Name: primary culture of mouse dopaminergic neurons
Uploaded: 2014-09-08T15:00:00.000+00:00
Duration: 11 min 58 s
Description: Watch this Scientific Journal Video about Primary Culture of Mouse Dopaminergic Neurons at JoVE.com

Supported by grants from CNRS and INSERM. PM acknowledges support from the Fondation pour la Recherche M&#233;dicale en France (Equipe FRM 2009). SC acknowledges support from the Fondation de France.

在这项工作中所用的小鼠照料并符合欧盟理事会（86/609 / EU）的使用实验动物的指导方针进行处理。 1，准备所需的解决方案<ol><li>库存解决方案<ol><li> 10X聚-L-鸟氨酸（巴解组织）溶液:称取10mg解放军氢溴酸盐（分子量= 30,000-70,000），溶于70毫升无菌水。筛选储存在-20℃下用0.2微米的针筒式过滤器，等分试样的溶液，和。 </li><li> 30％葡萄糖溶液:称取30克D（+） - 糖和无菌水溶解至100ml的总体积。过滤，分装，并储存在4℃。 </li><li> 5倍的Dulbecco改良的Eagle培养基（DMEM）/营养混合物F-12火腿:称取6克的DMEM / F-12火腿粉末和在无菌水中溶解至100ml的总体积。过滤，分装，并储存在4℃。 </li><li> 7.5％的碳酸氢钠（ 碳酸氢钠 ）Soluti于:称取7.5克的NaHCO 3和在无菌水中溶解至100ml的总体积。过滤，分装，并储存在4℃。 </li><li>的1M HEPES溶液:称取23.8克的HEPES和在无菌水中溶解至100ml的总体积。过滤，分装，并储存在4℃。 </li><li> 70％（V / V）乙醇稀释:将70ml无水乙醇溶液用30ml无菌水。 </li></ol></li><li>磷酸盐缓冲盐水与葡萄糖和抗生素（PBS-GAB）:加入10毫升30％的葡萄糖溶液和5毫升的青霉素 - 链霉素溶液至500ml Dulbecco氏磷酸盐缓冲盐水中。保存在4℃。 </li><li>灭活胎牛血清（IFBS）:解冻的牛血清一夜之间在4°C和灭活，在56℃下30分钟。等分入50ml并储存在-20℃。 </li><li>培养基:在灭菌水中，混匀依次40毫升5×DMEM / F12-火腿，加入1ml的1M HEPES，将2ml的200mM的L-谷氨酰胺，2个ml的青霉素 - 链霉素的加入4ml的30％的葡萄糖次加入3ml 7.5％的NaHCO 3，以180 ml ​​的终体积。过滤并使用相同的天。 </li><li>激素混合<ol><li>制备180毫升培养基。溶解200mg的脱辅基转铁蛋白的这种媒介。 </li><li>称取50毫克胰岛素的和溶解于2ml 0.1M的HCl中。加入慢慢加入8毫升无菌水混合的同时轻轻地。稀释此溶液在培养基中。 </li><li>称取19.3毫克腐胺二盐酸盐和溶解在10ml的无菌水。加入10 ml溶液的激素组合。 </li><li>制备在无菌水中的3mM的亚硒酸钠溶液并在无水乙醇中的2mM的孕酮溶液。加入20μl每个解决方案的激素组合。 </li><li>过滤激素混合，分装到10ml，并储存在-20℃。 </li></ol></li></ol> 2，准备培养板和仪器<ol><li>培养皿中的FBS涂层:先解剖当天，准备180毫升培养基。加入10毫升IFBS到90毫升培养介质中。加入300μl/孔的培养基/ 10％IFBS到聚-D-赖氨酸预涂布的24孔板。孵育过夜，在37℃。在4℃下储存剩余的介质（有或无IFBS）。 </li><li>消毒器械和巴氏吸管。聚集在材料表中列出，以及一个II级层流罩和CO 2培养箱设备。 </li></ol> 3，解剖小鼠中脑的<ol><li> （根据机构的指导方针）牺牲一个E13.5孕鼠。清洁鼠标，用70％乙醇的腹部和打开腹部壁。收集的子宫角，用细腻的剪刀打开它们，从子宫角解剖每个胚胎，取出羊膜。将胚胎在含有无菌PBS-GAB 100毫米的培养皿中，用血管钳在连续3个相同的浴用转移把它们洗干净。对于剥离，将胚胎由3 60mm的无菌培养皿中。 </li><li> MOVË在体视显微镜下最后的培养皿中。不要杀头的胚胎。使用Vannas剪刀，切除大脑。 <ol><li>小心地取出并丢弃fore-和后脑区域。以喙侧，切断靠近丘脑区域，以尾侧切口在峡部区域，除去上丘。 </li><li>一旦中脑腹侧被隔离，小心地取出使用的超精细镊子脑膜。收集解剖段，无脑膜，在无菌13毫升套管内填充的PBS-GAB。 </li></ol></li><li>清洁管彻底含有mesencephala用70％乙醇，并把它罩下。 </li></ol> 4，细胞分离<ol><li>洗mesencephala 3倍用无菌PBS-GAB。让大脑碎片每次冲洗之间解决，避免破坏组织。最后一次洗涤后，在3毫升胰蛋白酶/ EDTA的15分钟的仔细去除尽可能多的解决方案，尽可能孵育脑段，在37℃CO 2的培养箱中培养。 </li><li>火抛光的巴斯德吸液管以最大化神经元作为非抛光的玻璃移液管的端部的存活是锋利的，并且可以在解离步骤，损伤细胞。稍微减轻肢体的直径（最大0.5mm）的巴斯德吸管，而火抛光的。 </li><li>小心除去尽可能多的胰蛋白酶/ EDTA溶液尽可能和加入10 ml培养基/ 10％IFBS。洗脑段与3倍的培养基/ 10％IFBS。 </li><li>用火抛光的巴斯德吸液管，开始在6ml培养基/ 10％IFBS的mesencephala的离解。磨碎10X（避免产生气泡），使块定居，然后收集在一个新的无菌13毫升管中。 </li><li>加入6毫升培养介质中，重复前面的步骤一次，并收集含有解离的细胞在相同13毫升管的平台。 </li><li>离心细胞，在160 g下5分钟。弃上清，悬浮细胞培养液中添加了10％的激素组合。 </li></ol> 5，细胞接种<ol><li>计数细胞悬液中Malassez细胞和调节介质的体积的60万个细胞/ ml的浓度。周围1700000细胞可以从一个小鼠中脑获得。 </li><li>除去含FBS的涂覆​​介质从24孔板中，并添加于各孔的1 ml的细胞悬浮液（600,000个细胞/孔，TH +细胞的2-4％）。 </li><li>孵育板在37℃和5％CO 2/95％空气。无介质的改变是必要的。多巴胺能神经元后， 在体外各地5-7天（多巴胺转运体的表达相一致）的成熟。保持文化的长达15天不更换培养基细胞。 </li></ol> 6，免疫荧光协议<ol><li>盖玻片清洗<ol><li>解剖前一天，加入10毫升1 M盐酸到培养皿中。放置在所述液体12-15盖玻片的表面上，等待15分钟。 </li><li>沉封面潜入该液体，然后等待15分钟。 </li><li>除去HCl和水洗涤3次。 </li><li>一次，用纯乙醇快速洗涤盖玻片，然后加入纯乙醇的培养皿，并等待30分钟。 </li><li>引擎盖下，从乙醇中取出清洁的盖玻片上，并加入用无菌镊子12孔细胞培养板中的1盖玻片每孔。 </li><li>冲洗用无菌水（1ml /孔）盖玻片两次。然后，用无菌PBS洗涤一次它们。 </li></ol></li><li>盖玻片的涂布<ol><li>除去PBS中，加入500μl/ 2倍巴解组织以及（PBS稀释）。孵育4小时，在37℃下在CO 2培养箱中培养。 </li><li>删除巴解组织溶液和洗涤3次，用无菌的PBS。 </li><li>除去PBS，加入500微升/孔的20％IFBS / 1克/毫升的层粘连蛋白。在CO 2培养箱中孵育过夜，在37℃。 </li></ol></li><li>电镀的细胞免疫<ol><li>从12孔板中除去涂层介质。 </li><li>添加900,000个细胞/孔的2ml体积，并在培养箱中生长的细胞中，在37℃。细胞可以保持在培养最多15天而不更换培养基。 </li></ol></li><li>免疫组化<ol><li>从12孔板中除去培养基，并用500微升/孔的DMEM预热至37℃。 </li><li>加入500μl/孔的DMEM中预热至37℃，再加入微升轻轻160 /孔8％多聚甲醛（PFA，2％的最终浓度），并孵育10分钟，在37℃。 </li><li>除去PFA，洗3次，用PBS / 0.1M甘氨酸10分钟。 </li><li>除去PBS，加入300微升/孔的PBS / 0.05％的Triton X-100/20％山羊血清和温育30分钟，在室温下进行。 </li><li>除去培养基，加入300微升/孔的在PBS中稀释的第一抗体/ 0.05％的Triton X-100/1％山羊血清孵育2小时，在室温下进行。可用于多巴胺能神经元和培养特性的检测初级抗体被显示在材料表中。保持1以及无第一抗体，以评估不同的第二抗体的背景。 </li><li>除去培养基洗3次，用PBS / 0.2％明胶的10分钟。 </li><li>除去培养基，加入300微升/孔的在PBS中稀释的二级抗体/ 0.05％的Triton X-100/1％山羊血清孵育1小时，在室温下，在黑暗中。使用的二级抗体中注明的材料表中。 </li><li>除去培养基洗3次，用PBS / 0.2％明胶的10分钟。 </li><li>除去培养基，用PBS洗3次。 </li><li>装在载玻片上的细胞一侧的盖玻片下来一滴的Vectashield的。保持载玻片在室温下过夜，以让该安装介质干燥，然后将它们存储在4℃。 </li><li>掌握用共聚焦显微镜或装备表面荧光相差显微镜图像。被收购与徕卡SP2的紫外激光共聚焦显微镜的代表图像。 </li></ol></li></ol>

