The present work was supported by the German research foundation (Deutsche Forschungsgemeinschaft DFG: GRK 736, Fa 159/22-1; the research school of the Ruhr University Bochum (GSC98/1) and the priority program SSP 1172 "Glia and Synapse", Fa 159/11-1,2,3).

小鼠实验是按照德国法律和德国社会为畜牧业的神经科学的指导方针。在波鸿鲁尔大学的动物保护与利用委员会授予相应的许可证。 1.准备和皮层星形胶质细胞的培养注:进行下一个步骤，有准备的神经元前的星形胶质细胞的文化应该发展成融合单层之前完成这些步骤，该协议至少7天的。原发性星形胶质细胞从各地日龄（P），0-3的乳鼠获得的混合胶质细胞培养的。每T75烧瓶三大脑（6皮层）都可以使用。 <ol><li>通过用无菌条件下10％体积/体积马血清和0.1％体积/体积庆大霉素补充的Dulbecco改良的Eagle培养基（DMEM）制备星形细胞介质。储存在4℃的培养基中达2周。 </li><li>大衣3倍T75瓶无线第10微克/毫升聚-D-赖氨酸（PDL;在细胞培养级水中）在37℃下在6％的体积/体积的 CO 2的存在下至少1小时。涂布后，用磷酸盐缓冲盐水（PBS）洗烧瓶两次以除去未结合的阳离子，并添加9毫升星形细胞培养基。 </li><li>对于大脑的制备中，加入10毫升Hank氏平衡盐溶液（HBSS）为1x 10cm培养皿和1mL的HBSS至1x 15毫升管（无论将要使用的大脑的数量）。保持一双手术剪，2细镊子，并在过程中达到双目。 </li><li>快速斩首3幼仔的手术剪和收集元首在一个空的10cm平皿。 <ol><li>执行双目下的皮质的准备。使用两个细镊子，在中线删除从头骨的背部皮肤，从颈部，迈向眼睛的水平开始。此后，其血管，大脑颅骨下可见。 </li><li>在一双镊子的喙端固定头切开颅骨的中线，开始在后端。接着，夹断以暴露脑的右和头骨的左侧半部。 </li><li>关闭钳子的前端和脑下它腹侧的位置。解除脑出颅骨，并转移到HBSS填充10cm培养皿。与剩余的标本继续以同样的方式，直到所有的大脑中的菜收集。 </li></ol></li><li>收集大脑后，开始与皮质通过夹断使用一对像一把剪刀镊子的后脑的分离。一个钳子执行两半球之间的正中切口。仔细固定用钳子脑和切断脑和使用第二镊子嗅球与皮质半部到结束。 </li><li>修复一个半皮质与一个钳子和拆卸他们˚F剥离从半球脑膜ROM中的横向边缘，并使用第2钳子从皮质表面随后拉动它们。 </li><li>翻转皮质一半在其表面面向下来，对盘中;仔细剖析并删除月牙形海马使用一个钳子。转移单皮质成使用一个封闭的镊子进行个别皮质锥形15毫升管。 </li><li>收集所有皮质，过渡到无菌层流罩，并与筹备皮层组织的解离后开始。 </li><li>添加1毫升的DMEM到一个2毫升的反应管中，加入0.1％w / v的木瓜蛋白酶（30单位）。孵育在水浴悬浮液，在37℃直至澄清溶液开发（约3分钟）。添加0.24毫克/毫升的L-半胱氨酸和20微克/毫升DNA酶，轻轻摇晃。 注:对于消化，需要约1毫升酶液。 </li><li>过滤器（0.22微米孔径）将之前获得1毫升无菌溶液它的皮层组织。温育30分钟的管- 1小时，在37℃（ 即在水浴中）。 注意:步骤1.11 - 1.14是关键的，应在15分钟内完成。 </li><li>终止消化反应，加1毫升星形胶质细胞中，并用1毫升吸管仔细磨碎消化的组织。进行研制，轻轻移动吸移管柱塞，从而吸移和挤出皮层组织悬浮液中。 </li><li>一旦组织已被解离成单细胞悬浮液中，加入5毫升星形细胞介质和离心机在216×g下5分钟。 </li><li>从所得的沉淀小心吸出上清液并重新悬浮于1mL星形胶质细胞培养基中的细胞。 </li><li>细胞悬浮液加入到9毫升星形胶质细胞培养基（见1.2）补充T75瓶。 </li><li>孵育所述细胞在37℃下在6％的体积/体积的 CO 2的存在下孵化。 4 d后，执行第一次完全培养基变化（10毫升）中。此后，改变培养基，每2 - 3天。 </li><li> 7天后，检查细胞形成融合单层。如果没有，再等2 - 3天的文化完全融合直至达到。 注:星形胶质细胞显示大扁平细胞体，定位在烧瓶底部，不开发显着的细胞过程。他们从小相衬亮祖细胞和小胶质驻留在单层17的顶部有所不同。 </li><li>为了摆脱祖细胞，并获得纯星形胶质细胞文化，将T75瓶上轨道摇床，动摇文化O / N在250转。确保温度调节至37℃，并且将烧瓶中的过滤器是用实验室膜密封，以防止二氧化碳的蒸发。 </li><li>第二天，吸出培养基，并添加有20μM的胞嘧啶-1-β-D-阿拉伯呋喃糖苷（阿糖胞苷）补充10mL的新鲜培养基来eliminaTE残留分裂的细胞。 注:孵育阿糖胞苷2 -在37℃下在孵化3天，6％的CO 2，将导致一个纯星形胶质细胞培养。 </li><li>监视星形细胞培养的通过视觉检查在光学显微镜下的纯度。如果残留相衬亮祖细胞和小胶质细胞仍驻留在星形细胞单层17的顶部（见步骤1.16），重复步骤1.18和1.19。 注:汇合和纯星形胶质细胞单层可在继续下一步骤之前被保持在孵化器达14天（37℃，6％体积/体积的 CO 2）。一半的培养基改变每3天。不收集，冻结和解冻的星形胶质细胞，因为这些细胞会通过存储过程失去神经的支持性能。 </li></ol> 2.准备星形胶质细胞的间接共培养注:48 - 72小时准备神经元，星形胶质细胞转移到CEL前L-培养插入。通常，一个单一的T75烧瓶提供了足够数量的细胞，以支持与一种制备得到的神经元培养物。 <ol><li>根据需要于24孔板将尽可能多的插入。外套每个插入用10微克/毫升的PDL（溶于细胞培养级水中）下进行约1小时，在37℃，6％体积/体积的 CO 2。培养后，用PBS洗插入两次。在此期间，继续星形胶质细胞的准备。 注:填充有PBS中插入可以保持，直到星形胶质细胞悬浮液是准备使用。 </li><li>吸星形细胞培养基（与阿糖胞苷）中，用10毫升PBS洗涤培养一次，以确保任何剩余的血清被去除。 </li><li>加入3毫升0.05％胰蛋白酶-EDTA（乙二胺四乙酸）到烧瓶孵育胰蛋白酶将细胞在37℃，6％体积/体积的 CO 2约10分钟。 </li><li>控制通过目测用显微镜和facilita的细胞脱离通过敲击针对桌面烧瓶的侧TE上的细胞的分离。 注意:步骤2.5 - 2.8是关键的，并应15内完成 - 20分钟。 </li><li>继胰蛋白酶，轻轻重新悬浮在7mL的星形胶质细胞培养基中的细胞和细胞悬浮液转移到15毫升管中。离心机在216×g下5分钟。 </li><li>小心吸出上清液并重新悬浮于1mL星形胶质细胞培养基中的细胞沉淀。 </li><li>算使用计数室中的细胞。 </li><li>填补24孔板用500微升星形胶质细胞培养基的孔的底部和25,000个细胞在500μL的星形胶质细胞培养基转移到每个单独的插入物。孵育培养在37℃，6％体积/体积的 CO 2。 注:42后 - 72小时的培养将达到约100％汇合。在这一阶段，培养的纯度可以通过免疫细胞化学11进行验证。汇合培养物可维持到7天，直到继续到下一步。一半的培养基改变每3天。 </li></ol> 3.初级海马神经元的制备注:主小鼠海马神经元应从E15.5导出 - 定时孕鼠胚胎E16。 <ol><li>制备神经元培养基（MEM用10mM丙酮酸钠，0.1％w / v的卵清蛋白，2％体积/体积B27培养基补充剂，和0.1％体积/体积庆大霉素（无菌过滤）），并且制备培养基（HBSS用0.6％重量/ v葡萄糖和10mM的HEPES（4-（2-羟乙基）-1-哌嗪乙磺酸））。