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.

Les souris utilisées dans ce travail ont été pris en charge et traités en conformité avec les lignes directrices du Conseil de l&#39;Union européenne (86/609 / UE) pour l&#39;utilisation des animaux de laboratoire. 1 Préparation des solutions obligatoires <ol><li> Solutions mères <ol><li> 10x poly-L-ornithine (OLP) Solution: peser 10 mg de bromhydrate OLP (poids moléculaire = 30,000-70,000) et dissoudre dans 70 ml d&#39;eau stérile. Filtrer la solution à l&#39;aide de 0,2 um filtre de la seringue, aliquote, et conserver à -20 ° C. </li><li> 30% Solution de glucose: peser sur 30 g de D (+) - glucose et le dissoudre dans de l&#39;eau stérile jusqu&#39;à un volume total de 100 ml. Filtre aliquote, et conserver à 4 ° C. </li><li> Milieu de Eagle modifié de 5x Dulbecco (DMEM) / mélange nutritif F-12 Ham: peser 6 g de poudre DMEM / Ham F-12 et les dissoudre dans de l&#39;eau stérile pour un volume total de 100 ml. Filtre aliquote, et conserver à 4 ° C. </li><li> 7,5% de bicarbonate de sodium (NaHCO 3) Solutile: peser 7,5 g de NaHCO 3 et dissoudre dans de l&#39;eau stérile jusqu&#39;à un volume total de 100 ml. Filtre aliquote, et conserver à 4 ° C. </li><li> 1 M HEPES Solution: Peser 23,8 g d&#39;HEPES et dissoudre dans de l&#39;eau stérile jusqu&#39;à un volume total de 100 ml. Filtre aliquote, et conserver à 4 ° C. </li><li> 70% (v / v) d&#39;éthanol: Diluer 70 ml d&#39;éthanol absolu avec 30 ml d&#39;eau stérile. </li></ol></li><li> Phosphate Buffered Saline de glucose et les antibiotiques (PBS-GAB): ajouter 10 ml de solution à 30% de glucose et 5 ml de solution de pénicilline-streptomycine à 500 ml de solution saline tamponnée au phosphate de Dulbecco. Conserver à 4 ° C. </li><li> Sérum bovin fœtal inactivé (IFB): Décongeler sérum bovin pendant une nuit à 4 ° C et inactiver à 56 ° C pendant 30 min. Aliquote dans 50 ml et conserver à -20 ° C. </li><li> Milieu de culture: Dans l&#39;eau stérile, mélanger successivement 40 ml de 5x DMEM / F12-Ham, 1 ml de 1 M HEPES, 2 ml de 200 mM de L-glutamine, 2 ml de pénicilline-streptomycine, 4 ml de glucose à 30% unee 3 ml de 7,5% de NaHCO 3 à un volume final de 180 ml. Filtrer et utiliser le même jour. </li><li> Hormone Mix <ol><li> Préparer 180 ml de milieu de culture. Dissoudre 200 mg d&#39;apo-transferrine dans ce milieu. </li><li> Peser 50 mg d&#39;insuline et dissoudre dans 2 ml de HCl 0,1 M. Ajouter lentement 8 ml d&#39;eau stérile tout en mélangeant délicatement. Diluer cette solution dans le milieu de culture. </li><li> Peser 19,3 mg de dichlorhydrate de putrescine et de se dissoudre dans 10 ml d&#39;eau stérile. Ajouter 10 ml de la solution de la composition de l&#39;hormone. </li><li> Préparer une solution de sélénite de sodium à 3 mM dans de l&#39;eau stérile et une solution de 2 mM de progestérone dans l&#39;éthanol absolu. Ajouter 20 ul de chaque solution de la composition de l&#39;hormone. </li><li> Filtrer le mélange d&#39;hormones, en aliquote de 10 ml et conserver à -20 ° C. </li></ol></li></ol> 2 Préparation de plaques et instruments Culture <ol><li> FBS revêtement des plaques Culture: Le jour avant la dissection, préparer 180 ml de milieu de culture. Ajouter 10 ml d&#39;IFBS à 90 ml de milieu de culture. Ajouter 300 ul / puits de milieu de culture / 10% IFBS dans des plaques à 24 puits pré-enduites de poly-D-lysine. Incuber une nuit à 37 ° C. Conserver les médias restants (avec et sans les appels d&#39;offres) à 4 ° C. </li><li> Stériliser les instruments et les pipettes Pasteur. Rassemblez les équipements énumérés dans le tableau des matériaux ainsi que d&#39;une hotte de classe II à flux laminaire et un incubateur à CO2. </li></ol> 3. dissection