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First published online 2 March 2005
doi: 10.1242/dev.01702

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NF-B signalling regulates the growth of neural processes in the developing PNS and CNS

Humberto Gutierrez^*,, Valerie A. Hale^*, Xavier Dolcet and Alun Davies

School of Biosciences, Biomedical Building, Museum Avenue, PO Box 911, Cardiff, CF10 3US, Wales

View larger version (19K): [in a new window]   Fig. 1. Representative sample of reconstructed newborn nodose neurons. The illustrated neurons represent the range of morphologies observed in P0 control transfected cultures (upper row) and super-repressor IB- transfected cultures (lower row) after 24 hours incubation with 10 ng/ml BDNF. The neurons shown correspond to percentiles 25, 50, 75 and 100 of the sampled populations in terms of total neurite length. Scale bar: 50 µm.

View larger version (38K): [in a new window]   Fig. 2. Super-repressor IB- reduces neurite growth from neonatal nodose neurons but does not affect survival. P1 nodose neurons were transfected with a YFP expression plasmid together with either a super-repressor IB- plasmid or an empty control plasmid and were incubated in medium containing 10 ng/ml BDNF. Control transfected neurons were also grown without BDNF. (A) Percent survival 48 hours after transfection. (B) Total neurite length 24 hours after transfection. (C) Number of branch points in neurite arbors 24 hours after transfection. (D) Sholl analysis of neurite arbor morphology 24 hours after transfection. (E) Photomicrograph of a typical control transfected neuron grown for 24 hours with BDNF. (F) Photomicrograph of a typical super-repressor IB- transfected neuron grown for 24 hours with BDNF. Scale bar: 50 µm. The means and standard errors of data obtained from 40-70 neurons in each experimental condition shown. Statistical comparisons shown are with respect to the control transfected neurons, *P<0.05, **P<0.001.

View larger version (53K): [in a new window]   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.

View larger version (34K): [in a new window]   Fig. 4. Super-repressor IB- reduces neurite growth from nodose neurons in a developmentally dependent manner. E16, E18, P0, P1 and P3 nodose neurons were transfected with a YFP expression plasmid together with either a super-repressor IB- plasmid or an empty control plasmid and were incubated in medium containing 10 ng/ml BDNF. (A) Percent survival 48 hours after transfection. In addition to showing survival data for super-repressor IB- transfected and control transfected neurons grown with BDNF, survival data for control transfected neurons grown without BDNF are also shown at each age. (B) Total neurite length 24 hours after transfection. (C) Number of branching points in neurite arbors 24 hours after transfection. (D-H) Sholl analysis of neurite arbor morphology 24 hours after transfection at E16, E18, P0, P1 and P3, respectively. Statistical comparisons shown are with respect to the control transfected neurons, *P<0.05, **P<0.001.

View larger version (37K): [in a new window]   Fig. 5. BAY 11-7082 reduces neurite outgrowth from nodose neurons. P0 nodose neurons were incubated in medium containing 10 ng/ml BDNF and either the IB- phosphorylation inhibitor BAY 11-7082 at a concentration of 10 µmol/l or vehicle control. Vehicle-treated neurons were also grown without BDNF. (A) Percent survival after 48 hours incubation. (B) Total neurite length after 24 hours incubation. (C) Number of branching points in neurite arbors after 24 hours incubation. (D) Sholl analysis of neurite arbor morphology after 24 hours incubation. (E) Photomicrograph of a typical ßIII tubulin stained, vehicle-treated neuron grown for 24 hours with BDNF. (F) Photomicrograph of a typical ßIII tubulin stained, BAY 11-7082 treated neuron grown for 24 hours with BDNF. The means and standard errors of data obtained from at least 40 neurons in each experimental condition shown. Statistical comparisons shown are with respect to the control transfected neurons, *P<0.05, **P<0.001. Scale bars: 50 µm.

