Name: large-scale three-dimensional imaging of cellular organization in the mouse neocortex
Uploaded: 2018-09-05T15:30:00.000+00:00
Duration: 9 min 55 s
Description: Watch this Scientific Journal Video about Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex at JoVE.com

我们感谢淳宫胁和哈马为他们的建议在 AbScale 实验, 查尔斯横山为编辑手稿, eriko 公司 Ohshima 和美雪 Kishino 为他们的技术协助。这项工作得到了从山本理到金斗勋的研究资金和日本教育、文化、体育、科学和技术部 (下个) 为金斗勋 ("介观 Neurocircuitry" 的创新领域) 的科学研究资助; 22115004) 和(25890023)。

所有实验程序均经山本理瓦科动物实验委员会和山本理基因重组实验安全委员会批准, 并根据山本理脑科学动物设施的机构指南进行。研究所。
1. 成像室的制备
<ol><li>
成像室19
	<ol>
<li>使用硅橡胶板, 准备一个房间的厚度约5毫米和地板板的各种厚度。此外, 准备有和没有玻璃底部的培养皿 (图 1A)。</li>
	</ol>
</li>
	<li>
切片间隔
	<ol>
<li>准备垫片, 以保持样品, 使用硅橡胶板厚度为0.5 毫米 (图 1C)。</li>
	</ol>
</li>
</ol>2. 示踪剂注射
注: 注射入脑桥 (2.1) 或高级丘 (2.2)。注入脑桥标签的子脑投射神经元, 在大脑区域包括视觉和运动区域, 同时注入到上级丘标签子脑投射神经元的视觉区域。为控制实验, 注射生理盐水代替荧光标记的霍乱毒素亚单位 B。为不育条件的维护使用消毒的设备和塑料手套清洗用乙醇。
<ol><li>
对成年小鼠的脑桥进行注射。
	<ol>
<li>绘制1µL 荧光标记的霍乱毒素亚单位 B (25 µg/µL 在 PBS) 成26G 哈密尔顿注射器。</li>
		<li>在注射器上放置注射器泵。</li>
		<li>将注射器和泵放在放置在立体定向仪器上的机械手的刀架上。将机械手12°向后从垂直轴倾斜。</li>
		<li>麻醉注射戊巴比妥钠腹腔 (60 毫克/千克体重) 或管理异氟醚 (2–3%), 对雄性或雌性成年小鼠 (C57BL/6J 或Tlx3)。等待, 直到鼠标没有反应时, 它的尾巴被钳捏, 表明鼠标完全麻醉。</li>
		<li>把鼠标放在立体定向仪器上。</li>
		<li>小心去除头发使用剃刀刀片, 以防止感染和削减10毫米的头皮, 使 bregma 和 lambda 可见。用吸管管理0.1 毫升1% 利多卡因。通过调整在立体定向仪器上的喉舌的垂直位置来设置头部的角度, 使 bregma 和 lambda 具有相同的 z 电平。</li>
		<li>调整机械手的位置, 将其滑动到立体定向仪上, 使注射器尖端接近 bregma 并记录机械手的位置。通过移动机械手上的刀架来收回注射器。</li>
		<li>移动机械手5.4 毫米向后和0.4 毫米侧面。推进注射器, 使尖端接近头骨入口点。收回注射器并标记入口点。</li>
		<li>在标记的位置, 钻一个直径约1毫米的孔。</li>
		<li>插入注射器尖端的孔, 使尖端深度是6.9 毫米以上的测量在 bregma。</li>
		<li>使用泵在0.2 µL/分钟内注入1µL 的示踪剂。</li>
		<li>从大脑中取出注射器。</li>
		<li>如有必要, 用微纤丝止血和即时粘合剂的小片段覆盖裸露的大脑。</li>
		<li>使用用吸管输送的生理盐水冲洗裸露的大脑, 以防止感染和缝合头皮。</li>
		<li>从立体定向仪器中取出鼠标。允许鼠标在30˚C 的孵化器中从麻醉中恢复, 通常为1小时。不要让鼠标无人看管, 直到它恢复了足够的意识来维持胸骨卧床。在完全恢复后, 将鼠标退回到其他动物的公司。</li>
		<li>保持鼠标3–7天。</li>
	</ol>
</li>
	<li>
对成年小鼠的丘进行注射。
	<ol>
<li>准备一个30–50µm 尖直径的玻璃吸管。</li>
		<li>通过塑料管将玻璃吸管与哈密尔顿注射器连接 (图 2A)。</li>
		<li>用石蜡液填充玻璃吸管、塑料管和哈密尔顿注射器。</li>
		<li>在注射器上放置注射器泵。</li>
		<li>将玻璃吸管放在机械手上, 将机械手向后从垂直轴倾斜 (图 2B)。</li>
		<li>执行 2.1. 4–2.1 9。机械手的位置是1.4 毫米后, 0.5 毫米侧向 lambda。</li>
		<li>在头骨上放置一个小的塑料石蜡膜, 然后在它上放置大约1µL 的示踪剂溶液。快速推进玻璃吸管和填充它至少0.5 µL 的示踪剂溶液。</li>
		<li>插入玻璃吸管, 使尖端深度是3.0 毫米从大脑表面。</li>
		<li>使用泵在0.2 µL/分钟内注入0.5 µL 的示踪剂。</li>
		<li>从大脑中取出玻璃吸管。</li>
		<li>执行 2.1. 13–2.1 16。</li>
	</ol>
</li>
</ol>3. 固定和修整
<ol><li>注射戊巴比妥钠 (60 毫克/千克体重) 腹腔成鼠标 (C57BL/6J 或Tlx3/Ai9, 有或没有示踪剂注射如步骤2所述。等待, 直到鼠标没有反应时, 它的尾巴被钳捏, 表明鼠标完全麻醉。</li>
	<li>弄死老鼠人道地通过灌注老鼠 transcardially20与0.9% 生理盐水。</li>
	<li>修正鼠标20由灌注4% 多聚甲醛 (粉煤灰) 在0.1 米磷酸盐缓冲 (pH 7.5)。</li>
	<li>
用剪刀切开头皮, 使头骨暴露于20。
	<ol>
