Name: isolation, culture and long-term maintenance of primary mesencephalic dopaminergic neurons from embryonic rodent brains
Uploaded: 2015-02-19T15:00:00.000+00:00
Duration: 8 min 45 s
Description: Watch this Scientific Journal Video about Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains at JoVE.com

This work was performed using the Division of Brain Sciences, Department of Medicine, Imperial College London startup funds to K.N.A.

注:该动物保持并遵守处理与制度的指导方针，所有动物的程序批准了帝国学院的动物福利和伦理审查机构（AWERB）以及内政部和哈佛大学的机构动物护理和使用委员会（IACUC）符合联邦和州法规。 1，试剂和设备安装<ol><li>解冻，分装和存储1日-80ºC毫克/毫升层粘连蛋白的解决方案。溶解2微升在1ml DMEM / F12，导致在1-2微克/厘米2的涂布的浓度。 注:解冻层粘连慢慢在冰上并溶解在冷的DMEM / F12，并立即将其添加到板/盖玻片。 </li><li>稀释75毫升0.01％聚-L-鸟氨酸425毫升的PBS，并储存于4ºC。 注:工作液的保质期长达一个月。 </li><li>溶解25％（重量/体积）BSA的PBS，pH 7.4中，并储存在4ºC长达一个咋AR。 </li><li>制备50％FBS（在HBSS中）的失活的介质。 </li><li> （+） - - 葡萄糖（重量/体积），0.25％的BSA（重量/体积），以通过添加50U / ml的青霉素和链霉素，1×N2添加剂，5％（体积/体积）胎牛血清，0.36％D制备的完全培养基DMEM / F12，并储存在4ºC。 注:保质期是10天。 </li><li>在沸腾的70％（体积/体积）乙醇为至少30分钟的地方盖玻片以除去油脂和高压釜任何痕迹。 </li><li>在24孔板的地方盖玻片并加入500μl聚-L-鸟氨酸溶液到每个孔中。孵育所述组织培养罩下1小时，在室温下，然后，洗3次，用500μl水。加入500μl层粘连蛋白的解决方案，以每块板和孵化O / N在湿润的组织培养孵化器在37ºC。 注意:虽然没有洗涤是必要的层粘连蛋白处理后，洗涤该聚-L-鸟氨酸小于三倍导致细胞的死亡培养后24小时。 </li><li>擦亮玻璃吸管超过天然气或火炬f显示提示跛脚。 注意:在此步骤之后的尖端的直径应为约一半正常大小的。 </li><li>削减1.5 ml离心管的盖子，用剪刀蒸压釜和。 </li></ol> 2.解剖胚胎中脑腹侧的<ol><li> Narcotize定时怀孕（E12.5）的CO 2吸入水坝，然后由颈椎脱位安乐死。 </li><li>喷用70％乙醇的腹部，使腹部切口，直到子宫囊都暴露出来。使用镊子削减阴道附件，删除子宫。放置在冰冷的HBSS子宫，在100毫米培养皿中。 </li><li>除去从子宫和羊膜囊胚胎，使用镊子（ 图1C）。地方胚胎在新鲜的冰冷的HBSS。 注: 图1D的矩形表示胚胎中脑腹侧的位置。 </li><li>将解剖显微镜（10倍放大倍率）在胚胎和解剖脑拱通过切割大脑在峡部和中脑-间脑边界区域，使用bioscissor和镊子（ 图1E，请参考图1A，用于切口线）。 </li><li>后取出整个脑，做了脑内侧背（ 图1F）削减。 </li><li>除去脑膜，使用两个镊子（ 图1H）。 注:关键步骤:此步骤是为最佳的神经元的产量和存活至关重要。由于培养条件是最佳的脑细胞中，大部分从周围组织中的细胞死电镀后，并从该组织中的细胞凋亡信号可能损坏培养物的完整性。 </li><li>拼合在培养皿中脑组织，观察蝴蝶造型和削减大约各占一半机翼，用消毒的剃须刀片（ 图1H，I）。 注: 图1J描绘了孤立的腹侧中脑。 </li><li>转让一块腹脑入冰冷的HBSS在一个15毫升的锥形管，然后继续下一个胚胎。 注意:步骤2.1-2.8不应超过1小时。保持胚胎超出在冰上这个时间帧显著降低细胞活力。 </li></ol> 3.解离腹侧中脑细胞<ol><li>传送下一个层流罩腹侧中脑的部分。移除HBSS和加入1 ml预热（37℃）0.05％胰蛋白酶-EDTA入锥形管（体积足够容纳12个腹侧中脑）。孵育所述组织在37ºC5-10分钟。 </li><li>除去胰蛋白酶-EDTA罩下和加入1毫升去活化介质（血清将停用胰蛋白酶）来组织。 </li><li>取出失活介质和洗涤在1ml完全培养基中的组织的两倍。 注:避免除去从管的所有介质和吸取的组织，以防止废弃离解的细胞。 </li><li>加入1ml完全培养基和三叔尿酸盐用火抛光的玻璃吸管的组织。避免形成气泡，并继续直到捣碎单细胞悬浮液来实现的。 注:通常情况下，8-10通过限制提示所需的完整的解离。 </li><li>离心细胞，在400×g离心5分钟，在室温并取出介质。 </li><li>重新暂停通过缓慢吹吸在1ml完全培养基中的沉淀和向下多达四次。 </li></ol> 4.电镀中脑腹侧细胞<ol><li>混合10微升细胞悬浮液与90微升锥虫蓝溶液，并用血细胞计数器计数细胞数。 注:细胞的生存力，也可以在此阶段进行检查。 </li><li>放置无菌微量离心管帽在100mM培养皿转移聚-L-鸟氨酸/层粘连蛋白包被的盖玻片，使用镊子，一次一个上盖帽的顶部。 注意:不要洗盖玻片，避免干燥的层粘连蛋白，因为这可能会导致uneveñ文化和减少细胞的生存能力。 </li><li>加入完全培养基调节音量，以每1微升1500细胞并在每个盖玻片的顶部添加细胞悬浮液的100微升（总共150,000个细胞的）（体积为最佳，以覆盖盖玻片的表面而不溢出）。关闭培养皿孵育它们1小时，在湿润的组织培养箱（37℃，5％的CO 2）。 注:关键步骤:此步骤是必需的用于增强细胞的附着和生活力并增加每个胚胎盖玻片的数量。或者，所述细胞可以直接转移到孔中（含有盖玻片），但是单元的数目应增加（而不是150000），以350000每孔。换句话说，使用这种方法，8-10盖玻片能够获得，而直接转移产生，因为细胞，其附着到所述板（旁或盖玻片下）为约3-4盖玻片。平均viabiliTY培养物，使用该方法是在90％左右。 </li></ol> 5.文化成长和维护<ol><li> 1小时温育后，小心地转移盖玻片包括介质到24孔板，含有400微升预热（37℃）的完全培养基（总体积:500微升）。孵育细胞O / N为37ºC。 </li><li>内孔中温育24小时后，轻轻地加入500微升完全培养基到每个孔中。 注:第几个小时为多巴胺能神经元的存活和存活细胞的数目不显著超过该点改变的最关键的阶段。 </li><li>不附加媒体添加到培养的头两个星期或直至介质变为黄色。如果对于超过两周或介质颜色变为黄色，介质的交换一半（500微升）与新鲜的完全预热介质（典型地每两个星期）培养。 注:内多巴胺能神经元培养将生存在这些条件下6周以上。 </li></ol>

