作者感谢以前的实验室成员谁促成了这个协议的细化和国立卫生研究院（DA19808，DA15014到OM）多年来的支持。安娜阿布特1"在neuroAIDS跨学科和转化研究训练"（T32 - MH078795）研究员;因此，这项工作是支持部分露丝属Kirschstein国家研究服务奖5T32MH079785下由美国国立卫生研究院。

1。神经胶质细胞夹层（1〜2周电镀前神经元） <ol><li>要准备无菌工具清扫的地方，70％的乙醇，添加4毫升冷夹层中等至60毫米的菜（每一个脑盘），和2.5％胰蛋白酶和DNase冰解冻（见第六表的手术工具的详细信息）。 </li><li>使用大剪刀杀头100毫米的无菌培养皿中的2-4日龄仔兔和地方首长。 </li><li>使用介质剪刀通过皮肤中线切割和拉背部皮肤暴露的头骨。使用弯曲的剪刀中线切割和通过头骨的两个侧削减，使不损害脑组织下方。拆下上部的头骨，大脑暴露。 </li><li>提取的大脑，并放置在自己的60毫米，含4毫升冷夹层介质的菜（我看到的成分表）。将盘放在立体显微镜下（2000年徕卡变焦，变焦控制:7-30），并使用5号钳的半球分开，取出脑，剥离ØFF脑膜。 </li><li>收集在一个新的60毫米冷夹层介质菜的所有解剖的脑皮层，并直言不讳地用弯剪刀组织。 </li><li>肉末组织转移到无菌的15 mL试管。把4.5毫升的冷夹层介质的体积，并加入500微升2.5％胰蛋白酶。包装在封口膜管和漂浮在37℃水浴15分钟，每5分钟颠倒混合。 （注:在这里，我们描述了协议，我们一般使用最多5动脑筋时，如果使用更多的幼仔，卷可能需要进行适当调整） </li><li>组织转移到一个新的15毫升管，最大限度地减少含有胰蛋白酶清扫结转中等量。到4.8mL带来新鲜夹层介质的体积和添加200μL的DNA酶终浓度达到60微克/毫升（原液:3毫克的DNase和硫酸镁5毫克2毫升夹层介质）。 5毫升无菌吸管轻轻磨碎〜20倍TE神经胶质电镀液，再加入10毫升（成分表二）。 </li><li>允许组织的大块定居为4-5分钟，然后80％的细胞悬液转移至50ml管。重复一个额外的神经胶质电镀液2-3毫升（并使用一个较小的吸管如果需要的话）的组织trituration;再次，让组织来解决，并添加80％到以前的集合。神经胶质细胞电镀液和离心15分钟在280总量带来的20毫升克（或在IEC CENTRA CL2的离心机1300 RPM）。 </li><li>吸上清，重悬细胞沉淀在神经胶质细胞电镀液（大约用5-7毫升，取决于使用的幼仔数量），然后再添加额外的电镀液，以每个解剖的大脑有10毫升。搅拌均匀，板，每75厘米的 2瓶（即每一个烧瓶脑）10 ml的细胞悬液。 </li><li>将培养瓶中孵化器设置在37 ° C（5至10％的二氧化碳;比例的钠bicarbonaTE内容的介质）。更改后24-48小时，然后每隔3-4天的培养基。 </li></ol> 2。中学胶质电镀前神经元（48小时） <ol><li>准备加入3 paraplast蜡融化的小滴周围的每个清洗和灭菌thermanox圆周thermanox盖玻片（如神经元将被镀上的菜肴）。细胞将被镀上这一侧thermanox。我们thermanox是手工制作的可重用〜60毫米刀片切钻通〜50毫米的孔（见表六试剂）从塑料，虽然市售的文化以及插入可能被用来代替。 </li><li>洗净，主要的神经胶质细胞，PBS洗涤2次，大力摇晃每个烧瓶，以消除任何独立的细胞。每个烧瓶与融合的主要神经胶质细胞，将提供约10万星形胶质细胞，因此，我们经常使用2-4实验烧瓶。 </li><li>细胞分离tryspin - EDTA（1 mL10X 0.5％胰蛋白酶- EDTA原液9毫升的PBS稀释）为0.05％。 Combi机NE细胞从2烧瓶，并添加到一个50 mL锥形管等量的神经胶质细胞电镀液（见试剂表二）。离心15分钟，在280克吸弃上清液，并溶解在几毫升的神经胶质细胞电镀液的神经胶质沉淀。 </li><li>计数细胞，使用hematocytometer和稀释细胞悬液（〜1 × 10 6细胞/ mL）;板60毫米菜（如神经元将被镀上盖玻片）， 或 thermanox盖玻片（细胞，如果神经元将镀上60毫米的菜肴）。细胞应该是镀在使用神经胶质细胞电镀液，每盘或thermanox 50细胞。 thermanox盖玻片，板600μL细胞在滴加的方式，然后2小时后翻转thermanox淹没细胞和神经胶质细胞电镀液的情况下。 </li><li>如果准备次要的神经胶质细胞神经​​元盖玻片60 mm培养皿，改变介质N2.1晚上前神经剥离。 </li></ol> 3。神经解剖与文化<ol><li>准备每个无菌盖玻片的周长增加约3 paraplast蜡融化的小滴，以物理上独立的细胞层，防止细胞刮盖玻片（直径15毫米）。