Developmental switch in dominant expression of GFP from presynaptic to postsynaptic element. (A) Quantitative analysis on the distribution of GFP expression driven by Krox20 B enhancer/β- globin promoter in the MNTB and calyx of Held synapses from three age groups. Three age groups were analyzed: P6 to P10 (pre-hearing); P11 to P15 (intermediate); and P16 to P21 (post-hearing). Presynaptic terminals expressing Vglut1, but not GFP were also quantified. In the calyx, GFP expression is more robust in P6 to P10 and P11 to P15 age groups compared to P16 to P21. On the other hand, in the MNTB neurons expression of GFP is more prevalent in P16 to P21 compared with the two younger age groups. Data represents mean±s.e.m. Asterisks indicate significant difference at p < 0.05 (ANOVA) using ORIGIN 7.5 (Northampton, MA). N represents number of images from each age group from at least three animals. Examples of 3D images from the MNTB and calyx of Held synapses area: (B) GFP, (C) Vglut1 and (D) merge. Number of cells expressing GFP were counted as calyx only (purple arrow), MNTB only (yellow arrow) and both calyx and MNTB (blue arrow). Confocal and z stack images were acquired as described in Methods and 3D images were rendered using Volocity software (Perkin Elmer, USA). Image contrast enhancement was applied post acquisition for aesthetic reason.