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 border="0" cellpadding="0" cellspacing="0" width="1291">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td>14-801F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">DMEM 4.5g/L Glucose with L-Glutamine</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">0.05% Trypsin-EDTA (1X), Phenol Red&#160;</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life Technologies</td>
			<td style="width:139px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine, 200 mM Solution</td>
			<td>Life Technologies</td>
			<td>25030123</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium/Nutrient Mixture F-12 Ham</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D0547</td>
			<td>Powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Laminin - 1 mg/mL in Tris buffered NaCl</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">L2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-Ornithine hydrobromide</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin from porcine pancreas</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">I5523</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">apo-Transferrin human</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">T1147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Putrescine dihydrochloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">P5780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Progesterone</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">P8783</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Sodium selenite</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">S5261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">H4034&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">G7126</td>
			<td>Stock solution 1M in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">G9391</td>
			<td>Stock solution 2% (w/v) in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">T8532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde 16% in water</td>
			<td>Electron Microscopy Sciences</td>
			<td style="width:139px;">RT 15710-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydrogen carbonate (NaHCO3)</td>
			<td>Merck Millipore</td>
			<td style="width:139px;">106329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D(+)-Glucose, Monohydrate</td>
			<td>Merck Millipore</td>
			<td>4074-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Hydrochloric acid - c(HCl) = 1 mol/l (1 N) Titripur</td>
			<td>Merck Millipore</td>
			<td style="width:139px;">109057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Sterile water - Aqua B. Braun</td>
			<td style="width:176px;">Braun</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Ethanol absolute NORMAPUR analytical reagent</td>
			<td>VWR</td>
			<td style="width:139px;">20821.321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Sterile Petri Dishes</td>
			<td>VWR</td>
			<td>82050-566</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Pasteur pipettes plain glass - Wilhem Ulbrich GdbR.</td>
			<td>VWR</td>
			<td>612-2297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Counting chamber Malassez</td>
			<td>VWR</td>
			<td style="width:139px;">631-0975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serum Acrodisc Syringe Filter with Supor Membrane, Sterile, GF/0.2 &#181;m, 37 mm</td>
			<td>PALL Life science</td>
			<td style="width:139px;">4525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical Scissors - Straight, sharp-sharp, 14.5 cm long</td>
			<td style="width:176px;">Fine Science Tools</td>
			<td>14002-14</td>
			<td>To open the abdominal wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors - Straight, pointed, delicate, 10 cm long</td>
			<td style="width:176px;">MORIA</td>
			<td>4877A</td>
			<td>To open the uterine wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps - Curved, usual, serrated jaws 1 mm</td>
			<td style="width:176px;">MORIA</td>
			<td>2183</td>
			<td>To manipulate embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Vannas Scissors - Curved, pointed, 7 mm blades</td>
			<td style="width:176px;">MORIA</td>
			<td>MC50</td>
			<td>To take out the mesencephalon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Fine Forceps - Curved, delicate, 13 cm long</td>
			<td style="width:176px;">MORIA</td>
			<td>9987</td>
			<td>To remove meninges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD BioCoat Poly-D-Lysine 24-well Multiwell Plates</td>
			<td>BD Bioscience</td>
			<td style="width:139px;">356414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Falcon 12-well Cell Culture Plate, flat-bottom with lid</td>
			<td>BD Bioscience</td>
			<td style="width:139px;">353043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperFrost Microscope Slides, Ground edges 90&#186;</td>
			<td>MENZEL-GL&#196;SER</td>
			<td style="width:139px;">AG00008032E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precision cover glasses thickness No. 1.5H circular 18 mm &#216;</td>
			<td>MARIENFELD</td>
			<td>117580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyclonal Rabbit Anti-Microtubule-Associated Protein 2 (MAP2) Antibody</td>
			<td>Chemicon Millipore</td>
			<td>AB5622</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monoclonal Mouse Anti-Glutamate Decarboxylase (GAD67) Antibody, clone 1G10.2</td>
			<td>Chemicon Millipore</td>
			<td>MAB5406</td>
			<td>1/400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monoclonal Rat Anti-Dopamine Transporter (DAT) Antibody, clone DAT-Nt&#160;</td>
			<td>Chemicon Millipore</td>
			<td>MAB369</td>
			<td>1/500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monoclonal Mouse Anti-5-HT Antibody</td>
			<td style="width:176px;"></td>
			<td style="width:139px;"></td>
			<td>1/8,000 - Generous gift from Yves Charnay (Swizerland, Yves.Charnay@hcuge.ch)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Serum, New Zealand Origin</td>
			<td style="width:176px;">Life Technologies</td>
			<td style="width:139px;">16210-064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 405 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td style="width:176px;">Life Technologies</td>
			<td style="width:139px;">A-31556</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody</td>
			<td style="width:176px;">Life Technologies</td>
			<td>A-11001</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 Goat Anti-Rat IgG (H+L) Antibody</td>
			<td style="width:176px;">Life Technologies</td>
			<td>A-11007</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASHIELD HardSet Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Stereomicroscope</td>
			<td>Carl Zeiss microscopy</td>
			<td>Stemi-2000C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Bunsen Burner FIREBOY</td>
			<td style="width:176px;">VWR</td>
			<td style="width:139px;">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

37.2K 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

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

İlköğretim Rat Kortikal Nöronlar ve Glia bilaminar Co-kültür

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Fare Dopaminerjik ve Üç Boyutlu Collagen Matrix Tahliller striatal Eksplantlarından diseksiyonu ve Kültür

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

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 ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Modelleme nöronal ölüm ve fare birincil serebellar granül nöronlar dejenerasyon

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

GLUT4 protein Deconvolution mikroskobu kullanarak fare birincil hipotalamik nöronlarda ticareti canlı görüntüleri

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can be cultured in defined media for 16 days in vitro and are amenable to any analyses typically performed on dissociated cultures, including electrophysiological, morphological and survival analyses. The major advantage of these cultures is that specific cell types can be selectively studied in the absence of complex external influences, such as those arising from glial cells or other neuron types. When planning experiments with purified cells, however, it is important to note that neurons strongly depend on glia-conditioned media for their growth and survival. In addition, glutamatergic neurons further depend on glia-secreted factors for the establishment of synaptic transmission. We therefore also describe a method for co-culturing neurons and glial cells in a non-contact arrangement. Using these methods, we have identified major differences between the development of GABAergic and glutamatergic neuronal networks. Thus, studying cultures of purified neurons has great potential for furthering our understanding of how the nervous system develops and functions. Moreover, purified cultures may be useful for investigating the direct action of pharmacological agents, growth factors or for exploring the consequences of genetic manipulations on specific cell types. As more and more transgenic animals become available, labeling additional specific cell types of interest, we expect that the protocols described here will grow in their applicability and potential.

This protocol describes a cell sorting based method for the purification and culture of fluorescent GABAergic or glutamatergic neurons from the neocortex and hippocampus of postnatal mice or rats.

Floresans ile kurulan Purified Gabaerojik veya glutamaterjik nöronların primer hücre kültürü-aktif hücre sıralama

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...

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 ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

Beyin Dilimlerinde ve Birincil Hücre Kültüründe Piramidal Nöronların Balistik Etiketlemesi

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.