<ol>
	<li>Glowinski, J., Cheramy, A., Romo, R., Barbeito, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+regulation+of+dopaminergic+transmission+in+the+striatum.">Presynaptic regulation of dopaminergic transmission in the striatum.</a> Cell Mol Neurobiol. 8, 7-17 (1988).</li><li>Iversen, S. D., Iversen, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine:+50+years+in+perspective.">Dopamine: 50 years in perspective.</a> Trends Neurosci. 30, 188-193 (2007).</li><li>Dahlstroem, A., Fuxe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+Existence+of+Monoamine-Containing+Neurons+in+the+Central+Nervous+System+I.+Demonstration+of+Monoamines+in+the+Cell+Bodies+of+Brain+Stem+Neurons.">Evidence for the Existence of Monoamine-Containing Neurons in the Central Nervous System I. Demonstration of Monoamines in the Cell Bodies of Brain Stem Neurons.</a> Acta Physiol Scand Suppl. SUPPL. , 231-255 (1964).</li><li>Chinta, S. J., Andersen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+neurons.">Dopaminergic neurons.</a> Int J Biochem Cell Biol. 37, 942-946 (2005).</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>Hegarty, S. V., Sullivan, A. M., O'Keeffe, G. W. <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:+a+review+of+the+molecular+circuitry+that+regulates+their+development.">Midbrain dopaminergic neurons: a review of the molecular circuitry that regulates their development.</a> Dev Biol. 379, 123-138 (2013).</li><li>Samii, A., Nutt, J. G., Ransom, B. R. <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.">Parkinson's disease.</a> Lancet. 363, 1783-1793 (2004).</li><li>Santini, E., Heiman, M., Greengard, P., Valjent, E., Fisone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+mTOR+signaling+in+Parkinson's+disease+prevents+L-DOPA-induced+dyskinesia.">Inhibition of mTOR signaling in Parkinson's disease prevents L-DOPA-induced dyskinesia.</a> Sci Signal. 2, (2009).</li><li>Ohlin, K. E., 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=Vascular+endothelial+growth+factor+is+upregulated+by+L-dopa+in+the+parkinsonian+brain:+implications+for+the+development+of+dyskinesia.">Vascular endothelial growth factor is upregulated by L-dopa in the parkinsonian brain: implications for the development of dyskinesia.</a> Brain. 134, 2339-2357 (2011).</li><li>Obeso, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Missing+pieces+in+the+Parkinson's+disease+puzzle.">Missing pieces in the Parkinson's disease puzzle.</a> Nat Med. 16, 653-661 (2010).</li><li>Cooper, O., 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=Pharmacological+rescue+of+mitochondrial+deficits+in+iPSC-derived+neural+cells+from+patients+with+familial+Parkinson's+disease.">Pharmacological rescue of mitochondrial deficits in iPSC-derived neural cells from patients with familial Parkinson's disease.</a> Sci Transl Med. 4, (2012).</li><li>Michel, P. P., Toulorge, D., Guerreiro, S., Hirsch, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+needs+of+dopamine+neurons+for+stimulation+in+order+to+survive:+implication+for+Parkinson+disease.">Specific needs of dopamine neurons for stimulation in order to survive: implication for Parkinson disease.</a> FASEB J. 27, 3414-3423 (2013).</li><li>Decressac, M., Volakakis, N., Bjorklund, A., Perlmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NURR1+in+Parkinson+disease-from+pathogenesis+to+therapeutic+potential.">NURR1 in Parkinson disease-from pathogenesis to therapeutic potential.</a> Nat Rev Neurol. , (2013).</li><li>Jouve, L., Salin, P., Melon, C., Le Goff, L. K. e. r. k. e. r. i. a. n. -. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+brain+stimulation+of+the+center+median-parafascicular+complex+of+the+thalamus+has+efficient+anti-parkinsonian+action+associated+with+widespread+cellular+responses+in+the+basal+ganglia+network+in+a+rat+model+of+Parkinson's+disease.">Deep brain stimulation of the center median-parafascicular complex of the thalamus has efficient anti-parkinsonian action associated with widespread cellular responses in the basal ganglia network in a rat model of Parkinson's disease.