<ol>
	<li>Prasad, K. N., 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=Establishment+and+characterization+of+immortalized+clonal+cell+lines+from+fetal+rat+mesencephalic+tissue.">Establishment and characterization of immortalized clonal cell lines from fetal rat mesencephalic tissue.</a> In Vitro Cell Dev Biol Anim. 30A, 596-603 (1994).</li><li>Fall, C. P., Bennett, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+time+course+of+MPP++-induced+apoptosis+in+human+SH-SY5Y+neuroblastoma+cells.">Characterization and time course of MPP+ -induced apoptosis in human SH-SY5Y neuroblastoma cells.</a> J Neurosci Res. 55, 620-628 (1999).</li><li>Choi, H. K., Won, L., Roback, J. D., Wainer, B. H., Heller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+modulation+of+dopamine+expression+in+neuronal+hybrid+cells+by+primary+cells+from+different+brain+regions.">Specific modulation of dopamine expression in neuronal hybrid cells by primary cells from different brain regions.</a> Proc Natl Acad Sci U S A. 89, 8943-8947 (1992).</li><li>Alavian, K. N., Sgado, P., Alberi, L., Subramaniam, S., Simon, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+P75NTR+expression+causes+death+of+engrailed-deficient+midbrain+dopaminergic+neurons+by+Erk1/2+suppression.">Elevated P75NTR expression causes death of engrailed-deficient midbrain dopaminergic neurons by Erk1/2 suppression.</a> Neural Dev. 4, 11 (2009).</li><li>Chung, C. Y., 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=The+transcription+factor+orthodenticle+homeobox+2+influences+axonal+projections+and+vulnerability+of+midbrain+dopaminergic+neurons.">The transcription factor orthodenticle homeobox 2 influences axonal projections and vulnerability of midbrain dopaminergic neurons.</a> Brain. 133, 2022-2031 (2010).</li><li>Betarbet, R., Sherer, T. B., Greenamyre, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+Parkinson's+disease.">Animal models of Parkinson's disease.</a> Bioessays. 24, 308-318 (2002).</li><li>Schober, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classic+toxin-induced+animal+models+of+Parkinson's+disease:+6-OHDA+and+MPTP.">Classic toxin-induced animal models of Parkinson's disease: 6-OHDA and MPTP.</a> Cell Tissue Res. 318, 215-224 (2004).</li><li>Chesselet, M. F., Richter, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+of+Parkinson's+disease+in+mice.">Modelling of Parkinson's disease in mice.</a> Lancet neurology. 10, 1108-1118 (2011).</li><li>Takeshima, T., Johnston, J. M., Commissiong, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesencephalic+type+1+astrocytes+rescue+dopaminergic+neurons+from+death+induced+by+serum+deprivation.">Mesencephalic type 1 astrocytes rescue dopaminergic neurons from death induced by serum deprivation.</a> J Neurosci. 14, 4769-4779 (1994).</li><li>Sgado, P., 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=Slow+progressive+degeneration+of+nigral+dopaminergic+neurons+in+postnatal+Engrailed+mutant+mice.">Slow progressive degeneration of nigral dopaminergic neurons in postnatal Engrailed mutant mice.</a> PNAS. 103, 15242-15247 (2006).</li><li>Alavian, K. N., 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=The+lifelong+maintenance+of+mesencephalic+dopaminergic+neurons+by+Nurr1+and+engrailed.">The lifelong maintenance of mesencephalic dopaminergic neurons by Nurr1 and engrailed.</a> J Biomed Sci. 21, 27 (2014).</li><li>Simon, H. H., Bhatt, L., Gherbassi, D., Sgado, P., Alberi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midbrain+dopaminergic+neurons:+determination+of+their+developmental+fate+by+transcription+factors.">Midbrain dopaminergic neurons: determination of their developmental fate by transcription factors.</a> Ann N Y Acad Sci. 991, 36-47 (2003).</li><li>Bjorklund, A., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+neuron+systems+in+the+brain:+an+update.">Dopamine neuron systems in the brain: an update.</a> Trends Neurosci. 30, 194-202 (2007).</li><li>Dauer, W., Przedborski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease:+mechanisms+and+models.">Parkinson's disease: mechanisms and models.</a> Neuron. 39, 889-909 (2003).</li><li>Dexter, D. T., Jenner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson+disease:+from+pathology+to+molecular+disease+mechanisms.">Parkinson disease: from pathology to molecular disease mechanisms.</a> Free Radic Biol Med. 62, 132-144 (2013).</li><li>Schapira, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aetiopathogenesis+of+Parkinson's+disease.">Aetiopathogenesis of Parkinson's disease.</a> J Neurology. 258, S307-S310 (2011).</li><li>Chung, C. Y., 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=Cell+type-specific+gene+expression+of+midbrain+dopaminergic+neurons+reveals+molecules+involved+in+their+vulnerability+and+protection.">Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection.</a> Hum Mol Gen. 14, 1709-1725 (2005).</li></ol>