<li>用剪刀切开裸露头骨的中线。用镊子取出头骨。</li>
		<li>如果标记 bregma 和 lambda 的位置是必要的, 首先删除头骨的一个半球。将薄薄的钨针插入大脑中的 bregma 和 lambda 的位置上的头骨留在大脑, 然后取出剩下的头骨。 注意: 大脑可以存储在 PBS 4 ˚C。</li>
	</ol>
</li>
	<li>
对抑制神经元进行抗体染色, 将脑标本切成切片。
	<ol>
<li>把大脑样本放在 vibratome 上。</li>
		<li>在室温下切成500µm 厚的切片, 然后进行步骤 5 (AbScale 方法)。</li>
	</ol>
</li>
	<li>如果抗体染色是不必要的, 修剪的大脑样本块 (高达3毫米厚) 使用剃刀刀片 (图 3), 并继续步骤 4 (SeeDB 方法)。 注意: 切割或修剪后, 大脑可以在 PBS 中存储4摄氏度。SeeDB 方法是可取的, 当抗体染色是不必要的, 因为它需要较少的时间比 AbScale 方法。</li>
</ol>4. 无抗体染色清除 (SeeDB 法)
<ol><li>用刮刀将样品转移到含有20毫升0.5% α thioglycerol 和 20% (瓦特/v) 果糖的50毫升塑料管上, 并在室温下轻轻摇动, 将其孵化为 4 h。</li>
	<li>用刮刀将样品转移到含有20毫升0.5% α thioglycerol 和 40% (瓦特/v) 果糖的50毫升塑料管上, 并在室温下轻轻摇动, 将其孵化为 4 h。</li>
	<li>用刮刀将样品转移到含有20毫升0.5% α thioglycerol 和 60% (瓦特/v) 果糖的50毫升塑料管上, 并在室温下轻轻摇动, 将其孵化为 4 h。</li>
	<li>用刮刀将样品转移到含有20毫升0.5% α thioglycerol 和 80% (瓦特/v) 果糖的50毫升塑料管上, 并在室温下轻轻摇动, 将其孵化为 12 h。</li>
	<li>用刮刀将样品转移到含有20毫升0.5% α thioglycerol 和 100% (瓦特/v) 果糖的50毫升塑料管上, 并在室温下轻轻摇动, 将其孵化为 12 h。</li>
	<li>用刮刀将样品转移到含有20毫升0.5% α thioglycerol 和 80.2% (w/w) 果糖的50毫升塑料管上, 并在室温下轻轻摇动, 将其孵化为 24 h。 注意: 仔细处理样品, 使变形尽可能小。不要将样品孵化得比所示的长, 因为样品很快就会变得不透明。</li>
	<li>将样品嵌入80.2% 果糖溶液填充的成像腔内 (图 1B)。如有必要, 将小块橡胶粘合剂固定在样品上。如果房间太深, 在放置样品之前, 在房间里放一层底板。</li>
	<li>将培养皿放在成像室的玻璃罩上, 将水放在盘子中, 用共焦或双光子显微镜与水浸泡长工作距离目标进行图像处理。励磁波长和发射滤波器在表 1中描述。如有必要, 使用机动舞台。</li>
</ol>5. 用抗体染色清除 (AbSca 法)
<ol><li>准备试剂如表 2所述。</li>
	<li>使用刮刀将切片转移到含有4毫升 Sca/eS0 溶液的5毫升塑料管上, 并在37摄氏度的温和晃动下孵化12小时 (图 4B)。</li>
	<li>用吸管将溶液从试管中取出, 加入4毫升的 Sca/eA2 溶液, 并在37摄氏度时以温和的震动孵化36小时的切片。</li>
	<li>用吸管将溶液从试管中取出, 加入4毫升的 Sca/eB4 溶液, 并在37摄氏度时以温和的震动孵化24小时的切片。</li>
	<li>用吸管将溶液从试管中取出, 加入4毫升的 Sca/eA2 溶液, 并在37摄氏度时以温和的震动孵化12小时的切片。</li>
	<li>使用吸管将溶液从管中取出, 并加入4毫升 PBS, 在室温下进行温和摇动, 以6小时的方式孵化切片。</li>
	<li>用刮刀小心地将切片移到2毫升塑料管上。</li>
	<li>用原抗体 (表 3) 在1毫升的 AbSca 溶液中孵育48-72 小时37摄氏度 (图 4C), 并轻轻摇动。</li>
	<li>用刮刀小心地将切片移到5毫升塑料管上。</li>
	<li>在室温下4毫升的AbSca 溶液中孵育2小时2次, 并进行柔和的震动。</li>
	<li>用刮刀小心地将切片移到2毫升塑料管上。</li>
	<li>孵育与荧光标记的二级抗体 (材料表, 1:100) 在1毫升 AbScale 溶液48小时在37°c 与柔和的震动。</li>
	<li>小心地删除切片使用刮刀到5毫升的塑料管含有4毫升的 AbScale 溶液和孵化的切片为6小时, 在室温下轻轻摇动。</li>
	<li>用吸管将溶液从试管中取出, 加入4毫升的 AbSca 溶液,在室温下轻轻摇动, 将切片培养成2小时2倍。</li>
	<li>用吸管将溶液从试管中取出, 加入4毫升4% 的粉煤灰, 在室温下轻轻摇动, 将切片孵化1小时。</li>
	<li>使用吸管将溶液从管中取出, 并加入4毫升 PBS, 在室温下进行温和摇动, 以1小时的方式孵化切片。</li>
	<li>使用吸管将溶液从管中取出, 并加入4毫升的 Sca/eS4 溶液, 并在37摄氏度时以温和晃动的方式孵化12小时的切片。</li>
	<li>将垫片放在玻璃滑梯上, 将切片放在间隔内, 并将切片浸入 Sca/eS4 溶液中。用盖玻璃封住垫片 (图 1D)。</li>
	<li>使用共焦或双光子显微术, 用水浸泡长的工作距离目标, 将水放在盖子玻璃和图像上。如有必要, 使用机动舞台。 注: 检查样品的深部是否贴上类似于表面的部分, 以确认没有明显的标签偏差。 注: 检测到的抗体的穿透性在表 3中描述。</li>
</ol>6. 细胞位置测定
<ol><li>对于扫描图像中的每个位置, 使用三维图像过滤器14 (图 5A-5D) 计算相关值。</li>
	<li>确定相关值峰值的位置 (图 5D)。</li>
	<li>调查围绕峰值的图像以定位单元格 (图 5E)。</li>
</ol>

