Fig. 4

Long term cortical cultures demonstrate persistent GSI-induced enhancement of myelination and initiation of axonal node of Ranvier formation. On DIV5, cortical cultures were treated with DAPT or DMSO for 8 days, media was changed weekly thereafter without compound, and cells fixed on DIV28. a Left panels show triple immunostaining of MBP (green), Olig2 (red), and DAPI (blue). Red overlaid with blue appears pink. Right panels show digital masks created from MBP-stained images in the center panel. Masks were used for quantification of fiber length. Bars 100 µm. Arrows indicate areas with significant myelination. b Quantification of myelination showing raw data in 28 DIV cortical cultures as in a. Representative data shown is averaged from 16 image fields per concentration, mean ± SEM. Asterisk (*) denotes P values versus DMSO of < 0.0001; ANOVA with Dunnett’s post hoc test. There was a significant effect of three compound concentrations compared to DMSO [F(9, 27) = 17.50, P < 0.0001]. Post hoc comparisons indicated that the mean score for the concentrations 100 nM (M = 14.26, 2.433), 500 nM (M = 14.57, 2.32), and DAPT (M = 16.35, 1.39) was significantly different than DMSO. c Cortical co-cultures were grown for a total of 21 days, fixed, and immunostained for MBP (green, merged image) and the paranode-localized protein Caspr (red, merged image). Note the accumulation of Caspr protein at the edges of myelinated axon segments (arrows). Bar, upper panels 100 μm. Bar, lower panels 50 μm