This study was supported by an NIH/NIMH grant to S.Q. (R00MH087628).

涉及小鼠所有的实验过程是由亚利桑那大学的机构动物护理和使用委员会批准，并符合美国国立卫生研究院的指导方针。 1，组织来源的海马神经元文化<ol><li>要生成海马神经元的文化产前的乳鼠，使用时间怀孕小鼠（C57BL6 / J）是在17家饲养。与阴道塞检测这一天被指定为E0.5。计划中的收获时间为文化E16.5-E17.5。 注意:此协议描述了两个高密度的神经元与低密度神经元共培养两个24孔板的培养物（48盖玻片总数）。为多个板，原料和试剂可相应地缩放。 </li></ol> 2. 24孔板制备高密度培养的神经元注:胚胎的计划收获前执行以下步骤（2-3）的一天。 <ol><li>把两个24以及板进入细胞培养罩，打开的单个包装。 </li><li> 18G的注射器针头连接到1毫升的塑料单次使用的注射器。 </li><li>握住注射器，并瞄准左到井的底部的中心约1毫米的针尖。施加适度的力（〜250克），以蚀刻孔（约1毫米长）的底部。的槽将形成和塑料材料取代将形成升高的支撑平底孔表面的上方。蚀刻在约1毫米另一个〜1mm的长平行槽底部的以类似的方式的中心的右侧。 </li><li>重复此步骤的两个24孔板的所有孔中。用于高密度培养两个24孔板现在已准备好与聚D-赖氨酸（见下文步骤3.8）的涂层。 </li></ol> 3.准备盖玻片低密度神经元培养<ol><li>使用12毫米直径的1号圆形玻璃。 </li><li>将50盖玻片一个100毫米直径的无菌塑料培养皿内，并淹没盖玻片在20毫升70％的乙醇溶液。放置一个旋转平台上此培养皿与一小时中等（70转）速度旋转。除去70％乙醇，然后用新鲜批次的70％乙醇取代。旋转和洗涤一个小时。 </li><li>丢弃70％的乙醇清洗液。加入无菌水（通过0.2微米的过滤器过滤单元）。通过旋转用手培养皿冲洗盖玻片严格。冲洗三次，每次用新鲜无菌水代替。 </li><li>放置培养皿与层流无菌细胞培养罩内的盖玻片。通过真空管抽吸漂洗水，和加入10 mL无菌过滤水浸泡的盖玻片。盖玻片应当是无菌的并且表面应干净够聚D-赖氨酸涂层。 </li><li>从他们的个人包装打开两个24孔板，并将它们放置在引擎盖。 </li><li>打开罩内的真空管线。一个200μl的枪头连接到真空管线，并使用SCISSOR切尖创造一个更大的开放。 </li><li>使用细尖＃5镊子从培养皿，每次一个拾取盖玻片，并使用真空线完全干燥盖玻片。然后将一个盖玻片到每个24孔板的孔中。 50清洗盖玻片应该足以填满每两个24孔板的孔中。 </li><li>稀释聚D-赖氨酸母液（1毫克/毫升）与硼酸盐缓冲液（pH 8.5）工作溶液（0.1毫克/毫升）。对于4个24孔板（包括蚀刻的底部有两个高密度板），准备30毫升工作总的解决方案。 </li><li>添加300μl的0.1mg / ml的聚D-赖氨酸工作溶液到各孔用玻璃盖玻片。确保盖玻片完全浸没在溶液中。如果不是，则使用＃5镊子前端向下压盖玻片针对井的底部，并使用前端引导聚-D-赖氨酸溶液以覆盖盖玻片的所有的表面上。目视检查所有的盖玻片，使苏再没有空气被困在板和盖玻片之间。 </li><li>添加300μl的0.1mg / ml的聚D-赖氨酸的工作溶液与蚀刻底部的高密度培养板的每个孔中。用手旋转传播溶液以覆盖井的整个底部。 </li><li>返回所有与D - 聚赖氨酸四大板块的培养孵化器，孵化和O / N。 </li></ol> 4.洗涤和预调板文化注意:下面的步骤（4-9）中对组织收获的当天进行。 <ol><li>孵化/涂层盖玻片24孔板O / N后，把所有四大板块（两次与捐助者的盖玻片，用蚀刻胶底部的两个受高密度板材）到文化引擎盖。使用真空线以除去聚-D-赖氨酸溶液。 </li><li>紧接在除去聚-D-赖氨酸的溶液，添加约1毫升无菌水到各孔用塑料移液管。之后所有的孔填充以水，静置10分钟。通过真空除去水，然后在每孔加入300μl的洗涤介质以覆盖孔的底部（有或没有玻璃盖玻片）。不要让D - 聚赖氨酸涂层干燥。 </li><li>返回所有四大板块的细胞培养箱。该板已经准备好一旦分散的海马神经元准备使用。 </li></ol> 5.完全培养基的制备和胰蛋白酶解酶消化<ol><li>通过混合成分（见材料）准备60毫升完全培养基。使用50毫升注射器用0.2微米孔径的注射器过滤器相连，该完全培养基过滤到两个50毫升锥形管中。每管含有30 ml完全培养基。 </li><li>在新的50ml锥形管，加入约30ml洗涤介质（见材料），预热至37℃。这将用于酶消化后洗涤组织。 </li><li>准备酶切网上平台。在15微米升锥形管中，加入4.5毫升洗涤介质和0.5ml 2.5％的胰蛋白酶溶液和50微升100X DNA酶I. </li><li>放置完全培养基，洗涤介质，和在37℃水浴酶切介质。 </li></ol> 6.准备手术工具<ol><li>消毒的所有外科手术工具在灭菌袋:两个小剪刀，一对钳子的两个＃5镊子。 </li></ol> 7.脱除E16.5-E17.5小鼠胚胎和解剖海马脑进行<ol><li>制备两个10厘米培养皿中填充有冰冷无菌汉克氏平衡盐溶液（HBSS）。 </li><li>安乐死鼠标坝与苯巴比妥钠（100mg / kg的中的一个体积约0.5毫升）腹膜内注射。注射后，一旦大坝失去响应脚趾捏，用牺牲颈椎脱位。 </li><li>喷水坝的用70％乙醇腹部区域，并且使沿腹部中线2厘米长的切口，以暴露子宫。 </li><li>取出胚，并放置个别的人在用冷的解剖HBSS缓冲液10厘米直径的陪替氏培养皿。 </li><li>持有使用镊子颈部胚胎，并使用小剪刀，使下面穿过脑组织小脑水平，部分中线首次下调权利。不要杀头的胚胎。 <ol><li>插入来自尾区的小剪刀被剖开的右侧末端，所有的方式，通过胚胎脑的延髓部分（不交叉中线），并且在颅底水平做出左侧削减。 </li><li>再次插入左侧剪刀尖，使右侧削减。在延髓端离开未切割颅骨和皮肤组织的窄带，使整个大脑可以预留容易提取被翻转。 </li><li>用剪刀尖的胚胎脑舀出到HBSS解决方案。检查在解剖显微镜下大脑。大脑应该是完好的，没有粗切邻FF的区域。 </li><li>重复上述步骤，收集所有的胚胎大脑。 </li></ol></li><li>收集在含有20毫升冷HBSS缓冲区的新培养皿所有完整的大脑。放置一个解剖显微镜下的培养皿。 </li><li>查看从腹侧大脑。按住与尾端钳大脑，将锋利的＃5镊子角，使中间的喙点和尾部颞区之间的削减。重复此为另一半球。切割，分离两半球与海马完整的从脑干组织的休息后。 <ol><li>重复此为每个脑筋。 </li></ol></li><li>池中的所有解剖半球，然后取出使用两对＃5镊子脑膜。 </li><li>识别显影海马作为被颞叶内折叠弯曲的结构。交替两对镊子的海马从邻接皮质组织中分离出来。收集，集中所有的海马组织PI组织（ 见图1）。 </li></ol> 8.酶消化，分离成单个神经元<ol><li>使用塑料移液管，传输所有解剖海马到含有0.25％胰蛋白酶+ 1X DNA酶I在15毫升锥形管5 1ml洗涤培养基中的酶消化溶液。 </li><li>放置在管的水浴内，并且在37°C孵育25分钟，用在每5分钟的管的间歇平缓反相一次。 </li><li> 25分钟后，小心地取出使用通过抽吸手持塑料吸管酶溶液。温水洗涤介质加入到10毫升并通过缓慢颠倒管5倍轻轻洗净组织。取出洗净媒体，然后再额外洗净加入10 mL洗涤介质。 </li><li>取出洗净媒体，然后再加入3毫升的新鲜温暖的洗涤介质。用手摇动该管严格进行15次，以从组织去除消化神经元。该介质应该成为浑浊一旦细胞再从组织分离成洗涤介质。收集细胞悬液在一个新的15毫升锥形管，同时留下在管内的剩余组织。 </li><li>另外2 1ml洗涤培养基添加到组织，并通过一个塑料吸管为〜15倍捣碎剩余组织轻轻分开。让我们坐5分钟。池细胞悬液到包含收集"动摇过"细胞新的15毫升锥形管。 </li><li>计数神经元与血球的密度。通常情况下，可以根据解剖胚胎的数目来获得每微升3,000-5,000细胞。如果假设6胚胎解剖估计为2.5×10 7个神经元的平均数。 </li><li>计算出所需该细胞悬浮液时加入30毫升完全培养基，得到25万个神经元/ ml的密度的体积。这个体积添加到装有30ml完全培养基，得到高密度的神经元电镀介质两个锥形管中的一个。 </li> &lt;利&gt;这种高密度的神经元溶液转移1.2毫升到另外的30毫升完全培养基，得到的〜万神经元/ ml的浓度。使用这种稀释种子低密度盖玻片。 </li></ol> 9.神经和长期共培养电镀<ol><li>带来与高密度的神经元到细胞培养罩蚀刻残渣的两个24孔板中。通过真空取出300微升前提网上平台。 250,000元/毫升板0.6毫升高密度细胞悬液。 </li><li>带来与盖玻片其他两个24孔板进培养罩，并通过真空除去300μl的前提介质。板0.6毫升低密度细胞悬浮于在两个24孔板内的盖玻片万个神经元/毫升。 </li><li>返回所有的四个24孔板的孵化器。让我们坐2小时。 </li><li>与附着神经元向高密度培养翻转低密度盖玻片。确保神经元彼此面对（ 即 nOT由玻璃盖玻片分开）。然后用共培养两个板返回孵化器。 </li></ol> 10.共培养可持续<ol><li>通过加入300微升新鲜饲料培养基（见材料）在一天体外 （DIV）5.没有必要去除原始完全培养基的50％，如通过许多其他研究报道订阅共培养物。馈送介质还含有15μM阿糖胞苷（阿糖胞苷，得到5微米的终浓度），以尽量减少神经胶质污染和扩散的可能性。 </li><li>以下这种初始进料，而不阿糖胞苷每周添加300μl的新鲜进料介质到孔中。应的培养物中保持超过一个月，用新鲜饲料培养基每周更换一半培养基的超过一个月的时间点（与第一介质半替换在DIV33）。形态上，无论是高密度和低密度的神经元看起来很健康长达三个月（由EXPER观测到的时间最长imenters）。 </li></ol> 11.插图的实验操作低密度神经元的转染注意:当转染在低密度神经元是期望的，一个简单的磷酸钙协议可以通过。我们已发现，低密度培养物与DIV12之前磷酸钙协议更好的转染效率，但它们可以在DIV21以上低得多的效率，在这期间树突棘是突出被转染。培养的低密度神经元的转染的可行性通过用质粒pEGFP-C3质粒转染的神经元中所示（见下文）。 <ol><li>在DIV21，取出600微升从共培养的各孔中的条件培养基，并转移到一个新的24孔板中。然后600μl的新鲜进料培养基添加到共培养物，使其回到原来体积。 <ol><li>转移盖玻片与600μl的条件培养基的新24孔板中。确保低密度的神经元朝上。将高密度板材和盖玻片回到了孵化器的新板块。 </li></ol></li><li>准备2个1.5毫升无菌试管。在一个管中，加入600μl的2×HEPES缓冲盐水（HBS）溶液。计算12微克的pEGFP-C3的DNA基于质粒浓度的体积。在另一管中，添加74微升的CaCl 2（2M股票）和水的体积，加入的质粒后将使600微升。吹打混匀，再加入质粒DNA。慢慢拌匀。让双方坐在管子在室温5分钟。 </li><li>虽然手握住2X HBS管和适度搅拌在混合器内的溶液，通过拖放到600微升2X HBS管添加的所有600微升的DNA的氯化钙溶液2滴。至关重要的是，形成的沉淀物是在&#39;细砂&#39;颗粒的形状。混合过强，太快是不可取的，因为它更可能产生大量"雪flake&#39;状沉淀，whicH具有基于我们的观察显着降低的转染效率。 </li><li>让沉淀物继续在RT暗处，以形成30分钟。 </li><li> 100微升沉淀物添加到包含在盖玻片低密度神经元每口井，一滴，均匀地下降。返回板孵化器，并孵育2小时。假定大多数的 CaCl 2是游离形式，这导致在〜17.6毫米的Ca 2+的浓度，这可能是有毒的以下长时间培养的神经元。 </li><li>准备用50〜1ml洗涤介质的50毫升锥形管，暖机至37℃。 </li><li>在2小时末，使用DNA-磷酸钙低密度神经沉淀到罩。拿起用锋利的＃5镊子每个盖玻片，蘸了三次在50ml的洗涤介质简单地清洗。然后它返回到高密度的神经元原文共培养板，以朝下低密度神经元。 </li><li>继续培养，直到盖玻片上低密度培养神经元收获的研究。 </li></ol>

