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><tbody><tr><td>Fetal Bovine Serum</td><td>Lonza</td><td>14-801F</td><td></td></tr><tr><td>DMEM 4.5 g/L glucose with L-glutamine</td><td>Lonza</td><td>BE12-604F</td><td></td></tr><tr><td>0.05% Trypsin-EDTA (1x), phenol red</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>L-Glutamine, 200 mM solution</td><td>Life Technologies</td><td>25030123</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D8537</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium/Nutrient Mixture F-12 Ham</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D0547</td><td>Powder</td></tr><tr><td>Laminin - 1 mg/ml in Tris buffered NaCl</td><td>Sigma-Aldrich&amp;nbsp;</td><td>L2020</td><td></td></tr><tr><td>Poly-L-Ornithine hydrobromide</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P3655</td><td></td></tr><tr><td>Insulin from porcine pancreas</td><td>Sigma-Aldrich&amp;nbsp;</td><td>I5523</td><td></td></tr><tr><td>apo-Transferrin human</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T1147</td><td></td></tr><tr><td>Putrescine dihydrochloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P5780</td><td></td></tr><tr><td>Progesterone</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P8783</td><td></td></tr><tr><td>Sodium selenite</td><td>Sigma-Aldrich&amp;nbsp;</td><td>S5261</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich&amp;nbsp;</td><td>H4034&amp;nbsp;</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G7126</td><td>Stock solution 1 M in water</td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G9391</td><td>Stock solution 2% (w/v) in water</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T8532</td><td></td></tr><tr><td>Paraformaldehyde 16% in water</td><td>Electron Microscopy Sciences</td><td>RT 15710-S</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Merck Millipore</td><td>106329</td><td></td></tr><tr><td>D(+)-Glucose, Monohydrate</td><td>Merck Millipore</td><td>4074-2</td><td></td></tr><tr><td>Hydrochloric acid - c(HCl) = 1 mol/L (1 N) Titripur</td><td>Merck Millipore</td><td>109057</td><td></td></tr><tr><td>Sterile water - Aqua B. Braun</td><td>Braun</td><td></td><td></td></tr><tr><td>Ethanol absolute NORMAPUR analytical reagent</td><td>VWR</td><td>20821.321</td><td></td></tr><tr><td>Sterile Petri dishes</td><td>VWR</td><td>82050-566</td><td></td></tr><tr><td>Pasteur pipettes plain glass - Wilhem Ulbrich GdbR.</td><td>VWR</td><td>612-2297</td><td></td></tr><tr><td>Counting chamber Malassez</td><td>VWR</td><td>631-0975</td><td></td></tr><tr><td>Serum Acrodisc Syringe Filter with Supor Membrane, Sterile, GF/0.2 &amp;micro;m, 37 mm</td><td>PALL Life science</td><td>4525</td><td></td></tr><tr><td>Surgical Scissors - Straight, sharp-sharp, 14.5 cm long</td><td>Fine Science Tools</td><td>14002-14</td><td>To open the abdominal wall</td></tr><tr><td>Scissors - Straight, pointed, delicate, 10 cm long</td><td>MORIA</td><td>4877A</td><td>To open the uterine wall</td></tr><tr><td>Forceps - Curved, usual, serrated jaws 1 mm</td><td>MORIA</td><td>2183</td><td>To manipulate embryos</td></tr><tr><td>Vannas Scissors - Curved, pointed, 7 mm blades</td><td>MORIA</td><td>MC50</td><td>To take out the mesencephalon</td></tr><tr><td>Ultra Fine Forceps - Curved, delicate, 13 cm long</td><td>MORIA</td><td>9987</td><td>To remove meninges</td></tr><tr><td>BD BioCoat Poly-D-Lysine 24-well Multiwell Plates</td><td>BD Bioscience</td><td>356414</td><td></td></tr><tr><td>BD Falcon 12-well Cell Culture Plate, flat-bottom with lid</td><td>BD Bioscience</td><td>353043</td><td></td></tr><tr><td>SuperFrost Microscope Slides, Ground edges 90&amp;ordm;</td><td>MENZEL-GL&amp;Auml;SER</td><td>AG00008032E</td><td></td></tr><tr><td>Precision cover glasses thickness No. 1.5H circular 18 mm &amp;Oslash;</td><td>MARIENFELD</td><td>117580</td><td></td></tr><tr><td>Polyclonal Rabbit Anti-Microtubule-Associated Protein 2 (MAP2) Antibody</td><td>Chemicon Millipore</td><td>AB5622</td><td>1/200</td></tr><tr><td>Monoclonal Mouse Anti-Glutamate Decarboxylase (GAD67) Antibody, clone 1G10.2</td><td>Chemicon Millipore</td><td>MAB5406</td><td>1/400</td></tr><tr><td>Monoclonal Rat Anti-Dopamine Transporter (DAT) Antibody, clone DAT-Nt&amp;nbsp;</td><td>Chemicon Millipore</td><td>MAB369</td><td>1/500</td></tr><tr><td>Monoclonal Mouse Anti-5-HT Antibody</td><td></td><td></td><td>1/8,000 - Generous gift from Yves Charnay (Swizerland, Yves.Charnay@hcuge.ch)</td></tr><tr><td>Goat Serum, New Zealand Origin</td><td>Life Technologies</td><td>16210-064</td><td></td></tr><tr><td>Alexa Fluor 405 Goat Anti-Rabbit IgG (H+L) Antibody</td><td>Life Technologies</td><td>A-31556</td><td>1/200</td></tr><tr><td>Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody</td><td>Life Technologies</td><td>A-11001</td><td>1/1,000</td></tr><tr><td>Alexa Fluor 594 Goat Anti-Rat IgG (H+L) Antibody</td><td>Life Technologies</td><td>A-11007</td><td>1/1,000</td></tr><tr><td>VECTASHIELD HardSet Mounting Medium</td><td>Vector Laboratories</td><td>H-1400</td><td></td></tr><tr><td>Stereomicroscope</td><td>Carl Zeiss microscopy</td><td>Stemi-2000C</td><td></td></tr><tr><td>Bunsen Burner FIREBOY</td><td>VWR</td><td>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 ...

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Research

JoVE Journal

Neuroscience

37.8K Views.  Institut de Génomique Fonctionnelle, Montpellier. Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Video: Primary Culture of Mouse Dopaminergic Neurons

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

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

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

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

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Biology

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

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 ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

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 ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Culturing and Maintaining Dopaminergic Neurons from Mouse Embryonic Brain Tissue

Source: Weinert, M. et al., <a href="https://app.jove.com/v/52475">Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains.</a> J. Vis. Exp. (2015)
This video outlines a method for cultivating dopaminergic neurons from mouse embryonic brain tissue. The process includes treating the midbrain with a proteolytic enzyme, detaching the cells, and culturing them on polymer-coated coverslips in a nutrient-rich medium with antibiotics for their growth and long-term viability.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Studying Alpha-Synuclein Accumulation in Primary Embryonic Mouse Dopamine Neurons

Source: Er, S. et al., <a href="https://app.jove.com/v/61118">Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons.</a>&#160;J. Vis. Exp. (2020).
This video demonstrates the process of quantifying alpha-synuclein accumulation in embryonic mouse dopamine neurons pre-treated with alpha-synuclein pre-formed fibrils. It outlines the steps for fixing, staining, and quantifying the dopamine neurons containing alpha-synuclein aggregates using immunofluorescence.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1.&#160;Preparation

	Prepare ...

Primary Culture of Mouse Dopaminergic Neurons

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