de souris Mésencéphale <ol><li> Sacrifier une souris enceinte de E13.5 (selon les directives institutionnelles). Nettoyez l&#39;abdomen de la souris avec 70% d&#39;éthanol et d&#39;ouvrir la paroi abdominale. Recueillir les cornes utérines, ouvrez-les avec des ciseaux délicats, disséquer chaque embryon de cornes utérines, et enlever les membranes amniotiques. Placer les embryons dans une boîte de Pétri de 100 mm contenant stérile PBS-GAB et lavez-les par transfert dans 3 salles de bains successifs identiques en utilisant une pince. Pour dissection, placer les embryons par 3 dans une boîte de Pétri stérile de 60 mm. </li><li> Move le plat final Petri sous la loupe binoculaire. Ne pas décapiter les embryons. Avec des ciseaux Vannas, exciser le cerveau. <ol><li> Retirez délicatement et jeter les régions contremaîtres et du cerveau postérieur. Pour le côté rostral, couper près de la région du thalamus, de la coupe de côté caudal à la région de l&#39;isthme, retirez le colliculus supérieur. </li><li> Une fois le mésencéphale ventral est isolé, retirer délicatement les méninges à l&#39;aide de pinces ultra-fines. Recueillir les segments disséqués, sans les méninges, dans un tube de 13 ml stérile remplie de PBS-GAB. </li></ol></li><li> Nettoyez le tube contenant le mesencephala à fond avec 70% d&#39;éthanol et l&#39;amener sous le capot. </li></ol> 4. cellule de dissociation <ol><li> Laver mesencephala 3x avec PBS stérile-GAB. Autoriser les fragments du cerveau à régler entre chaque lavage et d&#39;éviter de perturber le tissu. Après le dernier lavage, éliminer soigneusement autant que possible la solution et incuber segments du cerveau dans 3 ml de trypsine / EDTA pendant 15 min à 37 ° C dans unIncubateur à CO 2. </li><li> Feu-polissez les pipettes Pasteur afin de maximiser la survie des neurones que la fin d&#39;une pipette de verre non poli est forte et peuvent endommager les cellules au cours des étapes de dissociation. Légèrement réduire le diamètre de l&#39;extrémité (jusqu&#39;à 0,5 mm) de la pipette Pasteur tandis que le feu-polissage. </li><li> Retirez délicatement autant solution / EDTA trypsine que possible et ajouter 10 ml de milieu de culture / 10% les appels d&#39;offres. Laver les segments du cerveau 3x avec milieu de culture / 10% les appels d&#39;offres. </li><li> Utilisation de la pipette Pasteur polie au feu, commencer dissociation de mesencephala dans 6 ml de milieu de culture / 10% les appels d&#39;offres. Triturez 10x (éviter les bulles d&#39;air), permettent aux morceaux de s&#39;installer, puis recueillir la moyenne dans un nouveau tube stérile de 13 ml. </li><li> Ajouter 6 ml de milieu de culture, répéter l&#39;étape précédente une fois et récupérer le milieu contenant les cellules dissociées dans le même tube de 13 ml. </li><li> Centrifuger les cellules à 160 g pendant 5 min. Jeter le surnageant et remettre en suspension les cellules dans du milieu de culture supplémenté avec10% mélange d&#39;hormones. </li></ol> 5. cellulaire Placage <ol><li> Compter les cellules en suspension dans une cellule de Malassez et ajuster le volume du milieu à une concentration de 600 000 cellules / ml. Environ 1,7 millions de cellules peuvent être obtenues à partir de l&#39;un mésencéphale de souris. </li><li> Retirer FBS contenant un milieu de revêtement de plaques à 24 puits et ajouter dans chaque puits 1 ml de suspension cellulaire (600 000 cellules / puits, 2-4% de cellules TH +). </li><li> Incuber la plaque à 37 ° C et 5% CO2 / 95% air. Pas de changement de milieu est nécessaire. Les neurones dopaminergiques sont matures après environ 5 à 7 jours in vitro (expression cohérente du transporteur de la dopamine). Maintenir les cellules en culture jusqu&#39;à 