View larger version (46K): [in a new window]   Fig. 6. Proteosome inhibition reduces neurite growth from nodose neurons. P0 nodose neurons were incubated in medium containing 10 ng/ml BDNF and either the proteosomal degradation inhibitor ALLN at a concentration of 10 µmol/l or vehicle control. Vehicle-treated neurons were also grown without BDNF. (A) Percent survival after 48 hours incubation. (B) Total neurite length after 24 hours incubation. (C) Number of branching points in neurite arbors after 24 hours incubation. (D) Sholl analysis of neurite arbor morphology after 24 hours incubation. (E) Photomicrograph of a typical ßIII tubulin-stained, vehicle-treated neuron grown for 24 hours with BDNF. (F) Photomicrograph of a typical ßIII tubulin-stained, ALLN-treated neuron grown for 24 hours with BDNF. Statistical comparisons shown are with respect to the control transfected neurons, *P<0.05, **P<0.001. Scale bars: 50 µm.

View larger version (49K): [in a new window]   Fig. 7. B decoy DNA reduces neurite growth from nodose neurons. P0 nodose neurons were transfected with a YFP expression plasmid together with either a B decoy DNA or a scrambled control variant and were incubated in medium containing 10 ng/ml BDNF. Control transfected neurons were also grown without BDNF. (A) Percent survival 48 hours after transfection. (B) Total neurite length 24 hours after transfection. (C) Number of branching points in neurite arbors 24 hours after transfection. (D) Sholl analysis of neurite arbor morphology 24 hours after transfection. (E) Photomicrograph of a typical neuron transfected with the scrambled control oligonucleotide and grown for 24 hours with BDNF. (F) Photomicrograph of a typical neuron transfected with the B decoy oligonucleotide and grown for 24 hours with BDNF. Statistical comparisons shown are with respect to the control transfected neurons, *P<0.05, **P<0.001. Scale bars: 50 µm.

View larger version (41K): [in a new window]   Fig. 8. Overexpression of p65 augments NF-B activation and promotes neuronal survival but does not enhance neurite growth. (A) Photomicrographs of representative fields of P0 nodose neurons co-transfected with the B-dependent GFP reporter plasmid, RFP plasmid and either the p65 overexpression plasmid (bottom) or the corresponding empty plasmid (top) and cultured with BDNF for 24 hours. Transfected neurons are outlined by the RFP (shown with a white filter, left panels). GFP fluorescence in the same fields (right panels) shows the marked increase caused by p65 overexpression. (B) Quantification of the level of NF-B-driven GFP fluorescence in p65 transfected and control transfected neurons after 24 hours incubation with BDNF. Fluorescence measurements are expressed as a percentage of the mean fluorescence of the control vector-transfected group (mean and standard error are shown). (C) Percent survival 48 hours after transfection. (D) Total neurite length after 24 hours incubation. (E) Number of branching points in neurite arbors after 24 hours incubation. (F) Sholl analysis of neurite arbor morphology after 24 hours incubation. Statistical comparisons shown are with respect to the control transfected neurons, **P<0.001. Scale bars: 100 µm.

View larger version (60K): [in a new window]   Fig. 9. Super-repressor IB- and B decoy DNA reduce the size and complexity of pyramidal dendritic arbors in cortical slice cultures. Slice cultures of the somatosensory cortex of P3/P4 neonatal mice were simultaneously bombarded with a mixture of two sets of gold particles, each carrying a different reporter (YFP or RFP) together with either an NF-B inhibitory system (super-repressor IB- plasmid or B decoy DNA) or the appropriate corresponding control (empty plasmid or scrambled B DNA, respectively). Forty-eight hours after transfection, between 40 and 50 individual neurons expressing each reporter were scanned, and the resulting Z-stack images of the dendritic trees were traced and analysed. (A,B,C) Sholl analysis, number of branching points and, total dendritic length, respectively, for neurons expressing super-repressor IB- or control plasmid. (D) Photomicrograph of representative pyramidal neurons transfected with the super-repressor IB- plasmid (arrow) or the control plasmid. (E,F,G) Sholl analysis, number of branching points and, total dendritic length, respectively, for neurons expressing B decoy DNA or control scrambled B DNA. (H) Photomicrograph of representative pyramidal neurons transfected with B decoy DNA (arrows) or the control scrambled DNA. The means, standard errors and statistical comparisons (*P<0.05, **P<0.001) are shown. Scale bars: 50 µm.

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