预先温暖和预平衡在过滤烧瓶神经元培养基中在37℃，6％体积/体积的 CO 2。 </li><li>放置玻璃盖玻片（12毫米直径）到24孔板的孔中（使用特殊的盖玻片，其缺口与细胞培养插入的使用）。 <ol><li>外套与15微克/毫升聚-DL-鸟氨酸在水（细胞培养级）在37℃下至少1小时。至少使用500微升每孔，以保证盖玻片完全覆盖。用PBS洗涤各孔两次。离开PBS中的孔，直到镀覆的细胞。确保孔不干燥。 </li></ol></li><li>解剖海马，准备3倍10厘米充满制备介质菜肴和1个2毫升管1毫升的准备中。准备好剪刀和2细镊子。 </li><li>通过颈脱位牺牲怀孕小鼠，消毒用70％V / V乙醇洗涤腹部，并用锋利的剪刀打开腹部。用剪刀仔细地切除子宫串珠和器官转移到1X 10cm皿充满了制备介质。 </li><li>接下来，请从子宫的胚胎。小心切开子宫，取出羊膜，而不使用2钳伤害胚胎。此后，斩首用剪刀的每个胚胎和磁头转移到与制备介质提供的第二10cm皿。 </li><li>与头的准备（类似prepar继续1.7） - 通货膨胀在1.4描述。 <ol><li>固定有一对镊子的头部和切割在颈部区域中的颅骨和皮肤靠近后脑，垂直于神经轴。使用第二对镊子解除两者头骨和覆盖皮肤和弯曲朝向头部的喙端软颅盖，从而露出大脑。 </li><li>为2钳子的帮助下，从颅腔分离大脑。要做到这一点，接近1钳子和脑启动在嗅球，并朝着移动后脑颅骨分离。收集再过10 cm培养皿充满制备介质的大脑。 </li></ol></li><li>如上对于星形细胞制备（步骤1.6）中描述从皮质部分解剖海马。从每个半球中取出海马，并收集他们在2毫升管中充满制备介质。 </li><li>在完成剥离之后，将管转移到无菌的层流工作台和diges吨，含木瓜蛋白酶1毫升消解液中的海马组织。如步骤1.9所述，准备消解液 - 1.10，但使用MEM代替DMEM。 注意:步骤3.9 - 3.12是关键的，应10内完成 - 15分钟。 </li><li>消化完成后，小心地撤回通过温和抽吸用移液器将消化溶液。 </li><li>依次添加，然后小心地吸每个洗涤周期1毫升新鲜培养基清洗海马神经元与介质3倍。不要离心细胞悬液。 </li><li>最后的洗涤步骤之后，请仔细磨碎在1毫升神经元中的组织。 </li><li>算使用计数室对细胞和板35,000个细胞在500μL的神经介质每一个24孔板（在3.2中提到的板）的单个孔。 注意:任选地，细胞等分试样可以用台盼蓝进行复染，以评估细胞制剂的活力。控制通过视觉检查接种密度离子使用显微镜（一般为90 - 每一个标准10X物镜的视场110细胞）。 </li><li>在孵化器处的神经元，在37℃，1小时6％体积/体积的 CO 2。 </li><li> （去籽与融合星形胶质细胞单层）从孵化器中孵化后，把准备好的插件（见步骤2.8）。通过抽吸星形胶质细胞中，用500微升新鲜的神经元中替换它交换媒介。 注意:应插入后2怀有星形胶质细胞融合单层- 3 DIV（天体外 ）。 </li><li>将带有星形胶质细胞刀片小心地插入包含神经元文化（3.13步），使用无菌镊子井。 </li><li>放置导致的间接神经元星形胶质细胞共培养放回培养箱中于37℃，6％体积/体积的 CO 2。 注意:在这些条件下，神经元可以在完全确定的培养基中培养长达4周。无介质的改变是必要的。对于长期培养物时，建议以填充ÈMPTY井用无菌水，以减少从24孔板的蒸发。 </li><li>进行体外细胞分析所需的培养后时段（ 例如，免疫细胞化学，PCR，Western印迹，电生理记录（膜片钳或者多电极阵列））7,15,18。 注:培养系统也适合于培养11急性和慢性药物治疗。 </li></ol>