No conflict of interest declared.

在中脑多巴胺能神经元是多巴胺在中枢神经系统中的主要来源。它们被分成三组，黑质致密部（黑质致密部），腹侧被盖区（VTA）和retrorubral字段（RRF）10，11。在黑质致密部和VTA的神经元产生重大多巴胺能通路，mesocortical，脑边缘和黑质纹状体，涉及的功能，如情感，动机和运动行为的控制。在黑质致密部和黑质纹状体系统的功能性破坏的神经元死亡是第二个最显着的神经变性疾病，帕?...

多巴胺能神经元从黑质损耗致密部导致帕金森氏病（PD），所述第二最常见的神经变性疾病的基本运动症状。这种脑的神经元群的灭亡的根本原因尚不清楚。研究负责开发和调制神经生理特性和mesDA神经元，几个细胞培养和动物模型系统的生存已使用的生化途径。永生化细胞系，包括大鼠多巴胺能细胞系1RB 3 AN 27（N27），所述的SH-SY5Y人的多巴胺能神经母细胞瘤细 ​​胞系，小鼠的多巴胺能杂交细胞系MN9D和人脑LUHMES细胞已被用于生化和有限的机理研究1 -5。对于mesDA神经元的具体损失的研究，几种神经毒素为基础和遗传模型已开发6-8。原发性脑腹侧文化，提供一个不可缺少的工具用于研究多巴胺能神经元的神经元和突触的属性和参与这一常见的疾病的发病机制中的通路。 这里我们提出了一个详细的协议，用于中脑多巴胺能神经元的分离，它含有导致更高的生存能力和每个胚胎盖玻片的增加的产量的修改。使用预成熟E12.5鼠脑（E14.5大鼠）提高了生存能力。在这个年龄段的神经元还没有开发轴突的是，它在清扫树叶细胞的完整，减少应力，从而提高显著生存能力。此外，小心解​​剖腹侧中脑，如在该协议的第2节描述的，进一步提高的生存能力。增加每个胚胎盖玻片的数量，另一种电镀方法，提出在该协议的第4条。这导致每个胚胎高达10盖玻片的收率相比，下4盖玻片标准电镀条件从而减少每个实验动物的数量。 在轴突和树突的文化展品生长的神经元，形成突触联系，揭示了神经元和突触的标记使这些文化适合活细胞成像，免疫细胞化学和电生理研究的存在。此外，使用的神经元培养物的促进基因和药理学操纵。 体外 2天的轴突生长允许发育研究。此外，培养物的长期存活（最多6周），使它们适合于这些神经元的速度慢，进行性变性的研究。

