Mechanisms of heterogeneity of calcium response to kainate and neuronal types in rat cortical primary culture

Abstract

The dynamics of intracellular Ca2+ signal in response to NMDA (N-methyl-D-aspartate, 30 μM) or KA (kainite, 30 μM), its dependence on extracellular Ca2+ and the mechanisms of KA-triggered Ca2+ entry into neurons have been tested in neurons of rat cortical primary cultures. The level of intracellular free Ca2+ concentrations ([Ca2+]i) was evaluated on Leica SP5 MF confocal microscope using Fluo-3 fluorescent dye, which resolves changes in [Ca2+]i in the micromolar range. The dynamics of [Ca2+]i increase in response to NMDA and KA was different but in both cases the [Ca2+]i increase required the presence of Ca2+ in the extracellular solution. The neuronal population was found to be heterogeneous, based on the response to KA applied together with either L-type calcium channel blocker nifedipine (3 μM) or IEM-1460 (3 μM), a blocker of Ca2+-permeable AMPAR (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) lacking GluR2 subunit. Experiments exhibited three types of calcium responses, characteristically belonging to interneurons (expressing Ca2+-permeable AMPAR), pyramidal neurons (with AMPAR containing GluR2, making them impermeable to Ca2+), and intermediate type of cells expressing both AMPAR types. Thus, we have demonstrated the role of AMPAR and L-type calcium channels in KA-triggered Ca2+ entry into neurons. The dynamics of [Ca2+]i during the KA treatment was shown to depend on subunit composition of particular AMPAR subtype expressed in neurons. The data suggest that neuronal types existing in adult cortical tissue are probably presented in primary culture, too.