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作者没有什么可透露的。

我们已经提出的程序, 以获得大规模的三维图像的细胞类型特定组织的主要细胞类型的小鼠皮层层5。与传统的切片染色相比, 该方法在确定大脑皮层的三维组织方面更为有用。该方法使图像获得从更广泛和更深层次的大脑区域相比, 典型的活体2 光子显微镜或常规共焦显微术, 从而可以让皮层细胞的综合分析组织。
该方法的一个关键步骤是抗体穿透。抗体的子集显示, 渗?...

哺乳动物大脑皮层由大量的细胞类型组成, 每一个有特定的基因表达模式, 电生理和生化特性, 突触连接, 和在体内功能1,2 ,3,4,5,6,7。这些细胞类型是否被组织成重复的结构是不清楚的。皮质柱, 包括视觉定向柱和体感桶, 有重复的结构, 但其细胞组织仍然不清楚8,9。这些是存在于特定的皮质区域, 不是大脑范围的系统。
在皮层5层, 大部分神经元被分为四大类。一种主要类型的兴奋神经元, 亚脑投射神经元, 项目轴突到皮层下靶, 包括桥脑, 脊髓, 和优越的丘, 因此, 代表了主要的皮质输出通路10。皮质投射神经元, 另一种主要类型的兴奋神经元, 支配皮层10。抑制神经元也包含两大类: parvalbumin 表达和生长抑素表达细胞11。
最近的分析表明, 四细胞类型被组织成重复结构12,13,14。次级脑投射神经元12、13、14和皮层投射神经元14组织成细胞型特异性 microcolumns, 有一直径的细胞。Parvalbumin 表达和生长抑素表达细胞与 microcolumns 的亚脑投射神经元, 而不是与 microcolumns 皮层投射神经元14。Microcolumns 自己周期性地排列形成一个六角格子阵列14并且在多皮层区域包括视觉, 感觉和马达区域在老鼠脑子12,14并且在语言人脑的区域13。神经元在个体柱陈列同步活动并且有相似的感觉反应14。这些观察表明, 5 层细胞类型组织成一个柱晶格结构, 代表第一个已知的大脑范围内重复功能模块的组织。
Microcolumns 有大约10µm 的半径并且有大约40µm 的空间周期性。此外, microcolumns 的方向与它们的顶端树突平行, 并根据它们在皮层14中的位置而变化。因此, 柱系统很难分析使用传统的皮质切片的典型厚度的几微米。此外, 周期性分析需要来自广泛脑区的三维数据, 因此, 共焦显微镜和活体2 光子成像的典型成像区域太窄。
最近, 技术已经发展到清除厚组织15,16。在这里, 我们描述了这些方法的应用, 以获得大型, 三维图像的主要细胞类型在小鼠皮层层5组成的柱系统。Subcerebral 投射神经元用逆行标记或增强绿色荧光蛋白在Crym-egfp转基因小鼠12中的表达进行标记, 皮质投射神经元用逆行标记标记或由 tdTomato 表达在Tlx3/Ai9小鼠17。Parvalbumin 表达和生长抑素表达细胞用免疫组化标记。(抗体标度 S) AbScale 方法18用于抗体染色实验, 而 (见深脑) SeeDB 方法19用于其他实验。这些方法克服了传统成像方法的难点, 揭示了5层14的精确细胞组织。