<ol>
	<li>Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cultureing Nerve Cells. , 339-370 (1998).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat Protoc. 1, 2406-2415 (2006).</li><li>Petersen, J. D., Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+microtubule-based+transport+of+dendritic+membrane+proteins+arises+in+concert+with+axon+specification.">Selective microtubule-based transport of dendritic membrane proteins arises in concert with axon specification.</a> J Neurosci. 34, 4135-4147 (2014).</li><li>Seibenhener, M. L., Wooten, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+hippocampal+neurons+from+prenatal+mice.">Isolation and culture of hippocampal neurons from prenatal mice.</a> J Vis Exp. , (2012).</li><li>Shi, Y., Ethell, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+control+dendritic+spine+plasticity+in+hippocampal+neurons+through+NMDA+receptor+and+Ca2+/calmodulin-dependent+protein+kinase+II-mediated+actin+reorganization.">Integrins control dendritic spine plasticity in hippocampal neurons through NMDA receptor and Ca2+/calmodulin-dependent protein kinase II-mediated actin reorganization.</a> J Neurosci. 26, 1813-1822 (2006).</li><li>Viesselmann, C., Ballweg, J., Lumbard, D., Dent, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+and+primary+culture+of+embryonic+mouse+hippocampal+and+cortical+neurons.">Nucleofection and primary culture of embryonic mouse hippocampal and cortical neurons.</a> J Vis Exp. , (2011).</li><li>Biffi, E., Regalia, G., Menegon, A., Ferrigno, G., Pedrocchi, 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+influence+of+neuronal+density+and+maturation+on+network+activity+of+hippocampal+cell+cultures:+a+methodological+study.">The influence of neuronal density and maturation on network activity of hippocampal cell cultures: a methodological study.</a> PLoS One. 8, e83899 (2013).</li><li>Crump, F. T., Dillman, K. S., Craig, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cAMP-dependent+protein+kinase+mediates+activity-regulated+synaptic+targeting+of+NMDA+receptors.">cAMP-dependent protein kinase mediates activity-regulated synaptic targeting of NMDA receptors.</a> J Neurosci. 21, 5079-5088 (2001).</li><li>Leal, G., Afonso, P. M., Duarte, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+induces+synaptic+delivery+of+hnRNP+A2/B1+by+a+BDNF-dependent+mechanism+in+cultured+hippocampal+neurons.">Neuronal activity induces synaptic delivery of hnRNP A2/B1 by a BDNF-dependent mechanism in cultured hippocampal neurons.</a> PLoS One. 9, e108175 (2014).</li><li>Clarke, L. E., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+roles+of+astrocytes+in+neural+circuit+development.">Emerging roles of astrocytes in neural circuit development.</a> Nat Rev Neurosci. 14, 311-321 (2013).</li><li>Kaneko, A., Sankai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+of+rat+hippocampal+neurons+at+low+density+in+serum-free+medium:+combination+of+the+sandwich+culture+technique+with+the+three-dimensional+nanofibrous+hydrogel+PuraMatrix.">Long-term culture of rat hippocampal neurons at low density in serum-free medium: combination of the sandwich culture technique with the three-dimensional nanofibrous hydrogel PuraMatrix.</a> PLoS One. 9, e102703 (2014).</li><li>Millet, L. J., Bora, A., Sweedler, J. V., Gillette, M. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cellular+peptidomics+of+supraoptic+magnocellular+and+hippocampal+neurons+in+low-density+co-cultures.">Direct cellular peptidomics of supraoptic magnocellular and hippocampal neurons in low-density co-cultures.</a> ACS Chem Neurosci. 1, 36-48 (2010).</li><li>Salama-Cohen, P., Arevalo, M. A., Meier, J., Grantyn, R., Rodriguez-Tebar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGF+controls+dendrite+development+in+hippocampal+neurons+by+binding+to+p75NTR+and+modulating+the+cellular+targets+of+Notch.">NGF controls dendrite development in hippocampal neurons by binding to p75NTR and modulating the cellular targets of Notch.</a> Mol Biol Cell. 16, 339-347 (2005).</li><li>Xu, S. Y., Wu, Y. M., Ji, Z., Gao, X. Y., Pan, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+technique+for+culturing+primary+fetal+rat+cortical+neurons.">A modified technique for culturing primary fetal rat cortical neurons.</a> J Biomed Biotechnol. 2012, 803930 (2012).</li><li>McCarthy, K. D., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+separate+astroglial+and+oligodendroglial+cell+cultures+from+rat+cerebral+tissue.">Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.</a> J Cell Biol. 85, 890-902 (1980).</li><li>Qiu, S., Weeber, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reelin+signaling+facilitates+maturation+of+CA1+glutamatergic+synapses.">Reelin signaling facilitates maturation of CA1 glutamatergic synapses.</a> J Neurophysiol. 97, 2312-2321 (2007).</li><li>Qiu, S., Lu, Z., Levitt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MET+receptor+tyrosine+kinase+controls+dendritic+complexity,+spine+morphogenesis,+and+glutamatergic+synapse+maturation+in+the+hippocampus.">MET receptor tyrosine kinase controls dendritic complexity, spine morphogenesis, and glutamatergic synapse maturation in the hippocampus.</a> J Neurosci. 34, 16166-16179 (2014).</li></ol>

