Fig. 6. BDNF and NT-4 regulate tangential migration of MGE-derived cells to the cortex. (A-C) E14 MGE-GFP explants were co-cultured with P2-P4 cortical slices for 1 day in the presence of control vehicle (0.1 M PBS, A), recombinant BDNF (50 ng/ml, B), or recombinant NT-4 (50 ng/ml, C) in the slice culture medium. Both BDNF and NT4 treatment lead to an increase the total number of MGE-GFP cells that migrate into the cortex. (D) Nuclear counterstaining (Hoechst 33258) reveals the cellular organization of the cortex in MGE-cortex co-cultures. (E) Quantification of the effects of NT4 on migration of MGE neurons into the cortex in MGE-cortex co-cultures (experiment shown in A,C). The migration index represents the density of GFP+ cells (number of cells per 104 µm2) found in 100 µm wide stripes of intermediate zone at indicated distances from the MGE-cortex interface (arrowhead on the inset). Note that NT4 treatment (blue triangles) leads to an increase in the number of cells that migrate into the cortex and in the maximum distance of migration. Quantification was performed on nine explants taken from three independent experiments. NT4 treatment showed a statistically significant increase of the migration index compared with the control according to an ANOVA test (P<0.0001). (F-K) Expression pattern of NT4 in the developing telencephalon. Coronal sections were processed for NT4 immunofluorescence (F,I) and counterstained with Hoechst (G,J). The double immunofluorescence images are shown in (H,K). (F-H) At low magnification, NT4 can be detected in the ventricular zone of the GE (double arrows in F), as well as in the cortical plate and marginal zone of the cortex (single arrow in F). (I-K) With a higher magnification image through the cortical intermediate zone, NT4 immunofluorescence can be detected in radially oriented processes (arrows in I), which are likely to be radial glial processes. (L) NT4 can acutely induce the motility of MGE-derived cells. Time-lapse video images of an E15 MGE-GFP cell located in the IZ of a P2 cortical slice. The cell was not moving for the first 60 minutes (red arrowhead) before the application of NT4. Application of NT4 (green arrowhead; 20 µl at 20 ng/ml) induces migration in the MGE-GFP cell. The arrowhead in each panel indicates the initial position of the cell body. Numbers at the bottom right indicate time in minutes. (M) Recombinant NT4 can acutely increase the rate of migration of MGE-derived cells. E14 MGE-GFP cells migrating in the IZ of P2 cortical slices were imaged by time-lapse video-microscopy for 70 minutes in the presence of control vehicle (PBS) and for 80 minutes after application of recombinant NT4 (20 ng/ml). The instantaneous rate of migration (µm per hour) of MGE-GFP cells (n=77) is plotted as a function of time. Quantification was performed on eight explants taken from two independent time-lapse video-microscopy experiments. Scale bar: 250 µm in A-C; 400 µm in D; 150 µm in F; 40 µm in L. *, P<0.05; **, P<0.003.
