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

Neurons dissociated from the brain of embryonic cockroaches (Periplaneta americana) can be maintained in culture for several weeks. The survival as well as the progressive organization of the neurons into a complex network was studied during a 5-week period under different culture conditions. About 10% of the dissociated cells adhered to the culture dish. This figure remained constant throughout the culture. The cell diameter ranged from 10 to 20 microns 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 cells exhibiting neurites increased to reach about 99% after 2 weeks. The different cells were then connected to each other, forming a network, which became 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 clusters were found to increase with time. The morphology of individual neurons within the network was visualized after intracellular injection of biocytin. Labeling with antibodies raised against serotonin or GABA indicated that neurons were able to differentiate and to acquire specific neurotransmitter fates. The serotonergic phenotype was found to appear progressively throughout the culture, in parallel with the formation of the network. Cell density, addition of fetal calf serum, and ecdysone were shown to influence the development of the network.

Effects of nicotinic and muscarinic ligands on embryonic neurones of Periplaneta americana in primary culture: a whole cell clamp study.

The pharmacological properties of acetylcholine (ACh) receptors of cultured neurones from embryonic cockroach brains were studied using the whole-cell configuration of the patch-clamp technique. More than 90% of the studied neurones responded to ACh by a monophasic inward current, the intensity of which varied from cell to cell. The sequence of potency of the five tested agonists was ACh > nicotine=carbamylcholine > suberyldicholine=oxotremorine. The dose-response relationship was complex, suggesting the existence of two populations of receptors: high-affinity receptors (extrapolated K(d) around 10(-7) M) and low-affinity receptors (extrapolated K(d) around 5x10(-5) M). The current-voltage relationship of the induced current was linear between -80 and -40 mV and the extrapolated reversal potential was not significantly different from 0 mV. The sequence of decreasing potency of the antagonists of the ACh response was: methyllycaconitine > alpha-bungarotoxin > mecamylamine > curare > strychnine > bicuculline > atropine > picrotoxin. These results show: (1) that, in embryonic brain neurones, the response to ACh corresponds to the opening of non-selective cationic channels; and (2) that the pharmacology of the ACh receptors is mainly but not solely nicotinic. The nature of the single events which underlie this response, as well as the structure of the channels (homo or hetero-oligomeric) remain to be investigated.