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://www-jove-com.remotexs.ntu.edu.sg/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://www-jove-com.remotexs.ntu.edu.sg/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://www-jove-com.remotexs.ntu.edu.sg/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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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+Transplantations+Reveal+Environmental+Influences+on+Interneuron+Diversity+and+Maturation.">Heterotopic Transplantations Reveal Environmental Influences on Interneuron Diversity and Maturation.</a> Cell Rep. 21 (3), 721-731 (2017).</li><li>Xu, Q., Tam, M., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+mapping+Nkx2.1-lineage+cells+in+the+mouse+telencephalon.">Fate mapping Nkx2.1-lineage cells in the mouse telencephalon.</a> J Comp Neurol. 506 (1), 16-29 (2008).</li><li>Thompson, L., Bjorklund, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival,+differentiation,+and+connectivity+of+ventral+mesencephalic+dopamine+neurons+following+transplantation.">Survival, differentiation, and connectivity of ventral mesencephalic dopamine neurons following transplantation.</a> Prog Brain Res. 200, 61-95 (2012).</li><li>Liang, Y., Agren, L., Lyczek, A., Walczak, P., Bulte, J. 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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醉时间 (< 4 分钟), 并保持幼犬的光加热是至关重要的。
移植的大脑可能有显著的变异性, 有些可能不包含任何移植的细胞, 而其他大脑将包含数以千计。结合几个 P0-P2 凋落物进行解剖, 可以增加采集的细胞数量, 以增加移植, 这使得一个既可以提高移栽细胞密度和/或增加嫁接幼崽的数量, 这将提高的可能性成功的移植。要瞄准合适的大脑区域, 重要的是, 幼崽的头部是稳定的, 不移动时, 降低微进入大脑。任何移位的头部或弯曲的微轴可能导致 mistargeting 和不准确的注射 (如失去细胞进入侧脑室)。另外, 每只幼崽执行多个注射点 (单边或双边) 可以提高成功率, 以防一个注射点 mistargeted。在未来, 最好是制定一个更有效的立体定向战略, 以稳定幼犬, 并提供更准确和一致的注射。
由于科学和技术上的原因, 选择了 P0-P2 时间框架。在这个年龄, 大多数 interneuron 前体已经迁移到他们的终端站点, 但很少的环境相互作用。这一时间框架可能不适用于其他神经细胞类型和大脑区域, 所以你可能想调整这些 timepoints 为他们的特定目的。例如, 比较这些 P0-P2 收获的 interneuron 前体与梅兰日兰收获细胞的治疗潜力是很有趣的: 一个年龄可能有更多的好处和/或不需要的副作用比其他。另外, 执行 heterochronic 移植 (例如, 在 P0-P2 收集细胞, 移植到 P7-10 大脑, 反之亦然), 以确定环境中的时间变化如何影响细胞的命运和成熟是很有趣的。在 P0-2 进行注射的好处是头骨相对较薄, 可以很容易地穿透尖锐的微。任何 P5 的注射都需要切除或稀释头骨。
本协议中描述的分析仅限于脑区间差异表达的 interneuron 亚型的具体特征。在未来, 人们可以利用单细胞测序的力量来表征移植细胞的全转录。这种无偏见的方法可以识别在移植细胞中强烈丰富或耗尽的特定基因 (或更大的信号叶栅) 与控制, 这将提供洞察的候选环境线索, 将影响命运测定或成熟。