<table><tbody><tr><td>Dulbecco&#039;s modified Eagle medium nutrient mixture F-12</td><td>Invitrogen</td><td>11330</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS) (1X), liquid</td><td>Invitrogen</td><td>24020-117</td><td></td></tr><tr><td>Fetal bovine serum, heat-inactivated (FBS)</td><td>Invitrogen</td><td>16140</td><td></td></tr><tr><td>N2 Supplement (100X), liquid</td><td>Invitrogen</td><td>17502-048</td><td></td></tr><tr><td>D-(+)-Glucose solution (45% (wt/vol) in water</td><td>Sigma</td><td>G8769</td><td></td></tr><tr><td>Bovine serum albumin BSA</td><td>Sigma</td><td>A9430</td><td></td></tr><tr><td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td><td>Sigma</td><td>L2020</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Invitrogen</td><td>15070</td><td></td></tr><tr><td>Trypsin (0.05% (wt/vol)</td><td>Invitrogen</td><td>25300</td><td></td></tr><tr><td>Bovine serum albumin (BSA) cell culture tested</td><td>Sigma&amp;nbsp;</td><td>A9418</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Sigma</td><td>P3813</td><td></td></tr><tr><td>Anti-Tyrosine hydroxylase (Th) antibody</td><td>Pel-Freez Biologicals</td><td>P60101-0&amp;nbsp;</td><td></td></tr><tr><td>Poly-L-ornithine, 0.01% solution</td><td>Sigma</td><td>P4957</td><td></td></tr><tr><td>Anti-Map2 (Microtubule associated protein-2A and -2B) antibody</td><td>Millipore</td><td>MAB3418</td><td></td></tr><tr><td>Anti-Synapsin-1 antibody</td><td>Millipore</td><td>AB1543P</td><td></td></tr><tr><td>Alexa Fluor 488 donkey anti-rabbit IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-21206</td><td></td></tr><tr><td>Alexa Fluor 594 donkey anti-rabbit IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-21207</td><td></td></tr><tr><td>Alexa Fluor 488 donkey anti-sheep IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-11015</td><td></td></tr><tr><td>Alexa Fluor 594 donkey anti-sheep IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-11016</td><td></td></tr><tr><td>Alexa Fluor 594 donkey anti-mouse IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-21203</td><td></td></tr><tr><td>Trypan blue solution (0.4% (wt/vol))</td><td>Biowhittaker</td><td>17-942E</td><td></td></tr><tr><td>Stereo Microscope</td><td>Carl Zeiss</td><td>Stemi 2000-C</td><td></td></tr><tr><td>Inverted phase contrast microscope</td><td>Carl Zeiss</td><td>Axiovert 40 C</td><td></td></tr><tr><td>Dumont Forceps</td><td>Fine Scientific Tools</td><td>May-45</td><td></td></tr><tr><td>Cover Slip Forceps &amp;ndash; Dumoxel</td><td>Fine Scientific Tools</td><td>11251-33</td><td></td></tr><tr><td>Two Dumont #45 Forceps &amp;ndash; Dumoxel</td><td>Fine Scientific Tools</td><td>11245-30</td><td></td></tr><tr><td>Blade Holder/Breaker Flat Grip - 11cm</td><td>Fine Scientific Tools</td><td>10052-11</td><td></td></tr><tr><td>Student Iris Scissors - Straight 11.5 cm</td><td>Fine Scientific Tools</td><td>91460-11</td><td></td></tr><tr><td>Fiber optic halogen illuminator</td><td>Nikon</td><td>MKII</td><td></td></tr><tr><td>Disposable Borosilicate Glass Pasteur Pipettes</td><td>Fisherbrand</td><td>13-678-20C</td><td></td></tr><tr><td>Hemocytometer</td><td>Proscitech</td><td>SVZ2NIOU</td><td></td></tr><tr><td>0.2 &amp;micro;m sterile filter units</td><td>Nalgene</td><td>NL-CE-156-4020</td><td></td></tr><tr><td>100x20 mm Petri dishes</td><td>BD Biosciences</td><td>351005</td><td></td></tr><tr><td>Round Cover Slip #1 Thickness German Glass 12 mm</td><td>Bellco Glass</td><td>1943-10012&amp;nbsp;</td><td></td></tr></tbody></table>

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.

免疫组织化学对酪氨酸羟化酶（TH）表明，该细胞在培养中的0.5-1％之间的多巴胺。电镀后的2小时的神经元突起出现并通过第一天，轴突和树突是可区分（ 图2），使用酪氨酸羟化酶（TH）和微管相关蛋白2（MAP2）抗体（ 图3）。神经元存活超过六个星期，并显示大量的产物。在培养的神经元 - 神经胶质细胞比直接相关的血清的培养基中的浓度，如前所示。同时消除FBS从培?...

Watch this Scientific Journal Video about Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains at JoVE.com

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

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 ...

isolation-culture-long-term-maintenance-primary-mesencephalic

Research

JoVE Journal

Neuroscience

13.9K 次观看.  Imperial College London. The causes of degeneration of midbrain dopaminergic neurons during Parkinson’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.

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

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

小学游离中脑多巴胺鼠类新生儿的细胞培养

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

科研

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 ...

神经科学

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

大鼠胚胎神经细胞的分离和培养：一个快速议定书“

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

动物的基因分型快速其次是建立大脑神经元原代培养

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

从E14-E16斯普拉格·道利大鼠胚胎分离的混合原发希马和皮质神经元生成神经球

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

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 ...

 用于 CNS 相互作用研究的原代小胶质细胞和神经元共培养方案

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

作者聚焦:用于研究疾病条件下小胶质细胞-神经元相互作用的体外共培养模型

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

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

摘要

探索更多视频

此视频中的章节

小学游离中脑多巴胺鼠类新生儿的细胞培养

从产前小鼠海马神经元的分离和培养

大鼠胚胎神经细胞的分离和培养：一个快速议定书“

小鼠多巴胺能神经元的原代培养

动物的基因分型快速其次是建立大脑神经元原代培养

从E14-E16斯普拉格·道利大鼠胚胎分离的混合原发希马和皮质神经元生成神经球

从胚胎小鼠小脑分离的神经元的主要培养

生成和共培养小鼠原代小胶质细胞和皮质神经元

生成和共培养小鼠原代小胶质细胞和皮质神经元

生成和共培养小鼠原代小胶质细胞和皮质神经元