Primary Culture of Mouse Dopaminergic Neurons

Summary

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.

Abstract

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'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.

Introduction

Dopamine, one of the essential brain neurotransmitters^1,2, is mainly released by midbrain dopaminergic (DA) neurons. The majority of DA neurons reside in the ventral part of the mesencephalon^2-6. Schematically, midbrain DA neurons can be divided in three anatomically and functionally distinct projection systems: mesostriatal, mesolimbic, and mesocortical pathways^2,5. 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.

Protocol

 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

1. Stock Solutions
   1. 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 µm syringe filter, aliquot, and store at -20 °C.
   2. 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
      ....

Results

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........

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fetal Bovine Serum	Lonza	14-801F
DMEM 4.5 g/L glucose with L-glutamine	Lonza	BE12-604F
0.05% Trypsin-EDTA (1x), phenol red	Life Technologies	25300-054
Penicillin-Streptomycin (10,000 U/ml)	Life Technologies	15140122
L-Glutamine, 200 mM solution	Life Technologies	25030123
Dulbecco’s Phosphate Buffered Saline	Sigma-Aldrich	D8537
Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham	Sigma-Aldrich	D0547	Powder
Laminin - 1 mg/ml in Tris buffered NaCl	Sigma-Aldrich	L2020
Poly-L-Ornithine hydrobromide	Sigma-Aldrich	P3655
Insulin from porcine pancreas	Sigma-Aldrich	I5523
apo-Transferrin human	Sigma-Aldrich	T1147
Putrescine dihydrochloride	Sigma-Aldrich	P5780
Progesterone	Sigma-Aldrich	P8783
Sodium selenite	Sigma-Aldrich	S5261
HEPES	Sigma-Aldrich	H4034
Glycine	Sigma-Aldrich	G7126	Stock solution 1 M in water
Gelatin	Sigma-Aldrich	G9391	Stock solution 2% (w/v) in water
Triton X-100	Sigma-Aldrich	T8532
Paraformaldehyde 16% in water	Electron Microscopy Sciences	RT 15710-S
Sodium bicarbonate (NaHCO3)	Merck Millipore	106329
D(+)-Glucose, Monohydrate	Merck Millipore	4074-2
Hydrochloric acid - c(HCl) = 1 mol/L (1 N) Titripur	Merck Millipore	109057
Sterile water - Aqua B. Braun	Braun
Ethanol absolute NORMAPUR analytical reagent	VWR	20821.321
Sterile Petri dishes	VWR	82050-566
Pasteur pipettes plain glass - Wilhem Ulbrich GdbR.	VWR	612-2297
Counting chamber Malassez	VWR	631-0975
Serum Acrodisc Syringe Filter with Supor Membrane, Sterile, GF/0.2 µm, 37 mm	PALL Life science	4525
Surgical Scissors - Straight, sharp-sharp, 14.5 cm long	Fine Science Tools	14002-14	To open the abdominal wall
Scissors - Straight, pointed, delicate, 10 cm long	MORIA	4877A	To open the uterine wall
Forceps - Curved, usual, serrated jaws 1 mm	MORIA	2183	To manipulate embryos
Vannas Scissors - Curved, pointed, 7 mm blades	MORIA	MC50	To take out the mesencephalon
Ultra Fine Forceps - Curved, delicate, 13 cm long	MORIA	9987	To remove meninges
BD BioCoat Poly-D-Lysine 24-well Multiwell Plates	BD Bioscience	356414
BD Falcon 12-well Cell Culture Plate, flat-bottom with lid	BD Bioscience	353043
SuperFrost Microscope Slides, Ground edges 90º	MENZEL-GLÄSER	AG00008032E
Precision cover glasses thickness No. 1.5H circular 18 mm Ø	MARIENFELD	117580
Polyclonal Rabbit Anti-Microtubule-Associated Protein 2 (MAP2) Antibody	Chemicon Millipore	AB5622	1/200
Monoclonal Mouse Anti-Glutamate Decarboxylase (GAD67) Antibody, clone 1G10.2	Chemicon Millipore	MAB5406	1/400
Monoclonal Rat Anti-Dopamine Transporter (DAT) Antibody, clone DAT-Nt	Chemicon Millipore	MAB369	1/500
Monoclonal Mouse Anti-5-HT Antibody			1/8,000 - Generous gift from Yves Charnay (Swizerland, Yves.Charnay@hcuge.ch)
Goat Serum, New Zealand Origin	Life Technologies	16210-064
Alexa Fluor 405 Goat Anti-Rabbit IgG (H+L) Antibody	Life Technologies	A-31556	1/200
Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody	Life Technologies	A-11001	1/1,000
Alexa Fluor 594 Goat Anti-Rat IgG (H+L) Antibody	Life Technologies	A-11007	1/1,000
VECTASHIELD HardSet Mounting Medium	Vector Laboratories	H-1400
Stereomicroscope	Carl Zeiss microscopy	Stemi-2000C
Bunsen Burner FIREBOY	VWR	451-0136