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The authors have nothing to disclose.

当前协议的主要目的是完全独立的神经元和星形细胞培养物，同时保持它们在共享介质。由于这个原因，所得到的培养物的纯度应在过程的开始进行验证。我们推荐使用神经元特异性微管蛋白，神经丝或蛋白质的NeuN神经元的标记，GFAP作为星形细胞标记，O4抗原少突胶质细胞前体标记，并Iba1蛋白识别小胶质细胞。 执行该协议的一个关键步骤的时候要特别注意，如由步骤说明之...

纵观几十年来，神经胶质细胞功能的一般性解释已经从一个单纯的支持对有关神经功能1积极调节作用的归属演变。因为对健康和疾病的2脑稳态其突出的影响，星形胶质细胞是科学界特别感兴趣的。在过去的几年中，研究一个多样性集中于神经胶质细胞的相互作用中体内和体外 3。然而，大多数的培养系统的不允许这两种细胞类型的单独分析和各自secretomes的。 有几种方法利用神经元和神经胶质细胞的直接共培养，实现长期持久的生存与生理有关的神经网络的发展4-6。本协议达到同样的目标，同时保持物理上分离的7两种细胞类型。相比conditioneD培养基接近8,9，我们的系统可以让研究神经细胞和星形胶质细胞之间的双向通信。分泌的信号分子的表达可以在细胞中的共享介质maturate进行监测。这个机会是特别相关的，因为星形细胞释放可溶性因子，如细胞因子，生长因子和细胞外基质分子10,11，从而调节神经元生长和功能7,12。因此，已经证明，另外在体外血小板反应蛋白视网膜神经节细胞的诱导突触13的形成。然而，其他未知因素是必要的，以使突触功能13。此外，通过星形胶质细胞释放分子具有以了解神经胶质细胞的相互作用的基础上被识别。 从小鼠和大鼠原代神经元和星形胶质细胞的培养先前已14-16所述。在这里，我们提出了一种优雅和多功能的工具，这两种细胞类型中的一种间接共培养方法结合起来。由于两个培养物物理分离但共享相同的介质，神经元，星形细胞和可溶性分子的影响，可以分别进行分析，从而为神经胶质细胞的相互作用的研究的强有力的工具。

<table border="0" cellpadding="0" cellspacing="0" width="608">
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	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:115px;">Reagents</td>
			<td style="width:152px;"></td>
			<td style="width:151px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">B27</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
			<td style="width:151px;">17504-044</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:115px;">Cell culture grade water</td>
			<td style="width:152px;">MilliQ</td>
			<td style="width:151px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:115px;">Cell culture grade water</td>
			<td style="width:152px;">MilliQ</td>
			<td style="width:151px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:115px;">Cytosine-&#223;-D arabinofuranoside (AraC)</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">C1768</td>
			<td style="width:191px;">CAUTION: H317, H361</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">DMEM</td>
			<td style="width:152px;">Gibco (Life Technologies)&#160;</td>
			<td style="width:151px;">41966-029</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">DNAse</td>
			<td style="width:152px;">Worthington</td>
			<td style="width:151px;">LS002007</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Gentamycin</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">G1397</td>
			<td style="width:191px;">CAUTION: H317-334</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Glucose</td>
			<td style="width:152px;">Serva</td>
			<td style="width:151px;">22700</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">HBSS</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
			<td style="width:151px;">14170-088</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">HEPES</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
			<td style="width:151px;">15630-056</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Horse serum</td>
			<td style="width:152px;">Biochrom AG</td>
			<td style="width:151px;">S9135</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">L-Cysteine</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">C-2529</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">MEM</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
			<td style="width:151px;">31095-029</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Ovalbumin</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">A7641</td>
			<td style="width:191px;">CAUTION: H334</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Papain</td>
			<td style="width:152px;">Worthington</td>
			<td style="width:151px;">3126</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">PBS</td>
			<td style="width:152px;">self-made&#160;</td>
			<td style="width:151px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Poly-D-lysine</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">P0899</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Poly-L-ornithine</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">P3655</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Sodium pyruvate</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">S8636</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">Trypsin-EDTA</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
			<td style="width:151px;">25300054</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Equipment</td>
			<td style="width:152px;"></td>
			<td style="width:151px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">24 well plates</td>
			<td style="width:152px;">Thermoscientific/Nunc</td>
			<td style="width:151px;">142475</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">24-wells plate (for the&#160; indirect co-culture)</td>
			<td style="width:152px;">BD Falcon</td>
			<td style="width:151px;">353504</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Binocular</td>
			<td style="width:152px;">Leica</td>
			<td style="width:151px;">MZ6</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Cell-culture inserts</td>
			<td style="width:152px;">BD Falcon</td>
			<td style="width:151px;">353095</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Centrifuge</td>
			<td style="width:152px;">Heraeus</td>
			<td style="width:151px;">Multifuge 3S-R</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Counting Chamber</td>
			<td style="width:152px;">Marienfeld</td>
			<td style="width:151px;">650010</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Forceps</td>
			<td style="width:152px;">FST Dumont (#5)</td>
			<td style="width:151px;">11254-20</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">glass cover slips (12 mm)</td>
			<td style="width:152px;">Carl Roth (Menzel- Gl&#228;ser)</td>
			<td style="width:151px;">P231.1</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Incubator</td>
			<td style="width:152px;">Thermo Scientific</td>
			<td style="width:151px;">Heracell 240i</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Micro tube (2 ml)</td>
			<td style="width:152px;">Sarstedt</td>
			<td style="width:151px;">72,691</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Microscope</td>
			<td style="width:152px;">Leica</td>
			<td style="width:151px;">DMIL</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Millex Syringe-driven filter unit</td>
			<td style="width:152px;">Millipore</td>
			<td style="width:151px;">SLGV013SL</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Orbital shaker</td>
			<td style="width:152px;">New Brunswick Scientific</td>
			<td style="width:151px;">Innova 4000</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Parafilm</td>
			<td style="width:152px;">Bemis</td>
			<td style="width:151px;">PM-996</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Petri dishes (10 cm)</td>
			<td style="width:152px;">Sarstedt</td>
			<td style="width:151px;">833,902</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">pipette (1 ml)</td>
			<td style="width:152px;">Gilson</td>
			<td style="width:151px;">Pipetman 1000</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Sterile work bench</td>
			<td style="width:152px;">The Baker Company</td>
			<td style="width:151px;">Laminar Flow SterilGARD III</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Surgical scissors</td>
			<td style="width:152px;">FST Dumont</td>
			<td style="width:151px;">14094-11</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Syringe</td>
			<td style="width:152px;">Henry Schein</td>
			<td style="width:151px;">9003016</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">T75 flask</td>
			<td style="width:152px;">Sarstedt</td>
			<td style="width:151px;">833,911,002</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">tube (15 ml)</td>
			<td style="width:152px;">Sarstedt</td>
			<td style="width:151px;">64,554,502</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Water bath</td>
			<td style="width:152px;">GFL</td>
			<td style="width:151px;">Water bath type 1004</td>
			<td style="width:191px;"></td>
		</tr>
	</tbody>
</table>