<table><tbody><tr><td>Crym-egfp transgenic mice</td><td>MMRRC</td><td>012003-UCD</td><td></td></tr><tr><td>Tlx3-cre transgenic mice</td><td>MMRRC</td><td>36547-UCD</td><td></td></tr><tr><td>ROSA-CAG-flox-tdTomato mice</td><td>Jackson Laboratory</td><td>JAX #7909</td><td></td></tr><tr><td>Silicone rubber sheet</td><td>AS ONE</td><td>6-611-01</td><td>0.5 mm thickness</td></tr><tr><td>Silicone rubber sheet</td><td>AS ONE</td><td>6-611-02</td><td>1.0 mm thickness</td></tr><tr><td>Silicone rubber sheet</td><td>AS ONE</td><td>6-611-05</td><td>3.0 mm thickness</td></tr><tr><td>Petri dishes</td><td>Falcon</td><td>351008</td><td></td></tr><tr><td>Cover glass</td><td>Matsunami</td><td>C022241</td><td></td></tr><tr><td>Cholera toxin subunit B (recombinant), Alexa Fluor 488 conjugate</td><td>Invitrogen</td><td>C22841</td><td></td></tr><tr><td>Cholera toxin subunit B (recombinant), Alexa Fluor 555 conjugate</td><td>Invitrogen</td><td>C22843</td><td></td></tr><tr><td>Cholera toxin subunit B (recombinant), Alexa Fluor 594 conjugate</td><td>Invitrogen</td><td>C22842</td><td></td></tr><tr><td>Cholera toxin subunit B (recombinant), Alexa Fluor 647 conjugate</td><td>Invitrogen</td><td>C34778</td><td></td></tr><tr><td>26 G Hamilton syringe</td><td>Hamilton</td><td>701N</td><td></td></tr><tr><td>Injector pump</td><td>KD Scientific</td><td>KDS 310</td><td>Pons injection</td></tr><tr><td>Injector pump</td><td>KD Scientific</td><td>KDS 100</td><td>Superior colliculus injection</td></tr><tr><td>Manipulator</td><td>Narishige</td><td>SM-15</td><td></td></tr><tr><td>Sodium pentobarbital</td><td>Kyoritsu Seiyaku</td><td>Somnopentyl</td><td></td></tr><tr><td>Isoflurane</td><td>Pfizer</td><td></td><td></td></tr><tr><td>Lidocaine</td><td>AstraZeneca</td><td>Xylocaine injection 1% with epinephrine</td><td></td></tr><tr><td>Drill</td><td>Toyo Associates</td><td>HP-200</td><td></td></tr><tr><td>Avitene microfibrillar hemostat</td><td>Davol Inc</td><td>1010090</td><td></td></tr><tr><td>Alonalfa</td><td>Daiichi-Sankyo</td><td>Alonalpha A</td><td></td></tr><tr><td>Surgical silk</td><td>Ethicon</td><td>K881H</td><td></td></tr><tr><td>Incubator</td><td>UVP</td><td>HB-1000 Hybridizer</td><td></td></tr><tr><td>Glass pipette</td><td>Drummond Scientific Company</td><td>2-000-075</td><td></td></tr><tr><td>Electrode puller</td><td>Sutter Instrument Company</td><td>P-97</td><td></td></tr><tr><td>Paraffin Liquid, light</td><td>Nacalai tesque</td><td>26132-35</td><td></td></tr><tr><td>Saline</td><td>Otsuka</td><td>1326</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Nacalai tesque</td><td>26126-54</td><td></td></tr><tr><td>Tungsten needle</td><td>Inter medical</td><td>&amp;Phi;0.1 *L200 mm</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000S</td><td></td></tr><tr><td>50 mL plastic tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>&amp;alpha;-thioglycerol</td><td>Nacalai tesque</td><td>33709-62</td><td></td></tr><tr><td>D(-) Fructose</td><td>Nacalai tesque</td><td>16315-55</td><td></td></tr><tr><td>BluTack</td><td>Bostik</td><td>CKBT-450000</td><td></td></tr><tr><td>Two-photon microscope</td><td>Nikon</td><td>A1RMP</td><td></td></tr><tr><td>Water-immersion long working distance objectives</td><td>Nikon</td><td>CFI Apo LWD 