Quantitative morphological analysis of embryonic cockroach (Periplaneta americana) brain neurons developing in vitro

Neurons dissociated from the brain of embryonic cockroaches (Periplaneta am ericana) can be maintained in culture for several weeks. The survival as we ll as the progressive organization of the neurons into a complex network wa s studied during a 5-week period under different culture conditions. About 10% of the dissociated cells adhered to the culture dish. This figure remai ned constant throughout the culture. The cell diameter ranged from 10 to 20 mu m and did not change significantly over time in culture. Whereas only a few cells exhibited neurites at the start of the culture, the number of ce lls exhibiting neurites increased to reach about 99% after 2 weeks. The dif ferent cells were then connected to each other, forming a network, which be came more and more complex. The number of cells per cluster as well as the length and the diameter of the "connectives" that Linked the different clus ters were found to increase with time. The morphology of individual neurons within the network was visualized after intracellular injection of biocyti n. Labeling with antibodies raised against serotonin or GABA indicated that neurons were able to differentiate and to acquire specific neurotransmitte r fates. The serotonergic phenotype was found to appear progressively throu ghout the culture, in parallel with the formation of the network. Cell dens ity, addition of fetal calf serum, and ecdysone were shown to influence the development of the network.