这项工作得到了国家卫生研究院（R01NS085215至K.F.，T32 GM107000和F30MH122146至A.C）的支持。作者感谢渡边奈女士娴熟的技术援助。

所有动物协议都经过马萨诸塞大学医学院机构动物护理和使用委员会（IACUC）的审查和批准。切片培养制备、质粒制备和电位也详细介绍了我们以前发布的方法，并可以参考其他信息10。
1. 切片文化准备
<ol><li>准备小鼠组织型海马片培养，如前所述11，使用产后6至7天大的男女小鼠。<ol>
<li>为由 （在 mM 中）: 238 蔗糖组成的有机切片培养物准备解剖介质， 2.5 KCl， 1 CaCl2， 4 MgCl2， 26 NaHCO3， 1 NaH2PO4， 和 11 葡萄糖在去离子水中， 然后气体与 5% CO2/95% O2 到 pH 值 7.4。</li>
		<li>准备有机片文化介质，包括: 78.8% （v/v） 最低基本中型鹰， 20% （v/v） 马血清， 17.9 m M NaHCO3， 26.6 mM 葡萄糖， 2 M CaCl2， 2 M MgSO4， 30m HEPES ， 胰岛素 （1 微克/mL）， 和 0.06 mM 抗坏血糖酸， pH 调整为 7.3.使用渗透计将渗透度调整到 310-330 渗透器。</li>
		<li>使用两把铲子将海马从整个大脑中分离出来，然后用组织斩波器切片（400μm）。使用两个钳子将切片分离，并转移到插入器下方充满培养介质 （950 μL） 的 6 井板中的 30 mm 细胞培养插入。</li>
	</ol>
</li>
	<li>将有机细胞切片培养物存储在组织培养孵化器（35°C，5% CO2）中，每两天更换一次切片培养介质。</li>
</ol>2. 普拉斯密德准备
<ol><li>为感兴趣的基因准备质粒。<ol>
<li>亚克隆增强绿色荧光蛋白（EGFP）基因成pCAG载体。</li>
		<li>用无内毒素净化套件净化 pCAG-EGFP 质粒，并溶解在内部溶液中，该溶液由含有 140 mM K-甲烷硫酸盐的二乙基热碳酸酯处理水组成， 0.2 mM EGTA、2 m M MgCl2和 10 mM HEPES，调整为 pH 7.3 与 KOH（质粒浓度:0.1 μg/μL）。</li>
	</ol>
</li>
</ol>3. 玻璃移液器制备
<ol><li>在微管式拉拔器上拉玻色硅酸盐玻璃移液器（4.5 - 8 M？）（图 1A）。 注:用于全细胞贴片夹录音的玻璃移液器非常适合电镀。</li>
	<li>在200°C下将玻璃移液器烘烤过夜进行消毒。</li>
	<li>在解剖显微镜下检查移液器尖端的大小，以接近电阻。<ol>
<li>可选:通过将移液器连接到电极电极，并使用微操纵器将移液器尖端操纵到含有（mM）的过滤消毒人工脑脊液（aCSF）中，验证移液器的电阻: 119 NaCl， 2.5 KCl， 0.5 CaCl2， 5 MgCl2， 26 NaHCO3， 1 NaH2PO4 和 11 葡萄糖在去离子水中， 气体与 5% CO2/95% O2 到 pH 值 7.4.使用电极器上的读数确认实际电阻。 注:移液器尖端越清晰，电阻越大。移液器阻力应低于 10 MΩ。具有高移液器电阻（图1B）的玻璃移液器在反复电镀时经常堵塞尖端。</li>
	</ol>
</li>
</ol>4. 电镀钻机设置
<ol><li>将电热器安装到标准的全细胞电生理学钻机上，配备直立显微镜，安装在换档台上，配有显微操纵器和永久泵。</li>
	<li>将电极器的头顶安装到微操纵器上，并将一对扬声器连接到电极器。将电击器连接到脚踏板，当准备就绪时，脚踏板可用于发送脉冲。 注:扬声器在打开时发出音调，这是电极电阻的指示器。这使得无需将注意力从程序中移开即可确定阻力的相对变化。</li>
</ol>5. 电镀制备
<ol><li>将切片培养物插入从 6 个井板转移到 3 厘米的 Petri 菜肴，里面装有 900 μL 的文化介质，并储存在桌面 CO2 孵化器中，直到准备好进行电镀。<ol>
<li>在 3.5 厘米的 Petri 菜肴中，用切片培养介质 （1 mL） 将新鲜培养物插入到电后培养切片中，至少 30 分钟。</li>
	</ol>
</li>
	<li>清洁并准备钻机进行电镀。<ol>
<li>在开始当天的实验之前，用 10% 漂白剂将线条香水 5 分钟，对管子和腔室进行消毒。</li>
		<li>用去离子化的自动压水灌注线条至少30分钟，以完全冲洗。</li>
