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.

Os ratos utilizados neste trabalho foram atendidas e tratadas de acordo com as diretrizes do Conselho da União Europeia (86/609 / UE) para o uso de animais de laboratório. 1 Preparação de soluções necessárias <ol><li> Soluções de Stock <ol><li> 10x de poli-L-ornitina (PLO) Solução: pesam-se 10 mg de bromidrato de PLO (peso molecular = 30,000-70,000) e dissolver em 70 ml de água estéril. Filtra-se a solução com uma seringa de filtro de 0,2 um, alíquota, e armazenar a -20 ° C. </li><li> 30% Solução de Glucose: pesam-se 30 g de D (+) - glicose e dissolve-se em água estéril para um volume total de 100 ml. Filter, alíquota, e armazenar a 4 ° C. </li><li> Meio de Eagle Modificado de 5x Dulbecco (DMEM) / Mistura nutriente F-12 de Ham: pesam-se 6 g de DMEM / Ham F-12 em pó e dissolve-se em água estéril para um volume total de 100 ml. Filter, alíquota, e armazenar a 4 ° C. </li><li> 7,5% de Bicarbonato de Sódio (NaHCO3) Solutiem: pesam-se 7,5 g de NaHCO 3 e dissolve-se em água estéril para um volume total de 100 ml. Filter, alíquota, e armazenar a 4 ° C. </li><li> Solução 1 M de HEPES: Pesar 23,8 g de HEPES e dissolvem-se em água estéril para um volume total de 100 ml. Filter, alíquota, e armazenar a 4 ° C. </li><li> 70% (v / v) de etanol: Diluir 70 ml de etanol absoluto, com 30 ml de água estéril. </li></ol></li><li> Salina tamponada com fosfato com glicose e antibióticos (PBS-GAB): adicionar 10 ml de solução de glucose a 30% e 5 ml de solução de penicilina-estreptomicina a 500 ml de fosfato de Dulbecco tamponado salino. Armazenar a 4 ° C. </li><li> De soro fetal bovino inactivado (ifbs): Descongelar soro bovino durante a noite a 4 ° C e inactiva a 56 ° C durante 30 min. Alíquota em 50 ml e armazenar a -20 ° C. </li><li> Meio de Cultura: Em água estéril, misturar sucessivamente 40 mL de 5x DMEM / Ham F12-, 1 ml de HEPES 1 M, 2 ml de 200 mM de L-glutamina, 2 ml de penicilina-estreptomicina, 4 ml de 30% de glicose umaº 3 ml de 7,5% de NaHCO 3 para um volume final de 180 ml. Filtra-se e usar o mesmo dia. </li><li> Hormônio Mix <ol><li> Preparar 180 ml de meio de cultura. Dissolve-se 200 mg de apo-transferrina neste meio. </li><li> Pesar 50 mg de insulina e dissolver em 2 ml de 0,1 M de HCl. Adicionar lentamente 8 ml de água estéril, enquanto se mistura com cuidado. Dilui-se esta solução no meio de cultura. </li><li> Pesar 19,3 mg de dicloridrato de putrescina e dissolver em 10 ml de água estéril. Adicionar a solução de 10 ml à mistura de hormona. </li><li> Prepara-se uma solução de selenito de sódio 3 mM em água estéril e uma solução 2 mM de progesterona em etanol absoluto. Adicionar 20 mL de cada solução para o mix de hormônios. </li><li> Filtrar a mistura hormonal, alíquota em 10 ml e armazenar a -20 ° C. </li></ol></li></ol> 2 Preparação de Pratos e Instrumentos Cultura <ol><li> FBS revestimento das placas de cultura: O dia antes da dissecção, preparar 180 ml de meio de cultura. Adicionar 10 ml deifbs para 90 ml de meio de cultura. Adicionar 300 ul / poço de meio de cultura / 10% ifbs em pré-revestidas placas de 24 poços com poli-D-lisina. Incubar durante a noite a 37 ° C. Armazene a mídia restante (com e sem OIF) a 4 ° C. </li><li> Esterilizar os instrumentos e pipetas Pasteur. Reúna os equipamentos listados na tabela de materiais, bem como uma capa de Classe II de fluxo laminar e uma incubadora de CO 2. </li></ol> 