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>

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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，即使没有突触连接，或在不同的密度培养的神经元之间的直接接触。这个协议消除了对未来的计划培养实验的制备胶质单层的需要，并且同时允许高密度和低密度培养物同时生长。神经胶质单层的制备可以是费时和​​Laborious，大部分文献使用的乳鼠小狗皮质2。因此，对于只小鼠工作的实验室中，这需要显著作出更多努力。更重要的是，在神经元的特定的机制进行了研究实验中，在共培养的神经胶质细胞的存在可能是不希望的，因为它可以防止归咎于观察到的效果具体地神经元，神经胶质细胞或两种细胞类型10之间的相互作用。此外，对于胶质细胞培养，血清补充是强制性的2,15，这可能污染胶质神经元共培养和引入额外的干扰因素。我们的协议中的一个重要特征是，共培养是确定的培养基条件下进行。我们严格使用无血清补充的完全培养基中，并且发现，如果在培养基交换时间表严格遵循，共培养可以超过三个月的持续，这应该满足大多数实验。因为实际上没有神经胶质细胞的存在，这种​​技术的一个限制是，应谨慎与文献的使用胶质神经元共培养物的结果进行比较时行使。 这个协议的另一个新颖的方面是，我们使用了一个简单的方法，以建立一个有效的生长在24孔板的底部，高密度的神经元之间，并附着在玻璃盖玻片上的正上方坐在低密度神经元微空间。以前的研究使用应用到涂盖玻片石蜡圆点〜胶质滋养层2,8,9高于500微米，以提高盖玻片。石蜡点的制备需要合适的尺寸和合适的温度，它要求的努力和实践的合理费用。相比较而言，在井底部塑料简单蚀刻用18G的针提供了神经元的两个层之间的盖玻片支持以及良好的分离。我们用膜片钳记录台数字仪表ž确定林通过集中在高密度，然后用60X水浸物镜的低密度层两个层的神经元之间的耳的空间，并且发现，该空间通常是150-200微米（179.4 +/- 25.3微米，每组5 ）。此较窄的空间（与由蜡点产生相比）是可能转化到更快的上升，以及神经营养因子浓度较高，因此提供用于低密度神经生长足够的支持。虽然这个较窄空间可以提高对培养基交换速率，以及是否较小的空间阻碍从得到的氧气和营养支持的神经元的问题，我们的研究结果表明，这是不太可能关注。相反，我们发现，在盖玻片下的高密度的神经元与来自相同的制备，但没有高密度的神经元相比，也长得更好（较高的存活率，更精细的树突树和由NR1染色更突触泪点，未示出数据）上覆COVerslips。因此，这种简单的协议不仅速度快，操作简单，而且在维持了高密度和低密度的神经元非常有效。 在体外培养的海马神经元需要生长促进基材如聚-D-赖氨酸和/或层粘连蛋白/胶原5,16的涂层。虽然细致选择和制备的玻璃盖玻片的是有效的神经元存活的关键，我们发现，与我们所选择的玻璃盖，用70％乙醇彻底清洗是足够的，从而否定严厉治疗的需要（ 例如 ，在酸的沸点）的盖玻片。盖玻片在乙醇清洗，并通过真空干燥之后，将玻璃似乎是疏水因原涂层。然而，为0.1毫克成功涂层/ ml聚-D-赖氨酸硼酸盐缓冲液应该呈现玻璃表面的亲水性，这样，当将盖玻片从清洗水，水均匀层拾取即均匀在整个玻璃表面的扩展可以被观察到。我们发现，这是极为重要的。否则，将神经元由于电镀涂层不足后最有可能死亡。虽然只有低密度海马神经元在这个协议中进行测试，预期其他神经元类型（ 例如 ，皮质神经元，小脑粒神经元，或专门的神 ​​经种群）也可以以低密度在高密度的神经元馈电的存在下持续层。最后，当这种简单的协议之后，可以期望收获健康超低密度的神经元。广泛的应用，如免疫细胞化学标记，实时成像，形态学研究和电记录都可以在按照标准步骤16,17这些神经元进行。