		<li>用含有0.001m四毒毒素（TTX）的过滤消毒的 ACSF 给生产线注入香水。 注:TTX将细胞毒性和死亡降到最低，因为内周10的过度兴奋。</li>
	</ol>
</li>
	<li>设置电击器的脉冲参数:振幅 +5 V、方脉冲、500 ms 的列车、50 Hz 的频率和 500 μs 的脉冲宽度。</li>
	<li>将玻璃移液器填充 5 μL 含质粒的内部溶液。<ol>
<li>通过多次轻拂和轻轻敲击尖端，从移液器尖端移除任何捕获的气泡。</li>
		<li>通过在解剖显微镜下可视化或重复步骤 3.3.1 来检查移液器的阻力，检查尖端是否损坏。 注:如果尖端损坏，必须丢弃玻璃移液器，并且此步骤必须重复使用以前在第 3 步中准备的新玻璃移液器。</li>
	</ol>
</li>
	<li>将移液器尖端牢固地连接到电极上并打开扬声器。当尖端与 aCSF 介质接触时，记录电风机的读数（移液器的电阻）。</li>
	<li>使用锋利的刀片切割培养件插入膜，并隔离一片培养。使用锋利的倾斜钳子小心地将切片培养体转移到电位室，并用切片锚固定其位置。<ol>
<li>不要将切片培养在孵化器外每次超过30分钟，以防止副作用，如神经元健康或功能12的变化。</li>
	</ol>
</li>
</ol>6. 感兴趣的电聚电池
<ol><li>用嘴或使用连接到管子上的 1 mL 注射器（0.2 - 0.5 mL 压力）对移液器施加正压力。</li>
	<li>使用微操纵器的三维旋钮控制装置在切片培养件表面附近操纵移液器尖端。</li>
	<li>选择目标细胞并接近目标细胞，保持正压力，直到细胞表面形成酒窝，在显微镜上可见。</li>
	<li>执行压力循环。<ol>
<li>通过口腔快速施加轻微的负压，使移液器尖端和等离子体膜之间形成松散的密封，通过膜向上进入移液器尖端来视觉显示。通过听扬声器音调的增加，观察移液器阻力的增加（+2.5 倍的初始阻力）。快速重新施加正压，使酒窝重新形成。</li>
		<li>立即完成至少两个压力周期而不停顿，然后保持负压力为 1s。 注:在周期之间暂停、施加过大的压力或将负压保持太久可能会导致严重的细胞损伤，并可能导致细胞在电电过程中死亡。</li>
	</ol>
</li>
	<li>当扬声器的音调达到音高中稳定的顶点时，使用脚踏板快速脉冲电极器，表示峰值电阻。在发送脉搏之前，不要在峰值阻力超过 1s 时等待。 注:我们在使用本协议10时未观察到任何脱靶电位。只有在压力循环期间与玻璃移液器接触的细胞被转染。将移液器定位到其他神经元附近不会导致基因转染。</li>
	<li>轻轻地将移液器从细胞中收回约 100 μm，而不施加压力。</li>
	<li>重新施加正压，验证电阻是否类似于第 5.5 步中录制的读出，然后接近下一个单元格。<ol>
<li>通过施加正压，去除电镀后通过视觉或显著增加（>15%）移液器电阻指示的潜在堵塞。 注意:如果没有可见的堵塞，并且阻力仍然显著提高，请丢弃移液器并使用新的移液器。平均而言，如果用户小心谨慎，移液器可用于多达20个电位事件。</li>
	</ol>
</li>
	<li>电镀后，将切片培养转移到新鲜培养件上，并在孵化器中以 35°C 孵育长达 3 天。</li>
</ol>7. 有机海马切片培养物的固定、染色和成像
<ol><li>修复电浸状有机细胞片培养物 2 × 转染后 3 天与 4% 的副甲醛和 4% 蔗糖在 0.01 M 磷酸盐缓冲盐水 （1x PBS） 在室温下 1.5 小时。</li>
	<li>在 0.1 M 磷酸盐缓冲区 （1x PB） 中取出 30% 蔗糖中的固定和孵化切片，使用 2 小时。</li>
	<li>将切片放在滑动玻璃上，并将滑梯玻璃放在碎干冰上将其冻结。在室温下解冻切片，并将它们转移到装满 1 倍 PBS 的 6 井板中。</li>
	<li>在 GDB 缓冲区（0.1% 明胶、0.3% Triton X-100、450 mM NaCl 和 32% 1x PB、pH 7.4）中用鼠标抗 GFP 和兔子抗 RFP 抗体在室温下 2 小时染色切片。</li>
	<li>在室温下用 1x PBS 清洗片片三次，每次洗 10 分钟。</li>
	<li>在室温下，在 GDB 缓冲区用抗鼠标 Alexa 488 结合的二级抗体和抗兔亚历克萨 594 结合的二级抗体孵育片片，持续 1 小时。在室温下用 DAPI （4 μg/mL） 在 1x PBS 中孵育片片 10 分钟。</li>
	<li>在室温下用 1x PBS 清洗切片三次，每次洗 3 分钟。</li>
	<li>使用安装介质在玻璃滑梯上安装切片并执行荧光成像。</li>
</ol>