3. Dissection of Mouse Mesencéfalo <ol><li> Sacrifique um rato grávida E13.5 (de acordo com as diretrizes institucionais). Limpe o abdômen do mouse com etanol 70% e abrir a parede abdominal. Recolha os cornos uterinos, abri-los usando uma tesoura delicada, dissecar cada embrião dos cornos uterinos, e remover as membranas amnióticas. Coloque os embriões em uma placa de Petri 100 milímetros contendo PBS estéril-GAB e lavá-los por transferência em 3 sucessivos banhos idênticos usando uma pinça. Para dissecção, coloque os embriões por 3 em um 60 milímetros placa de Petri estéril. </li><li> MovÊ O prato final Petri sob o microscópio estereoscópico. Não decapitar os embriões. Com uma tesoura Vannas, extirpar os cérebros. <ol><li> Cuidadosamente retire e descarte as regiões das letras e do rombencéfalo. Para o lado rostral, cortada perto do tálamo, para o corte lateral caudal na região do istmo, remover o colículo superior. </li><li> Uma vez que o mesencéfalo ventral é isolado, retire cuidadosamente meninges usando uma pinça ultrafinas. Recolher os segmentos dissecados, sem as meninges, em um tubo estéril 13 ml cheio de PBS-GAB. </li></ol></li><li> Limpe o tubo contendo o mesencephala exaustivamente com etanol 70% e trazê-lo sob o capô. </li></ol> A dissociação 4. celular <ol><li> Lavar mesencephala 3x com PBS estéril-GAB. Permitir fragmentos do cérebro para resolver entre cada lavagem e evitar a ruptura do tecido. Após a última lavagem, retirar cuidadosamente, tanto quanto possível solução e incubar segmentos cerebrais em 3 ml de tripsina / EDTA durante 15 minutos a 37 ° C numaIncubadora de CO 2. </li><li> -Polonês fogo as pipetas de Pasteur para maximizar a sobrevivência dos neurónios como a extremidade de uma pipeta de vidro não polido é afiado e pode danificar as células, durante os passos de dissociação. Reduzir ligeiramente o diâmetro da extremidade (+ 0,5 mm) da pipeta de Pasteur, enquanto fogo polimento-lo. </li><li> Remova cuidadosamente tanta solução / EDTA tripsina possível e adicionar 10 ml de meio de cultura / 10% OIF. Segmentos cerebrais Lavar 3x com meio de cultura / 10% OIF. </li><li> Utilizando a pipeta de Pasteur polida ao fogo, começam a dissociação de mesencephala em 6 ml de meio de cultura / 10% ifbs. Triturar 10x (evitar bolhas de ar), deixe os pedaços para resolver, em seguida, recolher o meio em um novo tubo estéril 13 ml. </li><li> Adicionar 6 ml de meio de cultura, repetir o passo anterior, uma vez e recolher o meio contendo células dissociadas no mesmo tubo de 13 ml. </li><li> Centrifugar células a 160 g por 5 min. Descartar o sobrenadante e ressuspender as células em meio de cultura suplementado com10% de mistura de hormônio. </li></ol> 5. celular chapeamento <ol><li> Contar as células em suspensão numa célula de Malassez e ajustar o volume do meio a uma concentração de 600.000 células / ml. Cerca de 1,7 milhões de células podem ser obtidas a partir de um mesencéfalo do rato. </li><li> Remover FBS contendo meio de revestimento de placas de 24 cavidades e adicionar a cada cavidade 1 ml da suspensão de células (600.000 células / poço, 2-4% das células TH +). </li><li> Incubar a placa a 37 ° C e 5% de CO 2/95% ar. Nenhuma mudança de meio é necessário. Neurônios dopaminérgicos estão maduros depois de cerca de 5-7 dias in vitro (expressão consistente do transportador de dopamina). Manter as células em cultura até 15 dias sem substituição médio. </li></ol> 