This work was supported in part by grants from the National Multiple Sclerosis Society (RG 5001-A-3 to S.J.C.), the National Institutes of Health (NS065808 to E.R.B.; NS078392 to S.J.C.), start-up funds from the UConn Health Center (to SJC) and the Kim Family Fund (UCHC in support of K.I.C.).

所有涉及动物的程序是按照人文关怀和使用实验动物的政策执行提出了实验动物福利办公室（NIH），只有从大学的机构动物照顾及使用委员会（IACUC）批准康涅狄格卫生服务中心。 1，制备混合胶质细胞培养<ol><li>消毒在使用前所有的仪器。加入3毫升含无阳离子的冰冷无菌Hank氏平衡盐溶液（HBSS）（Mg 2 +的或钙离子 ），以保持在冰上，以保持冷3无菌60毫米培养皿。 </li><li>隔离从出生后P0-P2小鼠幼仔的皮质，如前所述17,18。 注意：皮层下结构，如海马，不包括在这些培养物中。 </li><li>小心地取出脑膜，然后分离出皮层转移到新鲜的HBSS和游离组织酶，使用NE根据制造商的协议尤勒尔组织解剖工具包。 </li><li>板中的细胞在无菌的T-175烧瓶中，并在媒体孵育过夜[Dulbecco改良的Eagle培养基（DMEM）中，用10％胎牛血清（FBS）的含有1x青霉素/链霉素。 </li><li>第二天用新鲜培养基更换所有的媒体。继续培育文化，和普通媒体的变化，约3周，直到细胞单层成立。 注意：此单层将被包括星形胶质细胞和小胶质细胞与约10：1的比例19。 </li></ol> 2球细胞诱导混合胶质细胞培养<ol><li>准备ECM包被的盖玻片。 <ol><li>浸泡圆形盖玻片用在无菌培养皿中30分钟，无菌1N的盐酸（HCl）。用无菌水冲洗3次盖玻片，然后浸泡在95％EtOH中至少20分钟。然后让空气干燥。 </li><li>稀释外MATR的地方滴IX（ECM）的蛋白质涂层材料上的封口膜一张（层粘连蛋白10微克/ EG 150-250微升/滴毫升溶解在无菌磷酸盐缓冲液[PBS]）。分配液滴，使盖玻片不会碰的时候放在这些液滴。每个干盖玻片轻轻将上使用镊子液滴。 </li><li>留在液滴在盖玻片约。 4-6小时的培养罩的窗扇内关闭。 </li><li>将ECM包被的盖玻片（ECM涂覆的一面朝上），在每个24孔板的孔中。每次用无菌PBS 3X以及洗净，直到需要保持在4℃。 </li></ol></li><li>准备胶质细胞镀上盖玻片。 <ol><li>从胶质细胞培养物吸媒体，轻轻地用10ml无菌PBS洗胶质细胞单层3倍。接着，加入8-10毫升胰蛋白酶-EDTA溶液与T-175，并孵育5-10分钟，在37℃。 </li><li>当大多数的单层已经分离，加入20 ml完全传媒到该烧瓶中，以停止胰蛋白酶消化。收集含脱落细胞的培养基到50毫升锥形管中并旋转10分钟，在300 XG在室温下。 </li><li>吸出上清液，而不会干扰沉淀，然后轻轻吹打使用P1000吸管的溶液重悬所述细胞于2ml的新鲜完全培养基。 </li><li>确定使用血球细胞的数量，然后重新悬浮的细胞在完全培养基中在所希望的密度（ 例如，10 5个细胞/ ml）。板中的细胞上的细胞外基质包被的盖玻片（ 例如，500微升到24孔板的每孔）。添加额外的新鲜完全培养基中，以每孔3-5天的潜伏期，在37℃，或直至细胞生长至所需的视觉汇合。 </li></ol></li><li>治疗Psychosine。 <ol><li>每瓶psychosine A股浓度（108.3毫米）的二甲基亚砜（DMSO）。稀释此股票在100％乙醇作工作股票可以在n为加入到24孔板各孔中以达到10μM的最终浓度。 </li><li>在加入psychosine的时间，轻轻摇动培养板以混合psychosine到培养基中。通过加入隔日10μM的等效一周补充psychosine。 </li></ol></li><li>经过7天psychosine处理，收集媒体和吸管500微升冷的4％多聚甲醛（PFA）到每口井的。在室温下孵育20分钟，固定细胞。然后，洗涤​​各孔3次，用PBS，然后保持板在4℃下直到处理用于免疫细胞化学（见下文）。 注：细胞培养基可以被收集为酶促或酶联免疫吸附测定法在这一点上，如果需要的。 </li></ol> 3，免疫细胞化学（ICC）的可视化弧面细胞<ol><li>更换PBS以封闭缓冲液[PBS + 0.3％Tween-20的±5％正常山羊血清（NGS），并孵育细胞1小时，于室温下。 </li><li>在cubate固定细胞在初级抗血清伊巴-1 [（1：1,000）稀释在PBS +0.3％Tween-20的±2％NGS]为至少1小时，在室温下，然后用PBS 3×5分钟，洗每个孔/洗。 </li><li>添加适当的次级荧光缀合的抗体来识别第一抗体标记和反染色用4&#39;，6 - 二脒-2 - 苯基吲哚（DAPI，1:1000），以确定细胞核。孵育在二次抗体溶液1小时，在室温下，并用PBS中3×5分钟。 </li><li>挂载每个玻璃罩，以显微镜使用的安装介质的幻灯片。查看幻灯片上的荧光显微镜的视觉和定量分析。 </li></ol> 4，分析和弧面细胞的原代培养的表征<ol><li>使用以下三种形态的标准来确定文化球细胞： <ol><li>确定使用荧光显微镜球状细胞免疫阳性标记的小胶质细胞的标记，IBA-1 +。 </li><li>确定弧面细胞作为polynucleated（ 即含有两种或两种以上的DAPI阳性细胞核）。 </li><li>由一个圆形的形态，从静止小胶质细胞的高度支化的外观独特的识别球状细胞。 注：细胞也可通过流式细胞术从这些培养物中收集到的细胞表面标记物的分析和定量分析。 </li></ol></li><li>测量小胶质细胞和弧面细胞的吞噬活性<ol><li>评估psychosine处理的小胶质细胞的吞噬活性，按照制造商的说明添加FITC标记的胶乳珠的孔中。固定之前用PFA添加荧光标记的乳胶珠48小时。修复珠处理（如上文第2.4节）细胞和流程免疫细胞（如第3节所述）。 </li><li>选择荧光基团标记的第二抗体，使它们不重叠的荧​​光团标记的珠粒的荧光发射光谱。 </li><li> Analyzë伊巴-1 +小胶质细胞通过识别那些细胞的吞噬型材共定位与荧光标记的珠子。 注：Psychosine将激活小胶质细胞。吞噬的小珠将单核和多核伊巴-1 +细胞在此培养环境内识别。 </li></ol></li></ol> 5，感应距离纯化小胶质文化弧面细胞注：另一种方法来破译星形胶质细胞和小胶质细胞之间的细胞间信号是在体外球细胞模型的另一个优势。初级小胶质细胞为replating或共同培养的纯化可以通过降低粘附性的培养来实现，如下面的步骤（参见第5.2节）中描述。 <ol><li>收集从集流混合胶质细胞培养介质到50ml锥形管中，并于37℃培养箱中保持它。 </li><li>小胶质细胞的分离，从初级混合胶质细胞培养。<ol><li>从汇合混合胶质细胞培养T175烧瓶中除去培养基后，加入温水摇动媒体[组成的完全培养基+的25mM 4-（2 - 羟乙基）-1 - 哌嗪乙磺酸（HEPES）+的25mM碳酸氢钠（Sigma公司）]。摇动烧瓶3-4小时，在轨道摇床上以100rpm（37℃）。收集这些摇瓶媒体到50ml锥形管。 注：该媒体将包括从烧瓶少贴小胶质细胞。 </li><li>旋转介质，在300×g离心10分钟，在室温下进行。吸出上清液，然后重悬浮细胞于2ml的星形胶质细胞条件培养基（在步骤5.1收集）。 </li><li>建立使用血细胞计数器，然后板的小神经胶质细胞上衬底涂覆的盖玻片上获得的细胞数量（参见第2.1节），用于免疫细胞化学，或镀到超低粘附孔板弧面细胞共培养与其他细胞类型的将来采集，如少突胶质细胞（如递减在下面的部分进项税）。 注意：一旦镀金的小胶质细胞可以与如上所述（见2.3节）psychosine（10微米）来治疗。 </li></ol></li><li>共培养基层少突胶质细胞的细胞毒性分析。 注：收集从低粘附片用于随后的共培养到少突胶质细胞的原代培养物，或者少突胶质细胞祖细胞psychosine处理的小胶质细胞，可用于检查这些弧面状细胞在体外的潜在细胞毒性作用。 <ol><li>开发利用已建立的方法18,20初级少突胶质细胞的培养。收集psychosine处理的小胶质细胞，轻轻地吸取到从孔的底部分离的松散的贴壁细胞。收集细胞，离心分离机，在300×g离心10分钟，然后小心地重新悬浮在少突胶质细胞分化培养基[的Neurobasal介质组成，L-谷氨酰胺（1倍），B-27补充剂（1×），和三碘甲状腺氨酸（T3中，10纳克/毫升）]。 </li><li>计数使用血球细胞的数量，然后播种的小胶质细胞上的少突胶质细胞培养在1×10 5个细胞/ ml，从而以近似1:2的小胶质细胞/少突胶质细胞的比例。孵育作为共培养3天。 </li><li>固定细胞的免疫细胞化学（见2.4节）。收集的细胞培养基用于ELISA或根据需要进行化学分析。 </li></ol></li></ol>

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The authors declare no conflicts of interest.

