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.

Bu çalışmada kullanılan farenin bakım ve Avrupa Birliği Konseyi kurallarına laboratuvar hayvanlarının kullanımı için (86/609 / AB) uyarınca ele alınmıştır. Gerekli Çözümleri 1. Hazırlık <ol><li> Hazır Çözümler <ol><li> 10x Poli-L-Ornitin (PLO) Çözüm: PLO hidrobromid 10 mg tartılır (molekül ağırlığı = 30,000-70,000) doldurulmuş ve steril, 70 ml su içinde çözülür. -20 ° C de 0.2 um şırınga filtresi, kısım kullanılarak çözeltisi ve mağaza Filtre. </li><li> % 30 glükoz solüsyonu: 30 D (+) g tartın - Glukoz ve 100 ml&#39;lik toplam hacme kadar steril su içinde çözülür. 4 ° C&#39;de filtre, kısım ve saklayın. </li><li> 5x Dulbecco Modifiye Kartal Ortamı (DMEM) / Nutrient Mixture Ham F-12: DMEM / Ham F-12, 6 g toz tartılarak 100 ml&#39;lik toplam hacme kadar steril su içinde çözülür. 4 ° C&#39;de filtre, kısım ve saklayın. </li><li> % 7.5 Sodyum Bikarbonat (NaHCO3) Solutiile: NaHCO 3, 7.5 g tartılır ve 100 ml&#39;lik toplam hacme kadar steril su içinde çözülür. 4 ° C&#39;de filtre, kısım ve saklayın. </li><li> 1 M HEPES Çözüm: HEPES 23.8 g tartılır ve 100 ml&#39;lik toplam hacme kadar steril su içinde çözülür. 4 ° C&#39;de filtre, kısım ve saklayın. </li><li> % 70 (h / h) etanol: steril su, 30 ml mutlak etanol içinde 70 ml seyreltin. </li></ol></li><li> Glikoz ve Antibiotics (PBS-GAB) Fosfat Tamponlu Tuz Çözeltisi: 10% 30 glükoz solüsyonu ve Dulbecco Fosfat Tamponlu Salin, 500 ml penisilin-streptomisin çözeltisi 5 ml ilave ediniz. 4 ° C&#39;de saklayın. </li><li> Pasif hale getirilmiş fetal sığır serumu (iFBS) gece boyunca 4 ° C&#39;de bovin serum çözülme ve 30 dakika boyunca 56 ° C&#39;de etkisiz hale getirirler. -20 ° C&#39;de 50 ml kısım ve mağaza. </li><li> Kültür vasatı: steril suda, 5x DMEM / Ham F12-ardışık olarak 40 mi, 1 M HEPES 1 mi, 2 mi 200 mM L-glutamin, Penisilin-Streptomisin, 2 mi, 4 ml% 30 glikoz, bir karıştırınnd 180 ml&#39;lik bir nihai hacme kadar% 7.5 NaHCO3 3 mi. Filtre ve aynı gün kullanın. </li><li> Hormon Mix <ol><li> Kültür ortamının 180 ml hazırlayın. Bu ortamda apo-transferin 200 mg çözülür. </li><li> Insülin 50 mg tartılır ve 0.1 M HCI, 2 ml içinde çözülür. Yavaşça, karıştırma sırasında steril su içinde yavaş yavaş 8 ml ilave edilir. Kültür ortamında bu çözüm ile seyreltilir. </li><li> Putresin dihidroklorür 19,3 mg tartılır ve steril 10 ml su içinde çözülür. Hormon karışımı 10 ml&#39;lik bir solüsyon ilave edin. </li><li> , Steril su içinde bir 3 mM sodyum selenit çözeltisi ve mutlak etanol içinde bir 2 mM progesteron çözelti hazırlayın. Hormon karışımı her çözeltiden 20 ul ekle. </li><li> -20 ° C&#39;de hormon karışımı, bir kısım halinde, 10 ml ve mağaza Filtre. </li></ol></li></ol> Kültür Plakalar ve Araçların 2. Hazırlık <ol><li> Kültür levhalarını FBS Kaplama: diseksiyon önceki gün, kültür ortamının 180 ml hazırlar. 10 ml eklemekültür ortamı 90 ml iFBS. Ekle 300 ul / oyuk kültür ortamı / poli-D-lizin önceden kaplanmış 24 oyuklu plakalar içinde% 10 iFBS. 37 ° C&#39;de gece boyunca inkübe edilir. 