</a> J Neurosci. 30, 9919-9928 (2010).</li><li>Hirsch, E. C., Jenner, P., 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=Pathogenesis+of+Parkinson's+disease.">Pathogenesis of Parkinson's disease.</a> Mov Disord. 28, 24-30 (2013).</li><li>di Porzio, U., Daguet, M. C., Glowinski, J., Prochiantz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+striatal+cells+on+in+vitro+maturation+of+mesencephalic+dopaminergic+neurones+grown+in+serum-free+conditions.">Effect of striatal cells on in vitro maturation of mesencephalic dopaminergic neurones grown in serum-free conditions.</a> Nature. 288, 370-373 (1980).</li><li>Denis-Donini, S., Glowinski, J., Prochiantz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+heterogeneity+may+define+the+three-dimensional+shape+of+mouse+mesencephalic+dopaminergic+neurones.">Glial heterogeneity may define the three-dimensional shape of mouse mesencephalic dopaminergic neurones.</a> Nature. 307, 641-643 (1984).</li><li>Barbin, G., Mallat, M., Prochiantz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+studies+on+the+maturation+of+mesencephalic+dopaminergic+neurons.">In vitro studies on the maturation of mesencephalic dopaminergic neurons.</a> Dev Neurosci. 7, 296-307 (1985).</li><li>Marey-Semper, I., Gelman, M., Levi-Strauss, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+selective+toxicity+toward+cultured+mesencephalic+dopaminergic+neurons+is+induced+by+the+synergistic+effects+of+energetic+metabolism+impairment+and+NMDA+receptor+activation.">A selective toxicity toward cultured mesencephalic dopaminergic neurons is induced by the synergistic effects of energetic metabolism impairment and NMDA receptor activation.</a> J Neurosci. 15, 5912-5918 (1995).</li><li>Salthun-Lassalle, B., Hirsch, E. C., Wolfart, J., Ruberg, M., Michel, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+mesencephalic+dopaminergic+neurons+in+culture+by+low-level+stimulation+of+voltage-gated+sodium+channels.">Rescue of mesencephalic dopaminergic neurons in culture by low-level stimulation of voltage-gated sodium channels.</a> J Neurosci. 24, 5922-5930 (2004).</li><li>Toulorge, D., 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=Neuroprotection+of+midbrain+dopamine+neurons+by+nicotine+is+gated+by+cytoplasmic+Ca2.">Neuroprotection of midbrain dopamine neurons by nicotine is gated by cytoplasmic Ca2.</a> FASEB J. 25, 2563-2573 (2011).</li><li>Rousseau, E., Michel, P. P., Hirsch, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Iron-Binding+Protein+Lactoferrin+Protects+Vulnerable+Dopamine+Neurons+from+Degeneration+by+Preserving+Mitochondrial+Calcium+Homeostasis.">The Iron-Binding Protein Lactoferrin Protects Vulnerable Dopamine Neurons from Degeneration by Preserving Mitochondrial Calcium Homeostasis.</a> Mol Pharmacol. 84, (2013).</li><li>Orme, R. P., Bhangal, M. S., Fricker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcitriol+imparts+neuroprotection+in+vitro+to+midbrain+dopaminergic+neurons+by+upregulating+GDNF+expression.">Calcitriol imparts neuroprotection in vitro to midbrain dopaminergic neurons by upregulating GDNF expression.</a> PLoS One. 8 (e62040), (2013).</li><li>Choi, W. S., Kruse, S. E., Palmiter, R. D., Xia, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+complex+I+inhibition+is+not+required+for+dopaminergic+neuron+death+induced+by+rotenone,+MPP+,+or+paraquat.">Mitochondrial complex I inhibition is not required for dopaminergic neuron death induced by rotenone, MPP+, or paraquat.</a> Proc Natl Acad Sci U S A. 105, 15136-15141 (2008).</li><li>Trancikova, A., Ramonet, D., Moore, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mouse+models+of+neurodegenerative+diseases.">Genetic mouse models of neurodegenerative diseases.</a> Prog Mol Biol Transl Sci. 100, 419-482 (2011).