这里所描述的协议提供了一种新的模式系统，其中研究开发和激活小胶质细胞和球状细胞的功能特性。通过QQ，MSN 等前期工作。使用HEK293细胞系提供本协议的发展为球细胞形成21学习的模板。同样重要的是要指出，来自在该模型中的球状细胞来自原弧面细胞识别在GLD不同。举例来说，我们经常观察到小神经胶质细胞在我们的鼠文化quadranucleation，而球状细胞（GLD）经常被发现含有超?...

球细胞脑白质营养不良（GLD），也被称为克拉伯病，是一种致命的脱髓鞘疾病的功能突变的galatocerebrosidase（GALC）1基因造成的损失。政府物流服务署的最普遍的形式是由发生在儿童早期代表，往往前五年2,3岁特点是运动和认知能力下降，导致过早死亡的一个积极的临床过程中，婴儿的变体。基因检测是用来验证GLD 4的诊断。政府物流服务署的神经病理学发现广泛脱髓鞘，神经萎缩，星形胶质细胞增生和肿胀多有核小胶质细胞的存在被称为球状细胞5-7。弧面细胞的鉴定，常含在细胞质筒状包裹体，一直是政府物流服务署的定义特征，在过去97年虽然这些显眼细胞的特异功能一直难以实现。 非MYEL的参与GLD中的发病inating神经胶质细胞（小胶质细胞和星形胶质细胞），一直被认为是本病8到深刻的脱髓鞘二级响应。有趣的是，这种疾病的最初描述，在1916年5由克努兹克拉贝，包含已命名的脂质碎片多核巨噬细胞的报道形成“球状细胞&#39;并且是本病的决定性特征。 球状​​细胞病理学（GLD）的共同特征，虽然他们在政府物流服务署的角色长期以来被忽视了。有趣的是，这些细胞中，GLD的CNS组织中的最早的特性的变化。这种知识的缺乏可能是由于该多核巨噬细胞，称为巨核细胞中的其他疾病的形成中，通常认为是病理学的结果，而不是一个初始致病驱动力9的假设。因此，很少有研究调查了WHI机制CH弧面细胞是从吞噬细胞形成，特别是在政府物流服务署的中枢神经系统。本报告中所述的程序集中于球细胞的形成在CNS中的重要性和我们先前示范体外小胶质细胞以及这些细胞的那psychosine诱导的多核化表现出较高水平的细胞吞噬活性的。根据这些意见，球状细胞鸟类观察者的大脑经常含有PAS阳性的碎片，暗示高水平的吞噬活性。球状细胞也发现免疫阳性铁蛋白（一小胶质细胞标记物）10，KP-1 / CD68（单核细胞标记物），有的还积极为波形蛋白（一种中间丝蛋白，星形胶质细胞的标志物和激活的小胶质细胞），11， HLA-DRA（一个MHCII表面受体），和TNF-α7和IBA-112（用于识别的小胶质细胞钙结合蛋白）。基于标记这个集合，球状细胞从培养小胶质细胞起源独特的表型。 尽管他们的独特性，具体功能和选区于GLD发病的贡献已经在很大程度上被忽视。球状​​细胞已被认为是慢性脱髓鞘的次要结果。然而，过去的研究探讨球状细胞的时序关联到政府物流服务署的脑白质病变​​已经确定球状细胞在胚胎后期存在于出生后早期阶段;时间少突胶质细胞凋亡和公开脱髓鞘13前。因此，在GLD的神经病理学发展的时间顺序显示，球状细胞形成预先脱髓鞘在此疾病14。这导致我们在GLD早期形成球状的细胞可能代表一个决定性致病事件，而不是次要的，反应要少突胶质细胞受损15回应假设。此外，在政府物流服务署小胶质细胞活性的失调也被认为是事实上的R限流的hematopeotic干细胞疗法的远期疗效治疗本病16。因此，调查的细胞功能和小胶质细胞的调节，球状的细胞，以应对psychosine预计将提供在政府物流服务署的发病机制的新见解。 直到最近，缺乏在其中，研究球状细胞形成一个合适的模型已经限定的确切功能及这些细胞GLD的病理作用的了解。在最近的研究中，确定该弧面状细胞可被形成在直接响应psychosine，积聚在GLD病原脂质毒素。我们发现，小胶质细胞，但不巨噬细胞，被激活并转化成球状的细胞在初级胶质细胞培养响应于psychosine 15。该转化成球状的细胞被发现的胞外蛋白酶，基质金属蛋白酶（MMP）-3 15介导。最近，我们扩展了这些调查结果，并决定在此体外模型系统，开发出psychosine激活的小胶质细胞和球状细胞强效有毒的少突胶质细胞和少突胶质祖细胞。因此，当在GLD，psychosine和地层之前脱髓鞘将支持在该疾病的小胶质细胞的新兴初级和可能的致病作用弧面细胞的早期积累的上下文中考虑。 我们建议的球细胞形成的研究将揭示政府物流服务署的发病机制，这将有助于我们对这个疾病的认识新的信息。此外，政府物流服务署对这种新的细胞模型可提供一种新的形式从中新的治疗方法，以解决这种疾病的病理变化可以进行测试。因此，在本报告中，我们提供了体外发育非髓鞘神经胶质细胞的原代培养psychosine诱发球状细胞的详细协议。