The authors declare that they have no competing financial interests.

我们提出了无血清条件下的超低密度的海马谷氨酸能神经元的长期培养一个详细的协议。在〜2000个神经元/厘米2，密度比具有或不具有通过现有的文献2,3,11,13,14报道胶质支持最"低密度"培养制剂至少低两倍。除了作为超低密度，该协议是新颖的和在两个方式显著。第一，不需要胶质饲养层作为低密度的神经元得到从高密度的神经元是物理非常接近的范围内的营养因子的支持，即使?...

在体外条件下生长的海马神经元能够观察和这些神经元是否则不可能在体内的实验操作。此实验方法被广泛地用于揭示增长，极性，轴突说明书中，贩卖和蛋白质的亚细胞定位，突触形成和功能成熟1的神经元的机制。 这些体外培养海马神经元，从后期胚胎阶段时采收，是比较纯粹的金 ​​字塔形态2的（&gt; 90％），谷氨酸能细胞。因为神经元中的2-D表面在体外条件下生长的，这种方法可以方便地观察，如实时成像或免疫细胞化学（ICC）通过单个焦平面3染色;或操作，比如药物治疗和转染3-6。当高密度的增长，神经元往往因为较高的共同生存率高在除了消化道的支持从生长媒介和，也因为突起接触依赖性机制分泌7生长因子ncentrations。然而，低密度的海马神经元是可取的形态学研究，其中一个单独的神经元可在其全部被成像或染色的ICC分析。低密度的神经元是很难培养维持由于缺乏旁分泌的支持，因此往往需要从胶质（通常为皮质星形胶质细胞）饲养层，它具有神经元文化2之前做好准备营养支持。当共同培养与神经胶质细胞饲养层，低密度的神经元生长在盖玻片上，然后在神经胶质细胞层的顶部翻转，使得低密度的神经元和神经胶质彼此面对。是由放置在盖玻片的角落石蜡点，因此创造了"三明治"布局2,8,9创建胶质细胞和神经元之间的一个小的密闭空间。低密度的神经元将增长W¯¯ithin胶质细胞和盖玻片，创建由神经元和神经胶质细胞分泌的因素集中一个宽容的微环境之间的密闭空间。这种方法产生的低密度，通过合理隔开充分发展的神经元，因此促进IC卡的标签或活成像研究。 神经胶质细胞共培养的表观缺点，除了是费时和费力的，是防止神经元特异性的，或细胞自主，机制研究。虽然这个系统比远小于复合体内神经组织，通过分泌的，还未完全限定因素对神经元发育神经胶质细胞的影响可以混淆实验10。因此，在需要的神经元​​特异性机制查，确定的培养条件除去血清和胶质支撑层是必要的实验。先前的研究已经成功地培养使用次低浓度的神经元（〜9000个/厘米2）稀土元素维水凝胶基质11。由于相对较纯的神经元群体可以在无需胶质支持无血清条件下的高密度进行培养，我们推测海马神经元的超低密度可以在无血清定义培养基由生长共培养它们与高密度的神经元，在的方式，是类似于常规采用神经胶质细胞共培养。事实上，在"三明治"结构的高密度的海马神经元的文化已经被用来支持少数专业大细胞神经内分泌12。 因此，共培养具有高密度的神经元可以允许低密度的神经元得到的营养因子支持足以使长期生存。由此培养超低密度神经元的这个协议配制和验证。该协议可以在一个单一实验中实现，通过制备高密度（〜250,000个细胞/ ml）的DISsociated海马神经元，然后再制作稀释，得到密度〜万个神经元/毫升（〜3000个神经元/盖玻片，或〜2000个神经元/厘米2），这比大多数报道的低密度培养物低得多的2,3,9 ，11,13。这种培养条件是松散称为"超低密度的文化和与&#39;低密度&#39;间可变地使用。高密度的神经元接种于聚-D-赖氨酸涂覆的24孔板;而低浓度的神经元上的聚-D-赖氨酸涂覆的12毫米的玻璃盖玻片接种被放置在另一个24孔板内。与粘附的低密度的神经元的盖玻片2小时后翻转的高密度的神经元的顶部的神经元被下降并附着在盖玻片后。此外，代替使用石蜡点以提升高密度的神经元层上面的盖玻片，18G的注射器针头被用于蚀刻24孔板的底部，具有两个平行的条纹。由此产生的显示终端ACED，毗邻塑料提供的盖玻片升高支持。该空间是在150-200微米，允许足够的氧和培养基交换，同时提供用浓营养因子微环境一致地测量。在这种条件下，低浓度的神经元广泛生长，并可以超出培养3个月生存。当这些神经元在培养三周后与GFP质粒转染的树突大汗与树突棘云集。原则上证明，数据都表明，这种合作培养体系支持接种在​​聚-D-赖氨酸的"微孤岛"，这里的神经元形成可以促进细胞的调查autaptic连接海马神经元的超低密度文化-autonomous，独立于网络的机制。

