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>

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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>Plasmid preparation</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>Organotypic slice culture preparation</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>Electrode preparation</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>Single-cell electroporation and fluorescence imaging #1</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 &#38; 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>Fluorescence imaging #2</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

Research

JoVE Journal

Neuroscience

3.5K 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

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

海马脑片的器官型检查神经元损伤的文化模式

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

科研

神经科学

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

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 ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

从产前小鼠海马神经元的分离和培养

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

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 ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

神经血管重塑的分析Entorhino，海马脑切片培养

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Methods Article

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