Morphological differentiation of embryonic rat sympathetic neurons in tissue culture: II. Serum promotes dendritic growth

Abstract

In the preceding paper, we reported that embryonic rat sympathetic neurons formed axons, but not dendrites, when they were maintained in the absence of serum and nonneuronal cells. To assess the effects of serum-derived factors on cellular morphology, cultures were initially maintained in serum-free medium while nonneuronal cells were eliminated. Subsequently some cultures were chronically exposed either to fetal calf serum (10%) or to a high-molecular-weight ammonium sulfate fraction of serum (P40 material, 500 μg/ml). Phase-contrast microscopy revealed that serum and P40 material did not alter neuronal survival, but did cause flattening of the somata and fasciculation of processes. When neurons exposed to serum or P40 material were injected with Lucifer Yellow, it was found that the majority (⩾90%) had local, tapered processes that could be identified as dendrites by light microscopic criteria. These local processes also exhibited other dendritic characteristics in that (1) they reacted with monoclonal antibodies to nonphosphorylated forms of the M and H neurofilament subunits and to microtubule-associated protein 2; and (2) they had substantial amounts of RNA as determined by [ 3H]uridine autoradiography. Quantitative measurements of the effects of serum and P40 material on dendritic morphology revealed that (1) an 8-day exposure caused most neurons (>80%) to form dendrites; (2) neurons typically had more than one dendrite (mean of 4.1 ± 0.2 dendrites/cell after a 28-day exposure); and (3) the dendrites were relatively short with the maximum extent of the dendritic arbor being 110 ± 13 μm after 4 weeks. Serum and P40 material did not routinely cause the formation of supernumerary axons, did not alter radial axonal outgrowth from ganglion explants, and did not significantly increase [ 3H]leucine incorporation. Thus, serum contains a factor (or factors) which selectively stimulates the extension of dendrites, but not axons. If such a factor were operative in situ, it could play an important role in determining the morphology of sympathetic neurons. In examining the mechanism of serum-induced dendritic growth, we found that even high concentrations (5 μg/ml) of nerve growth factor failed to promote dendritic growth in the absence of serum; thus, nerve growth factor by itself is not a sufficient condition for the extension of dendrites. It was also observed that the percentage of cells forming dendrites in either serum-free or serum-containing media was unaffected by (1) agonists and antagonists known to bind to various cholinergic and adrenergic receptors; or (2) agents (25 m M K +, tetrodotoxin) known to affect electrical activity in vitro. Thus, serum-induced dendritic growth does not require normal electrical activity or afferent input.