25XW, NA 1.1, WD 2 mm</td><td></td></tr><tr><td>Water-immersion long working distance objectives</td><td>Nikon</td><td>CFI LWD 16XW, NA 0.8, WD 3 mm</td><td></td></tr><tr><td>Motorized stage</td><td>COMS</td><td>PT100C-50XY</td><td></td></tr><tr><td>Filter</td><td>Semrock</td><td>FF01-492/SP-25</td><td></td></tr><tr><td>Filter</td><td>Semrock</td><td>FF03-525/50-25</td><td></td></tr><tr><td>Filter</td><td>Semrock</td><td>FF03-575/25-25</td><td></td></tr><tr><td>Filter</td><td>Semrock</td><td>FF01-629/56</td><td></td></tr><tr><td>Filter</td><td>Chroma</td><td>D605/55m</td><td></td></tr><tr><td>5 mL plastic tube</td><td>AS ONE</td><td>VIO-5B</td><td></td></tr><tr><td>2 mL plastic tube</td><td>Eppendorf&amp;nbsp;</td><td>0030120094</td><td></td></tr><tr><td>Urea</td><td>Nacalai tesque</td><td>35905-35</td><td></td></tr><tr><td>Triton X-100</td><td>Nacalai tesque</td><td>35501-15</td><td></td></tr><tr><td>Glyserol</td><td>Sigma-aldrich</td><td>191612</td><td></td></tr><tr><td>D(-)-sorbitol</td><td>Wako</td><td>191-14735</td><td></td></tr><tr><td>Methyl-&amp;beta;-cyclodextrin</td><td>Tokyo chemical industry</td><td>M1356</td><td></td></tr><tr><td>&amp;gamma;-Cyclodextrin</td><td>Wako</td><td>037-10643</td><td></td></tr><tr><td>N-acetyl-L-hydroxyproline</td><td>Skin Essential Actives</td><td>33996-33-7</td><td></td></tr><tr><td>DMSO</td><td>Nacalai tesque</td><td>13445-45</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-aldrich</td><td>A7906</td><td></td></tr><tr><td>Tween-20 (1.1 g/mL)</td><td>Nacalai tesque</td><td>35624-15</td><td></td></tr><tr><td>Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555</td><td>Invitrogen</td><td>A21422</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555</td><td>Invitrogen</td><td>A21428</td><td></td></tr><tr><td>Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647</td><td>Invitrogen</td><td>A21235</td><td></td></tr><tr><td>Goat anti-Mouse IgG (H+L) Highly CrossAdsorbed Secondary Antibody, Alexa Fluor 488</td><td>Invitrogen</td><td>A11029</td><td></td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) Highly CrossAdsorbed Secondary Antibody, Alexa Fluor 488</td><td>Invitrogen</td><td>A21206</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr><tr><td>Water-immersion long working distance objectives</td><td>Olympus</td><td>XLUMPLFLN 20XW, NA 1.0, WD 2 mm</td><td></td></tr><tr><td>Anti-NeuN</td><td>Millipore</td><td>MAB377</td><td></td></tr><tr><td>Anti-NeuN</td><td>Millipore</td><td>ABN78</td><td></td></tr><tr><td>Anti-CTIP2</td><td>Abcam</td><td>ab18465</td><td></td></tr><tr><td>Anti-Statb2</td><td>Abcam</td><td>ab51502</td><td></td></tr><tr><td>Anti-GAD67</td><td>Millipore</td><td>MAB5406</td><td></td></tr><tr><td>Anti-GABA</td><td>Sigma</td><td>A2052</td><td></td></tr><tr><td>Anti-Parvalbumin</td><td>Swant</td><td>235</td><td></td></tr><tr><td>Anti-Parvalbumin</td><td>Frontier Institute</td><td>PV-Go-Af460</td><td></td></tr><tr><td>Anti-Parvalbumin</td><td>Sigma</td><td>P3088</td><td></td></tr><tr><td>Anti-Parvalbumin</td><td>Abcam</td><td>ab11427</td><td></td></tr><tr><td>Anti-Somatostatin</td><td>Peninsula Laboratories</td><td>T-4103</td><td></td></tr><tr><td>Anti-c-Fos</td><td>CalbioChem</td><td>PC38</td><td></td></tr></tbody></table>

