我们感谢卡琳Kindbeiter，穆里尔Bozon和FLORIE雷诺的帮助。这项工作是由法国国家科学研究中心，ANR YODA程序和Labex DevWeCan支持。

第1天 1。从E12.5小鼠胚胎脊髓地板板（FP）的解剖 注意:整个程序需要使用的无菌条件。最好是根据解剖罩进行解剖，以避免污染。机罩的表面应该用乙醇进行清洗。所有清扫工具必须消毒，并保持在无菌培养皿中。的液体（介质，夹层介质）必须保持封闭并置于冰浴中。每个胚胎必须被收集在单独的新鲜降。这对于组织保存重要。解剖与杜蒙＃5镊子进行。要传输的菜肴，握脐带或头部不至后脑的任何损害之间的胚胎。这一点非常重要，不要损坏脊髓顺利完成清扫。制备冷磷酸盐缓冲盐水（PBS）中，冰冷Hank平衡盐溶液（HBSS） -6.5％的葡萄糖和室温Neurobasal培养基。 1.1脊髓解剖动物是根据中心法国国家科学研究的以下欧洲指令动物保健指导治疗。安乐死的方法是颈椎脱位，它包括将压力施加到颈脱臼和从脑中的脊柱。被推荐用于小鼠动物模型中这种方法，并且可以在一个快速和有效的方式来进行。 <ol><li>安乐死怀孕小鼠在妊娠期（E0.5 =第一天以下的消光）的E12.5。将鼠标腹侧。 </li><li>浸泡腹部用70％乙醇。捏用阿德森镊子腹部的皮肤和切断通过皮肤和腹膜层用手术剪。做一个切口，并用它来切断对小鼠的两侧，以暴露腹腔。 </li><li>一个字符串，胚胎的存在于鼠标的每一侧。抓住UTE的一角RUS在两个胚胎，并剖析它从外观开始部分之间的组织。完整子宫转移到冰100毫米培养皿用冷PBS。 </li><li>提取胚外组织的胚胎。掌握胎盘组织（暗红色）与两对镊子和轻轻拉扯撕裂组织，并从子宫囊取出胚胎。 </li></ol> 注:为了更好的保护，收集他们的子宫囊逐个胚胎。 <ol start="5"><li>接下来的步骤是根据双目放大镜和解剖罩完成。将一个胚胎在下拉冷的HBSS-6.5％的葡萄糖。 </li><li>使用镊子，杀头的胚胎。 </li></ol> 注:切下颌和后脑之间，后脑的后部是下一个重要的步骤。 <ol start="7"><li>将胚胎腹面朝下面向实验者的前部。 </li><li>捏皮肤用一对镊子，并与另一对胚胎的抽离皮肤。 </li></ol> 注意:该步骤必须重复，直到皮肤完全从胚胎的前部移除。 <ol start="9"><li>把胚胎前侧到左侧。在的前部有一个镊子"牵制"胚胎。与其他钳子抓住皮肤和拉向胚胎的喙侧。脊髓是再访问。 </li><li>使用镊子的一侧切割仍然包裹脊髓脊膜。脊髓现已开放。 </li><li>把胚胎前侧到右边。 </li><li>从脊髓从胚胎的颈椎侧开始拆下组织。滑动脊髓（镊子应保持关闭，以避免脊髓损伤）下的镊子。镊子必须脊髓下可见。脊髓小下旋转动作都足以分离周围组织和背根神经节（DRG）。 </li></ol> 注意:如果保持附着周围组织添加重量至脊髓，从而增加了在下一步骤打破它的概率。 <ol start="13"><li>将分离的脊髓冷的HBSS-6.5％葡萄糖的新鲜滴。 </li><li>将脊髓在平坦的位置，在顶部和前侧离实验者脑膜。 </li><li> "牵制"使用剩余的后脑与一个钳脊髓，并与第2钳子抓住脑膜。撕下脊髓延髓身边做起脑膜。 </li></ol> 注意:该运动应该是缓慢和恒定，以避免对脊髓的任何断裂。振动运动可以促进脑膜支队。 1.2 FP解剖<ol><li>掐后脑有一个镊子。 </li><li>该FP是中央TRA组织的nsparent一部分。用手术刀切出的底板。 </li></ol> 注意:要切断两侧具有相同的质量，从嘴侧端开始，并交替地切右侧和所述底板的左侧。 <ol start="3"><li>保育解剖FP的Neurobasal培养基中于室温下进行。 </li></ol> 2。 FP文化 注意:应在无菌条件下在组织培养罩中执行的所有步骤。用新鲜培养基和刚解冻的补充和试剂。制备无菌的玻璃盖玻片上，温暖的Neurobasal + B27，血浆和HBSS-凝血酶。 2.1 FP文化<ol><li>将几个无菌盖玻片在一个100毫米的培养皿并在每个盖玻片的中心移液管20微升血浆。 </li><li>转让两到四个FP在每个等离子下降，并添加20微升的HBSS凝血酶旁边的血浆滴，仔细混匀。保持盖玻片最少10分钟在室温下，使血浆凝固。 </li></ol> 注:将两个心室肌的情况下，在部分情况下，夹层的完整解剖和更多。用枪头混匀缓慢，避免与计划生育外植体接触。 血浆凝块回缩几微米的凝结的迹象。凝固不能过早地停止，以避免血浆凝块脱落。 <ol start="3"><li>将每个盖玻片的24孔板中，加入500μl的Neurobasal + B27的。