The survival response of mesencephalic dopaminergic neurons to the neurotrophins BDNF and NT-4 requires priming with serum: comparison with members of the TGF-β superfamily and characterization of the serum-free culture system

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Abstract
The neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4), are established survival promoting molecules for dopaminergic (DAergic) neurons cultured from the fetal rat midbrain floor. We have cultured and compared the survival of embryonic day (E) 14 mesencephalic cells in fully defined, serum-free medium, with serum-primed cultures (one hour during dissociation). Cultures were characterized using antibodies against neuron-specific enolase (NSE), tyrosine hydroxylase (TH), vimentin, glial fibrillary acidic protein (GFAP), and the antigen A2B5. The absolute absence of serum did not reduce the survival of TH-positive DAergic neurons nor alter the percentages of cells staining for the above markers. Transforming growth factor-β3 (TGF-β3) and glial cell line-derived neurotrophic factor (GDNF), two members of the TGF-β superfamily, both promoted the survival of TH-positive cells (TGF-β3: 2-fold; GDNF: 1.6-fold) over the 8-day culture period. Survival mediated by TGF-β3 and GDNF was independent of whether or not the cells had been initially exposed to serum. In contrast, the survival promoting effects of BDNF and NT-4 were crucially dependent on serum priming. RT-PCR for the full-length trkB high affinity neurotrophin receptor revealed its presence in both culture systems. We conclude that priming with serum is important to make DAergic neurons 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 factors that synergistically act with neurotrophins in these cultures.