Fig. 3. NF-B-dependent gene expression in newborn nodose neurons is not affected by BDNF. (A) Photomicrographs of representative fields of neurons co-transfected with the GFP NF-B reporter plasmid, RFP plasmid and either the super-repressor IB- plasmid (right panels) or corresponding control plasmid (left panels) and cultured with 20 ng/ml BDNF for 12 hours. Transfected neurons and their dendritic morphology are outlined by the RFP (shown with a white filter) and can be seen in the upper panels. The effect of super-repressor IB- on neuronal morphology, seen in the previous figures, is also evident in these images. GFP fluorescence in the same fields (lower panels) shows the marked reduction caused by super-repressor IB-. (B) Quantification of the level of NF-B-driven GFP fluorescence in super-repressor IB--transfected and control-transfected neurons after the neurons have been incubated for 12 hours with and without 20 ng/ml BDNF. Fluorescence measurements were made from 40 to 60 neurons in each experimental condition, and the data are expressed as a percentage of the mean fluorescence of the No factor/Control vector-transfected group (mean and standard error are shown). Statistical comparisons shown are with respect to the control transfected neurons, **P<0.001. (C) Timecourse of NF-B-driven GFP fluorescence after BDNF stimulation. Neurons were co-transfected with the GFP NF-B reporter plasmid and the RFP plasmid, and were cultured without factors for 8 hours. Neurons were then imaged immediately before and at 0.5, 1, 2 and 3 hours after the addition of BDNF to the medium (20 ng/ml). An untreated group of neurons (No Factor) was imaged at the same times. The fluorescence of each neuron was quantified and expressed as a percentage of the initial (0 hour time point) measurement for each group. Scale bars: 50 µm.