<ol>
	<li>Washbourne, P., McAllister, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+gene+transfer+into+neurons.">Techniques for gene transfer into neurons.</a> Current Opinion in Neurobiology. 12 (5), 566-573 (2002).</li><li>Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+by+direct+microinjection+of+DNA+into+cultured+mammalian+cells.">High efficiency transformation by direct microinjection of DNA into cultured mammalian cells.</a> Cell. 22 (2), 479-488 (1980).</li><li>Rae, J. L., Levis, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+electroporation.">Single-cell electroporation.</a> Pflugers Archives: European Journal of Physiology. 443 (4), 664-670 (2002).</li><li>Teruel, M. N., Blanpied, T. A., Shen, K., Augustine, G. J., Meyer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+microporation+technique+for+the+transfection+of+cultured+CNS+neurons.">A versatile microporation technique for the transfection of cultured CNS neurons.</a> Journal of Neuroscience Methods. 93 (1), 37-48 (1999).</li><li>Rathenberg, J., Nevian, T., Witzemann, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transfection+of+individual+neurons+using+modified+electrophysiology+techniques.">High-efficiency transfection of individual neurons using modified electrophysiology techniques.</a> Journal of Neuroscience Methods. 126 (1), 91-98 (2003).</li><li>Tanaka, M., Yanagawa, Y., Hirashima, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+small+interfering+RNA+by+single-cell+electroporation+in+cerebellar+cell+cultures.">Transfer of small interfering RNA by single-cell electroporation in cerebellar cell cultures.</a> Journal of Neuroscience Methods. 178 (1), 80-86 (2009).</li><li>Wiegert, J. S., Gee, C. E., Oertner, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+Electroporation+of+Neurons.">Single-Cell Electroporation of Neurons.</a> Cold Spring Harbor Protocols. 2017 (2), 094904 (2017).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robot+for+high+yield+electrophysiology+and+morphology+of+single+neurons+in+vivo.">A robot for high yield electrophysiology and morphology of single neurons in vivo.</a> Nature Communication. 8, 15604 (2017).</li><li>Steinmeyer, J. D., Yanik, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+single-cell+manipulation+in+brain+tissue.">High-throughput single-cell manipulation in brain tissue.</a> PLoS One. 7 (4), 35603 (2012).</li><li>Keener, D. G., Cheung, A., Futai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+efficient+method+for+single-cell+electroporation+in+mouse+organotypic+hippocampal+slice+culture.">A highly efficient method for single-cell electroporation in mouse organotypic hippocampal slice culture.</a> Journal of Neuroscience Methods. 337, 108632 (2020).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> Journal of Neuroscience Methods. 37 (2), 173-182 (1991).</li><li>Ibata, K., Sun, Q., Turrigiano, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+synaptic+scaling+induced+by+changes+in+postsynaptic+firing.">Rapid synaptic scaling induced by changes in postsynaptic firing.</a> Neuron. 57 (6), 819-826 (2008).</li><li>Li, 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=Electroporation+on+microchips:+the+harmful+effects+of+pH+changes+and+scaling+down.">Electroporation on microchips: the harmful effects of pH changes and scaling down.</a> Science Reports. 5, 17817 (2015).</li><li>Karra, D., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+techniques+for+neuronal+cells.">Transfection techniques for neuronal cells.</a> Journal of Neuroscience. 30 (18), 6171-6177 (2010).</li><li>Taverna, E., Haffner, C., Pepperkok, R., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+manipulate+the+fate+of+single+neural+stem+cells+in+tissue.">A new approach to manipulate the fate of single neural stem cells in tissue.</a> Nature Neurosciences. 15 (2), 329-337 (2011).</li><li>Zhang, Y., Yu, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+microinjection+technology+in+cell+biology.">Single-cell microinjection technology in cell biology.</a> Bioessays. 30 (6), 606-610 (2008).</li><li>De Simoni, A., Griesinger, C. B., Edwards, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+rat+CA1+neurones+in+acute+versus+organotypic+slices:+role+of+experience+in+synaptic+morphology+and+activity.">Development of rat CA1 neurones in acute versus organotypic slices: role of experience in synaptic morphology and activity.</a> Journal of Physiology. 550, 135-147 (2003).</li></ol>