孵育48小时，在37°C </li></ol>第3天 3。 FP条件培养基（FP 厘米 ）收获 注:收获应在无菌条件下在组织培养橱中进行。如果上清液中的文化近郊污染或者如果楼板是浮动的（这意味着他们可能已经死了OD），那么不从这个收获的FP 厘米很好。 3.1 FP 厘米收获<ol><li>从每口井仔细收获的媒介。 </li></ol> 注意:不要吸取聚合血浆凝血酶混合或任何FP片段。 <ol start="2"><li>存储100μL分装于-80°C。 </li></ol>第四天 4。脊髓合神经元从E12.5小鼠胚胎解剖 注意:该制剂不需要在无菌条件下将被执行。请遵循上述收集胚胎的过程。准备冷PBS，冷的HBSS-6.5％葡萄糖和寒冷的neurobasal。 4.1脊髓解剖<ol><li>使用镊子，杀头的胚胎。 </li></ol> 注意:切下钳口和后脑之间，后脑的后部是重要接下来的步骤。 <ol start="2"><li>将胚胎腹面朝下在一个新的下降与面临的实验者的前部。 </li><li>捏胚胎的皮肤有一个镊子，并与另一个绝尘而去的皮肤。 </li></ol> 注意:该步骤必须重复，直到皮肤完全从胚胎的前部移除。 <ol start="4"><li>把胚胎前侧到左侧。在的前部有一个镊子"牵制"胚胎。与其他镊子，把握皮肤和拉向胚胎的喙侧，以暴露脊髓。 </li><li>使用镊子的一侧切割仍然包裹脊髓脊膜。脊髓现在已经公开和开放。 </li><li>胚胎前侧姿势要正确。 </li><li>从脊髓从胚胎的颈椎侧开始拆下组织。滑动脊髓（保持钳闭）下的镊子。为CEPS必须脊髓下可见。脊髓小下旋转动作都足以取下邻近组织和背根神经节（DRG）。 </li><li>在冷的HBSS，6.5％葡萄糖的新降转孤立脊髓。 </li><li>将脊髓在平坦的位置，在顶部和前侧离实验者脑膜。 </li><li> "牵制"利用剩余的后脑有一个镊子脊髓，与第二钳抓住脑膜。撕下脊髓延髓身边做起脑膜。 </li></ol> 4.2连合神经解剖<ol><li>掐后脑有一个镊子。 </li><li>在连合神经元位于脊髓（背侧）的最外侧部分。切出这部分外侧用细手术刀。 </li></ol> 注意:在脊髓的背侧部分可以从腹侧部分由单元d的差异区分密度，腹侧部分更加不透明。 注意:要切断两侧具有相同的质量，从嘴侧端开始，并交替地切一小长度的左侧的右侧和。 <ol start="3"><li>节约冰上立即使用的Neurobasal培养基的解剖组织。 </li></ol> 5。连合组织与计划生育厘米治疗 注意:此过程不要求无菌条件。该组织必须被收集在各个1.5毫升管。冰冻FP 厘米应该只能使用一次。避免多次重新冻结。准备温暖的FP 厘米 （37℃）。 5.1治疗方法连合神经元<ol><li> Dilacerate连合组织用小剪刀。 </li></ol> 注意:切组织尽可能地小，以使可能的单元的可访问性。 <ol start="2"><li>公平地分配不同的实验条件的控制处理和FP 厘米治疗之间的组织。 </li><li>离心分离的组织片段，在800×g离心1分钟，在4℃下</li><li>检查管之间的再分配。 </li></ol> 注意:如果需要，摇匀，离心，直到再次片段均匀分布。 <ol start="5"><li>除去上清液。 </li><li>添加温暖的FP 厘米和控制处理的组织。 </li></ol> 注意:音量必须调整到颗粒的量，并应至少两倍的颗粒的体积。 <ol start="7"><li>在37°C为30分钟。 </li></ol>注:轻轻摇动每次10-15分钟<ol start="8"><li>离心机在800×g离心1分钟，取出处理。 </li><li>加入裂解缓冲液</li></ol> 注意:该卷必须被调整到一个粒料的安装和应至少两倍的颗粒的体积。 <ol start="10"><li>吸管上下悬浮颗粒。 </li></ol> 注意:一个涡流也可以使用。 <ol start="11"><li>在冰上孵育15分钟。 </li><li>离心机以14,000×g离心15分钟，在4℃下</li><li>保留上清液，并测量用Bradford测定法9的蛋白质的量。 </li><li>添加6X Laemmli缓冲液。 </li><li>孵育在95℃下5分钟以变性蛋白含量。 </li></ol> 注意:样品可以立即使用，或者可以保存在-80℃。 <ol start="16"><li>评估通过Western印迹10的治疗效果。 </li></ol> 注:至少50总蛋白的微克应该为每个车道被加载。