</li><li>Gao, H. M., Liu, B., Zhang, W., Hong, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+of+microglial+NADPH+oxidase-derived+free+radicals+in+the+in+vitro+MPTP+model+of+Parkinson's+disease.">Critical role of microglial NADPH oxidase-derived free radicals in the in vitro MPTP model of Parkinson's disease.</a> FASEB J. 17, 1954-1956 (2003).</li><li>Lin, X., 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=Conditional+expression+of+Parkinson's+disease-related+mutant+alpha-synuclein+in+the+midbrain+dopaminergic+neurons+causes+progressive+neurodegeneration+and+degradation+of+transcription+factor+nuclear+receptor+related+1.">Conditional expression of Parkinson's disease-related mutant alpha-synuclein in the midbrain dopaminergic neurons causes progressive neurodegeneration and degradation of transcription factor nuclear receptor related 1.</a> J Neurosci. 32, 9248-9264 (2012).</li><li>Bye, C. R., Thompson, L. H., Parish, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birth+dating+of+midbrain+dopamine+neurons+identifies+A9+enriched+tissue+for+transplantation+into+parkinsonian+mice.">Birth dating of midbrain dopamine neurons identifies A9 enriched tissue for transplantation into parkinsonian mice.</a> Exp Neurol. 236, 58-68 (2012).</li><li>Ramonet, D., 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=Dopaminergic+neuronal+loss,+reduced+neurite+complexity+and+autophagic+abnormalities+in+transgenic+mice+expressing+G2019S+mutant+LRRK2.">Dopaminergic neuronal loss, reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2.</a> PLoS One. 6 (e18568), (2011).</li><li>Prestoz, L., Jaber, M., Gaillard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+axon+guidance:+which+makes+what.">Dopaminergic axon guidance: which makes what.</a> Front Cell Neurosci. 6, (2012).</li><li>Nunes, I., Tovmasian, L. T., Silva, R. M., Burke, R. E., Goff, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pitx3+is+required+for+development+of+substantia+nigra+dopaminergic+neurons.">Pitx3 is required for development of substantia nigra dopaminergic neurons.</a> Proc Natl Acad Sci U S A. 100, 4245-4250 (1073).</li><li>Ferri, A. L., 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=Foxa1+and+Foxa2+regulate+multiple+phases+of+midbrain+dopaminergic+neuron+development+in+a+dosage-dependent+manner.">Foxa1 and Foxa2 regulate multiple phases of midbrain dopaminergic neuron development in a dosage-dependent manner.</a> Development. 134, 2761-2769 (2007).</li><li>Rayport, S., 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=Identified+postnatal+mesolimbic+dopamine+neurons+in+culture:+morphology+and+electrophysiology.">Identified postnatal mesolimbic dopamine neurons in culture: morphology and electrophysiology.</a> J Neurosci. 12, 4264-4280 (1992).</li><li>Kim, K. M., Nakajima, S., Nakajima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+and+GABA+receptors+in+cultured+substantia+nigra+neurons:+correlation+of+electrophysiology+and+immunocytochemistry.">Dopamine and GABA receptors in cultured substantia nigra neurons: correlation of electrophysiology and immunocytochemistry.</a> Neuroscience. 78, 759-769 (1997).</li><li>Nefzger, C. M., 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=Lmx1a+allows+context-specific+isolation+of+progenitors+of+GABAergic+or+dopaminergic+neurons+during+neural+differentiation+of+embryonic+stem+cells.">Lmx1a allows context-specific isolation of progenitors of GABAergic or dopaminergic neurons during neural differentiation of embryonic stem cells.</a> Stem Cells. 30, 1349-1361 (2012).</li><li>Su, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+expression+of+Cdh2+is+essential+for+efficient+neural+differentiation+of+mouse+induced+pluripotent+stem+cells.">Immediate expression of Cdh2 is essential for efficient neural differentiation of mouse induced pluripotent stem cells.</a> Stem Cell Res. 10, 338-348 (2013).</li></ol>

