Grafting-from is the technique of choice to obtain polymer brushes. It is based on the growth of polymer chains directly from an initiator-functionalized surface, and its development gained momentum thanks to recent advances in controlled polymerization techniques. However, despite the great amount of work that has been performed on this subject, the influence exerted by the initiator layer on the characteristics of the resulting brushes has been almost completely overlooked. Our group has already demonstrated that positron annihilation spectroscopy (PAS) is a valuable analytical tool for the study of polymer brushes. Here, we applied this technique to show that differences in the organization of the initiator layer dramatically reflect on the characteristics of polymer brushes. Brushes made by surface-initiated atom transfer radical polymerization (ATRP) of a pH-responsive polymer, poly(dimethylaminoethyl methacrylate) (PDMAEMA), were investigated also in terms of the effects of protonation and of the incorporation of silver nanoparticles inside the brushes, shining a new light on the internal structure of such complex, fascinating systems.