4 ° C &#39;de (ve iFBS olmadan) kalan ortamı saklayın. </li><li> Aletleri ve Pasteur pipetler sterilize edin. Sınıf II laminar akış ve CO 2 inkübatör yanı sıra malzemeler tabloda listelenen ekipmanı toplayın. </li></ol> Fare mezensefalonun 3. Diseksiyon <ol><li> (Kurumsal kurallarına göre) E13.5 hamile fare Kurban. % 70 etanol ile fare karın temizleyin ve karın duvarı açın. , Narin makas kullanarak bunları açmak, rahim boynuzları toplayın rahim boynuzları her embriyo incelemek ve amniyotik membranlar kaldırmak. Steril PBS-GAB içeren bir 100 mm Petri kabındaki embriyolar yerleştirin ve forseps kullanarak 3 ardışık aynı hamam transferi ile yıkayın. Diseksiyon için, 60 mm steril Petri kabı içinde 3 embriyolar yerleştirin. </li><li> Movstereomicroscope altında son Petri e. Embriyoların kafasını etmeyin. Vannas makas kullanma, beyinleri kesip. <ol><li> Dikkatlice çıkarın ve ön ve arka beyin bölgeleri atın. Rostral tarafına, berzah bölgede kaudal yan kesim için, talamik bölgeye yakın kesim üstün kollikulusu çıkarın. </li><li> Ön orta beyin izole edildikten sonra, dikkatli bir şekilde ultra ince forseps kullanılarak meninks çıkarın. PBS-GAB ile dolu bir steril 13 ml tüp içinde, meninkslerde olmadan kesilmiş parçalarını toplamak. </li></ol></li><li> % 70 etanol ile iyice mesencephala içeren tüp temizleyin ve kaputun altında getirmek. </li></ol> 4. Hücre Ayrılma <ol><li> Yıkama steril PBS ile 3 kez GAB mesencephala. Beyin fragmanları Her yıkama arasında yerleşmek ve doku bozmamak için izin ver. Son yıkamadan sonra, 37 ° C&#39;de 15 dakika boyunca tripsin / EDTA, 3 ml beyin bölümleri mümkün olduğu kadar çok dikkatli bir şekilde çözeltinin ayrılması ve inkübe2 inkübatör CO. </li><li> Yangın-lehçe Pastör pipetleri olmayan bir parlatılmış cam pipet sonu olarak nöronların canlılığını maksimize keskin ve ayrışma adımları sırasında hücrelere zarar verebilir. Bunu yangın parlatma esnasında hafifçe ekstremite çapını Pasteur pipet (0,5 mm) azaltır. </li><li> Dikkatle mümkün olduğu kadar tripsin / EDTA çözeltisi çıkarın ve kültür ortamı 10 ml /% 10 iFBS ekleyin. Yıkama beyin bölümleri kültür ortamı /% 10 iFBS ile 3 kez. </li><li> Yangın cilalanmış Pasteur pipeti kullanarak, kültür ortamı /% 10 iFBS 6 ml mesencephala ayrılmasını başlar. Öğütün 10x daha sonra yeni bir steril 13 ml tüp içinde orta toplamak parçalar oturmasına izin verin, (hava kabarcıklarını önlemek). </li><li> , Kültür ortamı, 6 ml ilave kez önceki adımı tekrar ve aynı 13 ml&#39;lik bir tüp içinde çözülmüş hücreleri içeren ortamın toplanması. </li><li> 5 dakika boyunca 160 g&#39;de santrifüje hücreleri. Süpernatant atılır ile takviyeli kültür ortamında tekrar süspansiyon hücreleri10% hormon karışımı. </li></ol> 5. Hücre Kaplama <ol><li> Bir Malassez hücre içinde süspansiyon halinde hücrelerin sayısı ve 600,000 hücre / ml &#39;lik bir konsantrasyon ortamın seviyesini ayarlar. Çevresi 1,700,000 hücreleri bir fare mesensefalonundan elde edilebilir. </li><li> 24-delikli plakadan boyama maddesini FBS içeren çıkarın ve her bir kuyu hücre süspansiyonu 1 ml eklemek (600,000 hücre / göz, TH + hücrelerinin% 2-4). </li><li> 37 ° C&#39;de inkübe edin ve% 5 CO 2/95% hava. Hiçbir orta değişim gereklidir. Dopaminerjik nöronlar in vitro yaklaşık 5-7 gün (dopamin taşıyıcı tutarlı ifadesi) sonra olgun. Orta değiştirmeden 15 gün kültür up hücreleri tutun. </li></ol> 6. İmmünofloresan Protokol <ol><li> Lameller