<table border="0" cellpadding="0" cellspacing="0" width="884">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:500px;">Hank&#8217;s balanced salt solution (HBSS) containing no cations (Mg2+ and Ca2+).&#160;</td>
			<td style="width:297px;">Life technologies</td>
			<td style="width:87px;">14175-095</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neural Tissue Dissociation Kit</td>
			<td>Miltenyi</td>
			<td>130-092-628</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 uM cell-strainer</td>
			<td>Fisherbrand</td>
			<td>22363547</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#8217;s balanced salt solution (HBSS) containing cations (Mg2+ and Ca2+).&#160;</td>
			<td>Gibco</td>
			<td>14025-092</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s modified eagle medium (DMEM)</td>
			<td>Gibco</td>
			<td>11995-065</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal bovine serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicilin/Streptomycin</td>
			<td>Life technologies</td>
			<td>15070-063</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA solution</td>
			<td>Life technologies</td>
			<td>25299-056</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Psychosine</td>
			<td>Sigma</td>
			<td>P9256</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Science</td>
			<td>19208</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Goat Serum (NGS)</td>
			<td>Invitrogen</td>
			<td>PCN5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iba-1</td>
			<td>WAKO</td>
			<td>019-19741</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor conjugated antisera</td>
			<td>Life Technologies</td>
			<td>Various</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting Media</td>
			<td>Southern Biotech</td>
			<td>OB100-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phagocytic Assay Kit</td>
			<td>Cayman Chemicals</td>
			<td>500290</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma</td>
			<td>BP310-500</td>
		</tr>
	</tbody>
</table>

an in vitro model for the study of cellular pathophysiology in globoid cell leukodystrophy

多成核的小神经胶质细胞的确切功能，称为球状的细胞，即是在球状细胞脑白质营养不良（GLD）的中枢神经系统中唯一地丰富目前还不清楚。在认识这个差距已经阻碍了缺乏适当的体外研究模型。在这里，我们描述了一个主要的鼠神经胶质培养体系中，治疗psychosine结果在小神经胶质细胞的多核类似GLD中发现的特征球状细胞。使用这个新系统，我们定义的条件和分析模式弧面细胞的研究。可能利用此模型系统在我们以前的研究，其中确定了基质金属蛋白酶（MMP）的潜在作用进行了验证-3 GLD。这种新颖的体外系统可以是其中以研究这些独特的细胞的形成和功能，而且还潜在治疗操纵，一个有用的模型。

这个协议，因为写的，预计需时约36天，从开始到结束（参见图1：实验工作流程计划）来完成。一直以来，我们经历了&#39;弧面状“细胞的这种原代培养体系的发展是既可靠和可重复性：多核细胞的响应psychosine的形成始终7天的治疗观察。 使用IBA-1在用核反染色结合将使识别的大小胶质细胞的免疫细胞化学染色，圆形多核细胞（ 图1b）。在某些情况下?...

Watch this Scientific Journal Video about An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy at JoVE.com

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 ...

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Research

JoVE Journal

Neuroscience

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

7.8K 次观看.  University of Connecticut Health Center. 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. 

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

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

一个视神经挤压伤的小鼠模型，研究视网膜神经节细胞生存

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

科研

神经科学

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

代的永生化小鼠脑微血管内皮细胞株作为<em在体外</em血脑屏障型号

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

背根神经节神经元和区分脂肪干细胞：一种<em&gt;在体外</em&gt;共培养模型来研究周围神经再生

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

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 ...

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

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

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

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

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 ...

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

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

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

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

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

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

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

在体内成像完全活跃的脑组织在清醒斑马鱼幼虫和青少年由骷髅和皮肤去除

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...