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

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

&#160;The mice used in this work were cared for and handled in accordance with the guidelines of the European Union Council (86/609/EU) for the use of laboratory animals.
1. Preparation of Required Solutions
<ol>
	<li>Stock Solutions
	<ol>
		<li>10x Poly-L-Ornithine (PLO) Solution: weigh out 10 mg of PLO hydrobromide (molecular weight = 30,000-70,000) and dissolve in 70 ml of sterile water. Filter the solution using 0.2 &#181;m&#160;syringe filter, aliquot, and store at -20 &#176;C.</li>
		<...

<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>

This protocol presents the procedures and reagents necessary to prepare a primary culture of mesencephalic neurons from the embryonic mouse and the immunofluorescence procedure to detect dopaminergic neurons. Critical steps of the procedure are the dissection of the embryos and the mechanical dissociation of the collected brain fragments. High quality dissection instruments helps to master the dissection technique. DA neurons constitute a small proportion of mesencephalon. Accordingly, collecting the right part of the ve...

Dopamine, one of the essential brain neurotransmitters1,2, is mainly released by midbrain dopaminergic (DA) neurons. The majority of DA neurons reside in the ventral part of the mesencephalon2-6. Schematically, midbrain DA neurons can be divided in three anatomically and functionally distinct projection systems: mesostriatal, mesolimbic, and mesocortical pathways2,5. The nigrostriatal pathway is involved in motor behavior, the mesolimbic pathways play an important role in reinforcement, motivation, and learning, whereas the dopaminergic pathways projecting to the prefrontal cortex are implicated in cognition2.
DA neurons are involved in several human neurological disorders such as schizophrenia, attention deficit, hyper activity disorder, and Parkinson&#8217;s disease (PD)2,4. PD is characterized by a progressive and selective degeneration of DA neurons connecting substantia nigra pars compacta (SNc) to the striatum. The loss of nigro-striatal DA neurons results in severe dopamine depletion in the striatum that is responsible of the motor symptoms of PD (bradykinesia, resting tremor, and rigidity)7. The initial cause of the idiopathic PD has not been established and the current treatments are only symptomatic, aiming at restoring dopamine level in the striatum. The most prescribed drug is L-Dopa (Levodopa), the natural precursor of dopamine. Though administration of Levodopa compensates for the loss of dopamine for a certain time, motor complications occur after long-term treatments (dyskinesia and on/off states)8,9.
Research on dopaminergic neurons and PD is in constant progression and intense efforts are being made to develop treatments based on cell transplantation, gene therapy, or neuroprotective agents10,11. However, a major issue remains non-elucidated: what is the cause of the extreme vulnerability of DA neurons? Part of the answer can be found in the activity of DA neurons. A reduction in the electrical activity and of the excitability of DA neurons seems to augment their propensity to degenerate12. Nevertheless, the complexity of PD pathogenesis requires further studies to identify the mechanisms involved in DA neurons degeneration13-15.
Primary cultures are especially relevant to study DA neuron properties16-19 and to challenge these neurons to various stresses for evaluation of neuroprotective agents20-24. Rat culture models are most often used, as the dissection of rat embryo mesencephalon is easier, compared with the mouse, and higher amounts of neurons can be obtained in the rat. However, generation of transgenic mouse models of the disease25 has considerably increased the interest of the neuroscientist community for primary cultures from the mouse26-29. Although cultures prepared from newborn animals can be used, it is better to prepare them from embryos at the post-mitotic stage (E13.5 for mesencephalon neurons), when neurons have retained their capacity to differentiate. The following protocol presents isolated mesencephalon neurons in primary culture from mouse embryos (E13.5), which are the most difficult to prepare. Notably, we provide a protocol using serum-free culture medium for a better reproducibility. The two most critical steps in culture preparation (dissection and mechanical dissociation) will be carefully detailed in the associated video.

<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

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

An illustrated flow chart of the mesencephalon culture steps is shown in Figure 1. Briefly, after collecting E13.5 embryos from a pregnant Swiss mouse, ventral mesencephalon is dissected from the entire embryo. The isolated brain fragments are successively submitted to enzymatic digestion and mechanical dissociation. Dissociated cells are pelleted by centrifugation, resuspended in culture medium and plated in pre-coated 12- or 24-well plates. Cells are maintained up to 15 days without medium replacement....

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.

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

Research

JoVE Journal

Neuroscience

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 ...

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

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 ...

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. ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...