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 border="0" cellpadding="0" cellspacing="0" style="width:776px;" width="777">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium chloride</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">S7653</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium bicarbonate</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">S6297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Potassium chloride</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium phosphate monobasic</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">S0751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Calcium chloride</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">C5080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Magnesium chloride</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">M2670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Glucose</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">G7528</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sucrose</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">S7903</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Brain Matrices</td>
			<td style="width:203px;">Roboz</td>
			<td style="width:136px;">SA-2165</td>
			<td>Only needed if harvesting striatum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fine point Dumont Forceps</td>
			<td style="width:203px;">Roboz</td>
			<td style="width:136px;">RS-4978</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Microdissecting scissors</td>
			<td style="width:203px;">Roboz</td>
			<td style="width:136px;">RS-5940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Razor blades</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">12-640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Pasteur pipettes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">1367820C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Nanoject III</td>
			<td style="width:203px;">Drummond</td>
			<td style="width:136px;">3-000-207</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Manual Manipulator w/ stand</td>
			<td style="width:203px;">World Precision Instruments&#160;</td>
			<td style="width:136px;">M3301R/M10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">5 ml round bottom plastic tubes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">149591A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">60 mm Petri dishes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">12556001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">100 mm Petri dishes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">12565100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Pronase</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">10165921001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">16140063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">DNase I</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">4716728001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Celltrics 50um filters</td>
			<td style="width:203px;">Sysmex</td>
			<td style="width:136px;">04-0042327</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Trypan blue</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">15-250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Hemocytometer</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">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 之间进行的, 但由于移植细胞的成熟可能会因解剖/离解过程而略有延迟, 因此建议额外等待5-10 天来弥补这一延迟成熟。要进行的分析的类型将决定正确的策略来收获大脑。值得注意的是, 我们没有观察到特定 interneuron 亚群的优先细胞死亡, 这些子组可能偏向或反对某些亚型20。
免疫组化分析, 小鼠4% 多聚甲醛灌注, 脑切除。50微米 vibratome 切片是通过靶脑区, 并储存在防冻液和/或处理染色如前所述20。有些大脑没有含有任何西红柿 + 细胞, 这可能是由于靶向不正确 (例如, 注射太深进入心室), 移植过程中的细胞丢失或发生凋亡, 或宿主排斥移植细胞。根据最终细胞计数, 估计只有2-5% 的移植细胞存活20, 这是符合其他移植程序22,23。
不足为奇的是, 在成功的移植中, 番茄 + 细胞的总数量有显著的变异性, 从很多到成千上万的西红柿 + 细胞不等 (图 4A)。移植细胞在正确的区域被定位, 有许多显示 interneuron 形态和良好的 interneuron 神经化学标记 (图 4B)。即使细胞移植到新的环境中进行异位移植 (图 4C), 也能观察到类似的细胞存活数和成熟剖面。
除免疫组化分析外, 还进行了接枝细胞的电生理分析, 以确认它们已集成到脑回路中, 并显示预期的内在和射击特性。大脑是从 P30-35 的老鼠身上收获的, 为生理记录准备的切片, 如前所述20。嫁接中间神经元呈现出成人样的生理特性和不同的射击模式, 具有典型的 interneuron 亚型 (图 5A), 提示嫁接中间神经元能够在宿主环境中适当成熟。为了验证移植细胞是否集成在神经元网络中, sEPSCs 也被记录下来 (图 5B)。此外, 移植的一个子集进行了中间神经元表达 ChR2 后, 记录从金字塔细胞本地化附近移植中间神经元。这些数据表明, 突触后 GABAergic 电流是由蓝光诱发的 (图 5C-D)。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57723/57723fig4.jpg"> 图 4: 接枝 interneuron 前体填充宿主脑区, P30 小鼠在 P1 移植了番茄 + interneuron 前体的代表部分.(A)在伦皮质对皮质移植中, 移植细胞填充所有皮质层, 并显示模仿内源性中间神经元的形态。所选图像突出显示不同移植细胞数的变异性, 左图与右侧的移植相比, 每节的番茄 + 细胞数量要大得多。(B)低放大 (左) 和高放大 (右) 代表部分从伦海马到海马移植。注意, 大部分的番茄 + 细胞在地层中 oriens (因此) 表达 SST (可能是 O LM 细胞), 而许多番茄 + 细胞在地层 pyramidale (SP) 表达 PV (可能篮子细胞), 类似于内源性海马中间神经元。(C)纹状体中有番茄 + 的异位移植 (皮质-纹状) 的例子。刻度条 = 200 微米在 a 在低 mag 板在 b, 50 微米在 C 和大功率 mag 在 b.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57723/57723fig4large.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/57723/57723fig5.jpg"> 图 5: 移植中间神经元 electrophysiologically 成熟, 在宿主神经元网络中集成 (A)接枝中间神经元记录的最高发射频率的代表性例子。左, 快速扣球 interneuron 从髋关节到 Ctx 移植, 注射当前步骤:-100 pa 和 520 pa;右, 非快速扣球 interneuron 从 Ctx 到 Ctx 移植, 注入当前步骤:-100 pa 和 360 pa。(B) sEPSCs 的例子, 记录在晚扣球 interneuron 从髋部到髋关节移植。(C)显示Nkx2.1 的代表形象;Ai32细胞 (YFP) 从 Ctx 到 Ctx 移植与 biocytin 填充 (红色) 锥体细胞。刻度条 = 50 微米。(D)在锥体细胞中记录的蓝色光脉冲诱发的 GABAergic 突触后电流的例子, 记录有 [145 毫米] Cl。黑色, 平均痕迹;在红色, 平均反应记录在 Gabazine 的存在。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57723/57723fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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 ...

homochronic-transplantation-interneuron-precursors-into-early

Nanyang Technological University

Research

JoVE Journal

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

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism 
and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their 
function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and 
systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, 
we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent 
protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of 
many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development,
 including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully 
in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of 
shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical 
imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice 
and rats.

An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.

利用基于病毒的Cre重组马赛克产后小鼠脑开发基因功能分析

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

科研

神经科学

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

小鼠神经干细胞移植手术与器官的诱向性鼠肝炎病毒感染小鼠的脊髓

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

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 ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

新生小鼠脑巴细胞基因调控的立体定向手术

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

新化合物调制伽玛-丁酸受体 a 型神经传递的发现方法

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

多鼠脑的同时 Cryosectioning

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

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 ...