large-scale three-dimensional imaging of cellular organization in the mouse neocortex

哺乳动物大脑皮层由许多类型的兴奋和抑制神经元组成, 每一种都有特定的电生理和生化特性, 突触连接和体内功能, 但其基本功能和解剖学组织从细胞到网络规模是不太了解。在这里, 我们描述了一个方法, 三维图像的荧光标记神经元跨大区域的大脑, 以调查皮质细胞组织。在转基因小鼠中, 用荧光逆行神经元示踪剂或荧光蛋白的表达来标记特定类型的神经元。块脑标本,例如,半球, 是在固定后准备的, 用组织清除方法透明, 并受到特定细胞类型的荧光 immunolabeling。大面积扫描使用共焦或双光子显微镜, 配备了较大的工作距离目标和机动阶段。该方法可以解决小鼠大脑皮层细胞型特定柱功能模块的周期性组织问题。该方法可用于研究不同脑区和其他复杂组织中的三维细胞结构。

我们标记皮层投射神经元的 tdTomato 在Tlx3/Ai9 转基因小鼠和可视化的亚脑投射神经元, 通过注射逆行示踪 CTB488 进入脑桥。大脑的左半球受 SeeDB 方法扫描, 使用双光子显微镜, 配备了水浸泡长工作距离目标 (25X, 1.1, 工作距离2毫米) 和机动阶段。一叠401张图像 (512 x 512 像素; 像素大小 = 0.99 µm) 在 z 步 = 2.8 µm 在每30个扫描位置被获得了。两种细胞的细胞体在广泛的?...

