N-[acetyl- 3H] wheat germ agglutinin: Anatomical and biochemical studies of a sensitive bidirectionally transported axonal tracer

Abstract

Both retrograde and anterograde autoradiographic axonal labeling were associated with the appropriate pathways after injections of N-[acetyl- 3H] wheat germ agglutinin within numerous structures in the central nervous system of mice. Sections processed for light microscopic autoradiography following the placement of injections mainly within the neocortex, neostriatum, or cerebellum have revealed patterns of bidirectional axonal labeling in various thalamic, monoaminergic, deep cerebellar and precerebellar nuclei that were similar to that seen after similarly placed horseradish peroxidase injections. Small injections of N-[acetyl- 3H] wheat germ agglutinin, by way of an extremely limited extracellular spread of the tracer, yield large amounts of autoradiographic retrograde and anterograde axonal labeling. High specific activities, in addition to the sensitivity displayed by this tracer, allow the use of relatively short autoradiographic exposure times that still lead to an extensive signal over labeled neurons. Biochemical analysis of this radiolabeled derivative of wheat germ agglutinin was carried out using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results from Coomassie blue staining and fluorography of the gels have revealed that affinity-purified N-[acetyl- 3H] wheat germ agglutinin migrates predominantly as a monomer (molecular weight 18,000 ± 1000 daltons) as well as a dimer (35,100 ± 1000 daltons) when compared with native wheat germ agglutinin and molecular weight standards. Gels run on the native preparation, prior to derivatization, radiolabeling and affinity purification, in addition to containing species that co-migrate with the monomeric and dimeric forms of the lagged lectin, also reveal several other bands with molecular weights above and below those values for the monomer and dimer. When scintillation counting was performed on fresh gels of N-[acetyl- 3H] wheat germ agglutinin, the predominant species was again found to be the monomeric and dimeric forms; however, small peaks were also present around 12,000 daltons as well as above 43,000 daltons. Coomassie blue staining and fluorography never revealed banding below 18,000 daltons, though some trace of labeling was present, but not in the form of discrete bands, above 36,000 daltons. The presence, albeit small in comparison to the monomer and dimer, of these other higher and lower molecular weight species might in part result from fragments produced by proleolytic digestion of intact subunits of N-[acetyl- 3H] wheat germ agglutinin that may or may not possess intact binding sites. These results are discussed in light of previous neuroanatomical and biochemical observations made on wheat germ agglutinin, including the notion that the heterogeneity of tagged wheat germ agglutinin molecules is responsible for previously reported anterograde or retrograde axonal labeling generated after injections of this lectin. The proven efficiency and sensitivity of N-[acetyl- 3H] wheat germ agglutinin for generating extensive autoradiographic labeling of afferent and efferent pathways is discussed with regard to its usefulness in axonal tracing studies for revealing precise patterns of interneuronal connectivity that exist within the central nervous system.