Fig. 6. The increase in the number of TH-positive cells in LC cultures does not involve proliferation but rather survival and a second mechanism. In A, a representative TH-positive cell (red cytoplasm) and a BrdU-positive cell (dark nucleus) from an E13.5 LCr culture treated with BrdU and BDNF for 24 hours is shown. TH-positive cells in the culture were negative for BrdU and the number of BrdU-positive cells did not increase by BDNF or NT4 treatment. The graph in B displays the number of TH-positive cells after treatment with BDNF, NT4, NT3, bFGF or control media for 2, 24 or 48 hours. A steep increase in the number of TH-positive cells was detected between 2 and 24 hours in BDNF and NT4-treated cultures, whereas bFGF only induced a significant increase in cell number after 48 hours (*P<0.01, #P<0.05, unpaired t-test). (C) When NT4 was added to E13.5 LC cells before tissue dissociation (NT4 early), a constant higher number of TH-positive cells were detected in the wells, at all stages analyzed, as compared to wells in which NT4 was added at the time of plating (NT4 late), suggesting that early administration of NT4 prevents the loss of a population of TH-positive cells that would otherwise die shortly after dissociation. Between 2 and 12 hours a parallel increase in the number of noradrenergic neurons was detected in both early (at the time of dissociation) and late (at the time of plating) NT4 treatment, indicating that an additional mechanism is involved. *P<0.01 with respect to control and P<0.05 with respect to late NT4 treatment, #P<0.01 with respect to control, by unpaired t-test. Scale bar: 50 µm.

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