作者宣称他们没有利益冲突。

我们在这里描述一种电击方法，它以高效率和高精度同时转导兴奋神经元和抑制神经元。我们优化的电化方案有三个创新突破，以实现高效的基因转染。我们的第一个修改是增加移液器的大小相比，以前公布的协议3，5，6。这种变化使我们能够电化许多神经元，而无需移液器堵塞。此外，与以前的方法相比，低电阻移液器允许...

在分子生物学中，研究者最重要的考虑因素之一是如何将感兴趣的基因传递到细胞或细胞群中，以阐明其功能。不同的分娩方法可分为生物（如病毒载体）、化学（如磷酸钙或脂质）或物理（如电浸、微注射或生物毒性）1、2。生物方法效率高，可以是细胞类型特异性，但受特定遗传工具的发展的限制。化学方法在体外非常强大，但变染通常是随机的:此外，这些方法大多只保留给原细胞。在物理方法中，从技术角度来看，生物处理是最简单和最简单的，但再次以相对较低的效率产生随机转染结果。对于需要转移到特定细胞而不需要开发遗传工具的应用，我们期待单细胞电聚变3，4。
过去二十年来，电聚变只指场电电，而已开发出多个体外和体内单细胞电电化方案，以提高5、6、7的特异性和效率，证明电电化可用于将基因转移到单个细胞，因此，可以非常精确。然而，这些程序在技术上要求高、耗时、效率低下。事实上，最近的论文已经调查了机械化电镀钻机8，9的可行性，这可以帮助消除其中几个障碍，调查人员有兴趣安装这样的机器人。但对于那些寻找更简单的方法的人来说，电透电的问题，即细胞死亡、转染失败和移液器堵塞，仍然是一个问题。
我们最近开发出一种电透法，它使用大倾玻璃移液器、较温和的电脉冲参数和独特的压力循环步骤，在兴奋神经元中产生的转染效率比之前的方法高得多，并使我们能够首次在抑制性内窥镜中转染基因，包括小鼠组织性切片培养10海马CA1区域的索马托他汀表达抑制内周素。然而，这种电位法在不同抑制性内膜类型和神经元发育阶段的可靠性尚未得到解决。在这里，我们证明了这种电位技术能够将基因转化为兴奋神经元和不同类别的内窥神经。重要的是，无论在体外（DIV）切片培养年龄测试天数，转染效率都很高。这种既定的、用户友好的技术强烈推荐给任何有兴趣在体外小鼠脑组织环境中使用不同细胞类型的单细胞电聚变的调查员。