细胞将被镀上盖玻片的这一边。 </li><li>涂层板或盖玻片与0.5 mg / mL的聚赖氨酸（无论是聚- L -赖氨酸或D -聚赖氨酸的硼酸盐缓冲液溶解;见表六），2小时或过夜。用无菌水冲洗2-3次，并允许完全干燥。注:盖玻片放置在疏水培养皿，以避免溢出/传播与多聚赖氨酸或细胞电镀，可以很容易地发生在组织培养皿中的盖玻片在涂层过程中的解决方案。 </li><li>准备为神经剥离的神经胶质细胞夹层（步骤1.1）。安乐死一个为期17天的孕鼠，喷用70％乙醇的皮毛，并穿过内侧的皮肤暴露子宫。删除所有的胎儿，并将其放置在无菌100毫米菜。 </li><li>迅速解剖自由和杀头每个胎儿，用冷夹层介质（4毫升）每头放置在自己的60毫米菜。我们正常解剖实验，每4-5个胎儿，约20万个神经元，从每个E17胎儿派生。如果需要更多的胚胎，确保整个过程（从3.4至3.12步）不超过90-100分钟。 </li><li>使用立体显微镜，No.1/No.5镊子隔离拉动皮肤和颅骨开放，提取大脑半球分离，去除脑和脑膜脑皮层。 </li><li>收集在一个新的60毫米冷夹层介质菜的所有解剖皮层，并直言不讳地用弯剪刀组织成〜1毫米件。 </li><li>肉末组织转移到无菌的15 mL试管。把4.5毫升的冷夹层介质的体积，并加入500微升2.5％胰蛋白酶。包装在封口膜管和漂浮在36℃水浴15阿米努工商业污水附加费，颠倒混合，每5分钟。 </li><li>组织转移到一个新的15毫升管，最大限度地减少含有胰蛋白酶清扫结转中等量。置于4.8毫升新鲜夹层介质，添加200μL的DNA酶达到终浓度为60微克/毫升。磨碎，用无菌的5或2毫升吸管大约10倍;重复使用一个火抛光（冷）的巴斯德吸管游离于组织的这一必要步骤。然后让组织的剩余件（一般很少，如果有的话）解决4-5分钟。 </li><li>拆下上部的单细胞悬液，同时落户件组织背后的离开，并将其放置在一个新的管的神经元电镀液等体积（见表三）解决方案。而是轻轻拌匀，然后在细胞计数板15毫米的盖玻片（35000细胞200μL）或60（1 × 10 6细胞每3毫升）mm培养皿需要。转移细胞培养箱（36.5℃，5-10％CO2）。 </li><li>经过3-4小时的COMBINE的神经元层和继发胶质细胞如下。 60 mm培养皿中神经元，用温暖的DMEM细胞，改变介质的无血清培养液4ml（氮气，见表四），并添加一个神经胶质细胞thermanox每块板（神经胶质细胞，应面临的神经元）。对于神经细胞的盖玻片，转让3至5盖玻片到每个盘60毫米二级神经胶质细胞，神经元朝下。 </li><li>电镀后24小时内的神经元，每道菜添加胞嘧啶-β- D - arabinofuranoside 10微米，为了防止神经胶质细胞的增殖。我们准备4毫米库存解决方案，用氮气中等稀释的1:10，并添加25 UL / mL的稀释液，每道菜。 </li><li>一般用于细胞实验后7-10天在文化，虽然确切的时间取决于所需的分化阶段，他们已经成长为没有生存的显著减少至4个星期。第一个星期后，这是建议媒体的二分之一量，每周更换一次。 </li></ol>几个小时后的神经元连接到基板，他们开始扩展lamelliopodia。进程继续拉长（图2A），过几天在文化和两极化，并延伸的轴突和树突清晰可见（图2B; MAP2染色）。由于神经细胞成熟树突状乔木变得更加详尽和发展突触联系;电镀许多树突棘后约2-3周可见（图2C）11。 <img alt="图1" src="/files/ftp_upload/3257/3257fig1.jpg" /> 图1。培养大鼠皮层神经元与胶质细胞的饲养层（二级神经胶质细胞）的程序流程图。首先，11天前神经清扫，一个主要的神经胶质细胞文化是准备从新生大鼠（P2 - 4）。当细胞融合（大约9天前神经剥离的主要神经胶质细胞和两天后电镀），星形胶质细胞是机甲anically少突胶质前体细胞和小胶质细胞和菜肴或thermanox镀要么（二级神经胶质细胞）分离。从E17的胚胎和菜（涂多聚赖氨酸）盖玻片或镀的皮层神经元，然后获得。电镀后4小时，神经细胞层被添加到次要的神经胶质细胞。 <img alt="图2" src="/files/ftp_upload/3257/3257fig2.jpg" /> 图2在不同阶段的文化的例子（如5小时，12格，21格）。数字盖玻片上的皮层神经元在不同时间点的代表图像。后神经细胞附着于基材的神经元开始延长lamelliopodia。相衬图像显示了一些电镀后5小时延长突起（一）。 （B，从库克等人，2010年，经许可）在中间的图像显示12格的固定和皮层神经元与神经元的树突状标记MAP2免疫染色。 Hoechst染色被用来作为一个counterstai宁。由于神经细胞成熟的树突棘，也成为可见（C）。标尺:A，B = 25微米，C = 10微米。