6. Imunofluorescência Protocolo <ol><li> A limpeza das Lamelas <ol><li> O dia antes da dissecação, adicionar 10 ml de HCl 1 M em uma placa de Petri. Colocar a superfície dos líquidos 12-15 lamelas de vidro e esperar 15 minutos. </li><li> Afundar a tampaescorregar para o líquido e esperar 15 min. </li><li> Remover HCl e lavar 3x com água. </li><li> Lavam-se as lamelas com etanol puro rapidamente uma vez, em seguida, adicionar etanol puro para o prato e para aguardar 30 minutos. </li><li> Sob o capô, remova lamelas limpas a partir de etanol e adicionar uma lamela por poço de 12 poços da placa de cultura de células usando uma pinça esterilizada. </li><li> Lavar lamelas duas vezes com água estéril (1 ml / poço). Em seguida, lavá-los uma vez com PBS estéril. </li></ol></li><li> Revestimento das Lamelas <ol><li> Remover PBS e adicionar 500 uL / ​​poço de 2x PLO (diluído em PBS). Incuba-se durante 4 horas a 37 ° C na incubadora de CO 2. </li><li> Remover a solução de PLO e lavar 3x com PBS estéril. </li><li> Remover PBS e adicionar 500 uL / ​​poço de 20% ifbs / 1 g / ml de laminina. Incubar durante a noite a 37 ° C na incubadora de CO 2. </li></ol></li><li> Plaqueamento das células de Imunofluorescência <ol><li> Remover o meio de revestimento a partir de placas de 12 poços. </li><li> Adicionar 900,000 células / cavidade em 2 ml de volume e crescer as células a 37 ° C na incubadora. As células podem ser mantidas em cultura 15 dias sem substituição médio. </li></ol></li><li> Imunocoloração <ol><li> Remover o meio de placas de 12 poços e lava-se com 500 ul / poço de DMEM pré-aquecido a 37 ° C. </li><li> Adicione 500 ^ l / poço de DMEM pré-aquecido a 37 ° C, em seguida, adicionar cuidadosamente 160 ul / poço de 8% de paraformaldeído (PFA, 2% de concentração final) e incubar durante 10 min a 37 ° C. </li><li> Remover PFA e lavar 3x com PBS / 0,1 M de glicina durante 10 min. </li><li> Remover PBS, adicionam-se 300 ul / poço de PBS / 0,05% de Triton X-100/20% de soro de cabra e incubar durante 30 min à temperatura ambiente. </li><li> Remover o meio, adicionam-se 300 ul / poço de anticorpo primário diluído em PBS / 0,05% de Triton X-100/1% de soro de cabra e incubar durante 2 horas à temperatura ambiente. Os anticorpos primários que podem ser utilizados para a detecção de neurónios dopaminérgicos e caracterização da cultura estão indicados na tabela Materiais. Mantenha1 bem sem anticorpo primário para avaliar a base dos diferentes anticorpos secundários. </li><li> Remover o meio e lava-se 3x com PBS / 0,2% de gelatina durante 10 minutos. </li><li> Remover o meio, adicionam-se 300 ul / poço de anticorpo secundário diluído em PBS / 0,05% de Triton X-100/1% de soro de cabra e incubar durante 1 hora à temperatura ambiente, no escuro. Anticorpos secundários utilizados são indicados na tabela de Materiais. </li><li> Remover o meio e lava-se 3x com PBS / 0,2% de gelatina durante 10 minutos. </li><li> Remover o meio e lava-se 3x com PBS. </li><li> Montar as lamelas do lado vidro desliza célula para baixo em uma gota de Vectashield. Mantenha as lâminas à temperatura ambiente durante a noite para deixar a montagem meio seco, em seguida, armazená-los, a 4 ° C. </li><li> Adquirir imagens usando um microscópio confocal ou um microscópio de contraste de fase equipado para epifluorescência. Imagens representativas foram adquiridos com um microscópio confocal Leica SP2 UV. </li></ol></li></ol>

