A biphasic increase in cytosolic Ca^<2+> concentration induced by NMDA in cultured hippocampal neurons

We have investigated the changes in cytosolic Ca^<2+> concentration ([Ca^<2+>]_1) induced by N-methyl-D-aspartate (NMDA) in cultured hippocampal neurons. NMDA produced a biphasic increase in [Ca^<2+>]_1,consisting of an initial increase (INC1) occurred at several seconds after the start of application and a subsequent increase (INC2) after the end of application. The latter INC2 found in the present paper was larger and longer lasting than INC1,and suggested playing an important role in the long-term potentiation (LTP). The time course of INC2 showed two different patterns

(i) a rise began to decrease within 30 sec after the end of application of NMDA, (ii) continuted its increase more than 3min. To analyse the characteristics of these two different increses, we tried to fit normal distribution functions to the histograms of the maximum increase in [Ca^<2+>]_1. These two increases in INC1 and INC2 were obtained by subtracting the resting values from the peak value in INC1 and INC2,respectively. The histogram for INC1 was fitted by a sum of the normal distribution functions with one large peak value and with two small peaks. On the other hand, INC2 was well represented by a function composed of two normal distribution ones. Pretreatment of neurons with islet-activating protein (IAP) suppressed INC2 completely in about one-third of experimented neurons. In the rest of neurons treated with IAP, one of the two normal distribution functions disappeared. The increase in [Ca^<2+>]_1 in INC2 was suppressed by 40% by IAP. From those findings, it was suspected that one of mechanisms producing INC2 was coupled with IAP-sensitive GTP-binding protein.