Development and survival of neurons in dissociated fetal mesencephalic serum-free cell cultures: I. Effects of cell density and of an adult mammalian striatal-derived neuronotrophic factor (SDNF)

Abstract

The use of CNS cultures for detection and quantification of neuronotrophic activity in the CNS has been analyzed. In particular the development, i.e., neurotransmitter uptake characteristics, and survival of dopaminergic and GABAergic neurons in fetal mouse (E13)- dissociated mesencephalic cells cultured in serum-free, hormone- supplemented medium have been assessed as a function of culture time and cell density. At all times, more than 98% of the cells were classified as neurons on the basis of immunocytochemical criteria. Results indicate that the increase of cell density in vitro significantly enhances specific high-affinity dopamine uptake per dopaminergic cell and cell survival. This effect is not limited to the dopaminergic cells and suggests that the development of neurotransmitter-related traits and cell survival are influenced by cell density-derived trophic signals. The above-mentioned cultures and parameters have also been used to detect neuronotrophic activity in adult mammalian brain extracts or more purified preparations. In particular, bovine striatal extracts contain activity capable of increasing high-affinity neurotransmitter uptake parameters and cell survival of at least the dopaminergic and GABAergic neurons present in the culture system. The neuronotrophic activity from bovine striatum has been partially purified and is associated with a fraction whose main component is a basic protein of approximately 14 kDa.

Development and survival of neurons in dissociated fetal mesencephalic serum-free cell cultures: II. Modulatory effects of gangliosides

“…Briefly, rostra1 mesencephalic tegmentum and corpus striatum from brains of 13-and 15-d-old mouse embryos, respectively, were dissected under the microscope in sterile conditions and dissociated mechanically. The cells were centrifuged at 45 x g for 4 min and resuspended in a serum-free medium consisting of equal volumes of Eagle's basal medium and Ham's F12, supplemented with components as reported in the preceding paper by Dal Toso et al (1988). Two milliliters of cell suspension containing the desired amounts of cells were added to collagen-coated 35-mm-diameter tissue culture dishes (Falcon).…”

“…Prior to uptake studies, culture medium was removed, cells washed extensively with PBS containing glucose (6 gm/liter), Ca*+ (1 mM), and Mgz+ (1 mM). Assay of)H-dopamine (DA) uptake was conducted as previously described (Prochiantz et al, 198 1;Dal Toso et al, 1988). With the exception of kinetic studies, 5 x lo-* M 3H-DA was routinely employed.…”

“…Histochemical visualization of catecholaminergic neurons in culture was performed following noradrenergic uptake using the glyoxylic acid-induced fluorescence (GIF) technique, as previously described (Dal Toso et al, 1988). The GIF' neurons were visualized with a Zeiss III photomicroscope equipped with catecholamine epifluorescence and phase-contrast optics.…”

“…In this paper we report the effects of GM1 addition on the behavior of dopaminergic neurons in dissociated embryonic mouse mesencephalic cells cultured in a serum-free, hormone-supplemented medium. In these cultures, more than 98% of the mesencephalic cells can be classified as neurons on the basis of immunocytochemical criteria (Dal Toso et al, 1988). Survival and expression of specific high-affinity neurotransmitter uptake of the dopaminergic neurons appear to be dependent on adequate levels of trophic influences, either produced in culture or exogenously supplied.…”

Effect of Cocaine in Early Gestation on Striatal Dopamine and Neurotrophic Activity

“…media die rapidly. Several laboratories (21)(22)(23)(24)(25)(26), including ours The plates were air dried, dehydrated, and then scored for the (16,17,19,20,(27)(28)(29)(30)(31), have demonstrated that the addition of number of NSE neurons by a n observer blinded to treatment striatal extracts containing trophic activity directed at mesencehistory. Using a phase-contrast, inverted microscope Fluovert phalic neurons reduces this ncuronal death rate and stimulates (Leitz, Leipsig, Germany) the center strip of each well was the growth of DA neurons.…”