In vitro growth of locust embryonic pars intercerebralis neurosecretory cells

Abstract

Neurosecretory brain cells from embryonic locusts cultured in serum-free medium failed to show any visible signs of growth. In contrast, the same neurons co-cultured with CNS explants (brain-retrocerebral complexes and thoracic ganglia) show excellent axonal growth: sprouting occurs after one day of co-culture and increases within the first week. These results indicate the production of an active neunte outgrowth stimulating factor by co-cultured CNS explants. The similarity of the stimulating effects by the two explants on neurite outgrowth rule out brain neurohormones as probable candidates for the stimulating factor. In addition, neither insulin nor neuroparsin added to the culture medium to test their trophic effect improves the growth of the cells. Conditioned medium derived from cultures of brain-retrocerebral complexes produced no neurite outgrowth, suggesting that the active factor released in the medium by brain explants does not remain free in solution but binds to the substratum. Finally, neurons co-cultured with CNS explants attached to the bottom of the culture dish develop neurites only when in close proximity to the explants. This observation strongly suggests the binding of an active neurite outgrowth stimulating factor to the substratum in the vicinity of the explants. As a control for CNS explants, the action of non-nervous tissue was tested: a similar, but less extensive neurotrophic effect, was observed with esophagus segments co-cultured with neurosecretory brain cells. These results demonstrate that locust neurosecretory neurons isolated in cell culture require combined explants for elaborating processes and suggest that the neurite promoting effect is mediated by a substrate-associated molecule(s). Furthermore, the present study describes a defined culture system of neurosecretory neurons for general use in physiological and biochemical experiments.