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 border="0" cellpadding="0" cellspacing="0" width="713">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Neurobasal medium</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:144px;">21103-049</td>
			<td style="width:200px;">Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">B27 supplement</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:144px;">17504-044</td>
			<td>aliquot, store in 0.6ml size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">GlutaMAX-I</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:144px;">35050-061</td>
			<td>dilute 100X&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">antibiotic-antimycotic (AA)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:144px;">15240-096</td>
			<td>dilute 100X&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Complete Culture medium</td>
			<td style="width:135px;"></td>
			<td style="width:144px;"></td>
			<td>Neurobasal medium with 1X B27, 1X AA, 1X GlutaMAX-I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Wash medium</td>
			<td style="width:135px;"></td>
			<td style="width:144px;"></td>
			<td>same as &#39;Neurobasal medium&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Feed medium</td>
			<td style="width:135px;"></td>
			<td style="width:144px;"></td>
			<td>Neurobasal with 1X B27 supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">DNAse I</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:144px;">D5025</td>
			<td>prepare 100X stock at 0.6mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-D-lysine</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:144px;">P6407</td>
			<td>M.W. 70000-150000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">borate buffer</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:144px;">B6768 (boric acid); 71997(borax)</td>
			<td>1.24g boric acid &#38; 1.9g borax in 400ml H2O, pH to 8.5 use HCl</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">12-mm round glass coverslips</td>
			<td style="width:135px;">Glasswarenfabrik Karl Hecht GmbH</td>
			<td style="width:144px;">1001/12</td>
			<td>No. 1 glass, purchase from Carolina Biological Supply</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">proFection transfection kit</td>
			<td style="width:135px;">Promega</td>
			<td style="width:144px;">E1200</td>
			<td>see&#160; protocol for details</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">2X HEPES buffered saline (HBS)</td>
			<td style="width:135px;">Promega</td>
			<td style="width:144px;">E1200</td>
			<td>see&#160; protocol for details</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Syringe filter</td>
			<td style="width:135px;">Pall Corporation</td>
			<td style="width:144px;">4192</td>
			<td>0.2um pore size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Endofree plasmid prep kit</td>
			<td style="width:135px;">Qiagen</td>
			<td style="width:144px;">12362</td>
			<td>for preparation of transfection grade plasmid DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">anti-MAP2 antibody</td>
			<td style="width:135px;">Millipore</td>
			<td style="width:144px;">MAB3418</td>
			<td>mouse antibody, clone AP20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">anti-p-Tau antibody</td>
			<td style="width:135px;">Millipore</td>
			<td style="width:144px;">AB10417</td>
