In vitro retrograde neuritic transport of horseradish peroxidase isoenzymes by sympathetic neurons

Superior cervical ganglia from newborn rats were cultured as expiants in compartmental culture dishes consisting of three growth chambers separated by a grease barrier. During 2 weeks of culture, extensive neurite sprouting occurred across the grease barrier. Uptake and retrograde transport of horseradish peroxidase isoenzymes by these sympathetic neurons were studied by selectively exposing one culture chamber to the protein for 2 h. The expiants were examined by electron microscopy after cytochemical processing, and morphometric analyses were performed on the samples. In the neurite terminals, the labeled organelles included coated and uncoated vesicles, tubules, and vacoules. With concentrations of 1 and 4 mg/ml of horseradish peroxidase, approx 4 times as many vesicles were labeled by horseradish peroxidase isoenzyme C as horseradish peroxidase isoenzyme A. When the neuronal somata were examined for retrograde transport of the protein (at 1 mg/ml), 10 of 86 cells showed labeling with horseradish peroxidase-C, and only one of 99 cells was labeled with horseradish peroxidase-A. Based on organelle counts of the labeled cells, eight times as many organelles were labeled by horseradish peroxidase-C as horseradish peroxidase-A. Thus, cultured sympathetic neurons express similar selectivity of retrograde neuritic transport of horseradish peroxidase isoenzymes as other in vivo axonal systems so far studied. This selectivity appeared to operate at the levels of both initial uptake and subsequent neuritic transport. The compartmental culture technique should be useful for studies requiring isolation of neuronal somata from neurite terminals.