<ol>
	<li>Nawabi, H., Castellani, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+commissures+in+the+central+nervous+system:+how+to+cross+the+midline.">Axonal commissures in the central nervous system: how to cross the midline.</a> Cell. Mol. Life Sci. 68, 2539-2553 (2011).</li><li>Placzek, M., Briscoe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+floor+plate:+multiple+cells,+multiple+signals.">The floor plate: multiple cells, multiple signals.</a> Nat. Rev. Neurosci. 6, 230-240 (2005).</li><li>Chedotal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+tales+of+the+midline.">Further tales of the midline.</a> Curr. Opin. Neurobiol. 21, 68-75 (1016).</li><li>Nawabi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+midline+switch+of+receptor+processing+regulates+commissural+axon+guidance+in+vertebrates.">A midline switch of receptor processing regulates commissural axon guidance in vertebrates.</a> Genes Dev. 24, 396-410 (2010).</li><li>Ruiz de Almodovar, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+mediates+commissural+axon+chemoattraction+through+its+receptor+Flk1.">VEGF mediates commissural axon chemoattraction through its receptor Flk1.</a> Neuron. 70, 966-978 (2011).</li><li>Charoy, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gdnf+activates+midline+repulsion+by+Semaphorin3B+via+NCAM+during+commissural+axon+guidance.">gdnf activates midline repulsion by Semaphorin3B via NCAM during commissural axon guidance.</a> Neuron. 75, 1051-1066 (2012).</li><li>Kiecker, C., Lumsden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+organizers+in+patterning+the+nervous+system.">The role of organizers in patterning the nervous system.</a> Annu. Rev. Neurosci. 35, 347-367 (2012).</li><li>Le Dreau, G., Marti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal-ventral+patterning+of+the+neural+tube:+a+tale+of+three+signals.">Dorsal-ventral patterning of the neural tube: a tale of three signals.</a> Dev. Neurobiol. 72, 1471-1481 (2012).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> J. Vis. Exp. , (2010).</li><li>Salinas, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphogen+sonic+hedgehog+collaborates+with+netrin-1+to+guide+axons+in+the+spinal+cord.">The morphogen sonic hedgehog collaborates with netrin-1 to guide axons in the spinal cord.</a> Trends Neurosci. 26, 641-643 (2003).</li><li>Bechara, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FAK-MAPK-dependent+adhesion+disassembly+downstream+of+L1+contributes+to+semaphorin3A-induced+collapse.">FAK-MAPK-dependent adhesion disassembly downstream of L1 contributes to semaphorin3A-induced collapse.</a> EMBO J. 27, 1549-1562 (2008).</li></ol>

