Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Summary

The causes of degeneration of midbrain dopaminergic neurons during Parkinson’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.

Abstract

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson’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.

Introduction

Loss of dopaminergic neurons from the substantia nigra pars compacta leads to the cardinal motor symptoms of Parkinson’s Disease (PD), the second most prevalent neurodegenerative disorder. The underlying cause of the demise of this mesencephalic neuronal population is not known. To study the biochemical pathways responsible for the development and modulating neurophysiological properties and survival of mesDA neurons, several cell culture and animal model systems have been used. Immortalized cell lines, including the rat dopaminergic cell line 1RB₃AN₂₇(N27), the human dopaminergic neuroblastoma cell line SH-SY5Y, the mouse dopaminergic hybr....

Protocol

NOTE: The animals were maintained and handled in compliance with the institutional guidelines and all animal procedures were approved by the Imperial College's Animal Welfare and Ethical Review Body (AWERB) and the Home Office and Harvard University Institutional Animal Care and Use Committee (IACUC), in compliance with federal and state regulations.

1. Reagent and Equipment Setup

1. Thaw, aliquot and store 1 mg/ml Laminin solution at –80 ºC. Dissolve 2 µl in 1 ml DMEM/F12, resulting in a coating concentration of 1-2 µg/cm².
   NOTE: Thaw Laminin slowly on ice and dissolve in cold DMEM/F....

Results

Immunohistochemistry against Tyrosine hydroxylase (Th) shows that between 0.5-1% of the cells in culture are dopaminergic. Neuronal projections appear within 2 hr after plating and by the first day, axons and dendrites are distinguishable (Figure 2), using Tyrosine Hydroxylase (TH) and Microtubule-Associated Protein 2 (Map2) antibodies (Figure 3). The neurons survive for more than six weeks and show extensive outgrowth. The neuron-glia ratio in the cultures is directly related to the con.......

Discussion

Dopaminergic neurons in midbrain are the main source of dopamine in the central nervous system. They are divided into three groups, substantia nigra pars compacta (SNpc), ventral tegmental area (VTA) and retrorubral field (RRF) ¹⁰,¹¹. The neurons in SNpc and VTA give rise to major dopaminergic pathways, mesocortical, mesolimbic and nigro-striatal, involved in functions such as control of emotion, motivation and motor behavior. Demise of the neurons in SNpc and functional disruption of the nigrostria.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's modified Eagle medium nutrient mixture F-12	Invitrogen	11330
Hanks' Balanced Salt Solution (HBSS) (1X), liquid	Invitrogen	24020-117
Fetal bovine serum, heat-inactivated (FBS)	Invitrogen	16140
N2 Supplement (100X), liquid	Invitrogen	17502-048
D-(+)-Glucose solution (45% (wt/vol) in water	Sigma	G8769
Bovine serum albumin BSA	Sigma	A9430
Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane	Sigma	L2020
Penicillin/streptomycin	Invitrogen	15070
Trypsin (0.05% (wt/vol)	Invitrogen	25300
Bovine serum albumin (BSA) cell culture tested	Sigma	A9418
Phosphate-buffered saline (PBS)	Sigma	P3813
Anti-Tyrosine hydroxylase (Th) antibody	Pel-Freez Biologicals	P60101-0
Poly-L-ornithine, 0.01% solution	Sigma	P4957
Anti-Map2 (Microtubule associated protein-2A and -2B) antibody	Millipore	MAB3418
Anti-Synapsin-1 antibody	Millipore	AB1543P
Alexa Fluor 488 donkey anti-rabbit IgG   antibody	Molecular Probes	A-21206
Alexa Fluor 594 donkey anti-rabbit IgG   antibody	Molecular Probes	A-21207
Alexa Fluor 488 donkey anti-sheep IgG   antibody	Molecular Probes	A-11015
Alexa Fluor 594 donkey anti-sheep IgG   antibody	Molecular Probes	A-11016
Alexa Fluor 594 donkey anti-mouse IgG   antibody	Molecular Probes	A-21203
Trypan blue solution (0.4% (wt/vol))	Biowhittaker	17-942E
Stereo Microscope	Carl Zeiss	Stemi 2000-C
Inverted phase contrast microscope	Carl Zeiss	Axiovert 40 C
Dumont Forceps	Fine Scientific Tools	May-45
Cover Slip Forceps – Dumoxel	Fine Scientific Tools	11251-33
Two Dumont #45 Forceps – Dumoxel	Fine Scientific Tools	11245-30
Blade Holder/Breaker Flat Grip - 11cm	Fine Scientific Tools	10052-11
Student Iris Scissors - Straight 11.5 cm	Fine Scientific Tools	91460-11
Fiber optic halogen illuminator	Nikon	MKII
Disposable Borosilicate Glass Pasteur Pipettes	Fisherbrand	13-678-20C
Hemocytometer	Proscitech	SVZ2NIOU
0.2 µm sterile filter units	Nalgene	NL-CE-156-4020
100x20 mm Petri dishes	BD Biosciences	351005
Round Cover Slip #1 Thickness German Glass 12 mm	Bellco Glass	1943-10012