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>

<ol>
	<li>Lein, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+atlas+of+gene+expression+in+the+adult+mouse+brain.">Genome-wide atlas of gene expression in the adult mouse brain.</a> Nature. 445 (7124), 168-176 (2007).</li><li>Defelipe, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+classification+and+nomenclature+of+cortical+GABAergic+interneurons.">New insights into the classification and nomenclature of cortical GABAergic interneurons.</a> Nature Reviews Neuroscience. 14 (3), 202-216 (2013).</li><li>Jiang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+connectivity+among+morphologically+defined+cell+types+in+adult+neocortex.">Principles of connectivity among morphologically defined cell types in adult neocortex.</a> Science. 350 (6264), aac9462 (2015).</li><li>Sorensen, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlated+gene+expression+and+target+specificity+demonstrate+excitatory+projection+neuron+diversity.">Correlated gene expression and target specificity demonstrate excitatory projection neuron diversity.</a> Cerebral Cortex. 25 (2), 433-449 (2015).</li><li>Zeisel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+structure.+Cell+types+in+the+mouse+cortex+and+hippocampus+revealed+by+single-cell+RNA-seq.">Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq.</a> Science. 347, 1138-1142 (2015).</li><li>Tasic, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mouse+cortical+cell+taxonomy+revealed+by+single+cell+transcriptomics.">Adult mouse cortical cell taxonomy revealed by single cell transcriptomics.</a> Nature Neuroscience. 19 (2), 335-346 (2016).</li><li>Zeng, H., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+cell-type+classification:+Challenges,+opportunities+and+the+path+forward.">Neuronal cell-type classification: Challenges, opportunities and the path forward.</a> Nature Reviews Neuroscience. 18 (9), 530-546 (2017).</li><li>Horton, J. C., Adams, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cortical+column:+a+structure+without+a+function.">The cortical column: a structure without a function.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 360 (1456), 837-862 (2005).</li><li>Costa, N. M., Martin, K. A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whose+cortical+column+would+that+be?.">Whose cortical column would that be?.</a> Frontiers in Neuroanatomy. 4, (2010).</li><li>Molyneaux, B. J., Arlotta, P., Menezes, J. R. L., Macklis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+subtype+specification+in+the+cerebral+cortex.">Neuronal subtype specification in the cerebral cortex.</a> Nature Reviews Neuroscience. 8 (6), 427-437 (2007).</li><li>Hioki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+inhibitory+inputs+to+dendritic+and+somatic+compartments+of+parvalbumin-expressing+neocortical+interneuron.">Cell type-specific inhibitory inputs to dendritic and somatic compartments of parvalbumin-expressing neocortical interneuron.</a> Journal of Neuroscience. 33 (2), 544-555 (2013).</li><li>Maruoka, H., Kubota, K., Kurokawa, R., Tsuruno, S., Hosoya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodic+organization+of+a+major+subtype+of+pyramidal+neurons+in+neocortical+layer+V.">Periodic organization of a major subtype of pyramidal neurons in neocortical layer V.</a> Journal of Neuroscience. 31 (50), 18522-18542 (2011).</li><li>Kwan, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-dependent+posttranscriptional+regulation+of+NOS1+by+FMRP+in+the+developing+cerebral+cortex.">Species-dependent posttranscriptional regulation of NOS1 by FMRP in the developing cerebral cortex.</a> Cell. 149 (4), 899-911 (2012).</li><li>Maruoka, H., Nakagawa, N., Tsuruno, S., Sakai, S., Yoneda, T., Hosoya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lattice+system+of+functionally+distinct+cell+types+in+the+neocortex.">Lattice system of functionally distinct cell types in the neocortex.</a> Science. 358 (6363), 610-615 (2017).</li><li>Treweek, J. B., Gradinaru, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracting+structural+and+functional+features+of+widely+distributed+biological+circuits+with+single+cell+resolution+via+tissue+clearing+and+delivery+vectors.">Extracting structural and functional features of widely distributed biological circuits with single cell resolution via tissue clearing and delivery vectors.</a> Current Opinion in Biotechnology. 40, 193-207 (2016).</li><li>Tainaka, K., Kuno, A., Kubota, S. I., Murakami, T., Ueda, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+Principles+in+Tissue+Clearing+and+Staining+Protocols+for+Whole-Body+Cell+Profiling.">Chemical Principles in Tissue Clearing and Staining Protocols for Whole-Body Cell Profiling.</a> Annual Review of Cell and Developmental Biology. 32 (1), (2016).