Watch this Scientific Journal Video about Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex at JoVE.com

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

小鼠大脑皮层细胞组织的大尺度三维成像

在这里, 我们描述一个程序, 组织清除, 荧光标记, 和大规模的小鼠脑组织成像, 从而使可视化的三维组织的细胞类型在大脑皮层。

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

large-scale-three-dimensional-imaging-cellular-organization-mouse

Research

JoVE Journal

Neuroscience

8.3K 次观看.  RIKEN Brain Science Institute. 在这里, 我们描述一个程序, 组织清除, 荧光标记, 和大规模的小鼠脑组织成像, 从而使可视化的三维组织的细胞类型在大脑皮层。

视频: Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

科研

神经科学

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

多层皮质Ca<sup&gt; 2+</sup&gt;使用棱镜探针和小型荧光显微镜成像自由移动的小鼠

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

行为学

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light3 do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.4 Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.

Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.

多光子显微镜的清除小鼠脑组织表达YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

急性片的成年小鼠前脑的神经细胞迁移的延时成像

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

在使用微深皮质层的活体成像

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

生物学

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 ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

修改一个Colliculo  - 丘脑鼠脑切片，收纳室部件的3-D打印和多尺度光学成像

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

植入颅窗以在清醒小鼠 中重复体内 成像

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei.
Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases.
We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

使用MRI，组织清除和光片显微镜对完整的新生小鼠大脑进行全脑单细胞成像和分析

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of ...

小鼠新生儿脑显微 CT 扫描和图像处理

Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains

Neuroimages are a valuable tool for studying brain morphology in experiments using animal models. Magnetic resonance imaging (MRI) has become the standard method for soft tissues, although its low spatial resolution poses some limits for small animals. Here, we describe a protocol for obtaining high-resolution three-dimensional (3D) information on mouse neonate brains and skulls using micro-computed tomography (micro-CT). The protocol includes those steps needed to dissect the samples, stain and scan the brain, and obtain morphometric measurements of the whole organ and regions of interest (ROIs). Image analysis includes the segmentation of structures and the digitization of point coordinates. In sum, this work shows that the combination of micro-CT and Lugol's solution as a contrast agent is a suitable alternative for imaging the perinatal brains of small animals. This imaging workflow has applications in developmental biology, biomedicine, and other sciences interested in assessing the effect of diverse genetic and environmental factors on brain development.

作者聚焦：小鼠新生儿大脑的高分辨率成像 – 使用 Lugol 溶液造影剂的显微 CT 方案

This study describes the steps for obtaining high-resolution images of neonatal mouse brains by combining micro-computed tomography (micro-CT) and a contrast agent in ex vivo samples. We describe basic morphometric analyses to quantify brain size and shape in these images.

小鼠新生儿脑的显微CT成像和形态分析

Neuroimages are a valuable tool for studying brain morphology in experiments using animal models. Magnetic resonance imaging (MRI) has become the standard ...

小鼠大脑皮层细胞组织的大尺度三维成像

摘要

探索更多视频

此视频中的章节

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

多层皮质Ca

多光子显微镜的清除小鼠脑组织表达YFP

急性片的成年小鼠前脑的神经细胞迁移的延时成像

在使用微深皮质层的活体成像

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

修改一个Colliculo - 丘脑鼠脑切片，收纳室部件的3-D打印和多尺度光学成像

植入颅窗以在清醒小鼠中重复体内成像

使用MRI，组织清除和光片显微镜对完整的新生小鼠大脑进行全脑单细胞成像和分析

小鼠新生儿脑的显微CT成像和形态分析

小鼠大脑皮层细胞组织的大尺度三维成像

摘要

探索更多视频

此视频中的章节

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

多层皮质Ca

多光子显微镜的清除小鼠脑组织表达YFP

急性片的成年小鼠前脑的神经细胞迁移的延时成像

在使用微深皮质层的活体成像

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

修改一个Colliculo - 丘脑鼠脑切片，收纳室部件的3-D打印和多尺度光学成像

植入颅窗以在清醒小鼠 中重复体内 成像

使用MRI，组织清除和光片显微镜对完整的新生小鼠大脑进行全脑单细胞成像和分析

小鼠新生儿脑的显微CT成像和形态分析

植入颅窗以在清醒小鼠中重复体内成像