<table><tbody><tr><td>&lt;strong&gt;Plasmid preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plasmid Purification Kit</td><td>Qiagen</td><td>12362</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Organotypic slice culture preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>6 Well Plates</td><td>GREINER BIO-ONE</td><td>657160</td><td></td></tr><tr><td>Dumont #5/45 Forceps</td><td>FST</td><td>#5/45</td><td>Angled dissection forceps for organotypic slice culture preparation</td></tr><tr><td>Flask Filter Unit</td><td>Millipore</td><td>SCHVU02RE</td><td>Filtration and storage of culture media</td></tr><tr><td>Incubator</td><td>Binder</td><td>BD C150-UL</td><td></td></tr><tr><td>McIlwain Tissue Chopper</td><td>TED PELLA, INC.</td><td>10180</td><td>Tissue chopper for organotypic slice culture preparation</td></tr><tr><td>Millicell Cell Culture Insert, 30 mm</td><td>Millipore</td><td>PIHP03050</td><td>Organotypic slice culture inserts</td></tr><tr><td>Osmometer</td><td>Precision Systems</td><td>OSMETTE II</td><td></td></tr><tr><td>PTFE coated spatulas</td><td>Cole-Parmer</td><td>SK-06369-11</td><td></td></tr><tr><td>Scissors</td><td>FST</td><td>14958-09</td><td></td></tr><tr><td>Stereo Microscope</td><td>Olympus</td><td>SZ61</td><td></td></tr><tr><td>Sterile Vacuum Filtration System</td><td>Millipore</td><td>SCGPT01RE</td><td>Filtration and storage of aCSF</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Electrode preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Capillary Glasses</td><td>Warner Instruments</td><td>640796</td><td></td></tr><tr><td>Micropipetter Puller</td><td>Sutter Instrument</td><td>P-1000</td><td>Puller</td></tr><tr><td>Oven</td><td>Binder</td><td>BD (E2)</td><td></td></tr><tr><td>Puller Filament</td><td>Sutter Instrument</td><td>FB330B</td><td>Puller</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Single-cell electroporation and fluorescence imaging #1&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3.5 mm Falcon Petri Dishes</td><td>BD Falcon</td><td>353001</td><td></td></tr><tr><td>Airtable</td><td>TMC</td><td>63-7512E</td><td></td></tr><tr><td>CCD camera</td><td>Q Imaging</td><td>Retiga-2000DC</td><td>Camera</td></tr><tr><td>Electroporation System</td><td>Molecular Devices</td><td>Axoporator 800A</td><td>Electroporator</td></tr><tr><td>Fluorescence Illumination System</td><td>Prior</td><td>Lumen 200</td><td></td></tr><tr><td>Manipulator</td><td>Sutter Instrument</td><td>MPC-385</td><td>Manipulator</td></tr><tr><td>Metamorph software</td><td>Molecular Devices</td><td></td><td>Image acquisition</td></tr><tr><td>Peristaltic Pump</td><td>Rainin</td><td>Dynamax, RP-2</td><td>Perfusion pump</td></tr><tr><td>Shifting Table</td><td>Luigs &amp;amp; Neuman</td><td>240 XY</td><td></td></tr><tr><td>Speaker</td><td>Unknown</td><td></td><td>Speakers connected to the electroporator</td></tr><tr><td>Stereo Microscope</td><td>Olympus</td><td>SZ30</td><td></td></tr><tr><td>Table Top Incubator</td><td>Thermo Scientific</td><td>MIDI 40</td><td></td></tr><tr><td>Upright Microscope</td><td>Olympus</td><td>BX61WI</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Fluorescence imaging #2&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>All-in-One Fluorescence Microscope</td><td>Keyence</td><td>BZ-X710</td><td></td></tr></tbody></table>