<ol>
	<li>Glowinski, J., Cheramy, A., Romo, R., Barbeito, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+regulation+of+dopaminergic+transmission+in+the+striatum.">Presynaptic regulation of dopaminergic transmission in the striatum.</a> Cell Mol Neurobiol. 8, 7-17 (1988).</li><li>Iversen, S. D., Iversen, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine:+50+years+in+perspective.">Dopamine: 50 years in perspective.</a> Trends Neurosci. 30, 188-193 (2007).</li><li>Dahlstroem, A., Fuxe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+Existence+of+Monoamine-Containing+Neurons+in+the+Central+Nervous+System+I.+Demonstration+of+Monoamines+in+the+Cell+Bodies+of+Brain+Stem+Neurons.">Evidence for the Existence of Monoamine-Containing Neurons in the Central Nervous System I. Demonstration of Monoamines in the Cell Bodies of Brain Stem Neurons.</a> Acta Physiol Scand Suppl. SUPPL. , 231-255 (1964).</li><li>Chinta, S. J., Andersen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+neurons.">Dopaminergic neurons.</a> Int J Biochem Cell Biol. 37, 942-946 (2005).</li><li>Bjorklund, A., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+neuron+systems+in+the+brain:+an+update.">Dopamine neuron systems in the brain: an update.</a> Trends Neurosci. 30, 194-202 (2007).</li><li>Hegarty, S. V., Sullivan, A. M., O'Keeffe, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midbrain+dopaminergic+neurons:+a+review+of+the+molecular+circuitry+that+regulates+their+development.">Midbrain dopaminergic neurons: a review of the molecular circuitry that regulates their development.</a> Dev Biol. 379, 123-138 (2013).</li><li>Samii, A., Nutt, J. G., Ransom, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease.">Parkinson's disease.</a> Lancet. 363, 1783-1793 (2004).</li><li>Santini, E., Heiman, M., Greengard, P., Valjent, E., Fisone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+mTOR+signaling+in+Parkinson's+disease+prevents+L-DOPA-induced+dyskinesia.">Inhibition of mTOR signaling in Parkinson's disease prevents L-DOPA-induced dyskinesia.</a> Sci Signal. 2, (2009).</li><li>Ohlin, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor+is+upregulated+by+L-dopa+in+the+parkinsonian+brain:+implications+for+the+development+of+dyskinesia.">Vascular endothelial growth factor is upregulated by L-dopa in the parkinsonian brain: implications for the development of dyskinesia.</a> Brain. 134, 2339-2357 (2011).</li><li>Obeso, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Missing+pieces+in+the+Parkinson's+disease+puzzle.">Missing pieces in the Parkinson's disease puzzle.</a> Nat Med. 16, 653-661 (2010).</li><li>Cooper, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+rescue+of+mitochondrial+deficits+in+iPSC-derived+neural+cells+from+patients+with+familial+Parkinson's+disease.">Pharmacological rescue of mitochondrial deficits in iPSC-derived neural cells from patients with familial Parkinson's disease.</a> Sci Transl Med. 4, (2012).</li><li>Michel, P. P., Toulorge, D., Guerreiro, S., Hirsch, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+needs+of+dopamine+neurons+for+stimulation+in+order+to+survive:+implication+for+Parkinson+disease.">Specific needs of dopamine neurons for stimulation in order to survive: implication for Parkinson disease.</a> FASEB J. 27, 3414-3423 (2013).</li><li>Decressac, M., Volakakis, N., Bjorklund, A., Perlmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NURR1+in+Parkinson+disease-from+pathogenesis+to+therapeutic+potential.">NURR1 in Parkinson disease-from pathogenesis to therapeutic potential.</a> Nat Rev Neurol. , (2013).</li><li>Jouve, L., Salin, P., Melon, C., Le Goff, L. K. e. r. k. e. r. i. a. n. -. