Spatial and temporal growth factor influences on developing midbrain dopaminergic neurons

Abstract

Despite the large number of growth factors shown to affect dopaminergic cell survival and differentiation in vitro, presently little is known about the role these growth factors play during normal in vivo development of dopaminergic neurons. To address this issue, glia and neurons of both the mesencephalic home region as well as the striatal and cortical target areas have been screened for effects on dopaminergic cell survival in serum-free dissociated cell cultures of the embryonic day (E) 15 and E17 rat mesencephalon. In E15 mesencephalic cultures, the number of surviving tyrosine hydroxylase-immunoreactive dopaminergic neurons maximally increased 2.6-fold with medium conditioned by glia of the E15-E20 mesencephalon, the E17-E20 striatum, or the E20 cortex. In marked contrast, all glial-conditioned media (CM) failed to affect dopaminergic cell survival in E17 mesencephalic cultures. Similarly, E17 dopaminergic cell survival was not affected by CM derived from striatal or mesencephalic neurons. This absence of survival-promoting effects was not due to a general lack of sensitivity of the late embryonic dopaminergic neurons to growth factors. Basal survival of cultured E17 dopaminergic neurons declined with PD98059 (20 microM), a potent inhibitor of growth factor-activated microtubule-associated protein (MAP) kinase cascade. Moreover, irrespective of the age of the cultured mesencephalic tissue, dopaminergic growth factors with potential autocrine functions such as brain-derived neurotrophic factor (BDNF; 50 ng/ml) and glial cell line-derived neurotrophic factor (GDNF; 10 ng/ml) promoted dopaminergic cell survival 1.5- to 1.9-fold. These findings suggest that dopaminergic cell survival is predominantly affected by, as yet unknown, growth factors derived from mesencephalic, cortical, and striatal glia during early embryonic development, and by autocrine-acting growth factors during late developmental stages.

Etomidate reduces glutamate uptake in rat cultured glial cells: involvement of PKA

“…The supernatant was collected and membranes were pelleted at 100 000 × g for 1 h at 4 °C. The pellet was resuspended in serum‐free N2 medium ( Engele, 1998 ) to obtain a final protein concentration of 1 mg mL −1 . Protein contents of both cell lysates and membrane fractions were determined with the BCA protein estimation kit (Pierce Chemical, Rockford, IL, USA).…”

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP), a Neuron-Derived Peptide Regulating Glial Glutamate Transport and Metabolism

“…Neuronal cultures were established from E17 cerebral hemispheres at 300,000 cells/cm 2 and were maintained with a modified serum-free N2 medium (Engele, 1998) for up to 4 d.…”

“…Whether PACAP also is provided by CNS glia is presently unknown. To address this issue, we initiated dissociated cell cultures from the E17 rat cortex, where 95% of the cells represent microtubule-associated protein-2 (MAP-2)-immunoreactive (IR) neurons (Engele, 1998). In addition, cultures were initiated from the P1 rat cortex; these cultures were passaged three times.…”

Modafinil Occupies Dopamine and Norepinephrine Transporters in Vivo and Modulates the Transporters and Trace Amine Activity in Vitro

“…Thereafter, several investigators questioned the contribution of dopamine neurotransmission to modafinil pharmacology, on the basis of overlapping, but not identical behavioral and neurochemical responses of modafinil compared with amphetamine, a psychomotor stimulant with robust dopaminergic effects (Duteil et al, 1990;Akaoka et al, 1991;Simon et al, 1995;Lin et al, 1996;Ferraro et al, 1997a,b;Engber et al, 1998;Engele, 1998). Modafinil was a weak inhibitor of the DAT (Ͼ1 M affinity) and displayed no affinity for dopamine receptor subtypes (Mignot et al, 1994;Bryan Roth, personal communication).…”