Differential Involvement of Ca2+ Channels in Survival and Neurite Outgrowth of Cultured Embryonic Cockroach Brain Neurons

Abstract

The contribution of voltage-gated calcium channels (VGCC) to the development of cultured embryonic cockroach brain neurons was assessed using pharmacological agents. VGCC currents were recorded using the patch-clamp technique and were found to be blocked dose-dependently by micromolar concentrations of mibefradil. The activation and inactivation properties of the calcium channels enable a sizeable calcium current to flow at rest (about −30 and −20 mV in high-potassium culture media). As expected, the cytoplasmic-free calcium concentration was found to rise when the extracellular potassium concentration was raised from 3 to 15 and 30 mM. The effects of VGCC blockers and calcium chelators were different in fresh and in mature cultures in which the neurons were connected to each other to form a defined network. In fresh cultures, the two non-selective VGCC blockers (verapamil and mibefradil) induced a dose-dependent cell death that was proportional to their blocking effect on I Ba. This effect could not be prevented by addition of fetal calf serum to the culture medium. A similar effect was obtained using intra- or extracellular calcium chelating agents (10 μM BAPTA-AM or 10 mM EGTA). Quite unexpectedly, blockade of the P/Q-like (ω-Aga WA-sensitive) component of the calcium current by 500 nM of ω-AgaTx IVA had no lethal effect, suggesting that the corresponding channels are not involved in the survival mechanism. As expected from their lack of effect on I Ba, isradipine, nifedipine, and ω-CgTx GVIA did not induce cell death. When the neurons started growing neurites, their sensitivity to calcium channel blockade by mibefradil decreased, indicating a correlation between neurite outgrowth and resistance to calcium depletion. In mature cultures, the neurons became resistant to mibefradil, verapamil, and BAPTA-AM. However, these agents, as well as ω-AgaTx IVA, had a significant inhibitory effect on the increase in diameter of the connectives that linked adjacent clusters of neurons. This effect has been shown to result, in the case of mibefradil, from an inhibition of neurite outgrowth characterized by a significant reduction of the number of primary neurites and secondary branchings but not to a significant modification of the diameter of individual neurites. These results support the view that, as in vertebrates, calcium influx through VGCC plays an important role in survival and neurite outgrowth of cultured embryonic insect neurons. The differential contribution of the P/Q-like and R-like (ω-Aga WA-sensitive) calcium channels in these processes is discussed.