Figure 5. p75 neurotrophin receptor (p75NTR) prevents cerebellar granule neuron (CGN) migration in vitro.
(A) Migration analysis using transwell assay. Dapi staining of cells obtained from WT or p75NTR-/- P7 rat pups. (B) Quantification of CGN migration expressed as the density of Dapi+ cells at the bottom of the filter after 24 hr. At the beginning of the experiment the same number of cells were plated on top of the filter. No ligand was added to stimulate the migration. Unpaired t-test, N=4, *p=0.01, error bars indicate SEM. (C) Migration analysis using transwell assay in granule cells obtained from P7 rat pups exposed to Shh in the top and bottom compartment and brain-derived neurotrophic factor (BDNF) in the bottom compartment. (D) Quantification of CGN migration expressed as the density of Dapi+ cells at the bottom of the filter after 24 h of BDNF exposure. Two-way ANOVA, N = 4, p = 0.0186, error bars indicate SEM. (E) Migration analysis using transwell assay in granule cells obtained from P7 rat pups transfected with Ctrl-GFP or p75-GFP construct and expose to BDNF in the bottom compartment. GFP immunostaining of the transfected cells. (F) Quantification of CGN migration expressed as the density of transfected GFP+ cells at the bottom of the filter after 24 hr of BDNF exposure. Two-way ANOVA, N=4, *p=0.0065, error bars indicate SEM. (G) Time-lapse pictures from cerebellar organotypic slices were obtained from P7 rat pups and transfected with Ctrl-GFP (top panels) or p75NTR-GFP (bottom panels). (H) Mean migration speed is expressed as the total distance migrated over the total time of the experiment. Unpaired t-test, N=3, p = 0.0001, error bars indicate SEM. (I) Total distance migrated is expressed as the mean distance migrated per cell. Unpaired t-test, N=3, p = 0.0003, error bars indicate SEM. The experiments were done using cells from P7 rat pups.
