Neuronal-enriched cultures from embryonic rat ventral mesencephalon for pharmacological studies of dopamine neurons.
The method described herein provides a convenient and rapid procedure to obtain enriched neuronal cultures containing reproducible numbers of dopamine (DA) cells. These cultures allow experimental paradigms designed to study the effect of drugs on DA neurons without astroglial mediation. Neuronal-enriched cultures are prepared from the mesencephalon of rat embryos at the 14th day of gestation (E14). At that moment, DA cells of the developing substantia nigra are located ventrally at the level of the mesencephalic flexure. Because the neurons of the pars compacta are mostly born between E12 and E15, E14 corresponds to an optimal stage for obtaining a high survival of DA cells. A defined medium (EF12) allows the maturation of DA neurons and reduces drastically the number of astrocytes. After 7 days in vitro (DIV) in EF12, the cultures contain 2-5% astrocytes (GFAP+ cells) and DA neurons represent 0.5-2% of the cells, as assessed by immunostaining to tyrosine hydroxylase (TH). The function of DA neurons is assessed by [3H]DA uptake and of those non-DA neurons by the high affinity [3H]GABA uptake. Cell survival is assessed by Trypan blue dye exclusion.
Pardo
B
B
Departamento de Investigación, Hospital Ramón y Cajal, Madrid, Spain.
Paíno
C L
CL
Casarejos
M J
MJ
Mena
M A
MA
D000818:Animals; D002478:Cells, Cultured; D003584:Cytological Techniques; D004298:Dopamine / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D008636:Mesencephalon / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D009462:Neurology / Q000379:methods*; D009474:Neurons / Q000378:metabolism*; D051381:Rats / Q000196:embryology; D017207:Rats, Sprague-Dawley; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D005680:gamma-Aminobutyric Acid; D004298:Dopamine