Retrograde tracing of neural pathways with a protein gold complex. II. Electron microscopic demonstration of projections and collaterals.

This paper describes a sensitive method for tracing neural connections at the electron microscopic (EM) level using a new compound produced through the coupling of colloidal gold particles to a wheat germ agglutinin horseradish peroxidase conjugate (the WGAHRP-gold complex). Visualization of retrogradely labeled cells at the EM level was achieved either directly by gold particles scanning or after silver enhancement. By using different sizes of gold particles individually coupled to WGAHRP and injected in different brain areas EM detection of multiple retrograde labeling was possible. Thus retrogradely labeled cells were first identified at the light microscopic level through HRP histochemistry with tetramethylbenzidine as a chromogen and then examined under the electron microscope after osmication and embedding. Gold particles were readily identified as electron dense, round dots in spherical grey vesicles. Identification of different sizes of gold particles often localized in the same vesicle established that the protein-gold complex can be used to study collateralisation of parental axons.