the indirect neuron-astrocyte coculture assay: an in vitro set-up for the detailed investigation of neuron-glia interactions

正确的神经细胞的发育和功能是发展中国家和成人大脑的先决条件。然而，复杂的神经元网络的高度控制形成和维护背后的机制尚未完全迄今理解。关于健康和疾病的神经元的开放式问题是多种多样的，从理解基本的发展，人类的调查相关疾病， 如阿尔茨海默氏症和精神分裂症深远。可以在体外进行的神经元的最详细的分析。然而，神经元细胞，要求和需要为他们的长期生存的额外支持星形胶质细胞。此细胞异质性是在与目标解剖神经元和星形胶质细胞的分析冲突。我们在这里提出一个细胞培养测定法，其允许纯原代神经元和星形胶质细胞，其共享相同的化学上确定的培养基的长期共培养，而在物理上分开。在此设置中，培养物存活多达四个星期，该测定是适于关于神经元的神经胶质细胞的相互作用研究的多样性。

通过间接共培养系统中的神经元培养物的分析是多方面的，可以在培养的成熟的不同阶段进行。由于这样的事实，该细胞可被保持长达4周，培养物的长期的调查是可能的。 在图1中的中间左侧面板概略说明了共培养设置。与使用该系统，可以执行这两种细胞类型的活细胞成像，由星形胶质细胞单层（左上面板）和?...