作者没有透露

地板板条件培养基的生产提供了一种有效，简便的方法，以评估底板释放信号的生物学特性。 血浆凝血酶矩阵提供了极好的条件，组织的生存。然而，这种丰富的环境可能对于某些类型的实验的限制。因此，底板的组织，也可以培养在琼脂糖基质。底板的条件培养基的质量可以通过已知的地板板释放信号，如GDNF（ 图2），Shh和netrin诱导11的检测来评估。?...

地板板是脊髓发育的一个众所周知的构图中心，祖细胞和有丝分裂后细胞谱系的规范扮演关键角色，并控制轴突导航7,8。这里描述生产的FP 厘米的实验程序允许调查评估底板源性信号在发育过程中的各种情况下的功能特性，从 ​​细胞图案和存活细胞及轴突迁移。 为了说明使用这样的FP 厘米 ，含有合神经元背侧脊髓组织被解剖，dilacerated和与FP 厘米刺激。组织然后可以处理用于Western印迹分析。这使得调查轴突导向机械按楼层板信号发布规定。治疗dilacerated新鲜组织中的保存方法保存细胞内日微环境的巨大优势Ë组织。因此，通过FP 厘米治疗或任何类型的治疗的后果被评估的多个生理方式比在细胞和组织培养条件。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS REQUIRED</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, Ca2+/Mg2+- free</td>
			<td>Invitrogen</td>
			<td>14170-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G-7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Invitrogen</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma</td>
			<td>Sigma</td>
			<td>P-3266</td>
			<td>1mg/ml (Reconstitute in H2O and filtrate)</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T-4648</td>
			<td>10mg/ml(Reconstitute in H2O and filtrate)</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>50X</td>
		</tr>
		<tr>
			<td>Hepes (260g/mol)</td>
			<td>Sigma</td>
			<td>H-7006</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E-5513</td>
			<td>0,5M pH8</td>
		</tr>
		<tr>
			<td>MgCL2</td>
			<td>Euromedex</td>
			<td>2189</td>
			<td>1M</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>VWR</td>
			<td>24388.295</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL</td>
			<td>Sigma</td>
			<td>I-3021</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Protease inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>04 693 116 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Orthovanadate (Na3VO4)</td>
			<td>Sigma</td>
			<td>S-6508</td>
			<td></td>
		</tr>
		<tr>
			<td>distilled H2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Reagents required</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>TOOLS AND MATERIAL</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson forceps</td>
			<td>Fine Science Tools</td>
			<td>11027-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Fine Tips forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>micro knives</td>
			<td>Fine Science Tools</td>
			<td>10136-14</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Tools and material</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>
			<table border="1">
				<tbody>
					<tr>
						<td colspan="2">Dissection and FP culture</td>
					</tr>
					<tr>
						<td>Ethanol 70%</td>
						<td></td>
					</tr>
					<tr>
						<td>dissection hood</td>
						<td></td>
					</tr>
					<tr>
						<td>dissection microscope</td>
						<td></td>
					</tr>
					<tr>
						<td>Petri dishes</td>
						<td></td>
					</tr>
					<tr>
						<td>glass coverslips</td>
						<td>18mm x 18mm</td>
					</tr>
					<tr>
						<td>Bunsen gas burner</td>
						<td></td>
					</tr>
					<tr>
						<td>24 well plate</td>
						<td></td>
					</tr>
					<tr>
						<td>tissue culture incubator (37&#176;C, 5% CO2, humidity controlled)</td>
						<td></td>
					</tr>
					<tr>
						<td>Centrifuge</td>
						<td></td>
					</tr>
					<tr>
						<td colspan="2">RECIPES</td>
					</tr>
					<tr>
						<td colspan="2">FP culture media</td>
					</tr>
					<tr>
						<td>Neurobasal</td>
						<td></td>
					</tr>
					<tr>
						<td>B27</td>
						<td>1/50e dilution from stock</td>
					</tr>
					<tr>
						<td colspan="2">HBSS - Thrombin</td>
					</tr>
					<tr>
						<td>HBSS</td>
						<td>1 ml</td>
					</tr>
					<tr>
						<td>Thrombin 10mg/ml</td>
						<td>30 &#956;l</td>
					</tr>
					<tr>
						<td colspan="2">Lysis Buffer pH7,4</td>
					</tr>
					<tr>
						<td>Hepes (260 g/mol)</td>
						<td>25mM</td>
					</tr>
					<tr>
						<td>EDTA (pH8)</td>
						<td>5mM</td>
					</tr>
					<tr>
						<td>MgCL2</td>
						<td>1mM</td>
					</tr>
					<tr>
						<td>Glycerol</td>
						<td>10%</td>
					</tr>
					<tr>
						<td>IGEPAL (NP40)</td>
						<td>0.10%</td>
					</tr>
					<tr>
						<td>Protease Inhibitor Cocktail</td>
						<td>1X</td>
					</tr>
					<tr>
						<td>Sodium Orthovanadate</td>
						<td>0,2M</td>
					</tr>
					<tr>
						<td>H2O</td>
						<td></td>
					</tr>
					<tr>
						<td colspan="2">Laemmli Buffer</td>
					</tr>
					<tr>
						<td>Tris 0,5M, pH 6,8</td>
						<td>375 mM</td>
					</tr>
					<tr>
						<td>SDS</td>
						<td>6%</td>
					</tr>
					<tr>
						<td>Glycerol</td>
						<td>60%</td>
					</tr>
					<tr>
						<td>DTT</td>
						<td>0,6M</td>
					</tr>
					<tr>
						<td>Bromophenol blue</td>
						<td></td>
					</tr>
					<tr>
						<td>H2O</td>
						<td></td>
					</tr>
				</tbody>
			</table>
			</td>
		</tr>
	</tbody>
</table>

