SELECTIVE-INHIBITION OF NEURONAL PROTEIN-SYNTHESIS BY RETROGRADELY TRANSPORTED RICIN

The ability of the lectin, ricinus communis agglutinin I (ricin(120)), to undergo retrograde axonal transport and cause degeneration of neuronal cell bodies has been frequently exploited to establish the origin of peripheral axons. Since this cytotoxic action of ricin results from its inactivation of ribosomes, the retrogradely transported lectin was employed in the present study to inhibit protein synthesis in dorsal root ganglion (DRG) neurons whose axons project into the lumbar nerve trunk of bullfrog tadpoles. The procedure was developed to examine, during tadpole metamorphosis, the ratio of fast-transported radiolabeled protein accumulating at the proximal side of a nerve trunk ligature to the total newly synthesized protein in the cell bodies of origin. The relatively small diameter and fragility of the developing lumbar nerve trunks necessitated introduction of ricin by bath application to the cut nerve end rather than by intraneural injection. Consistent uptake of ricin was achieved by pretreatment with the phospholipase A(2) inhibitor, mepacrine, that blocks resealing of severed nerve fibers. Optimal time and dosage of ricin were established by determining the maximal achievable inhibition of [S-35]methionine into DRG protein. In stage XVI tadpoles, maximal inhibition of approximate to 65% was observed after 16 h incubation in 2.5 mg/ml ricin. As evidence that neuronal protein synthesis was effectively suppressed, there was no detectable anterograde axonal transport of [S-35]protein subsequent to ricin treatment. That protein synthesis inhibition was restricted to neuronal cells within the DRG was demonstrated by a combined immunocytochemical and autoradiographic approach. Under conditions where retrogradely transported ricin markedly reduced the density of silver grains decorating neurofilament-positive cells (neurons), there was no significant difference in grain density in Nissl-counterstained, neurofilament-negative regions that contained Schwann cells and other satellite cells.