<table><tbody><tr><td>Neurobasal medium</td><td>Life Technologies</td><td>21103-049</td><td>Protect from light</td></tr><tr><td>B27 supplement</td><td>Life Technologies</td><td>17504-044</td><td>aliquot, store in 0.6ml size</td></tr><tr><td>GlutaMAX-I</td><td>Life Technologies</td><td>35050-061</td><td>dilute 100X&amp;nbsp;</td></tr><tr><td>antibiotic-antimycotic (AA)</td><td>Life Technologies</td><td>15240-096</td><td>dilute 100X&amp;nbsp;</td></tr><tr><td>Complete Culture medium</td><td></td><td></td><td>Neurobasal medium with 1X B27, 1X AA, 1X GlutaMAX-I</td></tr><tr><td>Wash medium</td><td></td><td></td><td>same as &#039;Neurobasal medium&#039;</td></tr><tr><td>Feed medium</td><td></td><td></td><td>Neurobasal with 1X B27 supplement</td></tr><tr><td>DNAse I</td><td>Sigma-Aldrich</td><td>D5025</td><td>prepare 100X stock at 0.6mg/ml</td></tr><tr><td>poly-D-lysine</td><td>Sigma-Aldrich</td><td>P6407</td><td>M.W. 70000-150000</td></tr><tr><td>borate buffer</td><td>Sigma-Aldrich</td><td>B6768 (boric acid); 71997(borax)</td><td>1.24g boric acid &amp;amp; 1.9g borax in 400ml H2O, pH to 8.5 use HCl</td></tr><tr><td>12-mm round glass coverslips</td><td>Glasswarenfabrik Karl Hecht GmbH</td><td>1001/12</td><td>No. 1 glass, purchase from Carolina Biological Supply</td></tr><tr><td>proFection transfection kit</td><td>Promega</td><td>E1200</td><td>see&amp;nbsp; protocol for details</td></tr><tr><td>2X HEPES buffered saline (HBS)</td><td>Promega</td><td>E1200</td><td>see&amp;nbsp; protocol for details</td></tr><tr><td>Syringe filter</td><td>Pall Corporation</td><td>4192</td><td>0.2um pore size</td></tr><tr><td>Endofree plasmid prep kit</td><td>Qiagen</td><td>12362</td><td>for preparation of transfection grade plasmid DNA</td></tr><tr><td>anti-MAP2 antibody</td><td>Millipore</td><td>MAB3418</td><td>mouse antibody, clone AP20</td></tr><tr><td>anti-p-Tau antibody</td><td>Millipore</td><td>AB10417</td><td>rabbit polyclonal antibody</td></tr><tr><td>anti-NR1 antibody</td><td>Millipore</td><td>MAB1586</td><td>mouse antibody, clone R1JHL</td></tr><tr><td>anti-GluR1 antibody</td><td>Millipore</td><td>AB1504</td><td>rabbit polyclonal antibody</td></tr><tr><td>Hank&#039;s balanced salt solution</td><td>ThermoFisher</td><td>14025092</td><td>500ml size</td></tr></tbody></table>

a simplified method for ultra-low density, long-term primary hippocampal neuron culture

