Development regulation of GFP expression in calyx of Held synapse. (A) Example of fluorescence intensity profile analysis used to determine relative fluorescence GFP intensity versus the synaptic marker, Vglut1 fluorescence intensity as determined from merge images indicating co-localization of GFP and Vglut1 (insert, yellow square). (B) Percent GFP expression from three age groups was analyzed: P6 to P10 (pre-hearing); P11 to P15 (intermediate); and P16 to P21 (post-hearing). Note that GFP intensity is more robust at P11 to P15 and the relative amount is approximately similar between pre- and post-hearing age groups. Data represents mean±s.e.m. Asterisks indicate significant difference at p < 0.05 (ANOVA) using ORIGIN 7.5 (Northampton, MA) and comparisons were made against P6 to P10 groups. P6 to P10 (n = 151); P11 to P15 (n = 145); and P16 to P21 (n = 103) cells taken from images as described in Figure 5 from at least three animals per age group. Images were acquired as described in Methods.