciferase-green fluorescent protein (GFP) in the mouse brain. After transplantation, the luciferase signal is repeatedly measured using in vivo bioluminescence and GFP-expressing grafted cells identified in brain sections using fluorescence microscopy.

小鼠大脑中人类神经祖细胞的脑内移植和 体内 生物发光跟踪

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point ...

Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using interneurons to replace or mitigate the effects of altered neuronal circuits is an attractive therapeutic avenue. To this end, more knowledge is needed about how human stem cell-derived GABAergic interneuron-like cells (hdINs) mature, integrate, and function over time in the host circuitry. Of particular importance in neurodevelopmental disorders is a better understanding of whether these processes in transplanted cells are affected by an evolving and maturing host brain. The present protocol describes a fast and highly efficient generation of hdINs from human embryonic stem cells based on the transgenic expression of the transcription factors Ascl1 and Dlx2. These neuronal precursors are transplanted unilaterally, after 7 days in vitro, to the hippocampus of neonatal 2-day-old mice. The transplanted neurons disperse in the ipsi- and contralateral hippocampus of a mouse model of cortical dysplasia-focal epilepsy syndrome and survive for up to 9 months after transplantation. This approach allows for investigating the cellular identity, integration, functionality, and therapeutic potential of transplanted interneurons over an extended time in developing healthy and diseased brains.

Transplantation of human pluripotent stem cell-derived GABAergic neurons generated by neuronal programming could be a potential treatment approach for neurodevelopmental disorders. This protocol describes the generation and transplantation of human stem cell-derived GABAergic neuronal precursors into the brains of neonatal mice, allowing long-term investigation of grafted neurons and evaluation of their therapeutic potential.

将人干细胞衍生的GABA能神经元移植到出生后早期小鼠海马体中以减轻神经发育障碍

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using ...

可视化发育中的小鼠大脑中的神经元和神经胶质细胞动力学

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

作者聚焦：探索发育中的大脑中的细胞迁移和基因作用

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

Interneuron 前体 Homochronic 移植早期小鼠脑的研究

摘要

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此视频中的章节

工程神经前体宫内移植,用于Vivo神经结构研究

新生儿脑室下区电穿孔

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

小鼠胚胎干细胞向皮质 Interneuron 前体细胞的分化

小鼠后脑作为研究胚胎神经再生的模型

将化学遗传工程皮质内神经内生前生移植到早期产后小鼠大脑

小鼠大脑中人类神经祖细胞的脑内移植和体内生物发光跟踪

将人干细胞衍生的GABA能神经元移植到出生后早期小鼠海马体中以减轻神经发育障碍

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

Interneuron 前体 Homochronic 移植早期小鼠脑的研究

摘要

探索更多视频

此视频中的章节

工程神经前体宫内移植,用于Vivo神经结构研究

新生儿脑室下区电穿孔

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

小鼠胚胎干细胞向皮质 Interneuron 前体细胞的分化

小鼠后脑作为研究胚胎神经再生的模型

将化学遗传工程皮质内神经内生前生移植到早期产后小鼠大脑

小鼠大脑中人类神经祖细胞的脑内移植和 体内 生物发光跟踪

将人干细胞衍生的GABA能神经元移植到出生后早期小鼠海马体中以减轻神经发育障碍

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

小鼠大脑中人类神经祖细胞的脑内移植和体内生物发光跟踪