The authors have nothing to disclose.

这个协议提出必要从胚胎小鼠和免疫荧光法检测多巴胺能神经元的制备中脑的神经元的原代培养物的方法和试剂。关键步骤的程序是在胚胎的清扫和收集的脑碎片的机械解离。高品质的解剖工具，有助于掌握解剖技术。 DA能神经元构成脑的一小部分。因此，收集的腹侧中脑的右侧部分是必不可少的，以获得含有多巴胺神经元的2-4％的培养。机械解离应仔细并轻轻进行。如果文化包含集群的非游离?...

多巴胺，基本脑神经递质1,2中的一个，主要是释放由脑多巴胺能（DA）神经元。大多数DA能神经元位于中脑2-6的腹侧部分。概略地，中脑多巴胺神经元可在三个解剖和功能划分不同的投影系统:mesostriatal，中脑边缘和mesocortical通路2,5。黑质纹状体通路参与运动行为，中脑边缘通路在加强，动机和学习中发挥重要作用，而多巴胺能途径投射到额叶前部皮质有牵连的认知2。 多巴胺神经元参与了几个人的神经系统疾病如精神分裂症，注意力不足，过度活动症和帕金森氏病（PD）2,4。帕金森病的特点是DA能神经元的进行性和选择性变性连接黑质致密部（SNC）的纹状体。的黑质-纹状体多巴胺神经元的结果在纹状体严重多巴胺耗竭的损失，是负责对帕金森病的运动症状（运动迟缓，静止性震颤，和刚性）7。的特发性帕金森病的初始原因尚未确定与当前治疗是唯一的症状，目的是恢复的多巴胺水平在纹状体。最常用的处方药物是左旋多巴（左旋多巴），多巴胺的前体自然。尽管左旋多巴的给药补偿多巴胺在一定时间内的损耗，运动并发症后发生的长期治疗（运动障碍和开/关状态）8,9。 研究多巴胺能神经元和PD是在不断发展和正在作出巨大努力来开发基于细胞移植，基因治疗，或神经保护剂10,11治疗。然而，一个重要问题仍然是非阐明:什么是极端vulnerab的原因DA能神经元的能性？部分答案可以在DA能神经元的活性被发现。在DA能神经元的兴奋性电活动和减速似乎增加他们的倾向退化12。然而，帕金森病发病机制的复杂性，需要进一步研究，以确定神经元变性13-15参与多巴胺的机制。 小学文化，特别是有关研究多巴胺神经元的特性16-19和对神经保护剂20-24评估挑战这些神经元的各种压力。大鼠培养模型是最常用的，如大鼠胚胎中脑的解剖比较容易，比较用鼠标，以及较高量的神经元可以在大鼠中获得。然而，新一代的25病转基因小鼠模型大大提高了社区的神经学家小学文化，从鼠标26-29的兴趣。虽然文化公关从新生动物epared都可以使用，最好是它们的胚胎在有丝分裂后的阶段（E13.5的中脑的神经元）制备，当神经元都保留其分化能力。以下协议呈现在来自小鼠胚胎（E13.5），这是最困难的，制备原代培养物分离的脑的神经元。值得注意的是，我们提供了使用无血清培养基更好的重现性的协议。在培养制备物（夹层和机械解离）的两个最重要的步骤将被仔细地详述于相关联的视频。

