Prolonged activation of the N -methyl- d -aspartate receptor–Ca ²⁺ transduction pathway causes spontaneous recurrent epileptiform discharges in hippocampal neurons in culture

Abstract

The molecular basis for developing symptomatic epilepsy (epileptogenesis) remains ill defined. We show here in a well characterized hippocampal culture model of epilepsy that the induction of epileptogenesis is Ca 2؉ -dependent. The concentration of intracellular free Ca 2؉ ([Ca 2؉ ] i ) was monitored during the induction of epileptogenesis by prolonged electrographic seizure activity induced through low-Mg 2؉ treatment by confocal laser-scanning f luorescent microscopy to directly correlate changes in [Ca 2؉ ] i with alterations in membrane excitability measured by intracellular recording using whole-cell current-clamp techniques. The induction of long-lasting spontaneous recurrent epileptiform discharges, but not the Mg 2؉ -induced spike discharges, was prevented in low-Ca 2؉ solutions and was dependent on activation of the N-methyl-D-aspartate (NMDA) receptor. The results provide direct evidence that prolonged activation of the NMDA-Ca 2؉ transduction pathway causes a long-lasting plasticity change in hippocampal neurons causing increased excitability leading to the occurrence of spontaneous, recurrent epileptiform discharges.Epilepsy or the occurrence of spontaneous recurrent epileptiform discharges (SREDs, seizures) is one of the most common neurological conditions, affecting more than 2% of children and 1% of adults (1). Between 30% and 50% of epilepsy is symptomatic (1, 2), being caused by a known etiology that produces a permanent plasticity change in a previously normal brain, causing recurrent seizures (3). Much of our current knowledge concerning the pathophysiology of epilepsy has been derived from studies of human brain tissue from patients undergoing epilepsy surgery and various animal models of epilepsy (4-7). In several models of epilepsy, SREDs can be induced to occur for the life of the animal or preparation. The process of inducing SREDs or symptomatic epilepsy in previously normal neuronal networks is called epileptogenesis.Calcium is a major second-messenger system that regulates many neuronal processes (8), and alterations in calcium homeostasis have been implicated in the induction of epileptogenesis (9). Furthermore, indirect evidence has suggested that N-methyl-D-aspartate (NMDA) receptor activation may contribute to the induction of altered neuronal excitability in the kindling (10), hippocampal slice (11), and hippocampal neuronal culture (7) models of epilepsy. Thus, it is important to determine whether the induction of epilepsy is clearly dependent on elevated intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) and NMDA receptor activation by directly measuring both [Ca 2ϩ ] i and neuronal excitability during epileptogenesis. This investigation was initiated to determine whether the induction of epileptogenesis in a well characterized in vitro hippocampal neuronal culture (HNC) model of epilepsy (7) was calcium-dependent. This model of epilepsy is well suited to study the role of calcium in epileptogenesis, since it utilizes an episode of continuous seizure activity for...

Cellular mechanisms underlying acquired epilepsy: The calcium hypothesis of the induction and maintainance of epilepsy

“…Inhibition of the AMPA, KA, and metabotropic glutamate receptors had no antiepileptogenic effects in either of these culture models ( Fig. 6; DeLorenzo et al, 1998;Sun et al, 2002). This in vitro model of SE-induced AE is a powerful tool to evaluate the effects of agents on the development of AE.…”

“…Blockade of the NMDA receptor with MK801 or APV in both of these primary culture models inhibited the future development of SREDs DeLorenzo et al, 1998;Sun et al, 2002; Fig. 6 and Fig 7).…”

Evidence that injury-induced changes in hippocampal neuronal calcium dynamics during epileptogenesis cause acquired epilepsy

“…In addition, SE without the development of AE did not result in long-term changes in [Ca 2ϩ ] i , further indicating a relationship between the development of AE and the alteration in Ca 2ϩ dynamics. These results in an intact animal model of AE support in vitro models of AE that directly demonstrated the role of elevated [Ca 2ϩ ] i in producing spontaneous recurrent seizures after SE and glutamate excitotoxic injuries in hippocampal neurons (8)(9)(10)(11)(12)(13). The in vitro models of AE allow for a rigorous control of environmental conditions to directly demonstrate the role of altered Ca 2ϩ dynamics in causing AE.…”

“…The molecular basis for developing AE is still not completely understood. However, there is growing evidence from the SE and glutamate injury-induced models of AE that elevated intracellular calcium concentration ([Ca 2ϩ ] i ) and altered Ca 2ϩ -homeostatic mechanisms (Ca 2ϩ dynamics) may play a role in the development of AE (6,(8)(9)(10)(11)(12)(13). In addition, altered Ca 2ϩ dynamics have been observed in the hippocampus of chronic epileptic animals as long as 1 year after the induction of seizures in the in vivo pilocarpine model of AE (14).…”

Models of drug-induced epileptiform synchronization in vitro

“…If they are plated at appropriate densities they form interconnected networks that can be used for epilepsy research (Dichter, 1978). As mentioned above, such primary neuronal cultures are susceptible to convulsant treatments (Giachello et al, 2013) or application of low-Mg 2+ medium (De Lorenzo et al, 1998;Wang et al, 2014). Growing cultures on multi-electrode arrays can help make long-term recordings from this preparation (Potter and De Marse, 2001).…”