Temizleme <ol><li> Diseksiyon bir gün önce, bir Petri kutusu içine 1 M HCI 10 ml ekleyin. Sıvı 12-15 cam kapak yüzeyinde yerleştirin ve 15 dakika bekleyin. </li><li> Kapağın Evyesıvı girebilir ve 15 dakika boyunca bekleyin. </li><li> , HCI yi uzaklaştırmak ve su ile 3 kez yıkanır. </li><li> Bir kez saf etanol ile hızlı bir şekilde lamelleri yıkayın, sonra tabak saf etanol ilave edin ve 30 dakika boyunca bekleyin. </li><li> Kapağın altında, etanolden temizlenmiş lamelleri çıkarın ve steril pensler kullanılarak bir 12-yuvalı hücre kültürü plakasının oyuk başına 1 lamel ekleyin. </li><li> Yıkama steril su (1 ml / oyuk) ile iki kere lamelleri. Daha sonra, steril bir kez PBS ile yıkayın. </li></ol></li><li> Lameller Kaplama <ol><li> PBS çıkarın ve 500 ul / 2x FKÖ&#39;nün (PBS içinde seyreltilmiş) ilave edin. CO2 kuluçka makinesi içinde 37 ° C de 4 saat süreyle inkübe edilir. </li><li> FKÖ çözüm çıkarın ve steril PBS ile 3x yıkayın. </li><li> PBS çıkarın ve 500 ul / gözenek% 20 iFBS / 1 ug / ml laminin ekleyin. CO2 kuluçka makinesi içinde 37 ° C&#39;de gece boyunca inkübe edilir. </li></ol></li><li> İmmünofloresan için Hücreleri Kaplama <ol><li> 12-delikli plakadan kaplama orta çıkarın. </li><li> 900,0 ekleme00 hücre / göz 2 ml hacim içinde ve inkübatör içinde 37 ° C&#39;de hücreler büyür. Hücreler ortam değiştirme olmadan 15 gün kültür kadar tutulabilir. </li></ol></li><li> İmmünoboyama <ol><li> 12-delikli plakadan Ortamı çıkarın ve DMEM, 37 ° C&#39;de önceden ısıtılmış / 500 ul ile yıkayın. </li><li> / Oyuk DMEM 500 ul, 37 ° C&#39;de önceden ısıtılmış eklenmekte, daha sonra yavaşça 160 | il / göz, 8% paraformaldehid (PFA,% 2 nihai konsantrasyon) ilave ediniz ve 37 ° C&#39;de 10 dakika inkübe edilir. </li><li> PFA çıkarın ve PBS ile 3x yıkama / 0.1 M glisin, 10 dakika boyunca. </li><li> , PBS çıkarın 300 ul / göz PBS /% 0.05 Triton X-100/20% Keçi serumu ilave edilir ve oda sıcaklığında 30 dakika inkübe edilir. </li><li> , Orta çıkarın 300 ul / göz PBS içinde seyreltilmiş birincil antikor /% 0.05 Triton X-100/1% Keçi serumu ilave edilir ve oda sıcaklığında 2 saat süreyle inkübe edin. Dopaminerjik nöron ve kültürün karakterizasyonu tespiti için kullanılabilecek primer antikorlar Malzeme tabloda belirtilmiştir. Keep1 kuyusu primer antikor olmadan farklı ikincil antikorların arka değerlendirmek için. </li><li> Ortamı çıkarın ve PBS ile 3x yıkama /% 0.2 jelatin 10 dakika karıştırıldı. </li><li> , Orta çıkarın 300 ul / göz PBS içinde seyreltilmiş sekonder antikor /% 0.05 Triton X-100/1% Keçi serumu ekleyin ve karanlıkta, oda sıcaklığında 1 saat boyunca inkübe edilir. Kullanılan ikincil antikorlar malzemeleri tabloda belirtilmiştir. </li><li> Ortamı çıkarın ve PBS ile 3x yıkama /% 0.2 jelatin 10 dakika karıştırıldı. </li><li> Orta çıkarın ve PBS ile 3x yıkayın. </li><li> Aşağı Vectashield bir damla cam slaytlar hücre tarafında lamelleri monte edin. Daha sonra 4 ° C&#39;de saklayın, montaj orta kuruması için oda sıcaklığında bir gece slaytlar tutun. </li><li> Konfokal mikroskop ya epifluoresan için donatılmış bir faz-kontrast mikroskobu kullanılarak görüntü kazanır. Temsilcisi görüntüleri Leica SP2 UV odaklı mikroskop ile elde edildi. </li></ol></li></ol>

