Effects of local anesthetics and tetrodotoxin on Cultured Neuron of Lymnaea stagnalis

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To know whether the increase in intracellular Ca^<2+> concentration by lidocaine produces neurotoxicity, we compared morphological changes and Ca^<2+> concentration using fura-2 imaging in the culture neuron of Lymnaea stagnalis.We used BAPTA-AM, a Ca^<2+> chelator, to prevent the increase of the intracellular Ca^<2+> concentration and Calcimycin A23187,a Ca^<2+> ionophore, to identify the relationship between increased intracellular Ca^<2+> concentration and neuronal damage without lidocaine. Morphological changes were confirmed using trypan blue to stain the cells.Increasing the dose of lidocaine increased the intracellular Ca^<2+> concentration ; however, there was no morphological damage to the cells in lidocaine at 3x10^<-3> M. Lidocaine at 3x10^<-2> M increased the intracellular Ca^<2+> concentration in both the saline (from 238±63 to 1038±156 nM) and the Ca^<2+>-free medium (from 211±97 to 1046±169 nM) and produced morphological damage and shrinkage with a rugged surface. By the addition of BAPTA-AM, lidocaine at 3x10^<-2> M moderately increased the intracellular Ca^<2+> concentration (from 150±97 to 428±246 nM) and produced morphological damage. These morphologically changed cells were stained to dark blue with trypan blue dye. Ca^<2+> ionophore resulted in an extreme increase of the intracellular Ca^<2+> concentration ; however, no morphological damage was observed.These results indicated that the morphological damage was induced by lidocaine itself rather than by the increase in the intracellular Ca^<2+> concentration. Controlling the increase in the intracellular Ca^<2+> concentration is not enough to prevent the neurotoxicity of lidocaine.