single-cell electroporation across different organotypic slice culture of mouse hippocampal excitatory and class-specific inhibitory neurons

电电已成为将特定基因转移到细胞中以了解其功能的关键方法。在这里，我们描述了一种单细胞电聚技术，它最大限度地提高了小鼠组织型海马切片培养中兴奋和类特异性抑制神经元的体外基因转染效率（+80%）。我们使用大玻璃电极、含四毒素的人工脑脊液和轻度电脉冲，将感兴趣的基因传递到培养的海马CA1金字塔神经元和抑制内窥镜中。此外，电聚可以在培养海马切片中进行长达21天的体外，而不会降低转染效率，从而可以研究不同的切片培养发育阶段。随着对检查不同细胞类型基因的分子功能的兴趣日益浓厚，我们的方法演示了一种可靠而直接的方法，可以在小鼠脑组织中进行体外基因转换，该方法可以使用现有的电生理学设备和技术进行。

我们的单细胞电位能够精确地将基因输送到视觉识别的兴奋和抑制神经元中。我们在三个不同的时间点对三种不同的神经元细胞类型进行电击。帕瓦尔布明 （Pv） 或车辆谷氨酸类型 3 （VGT3） 表达神经元通过交叉 Pvcre （JAX #008069） 或 VGT3cre （JAX #018147） 线与 TdTomato （红色荧光蛋白的变体） 记者线 （Jax #007905）， 分别命名为 Pv/TdTomato 和 VGT3/TdTomato 线。从C57BL/6J、Pv/TdTomato 和 VGT3/Td...

Watch this Scientific Journal Video about Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons at JoVE.com

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

这里介绍的是一个单细胞电位化协议，可以在一系列体外海马切片培养年龄内提供兴奋和抑制神经元的基因。我们的方法提供精确和高效的基因表达在单个细胞，可用于检查细胞自主和细胞间功能。

小鼠河马兴奋和类特异性抑制神经元的不同组织型切片培养的单细胞电位

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...

single-cell-electroporation-across-different-organotypic-slice

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

3.6K 次观看.  University of Massachusetts Medical School. 这里介绍的是一个单细胞电位化协议，可以在一系列体外海马切片培养年龄内提供兴奋和抑制神经元的基因。我们的方法提供精确和高效的基因表达在单个细胞，可用于检查细胞自主和细胞间功能。

视频: Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

器官切片文化为产后神经再生的研究

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

科研

发育生物学

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

小鼠海马组织的前子宫内电和器官切片文化

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

神经科学

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

从大脑皮层的器官型切片制备全细胞记录

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

神经元形态的研究方法：<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em&gt;在Utero</em&gt;电穿孔

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Photostimulation and Whole-Cell Patch Clamp Recordings of Neurons in Mouse Hippocampal Slices

Source: Richevaux, L., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59534">In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices.</a> J. Vis. Exp. (2019)
The video demonstrates a protocol to study synaptic interactions and neuronal responses in a transfected mouse hippocampal brain slice. It involves illuminating long-range sensory neurons to trigger action potentials and recording the resulting excitatory post-synaptic potentials in the recorded neuron using the whole-cell patch-clamp technique.

小鼠海马切片中神经元的光刺激和全细胞膜片钳记录

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Solutions for acute slice ...

小鼠河马兴奋和类特异性抑制神经元的不同组织型切片培养的单细胞电位

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