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+brain+stimulation+of+the+center+median-parafascicular+complex+of+the+thalamus+has+efficient+anti-parkinsonian+action+associated+with+widespread+cellular+responses+in+the+basal+ganglia+network+in+a+rat+model+of+Parkinson's+disease.">Deep brain stimulation of the center median-parafascicular complex of the thalamus has efficient anti-parkinsonian action associated with widespread cellular responses in the basal ganglia network in a rat model of Parkinson's disease.</a> J Neurosci. 30, 9919-9928 (2010).</li><li>Hirsch, E. C., Jenner, P., Przedborski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Parkinson's+disease.">Pathogenesis of Parkinson's disease.</a> Mov Disord. 28, 24-30 (2013).</li><li>di Porzio, U., Daguet, M. C., Glowinski, J., Prochiantz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+striatal+cells+on+in+vitro+maturation+of+mesencephalic+dopaminergic+neurones+grown+in+serum-free+conditions.">Effect of striatal cells on in vitro maturation of mesencephalic dopaminergic neurones grown in serum-free conditions.</a> Nature. 288, 370-373 (1980).</li><li>Denis-Donini, S., Glowinski, J., Prochiantz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+heterogeneity+may+define+the+three-dimensional+shape+of+mouse+mesencephalic+dopaminergic+neurones.">Glial heterogeneity may define the three-dimensional shape of mouse mesencephalic dopaminergic neurones.</a> Nature. 307, 641-643 (1984).</li><li>Barbin, G., Mallat, M., Prochiantz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+studies+on+the+maturation+of+mesencephalic+dopaminergic+neurons.">In vitro studies on the maturation of mesencephalic dopaminergic neurons.</a> Dev Neurosci. 7, 296-307 (1985).</li><li>Marey-Semper, I., Gelman, M., Levi-Strauss, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+selective+toxicity+toward+cultured+mesencephalic+dopaminergic+neurons+is+induced+by+the+synergistic+effects+of+energetic+metabolism+impairment+and+NMDA+receptor+activation.">A selective toxicity toward cultured mesencephalic dopaminergic neurons is induced by the synergistic effects of energetic metabolism impairment and NMDA receptor activation.</a> J Neurosci. 15, 5912-5918 (1995).</li><li>Salthun-Lassalle, B., Hirsch, E. C., Wolfart, J., Ruberg, M., Michel, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+mesencephalic+dopaminergic+neurons+in+culture+by+low-level+stimulation+of+voltage-gated+sodium+channels.">Rescue of mesencephalic dopaminergic neurons in culture by low-level stimulation of voltage-gated sodium channels.</a> J Neurosci. 24, 5922-5930 (2004).</li><li>Toulorge, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotection+of+midbrain+dopamine+neurons+by+nicotine+is+gated+by+cytoplasmic+Ca2.">Neuroprotection of midbrain dopamine neurons by nicotine is gated by cytoplasmic Ca2.</a> FASEB J. 25, 2563-2573 (2011).</li><li>Rousseau, E., Michel, P. P., Hirsch, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Iron-Binding+Protein+Lactoferrin+Protects+Vulnerable+Dopamine+Neurons+from+Degeneration+by+Preserving+Mitochondrial+Calcium+Homeostasis.">The Iron-Binding Protein Lactoferrin Protects Vulnerable Dopamine Neurons from Degeneration by Preserving Mitochondrial Calcium Homeostasis.</a> Mol Pharmacol. 84, (2013).</li><li>Orme, R. P., Bhangal, M. S., Fricker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcitriol+imparts+neuroprotection+in+vitro+to+midbrain+dopaminergic+neurons+by+upregulating+GDNF+expression.">Calcitriol imparts neuroprotection in vitro to midbrain dopaminergic neurons by upregulating GDNF expression.</a> PLoS One. 8 (e62040), (2013).</li><li>Choi, W. S., Kruse, S. E., Palmiter, R. D., Xia, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+complex+I+inhibition+is+not+required+for+dopaminergic+neuron+death+induced+by+rotenone,+MPP+,+or+paraquat.">Mitochondrial complex I inhibition is not required for dopaminergic neuron death induced by rotenone, MPP+, or paraquat.</a> Proc Natl Acad Sci U S A. 105, 15136-15141 (2008).</li><li>Trancikova, A., Ramonet, D., Moore, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mouse+models+of+neurodegenerative+diseases.">Genetic mouse models of neurodegenerative diseases.