<table><tbody><tr><td>Fetal Bovine Serum</td><td>Lonza</td><td>14-801F</td><td></td></tr><tr><td>DMEM 4.5 g/L glucose with L-glutamine</td><td>Lonza</td><td>BE12-604F</td><td></td></tr><tr><td>0.05% Trypsin-EDTA (1x), phenol red</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>L-Glutamine, 200 mM solution</td><td>Life Technologies</td><td>25030123</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D8537</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium/Nutrient Mixture F-12 Ham</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D0547</td><td>Powder</td></tr><tr><td>Laminin - 1 mg/ml in Tris buffered NaCl</td><td>Sigma-Aldrich&amp;nbsp;</td><td>L2020</td><td></td></tr><tr><td>Poly-L-Ornithine hydrobromide</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P3655</td><td></td></tr><tr><td>Insulin from porcine pancreas</td><td>Sigma-Aldrich&amp;nbsp;</td><td>I5523</td><td></td></tr><tr><td>apo-Transferrin human</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T1147</td><td></td></tr><tr><td>Putrescine dihydrochloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P5780</td><td></td></tr><tr><td>Progesterone</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P8783</td><td></td></tr><tr><td>Sodium selenite</td><td>Sigma-Aldrich&amp;nbsp;</td><td>S5261</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich&amp;nbsp;</td><td>H4034&amp;nbsp;</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G7126</td><td>Stock solution 1 M in water</td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G9391</td><td>Stock solution 2% (w/v) in water</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T8532</td><td></td></tr><tr><td>Paraformaldehyde 16% in water</td><td>Electron Microscopy Sciences</td><td>RT 15710-S</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Merck Millipore</td><td>106329</td><td></td></tr><tr><td>D(+)-Glucose, Monohydrate</td><td>Merck Millipore</td><td>4074-2</td><td></td></tr><tr><td>Hydrochloric acid - c(HCl) = 1 mol/L (1 N) Titripur</td><td>Merck Millipore</td><td>109057</td><td></td></tr><tr><td>Sterile water - Aqua B. Braun</td><td>Braun</td><td></td><td></td></tr><tr><td>Ethanol absolute NORMAPUR analytical reagent</td><td>VWR</td><td>20821.321</td><td></td></tr><tr><td>Sterile Petri dishes</td><td>VWR</td><td>82050-566</td><td></td></tr><tr><td>Pasteur pipettes plain glass - Wilhem Ulbrich GdbR.</td><td>VWR</td><td>612-2297</td><td></td></tr><tr><td>Counting chamber Malassez</td><td>VWR</td><td>631-0975</td><td></td></tr><tr><td>Serum Acrodisc Syringe Filter with Supor Membrane, Sterile, GF/0.2 &amp;micro;m, 37 mm</td><td>PALL Life science</td><td>4525</td><td></td></tr><tr><td>Surgical Scissors - Straight, sharp-sharp, 14.5 cm long</td><td>Fine Science Tools</td><td>14002-14</td><td>To open the abdominal wall</td></tr><tr><td>Scissors - Straight, pointed, delicate, 10 cm long</td><td>MORIA</td><td>4877A</td><td>To open the uterine wall</td></tr><tr><td>Forceps - Curved, usual, serrated jaws 1 mm</td><td>MORIA</td><td>2183</td><td>To manipulate embryos</td></tr><tr><td>Vannas Scissors - Curved, pointed, 7 mm blades</td><td>MORIA</td><td>MC50</td><td>To take out the mesencephalon</td></tr><tr><td>Ultra Fine Forceps - Curved, delicate, 13 cm long</td><td>MORIA</td><td>9987</td><td>To remove meninges</td></tr><tr><td>BD BioCoat Poly-D-Lysine 24-well Multiwell Plates</td><td>BD Bioscience</td><td>356414</td><td></td></tr><tr><td>BD Falcon 12-well Cell Culture Plate, flat-bottom with lid</td><td>BD Bioscience</td><td>353043</td><td></td></tr><tr><td>SuperFrost Microscope Slides, Ground edges 90&amp;ordm;</td><td>MENZEL-GL&amp;Auml;SER</td><td>AG00008032E</td><td></td></tr><tr><td>Precision cover glasses thickness No. 1.5H circular 18 mm &amp;Oslash;</td><td>MARIENFELD</td><td>117580</td><td></td></tr><tr><td>Polyclonal Rabbit Anti-Microtubule-Associated Protein 2 (MAP2) Antibody</td><td>Chemicon Millipore</td><td>AB5622</td><td>1/200</td></tr><tr><td>Monoclonal Mouse Anti-Glutamate Decarboxylase (GAD67) Antibody, clone 1G10.2</td><td>Chemicon Millipore</td><td>MAB5406</td><td>1/400</td></tr><tr><td>Monoclonal Rat Anti-Dopamine Transporter (DAT) Antibody, clone DAT-Nt&amp;nbsp;</td><td>Chemicon Millipore</td><td>MAB369</td><td>1/500</td></tr><tr><td>Monoclonal Mouse Anti-5-HT Antibody</td><td></td><td></td><td>1/8,000 - Generous gift from Yves Charnay (Swizerland, Yves.Charnay@hcuge.ch)</td></tr><tr><td>Goat Serum, New Zealand Origin</td><td>Life Technologies</td><td>16210-064</td><td></td></tr><tr><td>Alexa Fluor 405 Goat Anti-Rabbit IgG (H+L) Antibody</td><td>Life Technologies</td><td>A-31556</td><td>1/200</td></tr><tr><td>Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody</td><td>Life Technologies</td><td>A-11001</td><td>1/1,000</td></tr><tr><td>Alexa Fluor 594 Goat Anti-Rat IgG (H+L) Antibody</td><td>Life Technologies</td><td>A-11007</td><td>1/1,000</td></tr><tr><td>VECTASHIELD HardSet Mounting Medium</td><td>Vector Laboratories</td><td>H-1400</td><td></td></tr><tr><td>Stereomicroscope</td><td>Carl Zeiss microscopy</td><td>Stemi-2000C</td><td></td></tr><tr><td>Bunsen Burner FIREBOY</td><td>VWR</td><td>451-0136</td><td></td></tr></tbody></table>