culture of isolated floor plate tissue and production of conditioned medium to assess functional properties of floor plate-released signals

在开发过程中，祖细胞和有丝分裂后神经元的调节自己的命运毗邻地区接收信号。地板板是一组神经胶质细胞衬里的室管膜运河在腹位置。地板板表示键形态发生素促进细胞谱系的脊髓中的图案形成。发育后期，地板板调节轴突在脊髓导航，作为一个屏障，以防止患侧轴突和控制中线交叉的道口通过合轴突1。这些功能是通过各种指导线索的分泌来实现。其中一些线索作为引诱和驱避剂的轴突生长，而其他规范指导受体和下游信号的轴突的灵敏度调节到当地指导线索2,3。这里我们描述了一种方法，它允许调查的地板板衍生的信号中的各种德韦的属性lopmental上下文的基础上，生产地板板条件培养基（FP 厘米）4-6。然后，我们举例说明在轴突导向的情况下使用这种FP 厘米 。首先，脊髓从小鼠胚胎中分离在E12.5和地板板被解剖出来并培养在血浆凝血酶基质（ 图1）。第二，两天后，连合组织是从E12.5胚胎解剖出来，研碎并暴露在FP 厘米 。三，组织处理对连合标记Western blot分析。

连合组织是由FP 厘米处理和对样品进行处理以供在Western印迹受体水平的分析。结果表明，增加的指导受体，PlexinA1，该交叉（ 图2），后介导合轴突的中线斥Semaphorin3B灵敏度水平的FP 厘米的应用。这表明，由FP释放信号调节PlexinA1水平4,6。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50884/50884fig1highres.jpg" src=...