</a> Prog Mol Biol Transl Sci. 100, 419-482 (2011).</li><li>Gao, H. M., Liu, B., Zhang, W., Hong, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+of+microglial+NADPH+oxidase-derived+free+radicals+in+the+in+vitro+MPTP+model+of+Parkinson's+disease.">Critical role of microglial NADPH oxidase-derived free radicals in the in vitro MPTP model of Parkinson's disease.</a> FASEB J. 17, 1954-1956 (2003).</li><li>Lin, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+expression+of+Parkinson's+disease-related+mutant+alpha-synuclein+in+the+midbrain+dopaminergic+neurons+causes+progressive+neurodegeneration+and+degradation+of+transcription+factor+nuclear+receptor+related+1.">Conditional expression of Parkinson's disease-related mutant alpha-synuclein in the midbrain dopaminergic neurons causes progressive neurodegeneration and degradation of transcription factor nuclear receptor related 1.</a> J Neurosci. 32, 9248-9264 (2012).</li><li>Bye, C. R., Thompson, L. H., Parish, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birth+dating+of+midbrain+dopamine+neurons+identifies+A9+enriched+tissue+for+transplantation+into+parkinsonian+mice.">Birth dating of midbrain dopamine neurons identifies A9 enriched tissue for transplantation into parkinsonian mice.</a> Exp Neurol. 236, 58-68 (2012).</li><li>Ramonet, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+neuronal+loss,+reduced+neurite+complexity+and+autophagic+abnormalities+in+transgenic+mice+expressing+G2019S+mutant+LRRK2.">Dopaminergic neuronal loss, reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2.</a> PLoS One. 6 (e18568), (2011).</li><li>Prestoz, L., Jaber, M., Gaillard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+axon+guidance:+which+makes+what.">Dopaminergic axon guidance: which makes what.</a> Front Cell Neurosci. 6, (2012).</li><li>Nunes, I., Tovmasian, L. T., Silva, R. M., Burke, R. E., Goff, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pitx3+is+required+for+development+of+substantia+nigra+dopaminergic+neurons.">Pitx3 is required for development of substantia nigra dopaminergic neurons.</a> Proc Natl Acad Sci U S A. 100, 4245-4250 (1073).</li><li>Ferri, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxa1+and+Foxa2+regulate+multiple+phases+of+midbrain+dopaminergic+neuron+development+in+a+dosage-dependent+manner.">Foxa1 and Foxa2 regulate multiple phases of midbrain dopaminergic neuron development in a dosage-dependent manner.</a> Development. 134, 2761-2769 (2007).</li><li>Rayport, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identified+postnatal+mesolimbic+dopamine+neurons+in+culture:+morphology+and+electrophysiology.">Identified postnatal mesolimbic dopamine neurons in culture: morphology and electrophysiology.</a> J Neurosci. 12, 4264-4280 (1992).</li><li>Kim, K. M., Nakajima, S., Nakajima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+and+GABA+receptors+in+cultured+substantia+nigra+neurons:+correlation+of+electrophysiology+and+immunocytochemistry.">Dopamine and GABA receptors in cultured substantia nigra neurons: correlation of electrophysiology and immunocytochemistry.</a> Neuroscience. 78, 759-769 (1997).</li><li>Nefzger, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lmx1a+allows+context-specific+isolation+of+progenitors+of+GABAergic+or+dopaminergic+neurons+during+neural+differentiation+of+embryonic+stem+cells.">Lmx1a allows context-specific isolation of progenitors of GABAergic or dopaminergic neurons during neural differentiation of embryonic stem cells.</a> Stem Cells. 30, 1349-1361 (2012).</li><li>Su, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+expression+of+Cdh2+is+essential+for+efficient+neural+differentiation+of+mouse+induced+pluripotent+stem+cells.">Immediate expression of Cdh2 is essential for efficient neural differentiation of mouse induced pluripotent stem cells.</a> Stem Cell Res. 10, 338-348 (2013).</li></ol>