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没有利益冲突的声明。

该协议提供了一个方法培养大鼠原代皮质神经元的神经胶质细胞的存在，同时使神经元容易被孤立的实验分析。一个健康的神经细胞表型的神经胶质细胞支持发展，同时也调节神经元的反应，在生理相关的方式的试验性治疗。此外，传代之前，培养这种细胞群成为在星形胶质细胞的选择性丰富与神经元的主要神经胶质细胞，从而阻止炎症的小胶质细胞的刺激。然而，对于某些类型的实验，可能所?...

<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>16 mM</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>22 mM</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>0.22 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.4</td>
		</tr>
		<tr>
			<td>Osmolarity</td>
			<td>310&plusmn;10 mOsm</td>
		</tr>
	</tbody>
</table>
Table 1. Dissection Medium.
<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>90%</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>50 &mu;g/mL</td>
		</tr>
	</tbody>
</table>
Tabe 2. Glia Plating Medium.
<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>90%</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>10%</td>
		</tr>
	</tbody>
</table>
Table 3. Neuron Plating Medium.
<table>
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>1M HEPES</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Ovalbumin</td>
			<td>50 mg/100mL</td>
		</tr>
	</tbody>
</table>
Table 4. N2 Medium.
<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>Sodium Borate</td>
			<td>12.5 mM</td>
		</tr>
	</tbody>
</table>
Table 5. Borate Buffer (for poly-lysine).
<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Company</th>
			<th>
				Catalogue number</th>
		</tr>
		<tr>
			<td>High glucose Dulbecco's Modified Eagle 
			Medium (DMEM)</td>
			<td>Invitrogen</td>
			<td>11995-073</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), Heat-inactivated</td>
			<td>Hyclone</td>
			<td>26400-044</td>
		</tr>
		<tr>
			<td>Horse Serum, Heat-inactivated</td>
			<td>Hyclone</td>
			<td>H1138</td>
		</tr>
		<tr>
			<td>Gentamicin (50mg/mL)</td>
			<td>Invitrogen</td>
			<td>15750-060</td>
		</tr>
		<tr>
			<td>N2 Supplement (100x)</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
		</tr>
		<tr>
			<td>HEPES buffer solution</td>
			<td>Invitrogen</td>
			<td>15630-080</td>
		</tr>
		<tr>
			<td>Albumin from chicken egg white, Grade VI 
			(Ovalbumin)</td>
			<td>Sigma-Aldrich</td>
			<td>A2512</td>
		</tr>
		<tr>
			<td>2.5% Trypsin</td>
			<td>Invitrogen</td>
			<td>15090-046</td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA (10X)</td>
			<td>Invitrogen</td>
			<td>15400-054</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas 
			(DNase)</td>
			<td>Sigma-Aldrich</td>
			<td>D-5025</td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Fisher</td>
			<td>12-646-106</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P1274</td>
		</tr>
		<tr>
			<td>Cytosine-&beta;-D-arabinofuranoside hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>C6645</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>Leica ZOOM 2000</td>
		</tr>
		<tr>
			<td>Cover glasses, Circles, 15 mm, Thickness 
			0.13-0.17 mm</td>
			<td>Carolina</td>
			<td>633031</td>
		</tr>
		<tr>
			<td>Thermanox sheets</td>
			<td>Grace BioLabs</td>
			<td>HS4550</td>
		</tr>
		<tr>
			<td>Large forceps</td>
			<td>Biomedical 
			Research 
			Instruments</td>
			<td>70-4000</td>
		</tr>
		<tr>
			<td>Fine-tipped No.5 forceps</td>
			<td>Fine Science 
			Tools</td>
			<td>91150-20</td>
		</tr>
		<tr>
			<td>Pattern No.1 forceps</td>
			<td>Biomedical 
			Research</td>
			<td>10-1400</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Instruments</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scissors, straight, sharp-blunt</td>
			<td>Biomedical 
			Research 
			Instruments</td>
			<td>28-1435</td>
		</tr>
		<tr>
			<td>Micro Dissecting scissors</td>
			<td>Biomedical 
			Research 
			Instruments</td>
			<td>11-2070</td>
		</tr>
		<tr>
			<td>Micro Dissecting Curved scissors</td>
			<td>Biomedical 
			Research 
			Instruments</td>
			<td>11-1395</td>
		</tr>
	</tbody>
</table>

