Induction of spontaneous recurrent epileptiform discharges causes long-term changes in intracellular calcium homeostatic mechanisms

Calcium and calcium-dependent systems have been long implicated in the induction of epilepsy. We have previously observed that intracellular calcium (Ca2+]i) levels remain elevated in cells undergoing epileptogenesis in the hippocampal neuronal culture (HNC) model. In this study, we employed the hippocampal neuronal culture (HNC) model of in vitro 'epilepsy' which produces spontaneous recurrent epileptiform discharges (SREDs) for the life of the neurons in culture to investigate alterations in Ca2+]i homeostatic mechanisms that may be associated with the 'epileptic' phenotype. Ca2+]i imaging fluorescence microscopy was performed on control and 'epileptic' neurons with two different fluorescent dyes ranging from high to low affinities for Ca2+]i. We measured baseline Ca2+]i levels and the ability to restore resting Ca2+]i levels after a brief 2-min exposure to the excitatory amino acid glutamate in control neurons and neurons with SREDs. Neurons manifesting SREDs had statistically significantly higher baseline Ca2+]i levels that persisted for the life of the culture. In addition, the 'epileptic' phenotype was associated with an inability to rapidly restore Ca2+]i levels to baseline following a glutamate induced Ca2+]i load. The use of the low affinity dye Fura-FF demonstrated that the difference in restoring baseline Ca2+]i levels was not due to saturation of the high affinity dye Indo-1, which was utilized for evaluating the Ca2+]i kinetics at lower Ca2+]i levels. Peak Ca2+]i levels in response to glutamate were the same in both 'epileptic' and control neurons. While Ca2+]i levels recovered in approximately 30 min in control cells, it took more than 90 min to reach baseline levels in cells manifesting SREDs. Alterations of Ca2+]i homeostatic mechanisms observed with the 'epileptic' phenotype were shown to be independent of the presence of continuous SREDs and persisted for the life of the neurons in culture. Epileptogenesis was shown not to affect the degree or duration of glutamate induced neuronal depolarization in comparing control and 'epileptic' neurons. The results indicate that epileptogenesis in this in vitro model produced long-lasting alterations in Ca2+]i regulation that may underlie the 'epileptic' phenotype and contribute to the persistent neuroplasticity changes associated with epilepsy.