</li><li>Gerfen, C. R., Paletzki, R., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GENSAT+BAC+cre-recombinase+driver+lines+to+study+the+functional+organization+of+cerebral+cortical+and+basal+ganglia+circuits.">GENSAT BAC cre-recombinase driver lines to study the functional organization of cerebral cortical and basal ganglia circuits.</a> Neuron. 80 (6), 1368-1383 (2013).</li><li>Hama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScaleS:+an+optical+clearing+palette+for+biological+imaging.">ScaleS: an optical clearing palette for biological imaging.</a> Nature Neuroscience. 18 (10), 1518-1529 (2015).</li><li>Ke, M. T., Fujimoto, S., Imai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeDB:+a+simple+and+morphology-preserving+optical+clearing+agent+for+neuronal+circuit+reconstruction.">SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.</a> Nature Neuroscience. 16 (8), 1154-1161 (2013).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Animal+Perfusion+Fixation+for+Rodents.">Whole Animal Perfusion Fixation for Rodents.</a> Journal of Visualized Experiments. (65), e3564 (2012).</li><li>Kim, S. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+electrotransport+selectively+enhances+the+transport+of+highly+electromobile+molecules.">Stochastic electrotransport selectively enhances the transport of highly electromobile molecules.</a> Proceedings of the National Academy of Sciences. 112 (46), E6274-E6283 (2015).</li><li>Murray, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple,+scalable+proteomic+imaging+for+high-dimensional+profiling+of+intact+systems.">Simple, scalable proteomic imaging for high-dimensional profiling of intact systems.</a> Cell. 163 (6), 1500-1514 (2015).</li><li>Ke, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+mapping+of+neuronal+circuitry+with+an+index-optimized+clearing+agent.">Super-resolution mapping of neuronal circuitry with an index-optimized clearing agent.</a> Cell Reports. 14 (11), 2718-2732 (2016).</li><li>Lee, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACT-PRESTO:+Rapid+and+consistent+tissue+clearing+and+labeling+method+for+3-dimensional+(3D)+imaging.">ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging.</a> Scientific Reports. 6, 18631 (2016).</li><li>Renier, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+Brain+Activity+by+Automated+Volume+Analysis+of+Immediate+Early+Genes.">Mapping of Brain Activity by Automated Volume Analysis of Immediate Early Genes.</a> Cell. 165 (7), 1789-1802 (2016).</li><li>Kubota, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+profiling+of+cancer+metastasis+with+single-cell+resolution.">Whole-body profiling of cancer metastasis with single-cell resolution.</a> Cell Reports. 20 (1), 236-250 (2017).</li><li>Li, W., Germain, R. N., Gerner, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex,+quantitative+cellular+analysis+in+large+tissue+volumes+with+clearing-enhanced+3D+microscopy+(Ce3D).">Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D).</a> Proceedings of the National Academy of Sciences. 114 (35), E7321-E7330 (2017).</li><li>Liu, A. K. L., Lai, H. M., Chang, R. C. C., Gentleman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+of+acrylamide+sodium+dodecyl+sulphate+(SDS)-based+tissue+clearing+(FASTClear):+a+novel+protocol+of+tissue+clearing+for+three-dimensional+visualization+of+human+brain+tissues.">Free of acrylamide sodium dodecyl sulphate (SDS)-based tissue clearing (FASTClear): a novel protocol of tissue clearing for three-dimensional visualization of human brain tissues.</a> Neuropathology and Applied Neurobiology. 43 (4), 346-351 (2017).</li></ol>

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>26G 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>&#934;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>&#945;-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 colspan="2">CFI Apo LWD 25XW, NA 1.1, WD 2 mm</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&#160;</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-&#946;-cyclodextrin</td>
			<td>Tokyo chemical industry</td>
			<td>M1356</td>
			<td></td>
		</tr>
		<tr>
			<td>&#947;-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

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

科研

神经科学

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

在麻醉大鼠的硅探头录制的大型神经元合唱

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

移动硅探针的神经行为鼠害的大型记录

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

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 ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

成像钙<sup&gt; 2+</sup&gt;在小鼠视网膜锥光感受器轴突终末动态

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

协议星形胶质细胞的三维共聚焦形态分析

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

人 Intraepidermal 神经纤维内线粒体三维成像及分析

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Three-Dimensional Motor Nerve Organoid Generation

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

三维运动神经器官生成

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.