Development of sympathetic neurons in compartmentalized cultures

Sympathetic neurons from the superior cervical ganglia of newborn rats were plated in the center compartments of three-compartment dishes and the effects of the local concentration of 7S nerve growth factor (NGF) on neurite extension and density in the side compartments were observed. Omission of rat serum and ascorbate from the culture medium in the side compartments proved favorable for the observation of neurite growth there, while these ingredients were supplied to the neurons via the center compartments. Under these conditions, 7S NGF concentrations ranging 0.01–10 μg/ml produced little difference in the rate of neurite extension (about 1 mm/day). However, a substantial influence on fiber density was observed, with higher densities produced in side compartments given 1–10 μg NGF/ml than in opposite side compartments of the same cultures given 0.1 μg/ml or less. This was observed irrespective of whether the center compartment containing the cell bodies was provided with 1 μg/ NGF/ml or no NGF. Furthermore, neurite growth was not apparent in NGF-deprived center compartments, while neurites exposed to NGF regenerated after neuritotomy in the side compartments. This suggests that the promotion of growth by NGF was largely (perhaps entirely) confined to the distal neurite regions directly exposed to extracellular NGF, while the growth of more proximal neurite regions (which may be presumed to contain NGF en route to the soma via retrograde transport) was promoted little or not at all in the absence of extracellular NGF. The results of this work show that the NGF concentration in the local environment of the neurites exerts important control over their local growth and strongly supports the hypothesis that tissue NGF levels regulate sympathetic innervation in vivo.