Watch this Scientific Journal Video about Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals at JoVE.com

Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals

底板是脊髓发育，以祖扩散提供信号和分化的神经元的重要结构。我们描述了一种方法，以产生地面板材的条件培养基，将它应用到新鲜脊髓组织，并通过生化，以评估所关注的蛋白质的后果。

隔离楼板组织条件培养基和生产，以评估楼板释放信号的功能性质的文化

During development, progenitors and post-mitotic neurons receive signals from adjacent territories that regulate their fate. The floor-plate is a group of ...

culture-isolated-floor-plate-tissue-production-conditioned-medium-to

Research

JoVE Journal

Neuroscience

7.9K 次观看.  University of Lyon 1. 底板是脊髓发育，以祖扩散提供信号和分化的神经元的重要结构。我们描述了一种方法，以产生地面板材的条件培养基，将它应用到新鲜脊髓组织，并通过生化，以评估所关注的蛋白质的后果。 

视频: Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

一个在培养皿中的功能电机组:脊髓植和肌肉细胞共培养

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

科研

神经科学

Recording Human Electrocorticographic (ECoG) Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), magnetoencephalography or functional magnetic-resonance imaging. These techniques have either inherently low temporal or low spatial resolution, and suffer from low signal-to-noise ratio and/or poor high-frequency sensitivity. Thus, they are suboptimal for exploring the short-lived spatio-temporal dynamics of many of the underlying brain processes. In contrast, the invasive technique of electrocorticography (ECoG) provides brain signals that have an exceptionally high signal-to-noise ratio, less susceptibility to artifacts than EEG, and a high spatial and temporal resolution (i.e., &lt;1 cm/&lt;1 millisecond, respectively). ECoG involves measurement of electrical brain signals using electrodes that are implanted subdurally on the surface of the brain. Recent studies have shown that ECoG amplitudes in certain frequency bands carry substantial information about task-related activity, such as motor execution and planning1, auditory processing2 and visual-spatial attention3. Most of this information is captured in the high gamma range (around 70-110 Hz). Thus, gamma activity has been proposed as a robust and general indicator of local cortical function1-5. ECoG can also reveal functional connectivity and resolve finer task-related spatial-temporal dynamics, thereby advancing our understanding of large-scale cortical processes. It has especially proven useful for advancing brain-computer interfacing (BCI) technology for decoding a user's intentions to enhance or improve communication6 and control7. Nevertheless, human ECoG data are often hard to obtain because of the risks and limitations of the invasive procedures involved, and the need to record within the constraints of clinical settings. Still, clinical monitoring to localize epileptic foci offers a unique and valuable opportunity to collect human ECoG data. We describe our methods for collecting recording ECoG, and demonstrate how to use these signals for important real-time applications such as clinical mapping and brain-computer interfacing. Our example uses the BCI2000 software platform8,9 and the SIGFRIED10 method, an application for real-time mapping of brain functions. This procedure yields information that clinicians can subsequently use to guide the complex and laborious process of functional mapping by electrical stimulation.
Prerequisites and Planning:
Patients with drug-resistant partial epilepsy may be candidates for resective surgery of an epileptic focus to minimize the frequency of seizures. Prior to resection, the patients undergo monitoring using subdural electrodes for two purposes: first, to localize the epileptic focus, and second, to identify nearby critical brain areas (i.e., eloquent cortex) where resection could result in long-term functional deficits. To implant electrodes, a craniotomy is performed to open the skull. Then, electrode grids and/or strips are placed on the cortex, usually beneath the dura. A typical grid has a set of 8 x 8 platinum-iridium electrodes of 4 mm diameter (2.3 mm exposed surface) embedded in silicon with an inter-electrode distance of 1cm. A strip typically contains 4 or 6 such electrodes in a single line. The locations for these grids/strips are planned by a team of neurologists and neurosurgeons, and are based on previous EEG monitoring, on a structural MRI of the patient's brain, and on relevant factors of the patient's history. Continuous recording over a period of 5-12 days serves to localize epileptic foci, and electrical stimulation via the implanted electrodes allows clinicians to map eloquent cortex. At the end of the monitoring period, explantation of the electrodes and therapeutic resection are performed together in one procedure.
