THE SURVIVAL RESPONSE OF MESENCEPHALIC DOPAMINERGIC-NEURONS TO THE NEUROTROPHINS BDNF AND NT-4 REQUIRES PRIMING WITH SERUM - COMPARISON WITH MEMBERS OF THE TGF-BETA SUPERFAMILY AND CHARACTERIZATION OF THE SERUM-FREE CULTURE SYSTEM

The neurotrophins, brain-derived neurotrophic factor (BDNF) and neurot rophin-4 (NT-4), are established survival promoting molecules for dopa minergic (DAergic) neurons cultured from the fetal rat midbrain floor. We have cultured and compared the survival of embryonic day (E) 14 me sencephalic cells in fully defined, serum-free medium, with serum-prim ed cultures (one hour during dissociation). Cultures were characterize d using antibodies against neuron-specific enolase (NSE), tyrosine hyd roxylase (TH), vimentin, glial fibrillary acidic protein (GFAP), and t he antigen A2B5. The absolute absence of serum did not reduce the surv ival of TH-positive DAergic neurons nor alter the percentages of cells staining for the above markers. Transforming growth factor-beta 3 (TG F-beta 3) and glial cell line-derived neurotrophic factor (GDNF), two members of the TGF-beta superfamily, both promoted the survival of TH- positive cells (TGF-beta 3: 2-fold; GDNF: 1.6-fold) over the 8-day cul ture period. Survival mediated by TGF-beta 3 and GDNF was independent of whether or not the cells had been initially exposed to serum. In co ntrast, the survival promoting effects of BDNF and NT-4 were crucially dependent on serum priming. RT-PCR for the full-length trkB high affi nity neurotrophin receptor revealed its presence in both culture syste ms. We conclude that priming with serum is important to make DAergic n eurons fully responsive to BDNF and NT-4. Underlying mechanisms might be sought at the level or distal of trkB receptor expression, without excluding the possiblity that serum elicits production of growth facto rs that synergistically act with neurotrophins in these cultures.