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>
		<li>30% Glucose Solution: weigh out 30 g of D(+)-Glucose and dissolve in sterile water to a total volume of 100 ml. Filter, aliquot, and store at 4 &#176;C.</li>
		<li>5x Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/Nutrient Mixture F-12 Ham: weigh out 6 g of DMEM/F-12 Ham powder and dissolve in sterile water to a total volume of 100 ml. Filter, aliquot, and store at 4 &#176;C.</li>
		<li>7.5% Sodium Bicarbonate (NaHCO3) Solution: weigh out 7.5 g of NaHCO3 and dissolve in sterile water to a total volume of 100 ml. Filter, aliquot, and store at 4 &#176;C.</li>
		<li>1 M HEPES Solution: Weigh out 23.8 g of HEPES and dissolve in sterile water to a total volume of 100 ml. Filter, aliquot, and store at 4 &#176;C.</li>
		<li>70% (v/v) Ethanol: Dilute 70 ml of absolute ethanol with 30 ml of sterile water.</li>
	</ol>
	</li>
	<li>Phosphate Buffered Saline with Glucose and Antibiotics (PBS-GAB): add 10 ml of 30% glucose solution and 5 ml of penicillin-streptomycin solution to 500 ml of Dulbecco&#8217;s Phosphate Buffered Saline. Store at 4 &#176;C.</li>
	<li>Inactivated Fetal Bovine Serum (iFBS): Thaw bovine serum overnight at 4 &#176;C and inactivate at 56 &#176;C for 30 min. Aliquot into 50 ml and store at -20 &#176;C.</li>
	<li>Culture Medium: In sterile water, mix successively 40 ml of 5x DMEM/F12-Ham, 1 ml of 1 M HEPES, 2 ml of 200 mM L-Glutamine, 2 ml of penicillin-streptomycin, 4 ml of 30% glucose and 3 ml of 7.5% NaHCO3 to a final volume of 180 ml. Filter and use the same day.</li>
	<li>Hormone Mix
	<ol>
		<li>Prepare 180 ml of culture medium. Dissolve 200 mg of apo-transferrin in this medium.</li>
		<li>Weigh out 50 mg of insulin and dissolve in 2 ml of 0.1 M HCl. Add slowly 8 ml of sterile water while mixing gently. Dilute this solution in the culture medium.</li>
		<li>Weigh out 19.3 mg of Putrescine dihydrochloride and dissolve in 10 ml of sterile water. Add the 10 ml solution to the hormone mix.</li>
		<li>Prepare a 3 mM sodium selenite solution in sterile water and a 2 mM progesterone solution in absolute ethanol. Add 20 &#956;l of each solution to the hormone mix.</li>
		<li>Filter the hormone mix, aliquot into 10 ml and store at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Preparation of Culture Plates and Instruments
<ol>
	<li>FBS Coating of the Culture Plates: The day prior the dissection, prepare 180 ml of culture medium. Add 10 ml of iFBS to 90 ml of culture medium. Add 300 &#956;l/well of culture medium/10% iFBS into poly-D-lysine precoated 24-well plates. Incubate overnight at 37 &#176;C. Store the remaining media (with and without iFBS) at 4 &#176;C.</li>
	<li>Sterilize the instruments and Pasteur pipettes. Gather the equipment listed in Materials table as well as a Class II laminar flow hood and a CO2 incubator.</li>
</ol>
3. Dissection of Mouse Mesencephalon
<ol>
	<li>Sacrifice an E13.5 pregnant mouse (according to institutional guidelines). Clean the abdomen of the mouse with 70% ethanol and open the abdomen wall. Collect the uterine horns, open them using delicate scissors, dissect each embryo from the uterine horns, and remove the amniotic membranes. Place the embryos in a 100 mm Petri dish containing sterile PBS-GAB and wash them by transfer in 3 successive identical baths using forceps. For dissection, place the embryos by 3 in a 60 mm sterile Petri dish.</li>
	<li>Move the final Petri dish under the stereomicroscope. Do not behead the embryos. Using Vannas scissors, excise the brains.
	<ol>
		<li>Carefully remove and discard the fore- and hindbrain regions. To the rostral side, cut close to the thalamic region, to the caudal side cut at the isthmus region, remove the superior colliculus.</li>
		<li>Once the ventral midbrain is isolated, carefully remove meninges using ultra fine forceps. Collect the dissected segments, without the meninges, in a sterile 13 ml tube filled with PBS-GAB.</li>
	</ol>
	</li>
	<li>Clean the tube containing the mesencephala thoroughly with 70% ethanol and bring it under the hood.</li>
</ol>
4. Cell Dissociation
<ol>
	<li>Wash mesencephala 3x&#160;with sterile PBS-GAB. Allow brain fragments to settle between each wash and avoid disrupting the tissue. After the last wash, remove carefully as much solution as possible and incubate brain segments in 3 ml of Trypsin/EDTA for 15 min at 37 &#176;C in a CO2 incubator.</li>
	<li>Fire-polish the Pasteur pipettes to maximize the survival of the neurons as the end of a non-polished glass pipet is sharp and can damage the cells during the dissociation steps. Slightly reduce the extremity diameter (up 0.5 mm) of the Pasteur pipette while fire-polishing it.</li>
	<li>Carefully remove as much Trypsin/EDTA solution as possible and add 10 ml of culture medium/10% iFBS. Wash brain segments 3x&#160;with culture medium/10% iFBS.</li>