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	<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.

Bu protokol, embriyonik fare ve dopaminerjik nöronlar tespit etmek için immünofloresan prosedürden mezensefalik nöronlar primer kültürü hazırlamak için gerekli prosedürler ve reaktif maddeler sunmaktadır. Prosedürün kritik aşamaları embriyoların diseksiyon ve toplanan beyin parçalarının mekanik ayrışma bulunmaktadır. Yüksek kaliteli diseksiyon aletleri diseksiyon tekniği master yardımcı olur. DA nöronlar mezensefalonun küçük bir kısmını oluşturmaktadır. Bu duruma göre, ventral mezens...

Dopamin, temel beyin nörotransmitter 1,2 biri, esas olarak orta beyin dopaminerjik (DA) nöronlar tarafından serbest bırakılır. DA nöronlarının çoğunluğu mezensefalon 2-6 ventral kısmında bulunur. Mesostriatal, mezolimbik ve mezokortikal yollar 2,5 şematik olarak, orta beyin DA nöronlar anatomik ve fonksiyonel üç ayrı projeksiyon sistemleri ayrılabilir. Prefrontal korteks projelendirme dopaminerjik yollar biliş 2 karıştığı oysa nigrostriyatal yolu, mezolimbik yollar takviyesi, motivasyon ve öğrenme önemli bir rol oynar, motor davranışları yer almaktadır. DA nöronlar örneğin, şizofreni, dikkat eksikliği, hiper aktivite bozukluğu, ve Parkinson hastalığı (PD) 2,4 gibi bazı insan nörolojik hastalıklarda rol oynamaktadır. PD substantia nigra bağlantı DA nöronlarının bir ilerici ve seçici dejenerasyonu ile karakterizedirstriatum pars compacta (SNc). PD motor semptomların sorumlu striatum şiddetli dopamin tükenmesine nigro-striatal DA nöronlar sonuçlarının kaybı (bradikinezi, dinlenme tremor ve rijidite) 7. Idiyopatik PH başlangıç ​​nedeni kanıtlanmamıştır ve güncel tedaviler striatum dopamin seviyesini yeniden amaçlayan, sadece semptomatik vardır. En reçete ilaç, L-dopa (levodopa), dopamin doğal öncüsüdür. Levodopa yönetimi belirli bir süre için dopamin kaybı telafi rağmen, motor komplikasyonlar sonra (üzerine diskinezi ve / kapalı devletler) uzun süreli tedaviler 8,9 oluşur. Dopaminerjik nöronlar ve PH ilgili araştırma sürekli ilerlemesi ve yoğun çabalar hücre nakli, gen tedavisi veya nöroprotektif ajanların 10,11 dayalı tedaviler geliştirmek için yapılıyor. Ancak, büyük bir sorun olmayan aydınlatılamamıştır kalır: aşırı vulnerab nedeni nedirDA nöronların lü k? Cevap kısmı DA nöron aktivitesi bulunabilir. Elektriksel aktivitesinde ve DA, nöronların uyarılabilirliğinin bir azalma 12 dejenere için eğilimlerini artırmak gibi görünüyor. Bununla birlikte, PD patojenezin karmaşıklığı DA katılan mekanizmaları nöronlar 13-15 dejenerasyonu tespit için ileri çalışmalara gerek. Primer kültürler DA nöron özelliklerini 16-19 incelemek ve nöroprotektif ajanların 20-24 değerlendirilmesi için çeşitli streslere bu nöronların meydan özellikle alakalı. Sıçan kültürü modeli, genellikle fare embriyo mezensefalon ayrılmasının daha kolay olduğu için, fare ile karşılaştırıldığında, nöronların ve daha yüksek miktarlar da Fare içinde elde edilebilir kullanılır. Ancak, hastalığın 25 transgenik fare modelleri nesil fare 26-29 primer kültürler için nörolog toplumun ilgi artmıştır ölçüde etti. Pr kültürlerin rağmenyeni doğmuş hayvanların epared bu nöronların ayırt etme kapasitelerini muhafaza zaman, mitotik sonrası aşaması (mezensefalon nöronlar için E 13.5) embriyo bunları hazırlamak için daha da kullanılabilir. Aşağıdaki protokol hazırlamak için en zor olan fare embriyoları (E 13.5), birincil kültürde izole mezensefalon nöronlar sunar. Özellikle, daha iyi bir yeniden üretilebilirlik için, serum içermeyen kültür ortamı kullanılarak bir protokol sağlar. Kültür hazırlama (diseksiyon ve mekanik ayrışma) olarak iki en kritik adımlar dikkatle ilişkili videoda detaylı olarak açıklanacaktır.

