Ca2+ and pH affect the neurite formation in cultured mollusc isolated neurones

Neurite formation in neurones isolated from adult molluscs in culture has been shown to depend on the total content of Ca in the cells, intracellular Ca2+ concentration, intracellular acid-alkaline balance, extracellular pH, and the capacity and composition of buffers. The neurones with a low total Ca content prior to cultivating (1.2 mmol/kg) and low buffer capacity of cytoplasm (pH artificially shifted to the acidic level) possess the most pronounced capability of neurite regeneration. Optimal media for neurite regeneration appear to contain sodium bicarbonate as a buffer either alone or with small additions of organic buffers (1.5-3 mM) at pHs increasing from 7.6 to 8.2 under equilibrium with air. In the absence of sodium bicarbonate, when only organic buffers are used (Tris-HCl; HEPES-Na2CO3), at constant pH values ranging from 7.5 to 8.2, no neurites are formed. Artificial enhancement of intracellular Ca2+ concentration at the beginning of culture completely inhibits neurite outgrowth, and when applied on the third to fifth days of culture, it causes retraction of the neurites already formed. Neurones loaded with calcium (10 mmol/kg) form no neurites regardless of medium composition and concentrations of buffers used at pHs ranging from 7.5 to 8.2. The results obtained allow to suggest that neurite regeneration is controlled by Ca2+- and pH-regulating intracellular systems.