	<li>Using the fire-polished Pasteur pipette, begin dissociation of mesencephala in 6 ml of culture medium/10% iFBS. Triturate 10x&#160;(avoid air bubbles), allow the chunks to settle, then collect the medium in a new sterile 13 ml tube.</li>
	<li>Add 6 ml of culture medium, repeat the previous step once and collect the medium containing dissociated cells in the same 13 ml tube.</li>
	<li>Centrifuge cells at 160 g for 5 min. Discard the supernatant and resuspend the cells in culture medium supplemented with 10% hormone mix.</li>
</ol>
5. Cell Plating
<ol>
	<li>Count cells in suspension in a Malassez cell and adjust the volume of medium to a concentration of 600,000 cells/ml. Around 1,700,000 cells can be obtained from one mouse mesencephalon.</li>
	<li>Remove FBS-containing coating medium from 24-well plates and add in each well 1 ml of cell suspension (600,000 cells/well, 2-4% of TH+ cells).</li>
	<li>Incubate the plate at 37 &#176;C and 5% CO2/95% air. No medium change is necessary. Dopaminergic neurons are mature after around 5-7 days in vitro (consistent expression of the dopamine transporter). Keep cells in culture up to 15 days without medium replacement.</li>
</ol>
6. Immunofluorescence Protocol
<ol>
	<li>Cleaning of the Coverslips
	<ol>
		<li>The day before the dissection, add 10 ml of 1 M HCl into a Petri dish. Lay at the surface of the liquid 12-15 glass coverslips and wait 15 min.</li>
		<li>Sink the coverslip into the liquid and wait for 15 min.</li>
		<li>Remove HCl and wash 3x with water.</li>
		<li>Wash the coverslips quickly with pure ethanol once, then add pure ethanol to the dish and wait for 30 min.</li>
		<li>Under the hood, remove cleaned coverslips from ethanol and add 1 coverslip per well of a 12-well cell culture plate using sterilized forceps.</li>
		<li>Wash coverslips twice with sterile water (1 ml/well). Then, wash them once with sterile PBS.</li>
	</ol>
	</li>
	<li>Coating of the Coverslips
	<ol>
		<li>Remove PBS and add 500 &#956;l/well of 2x PLO (diluted in PBS). Incubate for 4 hr at 37 &#176;C in the CO2 incubator.</li>
		<li>Remove the PLO solution and wash 3x with sterile PBS.</li>
		<li>Remove PBS and add 500 &#956;l/well of 20% iFBS/1 g/ml Laminin. Incubate overnight at 37 &#176;C in the CO2 incubator.</li>
	</ol>
	</li>
	<li>Plating the Cells for Immunofluorescence
	<ol>
		<li>Remove coating medium from 12-well plates.</li>
		<li>Add 900,000 cells/well in 2 ml volume and grow the cells at 37 &#176;C in the incubator. Cells can be kept in culture up to 15 days without medium replacement.</li>
	</ol>
	</li>
	<li>Immunostaining
	<ol>
		<li>Remove medium from 12-well plates and wash with 500 &#956;l/well DMEM preheated at 37 &#176;C.</li>
		<li>Add 500 &#956;l/well of DMEM preheated at 37 &#176;C, then add gently 160 &#956;l/well of 8% paraformaldehyde (PFA, 2% final concentration) and incubate for 10 min at 37 &#176;C.</li>
		<li>Remove PFA and wash 3x with PBS/0.1 M glycine for 10 min.</li>
		<li>Remove PBS, add 300 &#956;l/well of PBS/0.05% Triton X-100/20% Goat serum and incubate for 30 min at room temperature.</li>
		<li>Remove medium, add 300 &#956;l/well of primary antibody diluted in PBS/0.05% Triton X-100/1% Goat serum and incubate for 2 hr at room temperature. Primary antibodies that can be used for the detection of dopaminergic neurons and characterization of the culture are indicated in the Materials table. Keep 1 well without primary antibody to assess for background of the different secondary antibodies.</li>
		<li>Remove medium and wash 3x with PBS/0.2% gelatine for 10 min.</li>
		<li>Remove medium, add 300 &#956;l/well of secondary antibody diluted in PBS/0.05% Triton X-100/1% Goat serum and incubate for 1 hr at room temperature, in the dark. Secondary antibodies used are indicated in the Materials table.</li>
		<li>Remove medium and wash 3x with PBS/0.2% gelatine for 10 min.</li>
		<li>Remove medium and wash 3x with PBS.</li>
		<li>Mount the coverslips on glass slides cell side down in a drop of VECTASHIELD. Keep the slides at room temperature overnight to let the mounting medium dry, then store them at 4 &#176;C.</li>
		<li>Acquire images using a confocal microscope or a phase-contrast microscope equipped for epifluorescence. Representative images were acquired with a Leica SP2 UV confocal microscope.</li>
	</ol>
	</li>
</ol>

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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.

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 ...

primary-culture-of-mouse-dopaminergic-neurons

Research

JoVE Journal

Neuroscience

37.6K visualizações.  Institut de Génomique Fonctionnelle, Montpellier. Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Vídeo: Primary Culture of Mouse Dopaminergic Neurons

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

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

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

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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