15 jours sans remplacement du milieu. </li></ol> Protocole 6 de immunofluorescence <ol><li> Nettoyage des lamelles couvre- <ol><li> La veille de la dissection, ajoutez 10 ml d&#39;HCl 1 M dans une boîte de Pétri. Lay à la surface des liquides 12-15 lamelles de verre et attendre 15 min. </li><li> Coulez le couvercleglisser dans le liquide et attendre 15 min. </li><li> Retirer HCl et laver 3x avec de l&#39;eau. </li><li> Laver les lamelles rapidement avec de l&#39;éthanol pur une fois, puis ajouter de l&#39;éthanol pur à l&#39;antenne et attendre 30 min. </li><li> Sous le capot, enlever des lamelles nettoyés dans de l&#39;éthanol et ajouter 1 lamelle par puits d&#39;une plaque de culture cellulaire de 12 puits en utilisant des pinces stériles. </li><li> Laver deux fois avec de lamelles de l&#39;eau stérile (1 ml / puits). Ensuite, lavez-les une fois avec du PBS stérile. </li></ol></li><li> Revêtement des Lamelles <ol><li> Retirer PBS et ajouter 500 pl / puits de 2x OLP (dilué dans du PBS). Incuber pendant 4 h à 37 ° C dans l&#39;incubateur à CO 2. </li><li> Retirer la solution de l&#39;OLP et laver 3 fois avec du PBS stérile. </li><li> Retirer du PBS et ajouter 500 pl / puits de 20% les appels d&#39;offres / 1 g / ml laminine. Incuber pendant une nuit à 37 ° C dans l&#39;incubateur à CO 2. </li></ol></li><li> Étalement des cellules pour immunofluorescence <ol><li> Éliminer le milieu de revêtement de plaques à 12 puits. </li><li> Ajouter 900,000 cellules / puits dans un volume de 2 ml et croître les cellules à 37 ° C dans l&#39;incubateur. Les cellules peuvent être maintenues en culture jusqu&#39;à 15 jours sans remplacement du milieu. </li></ol></li><li> Immunocoloration <ol><li> Retirer moyen de plaques à 12 puits et laver avec 500 pl / puits de DMEM préchauffé à 37 ° C. </li><li> Ajouter 500 pl / puits de DMEM préchauffé à 37 ° C, puis ajouter délicatement 160 pl / puits de 8% de paraformaldéhyde (PFA, 2% concentration finale) et incuber pendant 10 min à 37 ° C. </li><li> Retirer PFA et laver 3 fois avec PBS / 0,1 M de glycine pendant 10 min. </li><li> Retirer PBS, ajouter 300 pl / puits de PBS / 0,05% de Triton X-100/20% de sérum de chèvre et incuber pendant 30 min à température ambiante. </li><li> Éliminer le milieu, ajouter 300 ul / puits de l&#39;anticorps primaire dilué dans du PBS / 0,05% de Triton X-100/1% de sérum de chèvre et incuber pendant 2 heures à température ambiante. Les anticorps primaires qui peuvent être utilisés pour la détection des neurones dopaminergiques et la caractérisation de la culture sont indiqués dans le tableau des matériaux. Garder1 bien sans anticorps primaire à évaluer pour le fond des différents anticorps secondaires. </li><li> Éliminer le milieu et laver 3 fois avec PBS / 0,2% de gélatine pendant 10 minutes. </li><li> Éliminer le milieu, ajouter 300 pl / puits d&#39;anticorps secondaire dilué dans du PBS / 0,05% de Triton X-100/1% de sérum de chèvre et incuber pendant 1 heure à température ambiante, dans l&#39;obscurité. Anticorps secondaires utilisés sont indiqués dans le tableau des matériaux. </li><li> Éliminer le milieu et laver 3 fois avec PBS / 0,2% de gélatine pendant 10 minutes. </li><li> Éliminer le milieu et laver 3 fois avec PBS. </li><li> Monter les lamelles sur le côté verre diapositives de cellule dans une goutte de Vectashield. Gardez les lames à température ambiante pendant la nuit pour laisser le milieu de montage à sec, puis les stocker à 4 ° C. </li><li> Acquérir des images à l&#39;aide d&#39;un microscope confocal ou un microscope à contraste de phase équipé pour l&#39;épifluorescence. Des images représentatives ont été acquises avec un microscope 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.