In addition to its primary clinical purpose, invasive monitoring also provides a unique opportunity to acquire human ECoG data for neuroscientific research. The decision to include a prospective patient in the research is based on the planned location of their electrodes, on the patient's performance scores on neuropsychological assessments, and on their informed consent, which is predicated on their understanding that participation in research is optional and is not related to their treatment. As with all research involving human subjects, the research protocol must be approved by the hospital's institutional review board. The decision to perform individual experimental tasks is made day-by-day, and is contingent on the patient's endurance and willingness to participate. Some or all of the experiments may be prevented by problems with the clinical state of the patient, such as post-operative facial swelling, temporary aphasia, frequent seizures, post-ictal fatigue and confusion, and more general pain or discomfort.
At the Epilepsy Monitoring Unit at Albany Medical Center in Albany, New York, clinical monitoring is implemented around the clock using a 192-channel Nihon-Kohden Neurofax monitoring system. Research recordings are made in collaboration with the Wadsworth Center of the New York State Department of Health in Albany. Signals from the ECoG electrodes are fed simultaneously to the research and the clinical systems via splitter connectors. To ensure that the clinical and research systems do not interfere with each other, the two systems typically use separate grounds. In fact, an epidural strip of electrodes is sometimes implanted to provide a ground for the clinical system. Whether research or clinical recording system, the grounding electrode is chosen to be distant from the predicted epileptic focus and from cortical areas of interest for the research. Our research system consists of eight synchronized 16-channel g.USBamp amplifier/digitizer units (g.tec, Graz, Austria). These were chosen because they are safety-rated and FDA-approved for invasive recordings, they have a very low noise-floor in the high-frequency range in which the signals of interest are found, and they come with an SDK that allows them to be integrated with custom-written research software. In order to capture the high-gamma signal accurately, we acquire signals at 1200Hz sampling rate-considerably higher than that of the typical EEG experiment or that of many clinical monitoring systems. A built-in low-pass filter automatically prevents aliasing of signals higher than the digitizer can capture. The patient's eye gaze is tracked using a monitor with a built-in Tobii T-60 eye-tracking system (Tobii Tech., Stockholm, Sweden). Additional accessories such as joystick, bluetooth Wiimote (Nintendo Co.), data-glove (5th Dimension Technologies), keyboard, microphone, headphones, or video camera are connected depending on the requirements of the particular experiment.
Data collection, stimulus presentation, synchronization with the different input/output accessories, and real-time analysis and visualization are accomplished using our BCI2000 software8,9. BCI2000 is a freely available general-purpose software system for real-time biosignal data acquisition, processing and feedback. It includes an array of pre-built modules that can be flexibly configured for many different purposes, and that can be extended by researchers' own code in C++, MATLAB or Python. BCI2000 consists of four modules that communicate with each other via a network-capable protocol: a Source module that handles the acquisition of brain signals from one of 19 different hardware systems from different manufacturers; a Signal Processing module that extracts relevant ECoG features and translates them into output signals; an Application module that delivers stimuli and feedback to the subject; and the Operator module that provides a graphical interface to the investigator.
A number of different experiments may be conducted with any given patient. The priority of experiments will be determined by the location of the particular patient's electrodes. However, we usually begin our experimentation using the SIGFRIED (SIGnal modeling For Realtime Identification and Event Detection) mapping method, which detects and displays significant task-related activity in real time. The resulting functional map allows us to further tailor subsequent experimental protocols and may also prove as a useful starting point for traditional mapping by electrocortical stimulation (ECS).
Although ECS mapping remains the gold standard for predicting the clinical outcome of resection, the process of ECS mapping is time consuming and also has other problems, such as after-discharges or seizures. Thus, a passive functional mapping technique may prove valuable in providing an initial estimate of the locus of eloquent cortex, which may then be confirmed and refined by ECS. The results from our passive SIGFRIED mapping technique have been shown to exhibit substantial concurrence with the results derived using ECS mapping10.
The protocol described in this paper establishes a general methodology for gathering human ECoG data, before proceeding to illustrate how experiments can be initiated using the BCI2000 software platform. Finally, as a specific example, we describe how to perform passive functional mapping using the BCI2000-based SIGFRIED system.

