Enhanced visualization of axonally transported proteins in the immature CNS by suppression of systemic labeling

In the neonate hamster, visualization of axonally transported proteins in the retinofugal pathway is obscured by high levels of systemic (background) labeling. Radiolabeled precursors injected into the eye diffuse rapidly into the general circulation and then across the immature blood-brain barrier to be incorporated into proteins that are synthesized throughout the brain. Systemic labeling can be suppressed, however, by i.p. injections of large amounts of either non-radioactive methionine 30 min after intraocular labeling with [35S]methionine, or non-radioactive leucine given at the time of intraocular labeling. Whereas the former competes with the radioactive precursor during incorporation into brain proteins (after most of the retinal labeling has already been achieved), the latter competes at the earlier stage of access to the brain. Both methods reduced background labeling by more than 60%, thereby allowing for unambiguous identification of axonally transported proteins. The pattern of rapidly transported proteins was found to be strikingly different between neonates and mature animals, including marked changes in an identified 'growth-associated protein' (50 kDa, pI 4.8).