Phenotypic properties of catecholamine-positive cells that differentiate in avian neural crest cultures

We have investigated several phenotypic features of the catecholamine-positive (CA+) cell population that develops in quail neural crest cultures. The number, spatial distribution, and morphology of CA+ and tyrosine hydroxylase-positive (TH+) cells are similar at all ages examined, suggesting that these 2 cell classes are identical. Neither CA+ nor TH+ cell bodies or processes were stained using antisera that recognize the 70 or 160 kDa subunits of chicken neurofilament protein. Other cell bodies and fibers in the cultures (which were CA- and TH-) were stained with these neurofilament antisera. The uptake and storage of 3H-norepinephrine by neural crest cultures containing CA+ cells were inhibited in the presence of desmethylimipramine and by incubation at 0 degrees C, but were unaffected by normetanephrine. Overnight treatment with reserpine eliminated histochemically detectable CA fluorescence from the cultures. Chronic reserpine treatment from day 2 to 7 in vitro prevented the appearance of CA+ cells, while normal numbers of TH+ and somatostatin-like immunoreactive (SLI) cells developed. The number and light-microscopic morphology of the CA+ cells that developed in these cultures were not dramatically altered by either exogenous NGF or 6-hydroxydopamine. Using the method of Grillo et al. (1974), we have demonstrated that the CA+ cells observed in the light microscope corresponded to cells containing abundant cytoplasmic granular vesicles (GV) characteristic of catecholamine storage granules observed in other systems. The GV diameters were quite similar in cells examined after 5, 7, 14, and 21 d in vitro. Most GV were 50-200 nm in diameter and were distributed in a unimodal manner, with the observed modal values in the range of 85-115 nm at the ages examined. The number of GV/micron2 of cytoplasmic area remained quite constant at all ages examined. These data, taken together with other available information, suggest that the CA+ cells that differentiate in our neural crest cultures resemble, in many respects, the small, intensely fluorescent cells found in autonomic ganglia and extra-adrenal chromaffin tissue of many species. At present, we do not know if the CA+ cells that differentiate in our neural crest cultures are a stable endpoint of development or whether they are a developmental intermediate in adrenergic differentiation that is normally observed only transiently during the development of avian sympathetic ganglia in vivo, but that can persist under our tissue culture conditions.