Recording Human Electrocorticographic ECoG Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

We present a method for collecting electrocorticographic signals for research purposes from humans who are undergoing invasive epilepsy monitoring. We show how to use the BCI2000 software platform for data collection, signal processing and stimulus presentation. Specifically, we demonstrate SIGFRIED, a BCI2000-based tool for real-time functional brain mapping.

神经科学的研究和实时功能皮层映射记录人力Electrocorticographic（ECOG）信号

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

解剖，文化与分析<em&gt;非洲爪蟾</em&gt;胚胎视网膜组织

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation of a given stimulus might be dependent on context and history. If this is actually the case, bi-directional interactions between the brain (or if need be a reduced model of it) and sensory-motor system can shed a light on how encoding and decoding of information is performed. Here an experimental system is introduced and described in which the activity of a neuronal element (i.e., a network of neurons extracted from embryonic mammalian hippocampi) is given context and used to control the movement of an artificial agent, while environmental information is fed back to the culture as a sequence of electrical stimuli. This architecture allows a quick selection of diverse encoding, decoding, and learning algorithms to test different hypotheses on the computational properties of neuronal networks.

In this paper, an experimental framework to perform closed-loop experiments is presented, in which information processing (i.e., coding and decoding) and learning of neuronal assemblies are studied during the continuous interaction with a robotic body.

闭环神经机器人实验来检验神经网络的计算性能

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

脑组织中的双管和同心微电极用于测量细胞外离子的信号

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young&#39;s elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

表征使用原子力显微镜，冲击压痕和流变测定脑组织的多尺度力学性能

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during development, homeostasis, and disease. Although macrophages have been studied in detail for decades, specialized features of microglia, the tissue-resident macrophages of the CNS, have remained largely mysterious, in part due to limitations in the ability to recapitulate mature microglial properties in culture. Here, we illustrate a straightforward procedure for the rapid isolation of pure microglia from the mature rodent brain. We also describe serum-free culture conditions that support high levels of microglial viability over time. Microglia cultured under these defined-medium conditions exhibit elaborate ramified processes and dynamic surveillance behavior. We illustrate some effects of serum exposure on cultured microglia and discuss how these serum-free cultures compare to both serum-exposed cultures as well as microglia in vivo.

Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions.

鼠小胶质细胞的分离培养促进无血清培养基动态分支形态学的研究

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

Presynapse Formation Assay Using Presynapse Organizer Beads and &ldquo;Neuron Ball&rdquo; Culture

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in appropriate order by transport from cell bodies and/or local translation in immature synapses. However, it is not fully understood how synaptic proteins accumulate in synapses in proper order. Here, we present a novel method to analyze presynaptic formation by using the combination of neuron ball culture with beads to induce presynapse formation. Neuron balls that is neuronal cell aggregates provide axonal sheets far from cell bodies and dendrites, so that weak fluorescent signals of presynapses can be detected by avoiding overwhelming signals of cell bodies. As beads to trigger presynapse formation, we use beads conjugated with leucine-rich repeat transmembrane neuronal 2 (LRRTM2), an excitatory presynaptic organizer. Using this method, we demonstrated that vesicular glutamate transporter 1 (vGlut1), a synaptic vesicle protein, accumulated in presynapses faster than Munc18-1, an active zone protein. Munc18-1 accumulated translation-dependently in presynapse even after removing cell bodies. This finding indicates the Munc18-1 accumulation by local translation in axons, not transport from cell bodies. In conclusion, this method is suitable to analyze accumulation of synaptic proteins in presynapses and source of synaptic proteins. As neuron ball culture is simple and it is not necessary to use special apparatus, this method could be applicable to other experimental platforms.

Presynapse formation is a dynamic process including accumulation of synaptic proteins in proper order. In this method, presynapse formation is triggered by beads conjugated with a presynapse organizer protein on axonal sheets of &#8220;neuron ball&#8221; culture, so that accumulation of synaptic proteins is easy to be analyzed during presynapse formation.

使用预突子形成测定使用预突子组织者珠和"神经元球"文化

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

从胚胎小鼠小脑分离的神经元的主要培养

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...