The authors have nothing to disclose.

Este protocolo apresenta os procedimentos e reagentes necessários para a preparação de uma cultura primária de neurónios do mesencéfalo do rato embrionário e o procedimento para a detecção de imunofluorescência neurónios dopaminérgicos. As etapas críticas do processo são a dissecção dos embriões ea dissociação mecânica dos fragmentos cerebrais coletados. Instrumentos de dissecção de alta qualidade ajuda a dominar a técnica de dissecação. Neurónios DA constituem uma pequena percentagem do mesenc...

A dopamina, um dos neurotransmissores cerebrais essenciais 1,2, é libertada principalmente por dopaminérgicos do mesencéfalo (AD) neurónios. A maioria dos neurónios DA residir na parte ventral do mesencéfalo 2-6. Esquematicamente, neurônios DA mesencéfalo podem ser divididos em três sistemas distintos de projeção anatômica e funcionalmente: mesostriatal, mesolímbico, e caminhos mesocorticais 2,5. A via nigroestriatal está envolvida no comportamento do motor, as vias mesolímbica desempenhar um papel importante no reforço, a motivação, aprendizagem e, ao passo que as vias dopaminérgicas que se projectam para o córtex pré-frontal estão implicados na cognição 2. Neurônios DA estão envolvidos em diversas doenças neurológicas humanas, como a esquizofrenia, déficit de atenção, transtorno de hiperatividade, e doença de Parkinson (DP) de 2,4. A DP é caracterizada por uma degeneração progressiva e selectiva dos neurónios DA ligar substantia nigrapars compacta (SNC) para o estriado. A perda de Nigro-estriatal da neurônios resulta na depleção de dopamina no corpo estriado grave que é responsável de os sintomas motores da DP (bradicinesia, tremor de repouso, rigidez e) 7. A causa inicial da DP idiopática não foi estabelecida e os tratamentos atuais são apenas sintomático, com o objetivo de restaurar o nível de dopamina no corpo estriado. A droga mais prescrita é L-Dopa (Levodopa), o precursor natural da dopamina. Embora a administração de Levodopa compensa a perda de dopamina por um certo tempo, complicações motoras ocorrer após tratamentos de longa duração (discinesia e liga / desliga estados) 8,9. Pesquisa sobre neurônios dopaminérgicos e PD está em progressão constante e estão sendo feitos intensos esforços para desenvolver tratamentos baseados em transplante de células, a terapia gênica, ou agentes neuroprotetores 10,11. No entanto, um grande problema permanece não elucidado: o que é a causa da extrema vulnerability de neurônios DA? Parte da resposta pode ser encontrada na actividade dos neurónios DA. A redução da atividade elétrica e da excitabilidade dos neurônios de DA parece aumentar sua propensão a se degenerar 12. No entanto, a complexidade do PD patogênese requer mais estudos para identificar os mecanismos envolvidos na degeneração de neurônios DA 13-15. As culturas primárias são especialmente relevantes para estudar propriedades DA neurônio 16-19 e desafiar esses neurônios para várias tensões para avaliação de agentes neuroprotetores 20-24. Modelos de rato de cultura são os mais frequentemente utilizados, como a dissecação do mesencéfalo de embriões de rato é mais fácil, em comparação com o rato, e maiores quantidades de neurónios podem ser obtidos na ratazana. No entanto, a geração de modelos transgênicos da doença 25 aumentou consideravelmente o interesse da comunidade neurocientista para culturas primárias do rato 26-29. Embora a cultura da prepared de animais recém-nascidos podem ser utilizados, é preferível prepará-los a partir de embriões na fase de pós-mitótico (E13.5 mesencéfalo de neurónios), quando os neurónios mantiveram a sua capacidade de se diferenciar. O protocolo a seguir apresenta os neurónios do mesencéfalo isoladas em cultura primária a partir de embriões de rato (E13.5), as quais são as mais difíceis de preparar. Nomeadamente, é proporcionado um protocolo utilizando meio de cultura isento de soro para uma melhor reprodutibilidade. As duas etapas mais críticas na preparação cultura (dissecção e dissociação mecânica) será cuidadosamente detalhado no vídeo associado.