primary culture of mouse dopaminergic neurons

多巴胺能神经元代表了在大脑中的神经元的总数的不到1％。这样低的神经元的数量调节重要脑部功能，如马达控制，动机和工作记忆。黑质纹状体多巴胺能神经元的选择性退化在帕金森氏病（PD）。这种渐进的神经元损失明确的病理症状电机（运动迟缓，静止性震颤和肌强直）有关。负责任的多巴胺能神经元退变主要代理仍是未知数。然而，这些神经元似乎是在各种条件下非常脆弱。原代培养物构成的最相关的模型之一来调查性能和多巴胺能神经元的特征。这些培养物可以以阻止或减缓神经元变性提交模仿PD病理各种应激剂和神经保护性化合物。 PD的大量的转基因小鼠模型中对已根儿在过去十年中ATED进一步增加研究者对多巴胺能神经元培养物的兴趣。在这里，视频协议侧重于胚胎小鼠大脑的细腻解剖。腹侧的精确切除是至关重要的，获得神经元培养足够丰富的多巴胺能细胞，使随后的研究。此协议可实现与胚胎转基因小鼠，并适合免疫荧光染色，定量PCR，第二信使量化，或神经元死亡/存活评估。

的中脑培养步骤的图示流程图如图1所示。简言之，从怀孕的瑞士小鼠收集E13.5胚胎后，腹侧被从整个胚胎解剖。在分离脑片段依次提交酶促消化和机械解离。解离的细胞，通过离心沉淀，重新悬浮在培养基中，并接种在预​​先包被12或24孔板中。将细胞保持15天不更换培养基。 腹侧中脑解剖的详细流程图，对应于步骤3.2中，示于图2。虚线红色线条表...

Watch this Scientific Journal Video about Primary Culture of Mouse Dopaminergic Neurons at JoVE.com

Primary Culture of Mouse Dopaminergic Neurons

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 ...

primary-culture-of-mouse-dopaminergic-neurons

Research

JoVE Journal

Neuroscience

37.7K 次观看.  Institut de Génomique Fonctionnelle, Montpellier. 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.

视频: Primary Culture of Mouse Dopaminergic Neurons

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

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 ...

生物学

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

丰富的少突胶质细胞培养和Myelinating产后小鼠组织联合培养的少突胶质细胞/神经元的推导

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

一个鼠标，两种文化：隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

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 ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

生活多巴胺能神经元可靠的鉴定中脑培养使用RNA测序和TH-启动子驱动的EGFP表达

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

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 ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

研究原发性胚胎小鼠中脑多巴胺神经元中预制纤维诱导β-合成素积累

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Culturing and Maintaining Dopaminergic Neurons from Mouse Embryonic Brain Tissue

Source: Weinert, M. et al., <a href="https://app.jove.com/v/52475">Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains.</a> J. Vis. Exp. (2015)
This video outlines a method for cultivating dopaminergic neurons from mouse embryonic brain tissue. The process includes treating the midbrain with a proteolytic enzyme, detaching the cells, and culturing them on polymer-coated coverslips in a nutrient-rich medium with antibiotics for their growth and long-term viability.

从小鼠胚胎脑组织中培养和维持多巴胺能神经元

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

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Studying Alpha-Synuclein Accumulation in Primary Embryonic Mouse Dopamine Neurons

Source: Er, S. et al., <a href="https://app.jove.com/v/61118">Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons.</a>&#160;J. Vis. Exp. (2020).
This video demonstrates the process of quantifying alpha-synuclein accumulation in embryonic mouse dopamine neurons pre-treated with alpha-synuclein pre-formed fibrils. It outlines the steps for fixing, staining, and quantifying the dopamine neurons containing alpha-synuclein aggregates using immunofluorescence.

研究原代胚胎小鼠多巴胺神经元中的 α-突触核蛋白积累

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1.&#160;Preparation

	Prepare ...