Ce protocole présente les procédures et les réactifs nécessaires à la préparation d&#39;une culture primaire de neurones mésencéphaliques de la souris embryonnaire et la procédure d&#39;immunofluorescence pour détecter les neurones dopaminergiques. Les étapes critiques de la procédure sont la dissection des embryons et la dissociation mécanique des fragments de cerveau prélevés. Instruments de dissection de haute qualité contribue à maîtriser la technique de dissection. Neurones dopaminergiques constit...

La dopamine, un des neurotransmetteurs du cerveau essentielles 1,2, est surtout rejeté par dopaminergiques du mésencéphale (DA) des neurones. La majorité des neurones dopaminergiques de séjourner dans la partie ventrale du mésencéphale 6.2. Schématiquement, les neurones dopaminergiques du mésencéphale peuvent être divisés en trois anatomiquement et fonctionnellement distinctes des systèmes de projection: mesostriatal, mésolimbique et les voies mésocorticales 2,5. La voie nigro est impliqué dans le comportement moteur, les voies mésolimbiques jouent un rôle important dans le renforcement, la motivation et l&#39;apprentissage, alors que les voies dopaminergiques projetant vers le cortex préfrontal sont impliquées dans la cognition 2. Neurones dopaminergiques sont impliqués dans plusieurs troubles neurologiques humains tels que la schizophrénie, déficit de l&#39;attention, trouble de l&#39;hyperactivité, et la maladie (le PD) de Parkinson 2,4. PD est caractérisée par une dégénérescence progressive et sélective des neurones dopaminergiques de la substance noire de liaisonpars compacta (SNC) dans le striatum. La perte de nigro-striée DA neurones entraîne un grave appauvrissement de la dopamine dans le striatum qui est responsable des symptômes moteurs de la maladie (bradykinésie, tremblement de repos, et la rigidité) 7. La cause initiale de la MP idiopathique n&#39;a pas été établie et les traitements actuels ne sont symptomatiques, visant à restaurer le niveau de dopamine dans le striatum. Le médicament le plus prescrit est la L-Dopa (lévodopa), le précurseur naturel de la dopamine. Bien que l&#39;administration de lévodopa compense la perte de dopamine pendant un certain temps, des complications motrices surviennent après un traitement de longue durée (dyskinésie et états ON / OFF) 8,9. La recherche sur les neurones dopaminergiques et PD est en constante progression et les efforts intenses sont déployés pour développer des traitements basés sur la transplantation de cellules, la thérapie génique, ou agents neuroprotecteurs 10,11. Cependant, reste un problème majeur non élucidé: quelle est la cause de l&#39;extrême vulnerabilité des neurones DA? Une partie de la réponse peut être trouvée dans l&#39;activité des neurones dopaminergiques. Une réduction de l&#39;activité électrique et de l&#39;excitabilité des neurones dopaminergiques semblent augmenter leur propension à dégénérer 12. Néanmoins, la complexité du PD pathogénie nécessite d&#39;autres études pour identifier les mécanismes impliqués dans la dégénérescence des neurones DA 13-15. Les cultures primaires sont particulièrement pertinentes pour l&#39;étude des propriétés des neurones DA 16-19 et de contester ces neurones à différentes contraintes pour l&#39;évaluation des agents neuroprotecteurs 20-24. des modèles de culture de Rat sont le plus souvent utilisés, comme la dissection du mésencéphale embryonnaire de rat est plus facile, par rapport à la souris, et des quantités plus élevées de neurones peuvent être obtenus chez le rat. Toutefois, la production de modèles de souris transgéniques de la maladie 25 a considérablement augmenté l&#39;intérêt de la communauté neuroscientifique pour des cultures primaires de la souris 26-29. Bien que les cultures prepared des animaux nouveau-nés peuvent être utilisés, il est préférable de les préparer à partir d&#39;embryons au stade post-mitotique (E13.5 pour les neurones de mésencéphale), lorsque les neurones ont conservé leur capacité de se différencier. Le protocole ci-dessous présente les neurones de mésencéphale en culture primaire isolés à partir d&#39;embryons de souris (de E13.5), qui sont les plus difficiles à préparer. En particulier, nous fournissons un protocole utilisant un milieu de culture sans sérum pour une meilleure reproductibilité. Les deux étapes les plus critiques dans la préparation de la culture (dissection et dissociation mécanique) seront soigneusement détaillés dans la vidéo associée.