<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

Neurónios dopaminérgicos representam menos de 1% do número total de neurónios no cérebro. Esta baixa quantidade de neurônios regula funções importantes do cérebro, como controle motor, motivação e memória de trabalho. Neurônios dopaminérgicos nigrostriatal degenerar seletivamente na doença de Parkinson (DP). Esta perda neuronal progressiva é inequivocamente associado aos motores sintomas da patologia (bradicinesia, tremor de repouso, rigidez muscular e). O principal agente responsável dos neurônios dopaminérgicos degeneração ainda é desconhecida. No entanto, esses neurônios parecem ser extremamente vulneráveis ​​em diversas condições. As culturas primárias constitui um dos modelos mais relevantes para investigar as propriedades e características de neurónios dopaminérgicos. Estas culturas podem ser submetidos a vários agentes estressores que imitam PD patologia e compostos neuroprotetores, a fim de parar ou abrandar a degeneração neuronal. Os inúmeros modelos de camundongos transgênicos do PD que foram generciados durante a última década aumentou ainda mais o interesse dos pesquisadores para as culturas de neurónios dopaminérgicos. Aqui, o protocolo de vídeo concentra-se na dissecação delicada de cérebros embrionárias de rato. Excisão precisa do mesencéfalo ventral é crucial para obter culturas neuronais suficientemente ricos em células dopaminérgicas para permitir estudos posteriores. Este protocolo pode ser realizado com camundongos transgênicos embrionárias e é adequado para imunofluorescência, PCR quantitativo, segundo quantificação mensageiro, ou avaliação morte / sobrevivência neuronal.

Um fluxograma que ilustra os passos de cultura de mesencéfalo é mostrado na Figura 1. Resumidamente, após a recolha de embriões a partir de um rato E13.5 Swiss grávida, mesencéfalo ventral é dissecado a partir de todo o embrião. Os fragmentos isolados do cérebro são submetidos sucessivamente a digestão enzimática e dissociação mecânica. As células dissociadas foram sedimentadas por centrifugação, ressuspensas em meio de cultura e semeados em placas de 12 ou de 24 poços pré-revestidos...

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.

Cultura primária de neurônios dopaminérgicos mouse

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

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Research

JoVE Journal

Neuroscience

37.2K visualizações.  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.

Vídeo: Primary Culture of Mouse Dopaminergic Neurons

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection e Cultura Primária de Neurônios do hipocampo do rato embrionárias e Cortical

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

Neurociência

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.

Bilaminar Co-cultura de neurônios de rato Primária Cortical e 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 ...

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.

Dissecção e Cultura do mouse e dopaminérgico do estriado explantes em três dimensões Ensaios matriz de colágeno

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.

Isolamento, cultura e Long-Term Manutenção da Primária mesencefálico Dopaminergic neurônios dos cérebros de roedores Embryonic

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.

Modelagem de morte Neuronal e a degeneração no Mouse grânulo cerebelar primária de neurônios

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.

Imagens ao vivo da proteína GLUT4 tráfico de Mouse de neurônios hipotálamo primário usando microscopia de deconvolução

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.

Cultura de células primárias de neurônios Gabaérgicos ou glutamatérgicos purificados estabelecidos através da triagem de células ativadas por fluorescência

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.

Cultura primária de neurônios isolados do cerebelo embrionário do rato

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.

Rotulagem Balística de Neurônios Piramidais em Fatias Cerebrais e na Cultura Celular Primária

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.