在体外培养初级海马神经元促进了神经发育的许多方面机械审讯。解离的胚胎海马神经元通常可以成功地在玻璃盖玻片上以高密度生长在无血清条件下，但低密度培养物典型地需要通过营养因子的供给共培养它们与神经胶质饲养层，制备其中可费时又费力。此外，神经胶质细胞的存在可能混淆对神经元特异性的机制的结果和预先排除研究解释。在这里，一个简单的方法，提出了超低密度（〜2000元/厘米2），长期（&gt; 3个月）原海马神经元的文化，是无血清条件下，无胶质细胞的支持。低密度的神经元生长在聚-D-赖氨酸涂覆的盖玻片，并翻转在一个24孔板生长高密度的神经元。除了使用石蜡点来创造一个空间betwee的两个N神经细胞层，实验者可以简单地蚀刻井的底部塑料，对其中的高密度居住的神经元，创造一个有利于低密度神经生长微小空间。共培养可以很容易地维护&gt; 3个月无低密度的神经元显著损失，从而促进这些神经元的形态和生理研究。为了说明这一点成功的培养条件下，提供的数据显示长期培养后满头突触形成的低密度细胞。此共培养体系还便于在聚-D-赖氨酸衬底并因此autaptic连接的形成的岛屿生长稀疏单个神经元的存活。

这里所描述的协议使成功超低密度，纯谷氨酸能神经元的长期培养，而不需要作为饲养层胶质细胞。该协议可图示在图1中，其涉及制备高密度的（在聚D-赖氨酸包被的24孔）和低密度的神经元（在聚D-赖氨酸包被的玻璃盖玻片）分开，和随后的共培养，可以维持长达三个月。 图2A和2B是在?...

Watch this Scientific Journal Video about A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture at JoVE.com

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

的简化方法超低密度，长期主要海马神经元文化

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

a-simplified-method-for-ultra-low-density-long-term-primary

Research

JoVE Journal

Neuroscience

15.7K 次观看.  University of Arizona College of Medicine-Phoenix. Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms. 

视频: A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

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

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

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

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

科研

神经科学

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

神经调节和线粒体运输：在较长的时间里，海马神经元的实时成像

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Primary Culture of Hippocampal Neurons from P0 Newborn Rats

The physiological properties of hippocampal neurons are commonly investigated, especially because of the involvement of the hippocampus in learning and memory. Primary hippocampal cell culturing allows neuroscientists to examine the activity and properties of neurons at the individual cell and single synapse level. In this video, we will demonstrate how to isolate and grow primary hippocampal cells from newborn rats. The hippocampus may be isolated from each newborn animal in as short as 2 to 3 minutes, and the cultures can be maintained for up to two weeks. We will also briefly demonstrate how to use these hippocampal neurons for ratiometric calcium imaging. While this protocol describes the process for the hippocampus, with little to no modification, it can be applied to other regions of the brain.