bilaminar co-culture of primary rat cortical neurons and glia

此影片将引导您通过培养大鼠皮层神经元胶质滋养层，称为一个二层或联合培养模式系统的存在的过程。该系统适用于各种需要的玻璃或塑料增长基板的实验需要，也可以用于其他类型的神经元的文化。 从后期的胚胎阶段（E17）中获得的大鼠皮层神经元均镀上盖玻片或组织培养皿中的神经胶质细胞饲养层菜肴或塑料盖玻片 （作为Thermanox），分别增长面临。具体的实验使用的技术，这可能需要，或不取决于两种配置的选择，即神经元生长在玻璃上（如钙成像与免疫印迹）。胶质细胞饲养层，混合胶质细胞星形胶质细胞，丰富了中学文化，分别是准备前的神经元新生幼鼠皮层（P2 - 4）清扫。 这个文化系统的一个主要优势相比，神经元的文化不仅是神经细胞生长，生存，从胶质滋养层，更准确地类似于大脑体内环境分泌的营养因子提供差异化的支持。此外，共培养可用于研究神经胶质细胞相互作用1。 与此同时，神经胶质细胞在神经元层的污染，是防止不同的手段，几乎是纯的神经元层（低密度文化，除了有丝分裂抑制剂，缺乏血清和使用优化培养基），可比其他方法1 -3。胶质层神经元可以很容易地分离过程中随时文化和不同实验应用范围从5，生物化学，细胞和分子生物学5-8电4，成像和麦克风使用roscopy 4,6,7,9,10。主要的神经元的延长轴突和树突，形成功能性突触的11，这是不是在神经细胞株的观察过程中，虽然一些细胞系扩展过程。 使用这种合作培养体系，培养大鼠海马神经元的一个详细的协议已被描述以前 4,12,13 。在这里，我们详细的修改协议，适合皮层神经元。从每只大鼠胚胎回收约20 × 10 6细胞，这种方法特别适用于需要大量的神经元（但不是一个高度同质的神经元群）的实验是有用的。编制的神经元和神经胶质细胞需要在一个具体时间的方式的计划。我们将提供一步一步培养大鼠皮层神经元的协议，以及培养的神经胶质细胞支持神经元。

Watch this Scientific Journal Video about Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia at JoVE.com

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

在这里，我们培养大鼠皮层神经元胶质滋养层的存在提供了一个协议。培养的神经元建立极性和创建的突触，可以用于各种应用，如电生理，钙成像，细胞存活率检测，免疫细胞化学，和RNA / DNA /蛋白分离，神经胶质细胞的分离。

二层共培养小学的大鼠皮层神经元和神经胶质

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

bilaminar-co-culture-of-primary-rat-cortical-neurons-and-glia

Research

JoVE Journal

Neuroscience

19.5K 次观看.  Drexel University College of Medicine. 在这里，我们培养大鼠皮层神经元胶质滋养层的存在提供了一个协议。培养的神经元建立极性和创建的突触，可以用于各种应用，如电生理，钙成像，细胞存活率检测，免疫细胞化学，和RNA / DNA /蛋白分离，神经胶质细胞的分离。

视频: Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

科研

神经科学

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 ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

活大鼠皮层神经元线粒体膜电位和活性氧的测定

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

神经元对神经胶质细胞在微流控文化互动平台（MCP）的神经元轴突和神经胶质细胞共培养系统的影像学分析

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

成像大鼠原代海马神经元树突棘使用结构照明显微镜

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

大鼠皮层神经元丝状肌动蛋白（F-肌动蛋白）泪点的量化

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

从E14-E16斯普拉格·道利大鼠胚胎分离的混合原发希马和皮质神经元生成神经球

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

人类干细胞衍生内皮细胞和GABAergic神经元的迁移、化学吸引和共培养测定

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

来自成人大鼠尿膀胱的原发神经元和胶质的隔离与培养

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

与嗅觉恩希廷格利亚合体培养的 Axotom 化大鼠视网膜甘利翁神经元，作为成人轴再生体外模型

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...