<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

Dopaminerjik nöronlar, beyindeki sinir hücrelerinin toplam sayısının en az% 1 temsil etmektedir. Nöronların Bu düşük miktarı, motor kontrolü, motivasyon ve çalışma belleği gibi önemli beyin fonksiyonlarını düzenler. Nigrostriatal dopaminerjik nöronlar selektif Parkinson hastalığı (PH) dejenere. Bu ilerleyici nöron kaybı tümden patolojinin motorlar semptomları (bradikinezi, dinlenme tremor ve kas sertliği) ile ilişkili. Dopaminerjik nöron dejenerasyonu sorumlu ana ajan hala bilinmemektedir. Bununla birlikte, bu nöronlar çeşitli koşullarda çok hassas olduğu görülmektedir. Primer kültürler özellikleri ve dopaminerjik nöronların özelliklerini araştırmak için en uygun modellerden birini oluşturmaktadır. Bu kültürler durdurmak ya nöronal dejenerasyonu yavaşlatmak amacıyla PD patolojiyi taklit çeşitli stres ajanları ve nöroprotektif bileşiklere sunulabilir. Jeneratör olmuştur PH çok sayıda transgenik fare modellerison on yılda ated başka dopaminerjik nöron kültürler için araştırmacıların ilgisini artırmıştır. Burada, video protokol embriyonik fare beyinleri hassas diseksiyon üzerinde duruluyor. Ventral mezensefalon hassas eksizyonu Daha sonraki çalışmalar, izin vermek için yeteri kadar dopaminerjik hücrelerin nöronal zengin kültürler elde etmek için önemlidir. Bu protokol, embriyonik transgenik fareler ile gerçekleştirilir ve immünofloresan boyama, nicel PCR, ikinci haberci miktarının ya da nöronal ölüm / hayatta kalma değerlendirme için uygundur edilebilir.

Mezensefalon kültür adımların bir akış şeması gösterilmektedir, Şekil 1 &#39;de gösterilmiştir. Kısaca, bir gebe fare E 13.5 İsviçre embriyolar alındıktan sonra, ventral mezensefalon tüm embriyo parçalanmıştır. Izole beyin parçaları arka arkaya enzimatik sindirim ve mekanik ayrılma sunulur. Ayrılan hücreler, önceden kaplanmış 12 ya da 24 oyuklu plakalar üzerinde kültür ortamı içinde yeniden süspansiyona alınmış ve kaplama, santrifüj ile pelet haline getirildi. Hü...

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.

Fare dopaminerjik nöronlar İlköğretim Kültür

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

37.8K Görüntüleme.  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.

Video: 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.

Embriyonik Fare Hipokampal ve Kortikal nöronlar primer Nucleofection ve Kültür

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

Nörobilim

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.

Kemirgen Yenidoğanlar İlköğretim grubuyla orta beyin dopamin Hücre Kültürleri

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

Biyoloji

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).

Post-natal murin Dokular Co-kültürler Myelinating zenginleştirilmiş oligodendrosit Kültürleri ve oligodendrosit / Nöron türetilmesi

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.

Bir Fare, İki Kültür: İzolasyon ve Yetişkin Nöral Kültür Bireysel Farelerde iki Nörojenik Bölgeler&#39;den Kök Hücre

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.

Embriyonik Kemirgen Brain itibaren İlköğretim Mesensefalik dopaminerjik Nöronlar İzolasyonu, Kültür ve Uzun Vadeli Bakım

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.

RNA sıralaması ve TH-organizatörü odaklı eGFP İfade Kullanımı orta beyin Kültürlerde Yaşayan dopaminerjik nöronlar güvenilir Kimlik

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.

Embriyonik Fare Beyincisinden İzole Edilen Nöronların Birincil Kültürü

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.

Primer Embriyonik Fare Orta Beyin Dopamin Nöronlarında Pre-formed Fibril Indüklenen α-Sinüklein Birikiminin İncelendirilmesi

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.

Fare Embriyonik Beyin Dokusundan Dopaminerjik Nöronların Kültürlenmesi ve Bakımı

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

Nöronal Kültür Teknikleri

Birincil Nöronal Kültür

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.

Primer Embriyonik Fare Dopamin Nöronlarında Alfa-Sinüklein Birikiminin İncelenmesi

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

	Prepare ...