The dissection and growth of cells from an individual brain area facilitates investigation of cellular and physiological parameters. We describe a method for primary cell culturing that produces neuron-enriched cultures in a serum-free environment.

从P0新生大鼠海马神经元的原代培养

The physiological properties of hippocampal neurons are commonly investigated, especially because of the involvement of the hippocampus in learning and ...

生物学

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

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

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

从产前小鼠海马神经元的分离和培养

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

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

低密度原海马神经元文化

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. Imaging studies are widely used to investigate their function and plasticity in physiological and pathological conditions. Because of their size and structure, localization studies of proteins require high-resolution imaging techniques. In this protocol, we describe a procedure to study in primary neurons the co-localization of target proteins with synaptic markers at a super-resolution level using structured illumination microscopy (SIM). SIM is a patterned-light illumination technique that doubles the spatial resolution of wide-field microscopy, reaching a detail of around 100 nm. The protocol indicates the required controls and settings for robust co-localization studies and an overview of the statistical methods to analyze the imaging data properly.

This protocol shows how to employ super-resolution microscopy to study protein co-localization in primary neuronal cultures.

超分辨率成像研究原发神经元中蛋白质和突触标记的共同定位

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. ...

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal neurons allows the study of individual neurons or subcellular components. We have shown subcellular protein localization within a neuron by immunocytochemistry, neuronal polarity, synaptic morphology, and its developmental change using a low-density primary hippocampal culture. Recently, ready-to-use frozen stocks of neurons have become commercially available. These frozen stocks of neurons reduce the time needed to prepare animal experiments and also contribute to the reduction of the number of animals used. Here, we introduce a reproducible low-density primary culture method using a 96-well plate. We used a commercially available frozen stock of neurons from the rat embryonic hippocampus. The neurons can be stably cultured long-term without media changes by reducing the growth of glial cells at particular timepoints. This high-throughput assay using low-density culture allows reproducible imaging-based evaluations of synaptic plasticity.

A ready-to-use frozen stock of neurons is a powerful tool for evaluating synaptic functions. Here, we introduce an easy low-density primary culture from frozen stock using a 96-well plate.

使用胚胎海马神经元冷冻原液进行简单且可重复的低密度原代培养

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal ...

A Technique to Culture Mouse Hippocampal Neurons

Source: Yang, R., et al. <a href="https://app.jove.com/v/61411">Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments.</a> J. Vis. Exp. (2021)
The video demonstrates a method for culturing neurons from murine hippocampi. Initially, the hippocampi undergo enzymatic digestion to break down the extracellular tissue matrix. Subsequently, the digested tissue is mechanically disrupted to isolate individual neurons. Finally, these neurons are cultured on glial feeder cells.

一种培养小鼠海马神经元的技术

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparing glia cell feeder dishes ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Generating a Low-Density Primary Neuron Culture From Frozen Embryonic Neurons

Source: Koganezawa, N. et al., <a href="https://app.jove.com/v/64872">Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons.</a>&#160;J. Vis. Exp. (2023)
This video demonstrates the method of culturing cryopreserved embryonic neurons, involving controlled thawing, dilution with a neurobasal medium, and seeding onto a poly-L-lysine-coated plate. Subsequent incubation supports the growth and maintenance of neuronal connections.

从冷冻胚胎神经元生成低密度原代神经元培养物

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Plate coating

	Coat a 96-well ...

An Indirect Neuron-Astrocyte Co-Culture Technique for Studying Neuron-Glia Interactions

Source: Gottschling, C. et al., <a href="https://app.jove.com/v/54757">The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions.</a>&#160;J. Vis. Exp.&#160;(2016)
This video illustrates a method for establishing an indirect neuron-astrocyte co-culture using a compartmentalized approach. In this setup, neurons and astrocytes are spatially separated but share a conditioned medium. Neurotrophic factors released by astrocytes promote neuron differentiation and the formation of synaptic connections, suggesting neuron-glia interactions.

一种用于研究神经元-胶质细胞相互作用的间接神经元-星形胶质细胞共培养技术

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...