Watch this Scientific Journal Video about The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions at JoVE.com

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

该协议描述了神经元的神经胶质细胞相互作用的条块分析间接神经元的星形胶质细胞共培养。

间接神经元的星形胶质细胞共培养试验:一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

the-indirect-neuron-astrocyte-coculture-assay-an-vitro-set-up-for

Research

JoVE Journal

Neuroscience

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

14.6K 次观看.  Ruhr-University. 该协议描述了神经元的神经胶质细胞相互作用的条块分析间接神经元的星形胶质细胞共培养。 

视频: The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Brain Imaging Investigation of the Neural Correlates of Observing Virtual Social Interactions

The ability to gauge social interactions is crucial in the assessment of others&#x2019; intentions. Factors such as facial expressions and body language affect our decisions in personal and professional life alike 1. These "friend or foe" judgements are often based on first impressions, which in turn may affect our decisions to "approach or avoid". Previous studies investigating the neural correlates of social cognition tended to use static facial stimuli 2. Here, we illustrate an experimental design in which whole-body animated characters were used in conjunction with functional magnetic resonance imaging (fMRI) recordings. Fifteen participants were presented with short movie-clips of guest-host interactions in a business setting, while fMRI data were recorded; at the end of each movie, participants also provided ratings of the host behaviour. This design mimics more closely real-life situations, and hence may contribute to better understanding of the neural mechanisms of social interactions in healthy behaviour, and to gaining insight into possible causes of deficits in social behaviour in such clinical conditions as social anxiety and autism 3.

This article demonstrates an experimental design in which whole-body animated characters are used in conjunction with functional magnetic resonance imaging (fMRI) to investigate the neural correlates of observing virtual social interactions.

观察虚拟社会交往中的神经关联的脑成像研究

The ability to gauge social interactions is crucial in the assessment of others&#x2019; intentions. Factors such as facial expressions and body language ...

科研

神经科学

Brain Imaging Investigation of the Memory-Enhancing Effect of Emotion

Emotional events tend to be better remembered than non-emotional events1,2. One goal of cognitive and affective neuroscientists is 
to understand the neural mechanisms underlying this enhancing effect of emotion on memory. A method that has proven particularly influential in the 
investigation of the memory-enhancing effect of emotion is the so-called subsequent memory paradigm (SMP). This method was originally used to investigate the 
neural correlates of non-emotional memories3, and more recently we and others also applied it successfully to studies of emotional memory (reviewed in4, 5-7). 
Here, we describe a protocol that allows investigation of the neural correlates of the memory-enhancing effect of emotion using the SMP in conjunction with 
event-related functional magnetic resonance imaging (fMRI). An important feature of the SMP is that it allows separation of brain activity specifically 
associated with memory from more general activity associated with perception. Moreover, in the context of investigating the impact of emotional stimuli, 
SMP allows identification of brain regions whose activity is susceptible to emotional modulation of both general/perceptual and memory-specific processing. 
This protocol can be used in healthy subjects8-15, as well as in clinical patients where there are alterations in the neural correlates of emotion perception 
and biases in remembering emotional events, such as those suffering from depression and post-traumatic stress disorder (PTSD)16, 17.

We present a protocol that uses functional magnetic resonance imaging to investigate the neural correlates of the memory-enhancing effect of emotion. This protocol allows identification of brain activity specifically linked to memory-related processing, contrary to more general perceptual processing, and can be used with healthy and clinical populations.

脑成像的情感记忆的促进作用调查

Emotional events tend to be better remembered than non-emotional events1,2. One goal of cognitive and affective neuroscientists is 
to understand the ...

Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional distractibility) effects (reviewed in 1). Complementing our recent protocol 2 describing a method that allows investigation of the neural correlates of the memory-enhancing effect of emotion (see also 1, 3-5), here we present a protocol that allows investigation of the neural correlates of the detrimental impact of emotion on cognition. The main feature of this method is that it allows identification of reciprocal modulations between activity in a ventral neural system, involved in 'hot' emotion processing (HotEmo system), and a dorsal system, involved in higher-level 'cold' cognitive/executive processing (ColdEx system), which are linked to cognitive performance and to individual variations in behavior (reviewed in 1). Since its initial introduction 6, this design has proven particularly versatile and influential in the elucidation of various aspects concerning the neural correlates of the detrimental impact of emotional distraction on cognition, with a focus on working memory (WM), and of coping with such distraction 7,11, in both healthy 8-11 and clinical participants 12-14.

We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

脑成像的情感对认知损害的影响调查

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

的斑马鱼胚胎神经上皮通透性的测定

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells.

一<em&gt;在体外</em&gt;模型细胞病理生理学的球细胞脑​​白质营养不良的研究

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

在癌神经侵袭的体外模型:背根神经节型号

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

The tau aggregation assay described in this manuscript mimics the anticipated features of in vivo tau misfolding and aggregation.

在体外陶氏蛋白聚集与药物筛选研究方法

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other ...

间接神经元的星形胶质细胞共培养试验:一个

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