			<td>rabbit polyclonal antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">anti-NR1 antibody</td>
			<td style="width:135px;">Millipore</td>
			<td style="width:144px;">MAB1586</td>
			<td>mouse antibody, clone R1JHL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">anti-GluR1 antibody</td>
			<td style="width:135px;">Millipore</td>
			<td style="width:144px;">AB1504</td>
			<td>rabbit polyclonal antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Hank&#39;s balanced salt solution</td>
			<td style="width:135px;">ThermoFisher</td>
			<td style="width:144px;">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是在铺板之后5天的高密度和低密度的神经元的插图。低密度文化约为30倍，密度较小。通过Kaech和银行家（2006年）所列出两个神经元发生的典型发展阶段。在第14天，无论是高密度和低密度的神经元显示精细树枝状结构，由DIC图像（ 图2C所揭示，D，注意两个面板从与不同的焦平面相同的字段，如由刻蚀的位置示出）。当这些神经元与MAP2抗体染色以显示树突，树突（ 叔 13 = 1.27，P&gt; 0.05;可以通过树枝状尖数目来衡量）的数量没有显著差异和总树突长度（ 叔 16 = 1.41，P&gt; 0.05 ）每高密度和低密度的神经元之间的神经元被找到（ 图2E中的F）。 免疫组化标记是用来揭示功能谷氨酸突触。我们使用双重染色标记的NMDA受体亚基NR1和AMPA受体亚基GluR1的（ 图3A），以及功能性突触（ 黄色共定位泪点）可以很容易地识别和使用ImageJ定量。低密度的神经元上，也有对树突状蛋白标志物MAP2抗体，在轴突蛋白标记对头组合。这双标记允许树突和轴突（ 图3B）的明显的区别。低密度培养物也可成功地与DNA质粒使用磷酸钙方法转染，虽然转染率通常是在稍后阶段（&lt;0.2％的神经元时在DIV21染）非常低。 图3C示出了EGFP的转染可以揭示神经元的形态，和渲染精细树突棘的结构可见。最后，作为概念证明，超低密度的神经元，可以促进autapse形成（ 图3D）的条件下生长。生长在D - 聚赖氨酸涂层"微孤岛"这些超低密度autaptic神经元可以通过高密度的进纸器神经元持续到超过2个月，这个共培养协议。 <img alt="图1" src="/files/ftp_upload/53797/53797fig1.jpg" /> &lt;STRONG&gt;图1.原理的高密度和低密度联合培养协议的（A）所示。E16.5-E17.5小鼠胚胎的大脑收集，海马解剖并用胰蛋白酶消化。 （B）消化海马洗涤，分离成单个神经元，并以高密度铺板（250,000个细胞/ ml）在24孔板;在此同时，低密度（10,000个细胞/ ml）的神经元被镀上盖玻片。对于高密度培养的孔用18G的注射器针头蚀刻，以提供对盖玻片升高的支持。 （C）共培养的插图。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53797/53797fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53797/53797fig2.jpg" /> 图2.两种高密度和低密度的神经元具有共同在共培养mparable形态发育。（A，B）显微照片示出了高密度和低密度层的铺板密度。 （C，D）的DIC图像，显示在高（℃）广泛的神经突生长和低密度（D）的神经元。盖玻片低密度的神经元都休息权高密度的神经元以上。请注意隆起塑料支撑的位置。树突状蛋白MAP2（ 五 ）免疫组化标记。 （F）定量（平均值±SEM）总树突长度和每个神经元的树突状尖号。无显著差（ 纳秒 ）被视为高密度和在共同培养中生长的低密度神经元之间。在（B，D），100微米。比例尺<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53797/53797fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> FO:保together.within页="1"&gt; <img alt="图3" src="/files/ftp_upload/53797/53797fig3.jpg" /> 图 3. 共培养种植密度低的神经元的实验操作。（A）的低密度培养免疫细胞化学标记允许通过的GluR1（ 绿色 ）和NR1（ 红色 ）的共同标签定义的功能突触的鉴定。 （B）神经元树突状蛋白MAP2（ 品红色 ）的联合标识和轴突蛋白P-头（ 绿色 ）。 （C），低密度海马神经元转染pEGFP-C3质粒转染，呈现出丰富的树突棘。 （D）聚维赖氨酸"微岛"准备低密度的神经元，并生长在高密度的神经元之上。的记录膜片电极填充用Alexa-555酰肼的神经元以显示神经元的形态。比例尺在公元20微米。7fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

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

Nanyang Technological University

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

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

抛光和钢筋，颅骨变薄窗口，为长期的小鼠脑成像

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

长期的嗅觉适应性的分子读数<em&gt; C.线虫</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

长时程增强一个非常稳定的和可重复的记录和保存海马鼠标片制备工艺的改进

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

自由游泳弱电鱼的长期行为跟踪

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

琼脂糖微室的长期钙成像<em&gt;秀丽隐杆线虫</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

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 ...

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-&#946; (A&#946;) peptide.

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

封闭系统中脑切片长期培养中精确定位和重复间歇成像的改进辊筒法

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

自由生育小鼠的长期感觉冲突

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...