<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

Les neurones dopaminergiques représentent moins de 1% du nombre total de neurones dans le cerveau. Cette faible quantité de neurones du cerveau régule les fonctions importantes telles que le contrôle moteur, la motivation et la mémoire de travail. Neurones dopaminergiques nigro-strié sélectivement dégénèrent dans la maladie de Parkinson (MP). Cette perte neuronale progressive est sans équivoque associée aux moteurs symptômes de la pathologie (bradykinésie, tremblement de repos, et une rigidité musculaire). Le principal agent responsable de la dégénérescence des neurones dopaminergiques est encore inconnue. Cependant, ces neurones semblent être extrêmement vulnérables dans diverses conditions. Les cultures primaires constituent l&#39;un des modèles les plus pertinents pour étudier les propriétés et les caractéristiques des neurones dopaminergiques. Ces cultures peuvent être soumis à différents agents de stress qui imitent PD pathologie et de composés neuroprotecteurs pour arrêter ou ralentir la dégénérescence neuronale. Les nombreux modèles de souris transgéniques de la MP qui ont été généATED au cours de la dernière décennie a encore augmenté l&#39;intérêt des chercheurs pour les cultures neuronales dopaminergiques. Ici, le protocole vidéo met l&#39;accent sur la dissection fine de cerveaux de souris embryonnaires. Excision précise de mésencéphale ventral est crucial d&#39;obtenir des cultures de neurones suffisamment riches en cellules dopaminergiques pour permettre des études ultérieures. Ce protocole peut être réalisée avec des souris transgéniques embryonnaires et est approprié pour la coloration par immunofluorescence, de PCR quantitative, la quantification de second messager, ou mort neuronale évaluation / de survie.

Un organigramme illustré les étapes de culture de mésencéphale est représenté sur la figure 1. Brièvement, après la collecte d&#39;embryons E13.5 de souris Swiss enceinte, mésencéphale ventral est disséqué à partir de l&#39;ensemble de l&#39;embryon. Les fragments isolés du cerveau sont successivement soumis à une digestion enzymatique et dissociation mécanique. Les cellules dissociées sont sédimentées par centrifugation, remises en suspension dans du milieu de culture et étalées da...

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.

Culture primaire de souris dopaminergiques neurones

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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JoVE Journal

Neuroscience

37.5K vues.  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.

Vidéo: 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.

Nucléofection et de la culture primaire de neurones embryonnaires de souris hippocampe et cortex

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

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Neurosciences

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.

Didermique co-culture de neurones de rat corticale primaire et des cellules gliales

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.

Dissection et de la culture de la souris dopaminergique et des explants striataux en trois dimensions dosages matrice de collagène

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

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Phosphate de calcium transfection de primaire neurones de l&#39;hippocampe

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

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.

Isolement, Culture et maintenance à long terme des mésencéphalique primaire dopaminergiques neurones à partir de cerveaux embryonnaires de rongeurs

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.

Modélisation de la mort neuronale et la dégénérescence de neurones granules primaires de cervelet de souris

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.

Images en direct de la protéine GLUT4 traite des neurones hypothalamiques primaire de souris au microscope de déconvolution

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.

Culture de cellules primaires de neurones GABAergiques ou glutamatergiques purifiés établis par tri cellulaire activé par fluorescence

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.

Culture primaire des neurones isolés du cervelet de souris embryonnaire

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.

Étiquetage balistique des neurones pyramidaux dans les tranches de cerveau et dans la culture cellulaire primaire

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