Developmental dependence of GFP expression in calyx of Held – MNTB synapse. Examples of images from postnatal pups taken from E12 – E13.5 in utero electroporated pregnant mouse injected with Krox20 B enhancer/β- globin promoter-GFP DNA construct. Brainstem slices from MNTB/calyx region from three age groups were analyzed. Transverse brainstem slices from P9 (A, B and C), P13 (D, E and F) and P21 (G, H and I) mice were obtained and processed to determine the expression of GFP (A, D and G) using Vglut 1 (B, E and H) as presynaptic marker and overlay images are also shown (C, F and I). Confocal images and z stack images were acquired as described in Methods. Image contrast enhancement was applied post acquisition for aesthetic reasons.